Avian Influenza Etiology, Pathogenesis and Interventions

PUBLIC HEALTH IN THE 21ST CENTURY SERIES
AVIAN INFLUENZA: ETIOLOGY,

PATHOGENESIS AND INTERVENTIONS
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AVIAN INFLUENZA: ETIOLOGY,

PATHOGENESIS AND INTERVENTIONS
SALOMON HAUGAN
AND
WALTER BJORNSON
EDITORS
Nova Biomedical Books

New York
Copyright 2010 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical
photocopying, recording or otherwise without the written permission of the Publisher.
For permission to use material from this book please contact us:
Telephone 631-231-7269; Fax 631-231-8175
Web Site: http://www.novapublishers.com
NOTICE TO THE READER

The Publisher has taken reasonable care in the preparation of this book, but makes no expressed
or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of
information contained in this book. The Publisher shall not be liable for any special,
consequential, or exemplary damages resulting, in whole or in part, from the readers use of, or
reliance upon, this material. Any parts of this book based on government reports are so indicated
and copyright is claimed for those parts to the extent applicable to compilations of such works.
Independent verification should be sought for any data, advice or recommendations contained in
this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage
to persons or property arising from any methods, products, instructions, ideas or otherwise
contained in this publication.
This publication is designed to provide accurate and authoritative information with regard to the
subject matter covered herein. It is sold with the clear understanding that the Publisher is not
engaged in rendering legal or any other professional services. If legal or any other expert
assistance is required, the services of a competent person should be sought. FROM A
DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE
AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
Library of Congress Cataloging-in-Publication Data
Avian influenza : etiology, pathogenesis, and interventions / [edited by] Salomon Haugan and Walter Bjornson.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-61761-566-5 (E-Book)
1. Avian influenza. I. Haugan, Salomon. II. Bjornson, Walter.
[DNLM: 1. Influenza in Birds. 2. Influenza, Human--prevention & control. 3. Disease Outbreaks--prevention & control.
WC 515 A9573 2009]
RA644.I6A946 2009
636.5'0896203--dc22
2009025487
Published by Nova Science Publishers, Inc. New York

Contents
Preface vii
Chapter I Interspecies Transmission of Avian Influenza Virus
(H3N2) to Dogs 1
Daesub Song, Bokyu Kang, Chulseung Lee, and Bongkyun Park
Chapter II Conventional and Experimental Vaccines against Avian Influenza 27
Ami Patel, Gary Wong, Mickey Sahib and Gary P. Kobinger
Chapter III Worldwide Preparedness to Prevent Eruption of Pandemic Flu and
to Control Pandemic Spread After its Emergence 49
Yoav Arnson and Yaron Bar-Dayan
Chapter IV Molecular Pathogenesis of Avian Influenza and Prospect of
Therapy Using Small Interfering RNA 69
Jeanne Adiwinata Pawitan
Chapter V Avian Influenza: Intervention and Therapy 83
Hongxuan He, Kai Zhou
Chapter VI Infection Control for Avian Influenza (H5N1) in Healthcare
Settings 97
Anucha Apisarnthanarak and Linda M. Mundy
Chapter VII U.S. and International Responses to the Global Spread of Avian Flu 115
Tiaji Salaam-Blyther and Emma Chanlett-Avery
Chapter VIII Avian Influenza: Agricultural Issues 159
Jim Monke
Chapter IX Potential Risks of Vaccination against Avian Flu Epidemics 167
Shingo Iwami and Yasuhiro Takeuchi
vi Contents
Expert Commentary
Preparation and Production of Prepandemic and Pandemic
Influenza Vaccine: A Personal View 195
Akikazu Sakudo, Toyokazu Ishikawa and Kazuyoshi Ikuta
Index 201
Preface
Avian influenza, sometimes avian flu, and commonly called bird flu, refers to "influenza
caused by viruses adapted to birds." Of greatest concern is highly pathogenic avian influenza
(HPAI). These influenza viruses occur naturally among birds. Wild birds worldwide carry the
viruses in their intestines, but usually do not get sick from them. However, avian influenza is
very contagious among birds and can make some domesticated birds, including chickens,
ducks, and turkeys, very ill. Infected birds shed influenza virus in their saliva, nasal
secretions, and feces. Susceptible birds become infected when they have contact with
contaminated secretions or excretions or with surfaces that are contaminated with secretions
or excretions from infected birds. The risk from avian influenza is generally low for most
people, because the viruses do not usually infect humans. However, confirmed cases of
human infection from several subtypes of avian influenza infection have been reported since
1997. The spread of avian influenza viruses from one ill person to another has been reported
very rarely, and has been limited, inefficient and unsustained. This important book gathers
the latest research from around the globe in this field.
Chapter I - Interspecies transmission is a crucial feature in the ecology and epidemiology
of influenza virus. Transmission of avian influenza virus to a new mammalian species is of
great concern, because it potentially allows the virus to adapt to a new mammalian host, cross
new species barriers, and acquire pandemic potential. Infection of an entire avian influenza
virus to an unrelated mammalian species is a rare event. Until now, several outbreaks of
avian influenza infection have occurred in mammals. Several cases of infection in mammals
by avian origin influenza viruses (H7N7, H4N5, H5N1, H3N2) have been reported.
Especially, avian influenza viruses are occasionally transmitted to other bird species,
particularly poultry, and to aquatic (seals, dolphins, whales) or terrestrial mammals (horses,
pigs, mink). Also in humans, cases of infection by a number of avian influenza viruses
transmitted main from poultry have been documented.
Here, the authors provide a current advance in our knowledge of interspecies
transmission of avian influenza virus to dogs at serological and molecular level, and give an
overview of available data on the intra- and interspecies virus transmission and pathogenicity.
Chapter II - Avian influenza H5N1 virus, family Orthomyxoviridae, naturally persists in
waterfowl and domestic bird reservoirs with sporadic outbreaks of highly pathogenic strains.
Several human cases were reported during the 1997 H5N1 avian epidemic in Hong Kong,
viii Salomon Haugan and Walter Bjornson
showing direct transmission from domestic poultry and the first occurrence of an H5
influenza subtype in humans. Highly pathogenic avian influenza (HPAI) H5N1 variants later
re-emerged following years of circulation in wild bird reservoirs and new human cases were
identified in Southeast Asia during 2003. Evidence suggests that the H5N1 virus is rapidly
evolving and although HPAI H5N1 has not yet adapted for efficient human-to-human
transmission, it is currently considered a major threat for a global influenza pandemic. The
World Health Organization (WHO) and several nations have prioritized improving available
inactivated or LAIV, and the development of alternative platforms against potential influenza
outbreaks. While currently approved vaccines have been successful against influenza viruses
of the same subtype, complete cross-protection has yet to be achieved. This chapter reviews
different vaccine strategies against avian influenza H5N1, reflects on the requirements for
effective vaccine development, and discusses the direction of future influenza vaccine
research. The rapid development of several experimental platforms in recent years has
enhanced protective efficacy and immunogenicity following immunization, additionally
benefiting understanding of influenza virus pathogenesis. The most promising platforms have
been evaluated successfully in ferrets and non-human primate models, with several
candidates currently in human clinical trials. The objective of influenza vaccine research will
be to develop a universal, single vaccine candidate capable of complete cross-protection
against divergent influenza subtypes.
Chapter III - Avian influenza or "bird flu" is causing increasing concern across the world
as experts are preparing for the possible occurrence of the next human influenza pandemic.
Countries worldwide are preparing for the arrival of the virus in wild birds and poultry within
their territories. All countries need to prepare for the possible arrival of human cases of
influenza imported through foreign travel.
Preparedness for biological threats requires awareness, planning, organization,
infrastructure and equipment stocking, education of personnel, and conducting drills as well
as availability, willingness and perceived self efficacy of the staff to respond in due time.
International collaboration has a key impact on successful medical preparedness. Cooperation
and coordination between countries is needed in the verge of a pandemic.
Most health authorities initiated disease prevention and containment policies. The World
Health Organization (WHO) is the basic coordinating and supervising force behind global
preparedness. The WHO has described the preparedness measures needed to be taken in the
pre-pandemic stage, during primary detection of highly pathogenic avian influenza (HPAI)
and at the pandemic stages. Countries worldwide have prepared multi-factorial programs
dealing with the subjects. The preparedness and contingency plans differ among different
countries and regions due to different resources availability, local experience with the
disease, specific local challenges and limitations. Many countries suffer from under-endorsed
and untested planes. In those areas suffering from lack of effective pandemic control plans,
the regional cooperation is also lacking.
This article reviews status of the worldwide preparedness to prevent eruption of
pandemic flu and to control pandemic spread after its emergence.
Chapter IV - Small interfering RNA (siRNA) technology is now available to switch off
a target gene. Many studies reported promising results of siRNA in combating viral infection
Preface ix
in animals, including avian influenza infection. This review will discuss the molecular
pathogenesis and the prospect of siRNA for the therapy of avian influenza infection.
Chapter V - In an avian flu pandemic, which methods could be used to treat or prevent
infection with influenza A (H5N1) virus? Foremost are antiviral drugs and vaccines, which
have already been used to prevent and treat human influenza A and B virus infections.
Although formally approved for other indications (i.e., treatment of hepatitis C), interferon
might also be useful for controlling avian flu. As has been shown for other viral infections,
RNA interference could be a powerful means with which to suppress the replication of avian
H5N1. Combined use of the currently available methods should be taken into account and
attempts should be made to develop new strategies directed at unexplored targets such as the
viral proteins hemagglutinin and viral polymerase (and endonuclease) and non-structural
protein.
Chapter VI - The re-emergence of avian influenza (H5N1) in Southeast Asia has
heightened concern for a potential influenza pandemic. Global pandemic preparation for
avian influenza (H5N1) has begun and it is imperative for healthcare workers (HCWs), who
in most cases serve as first responders, to be part of preparedness training. As relevant to
other transmissible agents, HCW preparedness training should include an understanding of
the modes and risks of avian influenza (H5N1) transmission and how to implement
appropriate infection control strategies to prevent and control of spread of avian influenza
(H5N1). In this chapter, the authors review the evidence for avian influenza (H5N1)
transmission, identified infection control strategies for both resource-adequate and resource-
limited settings, and post-exposure management of avian influenza (H5N1) for HCWs.
Healthcare epidemiology and infection control strategies include vaccination and
chemoprophylaxis of exposed HCWs, access to stockpiled oseltamivir, surveillance for
unrecognized cases and coordinated preparedness response plans by interdisciplinary groups
such as local and regional health departments, HCWs, healthcare administrators and
epidemiologists. The preparedness plans must include rapid creation of temporary isolation
facilities, restricted access to pre-identified HCWs, involvement of specialists for screening
and early case identification and continuous monitoring for optimal infection control
practices and regular feedback to involved HCWs. Although human-to-human transmission
of avian influenza (H5N1) has rarely occurred, vigilant preparedness and implementation
plans are essential in thwarting a potential avian influenza (H5N1) pandemic.
Chapter VII - One strain of avian influenza currently identified in Asia and Europe is
known as Influenza A/H5N1. Although it is a bird flu, it has infected a relatively small
number of people killing around 50% of those infected. Scientists are unsure if H5N1 will
cause the next influenza pandemic, but there is general consensus that one is overdue. Flu
pandemics have occurred cyclically, roughly between every 30 and 50 years. Since 1997,
when the first human contracted H5N1 in Hong Kong, the virus has resurfaced and spread to
more than a dozen countries in Asia and Europe infecting more than 140 people and
killing approximately half. Britain and Taiwan both reported avian flu cases of H5N1 in
2005. In the latter cases, the infected birds were identified as imports, and died in quarantine.
A global influenza pandemic could have a number of consequences. Global competition
for existing vaccines and treatments could ensue. Some governments might restrict the export
of vaccines or other supplies in order to treat their own population. Some countries might
x Salomon Haugan and Walter Bjornson
face a shortage of vaccines, antiviral medication, or other medical equipment, because of

limited global supply. Hospitality and airline industries, and international trade could be
negatively impacted. If global travel and trade were to suddenly drop, there could be
productivity losses and service disruptions. Essential workers might become ill or stay home
out of fear of contracting the virus. Such workers could include law enforcement, medical
personnel, mass transit drivers and engineers, and other crucial emergency personnel.
For FY2006, Congress has provided $25 million for global initiatives to prepare for
pandemic influenza through Foreign Operations appropriations; directed $33.5 million to
global disease detection through Labor, HHS, and Education appropriations; and reserved for
international avian flu efforts a portion of $3.8 billion through Defense appropriations.
Bills introduced in the 109th Congress would increase U.S. resources allocated to the
global fight against avian flu; develop a Pandemic Fund to augment ongoing U.S. and
international avian flu and pandemic preparedness initiatives; increase funding for preventing
the spread among animals of the H5N1 virus; and strengthen surveillance capacity within
affected countries.
This chapter provides an up-to-date account of global H5N1-related human infections
and deaths, outline U.S. government and international responses to the global spread of
H5N1, discuss situations in various countries affected by H5N1, and present some foreign
policy issues for Congress.
Chapter VIII - Since the fall of 2003, a strain of highly pathogenic avian influenza
(H5N1) has spread throughout Asia, infecting mostly poultry but also a limited number of
humans. In recent months, the virus has spread into parts of Europe. Controlling avian flu in
poultry is seen as the best way to prevent a human pandemic from developing, by reducing
the number of animal hosts in which the virus may evolve.
Avian flu can be highly contagious in domestic poultry. Strict biosecurity measures are
practiced among commercial poultry farms and are encouraged by governments. The
economic effects of any avian influenza outbreak can be significant, especially given
international trade restrictions. This report will be updated as events warrant.
Chapter IX - Highly pathogenic H5N1 influenza A viruses have spread relentlessly
across the globe since 2003. They are associated with widespread death of poultry,
substantial economic loss to farmers, and reported infections of more than 300 people with a
mortality rate of 60%. Influenza prevention and containment strategies can be considered
under the broad categories of antiviral, vaccine, and non-pharmaceutical measures. In
particular, using vaccination to reduce the transmission rate might provide an alternative to
mass culling by reducing both the susceptibility of healthy birds and the infectiousness of
infected birds. However, although vaccination can be a useful tool for control of avian
influenza epidemics, it might engender the emergence of a vaccine-resistant strain. Field and
experimental studies show thatsome avian influenza strains acquire resistance against
vaccination. The authors investigated, in the context of the emergence of a vaccine-resistant
strain, whether a vaccination program can prevent the spread of infectious disease. Our main
findings are that such a program might lead to an emergence and replacement of the vaccine-
resistant strain over a large geographical region, and that a vaccination to prevent the spread
of disease can instead spread the disease. Thus, if the vaccinations are not used appropriately,
prevention and control will be negatively affected by the vaccination program. Further, from
Preface xi
our theoretical studies, the authors propose how a vaccination against avian influenza should
be used.
Expert Commentary - Herein, the authors presented a personal view regarding the recent
advances and future perspectives on facilitating influenza virus isolation, vaccination
efficiency, and monitoring of vaccine production. Hopefully, readers such as researchers and
manufacturers involved in influenza vaccine production will be motivated by this personal
commentary, obtain information for their own research, and be inspired by new ideas for
future research on influenza vaccine.
Copyright 2010 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical
photocopying, recording or otherwise without the written permission of the Publisher.
For permission to use material from this book please contact us:
Telephone 631-231-7269; Fax 631-231-8175
Web Site: http://www.novapublishers.com
NOTICE TO THE READER

The Publisher has taken reasonable care in the preparation of this book, but makes no expressed
or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of
information contained in this book. The Publisher shall not be liable for any special,
consequential, or exemplary damages resulting, in whole or in part, from the readers use of, or
reliance upon, this material. Any parts of this book based on government reports are so indicated
and copyright is claimed for those parts to the extent applicable to compilations of such works.
Independent verification should be sought for any data, advice or recommendations contained in
this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage
to persons or property arising from any methods, products, instructions, ideas or otherwise
contained in this publication.
This publication is designed to provide accurate and authoritative information with regard to the
subject matter covered herein. It is sold with the clear understanding that the Publisher is not
engaged in rendering legal or any other professional services. If legal or any other expert
assistance is required, the services of a competent person should be sought. FROM A
DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE
AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
Library of Congress Cataloging-in-Publication Data
Avian influenza : etiology, pathogenesis, and interventions / [edited by] Salomon Haugan and Walter Bjornson.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-60741-846-7 (hardcover)
1. Avian influenza. I. Haugan, Salomon. II. Bjornson, Walter.
[DNLM: 1. Influenza in Birds. 2. Influenza, Human--prevention & control. 3. Disease Outbreaks--prevention & control.
WC 515 A9573 2009]
RA644.I6A946 2009
636.5'0896203--dc22
2009025487
Published by Nova Science Publishers, Inc. New York

In: Avian Influenza: Etiology, Pathogenesis and Interventions ISBN: 978-1-60741-846-7
Editors: S. Haugan and W. Bjorson, pp. 1-26 2010 Nova Science Publishers, Inc.
Chapter I
Interspecies Transmission of Avian

Influenza Virus (H3N2) to Dogs
Daesub Song, Bokyu Kang, Chulseung Lee, and Bongkyun Park1

Research Unit, Green Cross Veterinary Products, Kiheung, Yongin, South Korea
Department of Veterinary Virology, College of Veterinary Medicine and School of
Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea1
Abstract
Interspecies transmission is a crucial feature in the ecology and epidemiology of
influenza virus. Transmission of avian influenza virus to a new mammalian species is of
great concern, because it potentially allows the virus to adapt to a new mammalian host,
cross new species barriers, and acquire pandemic potential. Infection of an entire avian
influenza virus to an unrelated mammalian species is a rare event. Until now, several
outbreaks of avian influenza infection have occurred in mammals. Several cases of
infection in mammals by avian origin influenza viruses (H7N7, H4N5, H5N1, H3N2)
have been reported. Especially, avian influenza viruses are occasionally transmitted to
other bird species, particularly poultry, and to aquatic (seals, dolphins, whales) or
terrestrial mammals (horses, pigs, mink). Also in humans, cases of infection by a number
of avian influenza viruses transmitted main from poultry have been documented.
Here, we provide a current advance in our knowledge of interspecies transmission of
avian influenza virus to dogs at serological and molecular level, and give an overview of
available data on the intra- and interspecies virus transmission and pathogenicity.
Introduction
Transmission is the process by which the virus is shed from one animal and infects the
next, causing a serological response. Further, host to host transmission within a species may
occur or not. More important is adaptation, which means that the virus has become adapted to
2 Daesub Song, Bokyu Kang, Chulseung Lee et al.
a certain species so that it is fit for replication in that host and sustained interspecies
transmission. Most viruses infect and replicate in their specific host and establishment of
new, long-lived host-specific lineages of certain virus in certain new host is uncommon and
has rarely occurred, however, there are several viruses to adapt to more than two host species,
for example, influenza virus, rotavirus, parvovirus, human immunodeficiency virus, and
simian foamy virus. Infection to non-host species is occasionally acquired through
interspecies transmission from original hosts by chance or by specific natural living system.
They changes and evolve their nucleotide sequences and host ranges to survive efficiently.
They may exchange their nucleic information with other viruses or host cells, sometimes, lose
some part of sequence by environmental affect. The more frequent the virus contacts these
conditions, the more chances they have to evolve and adapt to non-host species. In case of
canine parvovirus, only a few amino acid substitutions located in receptor binding site make
their host range to be changed (Parrish, 1991, Truyen, 1999).
Influenza A viruses have a wide range of hosts, including birds as well as mammals
(Webster et al., 1992, Songserm et al., 2006a, Songserm et al., 2006b). The transmission
route in mammals is dependent on aerosols-nasal chains, in contrast to infection in birds
where infection by fecal-oral cycle prevails. In general, influenza virus does not produce
disease in their natural host, wild birds. Influenza virus that transmitted and infected domestic
poultry may be divided into two groups based on their clinical severity; highly pathogenic
avian influenza (HPAI) virus and low pathogenic avian influenza (LPAI) virus. Regardless of
pathogenicity of viruses, the cases of AI virus infection in domestic poultry seem to result
from the introduction of influenza virus from wild birds. Once introduced into domestic
poultry, the AI viruses may change their nucleotides and adapt to poultry species to ensure
sustained horizontal transmission within flocks. Low pathogenic AI viruses that have been
introduced to poultry from wild birds may mutate into high pathogenic viruses after certain
time of circulation in the poultry flock. There may be a lot of opportunity to infect from wild
birds to domestic poultry by low pathogenic AI viruses where domestic birds live freely,
share water with wild birds, or use water that might be contaminated by droppings from
infected wild bird (Murphy et al., 1982, Beare & Webster, 1991). Except open habitat of
domestic poultry flock, so-called live bird markets, where various kinds of live birds are
traded, are one of the most important sources of spread (Capua et al., 2003, Henzler et al.,
2003).
Transmission of avian influenza virus to the other species, overcoming species barrier
and leading to the development of clinical disease is a rare event (Shortridge et al., 1998,
Bulaga et al., 2003), however, their interspecies transmission has always been a great
concern. Although the influenza viruses have been transmitted to different mammal species
on several occasions, these events may make new epidemic lineages. For example, if an avian
influenza subtype, which has never infected to human or certain mammal, was introduced to
its new host, following cycles of replication and adaptation, it might spread efficiently among
the new hosts. Even though the mechanical process or molecular determinants to make
interspecies transmission possible has not fully elucidated, several studies indicated that the
relationship between the hemagglutinin protein of the virus and its receptor on the host cell is
essential to make the virus to infect a specific host (Ito, 2000, Ito & Kawaoka, 2000). In
particular, pigs are known to be involved in intermediate host for interspecies transmission of
Interspecies Transmission of Avian Influenza Virus (H3N2) to Dogs 3
influenza A viruses as mixing vessels for generation of reassortant viruses that have the
potential to jump from one species to another (Ito et al., 1998, Campitelli et al., 1997),
because they have receptors to both avian and human influenza strains (Ito et al., 1998). In
Europe, a H1N2 virus, a human-avian reassortant virus was first isolated in the U.K. in 1992
and continuously gaining ground (Brown et al., 1998), and avian-like H1N1 viruses are
highly prevalent in pig populations (Heinen, 2002). In the U.S., a triple reassortant H3N2
between the classical H1N1, the human H3N2 and avian subtypes is circulating (Olsen,
2002). Recently, swine-human-avian triple reassortant H1N2 and H3N2 subtype originated
from North America was reported to circulate in Korean pig farm (Lee et al., 2008). On the
basis of these evidences, it was thought that except pigs, AI virus infection to the other
terrestrial mammals including humans could be occurred only via the pig as mixing vessels
for a long time. In 1997, however, a highly pathogenic AI virus, H5N1 subtype, has crossed
species barrier and 18 H5N1 infected peoples died. Since then, several cases of H5N1
infection and one case of H7N7 infection to human have occurred. Natural infection with
H5N1 was first described in tigers and other large cats in zoo after feeding with virus-positive
chicken carcasses (Keawcharoen et al., 2004, Quirk, 2004), and cat to cat transmission has
occurred in the same zoo (Thanawongnuwech et al., 2005).
Previously, outbreaks of hemorrhagic pneumonia caused by equine influenza virus
H3N8) were noted in racing dogs (Chang et al., 1976) and a human influenza virus (H3N2)
was isolated from dogs (Crawford et al., 2005). Avian origin influenza (H5N1) infection was
identified in a dog after ingestion of a duck infected with subtype H5N1 during an outbreak
in Thailand in 2004 (Songserm et al., 2006b). Nevertheless of some cases of several different
subtype AI virus infections to dog, canine influenza virus was known to be originated from
equine influenza virus, subtype H3N8 until now. This virus, subtype H3N8, was proven to be
the etiology of respiratory disease of dog by experimental inoculation studies. Geographic
expansion, persistence of infection and evidence of pet dog infection supported the efficient
transmission of the virus among greyhounds. Molecular changes in hemagglutinin between
canine and equine virus was identified to suggest adaptive evolution in new host. Most direct
transmissions of whole influenza viruses from the original host species to a different one do
not result in adaptation in the new host species. As mentioned previously, more frequent
virus-host contacts are necessary for replication and horizontal transmission in the new host
(Webby et al., 2004). In that study, they reported an unprecedented interspecies transfer of a
complete equine influenza virus to the dog, and the emergence of a new canine specific
influenza virus associated with acute respiratory disease. In 2002, it was reported that an
outbreak of severe respiratory disease in a pack of English foxhounds in the United Kingdom
was caused by an equine influenza A virus, subtype H3N8 (Daly et al., 2008). The study also
demonstrated that dogs possess the relevant receptors for infection with equine influenza
virus in their respiratory tissues.
More recently, avian influenza virus, subtype H3N2, was first isolated from serial cases
of severe respiratory disease in dogs exhibiting severe respiratory disease, and transmission
among dogs was demonstrated by experimental reproduction of disease (Song et al., 2008).
We also demonstrated that dogs have large amount of avian influenza virus binding receptor
in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for
direct transmission of avian influenza virus (H3N2) from poultry to dogs. Not only
experimentally infected dogs but also contact-exposed dogs showed elevated rectal
temperatures, virus shedding, seroconversion, and severe necrotizing tracheobronchitis and
bronchioalveolitis (Song et al., 2009). Moreover, serological surveillance ascertained the
prevalence of the novel influenza virus in dog population in South Korea (Lee et al.,2009).
In this chapter, we aimed at presenting a novel AI virus causing clinical manifestation in
dogs and establishing intraspecies transmission, and genetic characteristics different from
equine infleuenza virus, subtype H3N8 or low pathogenic avian influenza virus, subtype
H3N2. On the basis of these evidences, we tried to explain the difference and new aspect of
interspecies transmission of avian influenza virus to dogs, which is different from previously
reported cases of avian influenza virus from wild birds to domestic poultry, equine infleuenza
virus from horses to dogs, or high pathogenic AI virus from birds to human.
Molecular Analysis for Avian Influenza Virus of

Interspecies Transmission
Pandemics of Influenza
There were 4 pandemics of influenza due to the emergence of antigenically different

strains in humans: 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1) (Reid et al.,
1999, Scholtissek et al., 1978, Schafer et al., 1993). And many fear that the Asian H5N1
avian influenza virus (AIV) in 1997 will become the next pandemic virus (Goldfield et al.,
1977). Cross-species transfers of swine and avian influenza to human have been documented
on several occasions. Besides human infections, most known mammalian infections with
highly pathogenic avian influenza virus H5N1 (HPAIV H5N1) have occurred in fields.
Sia-Gal Glycosidic Linkage for Avian and Human Influenza
Receptor specificity of the HA is important in determining host range and changes. The
HA protein mediates virus binding to sialic acid (SA)containing host cell surface molecules
and promotes the release of viral ribonucleoprotein complexes through membrane fusion.
There are 2 SA species influencing the viral infectivity: (N-acetylneuraminic acid [NeuAc]
and N-glycolylneuramic acid [NeuGc]) and the type of linkage to galactose
(sialyloligosaccharides terminated by SA linked to galactose by an 2,6 linkage [Ac2,6Gal]
or an 2,3 linkage [Ac2,3Gal]) on the host cell surface. Human influenza viruses
preferentially recognize sialyloligosacchrides containing SA2,6Gal(Rogers & Paulson,
1983, Rogers et al., 1983b), matched by mainly NeuAc2,6Gal linkages on the epithelial
cells of the human trachea (Couceiro et al., 1993). By contrast, avian viruses preferentially
recognize SA2,3Gal sialic acids(Rogers & Paulson, 1983, Rogers et al., 1983b), in
accordance with the predominance of sialyoligosaccharides with SA2,3Gal linkages on the
epithelial cells of duck intestine.
HA Cleavability
The HA protein is synthesized as a precursor protein that is cleaved into 2 subunits, HA1
and HA2 by host cell proteases. HA cleavability is a clear link with viral infectivity (Garten
& Klenk, 1999). Low pathogenic avian influenza viruses possess a single Arg residue at the
cleavage site, recognized by extracellular, trypsin-like proteases. These proteases are thought
to be secreted only by cells of the respiratory and intestinal tract and consequently limit
infections to these organs. By contrast, multiple basic amino acids at the HA1HA2
connecting peptide (RERRRKKR/G) have been appeared highly pathogenic avian influenza
viruses and contemporary H5N1 viruses (Webster et al., 2002). Multiple basic amino acids at
the cleavage are recognized by ubiquitous, intracellular, subtilisin-like proteases that thus
trigger systemic infection. In addition, HA cleavability is affected by the absence or presence
of a carbohydrate side chain near the cleavage site that may interfere with the accessibility of
host proteases to the cleavage site (Kawaoka et al., 1984). The gaining of a highly cleavable
HA converted an avirulent strain to virulence in Pennsylvania in 1983 (H5N2), Mexico in
1994 (H5N2), Italy in 1997 (H7N1), Chile in 2002 (H7N3), and Canada in 2004 (H7N3). HA
cleavability is, therefore, considered the major determinant of tissue tropism of avian
influenza viruses (Horimoto & Kawaoka, 1994).
Amino Acid Residues of the Sia-Gal Glycosidic Linkage for AIV
The H5N1 viruses which transmitted from chickens to humans in Hong Kong in 1997
were shown to retain specificity for SA2,3Gal (Matrosovich et al., 1999). Sequence
comparison, receptor specificity assays, and crystallographic analysis have identified amino
acid residues that determine receptor specificity: Gln-226 (found in avian viruses) determines
specificity for SA2,3Gal, whereas Leu-226 correlates with SA2,6Gal specificity in human
H2 and H3, but not H1, viruses (Rogers et al., 1983a, Matrosovich et al., 2000). In all human
viruses (with the few exceptions of early isolates from the Asian influenza outbreak
(Matrosovich et al., 2000), Leu-226 is associated with Ser-228, while Gln-226 is associated
with Gly-228 in avian viruses. For H1 viruses, Asp-190 (found in human and swine virus
isolates) or Glu-190 (found in avian virus isolates) determines preferential binding to 2,6 or
2,3 linkages, respectively (Gamblin et al., 2004, Stevens et al., 2004, Matrosovich et al.,
2000, Kobasa et al., 2004),
Internal Genes and Molecular Marker for AIV
Since 1997, studies reveal continued evolution of H5N1 that include changes in
antigenicity and the internal gene constellation, extended host range in avian species, an
ability to infect felids, enhanced pathogenicity in mice and ferrets, and increased
environmental stability (Beigel et al., 2005, Tiensin et al., 2005, WHO., 2005). Indeed, it is
the ability of the virus to extend its host range to migratory birds that is responsible for the
current rapid spread of the virus to birds in Central Asia, Europe, the Indian subcontinent,
and Africa.
Molecular markers located in viral internal genes have been used to predict the
transmissibility of H5N1 viruses in the mammalian species (Katz et al., 2000, Hatta et al.,
2001, Cheung et al., 2002, Seo et al., 2002). Highly pathogenic strains contained residues Ile-
223 in the NA, residue Ile-15 in M1 and residue Lys-198 in PB1 proteins, such as the Hong
Kong/483/97, Vietnam/1196/04 or Thailand/2(SP-33)/04. Residues Ile-223 in the NA,
residue Ile-15 in M1 and residue Lys-198 in PB1 proteins were identical to highly pathogenic
strains such as the Hong Kong/483/97, Vietnam/1196/04 or Thailand/2(SP-33)/04. Highly
pathogenic strains were identical to residues Ile-223 in the NA, residue Ile-15 in M1 and
residue Lys-198 in PB1 proteins such as the Hong Kong/483/97, Vietnam/1196/04 or
Thailand/2(SP-33)/04. In addition, Dk/CHN/E319-2/03 virus possessed Met-317 in PB1 and
Arg-355 in PB2, which were simultaneously observed in high and low pathogenic strains
(Table 3). Furthermore, Lys-627 in PB2 and Glu-92 in NS1 have been proposed to be
important determinants of the virulence of H5N1/97 viruses for mammals (Subbarao et al.,
1993, Hatta et al., 2001, Seo et al., 2002, Seo et al., 2004).
Receptor in Pigs
Pigs have an important role in interspecies transmission of influenza viruses. Swine

contain receptors for both human and avian viruses, and therefore, the potential for co-
infections with these viruses in swine can be occur (Ito et al., 1998). A receptor specificity
analysis indicated that all of the human and classic swine viruses preferentially recognize
NeuAc 2,6Gal, whereas most avian viruses prefer NeuAc 2,3Gal (Rogers & Paulson, 1983,
Rogers et al., 1983b). Surprisingly, the avian-like swine viruses showed a shift in receptor
specificity over time. Viruses isolated from European pigs up to 1984 recognized both SA-
galactose linkages, whereas those isolated after 1985 recognized only NeuA c 2,6Gal.
Amino Acid Residues of the Sia-Gal Glycosidic Linkage for SIV
Amino acid residues determine the shift in receptor specificity among avian-like swine
viruses. Comparison of the amino acid sequences of the HA molecules showed that an amino
acid change at residue 142 (145 in the H3 numbering system) was the only substitution that
occurred between 1983 and 1985 and was associated with loss of NeuAc 2,3Gal
recognition. Avian-like swine viruses isolated in 1985 or later (A/swine/Netherlands/12/85,
A/swine/Italy- Vir/671/87, A/swine/Germany/3/91, and A/swine/Schleswig- Holstein/1/92)
contained Leu at this position. On the other hand, those isolated earlier had different amino
acids: A/swine/Arnsberg/79 and A/swine/Netherlands/80, Ser; A/swine/Germany/2/81, His;
and A/swine/Belgium/83, Arg. Residue 142 (145 in the H3 numbering system) is located on
the loop of the HA near the receptor-binding pocket. A mutation at this position may have
contributed to a shift in receptor specificity (Ito et al., 1998).
Three Genotypes of the SIV
Three types of influenza viruses are circulating in pigs: classic H1N1, maintained in this
species for more than 60 years; human-like H3N2, present in pigs since 1969 (Kundin, 1970);
and avian-like H1N1, introduced into European pigs in 1979 (Garten & Klenk, 1999). The
first influenza A virus isolated from pigs was of the H1N1 subtype and related viruses of this
subtype are reported to have infected pigs in many countries (Roberts et al., 1987). Classical
swine H1N1 virus remained confined to North America until the 1970s when it was
introduced to Asia and Europe (Scholtissek et al., 1998). H1N2 viruses were isolated
previously in the United States in 1999, in France in 1987, in Japan from1978 to 1980, and in
the United Kingdom in 1994. Furthermore, since 1998, triple reassortant H3N2 influenza
viruses containing human, classical swine and avian virus lineage genes have been isolated
from pigs in the USA. These viruses had genes derived from human (HA, NA, and PB1) and
swine (NS, NP, and M) and avain (PB2 and PA) (Webby et al., 2000).
Swine H3N2 Triple-Reassortant Influenza Viruses
Recently, two antigenically distinct H3N2 reassortants were isolated from infected
animals: a double-reassortant virus containing genes similar to those of human and swine
influenza viruses, and a triple-reassortant virus containing genes similar to those of human,
swine and avian influenza viruses (Zhou et al., 1999). The triple-reassortant H3N2 viruses are
now endemic in swine population in North America (Webby et al., 2000). The triple
reassortant H3N2 influenza viruses containing human, classical swine and avian virus lineage
genes have been isolated from pigs in Canada, China, and Korea (Karasin et al., 2006, Yu et
al., 2008, Lee et al., 2008).
Amino Acid Residues of the Sia-Gal Glycosidic Linkage for H3N2 Triple-
Reassortant Viruses
H3N2 triple-reassortant viruses, which have the HA gene from human lineage viruses,
retain the receptor binding specificity to NeuAc2,6Gal receptors similar to human influenza
viruses. Val226 and Ser228 were expressed in the HA1 molecules of both turkey and swine
triple reassortants, while Leu/Ile226 and Ser228 are usually expressed in the human viruses
(Lindstrom et al., 1996). Leu, Ile, and Val are neutral non-polar amino acids, and
substitutions between them most likely maintain the hydrophobic interactions and the proper
conformation at the binding domain (Vines et al., 1998). Gln226 and Gly228 are usually
found in the HA1 molecules of avian viruses amino acids at these positions and are known to
play a critical role in determining the receptor binding specificity (Vines et al., 1998).
Isolation and Characterization of Avian Origin Canine Influenza Virus
Interspecies transmission is a crucial feature in the ecology and epidemiology of

influenza virus (Webster, 1998). The emergence of new virus subtypes and interspecies
transmission is of great concern, and measures adopted to counteract their spread are vital for
preventing influenza epidemics and pandemics. Among basic mechanisms of interspecies
transmission of influenza virus, direct transfer of an essentially unaltered virus from one
species to another can occur (Crawford et al., 2005); however, there are significant restricting
factors, in particular the presence or absence of host species-specific influenza virus binding
receptors in upper and lower respiratory tracts, that serve to prevent such cross-species or
zoonotic transmission events. Human influenza viruses bind to glycolipids or glycans that
contain terminal sialyl-galactosyl residues with 2,6 linkages (SA 2,6-gal), whereas avian
influenza viruses bind to residues with SA 2,3-gal linkages (Suzuki, 2005). Examples of
interspecies transmission of influenza viruses include recent infections in humans of the
H5N1 subtype of avian influenza virus, and in canines of the H3N8 equine influenza virus
(Crawford et al., 2005, Guan et al., 2004). However, most directly transmitted infections of
entire influenza viruses from a natural host species to a new host species do not result in
sustained transmission in the new host species (Crawford et al., 2005). Therefore,
establishing new, long-lived influenza virus lineage is uncommon and difficult (Webster et
al., 1992).
We reported an unprecedented interspecies transmission of a complete avian H3N2
influenza virus to dog, and the emergence of a new canine influenza virus associated with
acute respiratory disease. This occurred in South Korea where avian influenza viruses
(H3N2, H5N1, H6N1, and H9N2) currently circulate or were previously detected (Choi et al.,
2005). Pathogenicity of the isolated virus was investigated in experimental dogs, and
localization of SA 2,6-gal and SA 2,3-gal linkages was evaluated in upper and lower
canine respiratory tracts.
Transmission of avian influenza A virus to a new mammalian species is of great concern,
because it potentially allows the virus to adapt to a new mammalian host, cross new species
barriers, and acquire pandemic potential.
Transmission of an entire avian influenza virus to an unrelated mammalian species is a
rare event. There have been several outbreaks of avian influenza infection in mammals.
H7N7 influenza virus of avian origin was isolated from the lungs and brains of dead seals. In
addition, it was replicated to high titers in ferrets, cats and pigs, and caused conjunctivitis in
humans (Webster et al., 1981b, Webster et al., 1981a). Avian origin H4N5 was reported as
the cause of infection and mortality in harbor seals along the New England coastline
(Hinshaw et al., 1984), and avian origin H5N1 infection was identified in a dog after
ingestion of an H5N1-infected duck during an outbreak in Thailand in 2004 (Songserm et al.,
2006b).
Previously, outbreaks of hemorrhagic pneumonia caused by H3N8 equine influenza virus
were observed in racing dogs, and a human influenza virus (H3N2) was isolated from dogs.
However, these reports provided limited serological and virological evidence for influenza
virus infection in dogs (Chang et al., 1976, Houser & Heuschele, 1980). In this report, we
report the emergence of a new canine influenza virus that causes acute respiratory disease in
dogs and differs from previous outbreaks of H3N8 equine influenza virus infections.
Although, it was previously shown that dogs can be infected with influenza A viruses
(Crawford et al., 2005, Songserm et al., 2006b), this is the first report of avian H3N2
influenza infection in this species.
Concerning the possible mechanism of avian influenza virus transmission to dogs, we
posit that this transmission results from feeding dogs untreated minced meats of duck or
chickens. In Korea, untreated duck and chicken meats, including internal organs and heads,
have been widely used to feed dogs for fattening in local canine farms or kennels. In a
previous study, Korean H3N2 avian influenza virus was isolated from ducks and chickens
sold at live poultry markets. Live-bird markets are thought to constitute a missing link in the
epidemiology of avian influenza viruses, because they bring together numerous hosts, such
as chickens, ducks, turkeys, geese, and doves, in a high density setting which represents an
ideal environment for viral interspecies transmission (17, 18). S11 strain, whose HA and NA
genes showed the greatest identity to those of the canine A/canine/Korea/01/07 (H3N2)
isolate, was isolated from a tracheal swab of a healthy chicken, and is nonpathogenic in
poultry (Choi et al., 2005). These observations support the hypothesis that H3N2 avian
influenza viruses could be transmitted by feeding infected poultry by-products to dogs
(Webster, 1998).
It is also possible that cross-species transmission of influenza virus occurs by directly by
aerosol transmission from infected birds to susceptible dogs as a consequence of close
contact between the two species. Lectin staining results showed that canine upper (trachea
and bronchi) and lower (bronchiole) respiratory tract epithelium cells display SA 2,3-gal to
which avian influenza viruses bind, making possible a direct transmission of avian influenza
viruses from poultry to dogs.
Antigenic and phylogenetic analysis revealed that the HA and NA genes of the
A/canine/Korea/01/2007 (H3N2) isolate are closely related to Korean isolates identified in
2003 from chickens and doves. Furthermore, HA genes of canine influenza isolates were
different from recent Korean isolates from swine (Song et al., 2003). The other genes of the
canine influenza isolate are more closely related to those of the H9N2 isolate found in ducks
from Hong Kong, the H6N2 isolate from ducks in Japan, and several other avian influenza
strains from south eastern China in 2000 to 2005. This suggests that multiple variants of H3
influenza viruses may be circulating in these regions and causing diseases in pet dogs.
Experimental reproduction of the disease caused by this isolate induced severe
pathological changes in dogs consisting of necrosis and inflammation without extra-
pulmonary lesions. Additionally, the study showed that infected dogs excreted H3N2 virus in
nasal discharge but not in feces, suggesting that dog-to-dog transmission of H3N2 virus could
occur through the nasal route and that dog-to-dog transmission of the virus could play an
important role in the epizootiology of the disease.
In this study, virological, serological, pathological and phylogenetic analysis revealed
cross-species infection of an entire avian influenza A virus (H3N2) to another mammalian
species, dogs. Evidence of avian influenza virus infection in pet dogs raises the concern that
dogs may be become a new source of transmission of novel influenza viruses, especially
where avian influenza viruses are circulating or have been detected.
History
From May to September 2007, cases of severe respiratory disease were identified in
animals from three veterinary clinics located in Kyunggi Province and one kennel located in
Jeolla Province (southern part of Korea). Paired sera from 52 dogs from the kennel were
collected, and ninety percent (47/52) of these dogs seroconverted to canine H3N2 and 100%
were seropositive at the end of paired sera. The first case, which occurred in May, was
identified in a Miniature Schnauzer that exhibited symptoms of nasal discharge for 3 days
and sneezing for 2 days, after which the symptoms subsided and the dog recovered. Another
case, which occurred in August, was identified in a Cocker Spaniel that exhibited symptoms
of fever, cough, nasal discharge and anorexia, and died after the onset of clinical signs. In
September, severe respiratory disease was identified in two Jindo dogs, a native Korean breed
of hunting dogs known to have originated on Jindo Island, and one Yorkshire terrier. These
animals showed symptoms of severe cough, fever, and nasal discharge, and died 2 days after
visiting the same animal hospital. Finally, an outbreak of canine influenza occurred in an
animal clinic in which all of the 13 dogs housed in a shelter facility were shown to be
infected with the same virus and revealed clinical signs including nasal discharge, cough, and
high fever.
Figure 1. Phylogenetic relationship among hemagglutinin genes of canine influenza virus isolates. Tree
of HA genes from representative canine, human, avian, swine, and equine viral isolates. Phylogenetic
analysis indicated that the Korean canine influenza virus isolates belonged to a cluster different from
those of equine and canine H3N8 influenza viruses. The HA and NA genes of the canine isolate
(A/canine/Korea/01/07 (H3N2)) were closely related to those of Korean avian H3N2 viruses.
Reproduction of Pathgenicity in Dogs
Gross lesions were limited to the lungs, and were characterized by multifocal to
coalescing reddish consolidation. In DPI 3, 6 and 9 tissues, histopathological lesions were
observed in the trachea and lungs and in the absence of any extrapulmonary lesions, in
puppies infected with the isolate (A/canine/Korea/01/07 (H3N2)). Severe virus-induced
necrosis and inflammation of the upper (trachea and bronchi) and lower (bronchiole and
alveoli) respiratory tracts of dogs were observed by histological examination. Although minor
differences in the severity of the histological findings were observed among the 9 infected
dogs, all infected dogs shared the following histopathological features regardless of the
number of DPI: 1) moderate to severe multilobular or diffuse necrotizing tracheobronchitis
with suppurative inflammation in the lumina and squamous metaplasia of the
tracheobronchial epithelium (Figure 3B); 2) moderate to severe multilobular or diffuse
necrotizing bronchiolitis and alveolitis (i.e., bronchioalveolitis, occasionally accompanied by
chronic peribronchiolar and perivascular inflammation) (Figure 3D and E); and 3) mild to
moderate multilobular or diffuse thickening of alveoli septa by infiltrates of inflammatory
cells, such as interstitial pulmonary macrophages. At DPI 3, 6, and 9, large amounts of
influenza A virus antigens were found in bronchial and bronchiolar epithelium and lumens
(Figure 3F).
Overall, our analyses showed that avian-lineage H3N2 CIV had a narrow cellular tropism
for the respiratory tract as no extrapulmonary lesions and virus antigens were detected. The
detection of influenza virus antigens was limited to bronchial and bronchiolar epithelium and
lumens, occasionally involving alveolar septa and spaces (Song et al., 2008). This is different
from the multiorgan disease in the brain, spleen, lymph nodes, bone marrow, and liver that
H5N1 avian influenzae cause in humans and experimental animals (cats, ferrets and rodents)
(Korteweg & Gu, 2008). Notably, mild focal medullary renal hemorrhages were identified in
two of the nine infected dogs (22.2%). Since most avian influenza viruses are easily isolated
by using Madin-Darby canine kidney (MDCK) cells, we speculated that the H3N2 virus
would be able to damage kidney epithelial cells if the virus penetrates the pulmonary-blood
barrier (i.e. induces viremia). However, RT-PCR and virus isolation analyses revealed that
the fecal samples of the infected dogs were negative for the virus. Significantly, the kidney
lesions also lacked virus antigens. These observations, together with the mildness of the renal
lesions we observed, suggest that these lesions are not caused by CIV. Thus, it appears that
experimental H3N2 virus infection of dogs is limited to the respiratory tract.
The H3N2 virus caused a distinctively severe pneumonia to dogs that was unlike the
acute bronchopneumonia in pigs, ferrets and rodents that is induced by experimental swine
and human influenza virus infections (Jung et al., 2005, Svitek et al., 2008) and that is rapidly
followed by obvious recovery, such as the resolution of clinical symptoms (sneezing and
coughing) and pneumonic lesions. Instead, in dogs, CIV appears to cause a chronic, severe
pneumonia (Figures 1, 2a, 3 and 4). The severity of the disease may relate to the fact that CIV
is the result of recent interspecies transmission to a new host population that has never been
exposed to this virus previously and thus is seronegative.
The gross lung lesions induced by experimental H3N2 CIV infection were characterized
by severe reddish-tan consolidation, especially involving the intermediate lobes (Figure 1),
and were of similar severity at PIDs 3, 6 and 9. The histopathological changes were
particularly severe in the lower respiratory tracts (bronchiole and alveoli) and relatively
milder in the upper respiratory tracts (trachea and bronchi) (Figures 2a, 3 and 4). The findings
are consistent with the observation that canine bronchial and bronchiololar epithelial cells
bear larger amounts of the avian influenza-binding receptor [sialyl-galactosyl residues with
2,3-gal linkages (SA 2,3-gal)] than other areas of the respiratory tract (Song et al., 2008).
Severe suppurative, necrotizing tracheobronchitis that occurred diffusely in the upper
respiratory tract was observed in all dogs at PIDs 3, 6 and 9. In the initial stage of infection
(PID 3), the tracheal epithelium exhibited severe necrosis and exfoliation that was followed
by squamous metaplasia, and ciliated epithelial cells were rarely observed (Figure 2a).
Thereafter (PIDs 6 and 9), the tracheal epithelium exhibited recovery characterized by
hyperplasia of epithelial cells with dense nuclei but still accompanied mild necrotizing phase.
While ciliated epithelial cells were still rarely observed at PIDs 6 and 9, a few were observed
at PID 9. These histopathological changes probably facilitate the infection of respiratory
bacteria during the middle and later stages of influenza infection as well as during the initial
stage.
The tracheal epithelium was also infiltrated with mild to moderate numbers of
neutrophils that frequently had apoptotic bodies in their nuclei (Figure 2a). In contrast, the
propria-submucosa, including the tracheal glands, was infiltrated with large numbers of
lymphocytes and mononuclear leukocytes, and neutrophils to lesser extent; the mucus-
secreting cells of these tissues also exhibited mild necrosis. These findings suggest that the
H3N2 virus induces chronic-active persistent inflammation in the trachea, which is consistent
with the fact that nasal discharge and CIV shedding persist until PID 6 (Song et al., 2008).
We showed by in situ TUNEL assay and Toluidine staining that the neutrophils
infiltrating the tracheal epithelium were undergoing apoptosis (Figure 2b). In influenza
infections, neutrophil apoptosis generally occurs to maintain appropriate numbers of
neutrophils that can defend the body from secondary bacterial infections. Apoptosis plays an
important role in eliminating neutrophils from lesions without releasing hazardous
intracellular contents such as oxidants and myeloperoxidase. Our observations suggest that
neutrophils may be key inflammatory cells that drive the pathogenesis of H3N2 CIV in dogs.
The large numbers of neutophils in the tracheal tissues may be the result of excessive
production of neutrophil chemoattractant cytokines such as tumor necrosis factor- (TNF-),
interleukin (IL)-1, and IL-8, which are generally secreted by influenza-infected bronchiolar
epithelial cells and macrophages at the early and late stages of the infection, respectively
(Arndt et al., 2002). The cytokines rapidly attract neutrophils, which then act to remove
epithelial necrotic cell debris and defend the tissues from secondary infections.
The lower respiratory tract of all dogs at PIDs 3, 6 and 9 exhibited severe suppurative,
necrotizing bronchioalveolitis, i.e. bronchopneumonia. It was so severe that normal alveolar
spaces could not be observed even at the lowest microscopic magnification (x50) (Figures 3
and 4). Immunohistochemical analyses revealed that CIV antigens were mostly found in the
bronchial and bronchiolar epithelium and necrotic cells in the lumens and occasionally
alveolar epithelial cells such as type 2 pneumocytes; however, they were not identified in
neutrophils and macrophage-like mononuclear cells. This suggests that CIV is cytolytic to
pulmonary epithelial cells. The histological lesions were as severe as the proliferative and
necrotizing pneumonia (PNP) lesions found in porcine reproductive and respiratory

syndrome virus (PRRSV) disease, which is characterized by the accumulation of necrotic
debris in alveolar spaces, the thickening of alveolar septa by lymphohistiocytic inflammation,
and frequent lung fibrosis (Zimmerman, 2006). In CIV H3N2 infection, together with the
inflammatory (necrotizing) phase, a proliferative phase such as alveolar septa thickening
induced by inflammatory cell infiltrations was also observed concurrently in the pneumonic
lesions at PID 3, 6 and 9. It indicates that the H3N2 CIV induces atypical, chronic-active
bronchointerstitial pneumonia. The inflammatory cells that infiltrated the alveolar septa
consisted, in decreasing order of frequency, of alveolar macrophage-like mononuclear cells,
type 2 pneumocytes and, occasionally, lymphocytes. Neutrophils, most of which had a
bilobular nucleus but also frequently contained apoptotic bodies, mainly infiltrated the
alveolar spaces and, to a lesser extent, the lung parenchyma. In situ TUNEL assays confirmed
that the infiltrating neutrophils were undergoing the same apoptosis that was observed in the
tracheal epithelium. Our experimental dogs were negative upon culture for Bordetella
bronchiseptica, Pasteurella multocida and other bacterial pathogens that occur commonly in
the canine respiratory tract. These results suggest that the neutrophil infiltration is a
spontaneous response to H3N2 CIV rather than the consequence of bacterial superinfection.
In addition, diffuse mild to moderate pulmonary hemorrhage and hyaline membrane
formation were observed in four of nine infected dogs. In particular, mild pulmonary
vascultitis and perivascular hemorrhage and cuffing were observed infrequently (in three of
nine infected dogs).
120 40.5
Body te mp.( )
Virus she dding 40.0
100
Antibody tite r
Detection Rate (%), PI value
39.5
80
Body Temp.
39.0
60
38.5
40
38.0
20
37.5
0 37.0
0 1 2 3 4 5 6 7 8
Days Post Infe ction
Figure 2. Body temperature, virus shedding, and antibody seroconversion after challenge with canine
influenza virus. Body temperature was increased from DPI 1 and slowly decreased until DPI 7 to
normal temperature. Virus shedding was detected from DPI 1 to DPI 6 by RT-PCR. However, the
ELISA antibody titers were increased from DPI 6. Antibody titers were regarded as positive if PI value
was above 50.
Figure 3. Histopathological lesions in the trachea and lungs of (A and C) mock or (B and D to F)
influenza virus (A/canine/Korea/01/2007 (H3N2))-infected dogs at different post-inoculation days
(PID). (A) Mock-infected control dog at PID 9 showing normal pseudostratified columnar epithelium
lining of the trachea; original magnification 400. Hematoxylin and eosin (HE) stain. (B) Influenza-
infected dog at PID 9 showing necrotizing tracheitis characterized by necrosis (n), squamous metaplasia
(s), and hyperplasia of the epithelium and nonsuppurative inflammation (c) in the connective tissue;
original magnification 400. HE stain. (C) Mock-infected control dog at PID 3 showing normal alveoli;
original magnification 200. HE stain. (D) Influenza-infected dog at PID 3 showing severe diffuse
necrotizing bronchitis and bronchiolitis with suppurative inflammation in the lumina; original
magnification 100. HE stain. (E) Influenza-infected dog at PID 6 showing severe necrotizing
bronchiolitis; original magnification 200. HE stain. (F) Influenza-infected dog at PID 6 (serial section
of (E)) having large amounts of influenza A virus antigens (red stain; arrows) in the bronchiolar
epithelium and lumen. IHC; Fast red substrate; Mayers hematoxylin counterstain. (G) Influenza-
infected dog at PID 9 showing severe necrotizing alveolitis with accumulation of necrotic cells in
terminal bronchioles (tb) and alveoli (a); original magnification 200. HE stain.
The emergence of avian-lineage H3N2 CIV in dogs in South Korea is responsible not
only for economic loss and sorrow for pet owners; it is also a public health concern. We
reported previously that the dog populations in regions suffering an H3N2 CIV outbreak
exhibit high seropositivity (90 to 100%) to the virus, which indicates the ease with which this
virus is transmitted to other dogs. Our histopathology studies described here also suggest that
the severe, persistent pneumonia induced by H3N2 CIV may promote the severity (the
highest titer: mean 106.1 EID50/0.1 ml on PID 2 and 3) and duration (PID 1 to 6) of virus
shedding (Song et al., 2008), which ultimately promotes the efficacy of transmission to other
dogs. Our studies here also suggest that neutrophils and related chemoattractant cytokines
(TNF-, IL-1 and IL-8, etc.), which are normally elicited to provide defense against
secondary bacterial infections, may play a role in the pathogenesis of H3N2 CIV.
Figure 4. Lectin staining (red stain) for SA 2,3-gal (avian influenza virus receptors) and SA 2,6-gal
(human influenza virus receptors) in canine trachea, bronchus, and bronchioles, together with porcine
tissues as a positive control. Original magnification all x300. , no staining;, rare or few positive cells;
+, moderate numbers of positive cells; and ++, many positive cells.
Dog to Dog Infection with Avian Origin Canine

Influenza Virus (H3N2)
Susceptible dogs were brought into contact with dogs that had been experimentally
infected with an influenza A virus (H3N2) of avian origin designated A/canine/01/2007 that
had been isolated from a pet dog with severe respiratory syndrome. The experimentally
infected and contactexposed dogs all showed increased rectal temperatures, viral shedding,
seroconversion, and severe necrotizing tracheobronchitis and bronchioalveolitis.
Highly pathogenic avian origin canine influenza viruses (H3N2) have spread across
South Korea as from May 2007 through to December, 2007, transmission of these viruses in
South Korean animal clinics was observed repeatedly (Song et al., 2008). These viruses share
97% nucleotide sequence homology, which suggests the entire viruses were transmitted
directly from birds to dogs. To determine whether other dogs can be infected with these
viruses upon contact with an infected dog, we sought to experimentally contact-infect beagle
dogs. The dog to dog transmission of the virus raises questions about the interspecies
transmission of avian influenza viruses and the adaptation of these viruses to canine
physiology.
Transmission of virus from one host to another species is a important feature of the
ecology of the influenza virus (Webby et al., 1998). The influenza virus is generally
transmitted in an essentially unaltered form to other species by direct transfer. Examples of
this interspecies transmission mechanism include the recent human infections with the H5N1
subtype of avian influenza virus (Guan et al., 2004, Subbarao et al., 1998). Dogs infected
with avian subtype H3N2 have recently been identified in Korea, which suggests that an
avian influenza virus with high pathogenicity that can rapidly spread from dogs to dogs has
made the interspecies leap. It has been shown that most whole influenza viruses that are
directly transmitted from the natural host species to a different species do not achieve
sustained transmission in the new host species (Crawford et al., 2005). This suggests that
multiple virus-host interactions are needed before the virus can replicate and be transmitted
horizontally in a new host species (Webby et al., 2004). Here we show that close contact
between canine influenza virus-infected and uninfected dogs results in the spread of the virus
to the uninfected dogs, which then develop clinical signs of the disease.
We show that an avian origin canine influenza virus that was isolated from a pet dog can
spread from dog to dog by contact infection. A transient rise in rectal temperature was
observed in the challenge and exposure dogs. In addition, sero-conversion of the exposure
dogs was observed. These dogs also exhibited viral RNA in their nasal swabs and
histopathological changes in their upper and lower respiratory tracts. Our results demonstrate
that the avian origin canine influenza virus has adapted to canine physiology and can be
readily transmitted between dogs.
Figure 5. Virus shedding and the serological response of beagle dogs after contact transmission of
canine influenza virus.
Figure 6. Histopathology of dogs exposed to canine influenza virus (CIV) by contact with CIV-infected
dogs. Severe necrotizing, suppurative tracheitis and bronchioalveolitis were observed in the contact-
exposure group on days post inoculation (DPI) 13. However, CIV-associated lesions were not yet
present in these dogs on DPI 7. Original magnification all x200. Hematoxylin and eosin stain.
Genetic Characterization of Canine Influenza

Virus
The present study suggests that the H3N2 CIV [canine/Korea/01/07 (H3N2)] isolate has
2 surface protein (HA and NA) genes and 3 internal protein (M, NP and PB2) genes that
originated from the Ck/Korea/S06/03-like lineage LPM H3N2 viruses (genogroup A), an NS
gene that originated from Ck/Korea/LPM03/04-like lineage LPM H3N2 viruses (genogroup
C), a PA gene that originated from the Ck/Korea/LPM91/06-like lineage LPM H3N2 viruses
(genogroup D), and a PB1 gene that originated from the Dk/Hokkaido/120/01-like lineage of
wild, aquatic bird isolates (Figure 3). In summary, the CIV isolate might have evolved from a
novel Ck/Korea/S06/03-like LPM H3N2 virus that harbored at least 2 reassortment events of
the NS and PA genes between LPM viruses of the genogroups A, C, and D and 1
reassortment event of the PB1 gene that originated from Dk/Hokkaido/120/01-like lineage
viruses.
LPMs are places that are likely to be critical for the ecology and evolution of AIVs (Choi
et al., 2005). The dramatic evolution of H3N2 AIVs in the Korean LPM was not considered a
real threat, although it did raise some concern with regard to human public health, until
interspecies transmission generated the novel Ck/Korea/S06/03-like LPM H3N2, which
successfully infected dogs and induced fatal disease (Song et al., 2008). To our knowledge,
this is first time that a complete AIV has been infected and continuously transmitted to a new
species (avian to mammals) in nature where it evolved to become an epidemic in its new host,
i.e. dogs (Song et al., 2008, Song et al., 2009), although infections of H5N1, H7N7 and
H9N2 AIVs in humans and H4N6 AIV in pigs have been sporadically observed (Koopmans
et al., 2004, Peiris et al., 1999, Olsen, 2002, Peiris et al., 2004).
Figure 7. Nucleotide phylogenetic trees of the surface and internal protein genes of
A/canine/Korea/01/07 (H3N2) influenza virus. Abbreviations: Ab, aquatic bird; Ck, chicken; Dk, duck;
Md, migratory duck; Tk, turkey; Pb, pet bird.
Previous reports revealed that H3 AIVs in the Korean LPM have evolved dramatically by
undergoing frequent reassortments between aquatic bird isolates from south-eastern China
and dominant H3 AIVs in Korean chickens (Choi et al., 2005, Choi et al., 2004). This has
probably been aided by the migration of wild, aquatic birds from China to Korea during the
winter season. In fact, a recent report showed that H3N2 AIVs of four different genogroups
(A to D) emerged in Korean LPMs in poultry. The Ck/Korea/S06/03-like LPM H3N2
(genogroup A), which is dominant in Korean poultry, underwent continuous reassortment
events with wild aquatic bird isolates and created three different genogroups (B, C and D)
within H3N2 AIVs. In addition, our study further suggests the presence of a novel
Ck/Korea/S06/03-like LPM H3N2 virus generated through reassortment events between
viruses of genogroups A, B, and D. However, further monitoring is needed to see if the novel
virus is still circulating in domestic poultry, as well as in Korean dogs. In our study, we could
demonstrate neither the antigenic characteristics of the CIV isolate nor the antigenic
relationships with 4 different genogroup LPM H3N2 isolates. We tried to isolate H3N2 AIVs
from Korean LPM for the antigenic characterization of the CIV isolate but were unable to do
so. However, considering the similarities of the HA amino acid sequences between the CIV
and each representative LPM H3N2 isolate of genogroups A to D, we could speculate that
there are no significant differences in antigenicity between the CIV and LPM H3N2 isolates.
In summary, a novel Ck/Korea/06/03-like LPM H3N2 AIV was transmitted to dogs and
induced fatal respiratory disease in South Korea in 2007. Successful transmission of a whole
AIV to mammals was identified (Song et al., 2008). The remarkable evolution and perfect
adaptation of AIV to other mammal hosts, i.e. dogs, suggest the possibility of transmission of
AIVs to other mammal hosts such as humans. Our study advises continuous serological and
virological surveillance of H3N2 AIV and CIV in dog and human populations, as well as in
domestic poultry in LPM.
Figure 8. Genetic characterization of the full sequences of the eight gene segments of
canine/Korea/01/07 (H3N2) virus as compared with those of recent Korean LPM isolates during the
period 2003-2006. Abbreviations: Ab, aquatic bird; Ck, chicken; Dk, duck; Tk, turkey.
Prevalence of Canine Influenza Virus Infection in

Korea
Here, we report for the first time that avian H3N2 influenza is present at low but
detectable frequencies in farmed and pet dogs, as indicated by serological testing.
We observed that CIV-influenza was significantly more frequently prevalent in farmed
dogs than pet dogs (19% vs. 0.5%), as determined by the ELISA. However, it should be noted
that the 361 farmed dog samples included 52 samples from a farm in Cheonbuk that reported
an acute outbreak of CIV before sample collection. Anti-CIV antibodies were detected in
100% and 35% of these 52 dogs by ELISA and HI assays, respectively. In contrast, the
remaining dog farms in Chungbuk, Gangwon, Gyeongbuk, and Gyeongnam, which had not
suffered from CIV outbreaks, had seropositivity rates of 0-6%. Moreover, the other area that
evinced relatively high rates of seropositivity, the Moran market (its seropositivity rate was
11%), is where many domestic animals from diverse areas of South Korea are sold. Thus, it
appears that CIV presently tends to infect dogs in Korea in a sporadic fashion. Nevertheless,
the 100% seropositivity of the one farm suffering a CIV outbreak and the fact that most of the
farmed dogs lacked protective antibodies against the canine H3N2 virus strain that was used
in testing suggests the potential for an epidemic increase in canine H3N2 influenza virus
infections in dog farms.
The vast majority of the pet dogs that we examined lacked evidence of prior exposure to
CIV, as only 0.5% of the randomly selected 419 samples from animal hospitals were positive
for anti-CIV antibodies, as determined by both ELISA and HI assays. However, the 49
samples from four animal hospitals reporting an outbreak of CIV showed much higher
seropositivity rates (14.3%), as determined by both the HI and ELISA assays. Notably, while
none of the 12 serum samples from the S animal hospital initially had anti-CIV antibodies, as
determined by the HI and ELISA assays, all 12 animals seroconverted when we tested them a
week later, as determined by the ELISA assay. This resembles the third outbreak of canine
influenza in Korea that was reported by a recent paper (Song et al., 2008). This outbreak took
place in an animal clinic after two Jindo dogs and a Yorkshire terrier that were infected with
CIV arrived, after which all 13 dogs in the shelter facility were infected with the same virus
and revealed the typical clinical signs of CIV infection, including nasal discharge, cough, and
high fever (Song et al., 2008). These observations suggest that commercial CIV vaccines
must be developed and used in the Korean pet dog population.
We found that the seropositivity rates of the dogs occasionally differed depending on
whether an HI test or ELISA was performed. For example, while ELISA found that 11% and
100% of the dogs from the Moran market and the dog farm in Cheonbuk province had been
exposed to CIV, the HI test detected seropositivity rates of 0% and 35%, respectively.
Moreover, when we experimentally infected puppies with CIV and monitored their
seroconversion, we found the NP-based ELISA detected anti-CIV antibodies 2 days earlier
than the HI test. This suggests that the HI test is less sensitive than the ELISA. Moreover,
while the HI assay is often used to detect antibodies against viral hemagglutinin (HA) in
animal and human sera, it is not very reliable in detecting antibodies to avian influenza
viruses in mammalian sera because nonspecific hemagglutination inhibitors in the sera can
result in false positives (Lu et al., 1982). These observations suggest that the NP-based
ELISA is a better tool for the serological diagnosis of CIV infections in dogs.
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Chapter II
Conventional and Experimental

Vaccines against Avian Influenza
Ami Patel*1,2, Gary Wong*1,2, Mickey Sahib1,2

and Gary P. Kobinger 1,2
Special Pathogens Program, National Microbiology Laboratory,
Public Health Agency of Canada1
Department of Medical Microbiology, University of Manitoba,
Winnipeg, MB, Canada2
Abstract
Avian influenza H5N1 virus, family Orthomyxoviridae, naturally persists in

waterfowl and domestic bird reservoirs with sporadic outbreaks of highly pathogenic
strains. Several human cases were reported during the 1997 H5N1 avian epidemic in
Hong Kong, showing direct transmission from domestic poultry and the first occurrence
of an H5 influenza subtype in humans. Highly pathogenic avian influenza (HPAI) H5N1
variants later re-emerged following years of circulation in wild bird reservoirs and new
human cases were identified in Southeast Asia during 2003. Evidence suggests that the
H5N1 virus is rapidly evolving and although HPAI H5N1 has not yet adapted for
efficient human-to-human transmission, it is currently considered a major threat for a
global influenza pandemic. The World Health Organization (WHO) and several nations
have prioritized improving available inactivated or LAIV, and the development of
alternative platforms against potential influenza outbreaks. While currently approved
vaccines have been successful against influenza viruses of the same subtype, complete
cross-protection has yet to be achieved. This chapter reviews different vaccine strategies
against avian influenza H5N1, reflects on the requirements for effective vaccine
development, and discusses the direction of future influenza vaccine research. The rapid
development of several experimental platforms in recent years has enhanced protective
*
These authors contributed equally to this work
28 Ami Patel, Gary Wong, Mickey Sahib et al.
efficacy and immunogenicity following immunization, additionally benefiting

understanding of influenza virus pathogenesis. The most promising platforms have been
evaluated successfully in ferrets and non-human primate models, with several candidates
currently in human clinical trials. The objective of influenza vaccine research will be to
develop a universal, single vaccine candidate capable of complete cross-protection
against divergent influenza subtypes.
Introduction
Low pathogenic avian influenza (LPAI) viruses are frequently isolated from wild bird
reservoirs, but have limited pathogenesis in humans. In 1997, a highly pathogenic avian
influenza (HPAI) H5N1 variant was isolated from infected birds in Hong Kong and several
clinical cases of direct bird-to-human transmission were associated with contact of infected
poultry. This prompted the Hong Kong government to begin the immediate culling of
millions of chickens, resulting in substantial economic losses. While these measures were
successful in controlling the spread of the virus, HPAI H5N1 re-emerged during 2003 in
Southeast Asia. Since then, HPAI H5N1 has spread among birds throughout the Eastern
hemisphere and human cases have been reported in Asia, the Middle East, and Africa. More
recently, human-to-human transmission of H5N1 has been reported in Indonesia, Pakistan [1]
and China [2], although spread has only been limited to close family members. The high
pathogenicity associated with avian influenza H5N1 infection and the possibility of further
cross-transmission of the virus into humans makes this subtype a dangerous candidate for the
next influenza pandemic.
Pandemic Influenza and H5N1
Influenza A viruses are known to infect a broad host range but are most pathogenic to
humans, birds and swine. While seasonal influenza epidemics are localized and sporadic,
HPAI is responsible for some of the most devastating pandemics in recent history (Table 1).
Table 1. Major pandemic avian influenza outbreaks since the 20th century.
Name Year Virus strain Deaths Origin References

Spanish Flu 1918-1920 H1N1 ~50 million Unknown, perhaps the US [13, 92, 95]
Asian Flu 1957-1958 H2N2 ~1 million China [13, 95, 96]
Hong Kong Flu 1968-1969 H3N2 ~500,000 Hong Kong [13, 95, 96]
The most well known influenza pandemic is the Spanish Flu which occurred from 1918
to 1919 [3]. This pandemic was caused by an H1N1 influenza virus which killed
Conventional and Experimental Vaccines Against Avian Influenza 29
approximately 50 million people worldwide from both primary and secondary infections [4].
Following the Spanish Flu, the next pandemic was the 1957 1958 Asian Flu, which was
caused by an H2N2 influenza A virus and killed over 1 million people. An H3N2 virus was
responsible for the following pandemic in 1968 1969 (the Hong Kong Flu pandemic) where
the death toll was estimated at 500,000 worldwide. Although the origins of the Spanish Flu
were uncertain, the other two pandemics originated from avian influenza isolates that
acquired enhanced specificity for the human host through the exchange of genes and
evolution via mutations [3, 5].
Influenza Antigens and Host Specificity
Influenza A contains an eight-segmented, negative-sense, single-stranded RNA genome

encoding for 10 proteins: hemagglutinin (HA), neuraminidase (NA), two matrix proteins
(M1, M2), two non-structural proteins (NS1, NS2), the nucleoprotein (NP), and three
polymerase gene products (PB1, PB2, PA) [6-8]. There are currently 16 HA (H1-H16) and 9
NA (N1-N9) subtypes that have been characterized [8] and most persist in wild birds. Internal
proteins are known to be highly conserved between divergent influenza viruses, while the
most diversity occurs between envelope glycoproteins. The HA surface glycoprotein mediates
virus entry through binding to sialic acid receptors on host epithelial cells [9], while the NA
surface glycoprotein acts as a sialidase, facilitating budding of progeny virus [9, 10].
Human influenza viruses bind preferentially to alpha-2,6-linked sialic acid residues to
galactose located in the human respiratory tract epithelia. Avian influenza favour alpha-2,3-
linked sialic acid receptors found on avian gut epithelial cells. Pig epithelial cells express
both receptors, making them the ideal intermediate hosts for avian and human influenza
viruses. Co-infection of a same host by different influenza viruses can lead to genetic
reassortment and the generation of novel influenza virus strains containing both human and
avian genes.
Antigenic Drift and Antigenic Shift
Influenza virus evolution is continually driven via two major mechanisms: antigenic drift
(Figure 1) and antigenic shift (Figure 2). Antigenic drift occurs through point mutations in the
HA and NA surface glycoproteins that arise through selective pressure by the host immune
system and an error-prone RNA-dependent RNA polymerase [7], and is the mechanism
behind the creation of epidemic influenza strains. Antigenic shift occurs through genetic
reassortment of two influenza viruses through a common intermediate host and the exchange
of HA and/or NA genomic segments may result in the formation of novel recombinant
progeny belonging to a different subtype [7, 11]. Reassortment may also occur between other
structural and non-structural influenza gene segments, and result in changes to virus viability,
host receptor specificity and immunogenicity. Therefore, antigenic shift is the primary
mechanism for the creation of pandemic influenza strains.
Intermediate
Hosts
Figure 1. Antigenic drift: the mechanism for generation of epidemic influenza from avian influenza
strains. Spontaneous point mutations in the influenza viral genome may lead to progeny virus that is
slightly different genetically from the parent virus. Progeny may or may not be antigenically distinct
from the parent virus.
Mixing Vessel
Intermediate
Host
Figure 2. Antigenic shift: the mechanism for generation of pandemic influenza from avian influenza
strains. The genetic reassortment of two influenza viruses inside an intermediate mixing vessel
results in progeny virus that may have significant antigenic differences to the parent virus.
Pathogenesis and Treatment
Influenza infection can cause a highly contagious respiratory disease. General symptoms
including fever, chills, muscle aches, and headaches are often followed by sore throat, nasal
symptoms, hoarseness, cough, and/or diarrhea. The majority of people recover successfully,
but approximately 300,000 deaths per year occur worldwide. Secondary infections are
common in the young, elderly, and immunocompromised. Interestingly, during the 1918
Spanish Flu, young healthy adults (age 20-40) were the most severely affected. Evidence
suggests that disease severity and increased mortality in younger adults may have resulted
from an NS1 gene with increased virulence and uncontrollable up-regulation of the immune
response, generating a cytokine storm. Additionally, the absence of pre-existing antibodies to
the H1N1 subtype and increased secondary infections such as bacterial pneumonia may also
have been important factors [12-14].
Both mucosal and systemic immune responses play a role following influenza infection.
Secretory IgA (upper respiratory tract) and serum IgG (lower respiratory tract) are involved
in the protective immune response. There are also suggestions that the cellular response may
influence viral clearance and improve recovery from illness. The cytotoxic T-lymphocyte
(CTL) response is generated against conserved internal influenza proteins [15] [16] and may
be cross-reactive against divergent influenza viruses [17].
Antiviral drugs inhibiting the NA (oseltamivir, TamiFlu) or M2 (amantidine) proteins are
available, however vaccination has been the most effective prophylaxis against influenza
infection. There are several conventional platforms which include inactivated (INV) and live-
attenuated vaccines (LAIV). Additionally, experimental vaccines are also now being
evaluated as alternatives to offer broader protection against the more divergent H5N1 viruses.
Conventional Vaccines
The first conventional influenza vaccine was developed and licensed in 1945 for use by
the United States Army personnel [18]. This was a bivalent, formalin-inactivated vaccine
derived using whole influenza A and B viruses [19, 20]. Since then, several advances in
vaccine design and production methods have significantly improved conventional vaccines.
The World Health Organization (WHO) and the US Public Health Service decide the
influenza strains to be included in each seasonal vaccine based on the global prevalence of
the selected influenza species [8, 21, 22]. Two influenza A strains and one influenza B strain
are chosen each year for the annual vaccination program. Currently, trivalent INV and LAIV
are licensed for administration in humans (Table 2) [8].
Table 2. List of FDA-approved conventional avian influenza vaccines a
Inactivated influenza Year

Company Number of administrations Dosage Route
virus vaccine approved
Afluria CSL Limited 2007 1X 0.5mL IM
GlaxoSmithKline
Fluarix 2006 1X 0.5mL IM
Biologicals
IF Biomedical
FluLaval 2006 1X 0.5mL IM
Corporation of Quebec
Novartis Vaccines and
Fluvirin 1988 4-8 years old: 1X or 2X 0.5mL IM
Diagnostics Limited
9 years or older: 1X 0.5mL IM
6-35 months (primed or
Fluzone Sanofi Pasteur, Inc 1980 0.25mL IM
unprimed): 1X or 2X
36 months - 8 years (primed or
0.5mL IM
unprimed): 1X or 2X
9 years or older: 1X 0.5mL IM
Live-attenuated
influenza virus vaccine
0.1mL per
FluMist Sanofi Pasteur, Inc 2003 1X IN
nostril
Inactivated influenza
Sanofi Pasteur, Inc 2007 2X IM
virus vaccine, H5N1
None (Influenza Virus MedImmune Vaccines,
2007 2X 1mL IM
Vaccine, H5N1) Inc
CBER licensed product information. http://www.fda.gov/Cber/efoi/approve.htm#flu, accessed February
16th, 2009.
Inactivated Vaccines (Inv)
INV against influenza are currently available in one of three formulations: whole virus
(WV), subvirion (SV), and subunit (SU) [23]. The vaccines may contain trace amounts of egg
proteins and should not be administered to patients who may be allergic to eggs [24].
The WV vaccine is derived from intact influenza A and B viruses inactivated by formalin
or beta-propiolactone. The vaccine components are replicated in the allantoic cavity of
embryonated chicken eggs using seed viruses based on an H1N1 A/Puerto Rico/8/34
backbone and envelope proteins from chosen vaccine strains [23]. Although the WV vaccine
can be highly protective against closely matched influenza challenge, the vaccine has shown
adverse side reactions in children and is seldom used [23].
The SV vaccine was developed to reduce side effects associated with WV vaccines.
Similar to WV vaccines, harvested viral particles are first inactivated by formalin or beta-
propiolactone [21]. They are then split to produce subvirus particles by using solvents to
disrupt the viral envelope and inactivate any residual virus [8, 25]. Although SV vaccines
retain the immunogenicity of the virus, vaccine reactogenicity is reduced compared to WV
vaccines due to a lower quantity of non-viral components such as egg proteins, and non-
essential viral components such as proteins and lipid membrane [21]. SU vaccines contain
purified HA and NA and are produced by zonal centrifugation of SV vaccines in order to
separate the surface proteins from other viral antigens [21]. SU are less immunogenic than
WV, associated with less adverse side effects and thus along with SV are recommended for
use in the immunization of children under 9 years old [26].
While three SV vaccines have been approved for human use against H5N1 worldwide,
only one is approved by the Food and Drug Administration (FDA) for use in the United
States. Influenza Virus Vaccine, H5N1 (Sanofi Pasteur, approved in April 2007) is based on
the H5N1 A/Vietnam/1194/2004 isolate, where two aluminium hydroxide-adjuvanted doses
of 30g were sufficient to induce protection against the vaccine strain [27], as opposed to
Panvax (also 30g) [28] and Pandemrix (3.8g) [29].
There are other inactivated H5N1 vaccines currently in clinical trials. A recent clinical
study analyzed the effects of a WV vaccine against the wild-type, clade 1 H5N1
A/Vietnam/1203/2004 strain. Low doses of the vaccine (7.5g) without adjuvant produced
the best protective response against the vaccine strain and also induced immune responses
against divergent H5N1 viruses [30]. Since the vaccine is derived from cell-culture,
formulation time would be shortened for a pandemic vaccine and more importantly may be
useful for patients with anaphylactic responses towards egg proteins [30]. Furthermore, this
WV vaccine was found to have a comparable side effect profile to SV vaccines [30] and may
be recommended for young children.
Live-Attenuated Influenza Vaccines (LAIV)
LAIV candidates contain attenuated pathogens with minimal virulence but high levels of
immunogenicity. The objective of LAIV vaccination is to stimulate a systemic and mucosal
immune response that is analogous to natural infection [24].
The LAIV is created through reassortment, resulting in a virus expressing the surface
glycoproteins of the vaccine influenza strain as well as six internal proteins genes from the
donor influenza strain H2N2 A/Ann Arbor/6/60 or from B/Ann Arbor/1/66 [24]. Attenuation
is achieved through serial passage of the donor virus at decreasing temperatures in chicken
eggs, which generates a virus displaying temperature-sensitive (ts) and attenuation (att)
phenotypes [23]. The pathogenicity of live attenuated viruses is impaired since they only
replicate in the upper respiratory epithelium which has a temperature of 32oC 33oC [21].
The presence of multiple mutations enhances the stability of the ts, att phenotype and
decreases potential reassortment between the live attenuated vaccine and other circulating
influenza viruses.
Only one LAIV has been approved by FDA to date. FluMist (MedImmune) is a trivalent
cold-adapted (ca) live influenza vaccine [23] and is delivered by the intranasal route. There
are currently no approved LAIV against H5N1. However, a 2006 study has shown that a live-
attenuated H5N1 vaccine protected mice and ferrets from lethal homologous and
heterologous H5N1 challenges. The LAIV encoded a modified H5 hemagglutinin (HA) and a
wild-type N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and
Vietnam during 1997, 2003, and 2004. The remaining gene segments were derived from
H2N2 A/Ann Arbor/6/60. The safety, immunogenicity, and efficacy against divergent H5N1
viruses are promising and there are plans to further evaluate this vaccine in clinical trials [31].
Immune Response of Conventional Vaccines
INV can induce both local and systemic immune responses [24] which are mediated
through the production of anti-influenza serum antibody (IgG). In contrast, LAIV stimulate a
more localized immune response [24] and have lower IgG antibody titres than the inactivated
vaccine. However, they are able to generate a strong mucosal IgA response [32] and can
stimulate a cell-mediated immune response in addition to the B-cell response [24]. Although
the two types of vaccines activate different arms of the immune response, INV and LAIV
have similar protective efficacy and incidences of adverse reactions [32].
Safety Concerns Associated with Conventional

Vaccines
Most common side effects associated with INV include transient local inflammatory
reactions such as pain, erythema, and induration which last for 1-2 days. Systemic responses
may include fever, myalgia, arthralgia and headaches, although these are less frequent
symptoms and treatment is usually unnecessary [21].
The oculo-respiratory syndrome (ORS) is an unusual complication of the INV, where
symptoms include respiratory and/or ocular complications in vaccine recipients [33]. The
complication was eventually traced to a preparation of the Fluviral SV vaccine containing an
abnormally large proportion of unsplit virions [34]. The unsplit virions formed large
aggregates which may have resulted in the observed syndromes. Although ORS recurrence
may occur in subsequent influenza vaccinations, symptoms have been mild and do not
contraindicate future vaccination [34].
Guillain-Barre Syndrome (GBS) is a rare but serious potential side effect of influenza
vaccination. The autoimmune damage is believed to be induced by endotoxins or other
cellular components of Salmonella and Campylobacter which may contaminate the chicken
egg during vaccine production. Egg proteins in the vaccine preparations have also been
suggested contributors towards the development of GBS post-vaccination [21].
LAIV also possess additional safety concerns. The intranasal administration site is close
to the central nervous system (CNS) and may increase the risk of CNS-related complications.
Additionally, the vaccine may go through spontaneous genetic changes and lose attenuation
[24]. However, intranasal administration of influenza vaccines has generally been well-
tolerated. FluMist has been associated with an increased risk in young children (18 to 35
months) with asthma [35], but older children with stable moderate-to-severe asthma have not
shown significant worsening of symptoms [36].
Conventional Vaccines and Avian Influenza
Licensed vaccines against human influenza viruses are currently produced in

embryonated chicken eggs and the manufacturing process can take six to nine months. One
concern is that the high morbidity and mortality of chickens during an avian influenza
pandemic may compromise the egg supply needed for vaccine production. Also, avian
influenza viruses are often highly pathogenic to the embryo and present a considerable
challenge for vaccines generated in chicken eggs [37]. Another issue concerns human
influenza A and B seed viruses which often develop mutations favouring growth in chicken
eggs. These variants will confer less protection against circulating viruses if they are
significantly mismatched from the wild-type strain [38].
Unfortunately, both INV and LAIV are not 100% effective and may have reduced
efficacy if there are antigenic differences between the predicted vaccine strain and the
prevalent wild-type strain. This recently occurred during the 2003-04 flu season when the one
of the strains included in the seasonal influenza vaccine was the H3N2 A/Panama/2007/99
but the prevalent circulating strain was H3N2 A/Fujian/411/2002 [39]. The protective
efficacy of the LAIV against culture-confirmed influenza illness was determined to be only
56% during this particular season [40]. Therefore, vaccine efficacy relies heavily on the
selection of a vaccine strain that will be similar the predominantly circulating strain in the
upcoming year. Additionally, strain differences arising from antigenic drift and shift make it
difficult to produce a conventional vaccine with broad-spectrum efficacy against rapidly
diverging influenza viruses such as H5N1.
Experimental Vaccines
The use of conventional influenza vaccines in combination with appropriate

immunization strategies has been relatively successful at reducing the incidence and severity
of annual influenza infections. Unfortunately, these vaccines require yearly reformulation and
accurate prediction of the next circulating strain in order to achieve optimal protection. This
presents a challenge towards protection against emerging avian influenza viruses such as
H5N1, since most populations are immunologically nave. The limitations of conventional
vaccines have encouraged research into the development of alternative experimental vaccine
platforms for the safe and effective delivery of fully protective influenza vaccines. Achieving
cross-protection against different influenza strains has been one of the major challenges of
influenza vaccine research and is particularly evident against avian influenza H5N1, where
vaccines against one clade are not fully protective against a different clade[41].
There are several different experimental vaccines in development including novel
subunit vaccines that contain one or more potential antigens from H5N1. In particular: HA,
NA, M1, M2, and NP have all been suggested as antigen candidates for either single or
combination vaccines[39, 41]. The majority of vaccines have focused primarily on HA since
it is capable of generating a robust neutralizing antibody response. The matrix proteins and
NP are well conserved across H5N1 isolates and therefore have been considered as targets for
improving virus-specific cell-mediated immune responses[42, 43].
Focus was initially placed on evaluating vaccines containing purified viral proteins.
Despite promising initial results, this was eventually determined to elicit poor immune
responses[39]. Many studies have instead focused on developing vaccines using consensus-
based genes[44-47]. In one experiment, a consensus-based HA gene was constructed from
over 20 H5 HAs from clades 1 and 2[44]. Another study evaluated other consensus-based
conserved H5N1 antigens[45, 47]. Overall, subunit vaccines based on DNA expression
vectors (DNA vaccines), virus-like particles (VLPs) and various recombinant viral vectors
have shown promise as potential vaccine platforms against avian influenza.
Vaccines in Development
Virus-Like Particles (VLPs)
Live virus-like particles (VLPs) have been suggested as alternatives to conventional and
DNA vaccines. These vaccines contain non-infectious virus particles which express one or
more structural proteins, but no nucleic acids. Several studies have developed baculovirus
systems expressing avian influenza HA, NA, and matrix (M1, M2) proteins and VLPs are
produced through self-assembly[48-50]. Traditional egg-based production methods can be
avoided and VLP vaccines have been shown to be safe and immunogenic in mice, ferrets,
non-human primates (NHPs), and humans[49, 50]. A VLP vaccine against human papilloma
virus (HPV, Gardasil) has been approved and demonstrated both systemic and mucosal
immune responses.
DNA Vaccines
Naked DNA was initially considered in the context of gene transfer and the potential for
long-term protein expression in muscle cells and tissues. It was soon determined that a
desired transgene could be incorporated into DNA expression vectors and delivered for both
gene therapy and vaccine applications[51]. The majority of naked DNA vaccines consist of a
plasmid expression vector containing an individual antigen under the control of a eukaryotic
promoter, rather than a whole virus particle.
The first DNA-based vaccines demonstrated promising levels of protection in small
animal models, but had poor immunogenicity in NHPs and humans. One of the first
considerations was to improve the DNA backbone through tissue targeting and enhance
overall antigen expression[51]. Although there are several DNA backbones, individual
vaccine immunogenicity is dependent on the selection of an appropriate expression vector.
An efficient promoter is necessary to ensure optimal expression in mammalian cells and is
generally followed by an appropriate polyadenylation signal to stabilize the mRNA
transcript. Additional enhancer elements are often included, as well as a Kozak sequence to
improve ribosome binding and protein translation[51]. Finally, codon optimization is often
used to increase gene expression[44, 51, 52]. Together, these elements result in expression
vectors capable of generating strong protein expression with the potential to stimulate a
broader and greater immune response.
DNA vaccines against influenza were first evaluated in the context of the HA gene.
Naked DNA containing a single H5HA antigen was sufficient for full protection against
homologous challenge, but did prevent infection by heterologous viruses[53]. Several
subsequent approaches examined multivalent DNA vaccines with mixed plasmids containing
HA genes from different influenza subtypes, including H5 and H7 in a single vaccine[54].
The results suggested that an antigenically related H5 gene may be sufficient for temporary
protection during a pandemic until a matched vaccine could be synthesized. Interestingly,
these experiments revealed that protection could be achieved despite no detectable antibodies
against HA. Similar evidence was observed in both ferrets and chickens[53]. Further studies
looked at ways of stimulating the cellular response to help augment vaccine efficacy[43, 46,
47, 55, 56]. Although not directly implicated in virus neutralization, the T-cell response may
play a role in improving virus clearance. This may explain why full survival was observed
even with undetectable antibody titers. Experiments evaluating the NP antigen demonstrated
that while NP does not provide full protection against lethal challenge, it was able to
stimulate a sufficient immune response to protect 50% of the infected animals[54]. An M2
and NP-based combination vaccine was also protective at 50% in mice[55]. Fusion of NP to a
nuclear localization signal (NLS) improved efficacy, but was still short of stimulating full
protection [57].
Recently, several studies have evaluated multi-dose regimens using electroporation or
gene gun to efficiently deliver naked DNA vaccines in ferrets, non-human primates, and
humans [47, 58, 59]. These results have been very promising and improved protection has
been observed. Additional carrier molecules have also been evaluated, including cationic
lipid administration for delivery of the vaccines[44]. Newer technology has also generated
linear expression cassettes (LECs) and other linear DNA vectors that can be amplified
through cell-free synthesis[60]. The removal of the origin of replication and selective
resistance marker may reduce vector-associated immunogenicity and improve the efficiency
of the linear fragment. Codon optimized H5HA and N1 genes were included in a backbone-
free vector and amplified using a mesophilic DNA polymerase in a similar fashion to
polymerase chain reaction (PCR)[60].
DNA vaccines have been considered for priming the immune response before
vaccination with traditional or experimental vaccines. The VRC-AVIDNA036-00-VP DNA
vaccine (NIAID) is currently in clinical trials for administration following the Sanofi Pasteur
H5N1 inactivated vaccine. Alternatively, DNA vaccines have been considered for priming
before administration of viral vectored vaccines[43, 61, 62].
Viral Vectors
Other H5N1 vaccines in development include experimental vaccines based on
recombinant viral vectors. Many of these vectors contain deletions in the early replication
genes that allow for insertion of foreign DNA and prevent uncontrollable expression of
heterologous genes. Alternative strategies involve priming with a DNA vaccine, followed by
a viral vector boost to improve protective efficacy and long-term immune responses. Several
platforms have been developed for human and animal vaccines including: adenovirus, fowl
pox, vaccinia, and vesicular stomatitis virus vectors [63-66].
Adenovirus Vectors
Adenovirus (Ad) vectors have shown promise as a recombinant vaccine platform against
H5N1. As with DNA vaccines, early adenoviral vectors were considered primarily for gene
therapy[67, 68]. Adenovirus is a non-enveloped virus containing a linear double-stranded
DNA genome approximately 30 to 40kb. The virus does not integrate into the host
chromosome and is maintained as an episome in infected cells.
The idea of using adenovirus as a vector for foreign genes seems to have been accidental.
Early studies identified an insertion of the simian virus 40 (SV40) T-antigen into the E3
region of adenovirus in contaminated virus stocks, which was one of the first indication that
adenovirus could be used to deliver foreign DNA to target cells[67]. Early Ad vectors were
developed to contain deletions in the immediate early E1 genes, which was sufficient to
render the virus replication-incompetent. Additional deletions could also be made to the E3
gene region, which allowed for up to 8kb of foreign DNA to be inserted into a single Ad
vector[67-69]. The replication-incompetent vector allows for transient expression of
heterologous genes (transgenes). Several adenovirus serotypes can infect humans, generally
causing mild gastroenteritis, upper respiratory tract infections, or conjunctivitis. Human
adenovirus 2 (AdHu2) and 5 (AdHu5) are well characterized, with AdHu5 currently being
evaluated as a vaccine vector against several pathogens including Ebola, SARS, and malaria.
Ad vectors can generate a robust, diverse immune response that stimulates both humoral and
cellular immunity. The rapid clearance of Ad vector by cytotoxic T lymphocytes was a
challenge to gene therapy approaches but an advantage for vaccine applications as the
antigen is not persist inside the host[67, 70, 71]. Adenovirus vectors have also been evaluated
for influenza[72]. Should a pandemic occur, Ad vectors will provide an alternative to

conventional vaccines.
Most strategies have focused on evaluating Ad vectors containing the H5HA gene.
Protection against homologous challenge can be achieved through administration of a
matching HA, however optimal cross-protection against heterologous H5 viruses requires
alternative strategies. Different levels of protection were observed depending on the HA
fragment included in the Ad vaccine. Both the HA0 (cleavage site deleted) and HA1 genes
were capable of generating the best hemagglutination inhibition, neutralizing antibody titers,
and greater robust T-cell responses[73]. Several approaches have also evaluated the
combination of Ad vectors expressing HA from one or more H5 viruses belonging to
different clades. Protective efficacy of combined Ad vectors containing two divergent HAs
was improved with the addition of AdNP[74]. Similar to DNA vaccines, partial heterologous
protection could be observed even in the absence of neutralizing antibodies, supporting the
role of the CTL response in protection.
Further studies have also looked at combining three antigens in the same vector rather
than a mixture of antigen-encoded vectors[75]. Partial cross-protection against clade 1 and 2
H5 viruses was observed following immunization with a single AdHu5 vector containing
HA, NA, and M1. Alternatively, an AdM2 vector was also developed[62]. While it was able
to generate partial protection against challenge on its own, better protective efficacy was
observed after a DNA-priming[62].
Despite promising levels of protection, the presence of pre-existing immunity against
adenovirus vectors substantially reduces transgene expression after re-administration of the
same vector[67]. This has additional implications in sequential immunizations whether for
the same antigen or antigens originating from different infectious agents. Approximately 30-
50% of the population has neutralizing antibodies against AdHu5. Therefore, different
mammalian adenovirus serotypes with lower seroprevalence in humans are now being
considered as alternatives to AdHu5. Several candidates include simian and bovine Ad
vectors. Chimpanzee adenovirus 7 (AdCh7) was shown to have similar protective efficacy
and immunogenicity to AdHu5. Although the AdCh7-NP vaccine was partially protective, its
efficacy was similar to AdHu5-NP, suggesting an alternative platform to H5 vaccines[76].
Other studies have evaluated bovine adenovirus 3 (BAd3) containing HA as an alternative
Ad vector which also had comparable efficacy to AdHu5[77, 78].
Overall, adenovirus vectors are able to induce strong immune responses and broad
protection against diverging H5 isolates. Despite pre-existing immunity, these vectors are
potential candidates for the prevention of pandemic influenza.
Vaccine Delivery and Adjuvants
The route of vaccine delivery may have significant impact on vaccine immunogenicity
and is important for evaluating optimal efficacy and possible complications following
immunization [79]. There are several routes of immunization for influenza vaccines:
intramuscular (IM), intranasal (IN), oral, and intradermal (ID) (Table 3). Additionally,
vaccines may be combined with an adjuvant to induce stronger immune responses and
augment overall protective efficacy.
Table 3. Various recommended and hypothetical routes of vaccination and their

strengths and weaknesses.
Route Pros Cons References

Stimulates strong, long-lasting
Intramuscular Occupational risks for health workers
systemic immune response
[80-82]
Ensures complete delivery of vaccine Patients may be adverse to needle-
in its native form based injections
Intranasal Simulates natural course of infection Potential adverse CNS effects

[21, 24, 32,
Similar efficacy to intramuscular Potential complications for patients
85]
injections with respiratory disease
Vaccine stability inside the

Oral Ease of handling and usage
gastrointestinal tract
[85, 86]
Need 100-fold more antigen for 100-
fold less immune response
More immunogenic than intramuscular

Intradermal Higher incidence of side effects
infections
[87, 88]
Only a partial dose needed for
protective immune response
Intramuscular
The recommended route of immunization for the conventional influenza vaccines is IM

[32]. IM vaccinations are known to elicit strong systemic and detectable mucosal responses,
where CD8+ T cell responses are present in both effector and memory phases [80].
Furthermore, IM offers a faster rate of absorption than other routes of delivery, and muscle
tissue can often hold a larger volume of fluid without discomfort. Although this method of
administration ensures the intact delivery of the entire vaccine dose into the recipient, there
are occupational risks for health care workers. Both children and adults may be anxious
concerning the pain associated with IM vaccination [81, 82].
Intranasal
Aerosol administration is the best method for mimicking a natural influenza infection [21,
24]. Lymphoid tissues in the upper respiratory tract are important sites for inducing immune
responses [83] and IN vaccines are ideal for inducing immune responses at the primary site of
influenza infection. The efficacy of IN influenza vaccines was shown to be similar to IM
vaccines despite differences in antibody response [32]. However, a weaker immune response
was observed in patients with respiratory illnesses with reduced levels of total serum
antibody levels and a lower detectable neutralizing antibody titre [84]. Nevertheless, further
assessment is necessary to establish the safety of IN vaccines in patients with respiratory
disorders. There has also been little evaluation of adverse CNS side effects following IN
immunization [85].
Oral
Oral vaccination is an attractive route of immunization due to its simplicity of handling

and usage [85]. However, the design of an oral influenza vaccine should assess: vaccine
stability in the varying pH conditions of the gastrointestinal tract, possible dilution of the
vaccine dose following ingestion, and facilitation of antigen interactions with intestinal M
cells to aid with vaccine absorption [85]. Oral immunization is generally quite inefficient and
100-fold more vaccine may often be required to induce a similar immune response by a
parenteral injection [86]. Therefore, there have been few oral influenza vaccines that have
progressed to clinical trials.
Intradermal
Other developments have shown that vaccines delivered by the ID route to be more
immunogenic than IM vaccination. Similar immune responses were detected following
administration of a trivalent influenza vaccine delivered by either ID or IM, at 40% and
100% of the recommended dosage respectively [87]. Another study in which the ID dosage
was 20% of the administered IM dose also reported similar efficacy [88]. Although local
reactions were more frequent among ID vaccine recipients compared to IM, these reactions
were mild, transient, and did not cause complications [88]. The skin contains numerous
Langerhans dendritic cells which may stimulate both systemic and mucosal immune
responses. They may induce stronger antibody production by B-cells and also activate the
cellular immune response.
Adjuvants
Several adjuvants have been shown to enhance immunogenicity when used in

combination with an inactivated vaccine. One is MF59 (Fluad, Chiron Vaccines, Emeryville
[CA], US). Fluad has been shown to be more immunogenic than non-adjuvanted INV, with a
higher neutralizing antibody titre post-vaccination [89, 90]. However it was shown to be
significantly more reactogenic than non-adjuvanted vaccines and was associated with
increased local side-effects following immunization [21]. Another adjuvant is known as
immunopotentiating reconstituted influenza virosomes (Inflexal V, Berna Biotech,
Switzerland). Lipid virosomes containing HA and NA enter antigen presenting cells (APCs)
through HA-mediated endocytosis and are presented by MHC class I and II molecules similar
to natural infection [21]. The virosome displays comparable immunogenicity to conventional

vaccines, but with less adverse reactions [91]. A recent adjuvant is the proprietary system
developed by GlaxoSmithKline (GSK) for combination with a pre-pandemic split-virion
H5N1 vaccine [92]. This is an oil-in-water emulsion and can be mixed with low doses of the
H5 HA, effectively reducing the amount of antigen in the vaccine with the benefit of
increased immune responses in comparison to non-adjuvanted split-virion vaccines.
Additionally, cross-clade protection was also observed even at the lowest dose of antigen in
combination with the proprietary adjuvant system [29, 93, 94].
Table 4. Pros and cons of various conventional and experimental vaccines.
Concentional influenza
Pros Cons References
vaccines
Stimulates both local and
Inactivated Unusual side effects (GBS, ORS)
systemic immune system
Elicit protective Ab levels after Need HA matching for optimal [21, 24, 33,
two doses protection 34, 37]
Virus made in eggs, take long time
to manufacture
Associated with very little

Live-attenuated Virus may revert to virulence
adverse effects thus far
Stimulates adaptive cell- Virus may recombine with other
mediated responses viruses to create pandemic strains
[24, 37]
Need HA matching for optimal
protection
Virus made in eggs, take long time
to manufacture
Experimental influenza
vaccines
Particle size limits large inserts of
VLPs Non-infectious
foreign DNA
Safe and immunogenic (in [48-50]
mice)
Egg-based production avoided
Retain antigen conformation
Poor immunogenicity in NHPs
DNA Stable, safe
and humans [41, 47]
Immunogenic (in mice)
Stimulates humoral and cellular
immune responses
Induce strong immune
Adenovirus Pre-existing immunity in humans
responses
[67, 76, 78]
broad protection against
diverging H5 isolates
Future Prospects
The emergence of HPAI in birds and increased human cross-transmission suggests that
the next pandemic influenza may be of zoonotic origin. Although current conventional
vaccines are relatively inexpensive and effective, their protection relies on the appropriate
matching of vaccine with the predominantly circulating strain. In order to provide optimal
protection against a future pandemic virus, it will be necessary to develop a vaccine with
broad spectrum efficacy against diverging influenza isolates. As an alternative, there is
increasing evidence that experimental vaccines may address several weaknesses associated
with conventional vaccination. The development of experimental vaccines will necessitate
both rapid formulation and extensive clinical assessment before approval for human
administration. Along with appropriate immunization regimens and vaccination programs, it
should be possible to develop a vaccine against avian influenza with broad-spectrum
protection against divergent clades.
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2006. 105(1): p. 1-6.
Chapter III
Worldwide Preparedness to Prevent

Eruption of Pandemic Flu and to
Control Pandemic Spread After its
Emergence
Yoav Arnson1 and Yaron Bar-Dayan*1,2,3

Department of Medicine 'D', Meir Medical Center, Kfar Saba, Israel. Affiliated to the
Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel1
IDF Home Front Command, Ramle, Israel2
Department of Disaster and Emergency Medicine, the Faculty of Health Sciences,
Ben Gurion University of the Negev, Beer Sheva, Israel3
Abstract
Avian influenza or "bird flu" is causing increasing concern across the world as
experts are preparing for the possible occurrence of the next human influenza pandemic.
Countries worldwide are preparing for the arrival of the virus in wild birds and poultry
within their territories. All countries need to prepare for the possible arrival of human
cases of influenza imported through foreign travel.
Preparedness for biological threats requires awareness, planning, organization,
infrastructure and equipment stocking, education of personnel, and conducting drills as
well as availability, willingness and perceived self efficacy of the staff to respond in due
time. International collaboration has a key impact on successful medical preparedness.
Cooperation and coordination between countries is needed in the verge of a pandemic.
Most health authorities initiated disease prevention and containment policies. The
World Health Organization (WHO) is the basic coordinating and supervising force
*
Col. Dr. Yaron Bar-Dayan MD, MHA, Department of Medicine D, Meir Hospital, Kfar Saba, Israel and Chief
Medical Officer (ret), IDF Home Front Command, Ramle, Israel and Department of Disaster and Emergency
Medicine, Faculty of Health Sciences, Ben Gurion University, Beer Sheva, Israel, Home Address: 16 Dolev
St. Neve Savion, Or-Yehuda, ISRAEL, Mobile Phone: 009725578886215, E- mail: bardayan@netvision.net.il
50 Yoav Arnson and Yaron Bar-Dayan
behind global preparedness. The WHO has described the preparedness measures needed
to be taken in the pre-pandemic stage, during primary detection of highly pathogenic
avian influenza (HPAI) and at the pandemic stages. Countries worldwide have prepared
multi-factorial programs dealing with the subjects. The preparedness and contingency
plans differ among different countries and regions due to different resources availability,
local experience with the disease, specific local challenges and limitations. Many
countries suffer from under-endorsed and untested planes. In those areas suffering from
lack of effective pandemic control plans, the regional cooperation is also lacking.
This article reviews status of the worldwide preparedness to prevent eruption of
pandemic flu and to control pandemic spread after its emergence.
Introduction
Influenza pandemics have historically taken the world by surprise, giving health services
little time to prepare for the abrupt increase in the incidence of now cases and deaths that
characterize these events. Since late 2003 the world has moved closer to a pandemic than at
any time since 1968, when the last of the previous centurys three pandemics occurred.
Countries across the world should be preparing for the possible occurrence of the next
human influenza pandemic. Health organizations and experts worldwide are preparing for the
possible arrival of the highly pathogenic avian influenza A (HPAI) in infected wild birds and
poultry or with infected human by local spreading or via international travel.
The most effective management method for pandemic influenza relays on three efforts
detection of eruption in animals or human, treatment and isolation of sick individuals and
prevention of the pandemic from spreading once a disease has begun. Effective control and
management of the three steps requires significant cooperation efforts - interdisciplinary and
internationally.
Most health authorities, both local and international, have established policies dealing
with disease containment and pandemic prevention. Most of the data is publicly accessible.
For this review, public data from different countries, the World Health Organization (WHO)
principles and the UN System Influenza Coordination (UNSIC) progress report were
reviewed. In addition the PubMed database was searched for current reviews and publications
concerning avian influenza pandemic control.
We discuss the principles of preparedness as a concept, and the specific requirements for
pandemic prevention, we discuss the WHO basic principles of pandemic preparedness, and
the requirements for the desired preparedness, and we review the worldwide situation, and
present various national prevention and management strategies.
Preparedness towards an Emerging Outbreak
Throughout history mankind has faced global threats and disasters that rendered local
and national medical systems helpless. Large scale disasters or pandemics are declared when
the local health systems cannot cope with the severity or quantity of affected individuals, and
Worldwide Preparedness to Prevent Eruption of Pandemic Flu... 51
a larger scale of intervention is required - either interdisciplinary or internationally. This

situation is either due to lack of resources, lack of knowledge or lack of preparedness.
Planning for large scale biological threats requires preparedness and awareness.
Preparedness for any event is composed of two elements - Readiness and Alertness: (1)
Readiness - requires the development of doctrine, standards of procedures, effective and
efficient organization, qualified and sufficient personnel, logistics (supplies and
infrastructure), education and drills. (2) Alertness - requires availability, willingness and
perceived self efficacy.
The most effective management strategy for pandemic influenza will be through the
implantation of emergency response plans that include the activation of an incident command
system, a continuity of operation plans, cooperation with other health care institutions, a
unified command with local public health and local government agencies, and the appropriate
use of limited resources.
Preparedness for a pandemic is expensive and usually is beyond the ability of any single
nation. International collaboration has a key impact on successful medical preparedness.
Cooperation and coordination between countries is needed in the verge of a pandemic.
International preparedness requires the implantation of comprehensive and continuous
preparedness programs. The preparedness process must contain an international common
knowledge and common language database, and a unified doctrine for disaster management.
The doctrine must answer the following requirements: an international structure and
functional organization, an international management system, plans for use of existing
resources, effective international command and control systems, globally accessible
education programs.
Efforts concerning avian influenza preparedness must include understanding, monitoring
and altering disease related perceptions and psychological response. During the Severe acute
respiratory syndrome (SARS) epidemic, such perceptions affected the practice of public
health behaviors such as frequent hand washing and wearing of facemasks, which, in turn
contributed to the control of the epidemic. Effective public health action is dependent on
public psychological responses and the prevailing perceptions and beliefs of the community.
It is very likely that even at the onset of a human avian influenza epidemic, widespread
distress, panic and avoidance behaviors would occur in the affected communities.
Unconfirmed beliefs and misconceptions of newly emerging infectious disease are inevitable.
Raising population anxiety levels by warnings about a disease, produces only transient,
inconsistent and therefore, often ineffective results 1
Principles of Detection, Prevention and Treatment

of the Emerging Avian Influenza Pandemic
While dealing with pandemics the goals are detecting, containing and treating the disease
upon eruption. Historically, no attempt has been ever made to alter the natural course of a
pandemic near its start.
Since pandemics are remarkable events in that they affect all parts of the world, and once
international spread begins, each government will presumably make protection of its own
population the first priority, the best opportunity for international collaboration in the
interest of all countries is now, before a pandemic begins. In light of recent episodes of
human infection with H5N1 virus, the World Health Organization reiterated its 1997 call for
all countries to prepare for the next pandemic, which it termed inevitable, and possibly
imminent, and updated its own pandemic plan in April 20052. The basic strategic and
operational planning is based on six assumptions:
The risk of a pandemic is great. Since late 2003, the world has moved closer to a
pandemic than at any time since 1968. All prerequisites for the start of a pandemic
have now been met except one: the establishment of efficient human-to-human
transmission.
The risk will persist. Evidence shows that the H5N1 virus is now endemic in parts of
Asia, having established an ecological niche in poultry. The risk of further human
cases will persist, as will opportunities for a pandemic virus to emerge. Outbreaks
have recurred despite aggressive control measures, including the culling of more
than 140 million poultry.
Evolution of the threat cannot be predicted. Given the constantly changing nature of
influenza viruses, the timing and severity of the next pandemic cannot be predicted.
The final step improved transmissibility among humans can take place anytime
and at any rate.
The early warning system is weak. As the evolution of the threat cannot be predicted,
a sensitive early warning system is needed to detect the first sign of changes in the
behavior of the virus. In risk-prone countries, disease information systems and
health, veterinary, and laboratory capacities are weak.
Preventive intervention is possible, but untested.
Reduction of morbidity and mortality during a pandemic will be impeded by
inadequate medical supplies.
The WHO has published strategic global recommendations for disease prevention. These
recommendations respond to three projected stages in the pandemic progress. The stages
respond to the predicted course of the outbreak and presume disease eruption and spreading
will comply the patterns identified in previous pandemics: the pre-pandemic stage,
emergence and identification of the pandemic virus and pandemic spreading 2. The main
measures taken and obstacles encountered can be addressed according to the steps in the
disease progress.
Stage I: The Pre-Pandemic Phase
The pre-pandemic or inter-pandemic period refers to the period when no new influenza
subtypes have been detected in human. Animal infection with infective subtypes may be
present. The preparedness goals and challenges during the inter-pandemic phase are:
Reduce opportunities for human infection: The emergence of the pandemic depends on
the opportunities of human exposure to the virus. These opportunities persist depending on
the presence of H5N1 virus within wild animals. Control of the disease in animals is the most
effective method of reducing exposure. Prevention of behaviors that contribute towards
exposure increasing is the second way.
The efforts made at eliminating the disease in poultry seem futile by this point. The virus
is now endemic in many parts of Indonesia and Vietnam, and in some parts of Cambodia,
China, Thailand and possibly the Lao People's Democratic Republic3. Domestic ducks are
now known to be able to excrete large quantities of highly pathogenic virus without showing
clinical signs. Mammalian species not thought to be susceptible to infection have recently
developed a similar disease. Complete eradication of H5N1 in Asia is probably precluded by
its presence in wild bird populations; control of infection in wild birds is not a feasible
option. Advice to farmers and their families on how to avoid exposure is the second method
of reducing the probability that a pandemic virus will emerge. This option has likewise
become more difficult. The fact that domestic ducks can act as a silent reservoir has
removed the warning signal of a risk, especially for rural farmers and families, and increased
opportunities for unwitting human exposure. An inability to adequately compensate farmers
for lost birds reduces the incentive to report outbreaks.
Early warning and prediction of pandemic outburst: There are several factors
collaborating to make the early and effective detection of disease eruption difficult, especially
in the rural areas where the pandemic spread is thought to erupt. For H5N1, conclusive
diagnostic tests are technically difficult and costly, and can be conducted safely only in
specially equipped facilities. Surveillance is impaired by the fact that most cases have
occurred in rural areas. Case detection is complicated by the high prevalence of other severe
respiratory diseases having similar symptoms.
Current surveillance for human cases involves identifying potential exposure to A/H5N1
through recent travel to or from areas with known avian influenza activity. This information
enables the health care team to determine appropriate isolation and quarantine required while
providing care to the patient. Surveillance also enables health departments to track exposures
and initiate quarantine and treatment.
The roles and responsibilities of countries during phase 1 are in the level of preparedness
and planning. Countries should ensure that their national pandemic preparedness and
contingency planning is consistent with the coordinating role of WHO and partners during
international response. National pandemic preparedness plans should address the issue of
integration of national resources for rapid response and containment. These plans should be
made flexible and should continuously be updated to incorporate national and international
developments.
During the pre-pandemic Phase, the WHO responsibilities will be assisting and
supporting the preparedness effort of each country, developing and implementing training
programs for national and international members of rapid response and containment teams
and processing a global stock of antiviral medication, with an accessible and reliable method
of drug administration on demand.
Stage II: The Emergence of a Pandemic Virus
This stage is declared when human infections with new subtypes have been detected,
with no human to human infection, or with evidence of human spreading only in small
clusters. During this initial stage, the efforts should be directed at containing the spread of the
disease at the source. Aggressive measures, mainly the prophylactic use of antiviral drugs,
might contain a pandemic at its source or at least slow its spread, thus gaining time to put
emergency measures in place and augment vaccine supplies. For antiviral prophylaxis to be
effective, it should reach 80%-90% of the initially affected population within two to three
weeks at most after initial symptom onset4. Mass administration of drugs should be combined
with other measures, including area quarantine.
During the rapid response and containment stage, the countries responsibilities are to
coordinate national rapid response and containment operations, investigate potential
pandemic signals rapidly and facilitate the risk assessment, to mobilize national resources for
rapid response and containment, to intensify surveillance for cases of respiratory illness
inside and outside the containment zone, to evaluate the effectiveness of rapid response and
containment operations and to ensure the safety and security of international staff who are
assisting with rapid pandemic response activities
The WHO responsibilities during this stage are Coordinating the international response
including the deployment of international field teams to affected countries (upon request),
assisting countries in their assessment of signals of the possible emergence of pandemic
influenza, mobilizing international technical partners to support countries in rapid response
and containment if required, mobilizing and dispatching the resources (antiviral, other
materials and logistics) for rapid containment operations, mobilizing financial resources for
rapid response operations and ensuring appropriate control and accountability is in place for
material and financial resources.
Stage III: Pandemic Declared and Spreading

Internationally
Pandemic is declared when increased and sustained transmission from human to human
is detected in the general population. No attempt has ever been made to alter the natural
course of a pandemic near its start. Moreover, given the unpredictable behavior of influenza
viruses, no one can know in advance whether the start of a pandemic will begin gradually,
following the emergence of a virus not yet fully adapted to humans, or be announced by a
sudden explosion of cases, thereby precluding any attempt at containment. The actions to be
taken need to cover the following goals:
Local disease control and prevention: The most effective defense against the influenza
pandemic would be a directed vaccine to elicit a specific immune response against the strains
of influenza virus. However, until there is an actual influenza pandemic, there is no proof that
vaccines will actually effect morbidity and mortality, or delay the pandemic spread. Using
modern vaccine production methods, it is estimated that effective production of influenza
vaccine would take around six months from identification of the pandemic strand. In this time
the initial wave of pandemic outbreak would have past 5. Antiviral drugs assume a critical
early role in early disease treatment. Neuraminidase inhibitors are considered the drug of
choice for disease control, as no resistance to the drug has been identified6. The medication
works by inhibiting viral replication, specifically the release of newly formed virions from
infected host cells. They need to be administered as early as possible, ideally within 48 hours
of infection, to be effective 7. The main setback would be inadequate supplies of medication
in the rural infected regions and inequitable access to them. Not all countries can afford
stockpiling enough drugs 8. Furthermore, concerns about the overreliance of a
pharmaceutical solution, suggesting it may not be sufficient or effective, have been
expressed 9.
Pandemic presumably will not affect all countries or all parts of a country at the same
time. If efforts to contain an emerging virus at the source fail, health authorities will have at
least some opportunities to intervene to forestall spread within an affected community, within
a country, and internationally. Actions aimed at delaying spread must, however, take place
rapidly.
Since global availability of vaccine and anti-viral agents against influenza caused by
novel human subtypes are likely to be insufficient, the WHO recommends non-
pharmaceutical public health interventions to contain infection, delay spread and reduce the
impact of pandemic disease. The evidence base for non-pharmaceutical public health
interventions was recently reviewed by Aledort and colleagues 10. Their recommendations
include mainly hand hygiene, respiratory etiquette, human surveillance and case reporting,
rapid viral diagnosis, provider and patient use of masks and other personal protective
equipment and isolation of the sick.
On February 2007, the US Centers for Disease Control and Prevention released a
guidance document for the use of non-pharmaceutical interventions during an influenza
pandemic entitled Interim Pre-pandemic Planning Guidance: Community Strategy for
Pandemic Influenza Mitigation in the United StatesEarly, Targeted, Layered Use of Non-
pharmaceutical Interventions 11. Their use of non-pharmaceutical interventions is intended to
result in Delaying the exponential growth in incident case, decreasing the epidemic peak, and
reducing the total number of incident cases, thus reducing community morbidity and
mortality. The guidance proposes four non-pharmaceutical interventions to be used by
communities in order to mitigate the effects of a pandemic: isolation of the sick at home or in
a hospital, home quarantine of potentially exposed family members, child social distancing,
including dismissal of students from schools, and adult social distancing, including
cancellation of large public gatherings and alteration of workplace environments and
schedules.
To guide the use of such interventions, the CDC developed a Pandemic Severity Index to
categorize the severity of a pandemic, with the intent of allowing communities to consider
different recommendations under different pandemic circumstances. The Pandemic Severity
Index uses a five point scale that is based on a pandemics case fatality ratio; that is, the
percentage of pandemic influenza cases that die. A category 1 pandemic would have a case
fatality rate of <0.1%, category 3 would have a case fatality rate of 0.51.0%, and category 5
would be >2.0%. Based upon the Pandemic Severity Index level of a pandemic, some or all
of the non-pharmaceutical interventions would be recommended. For example, in a category

1 pandemic, only isolation of the sick would be recommended. In a category 4 or 5 pandemic,
all four non-pharmaceutical interventions would be recommended. The CDC also uses the
Pandemic Severity Index to guide decisions about how long measures need to be
implemented. For example, for a category 2 or 3 pandemic, the CDC suggests that measures
be implemented for 4 weeks or less; for category 4 or 5 pandemics, the CDC recommends
that measures be implemented for up to 12 weeks. The document recognizes that use of non-
pharmaceutical interventions during a pandemic could result in unintended social and
economic consequences. It is also recognized that more research is needed in areas such as
improved surveillance systems that are timely and sensitive to allow for the prompt
determination of the Pandemic Severity Index level, rapid and reliable diagnostic tools, the
feasibility of implementation of the non-pharmaceutical interventions, the efficacy of the
non-pharmaceutical interventions, and the unintended social consequences of the
interventions.
Conduct research to guide response measures: upon pandemic burst, research efforts
should focus on several targets: as the pandemic is identified, policy-makers will face an
immediate need for epidemiological data on the principal demographic groups affected,
modes of transmission, and pathogenicity. Such data will support urgent decisions about
target groups for vaccination and receipt of antiviral drugs. Monitoring the effectiveness of
health interventions is crucial, since many of these interventions have proved useful in the
prevention and control of infections, but their effectiveness during a pandemic has never been
comprehensively evaluated. Constant evaluation of medical and economic consequences is
important as a policy guide for the allocation of resources.
Key points:
The WHO has prepared a pandemic influenza protocol for rapid response and
containment program, which is divided to pre-pandemic phase, the verge of eruption
and pandemic spreading.
Each phase contains its own challenges and obstacles.
Every country has its own responsibility in each step. The WHO is responsible of
coordinating, teaching and controlling local preventive programs. The WHO also
prepares a global network of trained experts and stocks antiviral medication ready
for rapid distribution.
The authors believe that this plan will not prevent the development and spread of
pandemic influenza. In order to access this challenge effectively the world must
conduct a comprehensive cyclic repetitive process of preparedness (applying all the
elements of preparedness discussed above) that will be held in all the levels from the
global level to the local level.
Evaluation of Global Preparedness to Face a

Probable Eruption of Avian Influenza Pandemic
Throughout the world, plans addressing threats posed to poultry and humans by avian
influenza are being developed and implemented. Countries, worldwide, develop these
programs irrespective of whether they have actually faced outbreaks. Overall, the recognition
of the need for national level planning appears to be nearly global. The UN System Influenza
Coordination (UNSIC) published the forth global progress report in October 2008 12. The
report data was obtained by passing national surveys. Of 178 countries surveyed, 83% have
replied to the questions. 96% (140/146) of national authorities have published a national
action plan which includes an integrated response to address avian influenza in animals and
humans (figure 1). UNSIC data confirm that the level of preparedness planning is better in
countries directly affected by HPAI than countries without infection due to the management
of real outbreaks that help to improve and update plans. Specific progress can be identified in
the Africa region: now 92% (33/36) of countries in the region reported having an avian
influenza pandemic control plan. The queries passed enable comparing national preparedness
plans to the steps addressed by the WHO.
Figure 1. Existence of a National Action Plan Which Includes an Integrated Response to Avian and
Human Influenza.
Global preparedness for the eruption and spreading of the pandemic: Surveillance is
crucial for early detection of disease in humans and animals. Controlling the pathogens at the
animal source is the key issue in the fight against zoonotic diseases; animal cases should
trigger immediate investigation to limit the risk of animal to human transmission. UNSIC
data obtained from national authorities indicate that 75% (105/140) of the countries report
having a surveillance system which is operational and capable of detecting HPAI (figure 2).
All countries directly affected by HPAI infection (43 responding) report having a
surveillance system, of which 6 are not yet operational. In non-infected countries 30 of 96

report the absence or the presence of a non operational surveillance system.
Figure 2. Existence of an Animal Disease Surveillance System Capable of Detecting HPAI and Risk
Asessment.
Despite the importance of targeted wildlife surveillance, relatively few countries are
currently implementing activities due to financial, technical, or human resource and expertise
limitations. Wildlife surveillance is primarily carried out at the regional and global level by
international organizations and 31 non-governmental organizations, guided by predictive
species risk tools and spatially oriented to include the most important migratory flyway,
breeding habitats, or stop-over sites.
Reports from national authorities suggest that around 70% (102/144) of the countries are
reporting adequate laboratory access in the country for detecting HPAI in animals, although
the quality and availability of this access has not been assessed. National capacities seem
more developed in countries with experience of H5N1 infection, where significantly higher
proportionate levels of access to laboratory capacity within the country are reported, than
from non-infected countries (44/45 previously affected countries have reported access
compared to 83/98 non-affected). Challenges with animal health laboratory capacity, both at
national and at regional (sub continental) level, are due to a lack of adequate equipment,
management, personnel training and budget. The presence of modern equipment, often not
adapted to local conditions (such as no water or no electricity), is rare. The conditions of
collection and shipment of biological samples to the laboratories is also a limiting factor. Of
the 54 countries surveyed by FAO, only few have capacities to proceed to the final
characterization of the virus, in order to perform sequencing and phylogeny of the circulating
strain.
The major obstacles that compromise surveillance capacities are delays between the
disease occurrence and reporting, possibly resulting in disease spread and the need for
heavier control measures. Average reporting times were measured by the World Organization
for Animal Health (OIE). The average reporting time between the observation of the
suspected HPAI outbreak and laboratory confirmation reported to OIE was around 9 days in
2007, and 24 days in 2008 (although four records of one country reporting delays of 91 to
125 days are eliminated, the average is 12 days). According to data transferred to the UNSIC,
forty three percent (16/28) of the Americas and fifty percent (17/35) of Africas respondents
reported having no national capacity to identify the HPAI strands in human. Some
respondents indicated that they neither have national capacity nor access to a regional
laboratory capacity (3/35 in Africa and 4/42 in Eastern Europe and Central Asia). Out of the
infected countries responding to the survey, only a small number 11% (5/45) reported not
having the capacity to detect H5N1 in humans compared to that of non infected countries
where 42% (40/96) report no capacity.
Vast vaccination schemes and policies have been adopted by few countries worldwide.
The reasons why only few countries have opted for a vaccination strategy include no
justification for the use of the vaccination (country free from the disease, very rare outbreaks,
control and eradication possible without vaccination); the cost of large scale vaccination
campaigns; the lack of trained human resources; the limited ability to monitor the efficiency
of the vaccination campaign and logistical constraints (such as the lack of a cold chain).
Biosecurity assessment measures have been adopted by over 80% of responding
countries. Africa and Asia Pacific report the lowest number of assessments taking place.
Effective biosecurity measures ought to control all food production steps from the source to
the marketplace. The more complex the market chain (i.e. the more steps and people
involved) the harder it seems to be to control and eradicate HPAI. Biosecurity levels are
generally fair to good in larger scale commercial production systems, but poor in small scale
and backyard production. In live bird markets, slaughter houses and processing facilities,
biosecurity levels are also poor and very few countries actually implement biosecurity
measures. In most cases, such measures are not regulatory and therefore not enforceable.
Most of the commercial sector is implementing biosecurity measures on a voluntary basis but
farmers, especially small farmers, and small traders have limited incentives. There is a
positive connection between country income level and levels of biosecurity, but only 16% of
governments have reported to implement biosecurity measures in all production sectors
including villages and backyards.
The other factor which affects preparedness is motivation. Motivation drives from
knowledge, awareness and sense of effectiveness in the battle against pandemic spread.
Although this factor isn't easily measurable, it can be assumed that an existing compensation
scheme can be used to asses motivation of local farmers, and the existence of a national
broadcasted campaign will affect awareness.
Compensation scheme in place for poultry owners whose birds have been culled for the
control of HPAI were reported in 68% of responding countries. African and American
countries report a low number of compensation schemes, as expected by the lack of resources
(Figure 3). Previously infected countries and high income countries report a proportionally
larger prevalence of compensation schemes.
Figure 3. Implementation of compensation schemes for poultry owners whose birds have been culled.
A report published by the United Nations Childrens Fund (UNICEF) assessing

communication initiatives addressing prevention and control of avian influenza distinguishes
between awareness about avian influenza, knowledge about the diseases (routes of
transmission and forms of prevention) and behaviors that people practice. The four key
behaviors essential for disease control and containment were defined by the concerned
international organizations (UNICEF, WHO and FAO) in March 2006. They include hand
washing, cooking thoroughly, separation of poultry new flocks, different poultry species and
from living quarters and reporting all suspected cases of avian influenza infection among
poultry and humans. Awareness can be achieved by mass media or by direct guidance. A
large number of countries and regions launched national mass-media campaigns alerting from
pandemic spread, ranging from 60% in the Americas to 100% in the Middle East and North
Africa. A noticeable smaller proportion of the countries reported implementing assessment
measures as to effectiveness of the campaigns (Figure 4). While the report implies that there
is relative high awareness about avian influenza, the level of knowledge about the disease is
generally low. In addition, the report refers to data suggesting that all of the four behaviors
are rarely practiced in surveyed communities.
National authorities indicate some kind of collaboration across borders takes place. 86%
(126/146) of countries report some kind of cross border collaboration. However, only 52 of
those countries report participation in a sub regional strategy. These results are particularly
low in Asia Pacific (5/25) (Figure 5).
Figure 4. Launching and evaluating National Communications campaigns to Educate About Risks and
Prevent Transmissions to Humans. Campaign Launched 2008. Assessment of National Communications
Campaign Conducted.
Figure 5. Collaboration for Cross Border Prevention and Control of HPAI
Preparedness for the crucial stages of containing and diminishing the pandemic required
pre-existing plans, financing, cooperation and motivation. Quality and comprehensiveness of
national plans vary significantly between countries. The UNSIC survey shows that only
approximately 70% (106/145) of national plans have been endorsed at the top executive level
highlighting concerns regarding their implementation. Of the 35 reported plans which remain
un-endorsed 28 are from non-infected countries. Ninety percent (128/142) of respondents
indicated plans to implement social distancing measures such as school closures or
prevention of mass gatherings. Planning and preparing for border closures was reported by
only 26% (37/142) of responding countries. Countries in the Asia & Pacific region have
reported intentions to implement border closures and restrict movement during a pandemic to
a greater extent than countries in the Americas, Europe & Central Asia. This may be due to
the prevalence of outbreaks in animals and human cases in the region. However, only 30%
(43/142) of the countries reported logistical and legislative provisions in place. The UNSIC
global survey indicates that there are differences in the approaches taken by governments in
various regions and income levels with regard to pharmaceutical intervention, with around
70% (103/142) reporting that they have achieved national endorsement for such interventions
(the scale of these interventions is unknown). In Africa, however, only around 50% of
countries reported such progress relating to a corresponding reliance only on social
distancing measures in plans.
It is essential that private and public organizations prepare for the potential disruption
that a pandemic will cause beyond the health impact, including those that caused by an
increased level of worker absenteeism. Less than half of the countries reporting evidence of
planning have specific sector plans (42/101). Progress in this area varies significantly by
region and income level and remains low. Little or no efforts are made addressing the rights
and interests of disadvantaged groups, despite the likelihood that these groups will be
disproportionately affected in a pandemic. None of the reviewed plans in North Africa and
the Middle East suggested any systematic attempt to identify such groups, and none made
references to any economically or socially disadvantaged groups (with the exception of
Egypt).
Cross border preparations appear to be mixed regionally. Middle East and North Africa
plans surveying indicates that only 44% (7/16) of the plans have included details about
regional or cross border preparations. Similarly, London School of Hygiene and Tropical
Medicine analyses of national plans identified that only a small minority of African countries
have entered into collaborative agreements with their neighbors 13. However, European
Centre for Disease Prevention and Control (ECDC) has identified that 64% of European
countries have undertaken joint policy work with neighboring countries.
All the planned measures and financing availabilities should be tested and improved on
regular bases. Figure 6 demonstrates the number of countries that actually simulated or table-
top tested the plans they have made. While directly comparing figure 1 at the preface of this
discussion to figure 6 at the end, it can be seen that while almost all countries claim to be
prepared for pandemic eruption, many areas and countries have unendorsed and untested
plans, and we can assume that if the disease will erupt and begin spreading in one of these
regions, global capability of fighting, containing and defeating the pandemic would be
diminished.
Figure 6. National pandemic plans and government endorsements.

Key points:
Countries ability to respond to influenza outbreaks is improving. However, critical

gaps in the overall global preparedness are expected to interfere with effective
prevention of the development of avian influenza pandemic.
Surveillance and laboratory systems had improved, plans for response had been
developed and control measures had improved. However, improvement is not
enough and actions must be done to cover every corner in the world in order to
supply effective surveillance that will enable adequate response to control the
development of avian influenza pandemic spread.
The main weaknesses identified are in governance and capacity for animal health
services; more investment in surveillance networks and biosecurity is needed.
Health system capacity to detect and respond to influenza threats to humans
increased, but it varies significantly between regions;
There is an increasing awareness of the threat posed by HPAI H5N1, but this was not
translating into behavior change;
95% of responding countries developed pandemic preparedness plans. Plan quality
varies greatly, many are not operational and pay insufficient attention to sectors
other than health
More efforts are needed to ensure that humanitarian actors are ready to respond to a
full blown influenza pandemic and give increased attention to communicable disease
threats, especially when they cross borders.
Examples of Approaching Pandemic Control in

Regions and Countries
Given the global variations regarding measures needed to be taken in different regions
and the diversity of available resources, different governments have adopted their policies
based on disease history and prevalence, local demographic and geographic conditions,
economic availabilities and community acceptance of the different measures.
Hong Kong has opted for universal vaccination and culling to contain the disease 14 with
some degree of success. After stamping out a major outbreak of H7N7 HPAI in 2003, the
Netherlands has adopted a preventive, voluntary vaccination program in the face of the
current threat of H5N1 15. Two recent studies in early 2006 have examined pandemic
influenza preparedness in Europe 16 and in the Asia-Pacific region 17. The European study
reported that government commitment in most European countries is strong, and levels of
preparedness are generally good, although there are gaps in planning and variation between
the European countries. Cooperation between neighboring European countries needs to be
improved. Regional approaches in the Asia-Pacific region were more polarized, with Hong
Kong (SAR of China), Australia and New Zealand comparing favorably with the best
European plans. The plans of these three countries concentrate on harnessing available
resources and deploying stockpiles of vaccines and antiviral medication. The more resource-
poor countries (Thailand, China and Vietnam) addressed issues which were largely
overlooked in the European plansmainly non-pharmaceutical interventions such as social
distancing, travel restrictions and screening measures.
The UK pandemic contingency plan 18 describes the UK strategy of considering a broad
range of measures: antivirals (of which the UK has now acquired 14.6 million treatment
courses); non-pharmaceutical interventions such as hand washing; voluntary isolation of
cases; effective handling of contacts; and limiting non-essential travel and mass gatherings of
people to minimize the impact of the pandemic while a vaccine is developed against the
pandemic virus.
Currently there were no documented cases of HPAI in the US. The US National strategy
for pandemic influenza 19 focuses on three main threads: 1) preparedness and communication;
2) surveillance and detection and 3) response and containment. In contrast to the UK, the US
does not yet have large stocks of antivirals available for use while a pandemic vaccine is in
development, and instead will rely heavily on similar non-pharmaceutical interventions to the
UK, supplemented with other measures such as school closure.
Pandemic Influenza Preparedness Programs in

Israel
Applying the various influencing factors regarding preparedness for a pandemic in the
unique Israeli setting leads to several important insights regarding local pandemic
preparedness, as shown in the following examples.
In March 2006 samples taken from a commercial turkey farm in southern Israel due to
unexpected mortality rates (>0.7% per day) were positive for avian influenza subtype H5 by
PCR. Eight more outbreak foci in commercial poultry farms in small settlements were
identified within 2 weeks 20. In February 2006, influenza virus (H5N1) was detected for the
first time in Egypt. In March 2006, outbreaks were detected simultaneously in the Palestinian
Authority's Gaza Strip and Israel. Later in March 2006, a single case was detected in Jordan.
The near-simultaneous detection of several outbreak foci specifically on turkey farms,
increase the likelihood that the virus disseminated through the use of shared vehicles or by
personnel. Alternatively, the fact that all 9 farms used open sheds may support the role of
migratory birds in disease transmission.
The key control measures taken and the guiding principles for anti-viral prophylactic
treatment were based on distance from eruption center. Israeli-Palestinian cooperation
allowed coordination of cross-border mitigation efforts. Overall, these control measures
enabled full outbreak containment within 17 days, without further recurrences.
The challenges faced by the Israeli authorities. Agent and vector factors are expected to
determine much of the local impact of the pandemic, but they generally cannot be influenced
by preparedness and mitigation efforts. As these factors will remain unknown until the first
stages of the pandemic, Israeli preparedness planners have taken into account a wide range of
scenarios with different attack and mortality rates 21. For instance, a highly transmissible
pandemic may render isolation and quarantine efforts largely futile 22 and will require the
unparalleled ability to rapidly mobilize medical equipment and personnel to meet the
increased demands for care in both primary and secondary care facilities. A less transmissible
strain may enable an effective containment approach and may require therapeutic measures
similar to those taken during severe seasonal influenza epidemics. Israel has ensured that a
legal and ethical framework for implementation of response measures exists. Including
pandemic influenza in the list of dangerous communicable diseases defined by Israeli
law will allow the Ministry of Health to uphold extreme measures such as involuntary
quarantine and isolation, if needed.
Stockpiled antivirals and antibiotics are considered an important strategy to Israel s
preparedness. The Israeli Ministry of Health has successfully used cost-benefit analyses to
persuade decision makers to invest the funds necessary for the rapid creation of a national
antiviral stockpile, and several strategies for the use of these drugs during the pandemic are
considered23. Prioritizing target groups for antiviral drugs and vaccines, expected to be in
short supply, requires the addressing of complex ethical, legal, social, and political
considerations. The choice of which groups to prioritize would derive, in part, from the
prioritizing of the various goals in using these drugs. If the focus is on reducing all mortality,
different groups may be prioritized than if the main attempt is to reduce social disruption. A
national ethics committee was appointed to address these issues.
Key Points:
Various countries opted for different approaches for pandemic control and
prevention.
Hong-Kong is an example of a country which was infected by avian influenza and
opted for population vaccination.
Other countries, mainly the European region (including Israel) are examples for
antivirals as the therapy of choice to prevent pandemic spreading.
The US, as well as less prosperous countries relay on population isolation for disease
prevention.
The authors believe that local preparedness will only give a little delay and only a
small level of protection against pandemic spread. The only method that can give a
durable prevention of spread despite local preparedness is vaccination but there is a
risk the disease will spread before the development of effective vaccine.
The authors believe different prevention methodology in different areas will not
produce a protective global network and the disease will spread through the gaps of
this network and eventually will not spare any area in the world.
The authors believe that the only way to prevent each phase of the pandemic
development and spread is coordinated global collaboration and comprehensive
implication of global preparedness strategy allover the world.
Summary and Conclusions
Avian influenza presents as a probable cause for the next pandemic influenza eruption.
HPAI is a serious poultry disease. In view of the devastating consequence it causes to the
poultry industry and the potential for the virus to mutate into a pandemic flu virus, countries
all over the world adopt various strategies best suited to their needs and poultry production
systems to prevent and control HPAI. A strong veterinary service with adequate technical
manpower and financial resources to devise strategies and implement surveillance and
control programs, and a well developed poultry industry with high standards of biosecurity
are key success factors in combating HPAI. Some are very successful while others less so.
Countries like Malaysia and the UK do not rely on vaccination but adopt import control and
biosecurity to keep out HPAI 24. They have also successfully stamped out occasional
incursions of HPAI.
While there has been worldwide progress with development of pandemic preparedness
plans, there are also great disparities in preparedness among countries. Political and financial
commitment to pandemic readiness tends to be greater in countries that have experienced
HPAI outbreaks and countries supported through regional political bodies. Three major
categories of country preparedness can be identified:
[1] Wealthier industrialized countries that have deepened and developed multi-sector
pandemic preparations, in sectors other than health.
[2] Middle income countries that have developed the animal health, communications
and human health components of their national plans, but have yet to prepare for
continuity in sectors beyond health, including the provision of essential services, to
mitigate the economic and social impacts of pandemic.
[3] Low income countries that have not, during the past year, had the resources needed
to advance their level of pandemic preparedness. They seek significant financial and
technical support from international actors. They also anticipate putting pandemic
preparedness within the context of wider crisis preparations.
Finally, Preparedness planning for avian influenza should account for the unique
challenges associated with a simultaneous multifocal outbreak, including personnel
recruitment and allocation; coordination of all parties involved in outbreak mitigation and
investigation; simultaneous culling and disposal in multiple sites; and coordinated central and
local risk communication efforts. Case definition and antiviral prophylactic policies may be
revised ad hoc according to the unfolding events and in response to the medical and
psychological needs of each population. Outbreak containment could be partially achieved
and the magnitude of the first wave of pandemic spread can be reduced by non
pharmacological methods and by antiviral medications even without the use of vaccines.
These measures might give the global community enough time to develop and produce a
vaccine that might have the chance to prevent the second wave of pandemic spread.
References
[1] Peltz R, Avisar-Shohat G, Bar-Dayan Y. Differences in public emotions, interest, sense

of knowledge and compliance between the affected area and the nationwide general
population during the first phase of a bird flu outbreak in Israel. J Infect 2007;
55(6):545-550.
[2] World Health organization. WHO Global influenza preparedness plan. The role of
WHO and recommendations for national measures before and during pandemics. 2005.
http://www.who.int/csr/resources/publications/influenza/GIP_2005_5Eweb.pdf.
accessed October 2008.
[3] Vijaykrishna D, Bahl J, Riley S et al. Evolutionary dynamics and emergence of
panzootic H5N1 influenza viruses. PLoS Pathog 2008; 4(9):e1000161.
[4] World Health organization. WHO pandemic influenza draft protocol for rapid response
and containment. 2006. http://www.who.int/csr/disease/avian_influenza/guidelines/
protocolfinal30_05_06a.pdf. accessed October 2008.
[5] Goodman C, Mucherjee D, Faulkner E. How effective would antiviral vaccination and
antiviral drug prevention and treatment strategies before reducing the impact of the
next influenza pandemic? WHO Regional Office for Europe's Health Evidence
Network. 2006.
[6] Centers for disease control and prevention. High levels of adamantane resistance aming
influenza (H3N2) viruses and interim guidelines for the use of antiviral agents - united
states 2005-06 influenza session. United states 2005-06 influenza session. MMWR
morb mort weekly rep. 55 ed. 2006 p. 44-46.
[7] Sellwod C, Asgari-Jirhandeh N, Salimee S. Bird flu: if or when? Planning for the next
pandemic. Postgrad Med J 2007; 83:445-450.
[8] Gani R, Hughes H, Fleming D, Griffin T, Medlock J, Leach S. Potential impact of
antiviral drug use during influenza pandemic. Emerg Infect Dis 2005; 11(9):1355-1362.
[9] Jefferson T, Demicheli V, Rivetti D, Jones M, Di PC, Rivetti A. Antivirals for
influenza in healthy adults: systematic review. Lancet 2006; 367(9507):303-313.
[10] Aledort JE, Lurie N, Wasserman J, Bozzette SA. Non-pharmaceutical public health
interventions for pandemic influenza: an evaluation of the evidence base. BMC Public
Health 2007; 7:208.
[11] Centers for Disease Control. Interim Pre-pandemic Planning Guidance: Community
Strategy for Pandemic Influenza Mitigation in the United States - Early Targeted
Layered use of Non-Pharmaceutical Interventions. Centers for Disease Control and
Prevention, Atlanta, GA, 2007 http://www.pandemicflu.gov/plan/community/
commitigation.html;Accessed November 2008.
[12] UN System Influenza Coordinator & The World Bank. Responses to Avian Influenza
and State of Pandemic Readiness - Fourth Global Progress Report 2008.. http://un-
influenza.org/files/ProgressReport2008.pdf. Accessed November 2008.
[13] Ortu G, Mounier-Jack S, Coker R. Pandemic influenza preparedness in Africa is a
profound challenge for an already distressed region: analysis of national preparedness
plans. Health Policy Plan 2008; 23(3):161-169.
[14] Ellis TM, Sims LD, Wong HKH et al. Use of avian influenza vaccination in Hong
Kong. In: Schudel A, Lombard M, editors. OIE/FAO International Conference on
Avian Influenza. 2006 p. 133-143.
[15] Ministry of Agriculture Nature and Food Quality the Netherlands. Action Plan:
preventive, voluntary vaccination of poultry in the Netherlands in response to the
current threat of Avian Influenza (H5N1). 2006. .www.minlnv.nl/cdlpub/servlet/
CDLServlet?p_file_id=14099. Accessed November 2008.
[16] Mounier-Jack S, Cocker R. How prepared is Europe for pandemic influenza? Analysis
of national plans. Lancet 2006;(367):1405-1411.
[17] Cocker R, Muunier-Jack S. Pandemic influenza preparedness in the Asia-Pacific
region. Lancet 2006;(386):886-889.
[18] UK department of health and cabinet office. Pandemic influenza - a national framework
for responding to an influenza pandemic. 2007. http://www.dh.gov.uk/en/
Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/DH_080734.
Accessed November 2008..
[19] Homeland Security Council, the white house Washington D.C. National strategy for
pandemic influenza implementation plan. 2006. www.whitehouse.gov/homeland/
nspi_implementation.pdf. Accessed November 2008.
[20] Balicer RD, Reznikovich S, Berman E et al. Multifocal avian influenza (H5N1)
outbreak. Emerg Infect Dis 2007; 13(10):1601-1603.
[21] Balicer RD, Huerta M, Davidovitch N, Grotto I. Cost-benefit of stockpiling drugs for
influenza pandemic. Emerg Infect Dis 2005; 11(8):1280-1282.
[22] Fraser C, Riley S, Anderson RM, Ferguson NM. Factors that make an infectious
disease outbreak controllable. Proc Natl Acad Sci U S A 2004; 101(16):6146-6151.
[23] Balicer RD, Huerta M, Grotto I. Tackling the next influenza pandemic. BMJ 2004;
328(7453):1391-1392.
[24] Department for Environment Food and Rural Affairs. Avian Influenza What
Government is doing? 2008. http://www.defra.gov.uk/animalh/diseases/notifiable/
disease/ai/policy/index.htm. Accessed November 2008.
Chapter IV
Molecular Pathogenesis of Avian

Influenza and Prospect of Therapy
Using Small Interfering RNA
Jeanne Adiwinata Pawitan

Department of Histology, Faculty of Medicine University of Indonesia
Abstract
Small interfering RNA (siRNA) technology is now available to switch off a target
gene. Many studies reported promising results of siRNA in combating viral infection in
animals, including avian influenza infection. This review will discuss the molecular
pathogenesis and the prospect of siRNA for the therapy of avian influenza infection.
Introduction
Influenza-A viruses - including the H5N1 avian influenza virus - are negative-sense RNA
viruses that belong to the family Orthomyxoviridae. The viral genome consists of eight
segments that encode 11 viral proteins i.e. two surface glycoproteins namely hemagglutinin
(HA) and neuraminidase (NA), four polymerase proteins (PB1, PB2, PA, and PB1-F2),
nucleocapsid protein (NP), two nonstructural proteins (NS1 and NS2 that is recently called
nuclear export protein, NEP),(1) and two matrix proteins (M1 and M2).
These proteins have various functions and some of them were shown to play a role in the
pathogenicity and virulence of H5N1 influenza virus.(2, 3) Avian influenza virus may adapt
to mammals by mutations in the polymerase proteins and nucleoprotein (NP), which causes a
considerably higher polymerase activity and thus increase virulence in mammalian cells such
as in highly pathogenic avian influenza virus H5N1.(4) Therefore, switching off the
70 Jeanne Adiwinata Pawitan
expression of certain proteins by means of small interfering RNA (siRNA) may be used in the
therapy of avian influenza.
Small Interfering RNA and RNA Interference
The technique of RNA interference was introduced nearly 11 years ago by Andrew Fire
from Stanford University School of Medicine and Craig Mello from University of
Massachusetts Medical School, who are Nobel Prize winners of the year 2006 in Physiology
or Medicine. They and their colleagues reported that using double-stranded RNA was far
more effective compared to sense or anti-sense RNA in silencing a target gene, and they
called the technique as RNA interference (RNAi). However, RNA interference rarely leads
to a complete silencing of the target gene, and therefore the technique is alternatively called
as a knockdown of gene expression.(5)
In nature, two machineries for RNA interference have been recognized. Both use small
double stranded RNA to control post transcriptional gene expression. The first is endogenous
and called micro RNA (miRNA) and the other may be endogenous or exogenous and called
small interfering RNA (siRNA).(6) In mammalian cells, endogenous miRNA differs from
endogenous siRNA in their biogenesis,(7) the complementary degree to their target, and
presumably mode of silencing. While siRNA is fully complementary, miRNA is only
partially complementary to the target. Therefore, siRNA usually induce mRNA degradation,
while miRNA usually repress translation.(8)
Figure 1. ds siRNA= double strand small interfering RNA, Ago= Argonaute endonuclease, RISC=
RNA-induced silencing complex, siRNA= small interfering RNA, mRNA= messenger RNA.
Molecular Pathogenesis of Avian Influenza and Prospect of Therapy... 71
Mechanism of mRNA Degradation by siRNA
In a cell, small interfering RNAs (siRNAs) in the cytoplasm are bound by RNA-induced
silencing complex (RISC). Then, one strand of the siRNA is degraded, while the unpaired
remaining strand will guide the RISC to mRNAs that contain complementary sequence to the
guiding siRNA. After that, the mRNA is degraded by an Argonaute endonuclease in the
RISC, and protein expression is decreased, (9) (Figure-1).
Targeting Viral RNA
Theoretically, every parts of the viral genome encoding viral proteins may become the
target of silencing using siRNA, thus disrupting viral ability to infect cells, viral life cycle, or
viral mechanism to evade host immune response.
Targeting Hemeagglutinin mRNA
Influenza viruses infect cells by firstly attaching to cells. This attachment is mediated by
the hemagglutinin found on the surface of the virus that binds the receptor on the target cell.
Hemagglutinin of avian influenza viruses prefers to bind to a receptor in avian intestinal and
respiratory epithelium. The receptor consists of sialic acid linked to galactose by an -2,3
linkage.(10) Hemagglutinin of human influenza viruses prefers to bind to human receptors
that mainly consist of sialic acid linked to galactose by an -2,6 linkage. This receptor is
mainly found in the respiratory epithelium and conjunctivae. However, both human receptors
(having the -2,6 linkage) and avian receptors (having the -2,3 linkage) can be found in
human, the latter especially found in the lower respiratory tract and conjunctivae; a fact that
allows human infections by avian subtype viruses.(10-13) Further, hemeagglutinin binding
efficiency to human receptors may influence viral attachment and thus viral entry into
cells.(14)
After attachment, viral entry is promoted by proteolytic digestion of hemeagglutinin by
host proteases, and virulence is determined by the number of arginine residues on the
hemeagglutinin molecule that is needed in the proteolytic cleavage.(3)
Further, a study showed that in vitro the hemeagglutinin of H5N1 viruses (H5)
suppressed CD8+ cytotoxic T lymphocytes perforin expression. Therefore, it is suggested
that this suppression may cause impaired cytotoxicity of the T lymphocytes and caused
failure in the clearance of cells that were infected by the H5N1 virus. Failure in the clearance
of infected cells may cause prolonged T lymphocyte stimulation that leads to excessive
interferon-gamma production, and finally up-regulation of pro inflammatory cytokines in
macrophages and severe manifestations.(3)
Targeting hemeagglutinin mRNA may be very beneficial in preventing virus entry into
cells and in reducing severe manifestation. However, there are 15 hemeagglutinin subtypes
among influenza A viruses, and the hemeagglutinins are not well conserved. In addition,
hemeagglutinins are subjected to antigenic drifts and shifts that make them unfavorable
candidates as siRNA target.(15) Therefore, there was no report concerning the use of siRNA
to target hemeagglutinin mRNA. However, hemeagglutinin is very important in the
construction of siRNA delivery system.(16, 17)
Targeting Neuraminidase mRNA
Neuraminidase is a sialidase that cleaves the hemeagglutinins on progeny virions that are
attached to sialic acid-containing receptors on the surface of infected cells in which the
virions are generated. Cleavage will free the virus particles to infect other cells.(3)
Neuraminidases are very variable; in H5N1 virus isolates from the 1997 outbreak, the
neuraminidase has a 19 amino acid deletion in the stalk region of the enzyme and that is
supposed to play a role in virus adaptation during transmission from aquatic to terrestrial
birds. In the first human isolates in 2003 the neuraminidase has no deletion in the stalk
region. However, similar deletion in the stalk region as in the 1997 outbreak was found in
recent human and chicken isolates. Viruses having a stalk deletion in their neuraminidase are
less capable to be freed from the cells, but this shortage is counterbalanced by an additional
glycosylation site in the hemeagglutinin that facilitates the release of virus particles.(3)
Neuraminidase inhibitors are effective for most H5N1 viruses.(3) Therefore, targeting
neuraminidase mRNA may be an effective way in prophylaxis and therapy of avian
influenza. However, neuraminidases are not well conserved, and among influenza A viruses
there are 9 subtypes. This variability makes neuraminidases unfavorable candidates as siRNA
target.(15) Further, a small change as a histidine to tyrosine substitution at position 274 in the
neuraminidase has caused resistance to the neuraminidase inhibitor oseltamivir in three
human H5N1 cases.(3)
Targeting Viral M Protein mRNA
Matrix (M) proteins are encoded by the M gene and located beneath the viral envelope.
They play a role in virus assembly and release from infected cells. The M1 is the most
abundant protein in influenza A virus particles. It is a small membrane-binding protein that is
critical for virus budding,(18) while M2 is a small trans-membrane protein with H+ ion
channel property that controls the pH in the Golgi complex during hemeagglutinin synthesis
and virion release. In isolates derived from Thai and Indonesia, the M2 encoding gene was
found to be under positive selection. This fact suggests that M2 protein might play a role in
virus adaptation in host cell.(3)
In most of the clade 1 viruses (viruses that are isolated from humans or birds in the Indo-
China peninsula), the M2 protein contains a serine to asparagine substitution at residue 31.
This substitution is supposed to play a role in the resistance to the inhibitor of ion channel
activity of M2 protein, amantadines. (3, 19) However, such substitution is only found in few
of the clade 2 viruses (that are isolated from human or birds in China, Indonesia, Japan, and
South Korea).(3)
Considering their role, targeting M protein mRNA may be beneficial as was shown in a
study on Madin-Darby canine kidney (MDCK) cells and BALB/c mice. This study used
siRNA against M protein that is packed in a plasmid (pS-M48) that could reduce virus titers
in the MDCK cells and in the lung of infected mice, and could partially protect the mice from
lethal challenge by highly pathogenic H5N1 avian influenza virus.(20) However, another
study using siRNA against another conserved region of the M protein (M-37) showed that in
vitro the siRNA was not so effective in MDCK cells and was ineffective in chicken
embryos.(15)
Targeting Viral Polymerase Complex and its Transporting Machinery
The polymerase complex consists of several subunits, namely the polymerase basic
proteins 1 and 2 (PB1 and PB2), and polymerase acidic protein (PA).(1) The heterotrimeric
complex is responsible for replication and transcription of the viral genome in the nucleus of
infected cells.(2)
The polymerase proteins are supposed to be transported from the cytoplasm into the
nucleus by a nuclear import factor that is called Ran binding protein 5 (RanBP5),
alternatively known as importin 5, importin 3, or karyopherin 3. This nuclear import factor
interact with the PB1 subunit either alone or with a PB1-PA complex, and is assumed to
transfer the complex into the nucleus.(1)
Replication of the virus does not need a primer and occurs in two steps. In the first step, a
negative-sense viral RNA (vRNA) is copied to a positive-sense RNA (cRNA). In the second
step, the cRNA serves as a template to make new vRNA. Transcription of viral mRNA needs
an RNA primer. This primer is provided by the polymerase complex whose endonuclease
activity cleaves host pre-mRNA into short capped RNA of 9-17 nucleotides. This primer
generating process is called cap snatching, and the primer is also called the cap.(2, 21)
The N-terminal region of PA is responsible for multiple functions, such as protein
stability, endonuclease activity, cap binding that is needed to begin transcription, and
promoter binding that is needed to begin replication (21) and to stabilize the polymerase
complex,(2) while PB2 is supposed to play a role in the ability of the virus to replicate in
human or mouse cells.(14) Further, polymerase complex plays a role in the lethality of highly
pathogenic H5N1 virus in ferrets and mice. Comparison of highly pathogenic with low
pathogenic viruses showed 11 amino acid differences in polymerase genes (4 in PA, 3 in
PB1, and 4 in PB2).(22)
Therefore, in targeting viral polymerase complex, focusing on the terminal region of PA
that has multiple functions may be promising. These assumptions were proven in vitro and in
vivo,(23, 24) In vitro, antiviral properties of a siRNA expression plasmid against PA were
evaluated in MDCK cells, chicken embryo fibroblast cells, and embryonated chicken eggs.
The plasmid was efficiently transcribed into short hairpin that could silence the expression of
H5N1 influenza virus PA protein in those cells and eggs. Further, in a transient replication
model, the plasmid showed an effect on influenza virus-induced apoptosis.(24) In another
study using MCDK cell line, siRNA against PA were transfected by electroporation and
greatly reduced the PA mRNA as well as virion RNA, bot not cellular RNAs.(15)
In vivo, administration of siRNA against PA 20872106 (siPA) significantly inhibited

virus replication that was represented by a 10 fold reduction in lung virus titers in H1N1
influenza virus infected mice.(23) Another study using chicken embryos showed that
inoculation of siRNAs against 21 nucleotides in the conserved region of PA and PB1 i.e. PA-
2087, and PB12257 combined with oligofectamine potently inhibited influenza virus
production in the chicken embryos. However, siRNA against another conserved region of
PB1 (PB1129) were ineffective in inhibiting influenza virus production in chicken embryos,
and no significant reduction of virus titer was observed when oligofectamine was omitted.
This result suggests that oligofectamine serves as a vehicle to deliver the siRNA into cells in
vivo.(15)
Further, as the polymerase should work in the nucleus, targeting its transporting
machinery may be advantageous, and was proven in an in vitro model using 293T cells that
harbored PA, PB1 and PB2 containing plasmids. In this model, knock-down of RanBP5
using siRNA decreased nuclear accumulation of PA-PB1 dimer and accumulation of viral
RNA in the 293T cells.(1)
Targeting Viral Non Structural Protein mRNA
Human influenza A virus is able to block host innate immunity i.e. type I interferon
(IFN) response that results in enhanced replication of the virus, as IFN in certain condition
causes abortive infection. However, in other condition, such as in human A549 cells, IFN
induction was not directly correlated with the replication of avian influenza viruses. Even so,
human cells deficient in type I IFN response showed enhanced avian influenza virus
replication, which suggests the role of human type I IFN response in limiting avian influenza
virus replication.(14)
This IFN blocking ability is the property of the nonstructural NS1, which is supposed to
work in several mechanisms: i.e. to sequester double-strand RNA (dsRNA) by its amino
terminus, to bind protein kinase R, and to inhibit posttranscriptional processing of cellular
antiviral mRNAs that is accomplish by binding the important factors for cleavage and
polyadenylation specificity, and poly(A)-binding protein II. Further, NS1 has several
domains that play a role in anti-IFN property. Therefore, amino acid sequence differences
between strains is supposed to determine the predominant mechanism used by a particular
influenza virus to counteract host IFN response.(14) Further, in mouse and pig models, NS1
protein was supposed to be needed for the high virulence of the 1997 H5N1 viruses.
However, in ferrets but not mice, NS gene was proven to contribute to the lethality of highly
pathogenic H5N1 virus.(22)
In addition to impairment in innate immune response, NS1 also functions in reducing
adaptive immunity by inhibiting host signal transduction and gene expression that is
important in host protection against influenza virus replication. The inhibition is supposed to
work in several mechanisms: i.e. NS1 inhibits the mRNA export machinery that consists of
NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5 by binding to them and prevents the
nucleoporins directed mRNA to go through the nuclear pore complex. Moreover, influenza
virus inhibits the expression of a nucleoprotein (Nup98) that serves as a docking site for
mRNA export factors. Two of the mRNA export machinery i.e. Nup98 and Rae1 are induced
by IFNs. Therefore, IFN blocking by NS1 also impairs mRNA export machinery.(25)
Therefore, targeting NS1 protein is very promising as has been shown in a study using
siRNA in vitro and in vivo. In vitro, siRNA against conserved region of NS1 given to cells in
culture prior to H5N1 virus infection caused a 5 fold reduction in hemeagglutinatin (HA)
titers compared to control. In vivo, siRNA could protect H5N1 virus infected chickens from
virus-induced death up to 87.5%. Further, there was a significant reduction in plaque forming
unit and viral RNA level in lung tissues of the siRNA protected animals as seen by plaque
assay and real time polymerase chain reaction analysis respectively. The result of this study
suggests that siRNA against conserved region of NS1 might be promising for prophylaxis
and therapy of the H5N1 influenza virus infection in human.(26)
Targeting Viral Nucleoprotein mRNA
Nucleoprotein (NP) together with the polymerase complex constitutes the

ribonucleoprotein complex. Recognition of viral RNA gene segments by polymerase
complex is facilitated by NP encapsulation of the RNA segment.(3) The ribonucleoprotein
complex first worked in the nucleus, and then exported to the cytoplasm. (27) Considering
the important role of NP in the activity of polymerase complex, targeting NP mRNA might
be promising as has been shown by several studies.
In a study on avian influenza virus infected cells in culture using siRNA expressing
plasmid against NP showed that the plasmid efficiently transcribed the siRNA as was
measured by northern blot analyses. Further, western blot analyses showed the inhibition of
NP expression.(24)
To be used in therapy, siRNA should be able to prevent subsequent infection by newly
released virus. This property was proven for siRNA against a conserved region of NP (NP-
1496) in vitro. Transfection of the siRNA to influenza virus infected MDCK cells showed
reduction in virus titer compared to mock transfection.(15)
Another study used siRNA against a highly conserved region of NP in vivo. In the study,
delivery of the siRNA to highly pathogenic avian influenza A virus (H5 and H7 subtypes)
infected mice significantly reduced lung virus titers and prevented the mice from lethality
that was not mediated by IFN response. This result indicate that siRNA against the highly
conserved region of NP is promising in controlling avian influenza infection.(23)
Further siRNA expression plasmid against both NP and PA simultaneously targeted NP
and PA segments in the influenza virus genome and inhibited the synthesis of new viruses
that was demonstrated by the reduced cytopathogenic effects and viral induced apoptosis in
influenza virus infected cells in culture.(24)
Targeting Hosts mRNA
Viral-host interaction may help the virus to replicate by using host machineries, and may
cause severe manifestation in host due to host immune response and up-regulation of host
proteases. Therefore, there are two purposes in targeting hosts mRNA, namely to prevent
viral replication and to alleviate severe manifestation.
Targeting Hosts mRNA to Prevent Viral Replication
Viral entry into host cell needs host proteases to cleave viral hemagglutinin precursor.
The proreases needed are trypsin type proteases, and for the highly pathogenic avian
influenza virus, some of the needed proteases are furin, pro-protein convertase, type II
transmembrane serine proteases of the cell membrane, mosaic serine protease large form
(MSPL) and its splice variant TMPRSS13. Therefore, the use of siRNA to target those
proteases mRNA may prevent viral entry, though side effects due to the decrease of the
enzymes may arise.(28)
Sulfatide, which is highly expressed in various mammalian cells, including the epithelial
cells of the intestines and trachea, is synthesized by two transferases, namely ceramide
galactosyltransferase and cerebroside sulfotransferase, and is degraded by arylsulfatase A.
Host sulfatide that is associated with viral hemeagglutinin plays a role in influenza A virus
replication by facilitating translocation of the virus newly synthesized ribonucleoprotein
complex from the nucleus to the cytoplasm. Therefore, targeting host sulfatide mRNA might
be promising as was shown in a study using antisulfatide monoclonal antibody. In the study,
antisulfatide monoclonal antibody treatment of influenza A virus-infected cells significantly
reduced viral replication and prevented accumulation of viral NP in the nucleus of host cells.
Further, the antibody could protect intra-nasally infected mice with pathogenic influenza
A/WSN/33 (H1N1) virus against lethal challenge.(27)
Targeting Hosts mRNA to Alleviate Severe Manifestation
In H5N1 influenza virus infection, pathogenesis and severe manifestations in humans are
supposed to be due to up-regulation of cytokines, chemokines, TNF related apoptosis
inducing ligand (TRAIL), and apoptosis in organs.(3)
In vivo, in most of H5N1 influenza patients, significantly elevated serum pro-
inflammatory cytokines and chemokines was detected, and the serum levels were correlated
to viral loads in pharyngeal specimens.(3) Further, the levels of cytokines and chemokines
were significantly higher in H5N1 patients who died compared to those who recovered.(19)
In addition, hemophagocytotic activity was reported in H5N1 influenza autopsy cases. These
findings suggest that high viral loads may induce up-regulation of cytokines and
chemokines.(3)
The up-regulated cytokines and chemokines are various interleukins, namely IL-6, IL-8
that acts as a neutrophil attractant, IL-10, interferon gamma that is a strong inducer of the
macrophage attractant chemokines, namely CXCL10 and CXCL9, and the monocyte chemo-
attractant protein 1 (CCL-2). From the various cytokines, IL-8 is supposed to play a role in
the development of acute respiratory distress syndrome (ARDS).(19) However, serum
cytokine and chemokine levels do not always reflect their local production in the lungs.(3)
Investigations on local expression of cytokines and chemokines in the lungs of human

H5N1 influenza autopsy cases were done using immunohistochemistry and RT-PCR. The
results showed high expression of tumor necrosis factor- (TNF-) in a case from Hong
Kong by immunohistochemistry, and in two other cases by RT-PCR. In addition, increased
expressions of macrophage inflammatory protein-1, regulated on activation normal T cell
expressed and secreted (RANTES), interferon-, interferon-, and interleukin-6 were detected
by immunohistochemistry in another case.(3 )
As data regarding serum cytokine levels and the local expression in human lungs are
limited, interpretation of the data in critically ill patients is difficult to conclude. Therefore, in
vitro and animal studies were conducted. In vitro studies showed that H5N1 avian influenza
viruses caused increased expression of pro-inflammatory cytokines and chemokines in human
macrophages and respiratory epithelial cells compared to human influenza viruses. Further,
H5N1 influenza infected macrophages showed a delayed onset of apoptosis compared to
those infected with H1N1 influenza. Therefore, immune-mediated pathology may be
increased due to a longer period of cytokine and chemokine secretions by macrophages; and
prolonged survival of infected macrophages.(3)
In addition to up-regulation of pro-inflammatory cytokines and chemokines in
macrophage, there is up-regulation of functional TRAIL in macrophages infected with the
H5N1 virus. TRAIL is a death receptor ligand that upon binding to its receptor on a target
cell will trigger apoptosis of the target cell. In vitro, H5N1 virus infected macrophages
showed increased expression of TNF and TRAIL, and caused increased apoptosis in T
lymphocytes that were co-cultured with the infected macrophages. This phenomenon may
play a role in the lymphopenia and lung injury that are frequently observed in H5N1
patients.(3)
Further, in human autopsies apoptosis was detected in the lungs especially in alveolar
epithelial cells and leukocytes, as well as in cells of other organs such as spleen and intestinal
tissues. Therefore, apoptosis either that is caused by direct viral replication or up-regulation
of cytokines, chemokines and TRAIL may play a major role in the pathogenesis of the
injuries in the lungs and other organs.(3)
Therefore, targeting cytokine (especially IL8), chemokine and TRAIL mRNA may be
promising in reducing severe manifestations.
Furthermore, influenza A viruses were proven to increase significantly the expression of
latent pancreatic trypsin ectopically and pro-matrix metalloprotease-9 in various organs, that
caused digestion of collagen type IV, and thus destruction of the basement membrane. The
enzymes also caused destruction of tight junction components in endothelial cells, and thus
caused severe edema and connective tissue damage in various organs that lead to multiple
organ failure.(28) Therefore, targeting the mRNA of those enzymes may alleviate severe
symptoms and prevent multiple organ failure.
Mode of Artificial siRNA Delivery
Small RNAs can be easily synthesized to target any gene. The gene targeted can be host
genes or viral gene.(9) Further, there are various mode of delivery, in vitro as well as in vivo.
In vitro, several studies used siRNA expression plasmids or electroporation of naked siRNA
to transfect cells in culture.(15, 24) In vivo, intra venous injection of naked siRNA and intra-
nasal oligofectamine-siRNA complex has been administered to laboratory animals. (23) In
addition, bi-layer liposomes and virosomes can be used to deliver siRNA in vitro or in
vivo.(16, 17, 29)
The use of siRNA for the therapy of avian influenza can use 2 kinds of RNA, ie. double
strand (ds) siRNA that has a transient effect, or the more complicated short hairpin (sh) RNA
transcription system that has a sustainable effect.(29) Avian influenza is not a chronic disease
and does not need a sustainable siRNA delivery. Therefore, the prospect of ds siRNA for the
therapy of avian influenza is promising, but some obstacles might be encountered.
Advantages and Disadvantages of the Various Mode of siRNA Delivery
When naked siRNA is delivered via the blood stream, it may be recognized as foreign
RNA and treated as viral infection; thus it will be rapidly degraded before it can function.
Further, when naked siRNA can survive and safely reach the target cell, the problem persists
as to whether it can go through the lipid bi-layer of the cell membrane; eventhough an
opinion stated that stable siRNA at sufficient dose will be readily taken up by cells.(30, 31)
However, delivery of high dose siRNA resulted in faster degradation of the siRNA and
rebound of the target gene expression.(32) Therefore, this mode of delivery alone might not
be suitable for the prevention and treatment of avian influenza.
In an animal study, delivery of naked siRNA is combined with another mode of delivery,
and showed a good result. In the study, naked siRNA was administered intravenously, and
after a lethal H1N1 influenza virus challenge, it was followed by a second dose of siRNA in a
lipid carrier (oligofectamine) intranasally. In this study, the mice that received combination of
siRNA against NP and PA showed a 100% survival, while only 60% of the control mice
survived. Therefore administration of siRNA-oligofectamine complex intra-nasally may be
promising in the prevention and therapy of avian influenza.(23)
The problem of cell penetration of naked siRNA may be solved by either wrapping the
siRNA in a lipid delivery system such as bi-layer liposome or by modifying the phosphate
backbone to lower its charge.(29-31)
Another problem is when siRNA is used against proinflammatory cytokine mRNA, and
reaches non target cells or when it switch off non-target genes and exerts toxic effects. In
this case, modifications may be introduced to target the siRNA to specific cell types. (30, 31)
Another mode of delivery is by using virosomes. Virosomes are vesicles that are
constructed from influenza virus envelopes and thus bear hemagglutinin. Cationic lipid
siRNA complex can be incorporated into the virosome. Then, hemeagglutinin will bind to a
cellular target membrane, and receptor mediated endocytosis causes the content to be release
into the cytoplasm of the target cell. This mode of delivery was proven to successfully deliver
siRNA to several cell lines in vitro. Furthermore, siRNA was delivered into the cells in
peritoneal cavity when the siRNA containing virosomes were injected into the peritoneal
cavity. Therefore, this mode of delivery is supposed to be very promising in vivo, such as for
topical administration to the respiratory tract.(16) Another advantage of avian influenza
therapy using virosomes to transport siRNA into the cell is the HAs in the virosomes will
compete with viral HAs, thus reducing viral entry.
Conclusion
In vitro and animal studies showed promising results in the use of siRNA to target highly
conserved viral RNA such as certain regions of PA, PB1, NS1 and NP. Further, in addition to
the several modes of delivery, targeting host mRNA may be promising though further
researches in the efficacy and side effects are needed.
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Chapter V
Avian Influenza: Intervention and

Therapy
Hongxuan He*, Kai Zhou

National Research Center for Wildlife Born Diseases, Institute of Zoology, Chinese
Academy of Sciences, Datun Road, Chaoyang District, Beijing, PR China
Abstract
In an avian flu pandemic, which methods could be used to treat or prevent infection
with influenza A (H5N1) virus? Foremost are antiviral drugs and vaccines, which have
already been used to prevent and treat human influenza A and B virus infections.
Although formally approved for other indications (i.e., treatment of hepatitis C),
interferon might also be useful for controlling avian flu. As has been shown for other
viral infections, RNA interference could be a powerful means with which to suppress the
replication of avian H5N1. Combined use of the currently available methods should be
taken into account and attempts should be made to develop new strategies directed at
unexplored targets such as the viral proteins hemagglutinin and viral polymerase (and
endonuclease) and non-structural protein.
Introduction
Avian influenza is a disease known since antiquity that continues to afflict large numbers
of chickens, animals and people, and causes many deaths throughout the world. The annual
epidemic and the continued threat of a pandemic constitute a major infectious-disease
problem [1-8]. Although the prevention of the spread of virus and the removal of virus in
transit in the environment could theoretically contribute to the control of avian influenza,
*
Tel: 86-10-64807118(O/F), Email: hehx@ioz.ac.cn
84 Hongxuan He, Kai Zhou
effective control requires intervention by scientists, which may include the optimal use of
vaccines, antiviral drugs, siRNAs and other therapies. (Figure 1).
Figure 1. Avian influenza control therapies include the use of vaccines, antiviral drugs, siRNAs and
other therapies.
Vaccines
Vaccines are effective at preventing influenza, but only if they target the relevant viral
subtypes. New vaccines against the annual epidemics of influenza A and B are prepared each
year, separately in the northern and southern hemispheres. These are designed to target the
subtypes predicted to be prevalent in any given flu season, but sometimes those predictions
are wrong, leading to the ineffectiveness of that years vaccine. A vaccine for a pandemic
strain of H5N1 could not be prepared until after the pandemic began, because only then
would the relevant subtype be known.
Antiviral Drugs
Drugs against influenza, stockpiled in advance of a pandemic, appear to be the best

preparation, given the limitations of vaccines. Billions of dollars have been spent on
pandemic preparedness throughout the world, and a large portion of these expenditures is
applied to stockpiling anti-influenza drugs. Similar expenditures have been made in many
developed countries. The World Health Organization is poised to distribute anti-influenza
Avian Influenza: Intervention and Therapy 85
drugs at the first sign of an epidemic of H5N1. There are several such drugs, as described
below.
1. Amantadine and Rimantadine
Amantadine and Rimantadine (Figure 2) are anti-viral drugs believed to work by

blocking an ion-channel (M2) required for viruses to infect cells. Ion-channel function
appears to be required for uncoating during endocytosis. Amantadine was approved for anti-
viral uses beginning in 1966 by the US FDA. Subsequent widespread use has given rise to
amantadine-resistant influenza in humans and birds. By 20052006, the US CDC found that
92% of H3N2 isolates were resistant, along with two of eight H1N1 isolates. In Asia,
resistance is close to 100%. The most common mutation responsible for resistance is S31N in
M2, which confers resistance to both amantadine and rimantadine [9-13].
Figure 2. The chemical structural formula of Amantadine and Rimantadine
In a recent research study, both amantadine and rimantadine were found to be ineffective
against H5N1 virus infection. H5N1 virus from Vietnam and Thailand could resist either drug
through mutation.
2. Tamiflu and Relenza
Tamiflu (oseltamivir) (Figure 3) is an inhibitor of influenza neuraminidase that binds to

the enzyme active site. Tamiflu is a transition state analog, and was the first orally active
neuraminidase inhibitor commercially developed. Because neuraminidase is required for the
viral life cycle, its enzymatic active site is highly conserved, and Tamiflu is effective for a
range of neuraminidase subtypes. It is indicated both for prophylaxis and for treatment within
two days of the onset of symptoms.
In 2004 researchers revealed that Roches Tamiflu works against the bird flu virus H5N1
strain. The drug is effective against avian and human forms of the virus. In a study, the
researchers said that Tamiflu is effective against the strain that is now hitting Vietnam and
Thailand. The World Health Organization (WHO) mentioned Tamiflu as the drug for tackling
bird flu in the event of a human pandemic. Tamiflu has been used effectively in other strains
of bird flu [14-18].
Figure 3. The chemical structural formula of Tamiflu (oseltamivir)
Figure 4. The chemical structural formula of Relenza (zanamivir)

Relenza (zanamivir) (Figure 4) is also an inhibitor of influenza neuraminidase that binds

to the enzyme active site. Unlike Tamiflu, which is given orally, Relenza is usually
administered by inhalation, or can be injected. Relenza was tested on a sample of the H5N1
virus. It stopped the virus from multiplying in the same way it stops the flu virus acquired by
humans. The drug inhibits the proteins on the surface of this part of the virus. This stops the
virus from reproducing or replicating. In about 80% of cases it stops transmission of the flu.
It is even effective in helping people who have already caught the flu, as it seems to reduce
the symptoms [19].
3. Emodin
A study to find herbal neuraminidase inhibitors discovered that Reynoutria elliptica

extract was a strong inhibitor of that enzyme. The research identified four compounds with
significant neuraminidase inhibiting properties: emodin, two emodin derrivatives, and
resveratrol.
Emodin (Figure 5) and related compounds are also found in the Aloe vera plant.
Unfortunately, emodin is an anthraquinone laxative; taking it orally has significant side
effects. Emodin is a cathartic known to stimulate muscle contraction in the intestines, and a
large dose can result in painful cramping. It is not clear how much is absorbed into the blood
where it can fight a virus. The studies done on the anti-viral properties of emodin were done
in cells in a test tube, not in live animals. Many Aloe vera juices on the market are made
using only the gel of the plant; they contain very little emodin. The outer part of an aloe leaf
contains most of the emodinit is in the milky sap portion close to the surface. The Aloe
vera juices on the market do contain polysaccharides like acemannan that stimulate the
immune system to produce IL-1 and TNF, but those inflammatory cytokines are not in short
supply in people infected by H5N1 avian flu. Most of the research conducted on acemannan
has involved HIV/AIDS or related animal viruses like feline leukemia virus. It is not clear
that aloe polysaccharides protect against influenza viruses in general, or the H5N1 virus in
particular [20].
Figure 5. The chemical structural formula of Emodin

4. Resveratrol
Resveratrol (Figure 6) is a compound found in large amounts in red wine, grape seeds,
and Japanese knotweed. Resveratrol is known to be absorbed into the blood. The root of
Japanese knotweed is the richest known source of resveratrol. It has long been used in a
variety of herbal medicines in China and Japan, where it is considered a tonic and life
prolonging plant. This plant has spread to many other countries and is naturalized in the US
and parts of Europe.
The effects of alcohol on a person infected with avian flu raises obvious concerns
drinking red wine might help prevent an infection, but once an infection does occur, the
alcoholic component of wine is probably not desirable. And resveratrol in wine is not stable:
After opening a bottle of red wine, the resveratrol begins to oxidize and much of it is
degraded within 24-48 hours.
In addition to inhibiting neuraminidase, resveratrol also sends a message to cells to stop
manufacturing viruses. This was described in terms of blocking the nuclear-cytoplasmic
translocation of viral ribonucleoproteins and reducing the expression of late viral proteins
seemingly related to the inhibition of protein kinase C activity and its dependent pathway.
This study found no toxic effects of resveratrol at levels that significantly inhibited influenza
virus [21].
Figure 6. The chemical structural formula of Resveratrol
Interferon
Virus-infected cells synthesize and secrete type I interferons (INFs), which warn the
body of the dangerous intruders. Secreted IFNs circulate in the body and cause susceptible
cells to express potent antiviral mechanisms that limit further viral growth and spread. IFN
was discovered by Isaacs and Lindenmann in 1957 as a cytokine interfering with virus
replication. Since then, much progress has been made in demonstrating how IFNs are induced
and how they work by activating IFN responsive genes that mediate cell-autonomous
resistance against viruses.
Interferon is one of the bodys many cytokines, inflammatory messenger proteins
produced by cells under attack that can warn neighboring cells of an impending viral assault.
Interferon acts as an early warning system, communicating the viral threat and activating in
the cell a complex self-destruct mechanism should nearby cells find themselves infected.
Interferon instructs cells to kill themselves at the first sign of infection and take the virus
down with them. They should take one for the team and jump on a grenade to protect the rest
of the body. This order is not taken lightly; false alarms could be devastating to the body.
Interferon pulls the pin, but the cell doesnt drop the grenade unless its absolutely sure its
infected.
IFNs are classified according to their amino acid sequence, mode of induction, receptor
usage, and biological activity (Figure 7). Type I IFNs are produced by cells in direct response
to virus infection and comprise a large number (at least 13) of IFN- subspecies and a single
IFN-, as well as some additional family members. Type II IFN (IFN-) is produced by
immune cells and plays an important role in immune regulation and viral clearance by T and
NK cells. Novel IFNs consist of the recently discovered IFN-1, IFN-2, and IFN-3 (also
termed IL-28A, IL-28B, and IL-29). They are strikingly similar to the type I IFNs in being
directly induced by virus infection and having antiviral activity. However, they use distinct
receptors [22-28].
Figure 7. Cellular response to IFNs.

About 50 years ago, interferon was discovered with influenza virus as the inducer. Baron
and Isaacs described the absence of interferon from the lungs in fatal cases of influenza.
Since then, interferon and its use have come a long way, and pegylated -interferon, in
combination with ribavirin, has become the standard therapy for HCV infection. Therefore,
extensive experience has been accumulated with this combination that could be readily
implemented in the therapy of avian flu, for which the duration of treatment would be much
shorter than for HCV. When using interferon for the prophylaxis or treatment of influenza,
one should, however, take into account the fact that interferon alone might cause flu-like
symptoms.
The H5N1 virus carries a trick up its sleeve called NS1 (for non-structural protein). If
interferon is the bodys antiviral warhead, then the NS1 protein is H5N1s antiballistic
missile. NS1 itself binds to the viruss own double-stranded RNA, effectively hiding it from
the cells PKR cyanide pill, preventing activation of the self-destruct sequence. Interferon can
pull the pin, but the cell cant let go of the grenade. NS1 essentially foils the bodys attempt
by covering up the viruss tracks. Influenza viruses have been called a showcase for viral
cleverness.
All influenza viruses have NS1 proteins, but H5N1 carries a mutated NS1 with enhanced
interferon-blocking abilities. The H5N1s viral countermove isnt perfect. The virus just
needs to buy itself enough time to spew out new virus. Then it doesnt care if the cell goes
down in flamesin fact, the virus prefers it, because the cells death may trigger more
coughing. This is a really nasty trick that this virus has learnt: to bypass all the innate
mechanisms that cells have for shutting down the virus, laments the chief researcher who
first unearthed H5N1s deadly secret. It is the first time this mechanism has shown up and
we wonder if it was not a similar mechanism that made the 1918 influenza virus so
enormously pathogenic [29].
Sirnas
RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) directs

sequence-specific degradation of messenger RNA (mRNA) (Figure 8). This phenomenon was
initially observed in plants, in Caenorhabditis elegans, and, recently, in mammalian cells. In
plants, it is an evolutionarily conserved response to virus infection. Naturally occurring RNAi
is initiated by the dsRNA-specific endonuclease, called Dicer, which processively cleaves
long dsRNA into double-stranded fragments between 21 and 25 nucleotides long, termed
short interfering RNA (siRNA). SiRNAs are then incorporated into a protein complex that
recognizes and cleaves target mRNAs. RNAi can be triggered in mammalian cells by
introducing synthetic 21-nucleotide siRNA duplexes, bypassing the requirement for Dicer-
mediated processing of long dsRNA.
Figure 8. Principle of generating a pool of siRNAs
RNAi appears to be ideal for inhibiting avian influenza virus infection. First, influenza
virus is an RNA virus, without any DNA intermediates during its entire life cycle. Besides
mRNA, both vRNA and cRNA could also be potential targets for siRNA-mediated
degradation. Second, the influenza virus genome consists of eight segmented RNAs,
encoding a total of 10 proteins. Each protein is either an integral component of the viral
structure or plays a critical role during the virus life cycle. Interfering with the production of
any one of them is likely to have severe consequences on viral replication and production.
Thus, there are multiple siRNA targets and combinations of siRNAs to different targets may
be used simultaneously. The use of two or more siRNAs simultaneously may be required to
prevent the emergence of resistant virus, analogous to the use of drug cocktails for treating
other infectious diseases (caused by Mycobacteria, HIV, etc.). Third, influenza virus
naturally infects epithelial cells in the upper respiratory tract and the lungs in humans. Thus,
siRNAs can be administered by inhalation, which would not only be convenient but may also
result in much higher local siRNA concentrations than could be achieved by parenteral
injection. Considering that the number of virions is probably small at the onset of a natural
infection, sufficient amounts of siRNA may be delivered to epithelial cells in the upper
airways and the lungs to inhibit virus replication or production, thus potentially achieving
preventive and/or therapeutic effects. Finally, unlike vaccines that require the recipients to
have a relatively normal immune system, siRNA-based treatment does not depend on a
functional immune system and should be as effective in the elderly or immuno-compromised

individuals as in immunocompetent individuals.
Among influenza A viruses, 15 HA subtypes and nine NA subtypes are known. There are
also extensive differences in nucleotide sequences of other genes among influenza virus
isolates from different species. To design siRNAs that remain effective despite antigenic
drifts and shifts, we must focus on regions of the viral genome that are conserved among
different subtypes and strains of virus from human, chicken, duck, equine, and swine.
To circumvent the high cost of synthetic siRNA and to establish stable gene knock-down
cell lines by siRNA, several plasmid vector systems were designed to produce siRNA inside
cells driven by RNA polymerase III-dependent promoters such as U6 and H1-RNA gene
promoters. With these plasmid vectors, the phenotypes of gene silencing could be observed
by stable transfection of cells. Nevertheless, transient siRNA expression, with low and
variable transfection efficiency, remains a problem for chemically synthesized and vector
derived siRNA. Recently, several virus vectors have been developed for efficient delivery of
siRNA into mammalian cells. Retroviral vectors were designed to produce siRNA driven by
either U6 or H1-RNA promoter for efficient, uniform delivery and immediate selection of
stable knock-down cells [30-41].
Previous work had proven that siRNAs could be delivered to host cells with siRNA
expression vector, which could be an effective method for H5N1 therapy [42]. Delivery
systems compatible with human use have demonstrated the potential use of siRNAs for
prophylaxis and therapy of influenza virus infections in humans. Similarly, siRNAs have
proven to be a powerful new method with which to combat other respiratory virus infections
such as those involving RSV and severe acute respiratory syndrome (SARS).
Conclusion
In addition to viral RNA polymerase and/or endonuclease, mentioned earlier as potential

targets for new anti-influenza-virus agents, there are some other clues regarding the virulence
of H5N1 viruses in humans that could be considered as points of attack for chemotherapeutic
intervention. First, the amino acid at position 627 in the viral polymerase protein PB2 is
mutated from glutamic acid to lysine in H5N1 viruses, and this might represent an adaptation
of H5N1 viruses for efficient replication in mammalian cells. Second, the HA of H5N1
viruses contains an unusual stretch of basic amino acids (RRRKKRG) that is cleaved by
ubiquitous intracellular proteases (including furin). Recombinant H5N1 viruses that lack
these basic amino acids are not virulent in mice. Third, the C-terminal domain of the non-
structural protein NS1 of avian H5N1 viruses contains a sequence motif (ESEV/EPEV) that
can be considered a virulence factor because it binds to human host proteins and disrupts
their morphology and functioning. In ferrets, however, the C-terminal sequence is not
required for the virulence of H5N1 viruses. Therefore, the role of the ESEV/EPEV motif and
other molecular determinants of the virulence of H5N1 viruses must be explored further.
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Chapter VI
Infection Control for Avian Influenza

(H5N1) in Healthcare Settings*
Anucha Apisarnthanarak1, and Linda M. Mundy2

Division of Infectious Diseases1, Faculty of Medicine, Thammasart
University Hospital, Pratumthani, Thailand
Saint Louis University School of Public Health2, St. Louis, MO, USA
Abstract
The re-emergence of avian influenza (H5N1) in Southeast Asia has heightened
concern for a potential influenza pandemic. Global pandemic preparation for avian
influenza (H5N1) has begun and it is imperative for healthcare workers (HCWs), who in
most cases serve as first responders, to be part of preparedness training. As relevant to
other transmissible agents, HCW preparedness training should include an understanding
of the modes and risks of avian influenza (H5N1) transmission and how to implement
appropriate infection control strategies to prevent and control of spread of avian
influenza (H5N1). In this chapter, we review the evidence for avian influenza (H5N1)
transmission, identified infection control strategies for both resource-adequate and
resource-limited settings, and post-exposure management of avian influenza (H5N1) for
HCWs. Healthcare epidemiology and infection control strategies include vaccination and
chemoprophylaxis of exposed HCWs, access to stockpiled oseltamivir, surveillance for
unrecognized cases and coordinated preparedness response plans by interdisciplinary
groups such as local and regional health departments, HCWs, healthcare administrators
and epidemiologists. The preparedness plans must include rapid creation of temporary
isolation facilities, restricted access to pre-identified HCWs, involvement of specialists
*
A version of this chapter was also published in Avian Influenza Research Progress edited by Ernesto P. Allegra
published by Nova Science Publishers, Inc. It was submitted for appropriate modifications in an effort to
encourage wider dissemination of research.
Corresponding author: Anucha Apisarnthanarak, M.D., Division of Infectious Diseases, Faculty of Medicine,
Thammasart University Hospital, Pathumthani 12120 Thailand; Tel: 662-926-9999; Fax: 662-332-8522; E-
mail: anapisarn@yahoo.com
98 Anucha Apisarnthanarak and Linda M. Mundy
for screening and early case identification and continuous monitoring for optimal
infection control practices and regular feedback to involved HCWs. Although human-to-
human transmission of avian influenza (H5N1) has rarely occurred, vigilant preparedness
and implementation plans are essential in thwarting a potential avian influenza (H5N1)
pandemic.
Keywords: infection control, avian influenza (H5N1), influenza, pandemic, healthcare

workers
Introduction
Three influenza pandemics occurred in the 20th century - 1918, 1957, and 1968. All three
pandemic viruses have been informally identified by their presumed site of origin as Spanish,
Asian, and Hong Kong influenza (1). They represent three different antigenic subtypes of
influenza A virus: H1N1, H2N2, and H3N2, respectively. These three major influenza
pandemics have shown no predictable periodicity or pattern, and all have differed from one
another (1). It is estimated that the next influenza pandemic will cause 20% of the worlds
population to become ill, one in every hundred of whom will be hospitalized, and for there to
be seven million deaths over a few months (2-3).
The ongoing H5N1 influenza epidemic in Southeast Asia poses risks to both human and
animal health (4-7). The potential exists for cross-species transmission to humans and
subsequent reassortment of avian and human influenza viruses in co-infected individuals (8).
Pandemic planning and worldwide surveillance are key factors in mounting an effective
global preparedness strategy for avian influenza (H5N1) (9). Therefore, it is important for
healthcare workers (HCWs), who in most cases serve as first responders, to understand the
modes and risks of avian influenza (H5N1) transmission and to recognize the appropriate
infection control strategies recommended for prevention and control of the spread of avian
influenza (H5N1). In this chapter, we review the evidence for avian influenza (H5N1)
transmission, identified infection control strategies for both resource-adequate and resource-
limited settings, and post-exposure management of avian influenza (H5N1) for HCWs.
Evidence of Avian Influenza (H5N1) Transmission
Theoretically, pandemic influenza may originate from at least two mechanisms:

reassortment between an animal influenza virus and a human influenza virus that yields a
new virus, and/or direct spread and adaptation of a virus from animals to humans. The
characterization of the 1918 Spanish influenza virus polymerase gene suggested that all eight
genes of the H1N1 virus were more closely related to avian influenza viruses than to
influenza from any other species (10-11). This evidence indicates that an avian virus was
likely to have infected humans and adapted to human-to-human transmission. However, in
both 1957 and 1968, the Asian and Hong Kong influenza viruses independently emerged via
Hospital Infection Control for Avian Influenza (H5N1) 99
ressortment of two influenza viruses. In each new influenza virus, there was the
hemaglutinin, the neuraminidase, and the gene for one of the polymerase proteins (PB1) from
the avian virus, along with the remaining five genetic segments from human influenza virus
(12). Theoretical concern currently exists for whether the avian influenza (H5N1) is capable
of adapting to humans with high efficiency through low-titer aerosol transmission and be the
source of the first influenza pandemic of the 21st century. Recent work by Taubenberger and
colleagues provide some insights into the genetic changes that may be required for such a
pandemic to evolve (10). The role of PB1 is considered necessary but not sufficient, given
that this gene was transferred along with hemagglutinin during genetic reassortment during
both the 1957 and 1968 influenza pandemics. The genetic sequences of the 1997 Hong Kong
H5N1 virus and the 2004 Vietnam H5N1 virus reveal that several human isolates of the
viruses contained one of the five animo acid changes in PB1 that were needed for the 1918
virus to infect humans. These data suggest that additional genetic changes must occur before
these viruses will begin to spread efficiently from person to person and that a global
surveillance program is warranted to monitor changes in genetic sequences of avian influenza
viruses in birds and in humans.
Human influenza is thought to be transmitted primarily via large droplets, by indirect
contact, and via self-inoculation into the respiratory system or conjunctival mucosa (13-15).
However, given the uncertainty about the exact modes by which avian influenza may first
transmit between humans, additional precautions for HCWs involved in the care of patients
with documented or suspected avian influenza (H5N1) seems prudent. The rationale for the
use of additional infection control precautions for avian influenza (H5N1) as compared with
human influenza include 1) the risk of serious disease and increased mortality from highly-
pathogenic avian influenza viruses may be significantly higher, 2) each human infection
represents a risk for influenza to further adapt to humans and transmit more easily among
humans, and 3) although rare, human-to-human transmission of avian influenza may be
associated with the possible emergence of a pandemic strain (16).
Current evidences suggest that human influenza A (H5N1) viral infection occurs via
bird-to-human, possibly the environment-to-human, and limited, nonsustained human-to-
human transmission (15). To date, animal-to-human transmission is thought to be the
predominant mode of avian influenza (H5N1) transmission (17-18). Reported risk factors
include the plucking and food preparation of ill birds, handling cocks for fighting events and
tourism, playing with poultry, consumption of ducks blood or undercooked poultry, and
exposure to live poultry within the week prior to the onset of illness (15,17). Interestingly,
inter-species transmission of avian influenza (H5N1) viruses has occurred in experimental
settings from chickens to tigers, chickens to leopards, and chickens to domestic cats (19-21).
Human-to-human transmission of avian influenza (H5N1) has occurred via intimate contact
without the use of precautions but not from casual, social contact (22). Findings from
serologic studies of avian influenza (H5N1) among exposed HCWs, household contacts, and
poultry cullers suggest that transmission to date has been inefficient and that protective
antibody may develop in asymptomatic, exposed persons (18, 22-26). Albeit rare, probable
human-to-human transmissions of H5N1 have been reported in several household clusters
and in one case of presumptive child-to-mother transmission (6, 8). Given the viability of
avian influenza (H5N1) in the environment, several other modes of transmission from
environmental sources are plausible. These transmission sources include oral ingestion of
contaminated water, direct intranasal or conjunctival inoculation through water exposure, and
self-inoculation via infected fomites (15).
Dynamic Transmission and Control Strategies
The traditional global health approach to anticipated annual influenza epidemics is based
on a three core-component plan of 1) vaccination of high-risk populations, 2) use of
chemoprophylaxis for exposed, high-risk populations, and 3) treatment of populations at high
risk for complication of influenza (27-28). In a recent study, investigators used a stochastic
influenza simulation model of rural Southeast Asia to investigate the effectiveness of targeted
antiviral prophylaxis, quarantine, and pre-vaccination in containing an emerging influenza
strain at the source (29). Investigators found that, if the basic reproductive number (Ro; the
average number of secondary infections caused by a single typical infectious individual in a
completely susceptible population) was below 1.60, a preparedness response of targeted
antiviral agents would have a high probability of containing the disease. In this scenario, an
antiviral agent stockpile on the order of 100,000 to 1 million courses for treatment and
prophylaxis would be sufficient. If pre-vaccination occurred, then targeted antiviral
prophylaxis could be effective for containing strains with an Ro as high as 2.1. Combinations
of targeted antiviral prophylaxis, pre-vaccination, and quarantine could contain strains with
an Ro as high as 2.4. These results demonstrate considerable variability in the potential size of
an epidemic in the absence of and in response to tiered interventions. These models
contribute to preparedness planning efforts and offer global health partners a framework for
the distribution of resources, be it for preventive health efforts or to thwart off a new
pandemic avian influenza virus.
Several avian influenza (H5N1) preparedness considerations are imperative for HCWs in
both resource-adequate and resource-limited setting (29). The rationale for the preparedness
plans include that: 1) it is unlikely that vaccines will be readily available in adequate
supplies, 2) the populations at high risk for complications may expand tremendously given
the high attack rates in young people, and 3) containing illness among HCWs during an
influenza pandemic will be challenging even if there is excellent compliance with infection
control practices. Influenza is generally spread through respiratory droplets and droplet
precautions are recommended to prevent and control the spread of the virus in healthcare
settings (13). The addition of airborne and contact isolation has been recommended for avian
influenza (H5N1), partially based on the effective infection control strategies utilized for
Severe Acute Respiratory Syndrome (SARS) in 2003 (16).
In general, influenza attack rates during outbreaks among unvaccinated HCWs are as
high as 59% (13). Influenza attack rates remain greater than 10% among unvaccinated HCWs
even when there are excellent infection control measures and behavioral compliance (28).
Furthermore, viral shedding of influenza can extend for 7 days after symptoms begin and for
weeks among infants and immunocompromised individuals. This makes environmental
control of influenza even more difficult in healthcare settings. Thus, the initial specific
protection of HCWs will need to include available antiviral agents (oseltamivir and
zanamivir) for chemoprophylaxis and treatment. A recently proposed university hospital-

based preparedness plan from the University of Michigan Health System has recommended
the stockpiling of oseltamivir (28). There are four potential strategies for using antiviral
agents during an influenza outbreak (30-31): chemoprophylaxis for the entire influenza
outbreak and season, postexposure chemoprophylaxis, treatment of ill patients, and a
combination of chemoprophylaxis and treatment. Although chemoprophylaxis has been used
to prevent the spread of influenza in limited populations (31), several studies support the use
of prophylactic viral agents (32-35). Furthermore, in a model of the Asian influenza
pandemic during 1957-1958, use of antiviral prophylaxis of close contacts to the index cases
for 8 weeks would have reduced the attack rate from 33% to 2% (36). Thus, targeted
prophylaxis of HCWs and patients would likely mean giving 6-8 weeks of prophylaxis to all
vulnerable groups. Although attractive, this strategy is prohibitively expensive for most
hospitals and other healthcare facilities in both developed and developing countries. An
alternative strategy that focuses primarily on treatment of ill HCWs, with some targeted
prophylaxis of heavily-exposed workers, would be more financially feasible while continuing
to offer treatment for HCWs employed in the direct care of patients infected with influenza
during a pandemic outbreak. Recent studies reported that neuraminidase inhibitors
administered as treatment within 48 hours of symptoms decrease not only the duration of
illness, but also the incidence of hospitalization, antibiotic use and mortality (35, 37-38).
Healthcare workers were easily tracked and monitored for influenza-like illness (eg, myalgia
and fever). It seems feasible that such monitoring would identify most of the ill HCWs and
thus allow timely administration of antiviral therapy. Despite these recommendations,
healthcare settings in developing countries may find it difficult to implement antiviral therapy
because of the high costs associated with these therapeutic interventions.
Healthcare Epidemiology and Infection Control
Influenza is a well-recognized nosocomial pathogen (13-14). The incorporation of

effective infection control strategies into healthcare settings are adopted from the basic
understanding of the types of transmission and control mechanisms (Table 1). The current
recommendation for prevention and control in healthcare settings are based on what are
deemed optimal precautions for protecting individuals who are involved in the care of
patients with highly-pathogenic avian influenza (H5N1) and for reducing the risk of viral
reassortment in non-epidemic settings (15, 39-40).
The infection control components of an avian influenza (H5N1) preparedness plan
include:
1) basic infection control inclusive of hand hygiene,

2) use of personal protective equipment (PPE),
3) vaccination with seasonal influenza vaccines,
4) administration of antiviral drugs for prophylaxis,
5) surveillance and monitoring for HCW exposures,
6) evaluation of ill HCWs,
7) precautions for household and close contacts, and

8) limited visitation, if not quarantine, of ill patients.
Table 1. Recommended type of isolation, infection control strategies for patients and
occupational health strategies for healthcare workers to control and prevent the spread
of potentially transmissible pathogens in healthcare facilities.
Type of Potential pathogens Infection Control Strategies

isolation
Airborne Mycobacterium tuberculosis Negative pressure, private room with >12

Yersinia pestis, measles, monkey pox, air change/hour and exhaust to outside or
small pox, varicella, viral hemorrhagic high-efficiency filter; door kept closed;
fevers, varicella, gowns, gloves, N95 mask, protective eye
SARS1, avian influenza (H5N1)1 gear, shoe covers
Droplet Influenza2, diphtheria, mumps, Private room (may cohort, if necessary);

pertussis, plague, rubella, parvo-virus dedicated use of non-critical patient-care
B19, SARS, avian influenza items to a single patient, surgical mask
when entering the room
Contact MRSA3 , VRE, Toxin-producing C. Private room (may cohort, if necessary);

difficile, MDR gram negative bacilli, dedicated use of non-critical patient-care
RSV, SARS, avian influenza, small items to a single patient, gowns and gloves
pox, varicella, viral hemorrhagic
fevers, scabies
NOTE:
RSV = Respiratory syncythial virus
SARS = Severe Acute Respiratory Syndrome
MRSA = methicillin-resistant Staphylococcus aureus
MDR = multidrug-resistant
C. difficile = Clostridium difficile
1
For patients who require aerosol-generating procedures
2
Occupational health: annual vaccination
3
Masks are recommended for care of patients with MRSA in Europe and Canada (61-62)
Basic infection control for avian influenza (H5N1).

Educate HCWs about the importance of strict compliance with appropriate hand
hygiene after contact with infected patients, exposure to poultry, contact with
contaminated surfaces, and after removing gloves. Hand hygiene should consist of
washing with soap and water for at least 15 seconds or using other standard hand-
disinfection procedures as specified by either the state government, industry, or
United States Department of Agriculture (USDA) outbreak-response guidelines (39-
40).
Ensure that HCWs have access to appropriate PPE, instructions and training in PPE
use, and respirator fit-testing.
Patients should be treated with a combination of standard, contact, droplet or

airborne isolation precautions and should be housed alone in a negative-pressure
room, if available, or in a single room with a closed door. If a single room is not
available, patients should be housed in designated multibed rooms or wards. The
beds should be at least 1 meter apart and preferably separated by a physical barrier.
When feasible, there should be a limitation to the number of HCWs with direct
patient contact and limited access to the infected environment. If possible, these
designated HCWs should not have direct care responsibilities for other patients
without suspected or confirmed avian influenza (H5N1).
Minimize visitors and provide them with proper PPE and instructions in its use.
Personal Protective Equipment (PPE) for avian influenza (H5N1).

Highly-efficiency masks (N95 mask), long-sleeved cuffed gowns, a face shield or
eye goggles, and gloves are recommended for all HCWs.
Use of disposable gloves made of lightweight nitrile or vinyl or heavy-duty rubber
work gloves that can be disinfected. To protect against dermatitis, which can occur
from prolonged exposure of the skin to moisture in gloves caused by perspiration, a
thin cotton glove can be worn inside the external glove. Gloves should be changed if
torn or otherwise damaged. Appropriate glove removal after use, and before touching
non-contaminated items and environmental surfaces, should be taught, enforced and
monitored.
Protective clothing, preferably disposable outer garments or coveralls, an
impermeable apron or surgical gowns with long-cuffed sleeves, plus an impermeable
apron should be worn.
Disposable protective shoe covers or rubber or polyurethane boots that can be
cleaned and disinfected should be worn.
Safety goggles should be worn to protect the mucous membranes and eyes.
Disposable particulate respirators (e.g., N95, N99, N100) are the minimum level of
respiratory protection that should be worn. This minimal level of respiratory
protection may already be in use in poultry operations due to other hazards that exist
in the environment (39-40). HCWs must be fit-tested to the respirator model that
they will wear and also know how to check the face-piece to secure a face seal.
Workers who cannot wear a disposable particulate respirator because of facial hair or
other fit limitations should wear a loose-fitting, powered air purifying respirator
equipped with high-efficacy filters.
Disposable PPE should be properly discarded, and non-disposable PPE should be
cleaned and disinfected as specified in state government, industry, or USDA
outbreak-response guidelines. Hand hygiene measures should be performed after
removal of PPE.
Vaccination with seasonal influenza vaccine to reduce risk of avian influenza

(H5N1).
Unvaccinated HCWs should receive the current seasons influenza vaccine to reduce
the possibility of dual infection with avian and human influenza viruses. There is a
small possibility that dual infection could occur and result in viral reassortment
followed by the emergence of a new virus. The resultant hybrid virus could be highly
transmissible among people and lead to widespread infections. Vaccination of all
residents of affected areas is not supported by current epidemiologic data.
At present no licensed vaccines are available for avian influenza (H5N1).
Infrastructure for vaccine development targeting highly-pathogenic avian influenza
viruses must be undertaken in biosecure facilities to protect workers and minimize
environmental contamination (28).
Administration of antiviral drugs for HCW prophylaxis to avian influenza

(H5N1).
Industry workers should receive an influenza antiviral drug daily for the duration of
time during which direct contact with infected poultry or contaminated
environmental surfaces occurs. The choice of the antiviral drug should be based on
viral sensitivity testing when possible. In the absence of viral sensitivity testing, a
neuraminidase inhibitor (oseltamivir) is the first choice since the likelihood is
smaller that the virus will be resistant to this class of antiviral drugs for influenza.
Surveillance and monitor of HCW exposures for avian influenza (H5N1).

Healthcare workers caring for patients with suspected or confirmed avian influenza
(H5N1) infection should self-monitor temperature twice daily and report any febrile
events. If unwell for any reason, HCWs should not be involved in direct patient care;
those with fever (temperature >38 C) and avian influenza (H5N1) exposure should
undergo appropriate diagnostic testing. If an alternative cause is not identified, they
should be treated immediately with oseltamivir on the assumption of influenza
infection.
Those who have had a possible exposure to infectious aerosols, secretions, other
body fluids or excretions because of a lapse in aseptic technique should be
considered for post-exposure chemoprophylaxis with oseltamivir 75 mg once daily
for 7 to 10 days.
Healthcare workers involved in high-risk procedures (e.g., aerosol-generating
procedures) should be evaluated for pre-exposure prophylaxis.
Instruct HCWs to be vigilant for the self-assessment of fever, respiratory symptoms,
and/or conjunctivitis for 1 week after last exposure to avian influenza-infected or
exposed humans, birds or potentially contaminated environmental surfaces.
Individuals who become ill should seek medical care and, prior to arrival, notify
their health care provider that they may have been exposed to avian influenza. In
addition, employees should notify their health and safety representative.
With the exception of visiting a health care provider, individuals who become ill
should be advised to stay home for at least 24 hours after resolution of fever, unless
an alternative diagnosis is established or diagnostic test results indicate the patient is
not infected with influenza A virus.
While at home, ill persons should practice good respiratory and hand hygiene to
lower the risk of viral transmission to others.
Evaluation of ill HCWs for avian influenza (H5N1).

Healthcare workers who develop a febrile respiratory illness should have a
respiratory sample (e.g., nasopharyngeal swab or aspiration) collected.
The respiratory sample should be tested by RT-PCR for influenza A, and if possible
for H1 and H3. If such laboratory resources are not available, or if the result of local
testing is positive, then the Centers for Disease Control and Prevention (CDC)
should be contacted and the specimens forwarded for genetic testing.
Virus isolation should not be attempted unless a biosafety level 3+ facility is
available to receive and process clinical specimens.
Optimally, the acute- (within 1 week of illness onset) and convalescent-phase (after
3 weeks of illness onset) sera should be collected and stored at -700C for subsequent
testing.
Precautions for household and close contacts of avian influenza (H5N1) .

Household contacts should use appropriate hand hygiene, avoid face-to-face contact
with suspected or confirmed index cases, not share utensils, and consider use of
high-efficiency masks and protective eye gear.
Contacts who have shared a defined setting (household, extended family, hospital or
other residential institution, or military service) with a patient with proven or
suspected avian influenza (H5N1) infection should
A. Self-monitor body temperature twice daily and check for influenza-like
symptoms for 7 days after the index exposures.
B. Receive post-exposure prophylaxis (PEP) with oseltamivir 75 mg once daily
for 7 to 10 days.
Foundations of Influenza Preparedness and

Response Guidance for Healthcare Facilities
The collective global experiences from SARS outbreaks provide some important lessons
for preparedness and response planning in healthcare facilities (41). Although an avian
influenza pandemic has not occurred, human-to-human transmission of avian influenza is of
theoretical concern and may be associated with the emergence of a pandemic strain. A
preparedness and response plan should target the following areas: 1) coordination of a
dynamic response by multiple interdisciplinary groups, 2) detection of unrecognized cases, 3)
restriction of access to healthcare facilities, 4) optimization of airborne infection control
isolation procedures, and 5) coordinated staffing needs and infrastructure support.
Coordination of a dynamic response by multiple interdisciplinary groups.
Triage and management of influenza patients during the outbreak may involve multiple
services within the hospital, including emergency departments, outpatient clinics, medical
units, intensive care units, radiology, laboratory services and others. Several issues may need
to be addressed including entry screening and access control, increased demand for PPE,
clear and rapid communications with local health department and continuous education and
monitoring of infection control measures for HCWs. Healthcare facilities must be able to
escalate response measures quickly, using a graded or stepped approach, as the outbreak
becomes more widespread, because recommendations that are appropriate in one setting may
not be adequate in another.
Detection of unrecognized cases of avian influenza (H5N1).
Unrecognized cases of avian influenza (H5N1) may be a significant source of viral

transmission. In general, coronavirus shedding during SARS outbreaks peaked at 7 to 10 days
after symptoms began (28, 42), while viral shedding of influenza has been documented to
continue for one to several weeks after symptoms began; prolonged carriage is more common
in infants and immunocompromised hosts. Persistence of viral shedding post-infection makes
transmission of influenza even more difficult to control in both healthcare and community
settings. Therefore, surveillance measures directed at each of these groups, along with
education and surveillance of contacts, were key strategies relevant to early case recognition
and outbreak abrogation.
Restriction of access to healthcare facilities where avian influenza (H5N1)

cases are in care.
Healthcare workers attack rates during influenza outbreaks have been estimated to be as
high as 59% (13). Even with excellent infection control practices, attack rates of greater than
10% are likely to occur among HCWs in the absence of vaccine (13). Therefore, limiting
healthcare-associated transmission is a key administrative and infection control issue for
interruption of an avian influenza (H5N1) outbreak at the earliest stages. Additionally,
limitations on visitors, non-essential staff, and new elective admissions to facilities where
known or suspected patients with avian influenza (H5N1) are in care seems prudent yet must
be balanced with the logistical issues underway at each healthcare facility and community
setting.
Optimization of airborne infection isolation control isolation procedures.
Although human influenza transmission occurs mainly via large respiratory droplets,
additional precautions in healthcare settings may be prudent for the care of avian influenza
(H5N1) patients. The rationale for more conservative infection control recommendations are
partially based on the global experiences of the SARS epidemics and, in addition: 1) the risk
of more serious morbidity and mortality from highly-pathogenic avian influenza (H5N1), 2)
each human infection represents the potential opportunity for influenza to further adapt to
humans, and 3) although rare, human-to-human transmission of avian influenza may be

associated with the emergence of a pandemic avian influenza strain. Historically, most
healthcare facilities have required a limited number of rooms for airborne isolation given the
select key indicators for such isolation. Clinical scenarios requiring assignment of empiric
isolation precautions is shown on Table 2. A small study of hospitals in the Midwestern
United States found that few facilities had airborne infection isolations in intensive care units,
and none had such rooms in their emergency departments (43). In a US survey of infectious
diseases subspecialists, 29% reported no airborne infection isolation rooms in the emergency
rooms of the affiliated hospital, 17% reported no airborne infection isolation rooms in the
entire referral hospital, and 25% reported limited supplies of respirators for HCWs during an
influenza outbreak (44). In a recent observational study of infection control practices in Lao
Peoples Democratic Republic, Taiwan, and Thailand, only 5 of 20 (25%) hospitals were able
to demonstrate implementation of infection control practices consistent with the World
Health Organizations recommendations on visitor policies, private negative-pressure rooms,
and PPE (45). Such observational data suggest that ongoing assessment of infection control
practices prior to or during influenza pandemic are needed and that global preparedness plans
need to address the limitations of facilities to provide airborne isolation for suspected and
confirmed cases of avian influenza (H5N1) and to provide PPE to HCWs.
Table 2. Clinical Scenarios Requiring Assignment of Empiric Isolation Precautions.
Airborne Isolation Droplet Isolation Contact Isolation
Vesicular rash.a Petichial/ecchymotic rash with fever in Diarrhea in patients with a

Maculopapular rash with coryza and patient with meningitis. history of recent antibiotic
fever. Cough/fever/upper lobe pulmonary Paroxysmal or severe persistent cough use.
infiltrate. during periods of pertussis prevalence. Vesicular rash.a
Cough/fever/pulmonary infiltrate in any Symptoms of respiratory illness with History of infection or
location in a HIV-infected patient (or fever. colonization with MDR
patient at risk for HIV/AIDS). Fever, respiratory symptoms in a organisms.
Fever, respiratory symptoms in a person person with recent contact with Skin, wound or UTI in a
with recent contact with SARS/Avian SARS/Avian influenza patient, or patient with a recent hospital
influenza patient, or recent travel to area recent travel to area with SARS /avian or nursing home stay in a
with SARS /avian influenza influenza transmission. facility where MDR
transmission.a,b organisms are prevalent.
Abscess or draining wound
that cannot be covered.a
Fever, respiratory symptoms
in a person with recent
contact with SARS
patient/avian influenza or
recent travel to area with
SARS transmission.a
NOTE:
SARS = Severe Acute Respiratory Syndrome
MDR = multidrug-resistant
HIV = human immunodeficiency syndrome
AIDS = acquired immune deficiency syndrome
UTI = urinary tract infection
a
= condition requires 2 types of precautions
b
= for patients who require aerosol-generating procedures
Coordinated staffing needs and infrastructure support.
Strict adherence to infection control practices is an essential component of a

preparedness plan for limiting avian influenza (H5N1) transmission to HCWs (46-47). Staff
members will need rapid education and training on the use of PPE and may require emotional
and logistical support (46, 48). Likewise, wearing extensive PPE, especially particulate
respirators, for prolonged periods of time, combined with the needs for careful attention to
how that equipment is donned and removed, enhances HCW fatigue (48). Together, the
increases in triage efforts, surveillance measures, time requirements to effectively
communicate with administration and regional leadership, and balancing of HCW fatigue and
furloughs will require higher administrative and staffing needs. These tensions and
anticipatory resources were duly noted experiences in multiple settings during the SARS
epidemics (49).
Infection Control for Healthcare Facilities in

Resource-Limited Settings and Developing
Countries
For preparedness planning in healthcare facilities with limited resources, such as those
encountered in developing countries, four practical issues relevant to the adoption and
modification of the above recommendations should be considered. The practical issues to
consider include: 1) healthcare administrative support, 2) involvement of specialists,
3)creation of temporary isolation wards during an epidemic, and 4) improvement of
suboptimal and inconsistent practices (50).
Healthcare administrative support.

The protection of HCWs in developing countries has not been optimally prioritized
compared to minimal standards promoted, regulated and monitored by the US Occupational
Health and Safety Administration (OSHA). Albeit practical and economic challenges exist,
efforts to promote implementation of effective infection control and occupational health
strategies are overdue and now-recognized global need. Given the global experience with the
SARS outbreaks that occurred in both designated SARS and non-SARS hospitals (51-
53), global preparedness plans should include facilitation of administrative, fiscal and
infrastructure support for routine occupational health and safety programs for HCWs,
appropriate infection control expertise and infrastructure in healthcare settings, availability of
PPE to HCWs, and epidemiological resources for the control and prevention of spread of
emerging infectious diseases. These expenditures should not be viewed as an increase in the
cost of health care, but as preventive health and safety measures that insure protection to
HCWS and anticipated return on investment to the healthcare institution.
Involvement of specialists.
As in developed countries, providers with the least experience are often the first
responders to evaluate patients with unrecognized emerging infectious diseases (54-55). Such
clinical scenarios may lead to a delayed recognition of disease and missed opportunities to
interrupt disease transmission (54-55). Several reports emphasize the added value of
specialists (ie., infectious diseases, pulmonary and emergency room specialists) in screening
for suspected cases of emerging infectious diseases and early recognition of atypical cases in
acute and ambulatory care settings (7, 26, 52, 56). Although the value of infection control and
healthcare epidemiology expertise has been formally recognized in the North America and
Europe (57-58), such recognition of need for interdisciplinary expertise has not yet been
incorporated into most acute care institutions in developing countries and resource-limited
settings.
Creation of a temporary isolation ward during an epidemic.

Rapid creation of a temporary isolation ward using existing functional hospital units is
readily applicable to clinical settings in developing countries and resource-limited regions
(59). Such units should be divided into clean zones for changing into and out of street cloths,
intermediate zones for removing the inner layer of PPE, and contaminated areas for entering
isolation areas. Exhaust fans could be installed above windows in each room, if access to
airborne infection isolation rooms is impossible. The distance between beds should be kept at
a minimum of one meter to reduce the risk of cross-transmission between patients
Improve sub-optimal and inconsistent infection control practices.

As in all settings, coordinated infection control practices may be difficult to orchestrate
without effective communication that clearly outlines the objectives for these practices. This
issue was emphasized by Yap et al. in a report of increased methicillin-resistant
Staphylococcus aureus (MRSA) acquisition rates in Hong Kong intensive care units during
SARS outbreak (60). The Hong Kong study findings suggested increased MRSA
transmission when HCWs participated in the non-standard practice of wearing gloves and
gowns all the time. Several infection control practices, such as proper hand hygiene and how
to apply PPE correctly may need to be serially monitored, with feedback to HCWs in a timely
manner, to optimize appropriate infection control practices and to reduce the transmission of
transmissible agents.
Conclusion
The cumulative global experience from the SARS epidemics, together with our current
understanding of influenza virus transmission, suggests that healthcare facilities will be focal
points of care in future outbreaks. With preparedness plans underway for a potential avian
influenza (H5N1) pandemic, healthcare facilities will benefit from adopting or modifying a
strategic plan and identifying local expertise to optimize control of an outbreak at its earliest
stages. The collective summary outlined in this chapter will require regular updates, yet
nonetheless offers a framework for the development of specific, institutional and regional
preparedness and response plans that will assist in minimizing the impact of future outbreaks
of emerging infectious diseases.
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Chapter VII
U.S. and International Responses to

the Global Spread of Avian Flu*
Tiaji Salaam-Blyther and Emma Chanlett-Avery
Summary
One strain of avian influenza currently identified in Asia and Europe is known as
Influenza A/H5N1. Although it is a bird flu, it has infected a relatively small number of
people killing around 50% of those infected. Scientists are unsure if H5N1 will cause the
next influenza pandemic, but there is general consensus that one is overdue. Flu pandemics
have occurred cyclically, roughly between every 30 and 50 years. Since 1997, when the first
human contracted H5N1 in Hong Kong, the virus has resurfaced and spread to more than a
dozen countries in Asia and Europe infecting more than 140 people and killing
approximately half. Britain and Taiwan both reported avian flu cases of H5N1 in 2005. In the
latter cases, the infected birds were identified as imports, and died in quarantine.
A global influenza pandemic could have a number of consequences. Global competition
for existing vaccines and treatments could ensue. Some governments might restrict the export
of vaccines or other supplies in order to treat their own population. Some countries might face
a shortage of vaccines, antiviral medication, or other medical equipment, because of limited
global supply. Hospitality and airline industries, and international trade could be negatively
impacted. If global travel and trade were to suddenly drop, there could be productivity losses
and service disruptions. Essential workers might become ill or stay home out of fear of
contracting the virus. Such workers could include law enforcement, medical personnel, mass
transit drivers and engineers, and other crucial emergency personnel.
For FY2006, Congress has provided $25 million for global initiatives to prepare for
pandemic influenza through Foreign Operations appropriations; directed $33.5 million to
global disease detection through Labor, HHS, and Education appropriations; and reserved for
international avian flu efforts a portion of $3.8 billion through Defense appropriations.
*
This is an edited, reformatted and augmented version of a Congressional Research Service publication, Report
RL33219, dated January 9, 2006.
116 Tiaji Salaam-Blyther and Emma Chanlett-Avery
Bills introduced in the 109th Congress would increase U.S. resources allocated to the
global fight against avian flu; develop a Pandemic Fund to augment ongoing U.S. and
international avian flu and pandemic preparedness initiatives; increase funding for preventing
the spread among animals of the H5N1 virus; and strengthen surveillance capacity within
affected countries.
This chapter provides an up-to-date account of global H5N1-related human infections and
deaths, outline U.S. government and international responses to the global spread of H5N1,
discuss situations in various countries affected by H5N1, and present some foreign policy
issues for Congress.
Background
Bird (or avian) flu outbreaks have occurred at various times around the world.[1] One
strain of avian influenza currently spreading across Asia and Europe is known as Influenza
A/H5N1. Although it is a bird flu, it has also infected a relatively small number of people
killing around 50% of those infected. Until 1997, there were no known cases of humans
contracting avian influenza. However, that year, 18 people in Hong Kong contracted the
virus; of those 6 died. To stop its spread, 1.5 million birds were killed. Since 2003, scientists
have closely monitored resurgent H5N1 outbreaks, which have infected chickens and ducks
in a growing number of countries. The World Health Organization is particularly alarmed
about the rapid spread of H5N1 in part, because this strain of bird flu has demonstrated the
ability to cause high mortality rates among humans.
According to WHO, the hallmarks of a pandemic are: 1) a novel influenza virus strain
emerges; 2) the strain causes human disease; and 3) person-to-person transmission is
sustained. The pandemic steps usually occur in six phases. Table 1 shows the phases of an
influenza pandemic, as described by WHO. The WHO considers the recent H5N1 outbreak to
be in phase three pandemic alert phase, which means a virus new to humans is causing
infections, but does not spread easily from one person to another.[2]
Since H5N1 is a bird flu, and has not commonly infected people, humans have no
immunity against it. If H5N1 were to become transmissible among humans, an influenza
pandemic (worldwide disease outbreak) could begin, potentially causing millions of deaths.
Skeptics argue that predictions that H5N1 might cause a global pandemic are exaggerated,
because if the virus were able to become efficiently transmissible among people it would
have already transformed.
Still a growing number of health experts underscore that it is critical for governments to
prepare for some form of an influenza pandemic, as the world is overdue for one. During the
influenza pandemic of 1918-1919 (Spanish flu), estimates are that between 20 and 50 million
people died, and between 200 million and 1 billion were infected around the world. If an
influenza pandemic were to occur on the same scale as the Spanish flu, some estimate that
between 30 million and 384 million people could die around the world,[3] of which 1.9
million deaths could occur in the United States.[4]
U.S. and International Responses to the Global Spread of Avian Flu 117
Global Prevalence
Since 1997, when the first human contracted H5N1 in Hong Kong, the virus has
resurfaced and spread to birds in fifteen countries, ten in Asia and five in Europe.[5] In 2004,
nine Asian countries reported H5N1 poultry outbreaks: Cambodia, China, Indonesia, Japan,
Laos, Malaysia, Republic of Korea, Thailand, and Vietnam. By August 2005, birds in
Mongolia had become infected with the virus. Two months later, in October, domestic birds
in Russia and Kazakhstan had contracted H5N1 reportedly through contact with wild
waterfowl at shared water sources. By late October 2005, H5N1 had spread progressively
westward to affect six other regions in Russia, and had infected bird populations in Romania,
Croatia, and Turkey. Although human infection has not been reported outside Asia, health
officials are wary about infection among migratory birds, as the birds are currently acting as
vectors of the virus. Also, infection can potentially be controlled among domestic birds, but
not among wild ones. The chart below shows the latest number of confirmed human H5N1
cases as reported by WHO as of December 30, 2005[6]. The map in the Annex (Chart 1)
illustrates the human H5N1 cases.
Table 1. Human Cases of Avian Influenza A/H5N1
Indonesia Vietnam Thailand Cambodia China Turkey

Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths
16 11 93 42 22 14 4 4 7 3 4 2
Total Cases = 146; Total Deaths = 76
Congressional Response
Congress provided $25 million to support ongoing U.S. efforts to prevent and contain the
spread of H5N1 through P.L. 109-13, FY2005 Emergency Supplemental Appropriations. The
act, which passed in May 2005, also provided funds for domestic pandemic preparedness.[7]
Additionally, the act directed U.S. agencies to develop a coordinated response to the global
spread of H5N1. Congress provided the funds to U.S. Agency for International Development
(USAID). Pursuant to the statute, USAID transferred $15 million of the $25 million
appropriation to CDC.
The conference report for P.L. 109-102, FY2006 Foreign Operations Appropriations,
urges the United States Executive Director to the World Bank to use the voice and vote of the
United States to increase support for using International Development Association (IDA)
funds to help eligible countries prepare for and combat a potential avian influenza epidemic.
Particularly, the report points out that funds could be used in Asia for programs to increase
surveillance capacity, compensate small-scale farmers for timely reports of bird die-offs,
modernize animal husbandry practices, and upgrade infectious disease infrastructure. The
report also underscores that the $25 million provided in P.L. 109-13 is the first step in a
multi-year effort to contain, prevent, and prepare for the spread of avian influenza.
H.Rept. 109-337 for H.R. 3010, FY2006 Labor, HHS, and Education Appropriations,
includes $63.58 million for the Public Health and Social Services Emergency Fund
(PHSSEF) to enhance federal, state, and local preparedness to counter potential biological,
disease, chemical, and radiological threats to civilian populations. Additionally, $33.5 million
is directed to global disease detection. The bill does not include funds to support the
Presidents FY2006 $7.1 billion emergency request for avian flu and pandemic influenza
preparedness. Instead, appropriators provided additional funds through FY2006 Defense
Appropriations. The FY2006 Defense, Disaster Assistance, and Avian Flu Preparedness
Appropriations conference report, H.Rept. 109-359, reserves a portion of the $3.8 billion
directed to avian flu activities for international avian flu efforts.[8] The Senate passed the
House version after removing a controversial provision related to oil drilling in Alaska. H.R.
3010 and H.R. 2863 were presented to the President on December 28, 2005.
Table 3 reflects FY2006 appropriations that include funding for global avian flu
activities.
Press reports quote a number of Members expressing concern about funding the
Presidents $7.1 billion avian flu and pandemic preparedness request. The Chairman of the
House Energy and Commerce Committee, Joe Barton, reportedly stated that he would not
support funding for the bill if the President did not provide offsets for avian flu and pandemic
preparedness spending.[9] Others proposed that Congress spread out funding the request over
a few years.
Some congressional Members argued that the Administration has allocated insufficient
resources to the global fight against H5N1 and pandemic planning. Of the $7.1 billion
requested, approximately $388 million would be reserved for global efforts. A number of
Members have introduced legislation to increase U.S. resources allocated to the global fight
against avian flu. Some bills, such as H.R. 4062,
Pandemic Preparedness and Responsibility Act and its Senate companion, S. 1821,
propose developing a Pandemic Fundto augment ongoing U.S. and international avian flu
and pandemic preparedness initiatives. Other bills, such as H.R. 4476,
Global Network for Avian Influenza Surveillance Act, and its Senate companion, S. 1912,
advocate greater support for initiatives that prevent the spread of H5N1 among animals. A
number of bills, such as H.R. 3369, Attacking Viral Influenza Across Nations Act, and its
Senate companion, S. 969, suggest the U.S. strengthen surveillance capacity within affected
countries. Bills, such as H.R. 813, Flu Protection Act, and its Senate companion, S. 375, aim
to boost influenza vaccine supply. Additionally, other legislation, such as H.R. 4245,
Influenza Preparedness and Prevention Act encourage greater international cooperation.
Some Members of Congress have also expressed support for greater spending on global
initiatives during congressional hearings. For example, during the House International
Relations Committee hearing on pandemic flu in December 2005, Chairman Henry Hyde
questioned whether the amount the President requested for international pandemic flu
preparedness was sufficient. A number of other committees have also held hearings on avian
flu and pandemic preparedness, including the Senate Foreign Relations and Appropriations
Committees, House Agriculture Committee and a joint hearing by the House Homeland
Security and Armed Services Committees.
U.S. Executive Branch Response
On November 1, 2005, the President released the National Strategy for Pandemic
Influenza. One day later, on November 2, 2005, the Administration released the U.S.
Department of Health and Human Services (HHS) Influenza Plan. The HHS plan provided a
detailed explanation of how the national strategy would be implemented. Some were
disappointed by the relatively small proportion of funds reserved for international efforts. It
has been argued that greater investment in pandemic influenza preparedness abroad could
enhance domestic pandemic preparedness efforts. Of the $7.1 billion requested,
approximately $388 million is reserved for global initiatives. Of the $388 million, $200
million is made available for HHS to bolster international surveillance capacity; $131.5
million for USAID to implement avian influenza containment efforts globally; an additional
$18.5 million for the State Department for avian flu and pandemic preparedness activities in
diplomatic arenas, $20 million for the potential evacuation of U.S. government personnel and
their dependents in the event of a pandemic; and $18.3 million for the Department of
Agriculture to provide technical assistance in international animal surveillance.[10] Table 2
summarizes the FY2006 emergency request.
HHS (and its relevant agencies), USAID, the Department of Agriculture, and the
Department of Defense are the key U.S. departments and agency involved in containing the
global spread of H5N1 and preparing for pandemic influenza. The Department of State plays
a complementary role by raising the issue in diplomatic arenas. The unique role that each
agency plays is described in order of presence on the ground.
Prior to 2005 when Congress provided $25 million for preventing the global spread of
avian influenza and preparing for pandemic influenza U.S. agencies had been enhancing
laboratory capabilities, training health care providers, strengthening surveillance systems, and
developing influenza pandemic plans. Through the FY2005 emergency appropriations,
Congress directed U.S. agencies to revisit international influenza initiatives and ensure that
there was a coordinated response to the global spread of H5N1. USAID and HHS (including
its relevant agencies) undertook country planning visits to Vietnam, Cambodia, and Laos.
After the trip, the team outlined in a report[11] a number of factors that have complicated
efforts to contain the spread of H5N1 in Vietnam, Cambodia, and Laos, which included:
Between 70% and 80% of poultry in the three countries are raised in small backyard
farms, hindering national governments ability to ensure health standards.
Between 50% and 80% of poultry die from other avian infections, complicating
efforts to identify unusual die-offs, and limiting farmers likelihood of reporting bird
deaths to authorities.
Although culling is an essential element of controlling the spread of H5N1, poorer
countries can not afford to systematically compensate farmers for lost stock, which
also increases reluctance to report signs of infection.
Wild birds and domesticated ducks are H5N1 reservoirs.
Low levels of awareness exist among local farmers.
There is little pandemic preparedness activity in the countries toured.
The capacity to monitor and respond effectively to animal outbreaks is limited.

Veterinary services are inadequate to deal with the scope, severity, and rapid spread
of H5N1 epidemics, which has resulted in the disease becoming increasingly
endemic among animal populations in the region. The lack of human resources for
disease surveillance, diagnostics, and response also severely limits the capacity of
human health systems, and continued human infections of avian influenza threaten to
overburden already fragile public health infrastructures.
The report also included an action plan, which outlined the activities that each agency
would implement. The agency-specific strategies are briefly described below. Table 4
provides a country-specific illustration of Department of Health and Human Services (HHS)
and USAID spending for the FY2005 Emergency Supplemental Appropriations.
U.S. Department of Health and Human Services (HHS)
CDC is the key agency at HHS responsible for implementing U.S. anti-influenza
activities around the world. The Coordinating Center for Infectious Diseases and the Field
Epidemiology Training Program a CDC-sponsored activity are also critical components
of HHS global pandemic preparedness initiatives. Activities with foreign governments or
populations include pandemic preparedness and planning; training in avian influenza
surveillance; laboratory safety and skills instruction; epidemiology training; developing and
training rapid response teams; stockpiling support; and deployment of expert disease control
teams.
It is not possible to disaggregate H5N1-specific funding, because H5N1, seasonal flu,
and pandemic preparedness initiatives are interlocked. A significant part of H5N1 and
pandemic influenza planning is funded through the Global Disease Detection (GDD)
Initiative at CDC. GDD aims to recognize infectious disease outbreaks faster, improve the
ability to control and prevent outbreaks, and detect emerging microbial threats. CDC
estimates that in FY2004, it spent approximately $5 million on activities related to
international influenza through both its Infectious Diseases Control and GDD programs. In
FY2005, CDC spent approximately $6 million through these activities, in addition to the $15
million emergency appropriations. In 2005, CDC expanded its GDD activities by creating
new sites, improving early warning systems, researching new viral strains, and supporting
international organizations. Congress provided $21.4 million for GDD in FY2005.The HHS
FY2006 budget request suggests increasing GDD funding by $12.1 million to $33.5 million,
of which a portion would be used for international bird flu initiatives.[12] H. Rept.109-337,
FY2006 Labor, HHS, and Education Appropriations directed $33.5 million to GDD.
U.S. Agency for International Development (USAID)
USAID coordinates its global H5N1 and influenza response with other U.S. agencies. It
also works closely with the WHO, the Food and Agriculture Organization of the United
Nations (FAO), and other international governments and organizations to support national
influenza and H5N1 prevention efforts. To date, the agency has spent $13.7 million on avian
influenza prevention and containment ($10 million of which was funded through the FY2005
emergency appropriations).[13] Specifically, the agency has:
dedicated $7.5 million to Cambodia, China, Indonesia, Laos, and Vietnam for
strengthening disease surveillance, laboratory diagnosis, and rapid containment of
animal outbreaks;
provided $2.85 million for communication campaigns in Laos, Cambodia, Vietnam,
and Indonesia aimed at reducing animal handling practices that place humans at risk;
committed over $1.6 million to enhance national planning efforts, strengthen avian
influenza control and outbreak response, and augment human disease surveillance
systems and laboratories;
granted WHO $300,000 for international coordination efforts and for improving
disease control and surveillance measures;
provided WHO an additional$250,000 for personal protective equipment (PPE) used
in handling and disposing of infected poultry; and
distributed some 10,000 sets of PPEs, which include manual sprayers to assist in
decontaminating hospital rooms and equipment, Tyvek suits (protective coveralls
used in hazardous situations), gloves, boots, masks, and eye protection in Cambodia,
Thailand, Laos, Vietnam, and Indonesia.
The Administrations FY2006 emergency supplemental request allocates $131.5 million

to USAID for avian flu and pandemic preparedness initiatives abroad. The funds would be
used to pre-position supplies and equipment that prevent and control the spread of avian
influenza; launch awareness raising campaigns; and accelerate international planning and
preparedness. The request proposes that $2 million of the USAID funds be reserved for
initiatives in Russia and Eastern Europe.
Department of State
On September 14, 2005, President Bush announced the International Partnership on

Avian and Pandemic Influenza (IPAPI) at the U.N. General Assembly High-Level Plenary
Session. IPAPI seeks to generate and coordinate political momentum and action for
addressing the threats of avian and pandemic influenza based on a set of core principles. The
principles are focused on enhancing preparedness, prevention, response, and containment
activities (see Table 5). The Partnership brings key nations and international organizations
together to improve global readiness by:
elevating the issue of avian and pandemic influenza preparedness to the national
level;
coordinating efforts among donor and affected nations;
mobilizing and leveraging resources;
increasing transparency in disease reporting and surveillance; and

building capacity to identify, contain, and respond to pandemic influenza.
The State Department works closely with regional organizations, including the
Association of Southeast Asian Nations (ASEAN) and the Asia Pacific Economic
Cooperation (APEC) forum, to address avian influenza and the threat of an influenza
pandemic. The work includes efforts to encourage comprehensive national pandemic
preparedness plans that address the multi-sectoral impacts of an influenza pandemic.[14]
In the FY2006 supplemental request, the President proposed that the State Department
receive $38.5 million in FY2006 for international response coordination; diplomatic
outreach; exchanges of U.S. and foreign medical personnel; and for avian and pandemic
influenza health support and protection of U.S. government employees and families at U.S.
missions overseas. About $20 million of those funds would be reserved for the potential
evacuation of U.S. government personnel and dependents from overseas missions.
Department of Agriculture (USDA)
U.S. Department of Agriculture (and its related agencies)works closely with other U.S.
agencies on the ground, as well as other international organizations to help nations take steps
to address and control the spread of avian influenza. Dr. Ron DeHaven, Administrator,
Animal and Plant Health Inspection Service (APHIS) of USDA stated that addressing avian
flu at its source in affected poultry abroad and participating in international eradication
efforts provide the best opportunity to reduce or eliminate the risk of an H5N1 pandemic.[15]
In that view, USDA and other analysts consider the departments efforts a critical element in
the global fight against the spread of H5N1.
Through $4 million in FY2003 emergency funding, APHIS launched an outreach
campaign called Biosecurity for the Birds, which provides poultry farmers with the latest
information on biosecurity to prevent the spread of avian infections on farms. USDA is
translating the brochures for use in southeast Asia.
Additionally, in the FY2006 emergency supplemental, the President requests $91.3
million for USDA, of which $18.3 million is reserved for international initiatives. The would
be allocated as follows:
$8.0 million for wildlife, poultry and swine surveillance and diagnostics;
$1.75 million for biosecurity enhancement through education and information;
$1.05 million for technical assistance through training and avian movement control;
$3.8 million for training and education related to industry changes and food safety
planning;
$1.05 million for training and education regarding poultry destruction and disposal
methods;
$0.6 million for testing and evaluation of vaccine formulations; and
$2.1 million for in country expertise for longer term assistance.
Department of Defense (DOD)
The Department of Defense Global Emerging Infections System (GEIS) delivers health
care to American armed forces around the globe.[16] GEIS has a network of overseas
medical research laboratories that track, prevent, and treat infectious diseases around the
world. The objective is to protect the U.S. military and strengthen its ability to address the
challenges related to a potential pandemic influenza, including compromised military force
health and readiness. GEIS is also a critical partner in the WHOs Global Outbreak Alert and
Response Network (GOARN) (described below). Key DoD-GEIS activities to combat the
spread of H5N1 and prepare for an influenza pandemic have included:
providing a DoD staff veterinarian to serve as a member of the WHO GOARN Team
in Laos, and to conduct training workshops in detecting and diagnosing avian flu
cases;
placing a U.S. Navy microbiologist at the Institute Pasteur in Ho Chi Minh City,
Vietnam, to hold training sessions on rapid diagnostic test methodology;
monitoring and preventing infectious disease emergence in southeast Asia through
its Armed Forces Research Institute of Medical Sciences (AFRIMS).[17]
The Naval Medical Research Unit-2 (NAMRU-2) is another critical part of DoDs effort
to prevent H5N1 from becoming a human pandemic and prepare for an influenza pandemic.
NAMRU-2 supports the GEIS mission through four programs: emerging diseases, enteric
diseases, parasitic diseases, and virology. NAMRU-2 is an overseas research laboratory based
in Jakarta, Indonesia with related activities in Southeast Asia and the Pacific Islands.
NAMRU-2 also supports a satellite laboratory in Phnom Penh, Cambodia, in collaboration
with the Cambodian National Institute of Public Health. Key activities include:
bolstering local, national, and regional diagnostic and epidemiological capacity;

assisting in the development of new surveillance strategies, such as the novel
syndromic surveillance initiative Early Warning Outbreak Recognition System
(EWORS);
implementing a comprehensive influenza surveillance project in Indonesia, which
provides prevalence data and temporal, genotype data of circulating strains;
collaborating with CDC in its FY2005 and FY2006 global influenza activities; and
facilitating the transformation of outbreak response structures into more effective,
multidisciplinary, centrally directed ones.[18]
The FY2006 emergency supplemental request would reserve $10 million of the $130
million allocated to the Department of Defense for procuring protective equipment;
laboratory diagnostic equipment; portable field assay testing equipment; and surveillance and
communication equipment.
International Response[19]
Overview of the Role of the World Health Organization
The World Health Organization, established in 1948, is the U.N. systems authority on
international public health issues. It assists governments to improve national health services
and establish worldwide standards for foods, chemicals, and biological and pharmaceutical
products. WHO concentrates on preventive rather than curative programs, including efforts to
eradicate endemic and other widespread diseases, stabilize population growth, and improve
nutrition, sanitation, and maternal and child care. WHO works through contracts with other
agencies and private voluntary organizations. The United States has been a member of WHO
since its inception.
WHO is a central actor in the global response to the outbreak of H5N1 avian influenza.
As in the case of SARS in 2003, WHO seeks to mitigate the risks avian influenza and
infectious diseases pose to international public health, and to assure the availability of
appropriate containment mechanisms, particularly since global travel has become the primary
means of spreading disease around the world. With the exception of SARS and HIV/AIDS,
H5N1 is viewed as the most serious challenge the WHO has faced in the last few decades.
Whos Global Health Security
The Epidemic and Pandemic Alert and Response system is a critical part of WHOs
global health security plan. Key aspects of the program include:
The Alert and Response Operations: systematically track the development of

diseases, share and disseminate information, and coordinate rapid outbreak response
and logistics.
The Global Outbreak Alert and Response Network (GOARN): provides an
operational framework and aims to create a standardized international outbreak
response system through 112 institutions and networks of people and technical
resources.[20]
The Global Public Health Intelligence Network (GPHIN): tracks Internet
communications through a customized search engine, which effectively picked up
telecommunicated alerts in China during the SARS outbreak. WHO also uses the
system to clarify or refute information that may create disruption or panic.
Who Global Influenza Preparedness Plan[21]
In September 2005, U.N. Secretary-General Kofi Annan appointed Dr. David Nabarro as
the Senior U.N. System Coordinator for Human and Avian influenza. Dr. Nabarro, seconded
from the WHO, is responsible for coordinating the avian influenza containment efforts of the
various U.N. agencies. Dr. Nabarro is also tasked with encouraging global support and
implementation of the WHO Global Influenza Preparedness Plan. The plan outlines WHO
goals and actions, as well as recommended actions for individual nations, at each pandemic
phase (see Table 1). The plan contains an annex of recommendations to nations for non-
pharmaceutical public health interventions, such as isolation, quarantine and travel
restrictions. The annex stresses the use of voluntary rather than compulsory measures.
Additionally, it stresses that nations implement infection-specific responses, noting the lack
of demonstrated utility of certain practices. For example, certain SARS control measures,
such as temperature screening at airports, would not be expected to effectively control
influenza spread.[22]
WHO has requested $150 million to establish a global stockpile of influenza vaccines
and treatments. WHO officials underscore that wealthy and poor countries must develop
pandemic preparedness plans collectively to reduce national and international viral
transmission. The organization envisions using the stockpile to arrest a potential pandemic by
containing the virus at the first sign of an outbreak. In the event of an outbreak, WHO asserts
that a pandemic could potentially be averted if antiviral drugs were quickly distributed in a
poor country without access to them.[23] To date, countries have pledged between $20
million and $30 million to fund the stockpile. Roche, the patent holder of Tamiflu,
announced that it would donate three million courses of the drug to WHO[24]. The company
estimates that the three million courses would be ready before mid-2006.
Similarly, the U.N. General Assembly has established an emergency fund Central
Emergency Response Fund (CERF) to provide quick initial funding during the early
stages of emergencies and to minimize extra costs related to funding delays. The U.N. aims to
have a $500 million revolving budget that could be used within three to four days of the start
of an emergency. To date, the United Nations has received more than $200 million for the
fund, which will be launched in mid-January and should be operational by March.[25]
Role of other International Health Organizations
The U.N. Food and Agriculture Organization coordinates global surveillance and
response activities for animal influenza strains with pandemic potential, such as H5N1.[26]
To accomplish its mission, FAO works closely with the World Organization for Animal
Health, known by its French acronym, OIE.[27] Rapid detection of avian influenza outbreaks
is key to controlling the disease both in poultry and in people, and is therefore key to
preventing and controlling a potential influenza pandemic. FAO, OIE, and WHO work
closely to prevent and respond to the threat of an avian influenza pandemic. FAO has spent
$7.5 million on H5N1 initiatives since 2004. USAID is granting the UN organization $6
million, and the German government has pledged $20 million for 2005 and 2006
activities.[28] FAO is requesting an additional $175 million from the international
community, due to the rapid global spread of H5N1.
The World Bank provides low-interest loans to countries heavily affected by H5N1.
Additionally, the Bank coordinates efforts between countries, and encourages them to
develop pandemic plans that connect sectors, such as health and rural development. In
September 2005, representatives from the WHO, FAO, OIE and the World Bank met with
health experts from the United Nations, European Commission and H5N1-affected countries
to discuss the global spread of H5N1, to emphasize the importance of pandemic planning,
and to prepare a coordinated response. On November 4, 2005, the World Bank announced
that it would provide $500 million in loans to poor southeast Asian countries that are
struggling to combat avian influenza. The funds will be used to supplement government
resources, strengthen veterinary systems, and assist in culling and animal vaccination
programs.[29] Although the World Bank has agreed to provide $500 million in loans to
affected countries, the Bank estimates that $1 billion could be needed over the next three
years.[30] The $1 billion does not include the cost of financing human or animal vaccine
development, purchasing antiviral medicine, or compensating farmers for loss of income.
The WHO, FAO, OIE, and the World Bank co-sponsored a meeting on avian influenza
and human pandemic influenza on November 7-9, 2005, in Geneva, Switzerland, to develop
an integrated global plan and to focus on funding initiatives.[31] Participants agreed to a six-
point global plan which called for:
Controlling the virus at its source in birds;

Strengthening surveillance, early detection, rapid response systems, and laboratory
capacity;
Training national staff in investigating animal and human cases, and planning and
testing rapid containment activities;
Building and testing national pandemic preparedness plans, conducting a global
pandemic response exercise, and enhancing health systems;
Developing integrated country plans that encompass all sectors; and
Ensuring factual and transparent communications.[32]
International Health Regulations
An outbreak of infectious diseases raises many public health questions including the
application of international law, particularly as it affects three main areas International
Health Regulations (IHR); public health measures and civil and political rights; and
principles of state responsibility.[33] This section will focus on the IHR because of its
relevance to WHO.
On May 23, 2005, the World Health Assembly revised the IHR, adding novel influenza
strains (those with pandemic potential) and SARS to the list of notifiable diseases that
WHO urges countries to report. In addition, the revised IHR include a provision requiring
notification of events of international concern. This mechanism could strengthen WHOs
ability to address emerging diseases, because it requires member States to report unusual
health events whether or not they are attributable to a known pathogen. The updated IHR also
include expanded requirements for disease surveillance and control activities at points of
international travel (airports, border crossings, etc.), and urge developed countries to assist
developing countries to gain the capacities needed to meet the new disease control
guidelines.[34]
The revised IHR are to replace the existing IHR (adopted in 1969) on June 15, 2007,
when the revised regulations come into force. Considered an international legal instrument,
the revised IHR will be binding on all WHO member States who have not stated a reservation
or rejected them altogether, and on non-member States that have notified the Director-
General of WHO that they agree to be bound by the revised IHR.[35] Between now and June
2007, WHO and Member States may take concrete steps towards implementation of the
revised IHR and to improve their capacity to respond to international health risks and
emergencies.[36] The revised IHR do not include an enforcement mechanism. However, for
states to respond appropriately and avoid potentially harmful consequences, much of the
encouragement to comply will likely come from international pressure, as the SARS outbreak
demonstrated.
Affected Countries Response
Degree and sophistication of preparation for avian influenza vary widely among the
affected countries. The more affluent governments have undertaken more extensive measures
as well as committed national resources to hedge against the risk of a pandemic. Japan and
Taiwan have reportedly both accumulated stockpiles of Tamiflu and are preparing to
manufacture their own supply. Singapore has reportedly stockpiled antivirals for 10% of its
population, enhanced surveillance, and put a detailed contingency plan in place. WHO
officials praised an exercise run by South Korea which simulated how the government would
respond to an outbreak.[37] On the other hand, the closed governments of Burma (Myanmar)
and North Korea offer little reliable information about the presence of bird flu within their
borders. Although both Yangon and Pyongyang have provided limited cooperation with the
FAO, their officially rosy outlooks are treated with skepticism by international health experts
and could constitute a weak link in the event of a pandemic.
The profiles below focus on countries that have had cases of human infection. Although
Russia has had no human cases to date, an analysis of H5N1 cases has been included,
because H5N1 has spread to other parts of Europe from there. H5N1 cases in birds have been
confirmed in Romania, Turkey, Kazakhstan, the Ukraine, and Croatia,[38] and Turkey
confirmed two deaths from the virus in early 2006. Britain and Taiwan both reported cases of
H5N1 in 2005. However, the incidences are not discussed here, as experts concluded that the
imported birds were identified and died in quarantine, and are believed unlikely to have
spread the disease.
Cambodia[39]
Between February and April 2005, four Cambodians were confirmed to have died from
the H5N1 avian flu virus. All four victims lived in Kampot province, an area where 600
poultry reportedly had fallen ill and died in March 2005. Despite warnings, many villagers
ate birds that had been sick because food is not plentiful. Health experts predict that more
cases in Cambodia are likely, though the WHO has not reported any additional human cases.
Health officials in Kampot are being taught how to identify symptoms of avian influenza and
instructed to notify the provincial health department. In September 2005, more than 1,000
water birds were reportedly found dead in poultry farms in Batambang and several other
provinces. None of the birds tested have been confirmed to have the H5N1 virus. The
Cambodian government has cooperated fully with the WHO, but the government has limited
capacity to contain outbreaks of the disease. Compared to Thailand, in Cambodia, poultry
farms are smaller but more numerous and many chickens roam freely, while transportation
and communications links are far less developed; hence monitoring the nations poultry
stocks is more difficult. The U.S. government assessment team that visited Laos, Cambodia,
and Vietnam in July 2005 reported that the U.S. government, FAO, and WHO have strong
working relationships with relevant ministries in the Cambodian government, while over 200
international donors and NGOs operating in the country could play an effective role in
mobilizing an effective response to an outbreak of avian flu. On October 12, 2005, U.S.
Secretary of Health and Human Services Michael Leavitt, on a visit to Southeast Asia, signed
a cooperation agreement with Cambodian officials pledging $1.8 million to Cambodia to help
the country guard against the spread of H5N1.[40] United Nations experts estimated that
Cambodia needs $18 million to develop programs to stem the spread of the virus. In
December 2005, Germany announced that it would provide $3 million to the kingdom to help
fight the disease.[41]
Peoples Republic of China, Including Hong Kong[42]
The November 2005 confirmation of the first human cases and deaths from H5N1 in
China in 2005 renewed fears that the spread of H5N1 could accelerate within China.[43] The
close proximity of millions of people, birds, and animals in southern China has made it a
common breeding ground for deadly types of influenza viruses, including the H5N1 avian flu
virus, that jump the species barrier to humans. Added to this, the PRCs poor public health
infrastructure and the traditionally secretive, un-transparent policy approach of its communist
government have made international health specialists particularly concerned about the PRC
as a possible contributor to an H5N1 flu global pandemic. Health care specialists have cited
the PRC governments early lack of cooperation during the outbreak of Severe Acute
Respiratory Syndrome, or SARS a previously unknown virus that surfaced in southern
China in 2003 as a principal cause for that virus quick global spread before it was
contained.[44] As of January 1, 2006, there have been 31 outbreaks of the H5N1 strain in
Chinese poultry since late October 2005, heightening international health concerns.
Hong Kong in late 1997 is where the H5N1 avian flu virus for the first time was recorded
as jumping directly from its traditional animal species to humans, infecting 18 people in
Hong Kong and killing six. Although the Hong Kong government responded aggressively at
that time, in three days exterminating its entire poultry population of 1.5 million birds, the
1997 outbreak marked the beginning of the cycle of H5N1 outbreaks that expanded on a
much wider scale throughout Asia in late 2003 and early 2004. On January 27, 2004, a WHO
official stated that a staggering number of birds, both migratory and domestic, were
infected with the virus in at least ten Asian countries. That same day in 2004, the PRC
became the tenth country to acknowledge ongoing outbreaks of avian flu within its borders.
According to WHO, H5N1 is now considered endemic in parts of China. In addition to
afflicting domestic poultry and migratory birds in isolated parts of China, H5N1 also has
been documented in parts of Chinas pig population.[45]
The 2003 SARS experience appears to have made PRC leaders more sensitive to
potential catastrophic health issues. Consequently, Beijing has been far more assertive in
enacting measures to combat the H5N1 virus. But even with the positive steps that have been
taken, PRC officials face enormous problems in implementation. The PRC Ministry of Health
reports it has established 63 influenza monitoring labs throughout most of China[46] and has
crafted and published an emergency plan for an influenza pandemic, including a four-color-
coded notification system.[47] On November 21, 2005, PRC agricultural officials at a press
conference further announced the adoption and immediate implementation of contingency
regulations to combat the spread of the disease and to punish government officials that delay
or obfuscate medical and scientific reports about the virus. The regulations include
requirements that provincial and municipal level officials notify the central government
within four hours after a new flu outbreak.
By November 2005, PRC officials confirmed that they had either destroyed or vaccinated
millions of healthy domestic poultry and that they were planning to inoculate the entire
Chinese poultry population, a massive effort which would include as many as 14 billion
chickens, geese, and ducks.[48] As a logistical effort, the initiative faces daunting difficulties
first among them the sheer size of Chinas poultry population and the fact that the poultry
industry is widely scattered, including millions of rural households with a dozen or fewer
chickens that roam free. Second, according to medical experts, the poultry vaccine to be fully
effective must be given in two separate doses about a month apart, meaning the entire
undertaking has to be performed twice for a single inoculation to be effective.[49] In
addition, some health officials have expressed concern that such a broad campaign could
backfire and actually contribute to spreading the disease further. Potential problems include
the use of unlicensed or substandard vaccines (a problem announced in Liaoning Province in
2005) which could mask flu symptoms in birds but leave them still contagious;[50] and the
possibility that vaccinators themselves could spread the virus on their clothing or shoes
unless rigid decontamination procedures are followed.[51]
In another anti-flu initiative, on November 2, 2005, the Chinese government announced
an earmark of 2 billion yuan ($420 million) from Chinas current budget to fight avian flu
and the banning of poultry imports from 14 countries affected by avian flu. The Swiss
manufacturer of Tamiflu, Roche, also announced it had reached an agreement with China on
developing a generic version of Tamiflu.[52]
Despite these preparations, some international health experts quietly continue to question
the PRCs transparency on avian flu issues. In late April and June 2005, for instance, PRC
officials reported an unknown cause for the suspicious sudden deaths of thousands of
migratory birds in western Chinas Qinghai Lake. In July 2005, a virology team from Hong
Kong reported in a scientific journal that their research showed the Qinghai bird deaths were
from an H5N1 strain genetically similar to that originating in south China. The Hong Kong
report was vigorously criticized as inaccurate by Jia Youling, an official with the PRC
Ministry of Agriculture charged with coordinating avian-flu eradication.[53] On June 18,
2005, the Washington Post reported that Chinese farmers had been using one of two types of
anti-influenza drugs (amantadine, a drug meant for humans) to treat poultry for the H5N1
bird flu virus, potentially rendering the drug ineffective against the virus strain in humans
a story that PRC officials also have denied.[54]
In its anti-flu efforts, China also remains burdened by perennial problems involving local
and regional compliance with central government directives. This takes on new dimensions
when potential remedies such as the mandatory destruction of infected poultry flocks
may rob indigent farming families of their principal source of food or cash.
U.S.-PRC Cooperation
President George Bush and PRC President Hu Jintao have discussed greater avian flu
coordination on several occasions during a meeting at the U.N. summit in September 2005
and during Bushs visit to Beijing in November 2005.[55] During the latter visit, the two
sides initialed a joint initiative on avian flu, promising to participate in joint research on
human and animal virus samples, establish a mechanism to share influenza strains for
research purposes, and cooperate actively on a number of regional and international levels,
including the WHO, the U.N. Food and Agriculture Organization, and the World
Organization of Animal Health. In spite of this Sino-U.S. agreement, WHO officials on
December 30, 2005 announced that as of that date, China still had not shared with
international health officials flu virus samples from its infected poultry a key step in
tracking the virus mutation and devising an effective vaccine.
A this point, the level of cooperation also appears uncertain in another key area of the
bilateral agreement that involving cooperation on influenza vaccine development. China
appears to have advanced on vaccine development the PRCs State Food and Drug
Administration approved clinical trials for a Chinese-developed human avian flu vaccine in
November 2005 and the United States is separately working on a vaccine of its own.
Indonesia[56]
Indonesia is viewed, along with Cambodia, Laos, and Vietnam, as a weak link in the
effort to curb an outbreak of avian flu. A lack of resources, expertise, and a slow recognition
of the problem has hindered Indonesias response. Indonesia has a population of some 1.3
billion chickens with as many as 400 million of those in informal settings. Indonesia has
resisted mass culling of bird populations. In 2003, when H5N1 was first seen in the bird
population, there was not much alarm in Indonesia as the virus was not generally viewed as a
significant threat to humans. The virus is now considered endemic in the bird population of
Indonesia and outbreaks in birds have so far been reported in 25 out of Indonesias 33 cities
and provinces.[57] Concern grew in June 2005, when Indonesia saw its first human H5N1
fatality. WHO later confirmed H5N1 as the cause of death in July 2005. In October 2005,
when a 38-year-old man and two of his children died of the disease in an affluent section of
Jakarta, some began to speculate that the virus could spread from person to person, but to
date this has not been verified.
There have been a number of questionable reports regarding the number of human cases
of H5N1 infection. One report claimed that 85 people had been admitted to hospitals in
Indonesia with suspected or confirmed cases of avian flu since the first case in June 2005
(though the man died in June 2005, the cause of death was not attributed to H5N1 until July,
as indicated above).[58] However, WHO has only confirmed 16 human cases of H5N1
infection, of whom 11 have died.[59]
While Indonesia was viewed as initially trying to cover up the outbreak, it has more
recently moved to address the problem. Plans to stem the spread of the disease, should it
mutate and spread more widely among human populations, involve rapid reaction and
vaccine distribution. Such an approach is dependent on early detection and reporting by local
health officials, and the availability of the resources necessary to treat an outbreak. On
December 19, 2005, Indonesia announced a three-year national strategic plan to contain the
avian flu virus. The plan will use such measures as culling, vaccination, and community-
based surveillance of bird populations. Critics of the plan have pointed out that it does not
address birds kept in informal settings.[60] In addition, the government plans to establish a
national commission for bird flu control that includes all ministries, private and non-
governmental agencies, and the Red Cross.[61]
The Indonesian government appears to be making limited progress in acknowledging and
dealing with a large scale outbreak. Foreign Ministry Spokesman Yuri Thamrin has stated
we need international cooperation to fight the virus.[62] Agriculture Minister Anton
Apriyanto has indicated that the government will slaughter poultry to stem serious outbreaks.
The minister had reportedly earlier stated that the government did not have enough money to
compensate farmers for their slaughtered animals. The government reportedly spent $13
million in 2005 to cull infected livestock.[63] According to WHO expert Gina Samaan,
Indonesian hospitals are increasingly prepared and the surveillance system has been
enhanced, in the sense that there has been lots of training undertaken to ensure that
surveillance of the health department in the provincial and district levels can respond and can
initiate an investigation.[64] Eleven companies in Indonesia account for 60% of Indonesian
poultry and are reportedly reluctant to allow government monitoring of their birds for fear
that they will not be compensated for birds killed to stem an outbreak of the H5N1 avian
influenza. Indonesias poultry industry generated $3.75 billion in revenue in 2004.[65]
Health experts believe Indonesia does not have a sufficient supply of antiviral treatments
for a country with more than 200 million people, and where H5N1 is endemic among the bird
population. In September, Indonesias Minister of Health asked for international assistance
and expressed concern that her country is not capable of containing the spread of H5N1.[66]
Since then, the international community has pledged $140 million in assistance, and the
Indonesian Government has allotted just over $60 million for bird flu prevention.[67] WHO
officials have also called for countries to donate antiviral drugs to Indonesia. Additionally,
Australian Foreign Minister Alexander Downer has warned that Indonesia is not prepared to
respond to an avian flu outbreak amongst its human population. Australia has planned a
meeting with Indonesian and WHO officials in Indonesia to strengthen Indonesias capacity
to deal with avian flu. Australia has also pledged funding to Indonesia for the purchase of
Tamiflu tablets to treat about 40,000 people.[68] India has also reportedly agreed to provide
1,000 doses, adding to Indonesias own supply of 10,000 doses.[69]
Reporting indicates that Indonesian officials were aware of bird flu in the bird
populations for two years but suppressed the information until humans began to become
infected. It has been asserted that the Indonesian government failed to take measures that
could have broken the chain, [of the spread of bird flu] while discouraging research into the
outbreak. The outbreak was evidently suppressed due to lobbying by the poultry industry in
Indonesia. There are also allegations that the Indonesian government has not funded its
announced policy to vaccinate poultry against the virus.[70]
Laos[71]
An outbreak of H5N1 avian flu in poultry was confirmed early 2004, but Laos has had no
known cases in humans, according to the WHO. There have been no reports of avian
influenza in birds or humans in Laos in 2005.[72] As of June 2005, the Lao government
estimated that 60,000 birds had been lost to the infection and another 98,000 to culling.
However, this number reflects only documentation from commercial farms; the vast majority
of poultry-rearing in Laos takes place in smaller, family-run farms.
Some experts argue that there is an urgent need for foreign health organizations to focus
upon and assist Laos, given its proximity to other countries with the disease and the lack of
government capacity, particularly its weakness in surveillance. The central and local
governments have limited capabilities for collecting and disseminating information,
monitoring avian populations, and conducting laboratory analysis to confirm cases of the
virus. In addition, according to a U.S. government assessment team that visited Laos,
Cambodia, and Vietnam, the countrys health care system faces severe limitations and
would be quickly overwhelmed in the event of a large-scale human outbreak.[73] The FAO
and the WHO reportedly have strong working relationships with the Lao government.[74] On
October 13, 2005, U.S. Secretary of Health and Human Services Michael Leavitt, on a visit
to Southeast Asia, signed a cooperation agreement with Lao officials pledging $3.4 million to
Laos for controlling outbreaks of avian flu.[75]
Russia[76]
The H5N1 strain spread into Central Asia in 2005 and was first diagnosed in southern
Russia (in the Novosibirsk region) as well as in northern Kazakhstan in July 2005. Outbreaks
in both countries were attributed to contact between domestic birds and waterfowl migrating
from Southeast Asia. There have been no confirmed human cases in Russia. The avian flu
spread to eight southern regions of Russia, including two regions bordering the Caspian
Sea,[77] but did not spread north toward Moscow.. Besides Russia, avian flu was reported in
2005 in other countries bordering the Black Sea, including Romania, Turkey, and Ukraine,
and human cases were reported in Turkey in early 2006. The WHO is concerned about the
widening geographical spread of the avian flu into Russia and neighboring countries, because
it increases opportunities for humans to catch the virus and for the virus to improve its
transmissibility through mutation or reassortment.[78] The WHOs National Flu Center in St.
Petersburg announced in August 2005 that it would work more closely with the Vektor
Virology Center in southern Russia, which had been monitoring flu viruses among wild
migratory birds for several years.
In response to the reports of outbreaks in Russia, the EU in late August raised serious
concerns that the virus could spread to Western Europe and called on member-states to step
up surveillance efforts. It also banned the import of poultry from Russia. Responding to
rumors that the avian influenza had spread into western Russia, Germany in October
temporarily ordered free-range poultry to be kept indoors, as did the Netherlands in August.
Iran, in September 2005, banned the import of Russian wheat as feedstock.[79]
Most observers judged Russia as fairly efficient in identifying avian influenza cases and
working with international health organizations, at least at the outset. The areas where the
outbreaks occurred were quarantined. No poultry or products were permitted to be exported
beyond the areas, poultry in these areas exposed to H5N1 were slaughtered, and many people
were examined and immunized. Russias Deputy Foreign Minister Alexander Yakovenko
asserted in early October 2005 that Russia had made a major contribution to countering the
spread of avian flu and pandemic flu worldwide.[80] Other observers raised concerns about
Russias ultimate capacity to respond to the spreading virus, or to deal with human cases.
They warned that since Russia has devoted few budgetary resources in recent years to
improving healthcare, it has not adopted many newer disease-control measures, such as
employing fewer and more highly trained staff, using advanced disease-detection equipment,
and relying more on primary healthcare. According to one commentator, pandemic control
requires prompt detection of cases and targeted interventions for the first clusters. But it
remains doubtful whether Russia has the necessary capacity.... The countrys huge size [also]
is an obstacle to those services that do function well.[81]
Among measures taken by Russian federal and local officials, Chief Health Inspector
Gennadiy Onishchenko issued a directive in August 2005 to implement the May 2005
recommendations of WHO on controlling a possible influenza pandemic. According to WHO
criteria, Onishchenko stated, Russia is in the second stage of the avian flu epidemic, when the
virus is spreading among fowl and can cause human illness, although it has not become easily
transmissible among humans (see Table 1). He called for regional officials to introduce the
necessary corrections into regional plans to prepare for a [human] flu pandemic, including
the allocation of additional funds for prevention and treatment, and to coordinate these
plans with the federal government. In October 2005, he issued instructions to regional and
health officials regarding the clinical pattern, differential diagnosis, and prevention and
treatment of H5N1 influenza in humans. Regional officials complained that the regions had
strained to shoulder the financial burden of compensating owners for the destruction of birds
and of other containment measures. Consequently, regional representatives have called on the
federal government to provide more funds for responding to possible new outbreaks among
poultry, as well as humans. Some observers have also noted that the federal government
could have played a greater role in coordinating regional outbreak responses. Analysts have
noted that responses in each region were often divergent and not coordinated.[82]
Some Russian doctors and officials have argued that the risk of a pandemic is low, but
that the best methods to hedge against such a possibility are better medical care to boost the
health of at-risk Russians, flu immunizations for these Russians, and reserve supplies of flu
vaccine.[83] They suggest that existing human flu vaccines may help protect the population if
H5N1 becomes readily transmissible among humans. In early September 2005, Vladimir
Fisinin, the Vice President of the Russian Academy of Agricultural Sciences, called for the
Russian government to allocate funds to produce 40 million doses of existing human flu
vaccines, as well as 20 million reserve doses. At the same time, the St. Petersburg Institute of
Influenza is working with WHO on the development of a human vaccine targeting the H5N1
influenza virus. The Institute in late 2005 reported promising tests in animals, and plans
human clinical trials in 2006. The Moscow newspaper Nezavisimaya gazeta in late October
2005 urged the Russian government to also consider buying Tamiflu to treat humans in case
of a pandemic.[84]
Russian President Vladimir Putin called in November 2005 for the legislature to approve
Russian membership in the U.N.s FAO, in order to facilitate cooperation with member
countries in combating epidemics, including avian influenza. Russias Federal Service for
Veterinary and Plant Control (VPC) in September 2005 proposed that OIE, the European
Commissions Health and Consumer Protection Directorate, and U.S. veterinary officials
launch a joint program in early 2006 to monitor avian influenza in water fowl as they migrate
from places where they spend the winter Southeast Asia, Africa, northern Australia and
Oceania to Europe, Asia and North and South America. The VPC warned that the H5N1
virus is likely to reappear in southern Russia in Spring 2006 and possibly infect birds
migrating towards Central and Eastern Europe.[85]
Thailand[86]
Thailand, among the earliest and hardest hit by the avian flu, has emerged as a leader in
fighting the spread of the virus. From the initial 2003 outbreak, 8 of Thailands 12 reported
human cases were fatal.[87] Fourteen of the 22 reported human cases have been fatal to date.
As a major poultry exporter, Thailands economy has suffered significantly from the impact
on the industry. After an initially sluggish response, including allegations by the press that
government officials covered up evidence of an outbreak[88], the Thai authorities have led
the effort to respond to the problem and particularly to facilitate regional cooperation. During
a meeting with Prime Minister Thaksin in September 2005, President Bush praised Thailand
as a leader in fighting the disease and pledged further U.S. cooperation.
Considerable economic damage from the news of the influenza has spurred Bangkok to
address the problem. Thailands poultry exports, the fourth-largest in the world, bring in over
$1 billion annually; the loss this year contributed to a 4.4% year-on-year contraction of the
agricultural sector in mid-2005.[89] Both domestic and international demand for chicken fell
due to fears of infection. Thailand needs 90 days without outbreaks in order to receive
certification from the World Organization for Animal Health (OIE) to resume exporting fresh
poultry.[90]
Thai authorities have taken several steps to contain the spread of avian influenza. The
Department of Livestock Development, Ministry of Agriculture and Cooperatives is the focal
point for combating the virus, while Department of Disease Control, Ministry of Public
Health is also a key player. The National Committee on Avian Influenza Control, under the
supervision of a Deputy Prime Minister, was established in 2004 to map out national strategy.
As part of the plan, over 40 million birds have been exterminated, and surveillance teams
have been deployed throughout the country. In December 2005, the Ministry of Public Health
announced that Oseltamivir, an antiviral treatment for influenza, would be produced and
distributed to the public at subsidized prices.[91] Bird smuggling from Cambodia was
targeted by border authorities.[92] By mid-2005, over 11,000 poultry farms reportedly met
the governments biosecurity standards. Thai officials acknowledge, however, that small
farms with open-air facilities, which increase the risk of contamination, remain less regulated.
Unlike China, Thailand bans the use of H5N1 vaccines in its poultry population. Law
enforcement authorities cracked down on illegally imported bird flu vaccines from China; the
H5N1 vaccine is prohibited because the government believes that its use in poultry could lead
to further mutation of the virus.[93]
After the re-surfacing of the flu in July 2005, the Agriculture and Cooperatives Ministry
established guidelines for poultry farmers to get permission from local leaders before moving
their flocks. The movement of fowl is considered to be a key concern of livestock officials.
Mobile checkpoints were set up in the provinces most affected to enhance scrutiny of such
movements.[94] Fighting cocks have been implicated as one of the main transmitters to
humans. The sport is intensely popular in Thailand, with up to 30 million spectators
annually.[95] The industry, resistant to any form of government control, eventually struck a
compromise with the Thai government which allows for the registration of the birds and the
stadiums, as well as measures to control their movement.[96]
Thailand has promoted regional cooperation on containing the flu, proposing an ASEAN
animal hygienic fund and pledging $300,000 to start the project. The resulting center would
enhance cross-border surveillance and control measures, as well as serve as an information
distribution center for all ASEAN countries on the spread of the virus[97]. Public Health
Minister Suchai Charoenratanakul pledged that Thailand would contribute a minimum of 5%
of its own supply to a proposed regional stockpile of antiviral drugs.[98] Thailand and
Indonesia pledged to exchange information on influenza prevention and vaccine
development. Thailand received one million baht ($25,000) from FAO to set up laboratories
and serve as a coordinating center for avian experts, and has received technical assistance
from the European Union to improve networking between laboratories working on the avian
influenza. Thailand also hosts platforms that are cited as key to the U.S. government
response; USAID lists two Bangkok-based organizations as crucial implementing
partners.[99]
Turkey[100]
In early January 2006, the WHO confirmed four cases of H5N1 virus in humans; two of
them, young siblings, were fatal.[101] The deaths were the first from the virus outside of
China and Southeast Asia. Other press reports, citing Turkish officials, claimed up to 15
suspected human cases, most of them children in the eastern rural district of Dogubayazit.
WHO officials sent a team to the region for further investigation, and praised the initial
response of the Turkish Health Ministry.[102]
Vietnam[103]
WHO reports that there have been 93 confirmed cases including 42 deaths of avian
influenza in Vietnam since late December 2003. According to USAID, the H5N1 virus is
believed to be endemic in Vietnams waterfowl population. The Vietnamese government
estimates the countrys total poultry population to be around 250 million birds, including 20
million to 60 million ducks and geese. Between 60% and 70% of the poultry population is
raised in backyard farms, in close proximity to other birds, and the government estimates
that 65 per cent of farm households nationwide raise poultry. Poultry generally is sold live in
local markets and is slaughtered at home. U.N. agencies have estimated that disease
containment, including culling of poultry, have cost the Vietnamese economy an estimated
$200 million.[104] The wartime and tsunami supplemental (P.L. 109-13), which the House
passed on May 5, 2005 and the Senate on May 10, 2005, provides $25 million to help combat
the disease, including approximately $7 million to be used in Vietnam.
In 2005, the Vietnamese government began intensifying its response to the disease by
establishing an interagency working group that includes the FAO and WHO. At the local
level, inter-ministerial steering committees have been established within the Vietnamese
Communist Partys peoples committees, which operate throughout the country. However,
the quality of inter-ministerial coordination, in addition to the capacity of Vietnams local
institutions to monitor, report, and handle disease outbreaks, have been called into question.
The central government in Hanoi is developing a national pandemic preparedness plan, and
as of mid-October 2005 had presented a draft to international health agencies and foreign aid
donors. Since the first outbreak of avian influenza was reported, over 40 million birds have
been culled, though low compensation for farmers appears to have acted as a disincentive for
farmers to report signs of infection. In August 2005, Vietnam began a mass poultry
vaccination program. In early January 2006, the Ministry of Agriculture and Rural
Development (MARD) declared that under the program, all provinces and cities had
completed two phases of vaccinations for over 240 million birds. Critics have called
Vietnams previous poultry vaccination programs ineffective. In October 2005, the
government signed a bilateral health cooperation agreement with the United States and
agreed with a number of U.N. agencies to conduct a joint prevention program.
There are conflicting reports on the willingness of the Vietnamese government to
cooperate with international health workers. Many accounts praise the government for
responding quickly and cooperatively, particularly in the winter and spring of 2005, when
two sets of initial blood tests by Vietnamese and WHO officials indicated that dozens, and
perhaps scores, of Vietnamese might have been infected with the virus. Subsequent testing
revealed that the initial test results had been false positives.[105] Other accounts, which
appear to be in the minority, have charged that the Vietnamese government has been
uncooperative with international health agencies, particularly in the first months of the
outbreak in 2004.[106]
Issues for Congress
Some experts point out that in order to effectively contain the spread of H5N1 and
prepare for pandemic influenza, the U.S. government would need to develop a plan that
integrates domestic and international policy. Some of the policy responses may originate
domestically, but resonate globally. For example, issues related to U.S. drug policy, such as
vaccine technology and intellectual property rights could impact access to antiviral drugs and
vaccines in countries where H5N1 is endemic particularly since some of the most affected
countries do not have the capacity to produce or purchase sufficient quantities of the
drugs.[107] One article in the Journal of Public Health Policy pointed out that almost 40%
of the worlds supply of interpandemic influenza vaccines is used in countries that do not
produce their own vaccines.[108]
Concurrently, some domestic issues are impacted by international developments. For
example, some are concerned that the United States might not have enough antiviral
medication if an influenza pandemic were to occur within the next year since it belatedly
ordered Tamiflu (a drug effective in mitigating the course of illness caused by H5N1
infection in most cases). Senator Barack Obama is quoted as expressing concern in an
interview that the United States would have to wait for its Tamiflu shipments after Britain,
France, and Japan.[109] Some countries in Europe have reportedly ordered enough antiviral
medication to treat 20% to 40% of their populations. For example, the Dutch Health Ministry
has reportedly ordered enough Tamiflu to treat one-third of the population (5 million doses),
and Britain is believed to have ordered enough Tamiflu to treat about 25% of its population
(15 million people). Canada reportedly has stocks for just over 5% of its citizens.[110]
Current reported U.S. stocks are sufficient to treat slightly more than 1% of all Americans.
However, in November 2005, the President announced through the National Strategy for
Pandemic Influenza that the United States would procure enough medicine by the end of
2006 to treat 25% of the U.S. population. Below are some issues that particularly impact the
most affected countries in Asia, and other parts of the world.
Patent Protections
Intellectual property rights have become an increasingly contentious issue in global

health, particularly since companies began threatening to ignore patents for HIV/AIDS
treatments. In an effort to expand global access to flu drugs, the United Nations had been
encouraging Roche the patent holder of Tamiflu to license other companies to produce
generic versions of the drug. Roche announced on October 21, 2005 that U.S. pharmaceutical
companies could manufacture a generic version of Tamiflu.[111] Legislation introduced in
the first session of the 109th Congress aims to permit the United States to invoke a
compulsory license and export generic versions of the drug to non-producing countries.[112]
Some speculate that Roche has been increasing efforts to license its products in other
countries, in part because an Indian pharmaceutical company, Cipla, has threatened to
manufacture a generic version of the drug in spite of Roches patent rights. Underscoring
that Tamiflu is too expensive for many of the least developed countries, a Cipla
representative said that the company would sell the generic version of Tamiflu at a
humanitarian price in developing nations, and not in the United States or Europe.[113] Two
Indian pharmaceutical companies are reportedly negotiating with Roche to produce generic
versions of Tamiflu.[114] Roche also reached an agreement with a Chinese pharmaceutical
company to make the drug.[115]
Health experts predict that patent protections will continue to be a contentious issue as
poorer countries seek to protect themselves against virulent diseases. Some analysts contend
that Congress faces an issue of whether to help countries where H5N1 is endemic gain
greater access to generic versions of Tamiflu and other antivirals if licensed drugs are not
accessible. Supporters assert that the precedent for greater access to generics by poorer
countries had already been established on December 6, 2005, when World Trade
Organization (WTO) members approved changes to the intellectual property agreement
making permanent a decision on patents and public health[116]. The General Council
decision means that for the first time a core WTO agreement will be amended. The decision
directly transforms the August 30, 2003 waiver to Section 31(f) of the Trade-Related Aspects
of Intellectual Property Rights (TRIPS)[117]. The waiver permits a country without
manufacturing capacity to obtain cheaper generic versions of patented medicines from
countries under compulsory licenses. The waiver enables the country to receive generic
versions of drugs in situations of national emergency or other circumstances of extreme
urgency.[118] A separate statement describes members shared understanding on how the
decision is interpreted and implemented. Particularly, the statement points out that the
decision will be used in good faith in order to deal with public health problems and not for
industrial or commercial policy objectives.[119] Although the waiver was seen as a tool to
enable largely poorer countries to import generic versions of licensed drugs, one piece of
legislation proposes that the U.S. Trade Representative inform WTO that the United States
declares itself an eligible importing member to import pharmaceutical products, largely
because Roche is unable to meet the public health needs of the United States.[120]
WTO members voted against delineating which drugs should be included in the waiver
agreement. Consequently, there is not consensus on which drugs are considered critical in
protecting public health. Advocates argue that in the event of a pandemic, the new WTO
amendment should apply to antiviral drugs and H5N1 vaccines for use in animals. Opponents
are concerned that some might abuse and undermine the agreement by reselling the drugs and
vaccines for profit. In the event of a pandemic, Congress might be faced with the decision on
whether to support or oppose the export of generic antivirals. Additionally, increased
pressure might be placed on Congress to encourage USDA to share with other countries some
of its H5N1 vaccine for use in animals.
Global Data Sharing
In spite of Tamiflu stockpiling efforts, it is unknown if the medicine will be broadly

useful in treating human H5N1 victims in a pandemic scenario. Some health experts were
reportedly alarmed when two patients in Vietnam who were infected with H5N1 and
aggressively treated with Tamiflu later died. Some are beginning to question if the
recommended dosage should be changed, as doctors reportedly adhered to the recommended

regimen when treating the two patients.[121] Health experts point out that more information
is needed on patients who have already been treated for H5N1 with Tamiflu. Data from the
subjects would help in determining if the drug remains effective in fighting H5N1 and if
changes to dosage regimens are required.
Those pressing for greater international data sharing point to new research that might
counter previous findings on the limited effectiveness of amantadine. The New York Times
reported in September 2005 that researchers found that amantadine was no longer effective
against H5N1. WHO reportedly spent $1.3 million to stockpile the drug when it was used
during the 1997 H5N1 outbreak. The Times article asserted that in 2005, laboratory research
found that all human viral samples of H5N1 were resistant.[122] Before 2000, almost no
influenza virus was resistant to the drug. Some experts speculated that viral resistence
occurred in part, because China reportedly used amantadine, intended solely for humans, on
animals. (See Affected Countries Response section). However, the Wall Street Journal
quoted Dr. Shu Yuelong, the Director of Chinas national influenza laboratory, as stating that
preliminary evidence indicates that amantadine might be effective in treating avian influenza
in people.[123] Dr. Shu reported that all of the viral samples that have been isolated from
patients in China were sensitive to amantadine. Those findings conflicted with previous
research on virus samples that were taken from patients in Indonesia and found to be resistant
to the drug. The new research has reportedly prompted WHO and other officials to consider
whether amantadine might eventually play a role in fighting H5N1. The article underscores
that there are currently too few samples to draw any firm conclusions.
Some believe that some countries are intentionally withholding viral samples of H5N1
cases. One article stated that countries with human H5N1 cases do not want to send viral
samples to the WHO or other industrialized countries, because they fear the samples will be
used to develop up-to-date vaccines which they will not have access to.[124] Others have
speculated that China is withholding its samples, because it is trying to produce an H5N1
vaccine.[125]
Some analysts propose that the United States and other countries should vote to provide
WHO with enforcement mechanisms. Supporters argue that WHO should be able to force
countries to share viral samples. Others contend that Congress should provide greater support
and resources to WHO, particularly for strengthening global laboratory and testing
capabilities. Skeptics point out that WHO has not provided transparent, detailed data on the
adequacy of funds or how funds are spent.
Global Disease Surveillance
A number of analysts have argued that due to insufficient investment in disease

surveillance and health care in many of the countries where H5N1 is endemic, a pandemic
may progress before it is discovered. In this view, ill-equipped surveillance systems will be
slow to determine the source of a pandemic, evaluate the rate of viral transmission, ascertain
whether H5N1 has become efficiently transmissible among humans, or rate the effectiveness
of anti-flu initiatives. Senate Majority Leader Bill Frist has proposed $1 billion for a real-time
international threat detection system.[126]
USAID and other U.S. government officials suspect that the lack of documented human
cases of H5N1 in Laos has more to do with inadequate surveillance and reporting systems
than an absence of infection.[127] Some health experts believe that H5N1transmission could
already be underway in Laos, since surrounding countries have already had human and
animal outbreaks. Key U.S. agencies and international organizations have determined that
Laos is a country that needs critical prevention, monitoring, and surveillance support in order
to prevent full-blown human-to-human transmission of H5N1 that could emerge and sweep
across the region without warning.[128]
Some experts have expressed increasing concern about the capacity of poorer countries
that have not yet had H5N1 cases to effectively contain the spread of the virus and plan for
pandemic influenza, particularly in sub-Saharan Africa. FAO has recently warned that the
risk of H5N1 spreading to the Middle East and Africa has markedly increased. FAO is
particularly wary of the virus reaching Eastern Africa, as the surveillance capacities and
veterinary services in those countries are limited. According to Reuters, a WHO
representative declared that an H5N1 outbreak would likely be missed in Africa, as bird
nutrition is poor and high mortality among poultry is common. Concurrently, human cluster
cases are likely to be missed due to poor surveillance systems. South Africa is reportedly the
only country in sub-Saharan Africa to have drawn up a pandemic preparedness plan.[129]
Some experts fear that an unabated H5N1 outbreak in East Africa could make the bird flu
endemic there. If the virus were to become endemic in eastern Africa, it could increase the
risk that the virus would evolve through mutation or reassortment into a strain that could be
transmitted to and between humans.[130]
The press reported on December 20, 2005 that a bird suspected of having contracted
H5N1 in Ethiopia, tested negative of the virus.[131] Experts are concerned that birds in
Ethiopia and other countries in the Rift Valley, including Kenya, Tanzania, and Uganda, are
at particular risk of avian flu infection due to the large numbers of migratory birds that fly to
the region during the European winter. Those concerned about insufficient surveillance and
diagnostic equipment and expertise, point out that Ethiopia had to use health experts and
equipment from Egypt to determine what caused a rash of bird deaths in December 2005.
USAID with support from the U.S. Navy Medical Research Unit (NAMRU) in Cairo
reportedly provided $15,000 in emergency funding to analyze the viral samples of dead
pigeons found in Addis Ababa and the Eastern Somali region for H5N1 infection.
Additionally, USAID has reportedly reprogrammed $600,000 from existing surveillance
funds for bird flu initiatives in Ethiopia.[132] The funds are to help provide technical
assistance to the Ministries of Agriculture and Health, develop laboratory and
communications capacity, and procure Personal Protective Equipment for first responders.
Many of the countries in which H5N1 is endemic have complained that they can not
afford to implement the strategies recommended by the international community.
Furthermore they are hesitant to divert their limited budgets already struggling to contend
with AIDS, child and maternal health, tuberculosis, and other health challenges to
something that might not occur. Advocates of greater assistance to the region, point out that
countries with more resources for pandemic planning than neighboring poorer countries have
also acknowledged difficulties in responding to the H5N1 threat. A news report cited a South
Korean health worker who stated that his country is ill-equipped to respond to a pandemic
citing insufficient supplies of medication, hospital beds, and ventilators.[133]
On December 22, 2005, the Senate passed S. 2170, which would help developing
countries bolster their disease surveillance programs, and establish fellowships for citizens of
those countries to study epidemiology and public health in the United States. Additionally,
some in Congress have advocated for greater U.S. spending on fighting the global spread of
H5N1 avian flu. Press reports quoted Representatives Henry Hyde and Tom Lantos,
Chairman and Ranking Member of the House International Relations Committee
respectively, stating concern about the level of funding the Administration proposes to
provide for global efforts in FY2006[134]. Advocates assert that the $388 million the
Administration requests for international H5N1 initiatives will not be enough to fund the
significant amount of assistance needed by countries with H5N1-endemic stocks.
Particularly, experts add that the threat of an H5N1 or other influenza pandemic illuminates
the neglect that health care systems in many southeast Asian countries have faced over the
last couple of decades. Proponents argue that if the United States would increase its funding
to support global health care systems the global community could benefit from efficient
outbreak reporting and control measures, accurate diagnoses, enhanced case management,
and improved disease surveillance and monitoring.
Global Pandemic Planning
Some experts caution that pandemic preparedness plans must extend beyond procuring
and stockpiling antiviral drugs and vaccines. In this view, governments must also develop
detailed vaccine and treatment distribution plans. Particular attention has been paid to H5N1-
affected countries that have communication and infrastructure barriers, especially between
urban and rural areas (where many of the backyard poultry farms exist). Many Asian
countries have significant income and infrastructure gaps between rural and urban areas. In
the rural areas, there are often few hospitals and treatment centers. Equipment can be
outdated or lacking. Veterinary and animal health services can be limited. Additionally, in
many cases rural governments operate independently from urban governments, which tend to
receive larger portions of national resources. Farmers in rural areas may not adhere to
government H5N1 initiatives, exacerbating the problem. One infectious disease expert in
Hong Kong asserted that the communication problem is particularly acute in China. I trust
and believe the central government has very good intentions, but unfortunately, it is a very
big country. At the district, regional levels, the failure to communicate continues.[135]
Responses by East Asian Regional Groupings

As Southeast Asias major multinational fora, the Association of southeast Asian Nations
(ASEAN) has taken some steps to improve transnational coordination in combating the
spread of a potential pandemic, and limiting the spread of the H5N1 virus. To this end,
ASEAN members have created a number of institutional arrangements, including a Highly
Pathogenic Avian Influenza (HPAI) Taskforce, an ASEAN Expert Group on Communicable
Diseases, the ASEAN Animal Health Trust Fund, and the ASEAN Plus Three[136] Emerging
Infectious Diseases Programme. At the eleventh ASEAN summit in Kuala Lumpur, Malaysia,
in December 2005, ASEAN leaders discussed establishing a regional network of stockpiles of
antiviral drugs.
Drafting an avian influenza declaration was the single tangible achievement of the
inaugural meeting of East Asias newest regional grouping, the East Asia Summit (EAS),
which met in Kuala Lumpur in December 2005 immediately following the ASEAN
summit.[137] In their Summit Declaration on Avian Influenza Prevention, Control and
Response, EAS leaders committed to ensure rapid, transparent and accurate
...communications, establish information sharing protocols among member countries and
multilateral organizations, create a regional network of stockpiles of antiviral, and to
establish regional avian influenza and pandemic preparedness strategies backed by
supporting national legislation.
Pandemic planners are warning that no country has the surge capacity to meet national
demands for consumer products and medical services for the full term of an influenza
pandemic (an estimated six months to a year). The United States, and other industrialized
nations, rely on a range of critical products from H5N1-affected countries, such as medical
supplies, military parts, and sanitation equipment. These supply chains are replenished just-
in-time to minimize costs. If an outbreak were to occur, hospitals, food and water systems,
and the military could all be vulnerable to interrupted supply due to absenteeism, border
closures, and other supply chain disruptions. Therefore, the private sector, as well as national
and international trade organizations, have been urged to participate in pandemic planning.
Some analysts argue that resources allocated to containing the spread of H5N1 have been
insufficient in part, because many countries have funded the response primarily through the
Ministries of Agriculture and Health. Some experts point out that an influenza pandemic will
likely impact the animal and health sectors, as well as trade, security, hospitality, and labor.
Consequently, they say, governments should develop pandemic plans that utilize the
resources of other ministries that are often better funded, such as Ministries of Trade,
Tourism, and Commerce. Some analysts note that U.S. officials, such as the U.S. Trade
Representative and the Secretary of Commerce should be engaged in U.S. international
pandemic influenza planning efforts. Others would like Congress to encourage public-private
partnerships that augment U.S. international avian flu and pandemic preparedness efforts.
Combating Bird Flu among Animals in Affected Countries
Most countries have used mass culling to prevent viral spread when avian influenza
outbreaks are detected. However, some countries have not been able to rely on this process as
a primary containment measure, because the governments might not have been able to
compensate farmers for slaughtering their stocks. Scientists have also found that mass culling
is sometimes not feasible when wild birds are involved in transmission. Some health experts
assert that there should be more research on more affordable methods of preventing
pandemics at their source in the animals that carry the virus. Strategies such as
implementing cleaning days (when all live markets are simultaneously emptied and cleaned),
and separating ducks and chickens in live poultry markets may decrease viral transmission
among animals. Some countries (including China) propose using vaccination to control avian
influenza in poultry. Skeptics warn that animal vaccination is a risky strategy, as it is often
difficult to distinguish infected from vaccinated animals, complicating efforts to track the
disease. Additionally, vaccination campaigns, if not carried out properly, could result in
entrenchment of the disease rather than eradication, further threatening public health.[138]
Cost of Culling
It has been suggested that a global fund should be established to compensate farmers for
culling their poultry in countries whose governments can not afford to compensate the
farmers. The WHO has already expressed concern that some farmers in poorer countries may
not cull their poultry, because their livelihoods depend on poultry farming. For example,
Indonesia has carried out only a limited culling drive, because it lacks the funds to
compensate farmers.[139] Farmers in some parts of Romania reportedly failed to cull their
birds despite government orders to do so. In some affected countries, public and animal
health authorities are reluctant to destroy their populations dominant protein source and
income. A number of bills, such as H.R. 4062 and its counterpart S. 1821, have been
introduced that support the concept of a Pandemic Fund, which could include funds for
farmer compensation.
The World Bank announced that it would provide $500 million in loans to poor countries
struggling to fund national avian flu and pandemic preparedness plans a portion of which
could be used to support poor farmers.[140] However, the Bank noted that $1 billion could
be needed over the next three years to help countries contain the spread of H5N1. The Asia
Development Bank (ADB) also announced that it is prepared to provide at least $470 million
to support Asian anti-H5N1 and pandemic preparedness efforts.[141]
Some have suggested that the United States target some foreign aid funds to help the
affected governments including Vietnam, Indonesia, Cambodia, and Laos cover the
cost of compensating individuals and companies for the destruction of their birds. In this
view, such assistance could help the image of the United States in the region by
demonstrating American concern and could minimize reluctance to slaughter infected flocks.
Others would like to see increased assistance to prevent the spread of H5N1 among animals.
Global Economic Impacts
The health and non-health related costs of a global influenza pandemic could be very
high, though difficult to estimate. For example, Canadian and Asian hospitality and tourism
sectors were considerably impacted during the SARS outbreak. In 2002 and 2003, SARS cost
the Asia-Pacific region about $40 billion.[142] Additionally, flights to the region fell by
about 45%, crippling the airline and hotel industries. Canada estimated that it lost
approximately $1.2 billion, with about $763 million spent on the health-care system.[143] In
the event of a flu pandemic, researchers expect Britain, Greece, Spain, Italy, and other
countries that rely heavily on tourism, to be most affected economically. One economist
estimated that a flu pandemic could force Britains GDP to fall by 8% or $168 billion (about
95 billion pounds), and result in the loss of almost 1 million jobs (about 3% of all
employment).[144]
The World Bank estimates that a global influenza pandemic could cost the global
economy about $200 billion in one quarter or $800 billion over a year (about 2% of the
global GDP). The Bank based its estimate on the economic losses induced by the SARS
pandemic, which caused GDP to fall by 2% in Asia over a three month period in 2003.
However, the Bank underscored that it is virtually impossible to accurately determine how
much a global influenza pandemic would cost the world, because experts assume that the
immediate shock during a flu epidemic could be larger and last longer than SARS. The 1918
pandemic, for example, came in three waves, and spread over two years.[145] Some
economists have advised the United States to identify source countries for key imports and
develop a detailed plan that would ensure continuity.
Economists point out that an Asian economy crippled by an influenza pandemic could
impact the U.S. economy, even if a significant number of Americans was not sickened or
killed by H5N1. According to U.S. Trade Representative (USTR) Robert Portman, South
Korea and Malaysia are the 7th and 10th largest trading partners for the United States,
respectively. The United States earned $72 billion and $40 billion from South Korea and
Malaysia, respectively, in 2004. Both countries have had H5N1 cases among their
flocks.[146] Additionally, U.S. exports to China, one of the most threatened countries, grew
76 percent between 2000 and 2003, while sales to the rest of the world declined by 9 percent.
China is now the sixth largest market for U.S. exports and Americas third largest trading
partner overall surpassing Japan in 2003.[147] In 2004, U.S. exports to China grew to $33
billion, more than double the level in 2001.[148] Therefore, any pandemic related disruption
of bilateral trade could have a large impact. Alternatively, some economists predict that U.S.
poultry exports could increase as countries move to ban imported birds from countries with
H5N1-endemic stocks.
CLSA Asia-Pacific Markets, the Asian investment banking arm of Crdit Agricole of
France, estimates that H5N1 has already cost the region between $8 billion and $12 billion,
citing the prolonged poultry ban by the European Union from eight Asian countries and the
death or destruction of some 140 million chickens and other poultry. The Prime Minister of
Thailand stated that the avian flu has already cost his country some $1.09 billion, in addition
to the $55.78 million the government paid to farmers for a mass chicken cull.[149]
Some analysts caution that Congress should be prepared to respond to the impact that
potential fluctuations in supply and demand from key Asian markets might have on the U.S.
economy. Particularly, some would like Congress to direct the U.S. Trade Representative to
prepare a report that comprehensively analyzes the potential economic gains and losses to the
U.S. economy in a pandemic scenario due to changes in Asias economy. Experts point out
that the Congressional Budget Office (CBO) report A Potential Influenza Pandemic: Possible
Macroeconomic Effects and Policy Issues focuses on possible supply and demand changes in
the U.S. economy if an H5N1 pandemic were to reach the United States.[150] The Wall
Street Journal reported that the U.S. poultry industry currently exports about 15% of its
chicken meat annually, earning $2.2 billion in 2004. The article asserted that some poultry-
industry executives are concerned that importing countries might reject poultry from states
that have vaccinated the animals.[151] Consequently, many executives in the poultry industry
are opposed to vaccinating chickens intended for export. Some would like Congress to
require USDA to present clear guidelines on how and when poultry would have to be
vaccinated.
Global Biosafety
In October 2005, scientists reported that the 1918 influenza pandemic that had killed
between 20 million and 50 million people worldwide may have emerged from an avian flu
strain. Health experts have debated whether the genetic sequence of the 1918 influenza
should be published. Some were concerned that the information could be used to construct a
biological weapon. However, other scientists argued that sharing such important findings is
critical to efficiently identifying dangerous viruses, and to finding ways to disable them.
Ultimately, the genetic sequence was published.[152] Dr. Anthony Fauci, Director of the
National Institute of Allergy and Infectious Diseases, and Dr. Julie Gerberding, Director of
the CDC, said in a joint statement, The new studies could have an immediate impact by
helping scientists focus on detecting changes in the evolving H5N1 virus that might make
widespread transmission among humans more likely. Furthermore, the HHS National
Science Advisory Board for Biosecurity voted unanimously that the benefits [to making the
results public] outweighed the risk that it would be used in a nefarious manner.[153]
However, the Administration acknowledged that the influenza virus could be used as a
biological weapon and added the virus to the Select Agent list on October 20, 2005.[154]
Congress authorized the Select Agent program in the late 1990s to track the movement of
certain bacteria and viruses that could potentially be used as bioterrorist weapons.[155]
Health specialists caution that lab safety must be a top priority as other countries begin to
develop their own research and vaccine capacities. Some are closely watching Taiwan in its
effort to build its own influenza vaccine factory.[156] Japan, already accomplished in viral
research, is reportedly helping Vietnam build a biosafety lab to work with the influenza
virus.[157] If global influenza vaccine production is to increase, disease experts caution that
some form of oversight must first be established. Some scientists advocate the development
of an international influenza research facility. Supporters envision a global laboratory that
could rapidly identify influenza threats, and produce appropriate vaccines. It also could, they
say, streamline existing flu monitoring systems. Opponents of this idea believe that current
technology, such as the WHOs Internet-based FluNet, is fully capable of obtaining the same
goal. Furthermore, critics believe that scientists might lose interest in sharing viral samples, if
they believe their analytical and research capacities will be taken away.[158]
S. 1873, The Biodefense and Pandemic Vaccine and Drug Development Act, would
address production of pandemic products. The bill would authorize funding for surge
capacity of manufacturing vaccines. It would also authorize funding for research and
development of flu vaccines, counter measures, and pandemic products.
Appendix
Source: Information Based on the World Health Organization (WTO) website, and the World
Organization for Animal Health (OIE) website. Adapted by CRS. (K Yancey 1/6/06).
Figure 1. Map of Human and Animal H5N1 Cases
Table 2. WHO Pandemic Phases
Phase Description Overarching Public Health Goals

Interpandemic Period
Phase 1 No new influenza virus strains have been detected in Strengthen global influenza pandemic
humans. A virus strain that has caused human preparedness at the global, regional and
infection may be present in animals. If so, the risk of national levels.
human infection is considered to be low.
Phase 2 No new influenza virus strains have been detected in Minimize the risk of transmission to
humans. However, a circulating animal influenza humans; detect and report such transmission
virus strain poses a substantial risk of human disease. rapidly if it occurs.
Pandemic Alert Period
Phase 3 Human infection(s) with a new strain, but no human- Ensure rapid characterization of the new
to-human spread, or at most rare instances of spread virus strain, and early detection, notification
to a close contact. and response to additional cases.
Phase 4 Small cluster(s) with limited human-to-human Contain the new virus within limited foci or
transmission, but spread is highly localized, delay spread to gain time to implement
suggesting that the virus is not well adapted to preparedness measures, including vaccine
humans. development.
Phase 5 Larger cluster(s), but human to human spread still Maximize efforts to contain or delay
localized, suggesting that the virus is becoming spread, to possibly avert a pandemic,
increasingly better adapted to humans, but may not and to gain time to implement pandemic
yet be fully transmissible (substantial pandemic risk). response measures.
Pandemic Period
Phase 6 Pandemic: increased and sustained transmission in the Minimize the impact of the pandemic.
general population
Source: World Health Organization.
Table 3. FY2006 Emergency Supplemental Request ($ millions)
AGENCY ACTIVITIES AMOUNT

Department of Health and Increase vaccine manufacturing capacity so that 25% of 6,700.0
Human Services Americans would have access to antiviral medication, and the
entire U.S. population would have access to pandemic
influenza vaccines within a six-month period.
Department of Agriculture Research and development, domestic surveillance and 91.4

diagnosis of live bird markets, wildlife and bird flyways,
smuggling and waterfowl, planning and preparedness training
and modeling of scenarios, and the production of 40 million
doses of animal vaccine.
Department of Defense Purchasing avian influenza vaccines, increasing world wide 130.0
surveillance of the virus, and upgrading surveillance,
laboratory, information management equipment. Additionally,
$10 million of the $130 million is intended to assist military
partner nations in procuring protective equipment, laboratory
diagnostic equipment, portable field assay testing equipment
surveillance, and essential communication equipment.
Department of Homeland Pandemic scenario modeling, personal protective equipment, 47.3

Security private sector pandemic assistance planning, and exercises
and training for DHS frontline staff.
Department of the Interior For the U.S. Geological Survey (USGS), the U.S. Fish and 11.6
Wildlife Service, the National Park Service, and other Federal
agencies to begin an interagency effort to detect avian
influenza in wild birds, with an initial focus on early detection
activities in Alaska and coastal areas.
Department of State International response coordination, including foreign 38.5

governments and non-governmental organizations, diplomatic
outreach, exchanges of U.S. and foreign medical personnel,
and for avian and pandemic influenza health support and
protection of U.S. government employees and families at U.S.
missions overseas. $20 million of the funds would be reserved
for the potential evacuation of U.S. government personnel and
dependents from overseas missions.
Department of Veterans Increasing avian influenza surveillance programs and 27.0

Affairs establishing real-time surveillance data links with CDC.
USAID Pre-position supplies and equipment to prevent and control 131.5

the spread of avian influenza(within one year of receiving
funds); increase awareness of risks and appropriate behaviors
to reduce transmission among humans and animals; improve
surveillance and response; and accelerate international
planning and preparedness. $2 million of the funds are
reserved for Russia and Eastern Europe.
GRAND TOTAL 7,177.3

Source: Prepared by CRS from FY2006 Emergency Request.
Table 4. FY2006 Appropriations Providing Funds for Global Initiatives
Legislation FY2006 Senate

Appropriations FY2006 House Appropriations
H.R. 3010, Labor, HHS, $60 million for global $36.5 million to combat the
Education surveillance. spread of the avian flu in Asia,
and to enhance global
surveillance and response
network for infectious diseases.
H.Rept. 109-337 provides $183.5 million for the Public Health and Social Services
Emergency Fund (PHSSEF), of which $120 million would be available until expended.
Presented to the President for signature on December 28, 2005.
P.L. 109-102, Foreign $10 million, control the No similar language.
Operations spread of the avian flu.
P.L. 109-1 02 provides $25 million for strengthening international surveillance, reporting,
and response capacity.
H.R. 2863, Department $33 million, avian flu No similar language.
of Defense global surveillance;
H.Rept. 109-359 provides $3.8 billion for avian flu initiatives, of which $3.3 billion is
directed to the Public Health and Social Services Emergency Fund. $267 million of the
$3.3 billion is reserved for international initiatives, disease surveillance, vaccine registries,
research, and clinical trials. An additional $500 million is reserved for international
assistance, monitoring and tracking, and research and development. The conference report,
H.Rept. 109-359, Emergency Supplemental Appropriations to Address Hurricanes in the
Gulf of Mexico and Pandemic Influenza, provides $3.8 billion for avian influenza
initiatives. Specifically, the bill directs $3.3 billion to PHSSEF, $131.5 million to USAID,
$130 million to the Department of Defense, $71.5 million to APHIS, $47.3 million to the
Department of Homeland Security, $20 million to FDA, $27 million to the Department of
Veterans Affairs, $31 million to the Department of State, and $11.6 million to the
Department of the Interior. Presented to the President for signature on December 28, 2005.
Source: Prepared by CRS from FY2006 appropriations legislation.
Table 5. Country Allocations for FY2005 Supplemental
COUNTRY CDC FUNDING USAID FUNDING

Vietnam $2.634 million $ 3.45 million
Indonesia $0.25 million $ 3.15 million
Cambodia $ 1.858 million $ 2.25 million
Laos $ 1.858 million $ 1.60 million
China $0.00 $ 0.50 million
Regional $3.35 million $ 2.75 million
TOTAL $9.95 million $13.7 million
Sources: USAID Press Release, October 27, 2005 and CDC Washington Office, November 8, 2005.
CDC Funding reflects spending from FY2005 Emergency Supplemental. USAID Funding includes
reallocated funds from other programs.
Table 6. International Partnership on Avian and Pandemic Influenza (IPAPI)

Core Principles
1. International cooperation to protect the lives and health of our people;

2. Timely and sustained high-level global political leadership to combat avian and
pandemic influenza;
3. Transparency in reporting of influenza cases in humans and in animals caused by
strains that have pandemic potential, to increase understanding, preparedness and,
especially to ensure rapid and timely response to potential outbreaks;
4. Immediate sharing of epidemiological data and samples with the World Health
Organization (WHO) and the international community to detect and characterize the
nature and evolution of any outbreaks as quickly as possible, by utilizing, where
appropriate, existing networks and mechanisms;
5. Rapid reaction to address the first signs of accelerated transmission of H5N1 and other
highly pathogenic influenza strains so that appropriate international and national
resources can be brought to bear;
6. Prevent and contain an incipient epidemic through capacity building and in-country
collaboration with international partners;
7. Work in a manner complementary to and supportive of expanded cooperation with and
appropriate support of key multilateral organizations (WHO, Food and Agriculture
Organization, World Organization for Animal Health);
8. Timely coordination of bilateral and multilateral resource allocations; dedication of
domestic resources (human and financial); improvements in public awareness; and
development of economic and trade contingency plans;
9. Increased coordination and harmonization of preparedness, prevention, response and
containment activities among nations, complementing domestic and regional
preparedness initiatives and encouraging where appropriate the development of
strategic regional initiatives;
10. Actions based on the best available science.
Source: State Department Press Release, U.S. Launches International Partnership on Avian and
Pandemic Influenza. September 22, 2005. [http://www.state.gov/r/pa/prs/ps/2005/53865.htm]
References
[1] For a list of past avian flu outbreaks see CRS Report RS21747, Avian Influenza:
Agricultural Issues, by Jim Monke.
[2] WHO, Avian Influenza New Areas with Infection in Birds Update 34, Disease
Outbreak News, Oct. 13, 2005, at [http://www.who.int/csr/don/en/].
[3] Osterholm, Michael, Pandemic Influenza: A Harbinger of Things to Come.
Presentation at the Woodrow Wilson International Center for Scholars, September 19,
2005. [http://www.wilsoncenter.org/index.cfm?fuseaction=news.itemandnews_id=
145329]
[4] HHS Pandemic Influenza Plan, November 2005, [http://www.hhs.

gov/pandemicflu/plan/]. For more information on issues related to domestic efforts to
address H5N1 and pandemic influenza preparedness, see CRS Report RL33145,
Pandemic Influenza: Domestic Preparedness Efforts, by Sarah A. Lister.
[5] To date, H5N1 has been identified among birds in Cambodia, China, Croatia,
Indonesia, Japan, Kazakhstan, Korea, Laos, Malaysia, Mongolia, Romania, Russia,
Thailand, Turkey, and Vietnam. A bird in Britain was diagnosed with H5N1, however
it was an isolated case found in an imported bird that died in quarantine. World
Organization for Animal Health (OIE), Update on Avian Influenza in Animals.
December 21, 2005. [http://www.oie.int/eng/en_index.htm]
[6] WHO, Cumulative Number of Confirmed Human Cases of Avian Influenza A/H5N1,
January 9, 2006. [http://www.who.int/csr/disease/ avian_influenza/country/en/]
[7] For more information on the domestic response to H5N1, see CRS Report RL33145,
Pandemic Influenza: Domestic Preparedness Efforts, by Sarah A. Lister, and CRS
Report RS21747, Avian Influenza: Agricultural Issues, by Jim Monke.
[8] The FY2006 Defense, Disaster Assistance, and Avian Flu Preparedness Appropriations
conference report, H.Rept. 109-359, contains $3.8 billion for avian influenza initiatives.
$3.3 billion of the $3.8 billion is directed to the Department of Health and Human
Services (HHS) (of which $267 million is reserved for international initiatives, disease
surveillance, vaccine registries, research, and clinical trials). An additional $500
million is reserved for international assistance, monitoring and tracking, and research
and development, of which $131.5 million is directed to USAID, $130 million to the
Department of Defense, $71.5 million to the Department of Agriculture, $47.3 million
to the Department of Homeland Security, $20 million to FDA, $27 million to the
Department of Veterans Affairs, $31 million to the Department of State, and $11.6
million to the Department of the Interior.
[9] CQ Today, Bush Flu Spending Request Meets Resistance from House Republicans.
November 8, 2005. [http://www.cq.com]
[10] FY2006 Emergency Request for Avian and Pandemic Influenza Preparedness.
[http://www.whitehouse.gov/omb/budget/amendments/supplemental_11_01_05.pdf]
[11] Report from Country Planning Visits, U.S. Government Emergency Response to
Avian Influenza: A Plan of Action for Vietnam, Laos, and Cambodia. July 11-24,
2005. This report was provided to CRS by USAID.
[12] Interviews with CDC staff, October 13, 2005 and November 1, 2005.
[13] USAID Fact Sheet Avian Influenza Preparedness, Response, and Containment.
October 27, 2005. [http://www.usaid.gov].
[14] The State Department also implements influenza pandemic preparedness initiatives
through the Office of International Health Affairs (OES/IHA), which works with
agencies throughout the U.S. government to facilitate policy-making regarding
bioterrorism and health security, environmental health, infectious diseases (e.g., SARS,
Avian Influenza, Pandemic Influenza, Polio), health in post-conflict situations, and
surveillance and response. [http://www.state.gov/g/oes/ c1874.htm].
[15] USDA, Transcript of Technical Briefing regarding Avian Influenza. October 26, 2005.
[http://www.usda.gov/birdflu]
[16] GEIS website, [http://www.geis.fhp.osd.mil/].

[17] DoD, Global Emerging Infections System Annual Report Fiscal Year 2004.
[http://www.geis.fhp.osd.mil/GEIS/aboutGEIS/annualReports/GEIS_AR_04.pdf].
[18] DoD, Global Emerging Infections System Annual Report Fiscal Year 2004.
[http://www.geis.fhp.osd.mil/GEIS/aboutGEIS/annualReports/ GEIS_AR_04.pdf].
[19] This section prepared by Rhoda Margesson, Analyst in Foreign Affairs.
[20] For more information on the Global Outbreak Alert and Response Network, see
[http://www.who.int/csr/outbreaknetwork].
[21] The WHO influenza pandemic preparedness Home Page is at
[http://www.who.int/csr/disease/influenza/pandemic/en/index.html].
[22] See WHO, Department of Communicable Disease Surveillance and Response Global
Influenza Programme, Responding to the Avian Influenza Pandemic Threat:
Recommended Strategic Actions, WHO/CDS/CSR/GIP2005.8.
[23] WHO, Donation of three million treatments of oseltamivir to WHO will help early
response to an emerging influenza pandemic. August 24, 2005.
[http://www.who.int/mediacentre/news/releases/2005/pr36/ en/index.html].
[24] Roche, Roche donates 3 million treatments of antiviral Tamiflu to the WHO for use in
an influenza pandemic. August 24, 2005. [http://www.roche.com/med-cor-2005-08-
24]
[25] United Nations, U.N. Establishes New Emergency Fund, December 16, 2005.
[http://www.irinnews.org]
[26] See FAO avian influenza home page at [http://www.fao.org/ag/againfo/subjects/en/
health/diseases-cards/special_avian.html].
[27] See OIE avian flu home page at [http://www.oie.int/eng/AVIAN_INFLUENZA/
home.htm]
[28] Interview with FAO official, October 31, 2005.
[29] World Bank Press Release, New Global Program to Deal with Avian Flu. November
4, 2005. [http://www.worldbank.org]
[30] UN News Service, Bird flu: UN-sponsored conference draws up six-point action
plan. November 9, 2005. [http://www.un.org/apps/news/]
[31] The World Bank Group, Partners Meeting on Avian Influenza and Human Pandemic
Influenza. [http://www.worldbank.org]
[32] UN News Service, Bird flu: UN-sponsored conference draws up six-point action
plan. November 9, 2005. [http://www.un.org/apps/news/]
[33] The American Society of International Law, SARS and International Law, April 2003,
see [http://www.asil.org/insights].
[34] The revised International Health Regulations, approved by the World Health Assembly
on May 23, 2005, are available at [http://www.who.int/csr/ihr/en/].
[35] If a State makes a reservation that is compatible with the object and purpose of IHR
(2005) and at least one-third of other States have not objected to the reservation within
six months of notification, the revised IHR will enter into force for that State, subject to
its reservation. See WHO, Frequently Asked Questions About IHR, at
[http://www.who.int/csr/ihr/howtheywork/faq].
[36] See WHO, Frequently Asked Questions About IHR, at [http://www.who.int/csr/ihr/

howtheywork/faq].
[37] The Front Lines in the Battle Against Avian Flu Are Running Short of Money, New
York Times. October 9, 2005.
[38] OIE, Update on Avian Influenza in Animals, January 9, 2006. [http://www.oie.int/]
[39] This section prepared by Thomas Lum, Specialist in Asian Affairs, 7-7616.
[40] U.S., Cambodian Health Ministers Sign Deal on Bird Flu Cooperation, Agence
France Press. October 11, 2005.
[41] Cambodia Needs $18 Million for Bird Flu Fight UN, Reuters, December 16,
2005.
[42] This section was prepared by Kerry Dumbaugh, Specialist in Asian Affairs, 7-7683.
[43] As of January 1, 2006, there were 7 confirmed cases of avian flu and 3 deaths in China.
[44] For more on SARS Severe Acute Respiratory Syndrome see CRS Report
RL32227, SARS, Avian Flu, and other Challenges for Chinas Political, Social, and
Economic Transformation.
[45] According to the U.S. Centers for Disease Control and Prevention. See website at
[http://www.cdc.gov/flu/avian/outbreaks/asia.htm].
[46] Beijing Liaowang in Chinese. Translated on September 26, 2005, in FBIS,
CPP20051018050001.
[47] PRC Ministry of Health, Preparations and Plan for an Influenza Pandemic
Emergency, September 28, 2005, translated in FBIS, CPP20051012335002 (October
12, 2005).
[48] Cody, Edward, China to vaccinate billions of birds; campaign aims to stem avian flu,
Washington Post, November 16, 2005, p. A15.
[49] Fountain, Henry, How to vaccinate 14 billion birds, The New York Times, November
20, 2005, p. 2.
[50] Sipress, Alan, bird flu experts warn against bad vaccines; improper poultry inoculation
may spread virus, Washington Post, November 22, 2005, p. A24.
[51] McNeil, Donald Jr., Health experts fear Chinese flu vaccination plan could backfire,
The New York Times, November 20, 2005, p. 12.
[52] Wright, Tom, Roche to Let Chinese Producer Make Flu Drug. New York Times,
December 13, 2005.
[53] The independent virology team was from the University of Hong Kong and included
Dr. Guan Yi, a co-author of the scientific report published in Nature magazine on July
7, 2005. For reference to PRC official Jia Youlings comments, see Sipress, Alan,
China has not shared crucial data on bird flu outbreaks, officials say, in the
Washington Post, July 19, 2005, p. A15.
[54] Washington Post, June 18, 2005, p. A01. Some sources also have suggested that the
virus apparent new resistance to known drugs may be the result of renegade
pharmaceutical labs in China dispensing the wrong anti-viral medications, raising
additional questions about the PRC governments ability to exert control over a
potential pandemic. International Herald Tribune, July 5, 2005, p. 3.
[55] King Jr., Neil, Hu pledges efforts to ease U.S. strains , Asian Wall St. Journal,
September 15, 2005, p. A1.
[56] This section prepared by Bruce Vaughn, Analyst in Asian Affairs, 7-3144.
[57] Indonesia Set to Form National Commission for Bird Flu Control, Thai News
Service. January 10, 2006.
[58] Indonesia Reduces Confirmed Bird Flu Death Toll to 3 from 6, AFX Asia. October 6,
2005.
[59] WHO, Confirmed Human Cases of Avian Influenza A (H5N1). Accessed on December
30, 2005. [http://www.who.int/csr/disease/avian_influenza/country/en/]
[60] Toll UP but Indonesia Ready for Bird Flu, ISI Emerging Markets. January 2, 2006.
[62] Indonesia Calls for Intl Cooperation to Combat Bird Flu, Dow Jones Newswire. Sept.
30, 2005.
[63] Alan Sipress, Indonesia Warns of Possible Bird Flu Epidemic, Washington Post.
September 21, 2005.
[64] Indonesia Making Progress in Fight Against Bird Flu - WHO, AFX Asia. September
30, 2005.
[65] Phelim Kyne and Fitri Wulandari, Indonesian Poultry Cos Hobble Avian Flu Control
Ops, Dow Jones Newswires. October 5, 2005.
[66] Reuters, Indonesia says bird flu outbreak may become epidemic. September 21,
2005. [http://www.alertnet.org].
[68] Nicholas Zaminska, Asian Nations Start Critical Bird Flu Preparations, The Wall
Street Journal. October 3, 2005.
[69] Eaton, Dan and Telly Nathalia, Indonesia says bird flu outbreak may become
epidemic. Reuters. September 21, 2005. [http://www.alertnet.org/thenews/newsdesk/
JAK58836.htm].
[70] Alan Sipress, Indonesia Neglected Bird Flu Until Too Late, The Washington Post,
October 20, 2005.
[71] This section prepared by Thomas Lum, Specialist in Asian Affairs, 7-7616.
[72] WHO Urges Laos to Prepare for Deadly Human Version of Bird Flu, Agence France
Presse, August 27, 2005.
[73] U.S. Government Emergency Response to Avian Influenza: A Plan for Vietnam, Laos
and Cambodia: Report from Country Planning Visits, July 2005.
[74] WHO Urges Laos to Prepare for Deadly Human Version of Bird Flu, Agence France
Presse. August 27, 2005
[75] Intl Donors Pledge More Than $17M to Help Southeast Asia Combat Bird Flu,
Forbes.com. October 13, 2005.
[76] This section prepared by Jim Nichol, Specialist in Russian and Central Asian Affairs,
7-2289.
[77] The eight administrative areas are the Astrakhan, Chelyabinsk, Kurgan, Novosibirsk,
Omsk, and Tyumen oblasts (regions), the Kalmyk republic, and the Altay kray
(territory).
[78] World Health Organization. Geographical Spread of H5N1 Avian Influenza in Birds:
Situation Assessment and Implications for Human Health, Update 28, August 18, 2005.
[79] Agence France Presse, August 22, 2005; Foreign Broadcast Information Service
(FBIS), September 3, 2005, Doc. No. IAP-11012.
[80] The Lancet, August 27-September 2, 2005, p. 689; Interfax, October 4, 2005.
[81] The Lancet, August 27-September 2, 2005, p. 689.
[82] FBIS, August 18, 2005, Doc. No. CEP-19027.
[83] FBIS, September 13, 2005, Doc. No. CEP-346004.
[84] ITAR-TASS, October 23, 2005; ITAR-TASS, October 27, 2005; FBIS, October 24, 2005,
Doc. No. CEP-346001; December 6, 2005, Doc. No. CEP-346002.
[85] FBIS, September 7, 2005, Doc. No. CEP-27067.
[86] This section prepared by Emma Chanlett-Avery, Analyst in Asian Affairs, 7-7748.
[87] WHO, Cumulative Number of Confirmed Human Cases of Avian Influenza A/H5N1
Reported to WHO, December 7, 2005. [http://www.who.int/csr/disease/
avian_influenza/country/en/].
[88] Thai Authorities Should Take Strong Action Against Bird Flu, Bangkok Post
editorial. October 4, 2005.
[89] Bird Flu: Asian Contagion? Economist Intelligence Unit. July 25, 2005.
[90] Thailand Has First Avian Flu Outbreaks in 3 Months, CIDRAP News at
[http://www.cidrap.umn.edu]. July 11, 2005.
[91] Thai Public Health Minister Announced that Thailand Can Produce Oseltamivir, Thai
News Service. December 8, 2005.
[92] Squawking at the Bird Flu Warning, Los Angeles Times. September 1, 2005.
[93] Thailand Cracks Bird Flu Vaccine Smuggling Syndicate, BBC Monitoring Asia
Pacific. September 20, 2005.
[94] Avian Flu - Fresh Outbreak, Bangkok Post. July 14, 2005.
[95] Squawking at the Bird Flu Warning, Los Angeles Times. September 1, 2005.
[96] Ministry cuts Deal of Fighting Cock Zoning, Bangkok Post. July 15, 2005.
[97] Thailand Proposes Regional Bird Flu Control Center, Thai News Service. September
27, 2005.
[98] Bird Flu Pandemic Risk Very High, CNN.com. October 11, 2005.
[99] U.S. Government Emergency Response to Avian Influenza: A Plan of Action for
Vietnam, Laos, and Cambodia - Report from Country Planning Visits July 11-24, 2005.
United States Agency for International Development.
[100] This section prepared by Emma Chanlett-Avery, Analyst in Asian Affairs, 7-7748.
[101] WHO, Cumulative Number of Confirmed Human Cases of Avian Influenza A/H5N1
January 7, 2006. [http://www.who.int/csr/don/2006_01_07/en/index.html]
[102] Bird Flu Spreads Across Turkey, CNN News Online. January 9, 2006.
[103] This section prepared by Mark Manyin, Specialist in Asian Affairs, 7-7653.
[104] USAID, U.S. Government Emergency Response to Avian Influenza: A Plan of Action
for Vietnam, Laos and Cambodia. Report from Country Planning Visits, July 11-24,
2005.
[105] See, for instance, Nicholas Zamiska, Pandemic Watch: Inside U.N. Agency, Flu Data
Sparked A Tense Debate, The Wall Street Journal, October 18, 2005.
[106] See, for instance, Adrian Levy and Cathy Scott-Clark, Flu on the Wing, The
Guardian, October 15, 2005.
[107] For more information on these issues see CRS Report RL31145, Pandemic Influenza:
Domestic Preparedness Efforts, by Mark Gurevitz.
[108] Fedson, David, Preparing for Pandemic Vaccination: An International Policy Agenda
for Vaccine Development. Journal of Public Health Policy 2005, Volume 26, Issue 1,
April 2005. p.11.
[109] Harris, Gardiner, Officials May Spend Billions to Stockpile Influenza Drug. New
York Times, October 7, 2005.
[110] Ibid.
[111] Alonso-Zaldivar, Ricardo, Roche agrees to generic version of Tamiflu drug.
Baltimore Sun, October 21, 2005. [http://www.baltimoresun.com].
[112] H.R. 4392, To provide for the importation of pharmaceutical products under a
compulsory license as provided for under the World Trade Organization.
[113] McNeil Jr., Donald, Indian Company to Make Generic Version of Flu Drug Tamiflu.
New York Times. October 14, 2005. [http://www.nytimes.com].
[114] Jack, Andrew, India drugs groups in Tamiflu talks. Financial Times. December 12,
2005. [http://news.ft.com/home/us/]
[115] Wright, Tom, Roche to Let Chinese Producer Make Flu Drug. New York Times.
December 13, 2005. [http://nytimes.com]
[116] WTO, Members OK amendment to make health flexibility permanent. December 6,
2005. [http://www.wto.org/english/news_e/pres05_e/pr426_e.htm]
[117] Article 31(f) of the TRIPS Agreement says that production under compulsory licensing
must be predominantly for the domestic market. The concern was that this could limit
the ability of countries that cannot make pharmaceutical products from importing
cheaper generics from countries where pharmaceuticals are patented.
[118] For more information on this issue see CRS Report RS21609, The WTO, Intellectual
Property Rights, and the Access to Medicines of Controversy, by Ian F. Fergusson.
[119] WTO, Members OK amendment to make health flexibility permanent. December 6,
2005. [http://www.wto.org/english/news_e/pres05_e/pr426_e.htm]
[120] H.R. 4392, To provide for the importation of pharmaceutical products under a
compulsory license as provided for under the World Trade Organization.
[121] Chang, Alicia, Bird Flu Victims Die After Drug Resistence. Washington Post.
December 21, 2005. [http://www.washingtonpost.com]
[122] Rosenthal, Elisabeth, Two Studies Find Flu Treatments Fall Far Short. September 22,
2005. [http://www.nytimes.com].
[123] Zamiska, Nicholas, Scientists Says Bird-Flu Virus Appears to Be Stable in China; No
Signs that Avian Strain Is Easily passed by People; Old Drug Shows Promise.
December 12, 2005.
[124] Globe and Mail, Chinese officials havent shared samples of H5N1: experts.
November 18, 2005. [http://www.theglobeandmail.com].
[125] China Daily, China: Bird flu vaccine for human use developed. November 15, 2005.
[http://www.chinadaily.com.cn/english/doc/2005-11/15/content_494593_3.htm].
[126] Honorable Bill Frist website, First Addresses National Press Club on Avian Flu
Pandemic: The Economy Killer. December 8, 2005. The Senate passed S. 2170 on
December 22, 2005.
[127] Interview with USAID official, October 11, 2005.
[128] Ibid.
[129] Reuters, Bird Flu in Africa Could Swamp Health Systems: WHO. November 27,
2005. [http://www.nlm.nih.gov/medlineplus/news/fullstory_27731.html]
[130] UN agency says risk of bird flu spreading to Middle East, Africa rises markedly.
U.N. News Center, October 19, 2005. [http://www0.un.org/apps/news/story.asp?
NewsID=16037andCr=birdandCr1=flu]
[131] Reuters Foundation, ETHIOPIA: Birds Test Negative for avian flu. December 20,
2005. [http://www.alertnet.org]
[132] SAID, H5N1 Avian Influenza (AI) Most Recent Developments and Actions. November
26 - December 19, 2005.
[133] Grudgings, Stuart, Rich-poor divide hoblles Asias bird flu plans. Reuters, September
13, 2005. [http://www.reuters.com]
[134] GovEXEC.com, House panel calls plan for tracking avian influenza inadequate.
December 7, 2005. [http://govexec.com/dailyfed/1205/120705cdpm2.htm]
[135] Grudgings, Stuart, Rich-poor divide hobbles Asias bird flu plans. Reuters.
September 13, 2005. [http://www.reuters.com]
[136] ASEAN Plus Three consists of the ASEAN countries plus China, Japan, and South
Korea.
[137] Participants in the first EAS included the ten ASEAN members (Brunei, Burma,
Cambodia, Indonesia, Laos, Malaysia, Philippines, Singapore, Thailand, and Vietnam),
the plus three states (China, South Korea, and Japan), as well as Australia, New
Zealand, and India. For more on the summit, see CRS Report RS22346, East Asian
Summit: Issues for Congress, by Bruce Vaughn.
[138] Ilaria Capua and Stafano Manganon, Vaccination for Avian Influenza in Asia,
Vaccine, Vol. 22, 2004, pp. 4137-4138.
[139] Perry, Michael, Poor Asian farmers are weak link in bird flu fight. Reuters.
September 19, 2005. [http://www.alertnet.org/thenews/newsdesk/SYD28137.htm]
[140] World Bank, New Global Program to Deal with Avian Flu. November 4, 2005.
[http://www.worldbank.org]
[141] ADB, ADB Support for Asias Fight Against Avian Flu Could Reach $470 Million.
November 9, 2005. [http://www.adb.org/Documents/News/2005/nr2005173.asp]
[142] Osterholm, Michael, Preparing for the Next Pandemic. Foreign Affairs, July/August
2005. [http://www.foreignaffairs.org].
[143] Ibid. Country-specific SARS-related information, including costs and fatalities can be
found in CRS Report RL32072, Severe Acute Respiratory Syndrome (SARS): The
International Response, by Rhoda Margesson and Tiaji Salaam.
[144] Nordland, Rod and George Wehrfritz, A Costly Disease: Europe and the rest of the
world braces for the economic fallout of a possible bird-flu pandemic. October 24,
2005. [http://msnbc.msn.com/id/9711926/site/newsweek/]
[145] World Bank East Asia and Pacific Region, Spread of Avian Flu Could Affect Next
Years Economic Outlook. November 2005. [http://siteresources.worldbank.org/I
NTEAPHALFYEARLYUPDATE/Resources/EAP-Brief-avian-flu.pdf]
[146] U.S. Trade Representative Robert Portman discussion at the US-ASEAN Business
Council Second Annual Asia Forum, November 1, 2005.
[147] USTR website, Americas Trade with China. April 21, 2004. [http://www.ustr.gov]
[148] USTR website, Real Results in U.S. Trade with China. September 9, 2004.
[http://www.ustr.gov]
[149] Bullion, Alan, Threats on the Wing. The World Today, August/September 2005.
Also see, Bradsher, Keith, Some Asian Bankers Worry About the Economic Toll
From Bird Flu. New York Times. April 5, 2005. [http://www.nytimes.com]
[150] CBO, A Potential Influenza Pandemic: Possible Macroeconomic Effects and Policy
Issues. December 8, 2005. [http://www.cbo.gov/ftpdocs/69xx/doc6946/12-08-
BirdFlu.pdf]
[151] Kilman, Scott, Vaccine Remains Sticking Point in U.S. Defense Against Bird Flu.
December 12, 2005. [http://www.wsj.com]. Basic screening tests for bird flu used by
many importing countries leave ambiguous whether a bird testing positive is infected
with H5N1 or has been vaccinated against it.
[152] Kolata, Gina, Experts Unlock Clues to Spread of 1918 Flu Virus. New York Times,
October 6, 2005. [http://www.nytimes.com]
[153] Ibid.
[154] CDC, Possession, Use, and Transfer of Select Agents and Toxins Reconstructed
Replication Competent Forms of the 1918 Pandemic Influenza Virus Containing Any
Portion of the Coding Regions of All Eight Gene Segments. 70 Federal Register
61407, October 20, 2005.
[155] For more information, see the CDC Select Agent Program page at
[http://www.cdc.gov/od/sap] and CRS Report RL31719, An Overview of the U.S.
Public Health System in the Context of Emergency Preparedness, by Sarah A. Lister.
[156] Altman, Lawrence and Keith Bradsher, Vaccine Alone Wont Stem Avian Flu,
Experts Warn. New York Times. August 8, 2005. [http://www.nytimes.com]
[157] Interview with State Department staff, October 18, 2005.
[158] Abbott, Alison, The flu HQ. Nature, Volume 414, November 1, 2001.
[http://www.nature.com]
Chapter VIII
Avian Influenza:
Agricultural Issues*
Jim Monke
Summary
Since the fall of 2003, a strain of highly pathogenic avian influenza (H5N1) has spread
throughout Asia, infecting mostly poultry but also a limited number of humans. In recent
months, the virus has spread into parts of Europe. Controlling avian flu in poultry is seen as
the best way to prevent a human pandemic from developing, by reducing the number of
animal hosts in which the virus may evolve.
Avian flu can be highly contagious in domestic poultry. Strict biosecurity measures are
practiced among commercial poultry farms and are encouraged by governments. The
economic effects of any avian influenza outbreak can be significant, especially given
international trade restrictions. This report will be updated as events warrant.
Status of Avian Influenza Outbreaks
In the United States
The highly pathogenic H5N1 strain of current global concern has not reached the United
States, neither in poultry nor humans. (This report primarily addresses avian flu in poultry,
although some human dimensions are discussed.) The most recent cases in domestic poultry
were in 2004, with three unrelated and less pathogenic strains.
*
This is an edited, reformatted and augmented version of a Congressional Research Service publication, RS21747,
dated November 10, 2005.
160 Jim Monke
To reduce the possibility that H5N1 enters U.S. borders, the U.S. Department of
Agriculture (USDA) has blocked imports of poultry and poultry products from affected
countries. The Department of Homeland Security helps with enforcement through Customs
and Border Protection. Surveillance of migratory birds is increasing.[1]
In the Rest of the World
Since December 2003, as many as nine Asian countries have had confirmed outbreaks or
instances of H5N1 in poultry, including Vietnam, Thailand, Indonesia, Cambodia, China and
Hong Kong, South Korea, Malaysia, Laos, and Japan. More recently, in the summer and fall
of 2005, H5N1 spread westward and has been confirmed in at least five new countries:
Russia, Kazakhstan, Turkey, Romania, and Croatia. Wild birds seem to be one of the main
carriers, but their role in spreading the virus is not completely understood. The risk and
likelihood of the virus spreading into Africa and the Middle East is increasing. Other
countries on migratory bird routes are increasing surveillance efforts.
As the virus spreads, and becomes endemic in countries with low levels of veterinary
services or animal husbandry practices that harbor the virus, the chances increase that the
virus will evolve through mutation or reassortment into a strain that could be transmitted
easily between humans. Thus, many experts call for the swift and coordinated control of
avian flu in poultry as the best way to prevent a human pandemic from developing, by
reducing the number of animal hosts in which the virus may evolve.[2]
The situation in Asia is historically unprecedented and extremely challenging. The
United Nations Food and Agriculture Organization (FAO) estimates that over 130 million
birds have died or been culled in Asia. Some countries were reluctant to acknowledge the
disease for fear of economic consequences. In other countries, lack of compensation for
farmers whose flocks are destroyed has been a disincentive to report outbreaks early. In some
parts of Asia, about 80% of the poultry are produced in small backyard farms scattered
throughout rural areas, further complicating control.
Two Forms with Many Strains
Avian influenza (AI) viruses exist throughout the world in many different strains. Avian
flu is an Influenza A virus that infects birds, and certain strains have been known to infect
both animals and humans. Avian flu is characterized by two forms in birds:
a low pathogenicity (LPAI) form that causes mild illness, and

a highly pathogenic (HPAI) form that is extremely contagious, causes severe illness,
and frequently has high rates of mortality.[3]
Both forms are possible in several strains. Strains are identified by two surface proteins
designated by the letters H and N.[4] Some low pathogenic strains (H5 and H7) are capable
of mutating into highly pathogenic strains, and are thus treated nearly as aggressively. For
Avian Influenza: Agricultural Issues 161
example, during a 1999-2001 epidemic in Italy, an LPAI virus (H7N1) mutated into HPAI
within nine months.
Because LPAI is endemic in wild bird populations, low pathogenicity outbreaks are not
uncommon. The 2004 outbreaks in the United States included low pathogenicity strains of
H7N2 in Delaware, Maryland, and New Jersey, and H2N2 in Pennsylvania. A strain
classified as highly pathogenic H5N2 was found in Texas, although it did not manifest as
highly pathogenic. Other recent outbreaks in U.S. poultry include low pathogenicity H7N2 in
Connecticut and Rhode Island in 2003, and in Virginia, West Virginia, and North Carolina in
2002. There have been only three highly pathogenic outbreaks in the United States (1924,
1983, and 2004).
Transmission
Wild birds are the primary natural reservoir for Influenza A viruses and are often the
vector that introduces new outbreaks into domestic flocks. Wild birds often are resistant to
the virus and do not show clinical symptoms. The role of migratory birds is of increasing
concern, although, in the past, scientists have not been sure that infected birds were able to
migrate long distances.
Avian flu can be highly contagious in domestic poultry. The virus is spread by contact
with infected feces, nasal, or eye excretions. Once present in domestic flocks, human activity
becomes a risk for further transmission as people, clothing, vehicles, and supplies move
between farms. Thus, strict biosecurity measures are practiced among commercial poultry
farms and are encouraged by USDA and international agricultural organizations such as the
FAO.[5]
In the United States, avian flu viruses have been common in live bird markets
concentrated in urban areas with ethnic communities. Biosecurity practices can often be
lacking or insufficient if birds and equipment intermingle in the market or move back to
farms. Thus sanitation of crates, periodic disinfection of the market, and restrictions on
moving birds back into general farm populations are needed. USDA has focused on these
markets as one of the first places to control the disease. Live bird markets are a small portion
of the U.S. poultry industry (about 1/4 of 1%), but the frequency of outbreaks is of concern to
the majority of commercial growers practicing tighter biosecurity protocols. In Asia, a larger
network of live bird markets and the much larger number of small backyard farms have posed
significant problems for eradicating the disease.
Human Infection
Avian flu can infect humans through poultry-to-human transmission, usually through
contact with fecal matter or other live bird excretions. The World Health Organization
(WHO) and the World Organization for Animal Health (OIE) conclude that avian flu is not a
food-borne disease since the virus is killed by the temperature reached in normal cooking.
162 Jim Monke
The Centers for Disease Control and Prevention (CDC) recommends standard food safety
practices.
The human disease caused by H5N1 differs from typical human flu. H5N1 can replicate
in a wide range of cells, more so than the usual flu virus. This can result in a severe
disseminated disease affecting multiple organs, which has caused high rates of mortality. The
human vaccine currently available for mass inoculation in the fall of 2005 is felt to offer little
protection against H5N1; vaccine trials and development are underway. Public health
professionals are concerned that the virus could mutate or combine with human flu viruses. If
such a mutation were to occur, allowing efficient human-to-human transmission, a more
serious public health problem would result.
The number of human cases of H5N1 confirmed by WHO during the current outbreak
(December 2003-November 9, 2005) totals 125, resulting in 64 deaths (a 51% mortality rate).
Four countries have had human cases: Vietnam (92 cases, 42 deaths), Thailand (20 cases, 13
deaths), Indonesia (9 cases, 5 deaths), and Cambodia (4 cases and 4 deaths). Some scientists
believe that if the virus evolves to allow human-to-human transmission, the mortality rate
may decline, but whether this happens remains unknown.
The first human cases of H5N1 were in Hong Kong in 1997 (18 cases, 6 deaths). Two
other strains are documented to cause human illness: H7N7 in the Netherlands in 2003 (83
cases, 1 death), and H9N2 in Hong Kong in 1999 and 2003 (3 cases).
In the United States, the 2002 low pathogenic outbreak in poultry in Virginia resulted in
limited evidence of one human case. A man involved in the poultry depopulation effort was
found to have antibodies for H7N2 avian flu. In the fall of 2003, a man from Westchester
County, New York, contracted and recovered from H7N2 avian flu. The case was not initially
diagnosed as avian flu, and CDC first confirmed diagnosis in April 2004.
Control
Controlling avian flu in poultry through prevention and eradication is done domestically
by individual farmers in cooperation with state and federal governments, and with industry
associations and international organizations. In the United States, the USDA Animal and
Plant Health Inspection Service (APHIS) is the lead federal agency.
Internationally, the U.N. Food and Agriculture Organization (FAO) has a joint response
plan with WHO for the current outbreak. The $140 million, three-year plan is being
implemented but is not fully funded by donor countries. The United States has contributed
about $25 million.[6]
Preventing Infection
Biosecurity practices are the most important means of preventing outbreaks in poultry.
This includes preventing access of wild birds to domestic flocks and limiting access to farm
buildings by outside conveyances. For example, delivery trucks and personnel are cleaned
and disinfected before entering a farms biosecure area. In Asia and other parts of the world,
the large number of small farms or backyard flocks without biosecurity practices has posed
greater problems for control. Such animal husbandry practices are slow to change.
Eradicating Outbreaks
Because the virus is highly contagious and easily spread in poultry, the most common
method of control after there is an outbreak is culling (also called stamping out,
depopulating) the infected flocks, and certain flocks in close proximity to the infected flock.
Federal statute allows such destruction of animals (9 CFR 53.4). Quarantines of surrounding
areas are imposed (usually by state authorities) until the disease is eradicated. Following
depopulation, buildings and equipment are rigorously disinfected before new birds are
allowed, a process that takes at least several weeks. The virus is killed by common
disinfectants or heat (about 160 degrees F).
Vaccines
While vaccination of poultry is possible and has been used on a small scale with some
success, it generally is not considered a viable or sufficient control method. Vaccination
poses problems for international trade as many countries will not import Poultry products
from other countries that use vaccination as a means of control, since animals will test
positive for antibodies. If vaccination is not administered and monitored correctly, it can also
allow the virus to become endemic and continue to spread or mutate.[7]
In November 2005, USDA has a stockpile of 40 million doses of vaccine (for two types
of H5 and two types of H7 viruses). The Administrations recent funding request for avian flu
(discussed below) includes a proposal to double USDAs stockpile.
Federal Response to Domestic Outbreaks

Domestic outbreaks usually are managed through joint federal, state, and industry
cooperation. States usually lead the response in terms of depopulation and quarantines.
APHIS provides personnel and equipment to advise and supplement state resources. In highly
pathogenic outbreaks, APHIS may take a larger role. The USDA National Veterinary
Services Lab (NVSL) in Ames, IA, conducts confirmatory tests on the pathogenicity and type
of virus. USDA also works to limit export restrictions to small geographic areas (such as
states or counties) and reopen export markets once outbreaks are eradicated.
Indemnities to Farmers
Compensation programs are desired to encourage farmers to report outbreaks and
cooperate with disease control programs. Indemnification programs for low pathogenicity
outbreaks generally are managed by the states. Some industry associations, such as those on
the Delmarva penninsula (Delaware, Maryland, and Virginia), have compensation funds. In
the past, USDA has not had a standing compensation program for low pathogenicity avian
influenza.[8] However, a new program is being developed following increased appropriations
for a low pathogenicity program in FY2005. When indemnification is offered by USDA, the
standard rate for low pathogenicity programs is 50% of fair market value. For highly
164 Jim Monke
pathogenic outbreaks of avian flu, statute allows USDA to offer 100% indemnification (9
CFR 53.2).
Economic Impacts
The economic effects of any avian influenza outbreak can be significant. Expenses to
conduct depopulation and quarantines, as well as the direct loss of production, affect local
farms and regions. However, bigger economic effects come from international trade bans.
Localized quarantines and bans on the sale or movement of birds can affect farmers outside
the immediate quarantine area.
The United States is the worlds largest producer and exporter of poultry meat and the
second-largest egg producer. USDA estimates that about 8.5 billion broilers were produced in
2003, and total poultry production was worth $23.3 billion (out of $105 billion for all
livestock, and $200 billion total of crops and livestock). Broiler production was valued at
$15.2 billion, followed by eggs at $5.3 billion, and turkeys at $2.7 billion. The U.S. exports
about 16% of its poultry production.[9]
No estimates of the potential effect from an H5N1 outbreak in the United States are
available because of the highly uncertain nature of any possible, hypothetical outbreak. The
1983-84 outbreak of highly pathogenic avian flu in the United States caused the destruction
of 17 million birds and cost $65 million. In the small 2004 domestic outbreak, about 400,000
chickens were depopulated in the United States. This was less than 1/200 of 1% of the 8.5
billion broilers slaughtered in the U.S. for food annually. Yet, the effect on local regions and
individual farms can was much greater.
Federal Appropriations to Control

Avian Flu in Poultry
Federal appropriations for avian influenza have grown significantly in recent years. In
FY2004, Congress provided APHIS with $994,000 for avian flu for monitoring and control.
Following the 2004 domestic outbreak, USDA used emergency authority to release $13.7
million of Commodity Credit Corporation (CCC) funds to accelerate its avian flu plans. In
FY2005, Congress appropriated APHIS $23.8 million for avian flu, with about half for
indemnities. For FY2006, the APHIS appropriation for avian flu is $13.8 million. The
conference agreement for agriculture appropriations (H.R. 2744, H.Rept. 109-255) notes that
$28.3 million is available, including carryover, with about $12 million for indemnities.
The Emergency Supplemental Appropriations Act of 2005 (P.L. 109-13) provided $25
million to the U.S. Agency for International Development (USAID) and CDC to combat the
spread of avian flu. Conferees encourage U.S. cooperation to support FAO and WHO on a
joint international plan (the FAO/WHO plan mentioned above).
On November 1, 2005, President Bush submitted a request to Congress for $7.1 billion in
emergency funding to address avian flu in both humans and poultry. Of this amount, $91
million would go to USDA ($73 million to APHIS for domestic activities, $7 million to the
Agricultural Research Service, and $11 million for international activities in the form of
technical assistance on surveillance, biosecurity, culling, vaccination, and control).
References
[1] For domestic issues related to avian flu in poultry, see the U.S. Department of
Agriculture (USDA) at [http://www.aphis.usda .gov/lpa/issues/avian_ influenza]. For
background on human issues, see the Centers for Disease Control (CDC) at
[http://www.cdc.gov /flu/avian].
[2] International organizations include the U.N. Food and Agriculture Organization (FAO)
[http://www.fao.org/ag/againfo/subjects/en/ health/diseases-cards/special_avian.html],
the World Health Organization (WHO) [http://www.who.int/csr/disease/avian_
influenza/en], and the World Organization for Animal Health (OIE)
[http://www.oie.int/eng/avian_influenza].
[3] Tests for pathogenicity are conducted in two ways. The first is through genetic (DNA)
sequencing. The other is by inoculating healthy chickens and monitoring their immune
response and mortality over a 10-day period. HPAI strains can result in greater or lesser
rates of mortality, perhaps ranging from 30-100%. LPAI strains typically do not exceed
10-20 percent mortality.
[4] The surface proteins are called hemagglutinin and neuraminidase, abbreviated H and N.
Fifteen H subtypes and nine N subtypes have been identified, and they can occur in any
combination.
[5] For biosecurity recommendations, see the USDA Biosecurity for the Birds website at
[http://www.aphis.usda.gov/vs/birdbiosecurity/hpai.html].
[6] A Global Strategy for the Progressive Control of Highly Pathogenic Avian Influenza
(HPAI), U.N. Food and Agriculture Organization (FAO) and World Organization for
Animal Health (OIE), in cooperation with the World Health Organization (WHO),
November 2005 [http://www.fao.org/ag/againfo/subjects/documents/ai/ HPAIGlobal
Strategy31Oct05.pdf].
[7] See two journal articles by scientists at the World Organization for Animal Health
(OIE): Ilaria Capua and Stephano Marangon, Vaccination for avian influenza in Asia,
Vaccine, 22 (2004), 4137-7138 [http://www.oie.int/eng/avian_influenza/ vaccination%
20in%20Asia.pdf], and Ilaria Capua and Stephano Marangon, The use of vaccination
as an option for the control of avian influenza, Technical Item of the 71st General
Session of the OIE, May 2003, [http://www.oie.int/eng/avian_influenza/
A_71%20SG_12_CS3E.pdf].
[8] A limited USDA indemnification program was created for an LPAI outbreak in
Virginia in 2002 (9 CFR 53.11). The Administrations FY2005 budget request includes
a proposal for an LPAI indemnification program.
[9] The top five states in broiler production are Georgia (15%), Arkansas (14%), Alabama
(13%), Mississippi (9%), and North Carolina (9%), totaling 60% of U.S. broiler
production.
Chapter IX
Potential Risks of Vaccination against

Avian Flu Epidemics
Shingo Iwami* and Yasuhiro Takeuchi

Graduate School of Science and Technology, Shizuoka University, Japan
Abstract
Highly pathogenic H5N1 influenza A viruses have spread relentlessly across the
globe since 2003. They are associated with widespread death of poultry, substantial
economic loss to farmers, and reported infections of more than 300 people with a
mortality rate of 60%. Influenza prevention and containment strategies can be considered
under the broad categories of antiviral, vaccine, and non-pharmaceutical measures. In
particular, using vaccination to reduce the transmission rate might provide an alternative
to mass culling by reducing both the susceptibility of healthy birds and the infectiousness
of infected birds. However, although vaccination can be a useful tool for control of avian
influenza epidemics, it might engender the emergence of a vaccine-resistant strain. Field
and experimental studies show thatsome avian influenza strains acquire resistance against
vaccination. We investigated, in the context of the emergence of a vaccine-resistant
strain, whether a vaccination program can prevent the spread of infectious disease. Our
main findings are that such a program might lead to an emergence and replacement of the
vaccine-resistant strain over a large geographical region, and that a vaccination to
prevent the spread of disease can instead spread the disease. Thus, if the vaccinations are
not used appropriately, prevention and control will be negatively affected by the
vaccination program. Further, from our theoretical studies, we propose how a vaccination
against avian influenza should be used.
*
E-mail address: yukitadahara@yahoo.co.jp
168 Shingo Iwami and Yasuhiro Takeuchi
Keywords: Avian influenza, Vaccination, Resistant strain, Mathematical model
1. Introduction
Although small in size and simple in structure, influenza viruses are sophisticated organ-
isms with highly mutagenic genomes and wide antigenic diversity [36]. Mutation and re-
assortment have resulted in newer viruses such as H5N1, with new resistance against anti-
viral medications, and this might lead to the emergence of a human-to-human transmissible
strain, as occurred in the 1918, 1957 and 1968 pandemics [11, 21, 31, 36, 45]. With the
emergence of the H5N1 strain, which is now widespread in Southeast Asia and which dif-
fused recently in some area of the Balkan region and Western Europe, the threat of an
influenza pandemic seems to be real and inevitable, but no one can predict where and when
it might happen. A major public health concern is establishing a plan for the next influenza
pandemic, yet it remains unclear how to control such a crisis.
Vaccination of domestic poultry against the H5N1 subtype of avian influenza has been
used in several countries such as Pakistan, Hong Kong, Indonesia, China, and Vietnam
[6, 25, 42]. Using vaccination to reduce the transmission rate might provide an alternative
to mass culling by reducing both the susceptibility of healthy birds and the infectiousness
of infected birds [4, 5, 6]. However, incomplete protection at the bird level can cause the
silent spread of the virus within and among birds [34]. Further, vaccines might provide
immunological pressure on the circulating strains, which might engender the emergence
of drifted or shifted variants with enhanced potential for transmissibility in humans [11].
Therefore, although vaccination programs have been recommended recently, some field
evidence indicates that vaccination alone will not achieve eradication [22, 29, 30, 37]. To
contain avian influenza infections effectively, vaccination should only be used as part of
a comprehensive control strategy that also includes biosecurity, quarantine, surveillance,
education, and elimination of infected and at-risk birds [25].
An important issue related to influenza epidemics is the potential for the emergence of
vaccine-resistant influenza viruses. The vaccine-resistant strain, in general, causes a loss
of protection effectiveness of vaccination [22, 29, 30, 37] (there is experimental evidence
of the loss of protection effectiveness for antiviral-resistant strains [14]). Consequently, a
vaccination program that engenders the emergence of the resistant strain might promote the
spread of the resistant strain and undermine the control of the infectious disease even if the
vaccination protects against the transmission of a vaccine-sensitive strain [29, 30, 37]. For
example, in China, despite a compulsory program for the vaccination of all poultry com-
mencing in September 2005, the H5N1 influenza virus caused outbreaks in poultry in 12
provinces from October 2005 to August 2006 [6, 25, 37]. Genetic analysis revealed that an
H5N1 influenza variant (Fujian-like, FJ-like), which is a previously uncharacterized H5N1
virus sublineage, had emerged and subsequently became the prevalent variant in each of the
provinces, replacing those previously established multiple sublineages in different regions
of southern China. Some data suggest that the poultry vaccine currently used in China might
only generate very low neutralizing antibodies to FJ-like viruses (seroconversion rates re-
main low and vaccinated birds are poorly immunized against FJ-like viruses) in comparison
to other previously cocirculating H5N1 sublineages [29, 37]. That evidence implies the pos-
sibility that the emergence and replacement of FJ-like virus was preceded by and facilitated
Potential Risks of Vaccination against Avian Flu Epidemics 169
by the vaccination program, although the mechanism remains unknown epidemiologically

and virologically (some researchers consider that the emergence and replacement of FJ-like
virus are questionable [12, 23]). In addition, H5N2 vaccines have been used in Mexico
since 1995 [4]. Phylogenetic analysis suggests the presence of (previously uncharacter-
ized) multiple sublineages of Mexican lineage isolates that emerged after the introduction
of the vaccine. Vaccine protection studies further confirmed in vitro serologic results indi-
cating that commercial vaccine was not able to prevent virus shedding when chickens were
challenged with the multiple sublineage isolates [22, 30]. Therefore, the vaccine protective
efficacy would be impaired and the use of this specific vaccine would eventually become
obsolete. That fact also implies that the vaccine promotes the selection of mutation in the
circulating virus.
The emergence of a vaccine-resistant strain presents the risk of generating a new pan-
demic virus that is dangerous for humans through an avian-human link because of the
spread of a vaccine-resistant strain. Understanding the dynamics of the spread of a vaccine-
resistant strain is therefore crucial for implementation of effective mitigation strategies.
However, the dynamics of competition between vaccine-sensitive and vaccine-resistant
strains is, in general, complex [24, 27]. Actually, outcomes of the dynamics might be
influenced by several factors, including a loss of protection effectiveness, the competitive
advantage of a vaccine-resistant strain, and vaccination coverage. Until now, several theo-
retical studies have investigated the impact of an emergence of a resistant strain of antiviral
drug such as M2 inhibitors and NA inhibitors during an influenza pandemic among hu-
mans [1, 8, 24, 26, 27, 32, 39]. But, to our knowledge, no study has used a mathematical
model to investigate the application of a vaccination program among poultry in the context
of an emergence of a vaccine-resistant strain. It remains unclear whether a vaccination pro-
gram can prevent the spread of infectious disease when the vaccine-resistant strain emerges
and how a loss of immunization by vaccination of birds infected with the vaccine-resistant
strain affects the spread of infectious disease among birds. Nobody can give a simple and
clear explanation to capture the problems described above in a theoretical framework (us-
ing numerical simulations, many qualitative and quantitative but sometimes very complex
studies have investigated the effects of antiviral drugs [8, 24, 26, 27, 32, 39]). Furthermore,
we remain skeptical that a vaccination program can reduce the number of total infectious
individuals even if the vaccination protects against the transmission of a vaccine-sensitive
strain. We developed a simple mathematical model to evaluate the effectiveness, as a strat-
egy to control influenza epidemic, of a vaccination program among poultry which could
engender the emergence of a vaccine-resistant strain.
2. Methods
Herein we describe a homogeneous population model of avian influenza and its control
using a vaccination program in the presence of a vaccine-resistant strain (Fig.1).
All birds in the effective population are divided into several compartments, including
susceptible birds (X), vaccinated birds (V ), birds infected with vaccine-sensitive strain ( Y ),
and birds infected with vaccine-resistant strain ( Z). We assume that susceptible birds are
born or restocked at a rate of c per day and that all birds are naturally dead or removed from
the effective population at a rate of b per day. In the absence of vaccination, transmission
occurs at a rate that is directly related to the number of infectious birds, with respective
transmission rate constants and from infected birds with the vaccine-sensitive strain
and with the vaccine-resistant strain. The infectiousness of vaccine-sensitive and vaccine-
resistant strains are assumed to be exponentially distributed, respectively, with mean du-
rations of 1/(b + my ) and 1/(b + mz ) days. Actually, my and mz respectively signify
virulence of vaccine-sensitive and vaccine-resistant strains. We implicitly assume that the
infected bird with one strain can not be infected with other strain. Actually, the infected
birds rapidly die before the infection of other strains because the mean infectious period of
infected birds is very short [38, 42, 43].
Figure 1. Model structure for the emergence of vaccine-resistant strain during a vaccination pro-
gram: all birds in the effective population are divided into several compartments, respectively in-
cluding susceptible birds ( X), vaccinated birds (V ), birds infected with vaccine-sensitive strain ( Y ),
and birds infected with vaccine-resistant strain ( Z). The susceptible birds become infected with
vaccine-sensitive and vaccine-resistant strains at rates in direct relation to the number of respective
infectious birds. The infectiousness of vaccine-sensitive and vaccine-resistant strains are assumed
to be exponentially distributed. We assume that the newly hatched birds are vaccinated with a vac-
cination coverage (p), the vaccinated birds can be protected completely from the vaccine-sensitive
strain, but are partially protected from vaccine-resistant strain with a loss of protection effectiveness
of the vaccination (). See the Mathematical model section for corresponding equations.
At the beginning of the vaccination program, X moves directly to V by the vaccination.

However, after some period after the initial vaccination, the direct movement might vanish
because almost all birds are vaccinated. Therefore, we can assume that vaccination is only
administered to the newly hatched birds. The newly hatched birds are vaccinated at the rate
0 p 1 (more appropriately, p is proportional). Actually, p represents the vaccination
coverage. To simplify the theoretical treatment, as described in [34], we assume that the
vaccinated birds can be protected completely from the vaccine-sensitive strain (note that
the assumption is not necessary for our results: see Supplementary Information in [18]).
Actually, in laboratory experience, many avian influenza vaccines confer a very high level
of protection against clinical signs and mortality (90100 % protected birds) [30]. However,
many factors determine whether a vaccinated bird becomes infected, including age, species,
challenge dose, health, antibody titre, infections of immunosuppressive diseases, and cross-
reactivity of other avian influenza serotypes [34, 35, 41, 44]. On the other hand, we assume
that the vaccinated birds are partially protected from the vaccine-resistant strain at the rate
(proportion) 0 1 1 because of cross-reactivity of immune systems [14, 22, 29,
37, 44] (e.g., = 0 represents complete cross immunity against vaccine-resistant strains).
Actually, represents a loss of protection effectiveness of the vaccination caused by a
vaccine-resistant strain.
2.1. Mathematical model

We extended the standard susceptibleinfective model [2] including the effect of a vacci-
nation program that can engender the emergence of a vaccine-resistant strain [18, 40]. Our
basic mathematical model is given by the following equations:
X 0 = (1 p)c bX (Y + Z)X,
V 0 = pc bV ZV,
(1)
Y 0 = Y X (b + my )Y,
Z 0 = Z(X + V ) (b + mz )Z.
Here we investigate the impact of the vaccination program in a homogeneous area (and
heterogeneous areas in later) and specifically examine the role of epidemiological param-
eters such as the vaccination coverage (p) and the loss of protection effectiveness of the
vaccination () in the spread of the disease.
2.2. Estimation of epidemiological parameters

Baseline values of model parameters and their respective ranges used for simulations are
presented in Table 1 and 2. These parameters are based on the H7N7 avian influenza epi-
demics among poultry in The Netherlands in 2003 [9, 10, 38]. The initial population size
was c/b = 984 birds at the 2003 epidemic [38]. Usually, the mean lifespan of poultry is
about 2 years. However, we assume that the mean duration of a bird being in effective pop-
ulation is about 1/b = 100 days because of migration and marketing. Therefore, the birth
or restocking rate of birds is c = 9.84 birds per day. Estimated infectious period and trans-
Symbol Description Value (Range) Reference

c/b Initial bird population size 984 birds [38]
1/(b + my ) Mean infectious period of V-S strain 13.8 days [3, 38]
Transmissibility of V-S strain 4.78 104 day 1individual 1 [38]
(b + my )/(b + mz ) Relative mean infectious period of V-R strain 1.32 [14, 32, 39]
/ Relative transmissibility of V-R strain 0.58 [14, 32, 39]
Loss of vaccine effectiveness by V-R strain variable ( 0 1)
p Vaccination coverage variable (0 1)
Table 1. Description of physical characteristics, transmission, infectious, and vaccination parame-

ters of model (1) with their baseline values and ranges used for simulations. These parameters are
based on the H7N7 avian influenza epidemics in The Netherlands in 2003 [9, 10, 38]. Actually, V-S
and V-R represent vaccine-sensitive and vaccine-resistant, respectively.
mission parameters are 1/(b + my ) = 13.8 days and = 4.78 104 day1 individual 1,
respectively, [38]. These pathogenic characteristics such as infectious and transmission pa-
rameters are used in model (1) as parameters of the vaccine-sensitive strain. And also, the
epidemiological and biological feature of antiviral drug-resistance is well reported in [14].
The transmissibility and virulence of drug-resistant strains are usually lower than those of
the wild strain because of its mutation cost [13, 14, 24, 32]. Actually, antiviral drugs are also
used for prophylaxis drug intervention as vaccination [24, 32, 39]. Herein, we use some re-
duced value of transmissibility ( / = 0.58) and the increased value of infectious period of
the vaccine-sensitive strain ((b + my )/(b + mz ) = 1.32) for parameters of vaccine-resistant
strain (sensitivity analyses are given in Supplementary Information in [18]).
2.3. Reproduction numbers

A measure of transmissibility and of the stringency of control policies necessary to stop
an epidemic is the basic reproduction number, which is the number of secondary cases
produced by each primary case [2]. We obtain basic reproduction quantities of vaccine-
sensitive strain Rs (0) and vaccine-resistant strain Rr (0) before vaccination program. In
fact, during the vaccination program, the basic reproduction numbers depend on the vacci-
nation coverage Rs (p) and Rr (p). We derived these basic reproduction numbers in [18].
With the estimated parameters in Table 1 the basic reproduction number of vaccine-sensitive
and vaccine-resistant strain are Rs (0) = 6.53 and Rr (0) = 4.99, respectively (note that
Rs (0) corresponds to an estimated value in [38]).
Symbol Meaning Value Reference

Rs (0) Basic reproduction numbers of vaccine-sensitive strain 6.53 [3, 38]
Rr (0) Basic reproduction number of vaccine-resistant strain 4.99 [14, 32, 39]
Rr (0) Invasive reproduction number of vaccine-resistant strain 0.76
Table 2. Basic reproduction numbers and invasive reproduction numbers before the vaccination
program. These values are based on the H7N7 epidemic in The Netherlands in 2003 [9, 10, 38].
Furthermore, to clarify the concept of competition among strains simply, we introduce

the invasive reproduction number for the vaccine-resistant strain before the vaccination pro-
gram Rr (0), which signifies an expected number of new infectious cases with the vaccine-
resistant strain after a spread of a vaccine-sensitive strain among birds. The invasive repro-
duction number is considered as a competitive condition (relative fitness), which represents
some advantage measure of the vaccine-resistant strain against the vaccine-sensitive strain.
The estimated invasive reproduction number of the vaccine-resistant strain is Rr (0) = 0.76.
During the vaccination program, the invasive reproduction number also depends on the vac-
cination coverage.
2.4. Epidemiological scenarios

We consider a scenario in which a vaccine-resistant strain can emerge (i.e., be eventually
selected) during a vaccination program designed to be effective against the spread of a
vaccine-sensitive strain. This implies that Rr (0) > 1: otherwise the vaccine-resistant strain
can not emerge at all because Rr (p) is a monotonically decreasing function of the vaccina-
tion coverage p (see Supplementary Information in [18]). Acquisition of resistance ability
usually engenders a strain which, in the absence of a pharmaceutical intervention, is less
fit than the sensitive strain [13, 24, 27, 39]. Therefore, Rr (0) < Rs (0). We generally
assume the following conditions for reproductive numbers before the vaccination program
(our baseline parameter values are satisfied with these assumptions):
Rs (0) > 1, Rr (0) > 1, Rr (0) < 1.
The assumption precludes the possibility that a pre-existing vaccine-resistant strain beats
the vaccine-sensitive strain before the vaccination program because Rr (0) < 1.
3. R
We investigate how the loss of protection effectiveness of vaccination impacts the vaccina-
tion program, describe various program risks, and propose how to use poor vaccines, which
have a large loss, to maximize program effects in the situation that the resistance presents
at low levels. The detailed mathematical analyses are given in [17, 18, 40].
3.1. Evaluation of the effect of a vaccination program

Although vaccination is an important tool to control epidemics, the use of vaccination might
engender a spread of a vaccine-resistant strain. To demonstrate the interplay between these
opposing effects, we simulate model (1) to determine the final size of an epidemic (total
infected individuals Y + Z at equilibrium level) over vaccination prevalence (vaccination
coverage: 0 p 1) in Fig.2 (we use our baseline parameter values except for mz ). We
assume that the loss of the protection effectiveness is 35% ( = 0.35: this value can be
chosen arbitrarily with little effect on the meaning of the results). The estimated infectious
period of the vaccine-sensitive strain is 13.8 days [38] (see Table 1). Therefore, the viru-
lence of vaccine-sensitive strain is my = 0.062 day1 . Results show that the patterns of
the final size can be divided into two cases, which depend strongly on the virulence of the
vaccine-resistant strain. If the virulence of the vaccine-resistant strain is lower than that
of vaccine-sensitive strain (e.g., we choose mz = 0.045), then increasing the vaccination
coverage from 13.5% to 30.3% can increase the final size (green line at top figure in Fig.2).
On the other hand, if the virulence is higher ( mz = 0.065), increasing the coverage al-
ways decreases the final size (bottom figure in Fig.2). These two patterns are qualitatively
preserved for different virulence of the vaccine-resistant strain.
In [24, 27], although they consider the emergence of an antiviral drug-resistant virus, a
similar tendency (increasing the treatment level increases the final size of the epidemic) was
obtained through complex models that are difficult to treat mathematically. The mathemat-
ical model (1) presented herein demonstrates that the patterns of final size over vaccination
coverage only depend on the virulence of the vaccine-resistant strain as follows. Increas-
ing the coverage increases the final size when only both strains co-exist if the virulence
of vaccine-resistant strain is lower than that of vaccine-sensitive strain (m y > mz ). That
is to say, the vaccination is effective when either a vaccine-sensitive or a vaccine-resistant

strain exists. On the other hand, if the virulence of vaccine-resistant strain is higher than
that of vaccine-sensitive strain (my < mz ), the final size always decreases as the cover-
age increases. The other parameters can not change these patterns. In fact, many studies
Lower virulence
200
Total infected individuals

175
150
125
100
75
50
25
0.2 0.4 0.6 0.8 1

Prevalence rate of vaccination
Higher virulence
200
175
150
125
100
75
50
25
0.2 0.4 0.6 0.8 1

Figure 2. Final size of epidemics related with the prevalence rate of the vaccination: the top figure
represents that the vaccination is not always effective in the case of lower virulence of vaccine-
resistant strain. The bottom figure represents that the vaccination is always effective in the case of
higher virulence of the vaccine-resistant strain. We assume that = 0.35, mz = 0.045 (top) and
mz = 0.065 (bottom). These values of and mz are not so influential on the result. The blue, green,
and red lines respectively signify situations in which only the vaccine-sensitive strain exists, both the
vaccine-sensitive and the vaccine-resistant strains exist, and only the vaccine-resistant strain exists.
have ignored the impact of the virulence of the vaccine-resistant strain. In [15, 16], we
also found that the virulence of mutant strain determines a choice of the optimal prevention
policy for avian influenza epidemic. Therefore, we suggest that, to monitor and investigate
the virulence evolution between the vaccine-sensitive and vaccine-resistant strain is impor-
tant to develop avian influenza epidemic plans. In fact, if the vaccine-resistant strain has
higher virulence than the vaccine-sensitive strain, the vaccination program is always effec-
tive, even though the program engenders the emergence of a vaccine-resistant strain. On
the other hand, if the vaccine-resistant strain has lower virulence, we must carefully manage
vaccination to prevent the spread of a vaccine-resistant strain.
3.2. Impact of loss of protection effectiveness of vaccination
To ensure an effective vaccination program, the vaccine must protect vaccinated animals
against clinical signs of the disease and prevent mortality [30]. However, the vaccine-
resistant strain causes a loss of the protection effectiveness of the vaccination [22, 29, 30,
37, 46]. We investigate an impact of the loss of the protection on change of final size of the
epidemic over the vaccination coverage. Assume, hereafter, that the virulence of vaccine-
200

175
150
125
100
75
50
25
0.2 0.4 0.6 0.8 1

200
175
150
125
100
75
50
25
0.2 0.4 0.6 0.8 1

200
175
150
125
100
75
50
25
0.2 0.4 0.6 0.8 1

Figure 3. Impact of the loss of the protection effectiveness of the vaccination on the change of
the final size of the epidemic: the losses of the protection in the top, middle, and bottom figure are
= 0.05, 0.15, and 0.8, respectively. The top (0 ) and middle ( ) figures
portray the possibility of eradication of the infectious disease through the vaccination program.
However, in the bottom figure ( 1), the vaccination engenders a failure to prevent the
spread of the disease. The patterns of the change are divisible into these three cases, depending on
the loss of the protection. The blue, green, and red lines respectively correspond to the situation in
which only the vaccine-sensitive strain exists, both the vaccine-sensitive and the vaccine-resistant
strains exist, and only the vaccine-resistant strain exists.
resistant strain is lower than that of vaccine-sensitive strain (m y > mz ): otherwise, the
vaccination is always effective (our baseline parameter values are satisfied with my > mz ).
Actually, a resistant strain seems to have reduced virulence in general [13, 14, 24, 32].
We conduct a simulation using model (1) to elucidate the change of the final size with
the loss of the protection effectiveness 5%, 15%, and 80% over vaccination prevalence in
Fig.3. Results showed that the patterns of the change are divisible into three cases. In
theory, we can estimate the threshold values of the loss of the protection which determines
the patterns:
Rs (0) 1 1
= s
, = r .
R (0) 1 R (0)
In fact, = 0.056 and = 0.200 in our simulation from Table 1. When the loss of the
protection is between 0% and = 5.6% (5%: the top figure in Fig.3), the vaccination
can control the epidemic with the vaccination coverage of 84.7% without the emergence
of a resistant strain (a vaccine-resistant strain never emerges in the population). Therefore,
increasing the vaccination coverage always decreases the final size of the epidemics. For
the loss of the protection is between = 5.6% and = 20.1% (15%: the middle figure in
Fig.3), the vaccination eventually prevents the spread of the disease with 94.1% of vaccina-
tion coverage in spite of the emergence of the resistant strain. Increasing the coverage from
31.5% to 44.1% increases the final size. Therefore, the vaccination is not always effective.
However, when the loss of the protection is between = 20.1% and 100% (80%: the bot-
tom figure in Fig.3), the vaccination no longer controls the disease (even if the prevalence
rate is 100%) and the vaccine-resistant strain spreads widely through the population instead
of the vaccine-sensitive strain. In this case, the vaccination only slightly provides benefi-
cial effects for preventing the spread of the disease. Therefore, the loss of the protection
effectiveness of vaccination plays an important role in preventing the spread of the disease.
3.3. Vaccination can facilitate spread of disease
Sometimes a considerable spread of the resistant strain partially compromises the benefits
of a vaccination program [22, 29, 37, 46]. For example, even if we can completely execute
the vaccination program (p = 1), the final size of the epidemic can become larger than
that before the vaccination program (p = 0) by the emergence of vaccine-resistant strain
(bottom figure in Fig.3). This implies that the vaccination, which is expected to prevent the
spread of the disease, can instead help the spread of the disease. If the loss of the protection
effectiveness of vaccination is high ( 1), the vaccination might increase the
final size over vaccination coverage compared with that before the vaccination program
(vaccination always decreases the final size if 0 (top figure in Fig.3)). Here we
can also calculate such a risk of help, which depends on the loss of the protection. Let
(Rr (0) 1)
= , c = min{, 1}.
(Rs (0) 1) (Rr (0) Rr (0))
Actually, c = 0.236 in our simulation is from Table 1. When the loss of the protection is
between 23.6% and 100%, we found that the vaccination program is attended by the risk
that the final size becomes larger than that before the vaccination program.
3.4. Difficulty of prediction of a prevalent strain

Vaccination is well known to engender silent carriers or excretors if the vaccine can not
completely protect the vaccinated animals against clinical signs of the disease [30, 42]. The
existence of silent carriers or excretors is dangerous because they become a virus reservoir
and shed the virus into their environment, causing potential outbreaks among their own and
other species. Furthermore, even if a vaccination is effective in a bird (individual level),
an incomplete vaccination program for all birds (population level) can engender the silent
spread of an infectious disease [11, 34]. Additionally, we found that it is difficult for us to
predict a prevalent strain even if we can completely estimate the basic reproduction number
of vaccine-sensitive and vaccine-resistant strains during the vaccination program (although
estimations, usually, are almost impossible). Even when the basic reproduction number of
the vaccine-resistant strain is less than that of the vaccine-sensitive strain ( Rr (p) < Rs (p)),
the vaccine-resistant strain can beat the vaccine-sensitive strain and spread widely through
the population. Therefore, a non-ideal vaccination program might make a prediction of
prevalent strain difficult.
3.5. Optimal vaccination coverage

In the absence of a vaccine-resistant strain, a goal of vaccination program is to reduce the
basic reproductive number of vaccine-sensitive strain Rs (p) to be less than 1. We assume
that Rs (0) = 6.53. Therefore, the vaccination can eradicate the vaccine-sensitive strain
if at least 84.7% of the birds in poultry are vaccinated effectively based on the fraction
of 1 1/Rs (0) [2]. However, in the presence of the resistant strain, the simple theory is
inapplicable to an optimal vaccination coverage. Here we define the optimal vaccination
coverage which minimizes both the final size of the epidemic and the coverage.
We calculate the optimal vaccination coverage, which depends on the loss of the pro-
tection effectiveness of the vaccination in Fig.4 (sensitivity analyses are given in Supple-
mentary Information in [18]). At the point where the loss of the protection effectiveness is
greater than some threshold value o , the optimal vaccination coverage changes catastroph-
ically from high coverage to a low coverage. Here
Rs (0)
o = .
Rs (0) Rr (0)
Actually, o = 0.461 in our simulation from Table 1. The optimal vaccination coverage is
84.6% when the loss of the protection effectiveness is between 0% and 5.6%. In addition, if
the loss rate is between 5.6% and 20.1%, then the optimal coverage increases from 84.6%
to 100%. Furthermore, if the loss rate is between 20.1% and 46.1%, then the optimal cover-
age must always be 100%. Consequently, as long as the loss of the protection effectiveness
is small (0% 46.1%), the loss can be compensated by a high optimal vaccination cover-
age. However, if the loss rate is greater than 46.1%, the loss is no longer compensated by
the high vaccination coverage. The optimal coverage changes catastrophically from 100%
to 10.2%. Afterward, as the loss rate increases from 46.1% to 100%, the optimal coverage
decreases from 10.2% to 4.72% (the low vaccination coverage becomes optimal). This is
true because the poor vaccine (with a large loss of the protection) engenders the emergence
Optimal prevalence rate of vaccination

1
0.8
0.6
0.4
0.2
0.2 0.4 0.6 0.8 1

Loss of vaccine effectiveness
Figure 4. Optimal vaccination coverage: increasing of the loss of the protection effectiveness
engenders a catastrophic change in the optimal vaccination coverage. The optimal rate increases as
the loss increases if the loss of the protection effectiveness is small ( 0 o ). This implies
that a small loss of the protection effectiveness can be compensated by a high optimal vaccination
coverage. On the other hand, if the loss is large ( o 1), the optimal rate decreases as the loss
of the protection effectiveness increases. This eventuality implies that a large loss of the protection
effectiveness is no longer compensated by the high optimal vaccination coverage. Therefore, a low
coverage, which does not engender the emergence of a vaccine-resistant strain becomes optimal
because the poor vaccine engenders the increase of final size of the epidemic because of the spread
of the resistant strain.
of the vaccine-resistant strain for the high coverage: in addition, the spread of the resis-
tant strain increases the final size of the epidemic. Therefore, the loss of the protection
effectiveness strongly impacts also on the optimal vaccination coverage.
3.6. Variation of final size of epidemic according to the vaccination program

In countries where poultry are mainly backyard scavengers, optimum vaccination coverage
might be difficult to achieve [30]. The final size of the epidemic might be increased and the
program might fail if the optimal vaccination coverage can not be achieved. However, if we
can achieve optimum vaccination coverage, the final size is greatly reduced. The final size
of the epidemics can be variable depending on the coverage. Here we calculate the optimal
(smallest) and worst (largest) final size of the epidemic over the vaccination prevalence
in Fig.5 (black and yellow bars respectively represent the optimal and worst final size).
The variation of the final size is between black and yellow bars shown in Fig.5 (sensitivity
analyses are given in Supplementary Information in [18]).
If the loss of protection effectiveness is small, then the variation is very large. The
vaccination program can eradicate the disease or reduce the final size of the epidemic to a
very small size if we can execute the vaccination program near the optimal coverage. The
variation is sensitive for the coverage. Therefore, we must carefully manage the vaccination
program to control the disease when the loss is small. However, as the loss of protection
effectiveness increases, the variation decreases. In particular, when the loss is medium, the
reduction of the variation is remarkable. In addition, the reduction of the variation remains
almost unchanged when the loss is large. This implies that the variation becomes insensi-
Variation of total infected individuals

160
140
120
100
80
60
40
20
0.2 0.4 0.6 0.8 1

Figure 5. Variation of the final size of the epidemic over the vaccination prevalence: the black bar
represents the optimal (smallest) final size of the epidemic. The yellow bar represents the worst
(largest) final size of the epidemic over the vaccination coverage. The variation of the final size
depending on the coverage is between black and yellow bars. If the loss of protection effectiveness
is small, then the variation is very large. On the other hand, if the loss becomes large, then the
variation decreases. Therefore, the final size of the epidemic is strongly affected by the vaccination
coverage and the loss of protection effectiveness: a bad vaccination program (far from the optimal
coverage) increases the final size and prevents eradication of the disease.
tive if the loss is high. In this case, even if we can execute the vaccination program near the
optimal coverage, the effect of the program is not large. Therefore, although the final size is
strongly affected by the vaccination coverage and a non-optimal vaccination program (far
from the optimal coverage) increases the final size, in general, good vaccine treatment with
small loss of protection effectiveness has a great possibility for disease control. Demonstra-
bly, poor vaccine application has little or no benefit.
3.7. Effects of non-pharmaceutical intervention

Avian influenza vaccination need not be used alone to eradicate the disease: additional non-
pharmaceutical intervention is beneficial. Additional interventions must include culling
infected animals, strict quarantine, movement controls and increased biosecurity, exten-
sive surveillance [30, 34, 38, 42, 46]. We investigate the effects of some additional non-
pharmaceutical intervention measures on the vaccination program. The effects are consid-
ered by changing parameters of model (1).
Threshold values
Loss of protection effectiveness c o
Before notification of avian influenza 5.6% 20.1% 23.6% 46.1%
After notification of avian influenza 37.2% 88.6% 100% 96.8%
Table 3. Threshold values of the loss of protection effectiveness of the vaccination. These values
are calculated using parameters based on the H7N7 epidemic in The Netherlands in 2003 before and
after notification of avian influenza [38].
In the European Union (EU), regulations for the control of avian influenza strains are
imposed by EU council directive 92/40/EEC [38]. Virus output is reduced by the killing
and removal of infected poultry flocks (culling). During the H7N7 epidemic in The Nether-
lands in 2003, this and other approaches were executed. To investigate the effectiveness
of the control measures, A. Stegeman et al. quantified the transmission characteristics of
the H7N7 strain before and after detection of the first outbreak of avian influenza in The
Netherlands in 2003 [38]. In Table 1, we present the chosen epidemiological parameters,
Optimal prevalence rate of vaccination 1
0.8
0.6
0.4
0.2
0.2 0.4 0.6 0.8 1

120
Optimal total infected individuals
100
80
60
40
20
0.2 0.4 0.6 0.8 1

Figure 6. Effects of non-pharmaceutical intervention: the top figure shows the optimal vacci-
nation coverage with (pink curve) or without (black curve) non-pharmaceutical intervention. The
non-pharmaceutical intervention readily achieves the optimal coverage and hinders the catastrophic
change. The bottom figure shows the optimal final size of the epidemic with (pink bar) or without
(black bar) the non-pharmaceutical intervention. The intervention also dramatically reduces the final
size of the epidemic.
which are estimated on the H7N7 epidemic before notification of the circulation of the avian
influenza (these parameters are not affected by the additional control measures). Here we
choose other epidemiological parameters for vaccine-sensitive strain which are estimated
by the H7N7 epidemic after the notification in [38] (these parameters are affected by the
additional control measures) to evaluate an effect of the non-pharmaceutical intervention on
the vaccination program. The estimate of the transmission parameter decreases consider-
ably from 4.78 104 day1 individual 1 to 1.70 104 day1individual 1 by the control
measures. Furthermore, the estimate of the infectious period 1/(b + my ) is also reduced
from 13.8 days to 7.3 days. Therefore, control measures can reduce the basic reproduction
number Rs (0) from 6.53 to 1.22 [38]. In addition, we assume, for example, that the relative
transmissibility of vaccine-resistant strains is / = 0.7 and that the relative infectious pe-
riod of vaccine-resistant strain is (b + my )/(b + mz ) = 1.32 (these values are not strongly
influential on our results).
We calculated the threshold values of the loss of protection effectiveness of the
vaccination and present them in Table 3 when the vaccination program accompanies
non-pharmaceutical intervention. Results show that the non-pharmaceutical intervention
markedly reduces the risk of the emergence of the vaccine-resistant strain because
changes from 5.6% to 37.2%. In addition, the possibility that the vaccination program
eventually eradicates the spread of the disease increases because changes from 20.1% to
88.6%. Furthermore, because c changes from 23.6% to 100%, the vaccination program
always decreases the final size of the epidemic compared with that before the vaccina-
tion program, even if the size increases when both strains co-exist. When the vaccination
program accompanies non-pharmaceutical intervention, even if the loss of protection ef-
fectiveness is increased considerably by the vaccine-resistant strain, the loss can almost be
compensated by the high optimal vaccination coverage: o changes from 46.1% to 96.8%.
Fig.6 portrays the optimal vaccination coverage (top figure) and the optimal final size
of the epidemic (bottom figure) with (pink curve and bar) or without (black curve and bar)
the non-pharmaceutical intervention. The non-pharmaceutical intervention makes it easy
to achieve an optimal coverage and to prevent the spread of the disease. Moreover, catas-
trophic change does not occur until the loss of protection effectiveness becomes very high
(top figure in Fig.6). Furthermore, the optimal final size is also dramatically reduced by the
additional intervention (bottom figure in Fig.6). Even if vaccination without the additional
intervention can not prevent the spread of the disease, the vaccination with the intervention
can eradicate the disease (for example = 60%). Therefore, non-pharmaceutical interven-
tion improves weak points of vaccination programs such as the difficult control of optimal
vaccination coverage, the small applicability of the program with respect to the loss of
protection effectiveness caused by the vaccine-resistant strain, and so on.
3.8. Time-course of the spread of the disease

We investigate the time-course of spread of the disease according to vaccination and non-
pharmaceutical interventions for 500 days in the presence of a vaccine-resistant strain.
The results are presented in Fig.7. We consider that the vaccination program and non-
pharmaceutical interventions are executed after the vaccine-sensitive strain spreads and be-
comes endemic (around 200 days). Furthermore, the vaccine-resistant strain is assumed
to occur in a few individuals after the start of the vaccination program (around 260 days).
We assume that the vaccination coverage is p = 50%, the loss of protection effectiveness
is = 80%: the other parameters are the same as those used in the descriptions above.
These values of p and are not influential on our results (sensitivity analyses are shown in
Supplementary Information in [18]).
The top figure in Fig.7 depicts the epidemic curve without the vaccination program. It
is apparent that the vaccine-sensitive strain (the blue curve) becomes endemic at around 200
days after a pandemic phase of the disease if we execute no intervention policy. The middle
figure portrays the time-course of spread of the disease, assuming the vaccination program
alone. A vaccine-resistant strain (the red curve) emerges and spreads widely through the
600
Vaccine-sensitive strain HBlueL
Vaccine-resistant strain HRedL

500
400
300
200
100
100 200 300 400 500

time HdaysL
Vaccination
600
Vaccine-resistant strain HRedL

500
400
300
200
100
100 200 300 400 500

time HdaysL
Vaccination+Nonpharmaceutical intervention
600
500 Vaccine-resistant strain HRedL
400
300
200
100
100 200 300 400 500

time HdaysL
Figure 7. Time-course of the spread of the disease with vaccination and non-pharmaceutical inter-
ventions: we calculate epidemic curves with a vaccination program for 500 days. The vaccination
program and non-pharmaceutical intervention are started after the vaccine-sensitive strain becomes
endemic (around 200 days). We assume that the vaccine-resistant strain occurs after the start of
vaccination (around 260 days). The top, middle, and bottom figures respectively depict time courses
of infection without the vaccination program, with only the vaccination program, and with both the
vaccination program and the non-pharmaceutical intervention. The blue and red curves respectively
represent the number of infected individuals with vaccine-sensitive and vaccine-resistant strains. We
assume that the vaccination coverage is p = 0.5 and the loss of protection effectiveness is = 0.8.
population by replacing the vaccine-sensitive strain. It becomes endemic at around 450

days. This result shows the possibility that the emergence and replacement of the resis-
tant strain can be facilitated by the vaccination program, as in some vaccination programs
[22, 30, 37]. We can observe that it takes about several months for the resistant strain to
beat the sensitive strain (see the middle figure in Fig.7). Actually, the replacement time
of the resistant strain was reported as several months in the China and Mexico epidemics
[22, 30, 37]. The final size of the simulated epidemic is larger than that before (without) the
vaccination program because the loss of protection effectiveness = 80% is greater than
= 20% (see Fig.3). In this case, the vaccination program negatively affects the control of
infectious disease. The bottom figure presents the time-course of the spread of the disease
with both the vaccination program and non-pharmaceutical interventions. The vaccine-
sensitive strain is dramatically reduced and the vaccine-resistant strain hardly spreads in
the population: therefore, both strains are eventually controlled at a low level by the in-
terventions. Thus, non-pharmaceutical interventions can help the vaccination program and
control the resistance to spread in the population.
3.9. Geographical spread of resistance

We consider about a large geographical spread of avian influenza strains. The mode of
H5N1 spread from Asia to Europe, Africa and the Far East is unclear: risk factors such
as legal and illegal domestic poultry and exotic bird trades, and migratory bird movements
have been documented [47]. Certainly an effect of migratory birds movement is considered
as one of important risk factors for the spread of avian influenza strains [20, 28, 33]. How-
ever, there are countries that have reported H5N1 infection in poultry in which infections
are not associated with migratory bird movements and that did not report poultry trade with
other reported infected countries [20, 47]. In some counties with H5N1 cases, where the
demand for poultry is high, despite known risks of H5N1 transmission, poultry is trans-
ported illegally (for example, authorities in Vietnam estimated up to 70% of poultry that
are illegally transported from China, go undetected [47]). Actually, in South Asia such as
Vietnam, Thailand and Malaysia, these illegal or improper trades are common and persis-
tent and these birds were not vaccinated legally against H5N1 because of their illegal status
[19, 47]. Therefore, some researchers suspect that illegal trade of poultry or poultry prod-
ucts is a source for H5N1 outbreaks [20] and the trade makes an avian influenza control by
several interventions difficult.
The interventions used to control disease such as culling, stamping out, cleaning and
disinfection, and vaccination have not been successful in eradicating H5N1 in Asia [29, 37],
but have been effective in Europe [9, 10, 38]. In particular, vaccination strategies in Asia
countries such as China, Indonesia, Vietnam, have failed to eradicate H5N1 [30]. Indeed,
for example, the vaccine-resistant (FJ-like) strain had transmitted from vaccinated area to
non-vaccinated area such as Hong Kong, Laos, Malaysia, and Thailand, resulting in a new
transmission and outbreak wave in Southeast Asia, after the execution of the H5N1 vaccines
in China [6, 25, 37]. Avian influenza vaccines have only a limited impact on the disease
control and might promote the spread of the resistant strain. As mentioned above, the illegal
trades in Asia might account for the large geographical spread of the vaccine-resistant strain
(the mechanism remains unknown epidemiologically and virologically). We investigate
the role of illegal poultry trade in the avian influenza control using a vaccination program
among poultry in the context of a pre-existence of a vaccine-resistant strain.
Although, actually, an occurrence of the vaccine-resistant strain might be caused by
immunological pressures of the vaccination, the vaccine-resistant strain is assumed to be
present at low levels in both areas before the program. We consider that the vaccination
is executed in Area 1 and not in Area 2, but these areas are combined by illegal trades of
poultry from Area 1 to Area 2. We regard that only susceptible and vaccinated birds can be
exported at the rate e because those strains can cause severe illness and high mortality for
birds (we can expect that the migration can affect a balance of prevalence between those
strains in Area 2). All birds in the effective population are divided into several compart-
Figure 8. Model schematic showing a vaccination program and illegal trades: we consider, in
the context of an pre-existence of the vaccine-resistant strain, whether the resistance is selected by
the program in each area. Note that the vaccine-resistant strain is assumed to be present at low
levels in both areas before the program. The vaccination is executed in Area 1 and not in Area 2,
but these areas are combined by illegal trades of poultry from Area 1 to Area 2. We mention that
only susceptible and vaccinated birds can be exported because those strains can cause severe illness
and high mortality for birds. Therefore the migration of susceptible and vaccinated birds affects
a balance of prevalence between those strains in Area 2. In each area, susceptible birds ( X1, X2 )
become infected with vaccine-sensitive (Y1 , Y2 ) and vaccine-resistant (Z1, Z2 ) strains at rates in
direct relation to the number of respective infectious birds. We consider that vaccinated birds ( V1 ,
V2 ) can be protected completely from the vaccine-sensitive strain, but are partially protected from
vaccine-resistant strains.
ments, respectively including susceptible birds ( Xi ), vaccinated birds (Vi ), birds infected
with vaccine-sensitive strain (Yi ), and birds infected with vaccine-resistant strain ( Zi ) in
Area i (i = 1, 2). In the absence of vaccination, transmission occurs at a rate that is directly
related to the number of infectious birds, with respective transmission rate constants i and
i from infected birds with the vaccine-sensitive strain and with the vaccine-resistant strain
in Area i. Other assumptions are assumed to be the same in model (1). Thus we extended
the homogeneous vaccination model (1) including the effect of the illegal trade of poultry
in heterogeneous areas (Fig.8). Our deterministic patch-structured mathematical model is
given by the following equations:
X10 = (1 p)c (b + e)X1 (1 Y1 + 1 Z1 )X1,

V10 = pc (b + e)V1 1Z1 V1,
Y10 = 1 Y1 X1 (b + my )Y1 ,
Z10 = 1Z1 (X1 + V1) (b + mz )Z1 ,
(2)
X20 = c + eX1 bX2 (2 Y2 + 2Z2 )X2,
V20 = eV1 bV2 2Z2 V2,
Y20 = 2 Y2 X2 (b + my )Y2 ,
Z20 = 2Z2 (X2 + V2) (b + mz )Z2 .
We do focus on the illegal trade of poultry but do not focus on a migration of wild birds in
model (2). Note that both infected birds with vaccine-sensitives strains ( Y1 ) and those with
vaccine-resistant strains (Z1 ) must directly move from Area 1 to Area 2, if we consider the
migratory birds movement (only susceptible and vaccinated birds can move here).
(I) Area 1 1 > Rs1 , 1 > Rr1 V-S and V-R are eradicated
Area 2 (i) 1 > Rs2 , 1 > Rr2 V-S and V-R are eradicated
(ii) {1 < Rs2 , 1 > Rr2 } or {1 < Rs2 , 1 < Rr2 , Rr2 < 1} V-S is selected
(iii) {1 > Rs2 , 1 < Rr2 } or {1 < Rs2 , 1 < Rr2 , Rs2 < 1} V-R is selected
(iV) 1 < Rs2 , 1 < Rr2 V-S and V-R are selected
(II) Area 1 {1 < Rs1 , 1 > Rr1 } or {1 < Rs1 , 1 < Rr1 , Rr1 < 1} V-S is selected
Area 2 (i) 1 < Rs2 , 1 < Rr2 , Rr2 < 1 V-S is selected
(ii) 1 < Rs2 , 1 < Rr2 , Rs2 < 1 V-R is selected
(iii) 1 < Rs2 , 1 < Rr2 V-S and V-R are selected
(III) Area 1 {1 > Rs1 , 1 < Rr1 } or {1 < Rs1 , 1 < Rr1 , Rs1 < 1} V-R is selected
(ii) {1 > Rs2 , 1 < Rr2 } or {1 < Rs2 , 1 < Rr2 , Rs2 < 1} V-R is selected
(IV) Area 1 1 < Rs1 , 1 < Rr1 V-S and V-R are selected
(ii) 1 < Rs2 , 1 < Rr2 , Rs2 < 1 V-R is selected
Table 4. Exhaustive study of the selection and eradication of prevalent strains during the vaccination
program: we investigated what strains are eventually selected by the program in each area comparing
the basic and invasive reproduction numbers. Our results imply that the emergence and spread of
the resistance over the large geographical region is a possible phenomenon. Further, interestingly,
we found a possibility that the program can eradicate both strains in both areas. Note that V-S and
V-R represent vaccine-sensitive and vaccine-resistant, respectively.
Assume that = 1 = 2 and = 1 = 2 for simulations in model (2). We

also use the estimated parameters in Table 1 and 2. The export rate e is sampled from the
range of [0, 0.03]. We consider that minimum mean duration that birds in Area 1 are not
exported to Area 2 is about a month (i.e., the rate of the birds in Area 1 exported to Area 2
is e = 0.03 day1 ). Actually we do not have any justification for e, because there are not
any data or paper about it, but the range is not biologically unreasonable and can be chosen
arbitrarily with little effect on the meaning of the results. With the estimated parameters
in Table 1 the basic reproduction number of vaccine-sensitive and vaccine-resistant strain
before the program are Rs1 (0)|e=0 = Rs2 (0)|e=0 = 6.53 and Rr1(0)|e=0 = Rr2(0)|e=0 =
4.96, respectively, if we do not consider any illegal trades (i.e., e = 0). And also, the
estimated invasive reproduction number of the vaccine-resistant strain (vaccine-sensitive) is
Rr1(0)|e=0 = Rr2(0)|e=0 = 0.76 (Rs1 (0)|e=0 = Rs2(0)|e=0 = 1.32). During the vaccination
program, the basic and invasive reproduction numbers depend on the vaccination coverage
(0 p 1). We consider the same scenario (Rsi (0) > 1, Rri (0) > 1 and Rri (0) < 1) in
each area i as previous sections. Note that parameter values in Table 1 are satisfied with
these assumptions for e [0, 0.03]. Further, because the resistance presents at low levels in
both areas and the sensitive strain has already spread widely through the populations before
the program, we assume that Zi (0) > 0 and Yi (0) is near some steady state in each area.
We exhaustively investigated what strains are eventually selected by the program in
each area as follows (see Table 4). When the program is executed, the patterns of the
selection and eradication of prevalent strains in Area 1 are divisible into four cases. (I) both
Vaccinated area
300
Sensitive strain HBlueL
Resistant strain HRedL

250
200
150
100
50
50 100 150 200 250 300 350

time HdaysL
Non-vaccinated area
300
Sensitive strain HBlueL
Resistant strain HRedL

250
200
150
100
50
50 100 150 200 250 300 350

time HdaysL
Figure 9. Time-course of the spread of the disease with the vaccination program: we assume that
the vaccination coverage is p = 0.8, the export rate is e = 0.01, and the loss of the protection
effectiveness is = 0.8. We calculate epidemic curves with the vaccination program for 365 days.
The blue and red curves respectively represent the number of infected individuals with vaccine-
sensitive and vaccine-resistant strains. The top and bottom figures respectively depict time courses
of infection in Area 1 and in Area 2. The program completely changes the prevalent strain in Area
1 (the resistant strain excludes the sensitive strain) and partially changes one in Area 2 (the both
strains coexist).
the vaccine-sensitive and vaccine-resistant strains are eradicated, (II) the vaccine-sensitive
strain is selected, (III) the vaccine-resistant strain is selected, (IV) both the vaccine-sensitive
and vaccine-resistant strains are selected ((III) and (IV), respectively, represent a complete
and partial selection of the resistance in Area 1). For each case, we evaluated the selection
and eradication in Area 2 where is not vaccinated but affected the vaccination program
through the illegal trade. Results in Table 4 show that the replacement and spread of the
resistance over the large geographical region is a possible phenomenon. In the case of (III-
ii), (III-iii), (IV-ii), and (IV-iii), the resistance eventually spreads in both areas. We set, for
example, that the vaccination coverage is p = 80%, the export rate is e = 1%, and the
loss of the protection effectiveness is = 80%, which correspond to the case (III-iii). We
calculate epidemic curves with the vaccination program for 365 days in Fig.9. The blue and
red curves respectively represent the number of infected individuals with vaccine-sensitive
and vaccine-resistant strains. The top and bottom figures respectively depict time courses
of infection in Area 1 and in Area 2. The resistant strain excludes the sensitive strain in
Area 1 and invades into Area 2. The program changes the prevalent strain over the large
geographical region. And also the program seems to promote a coexistence of multiple
strains. Note that some sensitivity analyses concerned about the change of prevalent strains
for p, e, and are referred to Fig.10. Furthermore, we can find a possibility that the program
can eradicate both strains in both areas. That is, only a complete eradication of both strains
in vaccination area can achieve the complete eradication in another area, which correspond
to the case (I-i). The eradication can not occur in the other situations. This is a very
important information for disease control to prevent and eradicate some disease spread.
Next, we conducted simulations using our baseline parameters to elucidate how the
vaccination program and the illegal trade affect the selection of the resistant strain at the
final phase of the epidemic with the loss of the protection effectiveness = 40%, 60%,
and 80% in the left, middle, and right figures in Fig.10, respectively. The top and bottom
figures, respectively, represent the outcomes of the vaccination program in the area with
the vaccination program (i.e., Area 1) and in the area without the program (i.e., Area 2).
The blue, green, red, and pink regions respectively correspond to the situation in which
only the vaccine-sensitive strain is selected, both the vaccine-sensitive and the vaccine-
resistant strains are selected, only the vaccine-resistant strain is selected, and both strains
are eradicated. Note that the resistance is partially and completely selected in the green
and red regions, respectively. Results showed that the final phase is significantly affected
by the vaccination program and the illegal trade. In general, the high vaccination coverage
leads to a spread of the vaccine-resistant strain at the final phase. However, as the export
rate e increases, the resistance becomes difficult to be selected in Area 1. Further, when the
loss of the protection rate is relatively small, the high coverage can eradicate both strains
in Area 1. On the other hand, in Area 2, as the export rate increases, the resistance tends
to be easily selected. From these asymmetrical effects of the program and the trade, we
could observe non-synchronized changes of the prevalent strain over the large geographical
region. For example, if the export rate is relatively high (e.g. e = 0.02), the resistant
strain is partially selected in Area 2 before the selection in Area 1, but the sensitive strain
is eradicated in Area 1 before the eradication in Area 2, as the coverage increases. Thus
the illegal trade can affect a balance law of the prevalence strain in non-vaccinated area and
make the avian influenza control difficult and complex over the large geographical region
[4, 22, 29, 30, 37]. Illegal trades in poultry are a serious social behavior in order to evaluate
the effect of vaccination programs more precisely.
4. Conclusion
A serious problem of vaccination strategy is the emergence of vaccine-resistant strains [22,
29, 30, 37]. Even if a resistant strain emerges, a vaccination program must be managed to
control the spread of the disease. In the absence of the resistant strain, our mathematical
Figure 10. The outcomes of the vaccination program over the large geographical region: we assumed
that the loss of the protection effectiveness in the left, middle, right figures are _ = 40%, 60%, and 80%,
respectively. The blue, green, red, and pink region respectively corresponds to the situation in which
only the vaccine-sensitive strain is selected, both the vaccine-sensitive and the vaccine-resistant strains
are selected, only the vaccine-resistant strain is selected, and both strains are eradicated. The top and
bottom figures, respectively, represent which strain is selected in Area 1 and Area 2. Although the
selection significantly depends on the vaccination coverage and the export rate, the high vaccination
coverage generally leads to a spread of the vaccine-resistant strain at the final phase. Further, we could
observe non-synchronized changes of the prevalent strain in both areas. Thus the illegal trade can affect
a balance law of the prevalence strain in non-vaccinated area and make the avian influenza control
difficult and complex over the large geographical region.
models (1) (2) certainly show that a large vaccination coverage might markedly reduce
an epidemic curve and the final size of the epidemic. Therefore, we can control infectious
diseases as in previous models [2]. However, in the presence of the emergence of a vaccine-
resistant strain, the vaccination program cannot simply control the spread of the disease.
The control of the infectious disease through vaccination becomes more difficult.
The paradoxical result obtained here is that if the virulence of a vaccine-resistant strain
is less than that of a vaccine-sensitive strain, the final size of the epidemic might increase
as the vaccination coverage increases (see Fig.2). A vaccination that is expected to prevent
the spread of the disease can instead foster the spread of the disease. Although qualitatively
similar results were obtained through more complex models [24, 27], which can be treated
analytically only to a slight degree, one of our important results is the clear and simple
concept illustrating the value and pitfalls of vaccination programs: the concept can help
farmers and administrators avoid negative effects from paradoxical phenomena. We inves-
tigated how the loss of protection effectiveness impacts a vaccination programs results in
the lower virulence case. If the loss of protection effectiveness is between 0 and , the
vaccination program can eventually eradicate the disease even if a vaccine-resistant strain
emerges (see Fig.3). In particular, if the loss is between 0 and , the program prevents even
the emergence of the resistant strain. However, when the loss is greater than , the program
no longer prevents the wide spread of the resistant strain in spite of the large vaccination
coverage. Furthermore, if the loss is between c and 1, the program presents the risk that
the final size will become larger than that without the program. Therefore, in the context of
the emergence of the resistant strain, we must carefully execute the program to exercise a
positive effect of the vaccine effectively. Additionally, we investigated the optimal vaccina-
tion coverage, its final size, and the worst-case final size (see Fig.4, 5 and Supplementary
Information in [18]). The catastrophic change of the optimal coverage and the variation of
the final size depending on the loss of protection effectiveness were confirmed.
Further, in the context of a pre-existence of the vaccine-resistant strain, the program can
change the balance of prevalence between vaccine-sensitive and vaccine-resistant strains in
both vaccinated and non-vaccinated areas through the illegal trade of poultry. Case (III-
ii) in Table 4 represents that the resistant strain excludes the sensitive strain and spreads in
both areas. Cases (III-iii), (IV-ii), and (IV-iii) indicate that the resistance invades both areas,
and Fig.10 shows which strain is selected by the program in each area using our baseline
parameters in Table 1. The program seems to be able to promote the spread of the resistant
strain. Here, interestingly, if the illegal export rate is low, both strains can be selected in both
areas (green region), but if the export rate is high, the resistance cannot be selected in the
vaccinated area (blue region) but can be partially selected in the non-vaccinated area (green
region) in Fig.10. The non-synchronized changes in the prevalent strain can be explained as
follows: As the export rate increases, the susceptible and vaccinated birds move from Area
1 to Area 2, which can increase some herd immunity of bird population against the resistant
strain in Area 1 but decrease that in Area 2. This is because, although the sensitive strain can
be maintained by infections of only the susceptible birds, the resistance must be maintained
by infections of both the susceptible and vaccinated birds. Actually, we assumed that the
fitness of the resistance strain is less than one of the sensitive strain at the beginning of the
program (Rri (0) < 1). The poor resource, because of high export, leads to some advantage
of the sensitive strain in the vaccinated area and the rich resource leads to some advantage
of the resistant strain in the non-vaccinated area in the context of the lower fitness of the
resistance. Thus, the program can affect the balance of prevalent strains in both vaccinated
and non-vaccinated areas asymmetrically.
Although vaccination is now being used extensively to aid the prevention of emer-
gence or to control the spread of avian influenza [6], the vaccination sometimes has sev-
eral negative effects [11, 22, 30, 34, 37]. As discussed above, when a vaccine-resistant
strain emerges, model (1) predicts various risks in the program. To eradicate the infectious
disease effectively by vaccination, early detection of the resistant strain, monitoring of its
virulence and loss of protection effectiveness of vaccination caused by the resistant strain,
and attendance of non-pharmaceutical interventions are all required. Moreover, actually,
extensive vaccination programs are ongoing in Southeast Asia to control the H5N1 epi-
demic and many experts worry about the change of prevalent strain of avian influenza after
the programs [7, 21, 30, 37, 45]. For example, genetic findings revealed that FJ-like viruses
were responsible for all recently reported human infection cases (22 H5N1 human infection
cases from 14 provinces in China since November 2005) in China [37]. The FJ-like viruses
(which have a resistant ability against the vaccination) prevailed among poultry around
Southeast Asia after the vaccination program in China since September 2005 [6, 25, 37].
We indicated that a vaccination program sometimes selects its resistant strain in a vacci-
nated area and illegal trade can spread resistance to neighboring non-vaccinated areas by
model (2). In fact, most human infections of avian influenza result from contact with in-
fected poultry or with the surfaces contaminated with the secretions/excretions of infected
birds [7, 11]. Therefore, if the new strain selected by vaccination programs may easily mu-
tate and obtain a sustained human-to-human transmission ability, then we might have a risk
of a global pandemic such as the 1918-1919 Spanish influenza. Uncontrolled vaccination,
including loose post-vaccine surveillance, insufficient vaccine delivery systems and the use
of bad vaccines, poses a greater threat in further outbreaks and raises the possibility of the
potential mutation of the virus to become a pandemic pathogen [30]. We must carefully
reconsider the use of vaccination in most countries worldwide and carefully manage any
vaccination program that might select a vaccine-resistant strain.
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Expert Commentary
Preparation and Production of

Prepandemic and Pandemic Influenza
Vaccine: A Personal View
Akikazu Sakudo*1, Toyokazu Ishikawa2 and Kazuyoshi Ikuta1

Department of Virology, Center for Infectious Disease Control, Research Institute for
Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan1
The Research Foundation for Microbial Diseases of Osaka University, Kanonji Institute,
2-9-41, Yahata-Cho, Kanonji, Kagawa, 768-0061 Japan2
Recently, reports that avian influenza viruses have crossed the species barrier and
infected or killed humans have increased [1]. As co-infection with avian and human influenza
viruses in humans or other hosts could produce new viruses, which have the ability to infect
both hosts, the emergence of pandemic influenza viruses is of particularly concern [2]. To
tackle these issues, the most important and effective approach in terms of medicine is
vaccination and medication. In this commentary, we comment on the current problems and
future perspectives for pandemic influenza vaccine production.
Current influenza vaccines are mainly produced by culturing embryonated chicken eggs
after virus inoculation [3]. This system has several problems as follows. As virus strains must
come from viruses proliferating in embryonated eggs, the compatibility of the proliferation
ability in eggs with antigenicity against circulating viruses is the most important factor for
achieving efficient vaccine production [4]. In particular, avian influenza viruses, which have
potential of pandemic influenza viruses, are usually difficult to propagate in embryonated
eggs [5]. The virulence to embryos of high pathogenic influenza viruses such as pandemic
influenza viruses causes problems in the propagation of viruses. The virulence of high
pathogenic avian influenza viruses is due to the presence of a specific cleavage site
(RERRRKKR) for furin, which is a protease ubiquitously expressed in most tissues [6, 7]. In
*
Department of Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka
565-0871, Japan Phone: +81-6-6879-8309, Fax: +81-6-6879-8310, E-mail: sakudo@biken.osaka-u.ac.jp
196 Akikazu Sakudo, Toyokazu Ishikawa, and Kazuyoshi Ikuta
low pathogenic avian influenza viruses, the site is a non-cleavable sequence (RETR) for furin
but is cleavable by a protease such as trypsin, which is expressed only in the intestinal tract.
Recently, to eliminate the virulence of a high pathogenic avian influenza virus and to produce
a high yield, the use of recombinant viruses complexed with donor strain A/Puerto Rico/8/34
[H1N1; PR8 (Cambridge)] has been examined for vaccine production [8]. Such virus strains
as NIBRG-14, which consists of six genes encoding matrix protein (M), non-structural
protein (NS), nucleoprotein (NP), and RNA polymerase subunits (PA, PB1, and PB2)] from
PR8 (Cambridge) and two genes encoding hemagglutinin (HA) and neuraminidase (NA)]
from A/Vietnam/1194/04 (H5N1), are being stockpiled as prepandemic H5N1 vaccines and
are recommended by the World Health Organization (WHO) [9]. Recent studies have shown
that the growth property of vaccine seed viruses in eggs depends on the genes encoding
internal proteins of the donor virus. Seed viruses with the PR8(UW) strain as a background
exhibit superior growth compared to NIBRG-14 seed virus in embryonated chicken eggs
(four to sevenfold enhancement) [10]. This is the case for the propagation of viruses in
Mardin-Darby canine kidney (MDCK) cells [11].
However, it remains unclear whether a vaccine including the NIBRG-14 strain has a
vaccination effect against pandemic influenza viruses, especially with the emergence of
viruses from other subtypes than H5N1. It is also known that titers of antibodies induced by
vaccination with H5N1 influenza virus is low compared to seasonal influenza vaccines [4];
therefore, a higher dose of vaccines is estimated to be required for an efficient vaccination
effect. Meanwhile, developing a recombinant influenza strain from a pandemic influenza
virus will take long time after the emergence of a pandemic influenza virus.
In other problems, recent studies have shown that the emergence of mutant viruses with
different antigenicity during the culture of embryonated eggs decreases the efficiency of the
vaccine effect [12]. Furthermore, vaccines from embryonated eggs sometimes cause an
allergy reaction; therefore, individuals with egg allergy cannot be vaccinated by a vaccine
produced from embryonated eggs. Recently, the culture of cells such as Mardin-Darby canine
kidney (MDCK), Asian African Green monkey kidney (Vero), and the human retinoblast cell
line (PER.C6) has been attempted to overcome problems with the vaccine production system
[12, 13]; however, the use of bovine serum in culture medium causes a risk of variant
Creutzfeldt-Jakob disease (vCJD), which can be transmitted via blood [14]. In addition, most
cell lines require typsin, which is usually derived from animals, for the high proliferation of
influenza viruses [15]. One approach to this issue is the use of serum-free media, although
most serum-free media contain additive proteins derived from animals or human sources.
Therefore, the replacement of additives with recombinant or plant-derived proteins to
produce protein-free media is desired [16].
The current vaccination procedure against influenza uses a subcutaneous injection of
vaccine, which induces the memory of immune systems to efficiently produce neutralizing
antibodies, viz. serum IgG, against influenza viruses but not secretory IgA, whereas influenza
viruses infect via mucosa. Therefore, the memory induced by a vaccine is not so effective
against influenza virus infection through mucosa, as the memory of mucosal immune
protection is not induced by vaccination. In contrast, recent studies have shown that
neutralizing antibodies effuse to mucosa, suggesting that slight protection against influenza
viruses in mucosa is induced by subcutaneous vaccine injection [17]; however, if the virus
Preparation and Production of Prepandemic and Pandemic Influenza Vaccine... 197
strain is different between the vaccination and infection, the prevention efficacy is very low.
Recently, the development of adjuvants, which are used to enhance immune responses and
vaccination effects, has been initiated. The conventional adjuvant is aluminium [18],
although a more effective adjuvant than aluminium has recently been found [19-21]. The use
of the new adjuvant would increase vaccination efficiency and decrease the quantity of
vaccine required. To improve the efficiency and broadness of the preventative effect of the
vaccine, intranasal vaccination has also been developed. In experiments using mice, the
effectiveness of intranasal vaccination has been proved for broad influenza strains [21]. The
live, attenuated influenza virus (LAIV) vaccine approach is also promising for pandemic
vaccination, because LAIV vaccines are highly immunogenic in unprimed populations and a
single dose will provide a protective immune response [22].
As infectious viruses are indispensable for vaccine production, the virus isolation step is
essential for vaccine production. In particular, early and efficient vaccine production is
important in a pandemic, because a newly emerged influenza virus cannot be obtained until a
pandemic virus has emerged. In addition, the spread of a pandemic virus will be enhanced
time-dependently after emergence. Currently, it is estimated that at least 4 - 6 months are
necessary from the time a pandemic is declared by WHO until vaccine seed stock is made
available, with current vaccine technology [23]. Therefore, detection and isolation as early as
possible is very important for pandemic vaccine production; however, currently, there has
been little development toward facilitating influenza virus isolation. To concentrate viruses,
ultracentrifugation and polyethylene glycol (PEG) precipitation are conventionally used;
however, both of these methods partially inactivate viruses by the concentration procedure.
Recently, the possibility of using magnetic beads coated with bioadhesive polymers to
concentrate infectious influenza viruses has been suggested. An example is anionic magnetic
beads coated with poly(methyl vinyl ether-maleic anhydride) [poly(MVE-MA)], which can
be used to concentrate broad and natural circulating influenza viruses derived from humans
and poultry [24, 25]. The recovered influenza viruses have full activity to infect chicken
embryonated eggs and MDCK cells. The most important points of this method are its
simplicity and rapidity (< 30min). In other systems, a recent study by another group has
shown that formalin-fixed erythrocytes can be used to isolate infectious H5N1 influenza virus
from natural water [26]. Hopefully, these magnetic bead- or erythrocyte-based concentration
methods will facilitate influenza virus isolation and may contribute to pandemic vaccine
production.
Efficient vaccine production by monitoring the virus concentration and bacterial
contamination during culture is also important. Visible and near-infrared (Vis-NIR)
spectroscopy has been used in the field of agriculture, medicine, and pharmacology [27], and
recent studies have shown that this method is a powerful tool for online monitoring of quality
control, especially for automation [28], which is indispensable for vaccine production. For
seasonal influenza, 50 million eggs are used for vaccine production in Japan and 3.4 billion
eggs throughout the world, which has potential ability to produce 6 billion doses.
Approximately one dose of vaccine is derived from one egg. In this schedule, 0.1 - 0.2
million doses are estimated per day by one vaccine company. A recent WHO Strategic
Advisory Group of Experts (SAGE) recommended 150 million stockpile doses of H5N1
vaccine [29]. To improve efficient vaccine production, monitoring the virus concentration
and bacterial contamination during culture is therefore important and Vis-NIR spectroscopy
may contribute.
Herein, we presented a personal view regarding the recent advances and future
perspectives on facilitating influenza virus isolation, vaccination efficiency, and monitoring
of vaccine production. Hopefully, readers such as researchers and manufacturers involved in
influenza vaccine production will be motivated by this personal commentary, obtain
information for their own research, and be inspired by new ideas for future research on
influenza vaccine.
We thank Dr. Tomo Daidoji (Osaka University, Osaka, Japan) for valuable comments.
This study was supported in part by the Japan Science and Technology Agency, Heiwa
Nakajima Foundation, and Kieikai Research Foundation.
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Index
agriculture, 164, 197

A
aid, 40, 136, 143, 144, 145, 190
AIDS, 107, 124, 137, 140
absorption, 39, 40
air, 102, 103, 135
access, ix, 97, 102, 103, 105, 106, 109, 125, 137,
airports, 125, 126
138, 139, 147, 162
alcohol, 88
acid, 2, 4, 5, 6, 19, 24, 25, 29, 44, 71, 72, 73, 74, 89,
allantoic, 32
92, 99, 199
allergy, 196
acidic, 73
aloe, 87
activation, 51, 77, 90
alpha, 29, 94
active site, 85, 87
alpha interferon, 94
acute, 3, 8, 11, 20, 51, 76, 92, 105, 109, 110, 112,
alternative, viii, x, 27, 35, 38, 42, 101, 104, 167, 168
113, 141
alternatives, 31, 35, 38
acute respiratory distress syndrome, 76
aluminium, 32, 197
adamantane, 24, 67, 93
alveolar macrophage, 13
adaptation, 1, 2, 3, 16, 19, 72, 79, 92, 93, 98
alveoli, 11, 12, 14
additives, 196
alveolitis, 11, 14
adenoviral vectors, 37
amendments, 150
adenovirus, 37, 38, 45, 46
amino acid, 2, 5, 6, 7, 19, 24, 25, 44, 72, 73, 74, 89,
adenoviruses, 46
92
administration, 31, 34, 36, 37, 38, 39, 40, 42, 53, 54,
analog, 85
74, 78, 101, 108, 199
Animal and Plant Health Inspection Service, 122,
administrative, 106, 108, 153
162
administrators, ix, 97, 189
animal health, 58, 63, 66, 98, 141, 143
adolescents, 44
animal husbandry, 117, 160, 163
adults, 30, 39, 43, 46, 67, 111
animal models, 36
aerosol, 2, 9, 99, 102, 104, 107
animal studies, 77, 79
affect, 117, 164
animals, ix, x, 7, 10, 11, 20, 36, 44, 50, 53, 57, 58,
Africa, 6, 28, 57, 59, 60, 62, 67, 134, 140, 156, 160,
61, 69, 75, 78, 83, 87, 98, 116, 118, 128, 131,
183
134, 138, 139, 142, 143, 144, 146, 147, 149, 160,
age, 30, 170, 174, 177, 200
163, 175, 177, 179, 196
agents, ix, 38, 55, 67, 92, 97, 100, 109, 112
annotation, 79
aggregates, 34
anorexia, 10
agonist, 199
antibiotic, 101, 65, 107
agricultural, 129, 134, 161
antibody, 13, 33, 35, 36, 38, 39, 40, 44, 47, 76, 99,
agricultural sector, 134
111, 170
202 Index
antigen, 29, 35, 36, 37, 38, 39, 40, 41, 46, 193
B
antigen presenting cells (APCs), 40
antigenic drift, 29, 34, 71, 92
Baars, 111
antigenic shift, 29
bacilli, 102
antigenicity, 5, 19, 195, 196, 198
backfire, 129, 152
anti-sense, 70
bacteria, 12, 145
antiviral, ix, x, 53, 54, 56, 63, 65, 66, 67, 73, 74, 83,
bacterial, 12, 13, 15, 30, 42, 43, 197
84, 88, 89, 90, 93, 94, 100, 101, 104, 112, 115,
bacterial contamination, 197
125, 126, 131, 135, 137, 138, 141, 142, 147, 151,
bacterial infection, 12, 15
167, 168, 169, 172, 173, 190, 191, 192
Barack Obama, 137
antiviral agents, 67, 100, 112
barrier, 2, 11, 22, 103, 128, 195
antiviral drugs, ix, 54, 56, 65, 83, 84, 101, 104, 125,
barriers, vii, 1, 8, 44, 141
131, 135, 137, 138, 141, 142, 169, 172
basement membrane, 77
antiviral therapy, 101
B-cell, 33, 40
anxiety, 51
behavior, 52, 54, 63, 187
APEC, 122
Beijing, 83, 129, 130, 152
APHIS, 122, 148, 162, 163, 164, 165
Belgium, 6
apoptosis, 12, 13, 73, 75, 76, 77
bilateral trade, 144
apoptotic, 12, 13
Bill Frist, 140, 156
application, 95, 126, 169, 179, 192
binding, 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 29, 36, 71, 72,
appropriations, x, 115, 118, 119, 120, 121, 148, 163,
73, 74, 77, 127
164
biogenesis, 70
Appropriations Committee, 118
biological activity, 89, 94
ARDS, 76
biosafety, 105, 145
arginine, 71
biosecurity, x, 59, 63, 66, 122, 135, 159, 161, 163,
Armed Forces, 123
165, 168, 179
Army, 31
bioterrorism, 150
arthralgia, 33
bird flu, vii, viii, ix, 49, 67, 86, 110, 115, 116, 120,
ASEAN, 122, 135, 141, 142, 156, 157
127, 130, 131, 132, 135, 140, 152, 153, 156, 157
aseptic, 104
Black Sea, 132
Asia Pacific Economic Cooperation, 122
blood, 11, 78, 87, 88, 99, 136, 196
Asian, 4, 5, 28, 29, 98, 101, 117, 122, 126, 128, 141,
BMA, 80
143, 144, 152, 153, 154, 156, 157, 160, 192, 196
body fluid, 104
Asian countries, 117, 126, 128, 141, 144, 160
body temperature, 105
aspiration, 105
bone marrow, 11
assessment, 40, 54, 59, 60, 104, 107, 128, 132
border crossing, 126
Association of Southeast Asian Nations, 122
bovine, 38, 196
assumptions, 52, 73, 173, 184, 186
breeding, 58, 128
asthma, 34, 44
Britain, ix, 115, 127, 137, 143, 150
asymptomatic, 99
broad spectrum, 42
attachment, 71, 80
broilers, 164
Australia, 63, 131, 134, 156
bronchioles, 14, 15
authority, 124, 164
bronchiolitis, 11, 14
autoimmune, 34
bronchitis, 14
availability, viii, 49, 50, 51, 55, 58, 108, 124, 131
bronchopneumonia, 11, 12
avoidance behavior, 51
bronchus, 15
awareness, viii, 49, 51, 59, 60, 63, 119, 121, 147,
budgetary resources, 133
149
buildings, 162, 163
Burma, 127, 156
bypass, 90
Index 203
chromosome, 37
C
chronic disease, 78
circulation, viii, 2, 27, 180, 193
Caenorhabditis elegans, 90, 94
cleaning, 142, 183
Cambodia, 53, 117, 119, 121, 123, 127, 130, 132,
cleavage, 5, 38, 71, 74, 192, 195
135, 143, 148, 150, 152, 153, 154, 156, 160, 162
clinical assessment, 42
Cambodian, 123, 127, 128, 152
clinical symptoms, 11, 161
campaigns, 59, 60, 61, 121, 143
clinical trial, viii, 28, 32, 33, 37, 40, 43, 130, 134,
Canada, 5, 7, 23, 102, 113, 137, 143
148, 150
candidates, viii, 28, 32, 35, 38, 72
clinical trials, viii, 28, 32, 33, 37, 40, 130, 134, 148,
capacity building, 149
150
carbohydrate, 5, 23
clinics, 10, 15, 105
carrier, 36, 78, 80
clusters, 54, 99, 133
Caspian, 132
CNN, 154
Caspian Sea, 132
CNS, 34, 39, 40
cats, 3, 8, 11, 99, 111
coastal areas, 147
CCC, 164
coding, 47
CD8+, 39, 43, 71
codon, 36, 45
cell, 2, 4, 5, 12, 13, 21, 32, 33, 35, 36, 37, 38, 41, 43,
cohort, 44, 102
71, 72, 73, 76, 77, 78, 79, 89, 90, 92, 193, 196,
collaboration, viii, 49, 51, 52, 60, 61, 65, 123, 149
199
collagen, 77
cell culture, 43, 199
colonization, 107
cell growth, 199
Commodity Credit Corporation (CCC), 164
cell line, 73, 78, 92, 196, 199
communication, 60, 64, 66, 109, 121, 123, 141, 147
cell lines, 78, 92, 196
Communist Party, 136
cell surface, 4
communities, 51, 55, 60, 161
cellular immunity, 37
community, 51, 55, 63, 66, 67, 93, 106, 112, 125,
Cellular response, 89
131, 140, 141, 149
Centers for Disease Control (CDC), 55, 67, 85, 105,
compatibility, 195
111, 112, 117, 120, 123, 145, 147, 148, 150, 152,
compensation, 59, 60, 136, 143, 160, 163
157, 162, 164, 165
competition, ix, 115, 169, 172
Central Asia, 6, 59, 61, 132, 153
compliance, 67, 100, 102, 130
central nervous system, 34
complications, 33, 34, 38, 39, 40, 100, 112
CERF, 125
components, 32, 34, 47, 66, 77, 101, 120
certification, 134
compounds, 87
chemicals, 124
concentrates, 124
chemoattractant, 12, 15
conduct, 123, 136, 164
chemokine, 76, 77
conflict, 150
chemokines, 76, 77
Congress, x, 115, 116, 117, 118, 119, 120, 137, 138,
chicken, 3, 9, 18, 19, 32, 33, 34, 72, 73, 74, 92, 134,
139, 141, 142, 144, 145, 156, 164
144, 195, 197
Congressional Budget Office, 144
chickens, vii, 5, 9, 19, 23, 24, 28, 34, 36, 45, 75, 83,
congressional hearings, 118
99, 116, 128, 129, 130, 143, 144, 145, 164, 165,
conjunctivitis, 8, 37, 104
169, 193
Connecticut, 161
children, 32, 34, 39, 44, 47, 93, 112, 130, 135
connective tissue, 14, 77
chimpanzee, 46
consensus, ix, 35, 44, 113, 115, 138
China, 7, 9, 19, 21, 23, 26, 28, 42, 53, 63, 72, 83, 88,
consolidation, 11
117, 121, 124, 128, 129, 130, 135, 139, 141, 143,
constraints, 26, 59
144, 148, 150, 152, 155, 156, 157, 160, 168, 182,
construction, 72
183, 190, 191, 193
consumption, 99
China Daily, 155
204 Index
contamination, 104, 135, 197 Department of Agriculture, 102, 119, 122, 147, 150,
contingency, viii, 50, 53, 64, 127, 129, 149 160, 165
continuity, 51, 66, 144 Department of Defense (DOD), 119, 123, 147, 148,
contracts, 124 150
conversion, 16 Department of Health and Human Services, 119,
cooking, 60, 161 120, 147, 150
coronavirus, 106, 112, 113 Department of Homeland Security, 147, 148, 150,
correlation, 22, 93 160
costs, 101, 125, 142, 143, 156 Department of State, 119, 121, 147, 148, 150
cotton, 103 Department of the Interior, 147, 148, 150
cough, 10, 20, 30, 107 derivatives, 24
coughing, 11, 90 dermatitis, 103
covering, 90 destruction, 77, 122, 130, 133, 143, 144, 163, 164
Creutzfeldt-Jakob disease, 196 detection, viii, x, 11, 50, 53, 57, 64, 105, 111, 115,
critically ill, 77 118, 125, 126, 131, 133, 140, 146, 147, 180, 190,
Croatia, 117, 127, 150, 160 191, 197
crops, 164 developed countries, 84, 109, 126, 137
cross-border, 64, 135 developing countries, 101, 108, 109, 113, 126, 141
CRS, 146, 147, 148, 149, 150, 152, 155, 156, 157 developing nations, 138
CSR, 151 diarrhea, 30
C-terminal, 92 differential diagnosis, 133
culture, 13, 32, 34, 75, 78, 196, 197 digestion, 71, 77
Customs and Border Protection, 160 dimer, 74
cyanide, 90 diphtheria, 102
cycles, 2 directives, 130
cytokine, 24, 30, 76, 77, 78, 88, 94 disaster, 51
cytokine receptor, 94 discomfort, 39
cytokine response, 24 diseases, 9, 53, 60, 91, 107, 109, 113, 123, 124, 126,
cytokines, 12, 15, 21, 71, 76, 77, 87, 89 150, 189
cytoplasm, 71, 73, 75, 76, 78 disinfection, 102, 161, 183
cytotoxic, 30, 37, 71 disseminate, 124
cytotoxicity, 71 distress, 51
distribution, 56, 100, 131, 135, 141
D
diversity, 29, 63, 168
DNA, 35, 36, 37, 38, 41, 44, 45, 91, 95, 165
damage, 134
DNA polymerase, 37
database, 50, 51
doctors, 133, 139
death, x, 29, 43, 75, 77, 90, 130, 131, 144, 162, 167
dogs, vii, 1, 3, 4, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19,
deaths, x, 30, 50, 83, 98, 116, 119, 127, 128, 129,
20, 21, 22, 23, 25
135, 136, 140, 152, 162
domestic resources, 149
decontamination, 129
donor, 33, 121, 162, 196
decontamination procedures, 129
donors, 128, 136
defense, 15, 54
dosage, 40, 93, 139
deficiency, 107
draft, 67, 136
definition, 66
drinking, 88
degradation, 70, 78, 80, 90, 91
drug interaction, 93
delivery, 35, 36, 38, 39, 45, 47, 72, 75, 77, 78, 79,
drug resistance, 190, 191, 192
80, 92, 95, 162, 190
drug use, 67
demand, 106, 134, 144
drug-resistant, 93
dendritic cell, 40
density, 9
Index 205
drugs, 31, 54, 55, 65, 68, 84, 85, 101, 104, 125, 130, epithelial cells, 3, 4, 11, 12, 21, 29, 76, 77, 91
131, 135, 137, 138, 141, 142, 152, 155, 192 epithelium, 9, 11, 12, 14, 21, 33, 71, 79
duration, 15, 90, 93, 101, 104, 171, 185 epitope, 45
equilibrium, 173
E
equipment, x, 101, 108, 115, 121, 123, 133, 140,
142, 147, 161, 163
early warning, 52, 89, 120
erythrocyte, 197
East Asia, 141, 142, 156, 157
erythrocytes, 197
Eastern Europe, 59, 121, 134, 147
Ethiopia, 140
Ebola, 37
etiology, 3
ecological, 52
etiquette, 55
ecology, vii, 1, 8, 16, 17, 26, 42
eukaryotes, 94
economic losses, 28, 144
Europe, ix, x, 3, 6, 7, 61, 63, 67, 68, 88, 102, 109,
edema, 77
115, 116, 117, 121, 127, 133, 134, 137, 138, 156,
education, x, 106, 108, 115, 118, 120, 148
159, 183
egg, 32, 34, 35, 44, 46, 164, 196, 197
European Commission, 126, 134
Egypt, 62, 64, 140
European Union, 135, 144, 180
elderly, 30, 47, 92
evacuation, 119, 122, 147
electroporation, 36, 44, 45, 73, 78
evidence, ix, 97, 98, 134, 139, 162
eligible countries, 117
evolution, 3, 5, 17, 19, 21, 24, 25, 26, 29, 42, 52,
ELISA, 13, 20
149, 174, 191, 192, 193
embryos, 34, 73, 74, 195
execution, 183
emergence, 123
Executive Branch, 119
emergency departments, 105, 107
exercise, 126, 127, 189
emergency response, 51
exfoliation, 12
Emergency Supplemental Appropriations Act, 164
expenditures, 84, 108
emotional, 108
experimental condition, 46
emotions, 67
expertise, 58, 108, 109, 122, 130, 140
employees, 104, 111, 122, 147
experts, 116, 126, 127, 129, 131, 132, 135, 137, 138,
employment, 144
139, 140, 141, 142, 144, 145, 152, 155, 160
encapsulation, 75
exporter, 134, 164
encoding, 29, 45, 71, 72, 91, 196
exports, 134, 144, 164
encouragement, 127
exposure, ix, 16, 17, 20, 53, 97, 98, 99, 102, 103,
endocytosis, 40, 78, 85
104, 105, 111
endonuclease, ix, 70, 71, 73, 83, 90, 92
eye, 102, 103, 105, 121, 161
endorsements, 62
endothelial cells, 77 F
endotoxins, 34
Energy and Commerce Committee, 118 failure, 71, 77, 141, 175
England, 24 false alarms, 89
environment, 9, 83, 99, 103, 177 false positive, 21, 136
environmental contamination, 104 family, vii, 27, 28, 55, 69, 89, 94, 105, 132
environmental control, 100 family members, 28, 55, 89
enzymatic, 85 FAO, 58, 60, 68, 121, 125, 126, 127, 128, 132, 134,
enzymes, 76, 77 135, 136, 140, 151, 160, 161, 162, 164, 165
epidemics, x, 8, 28, 65, 84, 93, 100, 106, 108, 109, Far East, 183
120, 134, 167, 168, 171, 173, 174, 176, 178, 182, farmers, x, 53, 59, 117, 119, 122, 126, 130, 131, 135,
191 136, 142, 143, 144, 156, 160, 162, 163, 164, 167,
epidemiology, vii, ix, 1, 8, 9, 42, 47, 93, 97, 109, 189
113, 120, 141 farming, 130, 143
epithelia, 29
206 Index
farms, x, 9, 20, 23, 64, 119, 122, 128, 132, 135, 136, gene, viii, 5, 7, 17, 19, 24, 25, 29, 30, 33, 35, 36, 37,
141, 159, 160, 161, 163, 164 38, 45, 46, 69, 70, 72, 74, 75, 77, 78, 79, 80, 81,
fatalities, 156 92, 94, 95, 98
fatigue, 108 gene expression, 36, 70, 74, 78, 81, 95
FBIS, 152, 154 gene promoter, 92
FDA, 31, 32, 33, 85, 148, 150 gene silencing, 79, 92
fear, x, 4, 94, 115, 128, 131, 134, 139, 140, 152, 160 gene therapy, 36, 37, 46
feces, vii, 9, 161 gene transfer, 36, 46
federal government, 133, 162 generation, 3, 22, 29, 30
Federal Register, 157 generics, 138, 155
feedback, ix, 98, 109 genes, 6, 7, 9, 10, 17, 18, 29, 33, 35, 36, 37, 38, 73,
feedstock, 133 77, 78, 80, 89, 92, 94, 98, 110, 196
fever, 10, 20, 30, 33, 101, 104, 107 genetic testing, 105
fibroblast, 73 genetics, 22
fibrosis, 13 genome, 29, 30, 37, 69, 71, 73, 75, 91, 92
financial resources, 54, 66 genomes, 168
financing, 61, 62, 126 genomic, 29
first responders, ix, 97, 98, 109, 140 genotype, 21, 123
Fish and Wildlife Service, 147 Gibbs, 21
fitness, 172, 189, 190 GIP, 67, 199
flexibility, 155 GlaxoSmithKline, 31, 41
fluctuations, 144 global economy, 144
fluid, 39 gloves, 102, 103, 109, 121
focusing, 73 glutamic acid, 92, 199
foils, 90 glycans, 8
food, 59, 99, 122, 127, 130, 142, 161, 164 glycol, 197
Food and Drug Administration (FDA), 32, 130 glycolipids, 8
food production, 59 glycoprotein, 24, 29, 81
food safety, 122, 162 glycoproteins, 24, 29, 33, 69
foreign aid, 136, 143 glycosylation, 72
Foreign Broadcast Information Service, 154 goals, 51, 52, 54, 65, 125
foreign policy, x, 116 Golgi complex, 72
foreign travel, viii, 49 good faith, 138
fowl, 37, 133, 134, 135 governance, 63
France, 7, 137, 144, 152, 153, 154 gram negative, 102
funding, x, 116, 118, 120, 122, 125, 126, 131, 140, Greece, 143
141, 145, 163, 164 grouping, 142
funds, 65, 117, 118, 119, 121, 122, 126, 133, 134, groups, ix, 2, 56, 62, 65, 97, 101, 105, 106, 155
139, 140, 143, 147, 148, 163, 164 growth, 34, 55, 88, 124, 196, 198, 199
fusion, 4, 80 guidelines, 67, 102, 103, 111, 112, 126, 135, 145
guiding principles, 64
G
Gulf of Mexico, 148
gastroenteritis, 37 H
gastrointestinal tract, 39, 40
Gaza, 64 H1, 5, 23, 25, 29, 92, 105
Gaza Strip, 64 H1N1, 3, 4, 7, 21, 23, 28, 30, 32, 44, 74, 76, 77, 78,
GDP, 143, 144 85, 98, 196
geese, 9, 26, 129, 136 H1N2, 3, 7, 21, 23
H2, 5, 23, 24
Index 207
H3N2, v, vii, 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, homology, 15

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, Hong Kong, vii, ix, 5, 6, 9, 22, 23, 25, 27, 28, 29, 33,
34, 67, 85, 93, 98, 193 43, 63, 68, 77, 98, 109, 111, 112, 113, 115, 116,
H5N2, 5, 23, 161, 169, 192 117, 128, 129, 141, 152, 160, 162, 168, 183, 193
H7N1, 5, 161 horses, vii, 1, 4
H7N2, 22, 161, 162 hospital, 10, 20, 55, 101, 105, 107, 109, 110, 111,
H7N3, 5 112, 113, 121, 141
H7N7, vii, 1, 3, 8, 17, 23, 63, 162, 171, 172, 179, hospital beds, 141
180, 191, 193 hospitality, 142, 143
HA1, 5, 7, 38 hospitalization, 101
handling, 39, 40, 64, 99, 121 hospitalizations, 93, 112
harmonization, 149 hospitalized, 98
Health and Human Services, 119, 120, 128, 132, hospitals, 20, 101, 107, 108, 111, 112, 113, 131, 141,
147, 150 142
Health and Human Services (HHS), 119, 120, 150 host, vii, 1, 2, 3, 4, 5, 8, 11, 16, 17, 21, 24, 25, 28,
health care, 39, 51, 53, 104, 108, 110, 111, 119, 123, 29, 37, 55, 71, 72, 73, 74, 75, 76, 77, 79, 92
132, 139, 141 host population, 11
health care system, 132, 141 House, 118, 136, 141, 148, 150, 156
health care workers, 39, 111 household, 99, 102, 105, 111
health problems, 138 households, 129, 136
health services, 50, 63, 124, 141 HPV, 35
healthcare, ix, 97, 98, 100, 101, 102, 105, 106, 108, human activity, 161
109, 110, 111, 112, 133 human dimensions, 159
helix, 80 human exposure, 52, 53
hemagglutinin, ix, 2, 3, 10, 20, 21, 22, 23, 24, 25, 29, human immunodeficiency virus, 2
33, 44, 45, 69, 71, 76, 78, 79, 83, 99, 165, 196, human papilloma virus, 35
198, 199 human resources, 59, 120
hemisphere, 28 humanitarian, 63, 138
hemorrhages, 11, 13 hunting, 10
hemorrhagic fever, 102 husbandry, 117, 160, 163
hepatitis, ix, 83, 94 hyaline, 13
hepatitis B, 94 hybrid, 104
hepatitis C, ix, 83, 94 hydrophobic, 7
herbal, 87, 88 hydrophobic interactions, 7
herbal medicine, 88 hydroxide, 32
heterogeneous, 171, 184 hygiene, 55, 101, 102, 103, 104, 105, 109
heterotrimeric, 73 hygienic, 135
HHS, x, 115, 118, 119, 120, 145, 148, 150 hyperplasia, 12, 14
high risk, 100 hypothesis, 9
high-level, 149
I
high-risk, 100, 104
high-risk populations, 100
IAP, 154
hip, 149
identification, ix, 52, 55, 98
histidine, 72
identity, 9
histological, 11, 12
IFN, 74, 75, 88, 89, 94
histopathology, 15
IFN-, 89
HIV, 87, 91, 107, 124, 137
IgG, 30, 33, 196
HIV/AIDS, 87, 107, 124, 137
IL-1, 15, 76, 87
Ho Chi Minh City, 123
IL-10, 76
Homeland Security, 68, 118, 147, 148, 150, 160
IL-2, 89, 94
208 Index
IL-6, 25, 76 infectious disease, x, 43, 47, 51, 68, 91, 93, 94, 107,
IL-8, 12, 15, 76 108, 109, 110, 112, 113, 117, 120, 123, 124, 126,
immune cells, 89 141, 148, 150, 167, 168, 169, 175, 177, 183, 189
immune regulation, 89 inflammation, 9, 11, 12, 13, 14
immune response, 30, 32, 33, 35, 36, 37, 38, 39, 40, inflammatory, 11, 12, 13, 33, 71, 76, 77, 87, 89
41, 43, 44, 45, 46, 54, 71, 74, 75, 165, 197 inflammatory cells, 11, 12, 13
immune system, 29, 41, 87, 91, 171, 196 influenza a, 32, 44, 76, 77, 100, 104, 111, 119, 120,
immunity, 38, 41, 43, 45, 46, 47, 74, 116, 171, 189, 121, 122, 123, 124, 126, 128, 142
193, 199 influenza vaccine, viii, xi, 27, 31, 33, 34, 35, 38, 39,
immunization, viii, 28, 32, 35, 38, 39, 40, 42, 44, 46, 40, 41, 43, 44, 46, 47, 55, 101, 103, 118, 125,
169, 199, 200 130, 137, 145, 147, 183, 192, 195, 196, 198, 199
immunocompromised, 30, 100, 106 information sharing, 142
immunodeficiency, 107 information systems, 52
immunogenicity, viii, 28, 29, 32, 33, 36, 37, 38, 40, infrared, 197, 200
41, 43, 45, 47, 198 infrared spectroscopy, 200
immunohistochemistry, 77 infrastructure, viii, 49, 51, 105, 108, 112, 113, 117,
immunological, 94, 168, 183 128, 141
immunosuppressive, 170 ingestion, 3, 8, 40, 100
implementation, ix, 56, 61, 65, 68, 98, 107, 108, 125, inhalation, 87, 91
127, 129, 169 inhibition, 38, 74, 75, 88
imports, ix, 115, 129, 144, 160 inhibitor, 24, 72, 85, 87, 104, 112
in situ, 12, 138 inhibitors, 20, 55, 72, 87, 94, 101, 169
in vitro, 71, 73, 74, 75, 77, 78, 93, 169 inhibitory, 21, 80
in vivo, 44, 73, 74, 75, 77, 78, 80, 93 inhibitory effect, 80
inactivation, 94 injection, 40, 45, 47, 78, 91, 196
incentive, 53, 59 injections, 39, 46
incidence, 35, 39, 50, 101 innate immunity, 74
income, 59, 61, 62, 66, 126, 141, 143 inoculation, 3, 14, 17, 74, 99, 100, 129, 152, 162,
index case, 101, 105 195
India, 131, 155, 156 insertion, 37
Indian, 6, 137, 155 inspection, 122, 162
indication, 37 institutions, 51, 109, 124, 136
indicators, 107 instruction, 120
Indonesia, 28, 53, 69, 72, 93, 117, 121, 123, 130, integration, 53
131, 132, 135, 139, 143, 148, 150, 153, 156, 160, intellectual property, 137, 138
162, 168, 183, 192 intellectual property rights, 137
inducer, 76, 90 intensive care unit, 106, 107, 109
induction, 25, 45, 74, 89 intentions, 61, 141
induration, 33 interaction, 75
industrial, 138 interactions, 7, 16, 40, 93, 94
industrialized countries, 66, 139 interdisciplinary, ix, 50, 51, 97, 105, 109
industry, 66, 102, 103, 122, 129, 131, 132, 134, 135, interest, 125, 145
144, 161, 162, 163 interference, ix, 70, 80, 81, 83, 90, 94, 95
ineffectiveness, 84 interferon, ix, 25, 71, 74, 76, 77, 83, 90, 94
infants, 100, 106 interferon (IFN), 74
infectious, x, 24, 35, 38, 41, 43, 47, 51, 68, 83, 91, interferon gamma, 76
93, 94, 100, 104, 107, 108, 109, 110, 112, 113, interferons, 88, 94
117, 120, 123, 124, 126, 141, 148, 150, 167, 168, interferon-, 77
169, 170, 171, 172, 173, 175, 177, 181, 183, 184, interleukin, 12, 21, 77
189, 190, 197, 199 interleukin-6, 77
Index 209
interleukin-8, 21 lead, 135, 162, 163

interleukins, 76 leadership, 108, 149
International Development Association (IDA), 117 legislation, 118, 138, 142, 148
international law, 126 lesions, 9, 11, 12, 14, 17
international trade, x, 115, 142, 159, 163, 164 leukemia, 87
interstitial, 11 leukocytes, 12, 77
intervention, 51, 52, 61, 84, 92, 110, 172, 173, 179, life cycle, 71, 85, 91
180, 181, 182 lifespan, 171
intestinal tract, 5, 196 ligand, 76, 77
intestine, 4 likelihood, 62, 64, 104, 119, 160
intramuscular, 38, 39, 45 limitation, 103
intramuscular injection, 39 limitations, viii, 35, 50, 58, 84, 103, 106, 107, 132
intravenously, 78 linear, 36, 37, 45
invasive, 172, 185 linkage, 4, 71
investment, 63, 108, 119, 139, 144 links, 128, 147
isolation, ix, xi, 11, 50, 53, 55, 56, 64, 65, 93, 97, lipid, 32, 36, 40, 78
100, 102, 103, 105, 106, 107, 108, 109, 112, 113, liposomes, 78
125, 197, 198, 200 liver, 11
Israel, 49, 64, 65, 67 livestock, 131, 134, 135, 164
Italy, 5, 6, 21, 143, 161 loans, 125, 143
lobbying, 132
J
local government, 51, 132
localization, 8, 36
JAMA, 93
location, 107
Japan, 7, 9, 23, 72, 88, 117, 127, 137, 144, 145, 150,
logistics, 51, 54, 124
156, 160, 167, 195, 197, 198
London, 24, 25, 62
Jefferson, 67
long distance, 161
JEM, 80
Los Angeles, 154
jumping, 128
losses, x, 22, 28, 115, 144, 175
Jung, 11, 23, 25
love, 103
justification, 59, 185
lumen, 14
K lung, 11, 13, 21, 73, 74, 75, 77
lungs, 8, 11, 14, 76, 77, 90, 91
Kazakhstan, 117, 127, 132, 150, 160 lymph, 11
key indicators, 107 lymph node, 11
kidney, 11, 73, 196 lymphocyte, 30, 43, 71
killing, ix, 115, 116, 128, 180 lymphocytes, 12, 13, 37, 43, 71, 77
kinase, 74, 88 lysine, 92
King, 152
Korea, 7, 9, 10, 11, 14, 16, 17, 18, 19, 20, 23, 117, M
127, 144, 150, 156, 160
M1, 6, 29, 35, 38, 69, 72, 80
Korean, 3, 9, 10, 15, 17, 19, 20, 21, 141
macrophage, 12, 21, 76, 77
L macrophage inflammatory protein, 77
macrophages, 11, 12, 21, 71, 77
lambda, 94 magnetic, 197, 199
Laos, 117, 119, 121, 123, 128, 130, 132, 140, 143, magnetic beads, 197, 199
148, 150, 153, 154, 156, 160, 183 malaria, 37
large-scale, 132 Malaysia, 66, 117, 142, 144, 150, 156, 160, 183
law, x, 65, 115, 126, 187, 188 mammal, 2, 19
LCS, 80 Mammalian, 53
210 Index
mammalian cells, 36, 69, 70, 76, 90, 92, 94, 95 Mississippi, 165
mammals, vii, 1, 2, 3, 6, 8, 17, 19, 23, 69, 94 Missouri, 97
management, ix, 50, 51, 57, 58, 97, 98, 105, 112, models, viii, 28, 36, 74, 100, 173, 189
141, 147 molecular biology, 23
manpower, 66 molecules, 4, 6, 7, 36, 40
manufacturing, 34, 88, 138, 145, 147, 199 momentum, 121
market, 20, 59, 87, 144, 155, 161, 163 money, 131
market value, 163 Mongolia, 117, 150
markets, 2, 9, 21, 59, 136, 142, 144, 147, 161, 163, monitoring, 123, 128, 129, 131, 132, 133, 140, 141,
193 145, 148, 150, 164, 165
mask, 102, 103, 129 monoclonal, 76
mass, x, 115, 130, 136, 142, 144, 162 monoclonal antibody, 76
mass media, 60 monocyte, 76
maternal, 124, 140 mononuclear cell, 12
matrix, 29, 35, 69, 77, 80, 196 mononuclear cells, 12
matrix protein, 29, 35, 69, 196 morbidity, 34, 52, 54, 55, 106
MDR, 102, 107 morphology, 92
measles, 102 mortality, x, 8, 30, 34, 42, 52, 54, 55, 64, 65, 93, 99,
measures, viii, x, 8, 22, 28, 50, 52, 54, 56, 59, 60, 61, 101, 106, 116, 140, 160, 162, 165, 167, 170, 175,
62, 63, 64, 65, 66, 67, 100, 103, 106, 108, 121, 183, 184
125, 126, 127, 129, 131, 132, 133, 135, 141, 145, mortality rate, x, 64, 116, 162, 167
146, 159, 161, 167, 179, 180, 181, 193, 199 mosaic, 76
media, 60, 196, 199 Moscow, 132, 134
medical care, 104, 133 motivation, 59, 61
medical services, 142 mouse, 73, 74, 81
medication, x, 53, 55, 56, 63, 115, 137, 141, 147, movement, 61, 122, 135, 145, 164, 170, 179, 183,
195 185
medications, 66, 152, 168 mRNA, 36, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
medicine, 126, 137, 138, 195, 197 90, 91, 95
membership, 134 MRSA, 102, 109
membranes, 103 mucosa, 99, 196
memory, 39, 196 mucous membranes, 103
meningitis, 107 mucus, 12
messenger RNA, 70, 90 multidisciplinary, 123
meta-analysis, 44 multilateral, 142, 149
methicillin-resistant, 102, 109, 113 multiplication, 80
methodology, 123 mumps, 102
Mexican, 169, 192 muscle, 30, 36, 39, 87
Mexico, 5, 148, 169, 182 muscle cells, 36
MHC, 40, 45 muscle contraction, 87
microbes, 47, 199 muscle tissue, 39
microbial, 120, 195 mutagenic, 168
Middle East, 28, 60, 62, 140, 156, 160 mutant, 174, 196
migration, 19, 21, 171, 184, 185, 191 mutation, 6, 85, 130, 132, 135, 140, 160, 162, 169,
migratory birds, 5, 64, 117, 129, 133, 140, 160, 161, 172, 190
183, 185 mutations, 29, 30, 33, 34, 69, 191
military, 43, 105, 123, 142, 147 myalgia, 33, 101
mimicking, 39 Myanmar, 127
misconceptions, 51 Mycobacterium, 102
missions, 122, 147 myeloperoxidase, 12
Index 211
N O
N-acety, 4 observations, 9, 11, 12, 20, 21

nanoparticles, 199 obsolete, 169
nation, 51, 128, 171, 172, 180 occupational health, 102, 108
national action, 57 occupational risks, 39
national emergency, 138 Oceania, 134
National Park Service, 147 oil, 41, 118
National strategy, 64, 68, 119, 137 oligonucleotides, 80
natural, 2, 8, 16, 32, 39, 41, 51, 54, 91, 161, 197, 200 optimization, 36, 105
Navy, 123, 140 oral, 2, 38, 40, 100, 112
necrosis, 9, 11, 12, 14, 77 organ, 77, 168
nefarious, 145 organization, 125
Netherlands, 6, 23, 63, 68, 133, 162, 171, 172, 179, organizations, 120, 121, 122, 124, 132, 133, 135,
180, 191, 193 140, 142, 147, 149, 161, 162, 165
network, 56, 65, 123, 142, 148, 161 oseltamivir, ix, 93, 97, 100, 104, 105, 111, 112, 135,
networking, 135 154
neuraminidase, 29, 33, 69, 72, 85, 87, 88, 94, 99, oxidants, 12
101, 104, 112, 165, 196
P
neuraminidases, 72
neutralization, 36
Pacific, 59, 60, 61, 63, 68, 122, 123, 143, 144, 154,
neutrophil, 12, 13, 76
157
neutrophils, 12, 15
Pacific Region, 157
New England, 8
pain, 33, 39
New Jersey, 21, 22, 161
Pakistan, 28, 42, 168
New York, 21, 43, 93, 139, 152, 155, 157, 162
Palestinian Authority, 64
New York Times, 139, 152, 155, 157
pancreatic, 77
New Zealand, 63, 156
Pandemic Influenza Preparedness, 64, 150
NGOs, 128
paradox, 193
NIR, 197, 200
paradoxical, 189
NK cells, 89
parameter, 173, 175, 180, 185
Nobel Prize, 70, 79, 81
parasitic diseases, 123
non-human, viii, 28, 35, 36
parenchyma, 13
non-human primates, 35, 36
parenteral, 40, 91
non-infectious, 35
particles, 32, 35, 72
North Africa, 60, 62
partnerships, 142
North America, 3, 7, 24, 109
patents, 137, 138
North Carolina, 161, 165
pathogenesis, viii, ix, 12, 15, 23, 28, 69, 76, 77
North Korea, 127
pathogenic, vii, viii, x, 2, 3, 4, 5, 6, 15, 21, 25, 27,
N-terminal, 73
28, 34, 42, 44, 45, 46, 50, 53, 69, 73, 74, 75, 76,
nuclear, 36, 69, 73, 74, 80, 88
80, 81, 90, 93, 99, 101, 104, 106, 149, 159, 160,
nuclei, 12
161, 162, 163, 164, 167, 172, 191, 192, 193, 195
nucleic acid, 35
pathogens, 13, 26, 32, 37, 57, 102
nucleoprotein, 29, 44, 45, 46, 69, 74, 196
pathology, 77
nucleotide sequence, 2, 15, 92
patient care, 104
nucleotides, 2, 73, 74, 90
Pb, 18
nucleus, 13, 73, 74, 75, 76
PCR, 11, 13, 37, 64, 77, 105
nursing home, 107, 112
PEP, 105
nutrition, 124, 140
peptide, 5
212 Index
PER, 196, 199 President Vladimir Putin, 134

perceptions, 51 press, 129, 134, 135, 140, 200
perforin, 71 pressure, 29, 102, 103, 107, 112, 127, 138, 168
periodic, 161 prevention, viii, x, 38, 49, 50, 52, 54, 56, 60, 61, 63,
periodicity, 98 65, 67, 78, 83, 98, 101, 108, 121, 131, 133, 135,
peritoneal, 78 136, 140, 149, 162, 167, 174, 190, 197
peritoneal cavity, 78 preventive, 56, 63, 68, 91, 100, 108, 124
personal, 101 preventive programs, 56
pertussis, 102, 107 primary cells, 95
pH, 40, 72 primate, viii, 28
pharmaceutical, x, 55, 56, 61, 64, 67, 124, 125, 137, primates, 35, 36
138, 152, 155, 167, 173, 179, 180, 181, 182, 183, priming, 37, 38
190 prion diseases, 199
pharmaceutical companies, 137 private, 62, 102, 107, 124, 131, 142, 147
pharmaceuticals, 155 private sector, 142, 147
pharmacokinetic, 93 probability, 53, 100
pharmacological, 66 probable cause, 66
pharmacology, 197 production, xi, 12, 22, 31, 33, 34, 35, 40, 41, 54, 59,
phenotypes, 33, 92 66, 71, 74, 76, 80, 91, 145, 147, 155, 164, 165,
Philadelphia, 43 195, 196, 197, 198, 199
Philippines, 156 productivity, x, 115
phosphate, 78 progenitors, 24
phylogenetic, 9, 18 progeny, 29, 30, 72
phylogenetic tree, 18 program, x, 31, 56, 63, 99, 124, 134, 136, 145, 163,
phylogeny, 58 165, 167, 168, 169, 170, 171, 172, 173, 174, 175,
physiology, 16 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
placebo, 44 186, 187, 188, 189, 190
plague, 102 pro-inflammatory, 21, 76, 77, 78
planning, viii, 49, 52, 53, 57, 62, 63, 66, 98, 100, proliferation, 195, 196
105, 108, 110, 118, 119, 120, 121, 122, 126, 129, promoter, 36, 73, 92
140, 142, 147 propagation, 195
plants, 90 property, 72, 74, 75, 137, 138, 196
plaque, 75 property rights, 137
plasmid, 24, 36, 45, 73, 74, 75, 78, 92 prophylactic, 45, 54, 64, 66, 101, 111
platforms, viii, 27, 31, 35, 37, 135 prophylaxis, 31, 54, 72, 75, 85, 90, 92, 100, 101,
pneumonia, 3, 8, 11, 13, 15, 30, 42, 43, 112 104, 105, 172
point mutation, 29, 30 proteases, 5, 71, 76, 81, 92
Poland, 192 protein, ix, 2, 4, 5, 17, 18, 36, 44, 45, 69, 71, 72, 73,
political leaders, 149 74, 75, 76, 77, 80, 83, 88, 90, 91, 92, 143, 196
polyethylene, 197 protein kinase C, 88
polymer, 197, 199 protein sequence, 44
polymerase, ix, 29, 37, 69, 73, 74, 75, 79, 80, 83, 92, proteins, ix, 6, 29, 31, 32, 33, 34, 35, 45, 69, 71, 72,
98, 110, 196 73, 80, 83, 87, 88, 89, 90, 91, 92, 99, 160, 165,
polymerase chain reaction, 37, 75 196
polysaccharides, 87 protocols, 56, 67, 142, 161
population growth, 124 prototype, 43, 47
population size, 171 public, 15, 17, 50, 51, 55, 62, 67, 110, 120, 124, 125,
prediction, 35, 53, 177 126, 128, 135, 138, 141, 142, 143, 145, 149, 162,
pre-existing, 30, 38, 46, 61 168
President Bush, 121, 134, 164 public awareness, 149
Index 213
public health, 15, 17, 51, 55, 67, 110, 120, 124, 125, resource allocation, 149
126, 128, 138, 141, 143, 162, 168 resources, viii, x, 50, 51, 53, 54, 56, 59, 63, 66, 67,
Public Health Service, 31 100, 105, 108, 116, 118, 120, 121, 124, 126, 127,
public-private partnerships, 142 130, 131, 133, 139, 140, 141, 142, 149, 163, 199
Puerto Rico, 32, 196 respirator, 102, 103
Pyongyang, 127 respiratory, 3, 5, 8, 9, 10, 11, 12, 15, 16, 19, 21, 25,
26, 29, 30, 33, 37, 39, 40, 43, 44, 51, 53, 54, 55,
Q
71, 76, 77, 78, 79, 80, 91, 92, 99, 100, 103, 104,
105, 106, 107, 112, 113
quality control, 197
respiratory disorders, 40
quarantine, ix, 53, 54, 55, 64, 100, 102, 115, 125,
responsibilities, 53, 54, 103
127, 150, 164, 168, 179
responsibility, 126
R Resveratrol, 88
Retroviral, 92, 95
radiological, 118 revenue, 131
range, 2, 4, 5, 22, 25, 28, 64, 85, 133, 142, 162, 185 ribosome, 36
RANTES, 77 RISC, 70, 71
rash, 107, 140 risk, vii, 34, 52, 53, 54, 57, 58, 65, 66, 99, 100, 101,
reactivity, 170, 171 103, 104, 106, 107, 109, 111, 121, 122, 127, 133,
receptor agonist, 199 135, 140, 145, 146, 156, 160, 161, 168, 169, 176,
receptors, 3, 6, 7, 8, 15, 29, 71, 72, 79, 89 181, 183, 189, 190, 196
recognition, 6, 22, 57, 106, 109, 130, 199 risk assessment, 54
recombination, 42 risk factors, 99, 111, 183
recovery, 11, 12, 30 risks, ix, 39, 97, 98, 124, 127, 147, 173, 183, 190
rectal temperature, 4, 15, 16 RNA, viii, ix, 16, 29, 69, 70, 71, 73, 74, 75, 78, 79,
recurrence, 34, 44 80, 81, 83, 90, 91, 92, 94, 95, 196
Red Cross, 131 RNAi, 70, 79, 81, 90, 91, 95
red wine, 88 Romania, 117, 127, 132, 143, 150, 160
regional, viii, ix, 50, 58, 59, 60, 62, 66, 97, 108, 110, rotavirus, 2
122, 123, 130, 133, 134, 135, 141, 142, 146, 149 rubella, 102
regional cooperation, viii, 50, 134, 135 rural, 53, 55, 100, 125, 129, 135, 141, 160
registries, 148, 150 rural areas, 53, 141, 160
regular, ix, 62, 98, 109 rural development, 125
regulation, 30, 71, 75, 76, 77, 89, 127, 129, 180 Russia, 117, 121, 127, 132, 133, 134, 147, 150, 160
renal, 11, 93 Russian, 133, 134, 153
reparation, 99
replication, ix, 2, 3, 37, 55, 73, 74, 76, 77, 80, 83, S
88, 91, 92, 94
safety, 33, 34, 40, 43, 54, 104, 108, 120, 122, 145,
reproduction, 3, 9, 172, 177, 181, 185
162, 199
research and development, 145, 148, 150
Salmonella, 34
reservation, 127, 151
sample, 20, 87, 105
reserves, 118
sanitation, 124, 142, 161
reservoir, 24, 53, 161, 177
scabies, 102
reservoirs, vii, 27, 28, 119
seals, vii, 1, 8, 22, 26
residues, 5, 6, 8, 12, 25, 29, 71, 80
Secretary of Commerce, 142
resistance, x, 24, 37, 55, 67, 72, 85, 89, 93, 94, 152,
Secretary-General, 124
167, 168, 172, 173, 183, 184, 185, 186, 187, 189,
secretion, 93
190, 191, 192
security, 54, 124, 142, 150
resistence, 139
Security Council, 68
resolution, 11, 104
seed, 32, 34, 196, 197, 198, 199
214 Index
self-assessment, 104 spectrum, 34, 42, 44

Senate, 118, 136, 140, 141, 148, 156 spleen, 11, 77
sensitivity, 24, 104, 172, 177, 178, 181, 187 sporadic, vii, 20, 27, 28
separation, 60 stability, 5, 33, 39, 40, 73
sequencing, 58, 165 stabilize, 36, 73, 124
serine, 72, 76 stages, viii, 12, 50, 52, 61, 64, 106, 109, 125
serum, 20, 30, 33, 40, 76, 77, 196, 199 standards, 51, 66, 108, 113, 119, 124, 135
services, 50, 63, 66, 105, 120, 124, 133, 140, 141, Staphylococcus, 102, 109, 113
142, 160 Staphylococcus aureus, 102, 109, 113
severe acute respiratory syndrome (SARS), 37, 51, State Department, 119, 122, 149, 150, 157
92, 100, 102, 105, 106, 107, 108, 109, 112, 113, State Food and Drug Administration, 130
124, 125, 126, 127, 128, 129, 143, 144, 150, 151, steady state, 186
152, 156 stochastic, 100
severe asthma, 34 stock, 53, 119, 197
severity, 2, 11, 12, 15, 21, 30, 35, 50, 52, 55, 93, 120 stockpile, 65, 100, 111, 125, 135, 139, 163, 192,
shock, 144 197, 200
short supply, 65, 87 stockpiling, 55, 68, 84, 101, 120, 138, 141
shortage, x, 72, 115 stomatitis, 37, 46
sialic acid, 4, 29, 71, 72 strategies, viii, ix, x, 27, 35, 37, 38, 43, 45, 50, 65,
siblings, 135 66, 67, 83, 97, 98, 100, 101, 102, 106, 108, 120,
side effects, 32, 33, 39, 40, 41, 76, 79, 87 123, 140, 142, 167, 169, 183, 193
signal transduction, 74 structural protein, ix, 29, 35, 83, 92, 196
signals, 54 subcutaneous injection, 196
signs, 10, 16, 20, 53, 119, 136, 149, 170, 175, 177, submucosa, 12
191 sub-Saharan Africa, 140
simulation, 100, 176, 177 substitution, 6, 72
simulations, 169, 171, 185, 187 subtilisin, 5
Singapore, 113, 127, 156, 198 suffering, viii, 15, 20, 50
siRNA, viii, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, sulfatide, 76
79, 80, 90, 91, 92, 95 supplemental, 121, 122, 123, 136, 150
skin, 40, 103 supply chain, 142
smuggling, 135, 147 suppression, 71
social behavior, 187 surgical, 102, 103
social consequences, 56 susceptibility, x, 47, 167, 168
social impacts, 66 SV40, 37
Social Services, 118, 148 Switzerland, 40, 126
Somali, 140 symptoms, 10, 11, 30, 33, 34, 53, 54, 77, 85, 87, 90,
South Africa, 140 100, 104, 105, 106, 107, 128, 129, 161
South America, 134 syndrome, 13, 15, 26, 33, 44, 51, 107, 112, 113
South Asia, 183 synthesis, 37, 72, 75
South Korea, 1, 4, 8, 15, 19, 20, 25, 72, 127, 141, systemic immune response, 30, 33, 39
144, 156, 160 systems, 119, 120, 121, 126, 139, 140, 141, 142, 145
Southeast Asia, viii, ix, 27, 28, 97, 98, 100, 122,
T
123, 128, 132, 134, 135, 141, 153, 168, 183, 190
Spanish flu, 116
T cell, 39, 77
Spanish influenza, 98, 110, 190
T lymphocyte, 37, 43, 71, 77
species, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 16, 17, 24, 31, 53,
Taiwan, ix, 21, 23, 107, 112, 113, 115, 127, 145
58, 60, 92, 98, 99, 128, 170, 177, 195
targets, ix, 35, 44, 56, 80, 83, 91, 92
specificity, 4, 5, 6, 7, 21, 24, 25, 29, 43, 74, 79
T-cell, 36, 38, 43, 199
spectroscopy, 197
technical assistance, 119, 122, 135, 140, 165
Index 215
technology, 137, 145 transmembrane, 76

temperature, 13, 33, 104, 105, 125, 161 trans-membrane, 72
Thai, 72, 111, 134, 135, 153, 154 transparency, 122, 129
Thailand, 3, 6, 8, 25, 53, 64, 85, 86, 97, 107, 110, transparent, 126, 128, 139, 142
112, 117, 121, 128, 134, 135, 144, 150, 154, 156, transport, 79
160, 162, 183, 193 transportation, 128
therapeutic interventions, 101 travel, viii, x, 49, 50, 53, 64, 107, 112, 115, 124,
therapy, ix, 46, 65, 69, 70, 72, 75, 78, 79, 90, 92, 101 125, 126
threat, viii, 17, 22, 27, 42, 44, 52, 63, 68, 83, 89, triage, 108
122, 125, 130, 140, 141, 168, 190 trophoblast, 94
threatened, 137, 144 tropism, 5, 11
threatening, 137, 143 trypsin, 5, 76, 77, 196, 198
threats, viii, 49, 50, 51, 57, 63, 118, 120, 121, 145 tsunami, 136
threshold, 176, 177, 181 tuberculosis, 102, 140
throat, 30 tumor, 12, 77
tiger, 25, 111 tumor necrosis factor, 12, 77
tight junction, 77 Turkey, 117, 127, 132, 135, 150, 154, 160
time, 104, 108, 109, 128, 134, 138, 140, 142, 146, turkeys, vii, 9, 164
147 type I IFNs, 89
tissue, 5, 14, 36, 39, 45, 77 tyrosine, 72
tissue plasminogen activator, 45
U
T-lymphocytes, 43
TNF, 12, 15, 76, 77, 87
U.S. Agency for International Development, 117,
TNF-, 12, 15, 77
120, 164
Toll-like, 199
U.S. Department of Agriculture, 122, 160, 165
tonic, 88
U.S. Department of Agriculture (USDA), 102, 103,
tourism, 99, 143
122, 138, 145, 150, 160, 161, 162, 163, 164, 165
toxic, 78, 88
U.S. Geological Survey, 147
toxic effect, 78, 88
U.S. military, 43, 123
toxicity, 46
Uganda, 140
trachea, 4, 9, 11, 12, 14, 15, 76
Ukraine, 127, 132
tracking, 130, 148, 150, 156
UNICEF, 60
trade, x, 115, 142, 144, 149, 159, 163, 164, 183, 184,
United Nations, 60, 121, 125, 126, 128, 137, 151,
185, 186, 187, 188, 189, 190
160
Trade Representative, 138, 142, 144, 157
United States, 7, 26, 31, 32, 55, 67, 93, 102, 107,
Trade-Related Aspects of Intellectual Property
110, 112, 116, 117, 124, 130, 136, 137, 138, 139,
Rights (TRIPS), 138
141, 142, 143, 144, 154, 159, 161, 162, 164
trading partners, 144
United States Agency for International Development
training, ix, 53, 58, 97, 102, 108, 119, 120, 122, 123,
(USAID), 117, 119, 120, 121, 125, 135, 136, 140,
131, 147
147, 148, 150, 154, 156, 164
training programs, 53
upper airways, 91
transcription, 73, 78, 80
upper respiratory tract, 12, 30, 37, 39, 91
transcriptional, 70
urban areas, 141, 161
transfection, 75, 92
urinary tract infection, 107
transfer, 3, 8, 16, 36, 46, 73
transformation, 123 V
transgene, 36, 38
transition, 85 vaccines, ix, 115, 125, 129, 134, 135, 137, 138, 139,
translation, 36, 70 141, 145, 147, 152
translocation, 76, 80, 88 variability, 72, 100
216 Index
variation, 25, 42, 63, 178, 179, 189

W
vector, 36, 37, 38, 45, 46, 64, 92, 95, 161
ventilation, 112
warning systems, 120
ventilators, 141
water, 2, 58, 100, 102, 117, 128, 134, 142, 197, 200
vesicles, 78
waterfowl, vii, 27, 117, 132, 136, 147
vessels, 3
western blot, 75
Vietnam, 6, 32, 33, 43, 44, 53, 64, 80, 85, 86, 93, 99,
Western Europe, 133, 168
110, 111, 117, 119, 121, 123, 128, 130, 132, 136,
wild animals, 53
138, 143, 145, 148, 150, 153, 154, 156, 160, 162,
wildlife, 58, 122, 147
168, 183, 196
wine, 6, 7, 24, 25, 88
viral envelope, 32, 72
World Bank, 67, 117, 125, 126, 143, 144, 151, 156,
viral hemorrhagic fever, 102
157
viral infection, viii, ix, 69, 78, 83, 99, 200
World Health Organization (WHO), viii, 27, 31, 49,
viral vectors, 35, 37
50, 52, 84, 86, 93, 107, 111, 112, 116, 124, 146,
viremia, 11
149, 154, 161, 165, 196
virological, 8, 9, 19
World Trade Organization (WTO), 138, 146, 155
virology, 123, 129, 152
virulence, 5, 6, 22, 23, 30, 32, 41, 69, 71, 74, 80, 92, Z
170, 172, 173, 174, 175, 176, 189, 190, 195
virus infection, ix, 2, 3, 8, 9, 11, 20, 21, 23, 42, 45, zoonosis, 22
75, 76, 83, 85, 89, 90, 91, 92, 193, 196, 199 zoonotic, 8, 42, 57
virus replication, 37, 74, 76, 80, 88, 91, 94 zoonotic diseases, 57
vRNA, 73, 91

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PUBLIC HEALTH IN THE 21ST CENTURY SERIES

AVIAN INFLUENZA: ETIOLOGY,

Family History of Osteoporosis Firefighter Fitness: A Health and

AVIAN INFLUENZA: ETIOLOGY,

Nova Biomedical Books

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. New York

face a shortage of vaccines, antiviral medication, or other medical equipment, because of

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. New York

Interspecies Transmission of Avian

Daesub Song, Bokyu Kang, Chulseung Lee, and Bongkyun Park1

Molecular Analysis for Avian Influenza Virus of

There were 4 pandemics of influenza due to the emergence of antigenically different

Sia-Gal Glycosidic Linkage for Avian and Human Influenza

Amino Acid Residues of the Sia-Gal Glycosidic Linkage for AIV

Internal Genes and Molecular Marker for AIV

Pigs have an important role in interspecies transmission of influenza viruses. Swine

Amino Acid Residues of the Sia-Gal Glycosidic Linkage for SIV

Three Genotypes of the SIV

Swine H3N2 Triple-Reassortant Influenza Viruses

Isolation and Characterization of Avian Origin Canine Influenza Virus

Interspecies transmission is a crucial feature in the ecology and epidemiology of

Reproduction of Pathgenicity in Dogs

necrotizing pneumonia (PNP) lesions found in porcine reproductive and respiratory

Dog to Dog Infection with Avian Origin Canine

Genetic Characterization of Canine Influenza

Prevalence of Canine Influenza Virus Infection in

Arndt, U., G. Wennemuth, P. Barth, M. Nain, Y. Al-Abed, A. Meinhardt, D. Gemsa & M.

Olsen, N. J. Cox, A. I. Klimov, J. M. Katz & R. O. Donis, 2005: Transmission of equine

Webby, R., E. Hoffmann & R. Webster, 2004: Molecular constraints to interspecies

Conventional and Experimental

Ami Patel*1,2, Gary Wong*1,2, Mickey Sahib1,2

Avian influenza H5N1 virus, family Orthomyxoviridae, naturally persists in

efficacy and immunogenicity following immunization, additionally benefiting

Pandemic Influenza and H5N1

Name Year Virus strain Deaths Origin References

Asian Flu 1957-1958 H2N2 ~1 million China [13, 95, 96]

Influenza Antigens and Host Specificity

Influenza A contains an eight-segmented, negative-sense, single-stranded RNA genome

Antigenic Drift and Antigenic Shift

Pathogenesis and Treatment

Table 2. List of FDA-approved conventional avian influenza vaccines a

Inactivated influenza Year

Inactivated Vaccines (Inv)

Live-Attenuated Influenza Vaccines (LAIV)

Immune Response of Conventional Vaccines

Safety Concerns Associated with Conventional

Conventional Vaccines and Avian Influenza

Licensed vaccines against human influenza viruses are currently produced in

The use of conventional influenza vaccines in combination with appropriate

Virus-Like Particles (VLPs)

for influenza[72]. Should a pandemic occur, Ad vectors will provide an alternative to

Vaccine Delivery and Adjuvants

Table 3. Various recommended and hypothetical routes of vaccination and their

Route Pros Cons References

Intranasal Simulates natural course of infection Potential adverse CNS effects

Vaccine stability inside the

More immunogenic than intramuscular

The recommended route of immunization for the conventional influenza vaccines is IM

Oral vaccination is an attractive route of immunization due to its simplicity of handling

Several adjuvants have been shown to enhance immunogenicity when used in

Ami Patel1,2, Gary Wong1,2, Mickey Sahib1,2