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Emerging Viral Diseases of Southeast Asia

Issues in Infectious Diseases


Vol. 4

Series Editors

Heinz Zeichhardt Berlin Brian W. J. Mahy Atlanta, Ga.

Emerging Viral Diseases of Southeast Asia

Volume Editor

Sunil K. Lal

New Delhi

17 figures, 1 in color, and 7 tables, 2007

Basel Freiburg Paris London New York Bangalore Bangkok Singapore Tokyo Sydney

Issues in Infectious Diseases Sunil K. Lal, PhD


Senior Research Scientist, Virology Group International Centre for Genetic Engineering and Biotechnology New Delhi 110067 (India)

Library of Congress Cataloging-in-Publication Data Emerging viral diseases of Southeast Asia / volume editor, Sunil K. Lal. p. ; cm. (Issues in infectious diseases, ISSN 1660-1890 ; v. 4) Includes bibliographical references and index. ISBN-13: 978-3-8055-8175-2 (hardcover : alk. paper) ISBN-10: 3-8055-8175-0 (hardcover : alk. paper) 1. Virus diseasesSoutheast Asia. I. Lal, Sunil K. II. Series. [DNLM: 1. Communicable Diseases, EmergingAsia, Southeastern. 2. Virus DiseasesAsia, Southeastern. 3. Severe Acute Respiratory SyndromeAsia, Southeastern. WC 500 E525 2007] RA644.V55E442 2007 362.196 9200959dc22 2006022529 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents and Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Copyright 2007 by S. Karger AG, P.O. Box, CH4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 16601890 ISBN 3805581750 ISBN 978805581752

Contents

VII Foreword Lal, S.K. (New Delhi) 1 The Singapore Contribution in the Battle against the Severe Acute Respiratory Syndrome Fielding, B.C.; Tan, Y.-J. (Singapore) 23 Avian Influenza in Thailand Puthavathana, P.; Auewarakul, P.; Buranathai, C.; Aungtragoolsuk, N.; Kitphati, R.; Chotpithayasunondh, T. (Bangkok) 35 Emerging Viral Diseases of Fish and Shrimp Wang, M.; Lin, X.; Ma, G.; Bai, X. (Qingdao) 59 Avian Influenza H5N1 Virus: An Emerging Global Pandemic Lal, S.K. (New Delhi); Chow, V.T.K. (Singapore) 78 Henipaviruses: New Threats for Southeast Asia and Australia McCormack, J. (South Brisbane); Smith, G. (Coopers Plains) 94 Ross River Virus: An Arthritogenic Alphavirus of Significant Importance in the Asia Pacific Tupanceska, D.; Zaid, A.; Rulli, N.E.; Thomas, S.; Lidbury, B.A.; Matthaei, K.I.; Ramirez, R.; Mahalingam, S. (Canberra) 112 Emerging and Re-Emerging Infectious Diseases in Our Global Village Heymann, D.L. (Geneva)

125 The Role of a Pathology Laboratory in SARS and Other Emerging Infections Nicholls, J.; Peiris, J.S.M. (Hong Kong, SAR) 136 The Fight against Emerging Viral Diseases in Asia Lam, S.K. (Kuala Lumpur)

144 Author Index 145 Subject Index

Contents

VI

Foreword

Infectious viral diseases have always afflicted mankind and always will. New infectious diseases emerge as microbes adapt to new hosts and new environments. Asia has been a breeding ground for viruses where severe epidemics of dengue hemorrhagic fever (1954) and a variety of flu pandemics have originated such as the Asian flu (H2N2; 1957), the Hong-Kong flu (H3N2; 1968), and the Russian flu (H1N1; 1977). During the last 10 years, however, very dangerous viruses have repeatedly originated from Southeast Asia, e.g. the avian flu (H5N1) in Hong-Kong (1997), Nipah virus encephalitis in Malaysia (1998,) and, above all, the SARS outbreak from Southern China (2002). An estimated 75% of emerging infectious diseases in humans are zoonotic in origin. Microbes usually evolved to reach an equilibrium with their natural hosts without causing any disease. This microbe-host equilibrium which is a delicate balance, gets disturbed by economic development and land use leading to perturbations in the natural microbial environment, human demographics and behavior, and international travel and commerce thus creating an imbalance and increased possibilities to trigger the emergence of new infectious diseases. Microbes possess the ability to adapt naturally to their hosts, their environment, and to new ecological niches offered to them as humans encroach upon their territory. They are also adept at circumventing efforts to suppress them, whether as a result of internal host pressures such as innate and adaptive immune responses or in reaction to external pressure applied by antibiotics, antivirals, or vaccines. In the face of our efforts to eliminate them, microbes almost always adapt successfully, thwarting our efforts to destroy them. A prototypic example of the constant struggle between microbes and man is the evolutionary success

VII

of influenza viruses as they adapt to their many hosts, including humans. Similarly, the destruction of rainforests has led to exposure of humans to viruses and other microbes that otherwise would not have occurred, for example, an outbreak of Nipah virus in Malaysia occurred when pigs penned near fruit orchards contracted the virus from the droppings of bats, whose habitat had shifted as a result of deforestation. The infected pigs readily transmitted the virus to their handlers. Also, the success viruses have gained in the emergence of new occurrences may be attributed to the development of large industry poultry flocks increasing the risks of epizootics, dietary habits, economic and demographic constraints, and negligence in the surveillance and reporting of the first cases. New viruses do not emerge against a background of established infectious diseases and hostmicrobe interactions that have existed for centuries. For example, the newly emerging infectious diseases like Severe Acute Respiratory Syndrome (SARS), Nipah virus encephalitis, Lassa fever, and most recently, human disease caused by the H5N1 strain of avian influenza virus were at some point emerging diseases that had never been observed previously in human populations but are now slowly becoming a part of the background infectious disease burden. Infectious diseases that have previously occurred in humans also can re-emerge or resurge in different forms or in different environments as has been exhibited by the West Nile; monkeypox, and dengue virus. The SARS virus although contained, poses a similar threat since it is still at large in its zoonotic hosts viz., bats, civet cats and pigs. Today the world faces a threat of a much more unpredictable pandemic influenza, caused by the emergence of a new strain of influenza virus to which humans have never been exposed. Pandemics occur when a new influenza virus variant emerges to which the human population has no immunity. Influenza A viruses are most dangerous to humans because of their wide host range, their rapid mutation rate, and their capacity to cause serious disease. Over the past 2 years, the risk of an influenza pandemic has grown as an exceptionally virulent form of the H5N1 avian influenza virus and has circulated widely among domestic poultry and wild migratory birds in Asia, Europe, the Middle East, and Africa. As of February 9, 2006, the virus also has infected more than 166 people since late 2003, of whom half have died (WHO). Whether the virus develops into a strain capable of spreading from human to human in an efficient and sustained manner, thereby triggering a human pandemic, will depend on how the virus evolves and adapts to new hosts. Since its re-emergence in Southeast Asia in 2003, the virus has appeared in poultry in at least 18 countries and in multiple species of migratory birds, pigs, tigers, and leopards. As the virus has infected chickens and other domestic poultry, it has become increasingly virulent and has achieved the capability of jumping

Foreword

VIII

species to humans and to other animals with lethal consequences. Most alarmingly, the virus now seems to be transmitted from poultry back to migratory birds and, for the first time, is causing disease in the migrating bird population. This unprecedented pattern of transmission is an important reason why public health officials are watching the H5N1 virus carefully because it is a strain with the potential to cause the next influenza pandemic. Compared to 1918, we are much better equipped scientifically. We have the tools to monitor genetic sequences of influenza viruses as they evolve in both humans and birds. We also have the capacity to develop and manufacture countermeasures against new strains of influenza. As we prepare for the possibility of the next pandemic influenza, it will be important to optimize the use of available public health measures and scientific tools and technologies. Ongoing efforts in basic biomedical research are also critical to the comprehensive pandemic preparedness effort, including studies to understand viral pathogenesis, the ongoing search for new antivirals, new platforms and targets for vaccines, such as recombinant DNA and vector approaches, as well as improved vaccine manufacturing methods. It is essential to have a multipronged approach including surveillance, public health measures, and biomedical research with the ability to isolate infectious agents, decipher pathogenic mechanisms, and develop appropriate diagnostics, therapies, and vaccines are all critical components of a multipronged response to emerging and re-emerging infectious diseases, including both seasonal and pandemic influenza to develop effective antiviral drug stockpiling and vaccine development and distribution. Sunil K. Lal, New Delhi

Foreword

IX

Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 122

The Singapore Contribution in the Battle against the Severe Acute Respiratory Syndrome
Burtram C. Fielding, Yee-Joo Tan
Institute of Molecular and Cell Biology, Proteos, Singapore

Abstract
Severe acute respiratory syndrome (SARS) emerged in November 2002 in Guangdong Province, China. The disease finally spread to more than 30 countries, with more than 8,000 people infected worldwide. Under the coordination of the World Health Organization, laboratories from all over the world, including Singapore, worked together to identify and characterize the causative agent. SARS first reached Singapore in mid-March 2003 and by the end of the outbreak more than 230 probable cases of SARS were recorded in the small island-nation. Subsequently, the contribution of research from Singapore to understanding this potentially lethal infection and its causative agent has been significant. This review aims to record the contribution made by researchers in Singapore to the current understanding of SARS, including the epidemiology in the Singapore setting. Also the development of diagnostic tests such as antibody detection and polymerase chain reaction will be discussed. Finally, a summary of the research of the proteins associated with the SARS-coronavirus genome will be given.
Copyright 2007 S. Karger AG, Basel

An unusual form of atypical pneumonia emerged in China in November 2002. On February 11, 2003, the World Health Organization (WHO) was first notified about 305 infectious atypical pneumonia cases in Guangdong Province, including 5 deaths. By March 2003, the disease had spread to Hong Kong, Vietnam, Taiwan, Singapore and Canada. In response to this global threat, the WHO provided a preliminary case definition and initiated a worldwide network of laboratories to investigate the cause of what is now called severe acute respiratory syndrome (SARS) [1, 2]. Singapore General Hospital (SGH) formed an integral part of the WHO network of laboratories, providing a vital up-to-date information resource for researchers in Singapore, as well as making viral samples available for research purposes internationally.

The causative agent of SARS was identified as a novel coronavirus, now known as coronavirus (SARS-CoV) (for reviews, see [36]). SARS eventually spread to about 30 countries on 5 continents, infecting more than 8,000 people and resulting in 774 deaths. Since SARS had a dramatic impact on the health system and economy of Singapore, researchers of the island-nation became involved in the fight against this deadly disease early on. Subsequently, the contribution of research from Singapore to understanding this potentially lethal infection and its causative agent has been significant. This review aims to record the contributions made by researchers in Singapore to the current understanding of SARS, including the epidemiology in the Singapore setting. Also the development of diagnostic tests will be discussed. Finally, a summary of the research of the proteins associated with the SARS-CoV genome will be given.

Epidemiology Singapore

By mid-March 2003, SARS reached Singapore and was subsequently traced to three travelers returning from a holiday in Hong Kong [7, 8]. The index patient, a 23-year-old woman was admitted to Tan Tock Seng Hospital, Singapore, on March 1 with fever and a dry cough. Over the course of a few days, the virus spread rapidly from this patient to medical staff, patients and visitors, infecting 20 people in total, thereby initiating the SARS outbreak in Singapore [9]. The remaining two travelers from Hong Kong recovered without any further infections. Based on conventional epidemiological investigation, the index patient was linked to the infection of 4 patients, resulting in the four additional clusters of infection (fig. 1a) during a 2-month period [7, 10]. Three of these were infected by direct contact and the fourth was believed to have been infected by the index patient through another primary patient; three of the clusters were nosocomial and a fourth centered on a vegetable wholesale centre [11]. Sequencing the genome of the SARS-CoV isolated from the Singapore index patient and comparing it to 13 other sequences, confirmed that the index patient acquired the disease while staying at Hotel M, Hong Kong [12]. In fact, sequence analysis showed that all cases associated with Hotel M formed a phylogenetic cluster that is distinct from other viral isolates that have no direct link to Hotel M. Interestingly, the Singapore viral isolates appeared to be distinct from all other isolates, the former grouping in an additional phylogenetic cluster within the Hotel M-cluster [12]. Later, using mass spectrometry mini-sequencing to determine the genetic relationship of 13 SARS-CoV isolates from Singapore SARS patients, Liu et al. [13] reported inconsistencies in the original reported clinical transmission relationships. Based on sequence comparisons, they hypothesized that the fourth patient, or an as yet unidentified Singapore patient

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Number of cases infected by direct contact

60

Suspected SARS Probable SARS

50

40

30

20

10

0 1* 2 3 Case number 4 5

4 5

b Fig. 1. a Probable cases of SARS by reported source of infection. The figure excludes 22 cases with either no or poorly defined direct contacts or who were cases translocated to Singapore and the seven contacts of one of these cases (taken from Centers for Disease Control and Prevention (CDC), 2003). Cases 15 are shown and represent superspreaders in Singapore. b Infection clusters directly linked to the five superspreaders in Singapore. Probable and suspected cases of SARS are shown (taken from Centers for Disease Control and Prevention (CDC), 2003). *Represents the index case.

that infected the former, is the index case of all the late-generation SARS patients in Singapore. These data need to be confirmed by additional viral genetic characterization, especially from early-generation SARS cases. Superspreaders (an affected individual that infects 10 or more contacts) were responsible for the rapid transmission of SARS in Singapore, with only

SARS Research in Singapore

5 individuals linked to the infection of 144 of the probable SARS cases [10] (fig. 1b). Interestingly, the phenomenon of superspreading was also observed in other countries, including China, Toronto and Hong Kong [1417]. The reason for the ability of a patient to become a superspreader is not well understood, but contributing factors may include host characteristics (such as immune status, underlying disease, or co-infection with a second pathogen), higher levels of virus shedding, behavioral and environmental factors. Following stringent transmission control measures, including home quarantine, the outbreak of SARS in Singapore ended in late May 2003. By then, however, 33 of the 238 SARS patients diagnosed in Singapore died. In Singapore more than 70% of SARS infections were linked to hospitals and about 42% of patients were healthcare workers [7]. About 4 months after Singapore was declared SARS-free, a 27-year-old graduate student was admitted to SGH with fever. Investigations revealed that he had been working in a Bio-Safety Level (BSL) 3 laboratory on West Nile virus. Even though the patients symptoms were mild and his radiological findings developed late, the clinical features and incubation time were consistent with those described in the initial SARS outbreak [18, 19]. Ironically, on December 17, 2004, a coronavirus researcher from Taiwan was also diagnosed with SARS. He was isolated and no subsequent cross-infections were reported [20]. In both cases, exhaustive investigations ruled out all non-laboratory sources. In fact, sequencing data from the viral isolates obtained from the Singapore researcher and his SARS-CoV contaminated West Nile virus samples were highly identical [18]. This confirmed that the student was infected in the BSL3 laboratory that was involved in live SARS-CoV work during the initial outbreak. These cases highlight the potential infection risk posed by laboratories and shows that effective biosafety practices need to be adhered to.

SARS-CoV Cultivation in Cell Culture

In order to understand SARS-CoV pathogenesis, various groups are studying SARS-CoV infection and replication in cell culture systems. Researchers from the National University of Singapore, SGH and the National Environment Agency, Singapore, have worked together to characterize the growth and infection cycle of SARS-CoV in Vero E6 cells. In one study they reported that SARSCoV attaches, enters and uncoats within 30 min of infection. Several SARS-CoV particles were seen attaching to the cell plasma membrane as soon as 5 min after infection [21]. Another study reported rather rapid growth of the virus in Vero E6, resulting in dramatic ultrastructural changes in the cells, including swelling of the Golgi sacs [22]. The swelling was due to the accumulation of mature virions

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within the Golgi lumens, where coronaviruses are known to assembly and bud [23]. Following a latent period of 5 h postinfection (p.i.), extracellular virus was present and by 1215 h p.i., large accumulations of both intracellular and extracellular virus particles were observed [22]. Later, the same group documented the topographic changes in SARS-CoV-infected Vero E6 cells during the late stages of infection [24]. Using scanning electron and atomic force microscopy, they examined the maturation of SARS-CoV at the cell surface and showed that the cellular cytoskeleton network is involved in this process. In order to understand the hosts response to SARS-CoV infection at the cellular level, various groups compared the gene expression profiles of infected cells to that of healthy cells. One such group in Singapore analyzed the expression pattern of about 8,700 genes in PBMCs isolated from SARS patients [25]. They noted no significant upregulation of MHC-1 class or major cytokine genes, including interferons that are usually activated in viral infections. Genes that were upregulated included lactoferrin and bactericidal permeability increasing protein, to name a few. Both of these genes have functions related to neutrophil activity. Another interesting gene upregulated was lipocalin 2, which belongs to a class of secreted proteins that are thought to trigger apoptosis in immune cells [26]. Hence, the upregulation of lipocalin 2 during SARS infection may be a host response to limit tissue damage and inflammation. Taken together, these results showed that the response of SARS infected patients is mainly an innate inflammatory response and does not appear to be a specific immune response against the virus. Due to the genetic similarity of monkey and human genomes, another group in Singapore used SARS-CoV-infected Vero E6 cells as an alternative model for studying gene responses to SARS-CoV infection in lieu of human cells [27]. They reported 70 transcripts with altered expression and classified these according to their function. The transcription of many functionally distinct genes were changed including genes involved in host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking and modulators, apoptosis, and cytoskeletal proteins. Interestingly, they reported the downregulation of ANXA2, a signal transduction molecule, and speculated that this could lead to decrease fibrinolysis during SARS-CoV infection (see also section on Group-specific genes for 3a-fibrinogen link). Not only has the identification of permissive cell lines led to the study of host response, it also enables us to study the dynamics of the SARS-CoV in cell culture and to screen for SARS-CoV antiviral drugs. Scientists from the Genome Institute of Singapore, the National Environment Agency, Singapore, and SGH collaborated to determine the mutation frequency and dynamics of the SARSCoV [28]. By comparing the whole genome sequences of SARS-CoV isolated after different passages in Vero E6, they concluded that the overall rate of

SARS Research in Singapore

mutation in culture is low, suggesting that the virus is well-adapted to growth in cell culture. These scientists also screened 19 clinically approved antiviral drugs for in vitro anti-SARS-CoV activity [29]. The commercially available antivirals included the following pharmacological classes: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors and neuraminidase inhibitors. Using a Vero E6 cell based SARS-CoV cytopathic endpoint assay, they reported that four interferon subtypes (interferon- -1b, interferon- -n3, interferon- -n1 and human leukocyte interferon- ) showed complete inhibitory activity to SARS-CoV Peglylated interferon- was also showed to protect cynomologus . macaques from SARS infection [30], but thus far, its efficacy and efficiency in humans have not been validated in clinical trials.

Diagnostics

Prompt SARS-CoV diagnosis is critical for the management of patients and control of the disease syndrome, as well as the containment of SARS. In the following section, we will focus on the diagnostic assays developed by researchers from Singapore. Detection of Antibodies The use of immunofluorescence to detect for antibodies to SARS-CoV in patients is known to be specific and is useful in confirmation of SARS-CoV infection, hence a whole-virus-based immunofluorescence assay (IFA) was identified as the gold standard by WHO. So, in order to minimize the handling of live SARS-CoV researchers have been developing IFAs that employ the , immunodominant domains of SARS-CoV structural proteins, in particular the spike (S) and nucleocapsid (N) proteins. These IFAs aim to be cost effective, reproducible and non-hazardous, while being sensitive and specific in high throughput screening. The immunodominant fragments of SARS-CoV S (amino acids (aa) 441700) [31] and N proteins [32] were used to create S protein-based [33] and N-S fusion protein-based IFAs [34]. Both IFAs showed high specificity and sensitivity and are comparable to a commercial SARS IFA kit (EUROIMMUN, Germany) and a conventional IFA that was routinely used in Singapore hospitals during the SARS outbreak. Whats more, due to the overexpression of the protein in the baculovirus expression system, the S protein-based assay requires only onetenth of the serum used in the commercial IFA, making it more sensitive [33]. Various groups in Singapore also reported the detection of antibodies against SARS-CoV in patient serum using Western blotting and enzyme-linked immunosorbent assay (ELISA) [32, 3538]. Our group was the first to analyze

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Device number 1 2 3

Control line N 3a

Control

Patient 1

Patient 2

Fig. 2. Examples of rapid immunochromatographic test devices (Genelabs Diagnostics). Devices number 2 and 3 (on the right) after an assay with samples from 2 confirmed SARS patients. The two lines in the viewing window of device 2 for a sample from Patient 1 represent the control-line and GST-N, respectively. The three lines in the viewing window of device 3 for a sample from patient 2 represent the control-line, GST-N and GST-3a, respectively. The device on the left (number 1) shows a device after an assay with a sample from a healthy individual. The control line, which contains protein A that can bind with the anti-human IgG, serves as an indication of proper sample addition and migration.

the range of antibody responses to SARS-CoV during an infection. We demonstrated that sera from SARS patients showed immunoreactivity to N, S and a protein unique to SARS-CoV called 3a (see section on Group-specific genes) , [39]. Whereas all patient sera samples tested showed reactivity to N, only 73% of them showed reactivity to 3a. The knowledge gained from this study was used to develop one of the first rapid immunochromatographic assays for the detection of SARS infection (fig. 2) as well as an ELISA kit [36]. Both tests assay for the immunoglobulin G (IgG) antibodies against the bacterially expressed SARS-CoV recombinant proteins GST-N and GST-3a. The assays detected the IgG antibodies to SARS-CoV from both late-convalescence-stage (more than 21 days after onset of symptoms), as well as from early-acute-phase (110 days from onset) samples with overall specificities in excess of 97%. Similarly, researchers from SGH collaborated with their counterparts in Japan to develop a rapid immunochromatographic test that detects for the SARS-CoV N antigen [40]. Their kit is a qualitative assay for respiratory aspirates and serum samples and is based on the sandwich format enzyme

SARS Research in Singapore

immunoassay which detects for N using monoclonal antibodies produced in mice. Using a sample size of 200, the researchers reported a specificity of 100%. The analytical sensitivity of the assay was found, as expected, to be lower than that of RT-PCR, but comparable to that of ELISA. Detection of Viral RNA Due to its high specificity and sensitivity, RT-PCR and other nucleic acid tests are the preferred methods for the diagnosis of SARS [3, 4, 6]. Our colleagues at the Institute of Molecular and Cell Biology, in collaboration with scientists at Tan Tock Seng Hospital, Singapore, developed a gel-based one-step RT-PCR assay for detection of SARS-CoV [41]. They selected the genetically stable nsp1 region of the proteinase gene to design the primers. The sensitivity of this gel based assay is comparable with the commercial Artus RealArt HPAcoronavirus RT-PCR kit (Germany) and an in-house real-time PCR assay. Its simplicity of use is especially suitable in laboratories without expensive realtime PCR equipment. On the other hand, researchers at the Genome Institute of Singapore have designed primers against the conserved polymerase gene (RdRp) which are used in a quantitative real-time PCR assay [42]. Using in vitro transcribed RNA and virus spiked samples including blood, sputum and stool suspension the sensitivity of the assay was determined to be less than 85 copies per reaction. Furthermore, this assay is effective in detecting SARS-CoV in blood samples from infected patients. In another study, Loon et al. [43] reported the use of RT-PCR to screen tear samples from suspected SARS patients in Singapore. Some of the samples collected early in the course of infection tested positive for SARS-CoV Obviously, . this possible source of infection has serious health implications for health-care workers that come in contact with SARS-CoV infected patients and the necessary precautions have to be taken. Clearly, there have been significant advancements in the assays used for the detection of SARS-CoV Tests have become more sensitive and specific, . thereby eliminating cross-reactivity with other viruses and hence reducing false-positive results.

Characterization of Viral Proteins Encoded by the SARS-CoV Genome

Basic research to understand the virus at the genetic level will have implications for the development of vaccine and antiviral therapeutics, and in this section, we will highlight research activities in this area. The SARS-CoV genome is approximately 30 kb in length and contains 14 potential open reading

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Replicase (1a/1b)

10

15

20

25

30 kb

3b S 3a E M 6 7a

7b 8a

8b

9b N

Fig. 3. Structural organization of the SARS-CoV genome. The replicase genes (ORFs 1a/1b), which constitute the first 2/3 of the genome, are indicated by black solid boxes. Open reading frames (ORFs) in the remaining 1/3 of the genome are translated from eight subgenomic mRNAs. Four of these encode the structural proteins (checked boxes), spike (S), membrane (M) and envelope (E) and nucleocapsid (N). Another eight SARS-CoV-unique ORFs (grey solid boxes) encode accessory proteins with no significance sequence homology to viral proteins of other coronaviruses (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b).

frames (ORFs) [44]. Some of these ORFs encode for the replicase polyproteins (pp1a and pp1ab) and the structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) which are common to all members of the genus coronavirus [45]. The other 8 ORFs encode for group-specific accessory proteins varying in length from 39 to 274 aa (fig. 3). In our laboratory, we have obtained specific antibodies against the structural proteins, as well as several of the accessory proteins (fig. 4) and used them to characterize the expression, cellular localization and processing of these viral proteins in SARS-CoV infected Vero E6 cells (see below). In order to consolidate the information from different publications, we will adopt the nomenclatures used by Ziebuhr [46] as they are most consistent with those used for other coronaviruses. Alternate names that have been used in specific publications were noted in a recent review [47]. Replicase Gene (ORFs 1a and 1b) Analogous to other coronaviruses, the first two thirds of the SARS-CoV genome encodes the viral replicase genes (ORFs 1a and 1b), which translates into two large polyproteins, pp1a (486 kDa) and pp1ab (790 kDa) [46]. Expression of the ORF 1b-encoded region of pp1ab involves ribosomal frameshifting into the 1 frame just upstream of the ORF 1a translation termination codon. Proteolytic processings of these polyproteins are mediated by viral cysteine proteinases and produces a minimum of 13 nonstructural proteins (also called nsps), some of which are responsible for replicating the viral genome and/or generating a nested set of subgenomic mRNAs to express all the

SARS Research in Singapore

j Fig. 4. Intracellular localization of over-expressed SARS-CoV structural and groupspecific proteins. Vero E6 cells were transfected with plasmid constructs encoding for specific untagged SARS-CoV proteins. At about 16 hours post transfection, the cells were fixed and stained with antibodies raised against S (a), E (b), M (c), N (d), N-terminal of 3a (e), C-terminal of 3a (f), 7a (g) and (h), 3b (i) and 8b (j) proteins. The antibodies used were either rabbit polyclonal (af) and (i, j), mouse polyclonal (g) or mouse monoclonal (h). FITC-conjugated goat anti-rabbit or anti-mouse (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was used as secondary antibody. All antibodies, except rabbit anti-3b (Abgent, San Diego, Calif., USA), were raised by the Collaborative Antiviral Research Group at Institute of Molecular and Cell Biology, Singapore.

ORFs downstream of ORF 1b. Unlike most coronaviruses, which uses three proteinases for polyprotein processing, SARS-CoV is predicted to have only two proteinases which are a papain-like (accessory) cysteine proteinase (termed as PL2pro) and a 3C-like (main) proteinase (termed 3CLpro or Mpro). Shi et al. [48] at the National University of Singapore solved the three-dimensional structure of 3CLpro by NMR spectroscopy and found that 3CLpro exists as a dimer, which is consistent with earlier findings by another crystallography group [49]. They further showed that the extra C-terminal domain found in 3CLpro of

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10

Receptor-binding domain 300 -S 1 48 -S 3 168 362 1 Spike protein TMD 461 -S 2 790 HR1 HR2 CD 1255 798 358 1029 -S 9 1055 510 -S 10 1192

Fig. 5. Schematic diagram showing the five bacterially expressed regions of the spike (S) protein used by the Collaborative Antiviral Research Group at Institute of Molecular and Cell Biology, Singapore to raise antibodies. These antibodies were tested for their ability to block the interaction between S and its cellular receptor ACE-2, and to inhibit SARS-CoV propagation in Vero E6 culture. The number indicated at the ends of each black box represents the amino acid residue position of each fragment in the S protein. Also, the receptor binding domain is shown (a.a. 300510). CD cytoplasmic domain; HR1 Heptad repeat region 1; HR2 heptad repeat region 2; TMD transmembrane domain; anti. Adapted from Keng et al. [52].

SARS-CoV and other coronaviruses, but not found in the picornavirus 3C proteases, contributes to the formation of the active dimeric form of the protease. In addition, they also studied the fine conformational details of its interaction with substrates, thus providing the basis for rational drug design. Structural Proteins (S, E, M and N) Coronaviruses are positive-strand RNA viruses and the virion consists of a nucleocapsid core surrounded by an envelope containing three membrane proteins, S, E and M, while the RNA is packaged by the N protein into a helical nucleocapsid. The S protein is known to be responsible for inducing host immune responses and virus neutralization by antibodies [45]. Indeed, we demonstrated the presence of anti-S antibodies in the sera of SARS-CoV-infected patients by subjecting mammalian cells stably expressing the full-length S protein on the cell surface (CHO-SG cells) to immunofluorescence analysis [39, 50]. These Sexpressing cells associated tightly with Vero E6 cells that express a SARS-CoV receptor, the carboxypeptidase angiotensin-converting enzyme-2 (ACE2). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. Using rabbit anti-sera raised against five denatured recombinant S protein

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fragments expressed in Escherichia coli (fig. 5), we found that one of the sera obtained from the fragment encompassing aa 48358 significantly blocked the interaction between CHO-SG and Vero E6 cells, which is consistent with other publications that showed that the receptor binding domain of S is located around aa 300510 [see review, 51]. This work also describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its cellular receptor. In order to determine antigenic determinants that can elicit neutralizing antibodies, we further tested our rabbit anti-S polyclonal antibodies for their ability to inhibit SARS-CoV propagation in Vero E6 culture. Our results showed the anti-S 10 antibody, which was raised against aa 10291192 of S and included the heptad repeat 2 (HR2), has neutralizing activities that is comparable to the level detected in convalescent patients [52]. The interaction between the two heptad repeat domains (HR1 is located at aa 891975 and HR2 located at aa 11161193) in the C-terminal region of S brings the fusion peptide, predicted to be near the N terminus of HR1, into close proximity to the transmembrane domain, and this facilitates the fusion between viral and cellular membranes, allowing the virus to enter the cell [53, 54]. It may be postulated that the anti-S 10 antibody binds the HR2 domain with high affinity and blocks the interaction between HR1 and HR2, thus preventing SARS-CoV fusion with the host cells. Our results are consistent with the findings of several other laboratories that peptides from the HR2 region can block SARS-CoV infection [53, 55, 56]. As bacterially expressed proteins would be easy and cost effective to produce on a large scale, the S 10 fragment (aa 10291192) identified in this study may be an ideal vaccine candidate for SARS-CoV In future . studies, we would like to determine if the immunization of recombinant S 10 protein can prevent SARS-CoV replication and, more importantly, prevent disease in animal models. Numerous laboratories in Singapore have purified the SARS-CoV N protein using different expression systems and demonstrated its suitability as a diagnostic marker [32, 34, 39, 57; see section on Diagnostics]. Early work in our laboratory also showed that the N protein is phosphorylated and can be exported out of mammalian cells [58]. Indeed, later studies from other laboratories showed that N protein can be detected in patients sera, indicating that the extracellular export of N protein may occur in vivo [59, 60]. In addition, Singapore researchers also collaborated with their Indian counterparts to demonstrate self-association of the N protein using the yeast twohybrid system and co-immunoprecipitation experiments with mammalian cell lysates [61]. Also, they showed that the C-terminal 209 aa region constitutes the interaction domain responsible for self-association of the N protein. Further collaborative efforts from these scientists then revealed that N is capable of

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inducing apoptosis of COS-1 monkey kidney cells in the absence of growth factors by down-regulating ERK (extracellular-signal-regulated kinase), upregulating JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) pathways [62]. More recently, they showed that the phosphorylated N protein is translocated to the cytoplasm by binding to 143-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) [63]. A separate laboratory in Singapore showed that the N protein undergoes posttranslational modification by sumoylation [64]. The major sumoylation site was mapped to the lysine residue at position 62 of the N protein. The sumoylation of the N protein drastically promotes its homo-oligomerization and also modulates its ability to disrupt cell division. The same laboratory later showed that the SARS-CoV E protein can induce membrane permeability changes in Escherichia coli [65]. E protein expressed in bacterial and mammalian cells under reducing conditions exist as monomers, but form homodimer and homotrimer under nonreducing conditions. Using molecular dynamics simulations, they showed that E can form dimeric, trimeric, and pentameric aggregates through its putative transmembrane domain [66]. Taken together, these findings suggest that the SARS-CoV E protein could be a viroporin, which is a group of proteins that affects cellular trafficking and membrane permeability, thereby promoting the release of viral particles from cells [67]. Group-Specific Genes Of the group-specific SARS-CoV proteins (ORF 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b) (fig. 3), 3a is the largest and consists of 274 aa. Our work was the first to demonstrate the presence of anti-3a antibodies in SARS patients in Singapore [39], and this finding was subsequently verified in cohorts of patients from other countries [68, 69, 70]. Interestingly, in two separate cohorts of SARS patients, one from Taiwan [71] and one from Hong Kong [72], B cells recognizing the N-terminal region of 3a and before the first putative transmembrane domain, were isolated from patients. Taken together, these data strongly suggest that 3a could play an important immunological role as it is clearly presented to the host immune system during infection. We were also one of the three groups that first reported the expression of 3a in SARS-CoV infected cells [69, 70, 73]. We also determined the topology of 3a experimentally: its N terminus is facing the extracellular matrix and its C-terminus is facing the cytoplasm [73]. Together with our collaborators from Taiwan and China, we then showed that 3a is a structural protein as it is associated with virion purified from SARS-CoV-infected cells and it is incorporated into viral-like particles when co-expressed with M and E in the baculovirus system [74]. Indeed, our findings were independently verified by another group that detected 3a in purified virions and showed the association of 3a with intracellular

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virions especially at the plasma membrane before virus budding [75]. 3a is localized in the perinuclear region and is also transported to the cell surface, where it can undergo internalization [69, 73, 75]. Taken together with the experimental observation that 3a and S can interact [70, 73], it is possible to postulate that 3a may be able to modulate the expression of S on the cell surface [76]. In order to determine the effects of 3a on host functions, we analyzed the gene expression profile of 3a-expressing A549 cells, a lung epithelial cell-line. Our results showed that the expression of 3a upregulates the mRNA levels of all three subunits, A , B , and , of fibrinogen [77]. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells. Coincidently, upregulation of fibrinogen mRNA in PBMCs infected by SARSCoV was reported by another laboratory in Singapore [78]. The excessive production of fibrinogen and formation of fibrin at the site of injury may enhance cytokine production or imbalance procoagulant and/or fibrinolytic activities [79, 80]. Postmortem examinations of SARS victims in Singapore and elsewhere revealed extensive lung damage that is typical of acute respiratory distress syndrome [8184]. In addition, most SARS patients in Singapore and elsewhere have thrombocytopenia, elevated D-dimers, and prolonged activated partial thromboplastin time, which suggest dysregulation of the coagulation and fibrin polymerization pathways [7, 8589]. Our data demonstrated that expression of 3a alone can up-regulate the expression of fibrinogen, suggesting that 3a may contribute to SARS pathogenesis. We initially observed mutations in the 3a gene in a Singapore isolate after three passages in cell culture, resulting in the expression of several different forms of 3a protein in infected cells [73]. When we further examined viral RNA isolated directly from clinical samples, we found similar mutations suggesting that the mutation in 3a is not due to cell culture adaptation, but indicates the presence of quasi-species [90]. In fact, sequence comparison of isolates from different clusters of infection showed that both S and 3a showed positive selections during virus evolution, implying that these proteins play important roles in the virus life cycle and/or disease development [91]. The effects of these mutations on the biochemical properties of 3a remain to be determined. We also demonstrated the expression of another group-specific protein, 7a, in SARS-CoV-infected cells [92]. The 7a viral protein contains a cleavable signal peptide at the N-terminus and a transmembrane domain near the C-terminus. An endoplasmic reticulum (ER) retrieval motif (KRKTE), which is important for transport of proteins back to the ER [93], is located at the C-terminus cytoplasmic domain of 7a and mediates the recycling of 7a between the ER and Golgi apparatus such that 7a is present in the intermediate compartments, where coronaviruses are known to assemble and bud [23]. Interestingly, 7a can

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also interact with 3a, which can interact with M, E and S, suggesting that these viral proteins may form complexes during infection [73]. We further showed that the overexpression of 7a induces apoptosis via a caspase-dependent pathway, and in cell-lines derived from different organs, including lung, kidney and liver [94]. The ability of 7a to induce apoptosis in different cell-types is consistent with the clinical observation of apoptosis in different organs infected by SARS-CoV and suggests that 7a may contribute to viral pathogenesis. For example, lymphopenia, which is caused by the depletion of T lymphocytes by apoptosis, is one of the most common clinical abnormalities in SARS patients in Singapore and elsewhere [9598]. From the studies of other coronaviruses, accessory proteins are usually dispensable for viral replication in cell culture systems but may be important for viralhost interactions and thus contribute to viral stability and/or pathogenesis in vivo. It has not yet been established which of the SARS-CoV accessory proteins are essential for viral replication and/or for viral-host interactions. Nevertheless, the results from biochemical analysis of the 3a and 7a proteins suggest that they could play important roles in viral assembly or pathogenesis. Interestingly, a recent study showed that the SARS-CoV ORF6 protein can enhance the virulence of an attenuated murine coronavirus [99]. Future studies utilizing animal models and reverse genetics methods will be important to define the exact roles of these accessory proteins.

Concluding Remarks

In this chapter, we have described the various research activities on the SARS-CoV in Singapore since the outbreak of SARS here in March 2003. These virological studies are highly diverse and span the area of epidemiology, diagnostics and biochemical characterization of viral proteins. The findings of these scientific investigations have implications for diagnostics, vaccine and antiviral therapy developments, and form a critical part of our preparation for future outbreaks of infectious diseases. Although the SARS epidemic has been controlled by isolation, research on different aspects of this virus is not waning, as it is not known if the SARS-CoV will re-emerge, especially since its origins and potential reservoir(s) are unresolved. After the WHOs declaration of the end of the SARS epidemic, there were 4 confirmed SARS patients in Guangzhou, China, in late 2003 to early 2004 [100, 101]. These patients did not have any contact history with previously documented SARS cases. Sequence analysis of viruses isolated from these patients showed that they were not derived from the preceding epidemic in 2003 but likely from a common ancestor, suggesting that these cases represented new

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zoonotic transmissions [101]. Since this virus is still lingering in its yet to be identified natural host, there exists future opportunities for it to cross the species barrier and cause another human outbreak. Hence, it is important to continue to track viruses in wildlife and hunt for the natural reservoir of SARS-CoV . Asymptomatic infection or presentation of only mild symptoms makes the identification of SARS infection a challenge. In Singapore, several cases of atypical presentation were reported [88, 102105] and asymptomatic SARS infections in exposed healthcare workers were documented [106]. For the latter, there was no transmission probably because of the lower viral titer. Atypical presentations mainly arose because of complicated pre-existing conditions and in at least 2 of these cases, the failure to identify them early as SARS patients resulted in nosocomial transmissions [104, 105]. Clearly, the advances in the development of diagnostic assays for the detection of SARS infection (see above) will help to identify such cases. Nevertheless, the broad spectrum of clinical symptoms associated with SARS means that a high level of vigilance has to be maintained in order to catch a re-emergence promptly. In summary, the collaborations between different laboratories in Singapore as well as our collaborations with groups in different countries resulted in numerous publications in international peer-reviewed journals. A closer working relationship between hospitals, research laboratories and the industry has also resulted from these collaborations. Indeed, these research activities on SARS-CoV have allowed us to strengthen our interaction with our collaboration partners and also to build new networks with scientists all over the world. In addition, this SARS epidemic has also allowed Singapore scientists to realize our strengths and weakness, and provided a platform for us to strengthen our capabilities in infectious diseases and virus research. At the end of the day, Singapore has learnt to formulate better strategies to detect and effectively respond to the next event, be it a re-emergence of SARS, another flu pandemic or the emergence of another novel virus. Some of the lessons we have learnt in hospital management and community surveillance, which have been summarized in recent reviews [9, 107109], are very important and will help us handle future outbreaks of infectious diseases more effectively. Together with our collaborators in different countries, we hope to continue to identify new diseases and outbreaks as they occur, to study these infections and find ways to contain and treat them, and to implement the necessary measures to defeat them.
Acknowledgements
We thank members of the Collaborative Anti-Viral Research group, Institute of Molecular and Cell Biology, for critically reading the manuscript. We apologize to any investigators whose work we have inadvertently omitted.

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Idell S: Adult respiratory distress syndrome: do selective anticoagulants help? Am J Respir Med 2002;6:383391. Ware LB, Matthay MA: The acute respiratory distress syndrome. N Engl J Med 2000;342: 13341349. Chong PY, Chui P, Ling AE, Franks TJ, Tai DY, Leo YS, Kaw GJ, Wansaicheong G, Chan KP, Ean Oon LL, Teo ES, Tan KB, Nakajima N, Sata T, Travis WD: Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: challenges in determining a SARS diagnosis. Arch Pathol Lab Med 2004;128:195204. Franks TJ, Chong PY, Chui P, Galvin JR, Lourens RM, Reid AH, Selbs E, McEvoy CP, Hayden CD, Fukuoka J, Taubenberger JK, Travis WD: Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore. Hum Pathol 2003;34:743748. Hwang DM, Chamberlain DW, Poutanen SM, Low DE, Asa SL, Butany J: Pulmonary pathology of severe acute respiratory syndrome in Toronto. Mod Pathol 2005;18:110. Nicholls JM, Poon LL, Lee KC, Ng WF, Lai ST, Leung CY, Chu CM, Hui PK, Mak KL, Lim W, Yan KW, Chan KH, Tsang NC, Guan Y, Yuen KY, Peiris JS: Lung pathology of fatal severe acute respiratory syndrome. Lancet 2003;361:17731778. Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, Ahuja A, Yung MY, Leung CB, To KF, Lui SF, Szeto CC, Chung S, Sung JJ: A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003;348:19861994. Lew TW, Kwek TK, Tai D, Earnest A, Loo S, Singh K, Kwan KM, Chan Y, Yim CF, Bek SL, Kor AC, Yap WS, Chelliah YR, Lai YC, Goh SK: Acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome. JAMA 2003;290:374380. Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, Law KI, Tang BS, Hon TY, Chan CS, Chan KS, Ng JS, Zheng BJ, Ng WL, Lai RW, Guan Y, Yuen KY, HKU/UCH SARS Study Group: Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 2003;361:17671772. Singh K, Eong OE, Kumarsil B, Saw S, Sethi S: Severe acute respiratory syndrome without respiratory symptoms or abnormal chest radiograph findings. Clin Infect Dis 2004;38:585586. Wong RS, Wu A, To KF, Lee N, Lam CW, Wong CK, Chan PK, Ng MH, Yu LM, Hui DS, Tam JS, Cheng G, Sung JJ: Haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis. BMJ 2003;326:13581362. Tan THP, Barkham T, Fielding BC, Chou C-F, Shen S, Lim SG, Hong W, Tan Y-J: Genetic lesions within the 3a gene of SARS-CoV. Virol J 2005;2:51. Yeh SH, Wang HY, Tsai CY, Kao CL, Yang JY, Liu HW, Su IJ, Tsai SF, Chen DS, Chen PJ, National Taiwan University SARS Research Team: Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: molecular epidemiology and genome evolution. Proc Natl Acad Sci USA 2004;101:25422547. Fielding BC, Tan Y-J, Shen S, Tan THP, Ooi E-E, Lim SG, Hong W, Goh P-Y: Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome (SARS) coronavirus. J Virol 2004;78:73117318. Teasdale RD, Jackson MR: Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the Golgi apparatus. Annu Rev Cell Dev Biol 1996;12:2754. Tan Y-J, Fielding BC, Goh P-Y, Shen S, Tan THP, Lim SG, Hong W: Over-expression of 7a, a protein specifically encoded by the Severe Acute Respiratory Syndrome (SARS)-coronavirus, induces apoptosis via a caspase-dependent pathway. J Virol 2004;78:1404314047. Chng WJ, Lai HC, Earnest A, Kuperan P: Haematological parameters in severe acute respiratory syndrome. Clin Lab Haematol 2005;27:1520. ODonnell R, Tasker RC, Roe MF: SARS: understanding the coronavirus: apoptosis may explain lymphopenia of SARS. BMJ 2003;327:620. Panesar NS: Lymphopenia in SARS. Lancet 2003;361:1985. Singh K, Hsu LY, Villacian JS, Habib A, Fisher D, Tambyah PA: Severe acute respiratory syndrome: lessons from Singapore. Emerg Infect Dis 2003;9:12941298. Pewe L, Zhou H, Netland J, Tangudu C, Olivares H, Shi L, Look D, Gallagher T, Perlman S: A severe acute respiratory syndrome-associated coronavirus-specific protein enhances virulence of an attenuated murine coronavirus. J Virol 2005;79:1133511342.

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100 Liang G, Chen Q, Xu J, Liu Y, Lim W, Peiris JS, Anderson LJ, Ruan L, Li H, Kan B, Di B, Cheng P, Chan KH, Erdman DD, Gu S, Yan X, Liang W, Zhou D, Haynes L, Duan S, Zhang X, Zheng H, Gao Y, Tong S, Li D, Fang L, Qin P, Xu W, SARS Diagnosis Working Group: Laboratory diagnosis of four recent sporadic cases of community-acquired SARS, Guangdong Province, China. Emerg Infect Dis 2004;10:17741781. 101 Song HD, Tu CC, Zhang GW, et al: Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc Natl Acad Sci USA 2005;102:24302435. 102 Fisher DA, Lim TK, Lim YT, Singh KS, Tambyah PA: Atypical presentations of SARS. Lancet 2003;361:1740. 103 Ho K, Singh KS, Habib AG, Ong BK, Lim TK, Ooi EE, Sil BK, Ling AE, Bai XL, Tambyah PA: Mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in Singapore. J Infect Dis 2004;189:642647. 104 Tan TT, Tan BH, Kurup A, Oon LL, Heng D, Thoe SY, Bai XL, Chan KP, Ling AE: Atypical SARS and Escherichia coli bacteremia. Emerg Infect Dis 2004;10:349352. 105 Tee AK, Oh HM, Lien CT, Narendran K, Heng BH, Ling AE: Atypical SARS in geriatric patient. Emerg Infect Dis 2004;10:261264. 106 Wilder-Smith A, Teleman MD, Heng BH, Earnest A, Ling AE, Leo YS: Asymptomatic SARS coronavirus infection among healthcare workers, Singapore. Emerg Infect Dis 2005;11:11421145. 107 Gopalakrishna G, Choo P, Leo YS, Tay BK, Lim YT, Khan AS, Tan CC: SARS transmission and hospital containment. Emerg Infect Dis 2004;10:395400. 108 Quah SR, Hin-Peng L: Crisis prevention and management during SARS outbreak, Singapore. Emerg Infect Dis 2004;10:364368. 109 Wilder-Smith A, Low JG: Risk of respiratory infections in health care workers: lessons on infection control emerge from the SARS outbreak. Southeast Asian J Trop Med Public Health 2005;36:481488.

Yee-Joo Tan, PhD Collaborative Antiviral Research Group Institute of Molecular and Cell Biology, 61 Biopolis Drive Proteos 138673 (Singapore) Tel. 65 65869625, Fax 65 67791117, E-Mail mcbtanyj@imcb.a-star.edu.sg

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Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 2334

Avian Influenza in Thailand


P Puthavathanaa, P Auewarakula, C. Buranathaib, N. Aungtragoolsuka, . . c R. Kitphati , T. Chotpithayasunondhd
a Faculty of Medicine Siriraj Hospital, Mahidol University, bDepartment of Livestock Development, Ministry of Agriculture, cDepartment of Medical Science, dQueen Sirikit National Institute of Child Health, Ministry of Public Health, Bangkok, Thailand

Abstract
Thailand first recognized the massive die-offs of poultry by late 2003, and the government officially reported the outbreak of H5N1 pathogenic influenza virus on January 23, 2004. At present, the country had already passed three waves of the epidemics. Outbreaks mostly occurred in the central and eastern parts of the country where water reservoirs and wetlands are abundant and poultry population is dense. The outbreaks also happened when the weather was wet and cool. Poultry vaccination is prohibited in Thailand. Pre-emptive depopulation and several means of biosecurity measures have been applied to control the outbreaks. Free-grazing ducks, fighting cocks and backyard chickens remain to be the major problems of the country. Of all three outbreaks, there were 22 human cases with 14 deaths which made the fatality rate 63.6%. The disease was more fatal in children than adults. The infection also spread to other mammalian species: tigers, leopards and cats. Phylogenetic analysis revealed that all of the eight genomic segments of the viruses isolated from humans were of avian origin which suggested direct transmission of virus from avian to man. The Thailand H5N1 viruses are resistant to amantadine and their genetic properties are closely related to the Vietnam viruses, and grouped together in genotype Z.
Copyright 2007 S. Karger AG, Basel

Influenza viruses are divided into three immunological types: A, B and C. Based on antigenic difference of hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are further divided into 16 HA subtypes and 9 NA subtypes. Aquatic birds have been proposed to be the ancestors and natural hosts of all influenza A viruses existing in human and animals. Even though all influenza A subtypes can be found in aquatic birds, only 3 subtypes, H1N1, H2N2 (existed between 1957 and 1968) and H3N2, are recognized as human

influenza viruses [1]. Up to the present, transmission of highly pathogenic avian influenza virus (HPAI) to human has been reported with H5N1 [2, 3] H7N7 [4] and H9N2 [5] subtypes only, and the H5N1 virus is shown to be much more virulent than the others.

Epidemiology of Avian Influenza Viruses

Outbreaks of HPAI H5 viruses occurred in many regions of the world. In 1993, H5N2 virus caused a widespread outbreak in Mexico. The virus was originally low pathogenic and caused a reduction in egg production. Within a year, the virus acquired high pathogenicity through an insertion of multiple positively charged amino acids at the hemagglutinin cleavage site [6]. This virus belonged to American lineage and was only distantly related to the current H5N1 virus in Asia. In 19971998, multiple outbreaks of HPAI H5N2 were found in Italy. This Italian virus belonged to the Eurasian lineage and had hemagglutinin that was related to that of the Hong Kong 1997 viruses [7, 8]. Outbreak of the HPAI H5N1 virus in Hong Kong in 1997 was the first reported direct transmission of avian influenza (AI) from poultry to human. There were 18 cases with six deaths occurring in this outbreak [2, 9]. Despite a total depopulation of poultry in Hong Kong, the virus re-emerged and led to two human deaths in 2003 [3]. The return of H5N1 virus since 2003 is more devastating and cannot be eradicated until now. The virus swept across East to Southeast Asia where the virus persists. At present, the H5N1 virus spread over Asia to Europe and Africa.

Outbreaks of Avian Influenza in Thailand

After the first outbreak of H5N1 virus in Hong Kong in 1997, the Department of Livestock Development (DLD) had conducted both active and passive surveillance in avian and swine. However, the H5N1 virus had never been detected until the first recognition of the massive die-off of poultry in the Central and Northern provinces of the country in late 2003. The nationwide surveillance was implemented in December 2003 to detect human cases, and it was extended to poultry in mid-January 2004. Laboratory investigation for human cases was conducted by the National Institute of Health (NIH), Department of Medical Science (DMS), Ministry of Public Health, and also by the Faculty of Medicine Siriraj Hospital, Mahidol University. Antigen detection, virus isolation and molecular subtyping of viruses by conventional reverse transcription-polymerase chain reaction (RT-PCR) and

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real time RT-PCR were performed and the first two human cases were confirmed independently by both institutes on January 23, 2004. Meanwhile, DLD had sent the first avian influenza isolate from poultry to University of Hong Kong in January 2004 for subtype identification and led to the announcement of H5N1 infection in poultry by the Ministry of Agriculture also on January 23, 2004. At present, there are many laboratories in Thailand which can conduct H5N1 diagnosis. Those laboratories include the National Institute of Animal Health, DLD, the NIH, DMS and several regional laboratories of both institutes. University hospitals also support and transfer technology to strengthen those authorized laboratories. Up to the present, Thailand has suffered three major waves of epidemic of HPAI H5N1. The first official report for presence of HPAI came from a layer chicken farm in Suphanburi province, and the last report of HPAI was from a layer farm in Chiangmai. The first outbreak ended on May 24, 2004 after affecting 188 from a total of 71,864 villages or 146 of 7,409 subdistricts in 42 of 76 provinces. During this period, approximately 30 million birds were culled to confine the epidemic. The clearance of the first outbreak was probably due to both intensive intervention and the weather, which was dry and hot with average temperature of 30 C and peak temperature of higher than 40 C in April. The second wave of the epidemic was confirmed on July 3, 2004 in a layer farm in Pranakorn Sriayudhaya province and the last confirmed case was detected in a backyard flock in Lopburi province. The infected flock was stamped out together with depopulation and disinfecting process, which was completed on April 12, 2005. Lopburi province was declared HPAI free on May 3, 2005. Collectively, the second outbreak spread over 784 subdistricts in 51 provinces and a total of 6.6 million birds were culled. The third wave of the epidemic occurred even under the extensive surveillance for prevention and control, which have been conducted continuously. A small outbreak in poultry farms and backyard chicken lasted to the end of November and it is worse by the occurrence of 5 human cases with 2 deaths. The epidemic began on July 1, 2005 in a quail farm in Suphanburi province. The third outbreak spread over 56 subdistricts in 11 provinces and a total of 400,000 birds were culled during July to November 2005. The poultry population is dense in the Central and Eastern provinces of the country. Outbreaks are also concentrated in those regions including the southern part of the Northern region. The geographical location of those epidemic areas are wetlands with plenty of water reservoirs. Several provinces in the Central and the southern part of the northern regions such as Suphanburi, Kanchanaburi, Nakornpathom, Pranakorn Sriayudhaya and Kamphaengphet frequently face the situation of repeated outbreaks. Part of the details of these outbreaks and poultry management policy in Thailand were previously reported by Tiensin et al. [10] in 2005. Epidemiological findings indicated that the majority of infected animals

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were backyard poultry, fighting cock, and free-grazing duck. In contrast, poultry in the commercial system were rarely affected since most of them were certified standard farms and maintained in high biosecurity. Vaccination against AI is prohibited in Thailand. Therefore, some special conditions were applied to these susceptible groups of poultry such as pre-emptive stamping-out, disinfection and movement control. Depopulation of clinically suspected flock is carried out as fast as possible without requirement of laboratory investigation. Compensation for the cost of poultry is 75% of the local market price. Repeated cycles of the outbreak suggested that occurrence of avian flu is seasonal dependent. There are three seasons in Thailand: summer, which lasts from March to May, rainy season from June to October and cold season from November to February. The outbreaks occur when the weather is wet and cold.

Characterization of H5N1 Viruses

Origin of the H5N1 virus causing the outbreak in Hong Kong in 1997 was revealed by sequence analysis. The virus appeared to be a reassortant which acquired HA gene from the Goose/Guangdong/1/96(H5N1)-like virus (GsGd), NA gene from A/teal/Hong Kong/W312/97(H6N1)-like virus, and internal genes are probably derived from A/teal/Hong Kong/W312/97(H6N1) or A/quail/Hong Kong/G1/97(H9N2)-like virus [11, 12]. After a total depopulation of poultry in Hong Kong, the virus disappeared. However, the ancestors of this virus still existed in southern China and continued on reassortment with other avian viruses resulting in multiple reassortant forms, which are called genotypes. Several genotypes emerged and disappeared. The H5N1 virus that causes widespread outbreaks in many countries in Asia since December 2003 to the present was a new reassortant and was categorized as genotype Z. It contained polybasic amino acids at the HA cleavage site characteristic of HPAI; the same as the Hong Kong H5N1 virus 1997. HA and NA of the new virus are still in the GsGd lineage similar to all other H5N1 viruses found in this region. Although Thailand, Vietnam, and Indonesia viruses belonged to the same genotype Z, the Thailand and Vietnam viruses clustered closely together, while Indonesia virus formed a distinct clad in a phylogenetic analysis of HA. This indicated that the ancestor of Indonesia virus was not the same as that of the Thailand and Vietnam viruses [13]. The Indonesia viruses are closely related to current viruses isolated in Yunan and Hunan, while the Thailand and Vietnam viruses are closely related to Hong Kong viruses isolated in 2003. This indicated that all the viruses originated from Southern China. In June 2005, there was an outbreak in migratory birds at Qinghai Lake in Western China. The viruses of this outbreak have spread widely by migratory birds to Europe and Africa. Although this virus formed a separate cluster in phylogenetic

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analyses, it is more related to the Indonesia viruses than to the Thailand and Vietnam viruses. It has been proposed to call Thailand and Vietnam viruses as clade 1, and Indonesia and Qinghai Lake viruses as clade 2. The viruses in different sublineages differed not only genetically but also in their antigenic properties. This would make vaccine development for H5N1 influenza more complicated [14]. It is interesting that all the HPAI in this region maintain the HA and NA of GsGd lineage despite multiple reassortment events that provided the viruses with abundant genetic materials from other avian viruses. During the last several years the virus has exchanged several internal genes through reassortments, but the HA and NA are maintained. Therefore, it is suggested that this H5 and N1 combination has been optimized for rapid spreading in domestic poultry. It has been hypothesized that a gain of an N-linked glycosylation site in HA globular head and a deletion at the stalk of NA complemented each other and optimized the virus for efficient infection and spreading in land-based poultry [15]. As compared to Hong Kong H5N1 virus 1997, the new H5N1 virus possesses one more glycosylation site at the globular head and one more amino acid deletion in the NA stalk (20 amino acid deletion instead of 19 amino acid deletion) [16]. Viral Virulence H5N1 viruses which swept across East and Southeast Asia since 2003 were more virulent than H5N1 viruses of the Hong Kong outbreak of 1997. We found the overall fatality of about 63.6% (14 of 22 cases) which is similar to that of Vietnam, while the fatality of 33% (6 of 18 cases) was reported from the Hong Kong outbreak 1997 [9]. There has been a lot of interest in identifying genetic determinant of the highly pathogenic phenotype. The multiple positively charged amino acid sequence at the cleavage site of HA is clearly an important factor, but PB2 and NS genes have been also shown to influence the viral virulence. A mutation from glutamic acid to lysine at the amino acid position 627 on PB2 was shown to associate with virulence in mice. Most human viruses as well as viruses from other mammalians, such as tigers, contain lysine at this position, while avian viruses contain glutamic acid. Regarding the NS gene, it has been suggested that a mutation from glutamic acid to aspartic acid at position 92 confers the property of cytokine hyperinduction to the virus [17]. This mutation was found in Hong Kong H5N1 virus 1997 and in this new H5N1 virus, but it is absent in other nonpathogenic viruses. Highly pathogenic H5N1 viruses have been shown to hyper-induce the production of proinflammatory cytokines from human primary macrophages [18]. An immunopathogenesis model was raised to explain widespread damage despite limited viral replication in human. The high pathogenicity of this HPAI is therefore likely to be contributed by multiple genetic elements.

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Antiviral Drugs Amantadine and rimantadine are the first drugs for treatment and prophylaxis of influenza virus type A. It acts as the M2 ion channel blocker to prevent the release of the ribonucleoprotein complex from endosome into cytoplasm. A mutation in any one of the four amino acids in transmembrane protein at residues 26 (Leu to Phe), 27 (Val to Ala or Thr), 30 (Ala to Thr or Val) and 31 (Ser to Asn or Arg) can confer drug resistance property to the virus [19]. The H5N1 viruses from outbreak 1997 were reported to be sensitive to amantadine, while amantadine resistance was common during the present outbreak. We have demonstrated two point mutations in the M gene: the change from serine to asparagine at position 31, and the second position, which may not confer much effect, was the change from leucine to isoleucine at position 26. Amantadine resistance of three human H5N1 virus isolates had been also shown phenotypically [16]. While Thailand and Vietnam viruses are amantadine resistant, most viruses in China and the virus from Qinghai Lake that are currently spreading to Europe and Africa are amantadine sensitive. Oseltamivir has been brought into Thailand for treatment of the avian flu patients since the first outbreak. It is demonstrated that a higher concentration of oseltamivir is required to inhibit H5N1 virus replication in vitro [20].

H5N1 Avian Influenza in Human Cases in Thailand

There were 12 human cases with 8 deaths occurring during the first outbreak and 5 cases with 4 deaths during the second one. No case had been found after October 2004 until the re-emergence of the third outbreak. As of November 30, 2005, there were 5 cases with 2 deaths. Overall, there is a total of 22 cases with 14 deaths which give rise to the overall fatality rate of 63.6%. Nevertheless, the fatality rate in children is higher than in adults. Demographic Data of the Patients Of 22 Thai patients from the three waves of the epidemic, 12 were less than 14 years old with a male to female ratio of 1.4:1 (13 vs. 9). Most of these patients resided in the central and a few in the lower northern regions of the country. Geographic location where the patients lived correlated well with density of poultry population. Source of Infection Phylogenetic analysis revealed that all of the eight segments of H5N1 genomes derived from human isolates were of avian origin which suggested direct transmission of the avian virus to humans. Regarding 22 Thai patients,

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Table 1. Clinical description of H5N1 influenza in 17 Thai patients Symptom Fever, cough and dyspnea Sore throat Myalgia Diarrhea Rhinorrhea Vomiting Abdominal pain Number of cases (%) 17 (100) 12 (71) 9 (53) 7 (41) 9 (53) 4 (24) 4 (24)

10 had a history of contact with sick or dead chickens, 11 cases had no direct contact but lived in the villages with the event of poultry die-off; 1 case contracted the disease by providing bedside care of a probable case [21], Practices common to the Thai victims were preparing meat from the carcasses of the infected sick or dead chicken, playing with chickens, and handling fighting cocks. Although some countries reported consuming fresh duck blood or uncooked meat as risk factors to contract the infection, this mode of transmission is not generalized for Thailand. It is interesting that there were only 22 patients among several thousands of people at risk, e.g. poultry farmers, workers in slaughter houses and cullers. At the early phase of the first outbreak, these people did not wear or use any protective devices, but symptomatic H5N1 infection could not be found. Clinical Manifestation Severe symptoms and fatal outcome of influenza H5N1 have been reported [22]. However, the patients with mild symptoms had been recognized during the third outbreak. Detailed information of 17 patients from the first two outbreaks is presented here. The clinical presentation during hospitalization of these Thai patients is shown in table 1. Chest X-ray at early onset of disease showed lobar and patchy pneumonia in most of the cases and interstitial pneumonia was only found in 2 cases. At late onset, acute respiratory distress syndrome was found in all 12 dead cases, while it was present only in 1 of the 5 survivor cases. In contrast to ordinary human influenza virus infection, H5N1 viruses induce multi-organ failure which may be fatal. Outcomes of the disease in these patients are shown in table 2. An unusual manifestation of influenza H5N1 had been recognized in a Vietnamese patient who was hospitalized with acute encephalitis and coma and

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Table 2. Outcome of 17 Thai patients with H5N1 influenza Symptom Respiratory failure Cardiac failure Renal failure Liver failure Bone marrow failure Dead Number of cases (%) 13 (76) 7 (41) 5 (29) 3 (18) 1 (6) 12 (71)

died without development of pneumonia [23]. A Thai patient also developed diarrhea with led to hospitalization before subsequent development of pneumonia [24]. Blood profiles among 17 patients had shown 58% leucopenia, 58% lymphopenia and 33% thrombocytopenia without anemia. High levels of liver enzyme were found in 67%, and serum creatinine levels higher than 1.5 mg/dl were found in 33% of the cases. Part of this information was reported previously [22]. Pathogenesis of H5N1 Virus in Humans The pathogenesis of H5N1 avian influenza in humans is not well understood. In Thailand, despite 14 fatal cases, only 3 autopsy cases were investigated. Unlike other avian and mammalian species, H5N1 infection in humans is poorly disseminated to distal organs. Using RT-PCR to detect negative or positive stranded viral RNA demonstrated that virus replication was limited to lung, trachea and probably other organs including intestine and liver. We found by immunohistochemistry that the major target cells for viral replication are alveolar epithelial cells [25]. We also found an upregulation of TNF- in the lung of an autopsy case [25]. This is in agreement with the previously raised hypothesis that hyperinduction of pro-inflammatory cytokines by H5N1 viruses may be an important pathogenic mechanism especially in humans [17, 18]. The virus has been shown to survive the host innate defense by cytokines. The ability of the virus to continue replicating while the high level of cytokines induces inflammation and tissue damage may be the reason of its high virulence. The location of viral target cells in alveoli may explain the clinical feature, which is mainly pneumonitis. It is not clear whether the distribution of sialic acid receptor plays a role in the type of target cells.

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H5N1 Avian Influenza in Animals in Thailand

Poultry Despite the high pathogenicity in terrestrial domestic poultry, most HPAI are nonpathogenic in aquatic birds, which are the natural or reservoir hosts of influenza virus. The H5N1 virus which emerged in Hong Kong in 1997 affected mainly terrestrial poultry. The H5N1 variant which caused lethal infection in aquatic birds was recognized in Hong Kong in November 2002 from the death of egrets, gray heron, Canada geese and ducks [26]. The outbreak of H5N1 virus in Thailand produced massive deaths in both terrestrial and aquatic birds: poultry including layers, broilers, backyard chickens, fighting cocks, geese, quails and ducks. The overall epidemiological findings indicated that the majority of infected animals which are still the problems nationwide were freeranging ducks, backyard poultry and fighting cocks. Grazing duck flocks, which feed on rice grains after harvesting season, move from one rice field to several other rice fields over a distance of a hundred kilometers. This has been a common style of duck raising in Vietnam and Thailand for several decades. Moreover, most of the houses in villages in the rural areas keep backyard poultry for their daily food. This kind of life style is based on the purpose of self-sustained economics; in addition, in remote areas, to keep a cold chain of fresh food to the markets is not practical. Cock fighting has been a type of native sport for hundreds of years. This kind of cock is well taken care of. They live closely together with the owners, even sharing the same rooms. One common practice among some owners of fighting cocks is to suck secretion from the cocks throat by mouth to mouth in order to clear the respiratory airway of the cocks. Other Animals H5N1 viruses were also isolated from wild-living birds, especially the open-billed stork which shares the same paddled fields as the free-grazing ducks. Some of these birds are migratory, but small groups have settled and become permanent residents. It is also to be noticed that the viruses have now spread to domestic birds such as pigeons and sparrows. Unexpected species to acquire H5N1 infection are captive tigers (Panthera tigris) and leopards (Panthera pardus) through feeding on contaminated raw chicken carcasses. Horizontal transmission was also demonstrated in tigers kept in the same zone. H5N1 infection is fatal in tigers. The virus disseminated to several distal organs and the sick animals usually died of respiratory distress condition [27]. H5N1 virus infection in cats was also demonstrated [28]. It was also demonstrated that H5N1 viruses could infect pigs. Of 3,175 pig serum samples collected from slaughter houses in Vietnam in January 2004,

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8 (0.25%) had antibody to H5N1 Vietnam isolates as tested by neutralization test and Western immunoblotting [29]. Vietnam or Thailand H5N1 isolates could replicate at modest titer in the respiratory tract of the inoculated pigs, but no contact transmission was found under experimental conditions. Respiratory symptoms varying from mild cough to pneumonia were strain dependent [29].

Ducks as the Pandemic Threat

It is believed that influenza viruses in aquatic birds were in an evolutionary stasis. The viruses have reached an equilibrium that allows their existence in a natural host without causing disease. However, the currently spreading H5N1 viruses, as well as the viruses isolated in China after 2002, are highly pathogenic in ducks. The event led to the suggestion that equilibrium between the H5N1 virus infection and aquatic birds as the natural reservoir host was broken down. The long-term host-viral interaction was disturbed by the viral adaptation for rapid spread in domestic poultry. In vivo experimentation has shown that H5N1 viruses from Vietnam and Thailand replicated efficiently in the inoculating ducks and elicited a wide spectrum of disease symptoms ranging from asymptomatic infection to neurological symptoms and death. Pattern of viral shedding changed to support the efficiency of viral transmission via the respiratory route. Higher titers of viruses were shed from trachea rather than the digestive tract of the infected contact ducks. Therefore, it is likely that the pattern of transmission is also deviated from the primarily fecal-oral route to favor the respiratory route [30]. It is likely that the H5N1 virus will readapt to its natural host in the near future. This would result in a virus that is highly pathogenic in chickens but nonpathogenic in ducks. Such viruses would cause an enormous obstacle to the viral eradication attempt and ducks would play an important role as the viral reservoir.

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Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, Meijer A, van Steenbergen J, Fouchier R, Osterhaus A, Bosman A: Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 2004;363:587593. Peiris M, Yuen KY, Leung CW, Chan KH, Ip PLS, Lai RWM, Orr WK, Shortridge KF: Human infection with influenza H9N2. Lancet 1999;354:916917. Garcia M, Crawford JM, Latimer JW, Rivera-Cruz E, Perdue ML: Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico. J Gen Virol 1996;77:14931504. Donatelli I, Campitelli L, Trani LD, Puzelli S, Selli L, Fioretti A, Alexander DJ, Tollis M, Krauss S, Webster RG: Characterization of H5N2 influenza viruses from Italian poultry. J Gen Virol 2001;82:623630. Capua I, Marangon S, dalla Pozza M, Terregino C, Cattoli G: Avian influenza in Italy 19972001. Avian Dis 2003;47(3 suppl):839843. Chan PKS: Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in 1997. Clin Infect Dis 2002;34(suppl 2):S58S64. Tiensin T, Chaitaweesub P, Songserm T, Chaisingh A, Hoonsuwan W, Buranathai C, Parakamawongsa T, Premashthira S, Amonsin A, Gilbert M, Nielen M, Stegeman A: Highly pathogenic avian influenza H5N1, Thailand, 2004. Emerg Infect Dis 2005;11:16641672. Hatta M, Kawaoka Y: The continued pandemic threat posed by avian influenza viruses in Hong Kong. Trends Microbiol 2002;10:340344. Subbarao K, Shaw MW: Molecular aspects of avian influenza (H5N1) viruses isolated from humans. Rev Med Virol 2000;10:337348. Li KS, Guan Y, Wang J, et al: Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 2004;430:209213. Chen H, Smith GJD, Li KS, et al: Establishment of multiple sublineages of H5N1 influenza virus in Asia: Implications for pandemic control. Proc Natl Acad Sci USA 2006;103;28452850. Matrosovich M, Zhou N, Kawaoka Y, Webster R: The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 1999;73:11461155. Puthavathana P, Auewarakul P, Charoenying PC, Sangsiriwut K, Pooruk P, Boonnak K, Khanyok R, Thawachsupa P, Kijphati R, Sawanpanyalert P: Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J Gen Virol 2005;86:423433. Seo SH, Hoffmann E, Webster RG: Lethal H5N1 viruses escape host anti-viral cytokine responses. Nat Med 2002;8:950954. Cheung CY, Poon LLM, Lau AS, Luk W, Lau YL, Shortridge KF, Gordon S, Guan Y, Peiris JSM: Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 2002;360:18311837. Suzuki H, Saito R, Masuda H, Oshitani H, Sato M, Sato I: Emergence of amantadine-resistant influenza A viruses: epidemiological study. J Infect Chemother 2003;9:195200. The World Health Organization Global Influenza Program Surveillance Network: Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 2005;11:15151521. Ungchusak K, Auewarakul P, Dowell SF, et al: Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med 2005;352:333340. Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, et al: Human disease from Influenza A (H5N1), Thailand, 2004. Emerg Infect Dis 2005;11:201209. de Jong MD, Cam BV, Qui PT, Hien VM, Thanh TT, Hue NB, Beld M, Phuong LT, Khanh TH, Chau NVV, Hien TT, Ha DQ, Farrar J: Fatal avian influenza A (H5N1) in a child presenting with diarrhea followed by coma. N Engl J Med 2005;352:686691. Apisarnthanarak A, Kitphati R, Thongphubeth K, et al: Atypical avian influenza (H5N1). Emerg Infect Dis 2004;10:13211324. Uiprasertkul M, Puthavathana P, Sangsiriwut K, Pooruk P, Srisook K, Peiris M, Nicholls JM, Chokephaibulkit K, Vanprapar N, Auewarakul P: Influenza A H5N1 replication sites in humans. Emerg Infect Dis 2005;11:10361041.

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Ellis TM, Bousfield RB, Bissett LA, Dyrting KC, Luk GS, Tsim ST, Sturm-Ramirez K, Webster RG, Guan Y, Malik Peiris JSM: Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathol 2002;33:492505. Thanawongnuwech R, Amonsin A, Tantilertcharoen R, et al: Probable tiger-to-tiger transmission of avian influenza H5N1. Emerg Infect Dis 2005;11:699701. Kuiken T, Rimmelzwaan G, van Riel D, van Amerongen G, Baars M, Fouchier R, Osterhaus A: Avian H5N1 influenza in cats. Science 2004;306:241. Choi YK, Nguyen TD, Ozaki H: Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004. J Virol 2005;79:1082110825. Sturm-Ramirez KM, Hulse-Post DJ, Govorkova EA, Humberd J, Seiler P, Puthavathana P, Buranathai C, Nguyen TD, Chaisingh A, Long HT, Naipospos TS, Chen H, Ellis TM, Guan Y, Peiris JSM, Webster RG: Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia. J Virol 2005;79:1126911279.

Dr. Pilaipan Puthavathana Department of Microbiology Faculty of Medicine, Siriraj Hospital Mahidol University Bangkok 10700 (Thailand) Tel. Fax 66 2 4184148, E-Mail siput@mahidol.ac.th

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Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 3558

Emerging Viral Diseases of Fish and Shrimp


Min Wang, Xiaoting Lin, Guangyong Ma, Xiaoge Bai
Department of Biology, College of Marine Life Sciences, Ocean University of China, Qingdao, China

Abstract
Generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. Fish and shrimp, the main aquaculture product sources, have gained the most attention. Many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. Among various causative agents of fish and shrimp diseases such as bacteria, fungi, parasites and so on, viruses are one of the most destructive pathogens. Until now, approximately 60 different viruses have been detected in fish. This chapter describes 25 viral diseases (caused by 29 different fish viruses) which have been classified according to virus families, and has described in detail 5 extensively studied emerging fish diseases namely infectious pancreatic necrosis virus (IPNV), channel catfish virus disease (CCVD), infectious hematopoietic necrosis (IHN), infectious salmon anemia (ISA) and lymphocystis disease (LCD), pathogens of which are IPNV CCV, IHNV ISAV and LCDV, respectively. Also , , the latter half of the chapter focuses on five emerging shrimp viruses, namely white spots syndrome virus (WSSV), yellow-head virus (YHV), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV).
Copyright 2007 S. Karger AG, Basel

Emerging Viral Pathogens of Fish

This chapter describes the different viruses that have recently been detected in fish and shrimp. In the first half of the chapter we have described viruses of the fish and the latter deals with viruses of shrimp. For each virus we have described the biology of the virus, its clinical signs, transmission and current methods of prevention.

Infectious Pancreatic Necrosis Infectious pancreatic necrosis virus (IPNV) was first isolated in North America in 1960 and then confirmed as the pathogen of infectious pancreatic necrosis (IPN), a contagious, high-mortality disease of young, hatchery-reared salmonids. IPNV is the typical species of the genus Aquabirnavirus of the family Birnaviridae and these genus members infect fish, mollusks and crustaceans. IPNV is a naked, icosahedral, double-stranded RNA virus with a mean diameter of 60 nm [1]. The molecular weight of IPNV is 55 7 106 Da and IPNV possesses a sedimentation coefficient of 440 S and bands at a density of 1.32 g/ml in CsCl. IPNV replicates in a variety of fish cell lines at temperatures below 24 C and may be visible in 1620 h at 22 C [2]. IPNV exerts much of its cytopathic effects in cell culture through the induction of apoptosis and may contribute to the nature of the disease and increase mortality. IPNV persistence in an aqueous environment is at least 300 days at 4 C and 60 days at 14 C [3]. The IPNV virus genome consists of two segments of double-stranded RNA, which represents 8.7% of the virion mass. The larger segment, A, encodes a polyprotein of approximately 106 kDa which is autoproteolytically cleaved to form the viral capsid proteins VP2 and VP3, and a nonstructural (NS) protein. Recently, the active residues in the NS protein responsible for processing of the polyprotein have been identified, thus proving that NS represents the viral protease [4]. A 17-kDa (VP5) protein encoded by a second open reading frame (ORF) of segment A which precedes and partially overlaps the polyprotein gene has been detected in virus-infected cells [5]. Genome segment B encodes a 94-kDa protein, VP1, which represents the putative viral RNAdependent RNA polymerase [6]. Clinical Signs Affected fish exhibit a variety of external signs of disease and have depressed hematocrit values. Internally, the liver and spleen appear pale, and the stomach and intestine are devoid of food but filled with mucoid fluid. Petechial hemorrhages are evident throughout the pyloric and pancreatic tissues. Pancreatic acinar cells undergo massive necrosis and degenerative changes also occur in the renal hematopoietic tissue. Transmission Once IPNV has been introduced into a population, horizontal transmission occurs as a result of ingestion of contaminated feces, water, or other materials and when fish pass contaminated water through their buccal cavity and over their gills. Smith et al. [7] had assumed that the only possible mode of transmission

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is horizontal according to their study. However, Seeley et al. [8] injected zebra fish (Brachydanio rerio) with IPNV and found the virus was passed on to the eggs after spawning, which affirmed the vertical transmission of IPNV . Besides, this transmission was found to be via the female alone [8]. Mulcahy and Pascho [9] had found the adsorption to fish sperm of IPNV supporting the , existence of the vertical transmission of IHNV. Prevention The replication of IPNV was inhibited in vitro by guanine 7-N-oxide, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), halocyamine A (an antimicrobial substance isolated from hemocytes of the solitary ascidian Halocynthia roretzi), 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and pyrazofurin (the orotidine monophosphate decarboxylase inhibitor). However, some of the compounds were also found toxic to host cells, such as ribavirin, which suppressed cellular DNA and RNA synthesis within 23 h after addition of the drug [10]. Different strategies for developing IPN vaccines have been tested since the virus was first isolated in 1960. Vaccination with live virus has not been successful and is probably not an acceptable strategy for environmental risk reasons. Vaccination with inactivated virus has been tested in rainbow trout given by the oral route, by immersion and by injection. Protection against challenge was obtained only by injection. Oral and immersion vaccination of Brook trout fry with inactivated, purified virus were not protective. IPN vaccines based on inactivated virus may be effective but are expensive. Subunit vaccines seemed to be most effective among all the vaccines in the vaccination of fish against IPNV. Labus et al. [11] reported the recombinant antigen made from VP2 of IPNV were capable of inducing antibodies reactive with whole IPNV in ELISA. On DNA vaccination, recently Mikalsen et al. [12] reported a high level of protection induced by the plasmid expressing the fulllength large ORF polyprotein of Segment A of the IPNV Microteks researchers . also constructed a novel recombinant DNA based expression system in Escherichia coli, which allows producing large quantities of recombinant IPN vaccine [http://www.microtek-intl.com]. This vaccine is currently in experimental stages and laboratory challenge trials are being setup in Chile.

Channel Catfish Virus Disease Channel catfish virus (CCV; ictalurid herpesvirus 1) was first isolated by Fijan et al. [13] and was classified into Ictalurivirus in Herpesviridae on the basis of morphology. All herpesviruses are morphologically conserved and invariably

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exhibit a large (100 nm diameter) thick-walled nucleocapsid, surrounded by an envelope with an intervening proteinaceous layer, despite their diversity of host range and genome size. It has been shown that the pH and the culture medium is an important factor in maintaining virus infectivity. The temperatures higher than 4 C rapidly inactivate the virus while this one can be grown successfully after several months of storage at 75 C and at 20 C. In common with other herpesviruses, CCV is quickly inactivated by UV irradiation. CCV DNA displays a buoyant density of 1.715 g/cm3 in a cesium chloride solution, with a genome of 56.1% GC content and 85 106 Da molecular weight. The CCV genome (134 kb) is composed of a unique long region (97.1 kb) bordered by 18.5-kb direct repeats. The direct repeats contain ORFs 114, while the unique long region contains ORFs 1579. Repeat regions are a common feature of herpesviruses and often contain genes that encode products that are important in viral pathogenesis, including latency-associated transcripts (LATs) [14]. The 1,035-bp ORF59 of CCV codes for an abundant hydrophobic membrane glycoprotein considered to be the major envelope glycoprotein of the virus. So far, two susceptible cell lines, BB (brown bullhead) and CCO (channel catfish ovary) cell lines were available for the propagation of CCV. The CCO cell line was shown to be the more sensitive cell line for CCV research and diagnostics. Both cell lines yield cytopathic effect of cell fusion at 2448 h postinfection and the best replication temperature of CCV in BB cells is at 25 C. Growth of CCV did not occur at 37 C. Clinical Signs Fish with the disease swim erratically, eventually becoming lethargic, and often hang vertically in the water with their heads near the surface. Infected fish exhibit exophthalmia, hemorrhagic lesions at the base of fins, and hemorrhage and necrosis in the viscera, especially the liver and kidney [15]. Transmission The sites where the virus is most abundant during the course of overt infection are posterior kidney, skin, gills, spleen and intestine, respectively, in decreasing magnitude. The transmission of CCV is horizontal and vertical. Horizontal transmission may be direct or vectorial with water being the main abiotic vector. The virus has been shown to readily adsorb to pond sediments and interaction with suspended clay particles in pond water may influence horizontal transmission. Animate vectors and inanimate objects could also act in CCV transmission. Wise et al. [16] had successfully mated two CCV-positive fish and collected fertilized eggs. Offspring that hatched were tested and were shown to have CCV [16]. This result indicated vertical transmission of CCV,

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but the mechanism of vertical transmission is not known, as infectious virus has not been detected on the skin or in the sexual products of spawning adults. Prevention The incubation of eggs and rearing of fry and juveniles in facilities separated from carrier populations are critical to preventing the occurrence of CCVD in a CCV-free fish production site. Because virus is only detected during active outbreaks, defining CCV-free status has been done largely from historical data or identifying populations that are seronegative to the virus [17]. Acyclovir was very effective on inhibiting the replication of CCV in vitro. Interestingly, Chinchar et al. [18] found channel catfish reovirus (CRV), a double-stranded RNA virus, could inhibit CCV replication by two different mechanisms, directly as a consequence of its own replication and indirectly due to the induction of an antiviral factor. Only laboratory trials of vaccines against CCVD have been reported. Both vaccination of eggs of fry with a subunit vaccine and immunization of fingerlings with an attenuated virus vaccine have been successful in the protection of catfish from CCV infection [19]. Nusbaum et al. [17] have performed a DNA vaccination trial of CCV on channel catfish. Single injections of DNA expression constructs containing ORF 59 (38% in vaccine efficiency), encoding the envelope glycoprotein, or ORF 6 (15% in vaccine efficiency), encoding a presumptive membrane protein, were found to elicit the strongest resistance. Even more effective was a combination vaccine pair in which both ORF 59 and ORF 6 expression constructs (46% in vaccine efficiency) were injected. Both ORF 6 and ORF 59 were able to elicit virus neutralizing antibodies capable of an anamnestic response on viral challenge. This evidence provides adequate proof of principle for the use of DNA vaccines in channel catfish and the effectiveness of the resistance to viral infection they elicit.

Infectious Hematopoietic Necrosis Virus Infectious hematopoietic necrosis (IHN) is caused by an enveloped singlestranded RNA (ssRNA) Rhabdovirus, so-called infectious hematopoietic necrosis virus (IHNV). IHNV is classified into the genus Novirhabdovirus of the family Rhabdoviridae. The IHNV genome is comprised of six viral genes in the order 3 -N-P-M-G-NV-L-5 , which encode a nucleoprotein (N), a polymeraseassociated protein (P), a matrix protein (M), a glycoprotein (G), a nonstructural nonvirion protein (NV), and an RNA-dependent RNA polymerase (L). The NV gene is located between the G and L cistrons and is the characteristic gene that

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distinguishes Novirhabdovirus from other genera in Rhabdoviridae. The virus normally produces an acute infection in rainbow trout (Oncorhynchus mykiss) fry that eventually results in death, and mortality rates in hatcheries can reach as high as 8090% [20]. Clinical Signs The primary pathological lesion in diseased fish is extensive necrosis of the hematopoietic tissues. During an IHNV infection, the spleen and kidney are among the primary sites of infection and inflammation. In terminal cases, necrosis of the liver, pancreas, and granular cells of the lamina propria of the gut is also found [21]. Transmission IHNV is usually spread by survivors of infections, which carry subclinical infections. When such fish mature, they may shed the virus during spawning. Transmission of virus was demonstrated via both the horizontal and vertical routes. Beside feces, urine, spawning fluids and mucus secretions of clinically infected fish, contaminated equipment, blood sucking parasites (e.g. leeches, Argulus spp.) and fish-eating birds can also spread the disease horizontally. Eggs from infected fish are usually infective and lead to the vertical transmission. However, if eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. Otherwise, if fry subsequently became infected they were lifetime carriers. Mulcahy and Pascho [9] found the adsorption to fish sperm of IHNV providing another proof , for the vertical transmission of IHNV. Prevention Eggs, alevins and fry should be reared on virus-free water supplies in premises completely separated from possible IHNV-positive carriers. Broodstock from sources with a history of IHN outbreaks should also be avoided wherever possible. Simon et al. [22] reported the development of an IHNV subunit vaccine produced by a new protein production system based on the bacterium Caulobacter crescentus, which only provided relative percent survival of 2634% in rainbow trout fry. The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of IHNV in rainbow trout using DNA vaccine technology were assessed, and only the DNA vaccine encoding G protein induced significant protection and protective neutralizing antibodies. LaPatra et al. [23] reported that DNA vaccine pIHNw-G was shown to provide significant protection as soon as 4 days after intramuscular vaccination in 2 g rainbow trout (O. mykiss) held at 15 C. Nearly complete

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protection (higher than 90%) was observed at later time points (7, 14, and 28 days postvaccination) using a standardized waterborne challenge model (duplicate groups of 25 fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 1, 7, 14, 21 and 28 days). The levels of immunity elicited by the vaccine doses of 0.12.5 g were not significantly different [23]. Kim et al. [24] vaccinated rainbow trout against IHNV by a DNA vaccine encoding the G gene of the virus (IHNV-G), yielding mortality of only 5% or 12% at 30 or 70 days postvaccination, respectively. However, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx protein. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response, which supports the findings of LaPatra et al. [23].

Infectious Salmon Anemia The causative agent of infectious salmon anemia (ISA) is an enveloped virus of 45140 nm in diameter with a buoyant density of 1.18 g/ml in sucrose and CsCl gradients and showed maximum replication at 15 C, but no replication at 25 C. The virus may be cultured in various cell lines while until now the SHK-1 (salmon head kidney) cell line seems to be the most susceptible one and was widely used, with appearance of CPE between 3 and 12 days postinoculation. Others such as CHSE-214 (Chinook salmon embryo), TO, AS and ASK-2 (Atlantic salmon kidney) cell lines could also be used for the propagation of some strains of ISAV . ISAV is classified as a member of the Orthomyxoviridae due to its similarity in morphology, biochemical, and replication properties to influenza viruses. Low amino acid identity values (between 13% and 25%) of ISAV proteins compared with other orthomyxoviruses support the proposal to assign ISAV to a new genus within the Orthomyxoviridae, tentatively named Isavirus by Krossy in 1999 [25]. The ISA virus genome is a single-stranded RNA consisting of 8 negativesense segments ranging in size from 736 to 2,185 bp. The complete sequence of ISAV is 12,716 bp in size and encodes 10 viral polypeptides. The genomic segment 1 encoded product is assumed to correspond to the PB2 of the influenza viruses, a polymerase. Segment 2 also encodes a viral RNA polymerase called PB1, which is the most conserved protein of orthomyxoviruses. Segment 3 encodes a nucleoprotein (NP) found to be one of the major antigens of the virus. Segment 4 encodes a protein that is not recognized by polyclonal anti-ISAV and has a predicted cytoplasmic location, which indicates that this protein is the acidic part of the viral polymerase, the PA. Segment 5 is

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assumed to encode the receptor-destroying enzyme (RDE) which exerts an esterase activity instead of the neuraminidase activity of influenza A and B viruses. Segment 6 encodes the hemagglutinin (HA), which can be recognized by the ISAV-specific monoclonal antibody 3H6F8. Both the genomic segments 7 and 8 have two overlapping ORFs. Segment 7 includes two ORF of mRNA transcript, encoding two nonstructural (NS) matrix proteins. Segment 8 was the first ISAV segment that was cloned and sequenced, which encodes both a nonstructural protein (NS1) and a structural protein (NEP). Clinical Signs Pathological changes due to ISA are characterized by severe anemia, leucopenia, petechiae in the viscera, ascites, hemorrhagic necrosis of liver and kidney, and congestion of the liver, spleen, kidney and foregut [26]. Transmission Infected fish may transmit the disease weeks before they show apparent signs of infection. The virus may spread horizontally, from fish to fish, by shedding of virions from the blood, gut contents, urine, and epidermal mucus of infected salmon and so on, while blood and mucus contain large amounts of virus and more effectively transmit the disease than feces, plankton and salmon lice. Moreover, fish that survive epizootics may shed viral particles for more than one month into the surrounding water. Although spawning fluids may be infective, there is no proof for the vertical transmission of this virus. Melville and Griffiths [27] collected eggs from grilse that were individually identified as ISAV-positive based on the detection of pathogen in ovarian fluid by RT-PCR and fertilized, disinfected and reared the eggs under quarantine conditions. ISAV was not detected in eyed eggs, alevins or parr. No mortalities occurred among fish injected with the egg homogenates. These observations suggest the absence of a vertical transmission route for ISAV infection. Prevention It is recommended that culture sites be spaced no less than 56 km apart and wastewater from slaughter and processing facilities be thoroughly disinfected, because the dissemination of virus may readily contaminate culture facilities within 56 km of an infected site in a 6- to 12-month period. Further contagion may be managed by control of ship and personnel movements among sites, destruction of infected lots, and the closure and fallowing of contaminated sites. The first commercially available ISAV vaccine was an autogenous product using the ISAV isolate NBISA01 and licensed in Canada and the United States

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as Forte V1 by Novartis (Aqua Health), a second inactivated ISAV vaccine has also been marketed in New Brunswick since 2001. It had been recognized that the immune response in Atlantic salmon did not provide full protection against the disease, for that both fish that had previously recovered from experimental ISAV infection and those that were passively immunized with serum from fish that had recovered from ISA could also contribute to the mortality. So, the commercial vaccines mentioned above were not completely effective. For this reason, even more than 1 million of the vaccinated fish had to be removed early after some of them tested positive for ISAV .

Lymphocystis Disease Virus Lymphocystis disease virus (LCDV) is a large (200300 nm in diameter) DNA virus which belongs to the genus Lymphocystivirus of Iridoviridae. It consists of an icosahedral capsid, a linear, double-stranded DNA and an outer envelope. LCDV has a chemical composition of 1.6% DNA, 42.3% protein and 17.1% lipid, most of them being phospholipids. The 39% unidentified components may be constituted mostly by sugars. To date, two strains of LCDV , LCDV-1 isolated in the United States and LCDV-C isolated in China, have been extensively studied [28, 29]. The genome of LCDV-1 is 102,653 bp in length with a base composition of 29.07% G C and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. 38 of the 195 potential gene products of LCDV-1 show significant homology to functionally characterized proteins of other iridoviruses. 110 largely nonoverlapping ORFs of the 195 potential ORFs are likely to represent viral genes, e.g. genes encoding the viral DNA (cytosine-5) methyltransferase (ORF 005L), the major capsid protein (ORF 147L), the largest subunit of the DNA-dependent RNA polymerase (ORF 016L), and the DNA polymerase (ORF 135R) [30]. The LCDV-C genome is 186,250 bp in length with a base composition of 27.25% G C and contains 240 potential ORFs, which encode polypeptides ranging from 40 to 1193 aa. 176 nonoverlapping putative viral genes are assumed present in the 240 potential ORFs. The genome of LCDV-C has 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases [31]. Among the 8 genes, ORF002L encodes the caspase recruitment domain involved in apoptotic signaling. ORF016L encodes tumor necrosis factor receptor domains. ORF209R and ORF216L encodes an N-terminal domain of cell division protein 48 (CDC48) and a collagen triple-helix repeat.

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Fig. 1. Pearl-like particles (indicated by black arrows) emerging on the surface of organs of LCD infected flounder.

ORF011L, ORF047R, and ORF058L may encode thymidylate synthase, a sitespecific recombinase, and a transmembrane receptor, respectively [31]. Clinical Signs The main clinical signs are white (occasionally pale red), paraffin-like nodules covering the skin and fins of sick fish. Infections can also occur in the eyes, kidney, spleen, liver, heart, ovaries and mesenteries [32] and pearllike particles could be found on the surface of internal organs of sick fish (fig. 1). Transmission Horizontal contact and water-borne transmission appear to be the principal mechanism for lymphocystis virus spread. High population density and external trauma enhance transmission. External surfaces including the gills appear to be the chief portal of epidermal entry. Experimental transmission of LCDV-C through the oral route and scarification of the skin route was proved successful [33]. Sun et al. [34] found that eggs of Japanese flounder showed LCDV-positive using the diagnostic method of in situ hybridization. This finding may suggest the existence of vertical transmission of LCDV-C in Japanese flounder.

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Prevention At present, there is no known method of therapy or immunization. Avoidance of stocking with clinically infected fish, early detection through monitoring and sterile disposal, along with minimizing stocking densities and handling skin trauma, have proven to be effective controls. Some researchers had reported that LCD commonly broke out during the high temperature season, but some also reported the opposite conclusions [33]. Therefore, the relationship between the occurrence of LCD and water temperatures need further studies.

Emerging Viral Pathogens of Shrimp

Worldwide, there are approximately 50,000 farms that occupy almost 1.2 million hectares of land (approx. 3 million acres). Total revenues generated from the production of farm-raised shrimp worldwide were recently estimated at USD 56 billion annually. Virus is one of the major pathogens in cultured penaeid shrimp and cause large economic losses to the shrimp-culture industry. At present, at least 15 viruses are known to infect cultured and wild marine penaeid shrimp which can be classified into 8 virus families (table 1).

White Spots Syndrome Virus White spots syndrome virus (WSSV) causes White spot disease (WSD), which has been reported from many Asian countries where penaeid shrimp are pond reared. Original outbreaks were reported from China in 1993 and they spread rapidly thereafter to Japan, Thailand, Korea, India, USA, Central and South America. It causes a high mortality, accumulating to 100%. A nonoccluded virus, WSSV, was found to be the major pathogen for the disease. WSSV has a wide range of susceptible hosts. It infects not only the penaeid and nonpenaeid shrimp but also crab, crayfish, and lobster and perhaps artemia, copepod and insect larvae [35]. Clinical Signs The clinical signs of infected shrimps include red or pink body surface and appendages, loose shell, and white calcium deposits embedded in shell, white spots 0.52.0 mm in diameter for which the disease is named (but white spot disease can occur without these signs), lack of appetite and slow movement. Major targets of the virons are ecto- and mesodermal origin, such as the gills, lymphoid organ, cuticular epithelium. And virions are assembled and replicated in the nucleus.

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Table 1. Emerging viral pathogens of shrimp


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Family

Genus

Name

Acronym

Virion shape/ size (nm)

Genome

Baculoviridae Baculoviridae Baculoviridae unclassified Baculoviridae unclassified Baculoviridae Whispovirus Baculovirus panaei monodon baculo virus baculoviral midgut necrosis penaeid hemocyte rod-shaped virus white spots syndrome virus Used name hypodermal and hematopoietic necrosis baculovirus or baculovirus of penaeid chinese (from mainland china) systemic ectodermal mesodermal baculovirus (from Tailand) white spot baculovirus (from Taiwan, China) penaeid rod-shaped DNA virus or formerly (from Japan) BP MBV BMN (PjNOB) PHRV WSSV rod, 75 rod, 69 rod, 72 228 275 310 114 kb dsDNA, unknown dsDNA dsDNA unknown rod, 80120 250380 rod, 120 265 292,967 bp dsDNA

Nimaviridae (tentatively)

HHNBV Or PcBV

SEMBV

rod, 121

276

WBV PRDV (formerly RV-PJ)

rod, 87 rod, 84

330 226

Iridoviridae
Viral Diseases of Fish and Shrimp 47

Parvoviridae

unclassified Iridovirus Brevidensovirus

Reoviridae

unclassified parvoviridae unclassified parvoviridae Unclassified Reoviridae Okavirus unclassified Rhabdobviridae unclassified Togavirida Enterovirus

Roniviridae

iridovirus of penaeid shrimp infectious hypodermal and hematopoietic necrosis virus hepatopancreatic parvovirus lymphoidalaprvolike virus type III reolike virus type IV reo-like yellow-head virus Rhabdovirus of penaeid shrimp lymphoid organ vacuolization virus taura syndrome virus

RIDO IHHNV

136 icosahereon, 2225

unknown 4.1 kbp

ssDNA ssDNA

HPV LPV REO-III REO-IV YHV RPS LOVV TSV

Togaviridae Picomaviridae

Icosahereon 2224 Icosahereon 1820 icosahereon 5070 icosahereon 5070 bacilliform 3850 160168 bacilliform 45 160 icosahereon 5254 icosahereon 3132

6,321 bp unknown unknown

ssDNA ssDNA dsRNA

unkonwon dsRNA unknown ssRNA unknown unknown 10,205 bp ssRNA ssRNA ssRNA

a Fig. 2. Electron micrograph of WSSV a Negatively stained intact WSSV virions. . b negatively stained WSSV nucleocapsids.

Fig. 3. Tail-like appendages of WSSV [36].

Electron-microscopic studies revealed that virions of WSSV are enveloped, nonoccluded and rod-shaped. Its size is about 80120 250380 nm (fig. 2). WSSV has tail-like appendages which can be visible after negative staining (fig. 3) [36].

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Genome and Proteome The genome of WSSV is a double-stranded circular DNA. Up to now, the complete genomes of three different isolates of WSSV have been reported [37]. In 2001, Yang et al. [37] reported the complete genome of WSSV. Intact WSSV genomic DNA was isolated from dead and moribund WSSV-infected Penaeus japonicus shrimp collected from shrimp ponds in Tongan, Xiamen, east China. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). 9 homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes have been identified. WSSV is the largest animal virus which has been completely sequenced and the first complete genome sequence of a marine invertebrate virus. Only 6% ORFs of the complete sequence of WSSV show homology to known genes, such as protein kinase, DNA polymerase, thymidylate synthase, dUTPase, ribonucleotide reductase (large subunit), ribonucleotide reductase (small subunit), endonuclease, chimeric thymidine kinase-thymidylate kinase, TATA box binding protein, class I cytokine receptor, collagen protein. The most unique feature of WSSV is the presence of an intact collagen gene- encoding an extra cellular matrix protein of animal cells that has never been found in any viruses. The other ORFs (94%) remain unassigned, as they lack homology to known proteins in public databases. Unique features of the WSSV genome are the presence of a very long ORF of 18,234 bp, with unknown function, a collagenlike ORF, and nine noncoding regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. Because of the large size of the genome and its uniqueness of the proteins that the WSSV open reading frames (ORFs) encode, WSSV has not yet been fully characterized. There are a total of at least 39 structural protein genes in WSSV. VP28, VP26, VP19, VP76, VP68, VP281 and VP466 are the major envelope proteins of WSSV. Although VP26 was initially thought to be a major capsid protein, immunogold electron microscopy with an antibody specific to VP26 has subsequently shown that VP26 is an envelope protein [38]. VP28 is envelope protein which has 3 forms with apparent sizes of 25, 26 and 29 kDa. In native hosts, VP28 has been reported to occur only in its nonglycosylated form, although VP28 is also predicted to have the signal peptide sequence as well as five potential glycosylation sites that have been shown to become glycosylated in insect cells. Compared to some known membrane proteins, VP281 lacks a predominant transmembrane region. The absence of transmembrane domains may suggest that this protein was produced in soluble form. Such forms are well documented in some membrane fusion proteins, pathogen receptors and cell adhesion molecules, functioning to anchor the polypeptides to the

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membrane hydrophobic phase by means of locating aqueous activities or possibly interacting with some membrane-spanning components. VP24, VP15, VP35 and VP664 are the nucleocapsid proteins of WSSV. A putative function of VP15, a highly basic protein with no hydrophobic regions, is that of a histone-like, DNA-binding protein. Using bacterially expressed VP15 [39] fusion proteins in ELISA experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. WSSV VP15 binds nonspecifically to double-stranded DNA but has a clear preference to supercoiled DNA, suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. A collagen-like ORF VP664 is the largest viral capsid protein of WSSV ever found, and the antibody against VP664 can be found to bind specifically to the globular subunit of the WSSV nucleocapsid. Transmission The major mode of transmission is horizontal. The virus initially appears in the shrimp in the stomach, gill, cuticular epidermis and connective tissue of the hepatopancreas. Chou et al. [40] indicated that under experimental conditions the virus can infect the shrimp via water and oral inoculation. From this study, some shrimp sampled at 16 h showed virus in the stomach but not in the gills, while others sampled at the same time showed virus in the gills but not in the stomach. This suggests that the virus infection could be either via the oral pathway or via water to the gill. Prevention and Control The sanitary prophylaxis are essential for prevention of WSSV, including increasing acclimation times before stocking; using nonspecific immune stimulants (NSIS), fortified mineral and vitamin diets to increase stress tolerance; using good quality diets and to continue the use of NSIS through out the life cycle; monitoring for the presence of vectors carrying WSSV in ponds. Vaccine Although invertebrates lack a true adaptive immune response, still some reports on the anti-virus vaccine. Witteveldt et al. [39] reported that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine (by injection or oral vaccination). Witteveldt et al. [39] also reported vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7 and 21 days after vaccination. A

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significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, and it will open the way to new strategies to control viral diseases in shrimp and other crustaceans.

Yellow-Head Virus Yellow-head virus (YHV) has been reported from pond-reared shrimp in India, the Philippines, Thailand, and Texas, United States of America. Original outbreaks were reported from Thailand in 1991, but subsequent reports have been sporadic. Disease outbreaks may occur at all seasons but usually at 5070 days after pond stocking. They seem to be favored by widely fluctuating environmental conditions. Other precipitating factors may include stress induced by chemical and insecticide residues. The infection generally resulted in a cumulative mortality of 100% within 35 days after the onset of the disease. YHV has a wide range hosts, all decapods crustaceans (order Decapoda) including prawns, lobsters and crabs from marine, brackish or freshwater environments, are considered susceptible to infection. However, the disease has mainly been a problem in farmed penaeid prawns. Clinical Signs The clinical signs of infected shrimp include white, yellow or brown gills; yellowing of the cephalothorax and general bleaching of body; yellow, swollen digestive gland makes the head appear yellow. Genome and Taxonomy YHV is a positive-sense, single-stranded RNA (ssRNA) virus [41], and there is no report of the complete genome sequence of YHV. YHV has now been formally classified in the new genus Okavirus and new family Roniviridae within the viral order Nidovirales (NCBI Taxonomy ID: 96029). Transmission The major source of infection for rearing ponds is animate vectors from pond inlet water. Direct transmission is thought to occur through several vectors including contaminated water, decomposing fecal matter or tissue, cannibalism of dying shrimp (in hatcheries), and from fluid from infected females. Direct transmission may occur between unrelated crustacean species. Indirect transmission is

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thought to occur by exposure to viable viruses from previous hatchery or pond growing cycles, an infected water supply (new or previously used), infected food (unusual), or on equipment surfaces that have consumed infected shrimp and carry the virus in the gut [42]. Prevention and Control Sanitary prophylaxis is necessary for prevention of YHV, including proper cleaning and disinfection of ponds before stocking, including removal of potential carrier crustaceans; elimination or screening of potential carriers from exchange water; avoiding exchange of equipment amongst ponds; avoiding use of fresh aquatic feeds; continuous removal and destruction of moribund and dead shrimp whenever they appear.

Infectious Hypodermal and Haematopoietic Necrosis Virus Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is known from both the Eastern and Western Hemispheres, especially among cultured penaeid shrimp. In the Western Hemisphere, IHHNV is commonly found in cultured and wild penaeid shrimp in the eastern Pacific from Peru to Mexico. IHHNV also has been identified in cultured penaeid shrimp in China, Japan, India and other Asian countries. Clinical Signs The clinical symptoms of infected shrimp include reduced and irregular growth in juveniles and sub-adults (runt-deformity syndrome), white to buff mottling of shell, especially at the junction of shell plates of the abdomen, giant black tiger prawn (Penaeus monodon) may appear blue and deformed rostrums grow to one side. Genome and Proteome IHHNV has been classified as a member of the family Parvoviridae, genus Densovirinae. IHHNV is the smallest of the known penaeid shrimp viruses. The IHHN virion is a 22-nm, nonenveloped icosahedron, with a density of 1.40 g/ml in CsCl, contains linear single-stranded DNA with an estimated size of 4.1 kb, and has a capsid with four polypeptides of molecular weight 74, 47, 39, and 37.5 kDa [43]. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The sequences of the 39 protein of IHHNV have been identified according to NCBI, including 3 coat protein, 5 capsid protein, 2 other structure protein

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and other non-structure protein. The left ORF of IHHNV genome most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest amino acid sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. The search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae [44]. Transmission Horizontal transmission of IHHNV is known to occur by cannibalism of infected carcasses, by direct contact between prawns, and by indirect contact via water. Cannibalism is known to be the most rapid and effective mechanism of infection and is the basis of the bioassay test for asymptomatic carriers in prawn populations. IHHNV-resistant penaeid species and early life stages may carry the virus latently and transfer it to more susceptible species and life stages. It is believed that IHHNV may be transmitted vertically from broodstock to their progeny. Poulos et al. [45] suggested vertical transmission may have contributed significantly to the rapid spread of IHHNV in aquaculture operations in Sonora and Sinaloa, Mexico, and could have played an important role in the apparent IHHNV epizootic in wild prawns. Prevention and Control Eradication methods are applied to certain aquaculture situations, such as the complete depopulation of all culture stocks, disinfection of the culture facility, avoidance of re-introduction of the virus (from other culture facilities, wild shrimp, etc.), re-stocking with IHHNV-free postlarvae that have been produced from IHHNV-free broodstock. Virus-free verification of Litopenaeus vannamei broodstock has become a general routine hatchery procedure to prevent the disease from spreading through vertical transmission [46]. Vertical transmission was a crucial factor for the increase of IHHNV prevalence in domesticated shrimps from generation to generation. In the case of females with the highest IHHNV infection, the embryos generally failed to develop and hatch. The lack of IHHNV in females identified as virus-negative on the basis of a nested-PCR analysis led to consider the application of this procedure to prevent the vertical transmission of the

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virus. The identification of IHHNV-free females would not only improve nauplius quality but also production of nauplii.

Taura Syndrome Virus Taura syndrome (TS), caused by TS virus (TSV), has resulted in serious disease epizootics throughout the shrimp-growing regions of the Western Hemisphere. The virus has recently been introduced into Asia with infected imported Pacific white shrimp, Penaeus vannamei, from Central and South American sources [47]. The principal host for TSV is the Pacific white shrimp, P vannamei, although other species can be infected and present disease. TSV . can also infect other Western Hemisphere penaeid species (i.e. P stylirostris, . P setiferus, and P. schmitti), sometimes resulting in disease and mortalities in . PL or yearly juvenile stages, but also in asymptomatic persistent infections. Other Western Hemisphere penaeids (P aztecus and P duorarum) and Eastern . . Hemisphere penaeids (P chinensis, P. monodon, and P. japonicus) have been . experimentally infected with TSV [48]. Clinical Signs The clinical signs of infected shrimp include pale red body surface and appendages, tail fan and redness at pleiopods, shell soft and gut empty, death usually at moulting and multiple irregularly shaped and randomly distributed melanized cuticular lesions. Genome and Taxonomy TSV particles are 32 nm, nonenveloped icosahedrons with a buoyant density of 1.338 g/ml. The genome of TSV consists of a linear, positive-sense single-stranded RNA of 10,205 nucleotides, excluding the 3 poly-A tail, and it contains two large open reading frames (ORFs). ORF 1 contains the sequence motifs for nonstructural proteins, such as helicase, protease and RNA-dependent RNA polymersae. ORF 2 contains the sequences for TSV structural proteins, including the three major capsid proteins VP1, VP2 and VP3 (55, 40 and 24 kDa, respectively) [49]. TSV has been characterized and tentatively assigned to the family Picornaviridae [49]. TSV was recently placed in the genus Enterovirus. Transmission TSV can be passed from shrimp to shrimp via the water, but with far less efficiency than by cannibalism. Vertical transmission is suspected, but this has yet to be conclusively demonstrated. In addition to human-mediated movement

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of subclinical carriers of TSV, aquatic insects and sea birds have been implicated in its transmission. The water boatman, Trichocorixa reticulata (Corixidae), feeds on dead shrimp and is believed to spread TSV by flying from pond to pond. Feces of laughing gull, Larus atricilla, collected from around TSV-infected ponds in Texas during the 1995 epizootic were also found to contain viable TSV. Survivors of acute TSV infection pass through a brief transitional phase and enter the chronic phase, which may persist for the rest of their lives. This subclinical phase of infection is believed to have contributed to the spread of the disease via carriage of viable TSV. Prevention and Control To date, there is no possible treatment. Selected stocks of TSV-free P vannamei . and P stylirostris with varying degrees of resistance to TS disease are commer. cially available. Screening broodstock is necessary method of preventing spread to eggs and nauplii. The sanitary prophylaxis include proper cleaning and disinfection of ponds and supply reservoirs and canals and before stocking, including removal of potential carrier crustaceans, elimination or screening of potential carriers from exchange water and avoiding exchange of equipment amongst ponds. The vaccine of TSV has not been reported. Environmental factors and poor water quality resulting from increased effluent discharge, movement of aquatic animals, inadequate farm management, rapid proliferation of farms, etc., have been implicated in major disease outbreaks occurring in epizootic conditions. However, the underlying causes of such epizootics are highly complex and difficult to pinpoint. An understanding of the relationship between host, pathogen and environment is important in this regard. Since aquatic animal disease is the end result of a series of linked events, treatment of disease should go beyond consideration of the pathogen alone.

References
1 2 3 4 Cohen J, Poinsard A, Scherrer R: Physicochemical and morphological features of infectious pancreatic necrosis virus. J Gen Virol 1973;21:485498. Malsberger RG, Cerini CP: Multiplication of infectious pancreatic necrosis virus. Ann NY Acad Sci 1965;126:320327. Baudouy AM: Experimental data on the remanence of the virus causing the infectious pancreatic necrosis in Salmonidae in water environment. Ann Rech Vet 1976;7:7582. Weber S, Fichtner D, Mettenleiter TC, Mundt E: Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon. J Gen Virol 2001;82: 805812. Heppell J, Tarrab E, Berthiaume L, Lecomte J, Arella M: Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus. J Gen Virol 1995;76: 20912096.

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Duncan R, Mason CL, Nagy E, Leong JA, Dobos P: Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: a putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif. Virology 1991;181:541552. Smith G, Bebak J, McAllister PE: Experimental infectious pancreatic necrosis infections: propagative or point-source epidemic? Prev Vet Med 2000;47:221241. Seeley RJ, Perlmutter A, Seeley VA: Inheritance and longevity of infectious pancreatic necrosis virus in the zebra fish, Brachydanio rerio (Hamilton-Buchanan). Appl Envir Microbiol 1977;34:5055. Mulcahy D, Pascho RJ: Adsorption to fish sperm of vertically transmitted fish viruses. Science 1984;225:333335. Jashes M, Gonzalez M, Lopez-Lastra M, De Clercq E, Sandino A: Inhibitors of infectious pancreatic necrosis virus (IPNV) replication. Antiviral Res 1996;29:309312. Labus MB, Breeman S, Ellis AE, Smail DA, Kervick M, Melvin WT: Antigenic comparison of a truncated form of VP2 of infectious pancreatic necrosis (IPN) virus expressed in four different cell types. Fish Shellfish Immunol 2001;11:203216. Mikalsen AB, Torgersen J, Alestrom P, Hellemann AL, Koppang EO, Rimstad E: Protection of Atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination. Dis Aquat Organ 2004;60:1120. Fijan NN, Wellbom TL, Naftel JP: An acute viral disease of channel catfish. US Department of Interior Bur. Sport Fish and Wildlife. Tech Paper 1970;43:11. Stingley RL, Griffin BR, Gray WL: Channel catfish virus gene expression in experimentally infected channel catfish, Ictalurus punctatus (Rafinesque). J Fish Dis 2003;26:487493. Stingley RL, Gray WL: Transcriptional regulation of the channel catfish virus genome direct repeat region. J Gen Virol 2000;81:20052010. Wise JA, Harrell SF, Busch RL, Boyle JA: Vertical transmission of channel catfish virus. Am J Vet Res 1988;49:15061509. Nusbaum KE, Smith BF, DeInnocentes P, Bird RC: Protective immunity induced by DNA vaccination of channel catfish with early and late transcripts of the channel catfish herpesvirus (IHV-1). Vet Immunol Immunopathol 2002;84:151168. Chinchar VG, Logue O, Antao A, Chinchar GD: Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor. Dis Aquat Organ 1998;33:7785. Dixon P: Immunization with viral antigens: viral diseases of carp and catfish. Dev Biol Stand 1997;90:221232. Biacchesi S, Thoulouze MI, Bearzotti M, Yu YX, Bremont M: Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. J Virol 2000;74:1124711253. Purcell MK, Kurath G, Garver KA, Herwig RP, Winton JR: Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination. Fish Shellfish Immunol 2004;17:447462. Simon B, Nomellini J, Chiou P, Bingle W, Thornton J, Smit J, Leong JA: Recombinant vaccines against infectious hematopoietic necrosis virus: production by the Caulobacter crescentus S-layer protein secretion system and evaluation in laboratory trials. Dis Aquat Org 2001;44: 1727. LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, Anderson ED: Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination. Vaccine 2001;19:40114019. Kim CH, Johnson MC, Drennan JD, Simon BE, Thomann E, Leong JA: DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish. J Virol 2000;74:70487054. Krossoy B, Hordvik I, Nilsen F, Nylund A, Endresen C: The putative polymerase sequence of infectious salmon anemia virus suggests a new genus within the Orthomyxoviridae. J Virol 1999;73:21362142. Rolland JB, Winton JR: Relative resistance of Pacific salmon to infectious salmon anaemia virus. J Fish Dis 2003;26:511520.

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Melville KJ, Griffiths SG: Absence of vertical transmission of infectious salmon anemia virus (ISAV) from individually infected Atlantic salmon Salmo salar. Dis Aquat Organ 1999;38:231234. Iwamoto R, Hasegawa O, LaPatra S, Yoshimizu M: Isolation and characterization of the Japanese flounder (Paralichthys olivaceus) lymphocystis disease virus. J Aquat Anim Health 2002;14: 114123. Zhang QY, Ruan HM, Li ZQ, Yuan XP, Gui JF: Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines. Dis Aquat Organ 2003;57:2734. Tidona CA, Darai G: The complete DNA sequence of lymphocystis disease virus. Virology 1997;230:207216. Zhang QY, Xiao F, Xie J, Li ZQ, Gui JF: Complete genome sequence of lymphocystis disease virus isolated from China. J Virol 2004;78:69826994. Marcogliese DJ, Fournier M, Lacroix A, Cyr DG: Non-specific immune response associated with infections of lymphocystis disease virus in American plaice, Hippoglossoides platessoides (Fabricius). J Fish Dis 2001;24:121124. Zhang YJ, Wu ZY: Primary studies on lymphocystis disease of marine fish. Fish Dis Res 1992;14:78. Sun XQ, Huang J, Liu YK, Qu LY, Hong XG, Zhang JX: The studies on diagnositic method of dot blot and in situ hybridization for lymphocystis disease of cultured Paralichthys olivaceus. High Technol Lett 2003;1:8994. Chen XF, Chen P, Wu DH: Study on a new bacilliform virus in cultured shrimps. Science in China Series C. Life Sci 1997;27:415420. Wang SY, Hong C, Lotz JM: Development of a PCR procedure for the detection of Baculovirus penaei in shrimp. Dis Aquat Org 1996;25:123131. Yang B, Song X, Huang J, Lei Z: Epidemiology and diagnosis of disease by infectious hypodermal and hematopoietic necrosis virus (IHHNV): a review. J Fish Sci China 2005;12: 519524. Tsai JM, Wang HC, Leu JH: Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol 2004:1136011370. Witteveldt J, Cifuentes CC, Vlak JM: Protection of Penaeus monodon against white spot syndrome virus by oral vaccination. J Virol 2004;78:20572061. Chou HY, Huang CY, Wang CH: Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis Aquat Organ 1995;23:165173. Tang KFJ, Lightner DV: A yellow head virus gene probe: nucleotide sequence and application for in situ hybridization. Dis Aquat Org 1999;35:165173. Walker PJ, Cowley JA, Spann KM, Hodgson RAJ, Hall MR, Withyachumnarnkul, B: Yellow head complex viruses: transmission cycles and topographical distribution in the Asia-Pacific Region; in Browdy CL, Jory DE (eds): The New Wave. Proceedings of the Special Session on Sustainable Shrimp Culture, Aquaculture 2001. Baton Rouge, The World Aquaculture Society, 2001, pp 292302. Bonami JR, Brehelin M, Mari J, Trumper B, Lightner DV: Purification and characterization of IHHN virus of penaeid shrimps. J Gen Virol 1990;71:26572664. Shike H, Dhar AK, Burns JC, Shimizu C: Infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito Brevidensoviruses. Virology 2000;277:167177. Poulos BT, Pantoja CR, Bradley-Dunlop D: Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp. Dis Aquat Organ 2001;47:1323. Emmerik Mottea, Edwin Yugchaa, Juan Luzardoa: Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei. Aquaculture 2003;219:5770. Aguirre Guzman G, Ascencio Valle F: Infectious disease in shrimp species with aquaculture potential. Recent Res Dev Microbiol 2000;4:333348. Lightner DV (ed): A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. Baton Rouge, World Aquaculture Society, 1996, pp 1304.

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Overstreet RM, Lightner DV, Hasson KW, McIlwain S, Lotz J: Susceptibility to TSV of some penaeid shrimp native to the Gulf of Mexico and southeast Atlantic Ocean. J Invertebr Pathol 1997;69:165176.

Min Wang Department of Biology, College of Marine Life Sciences Ocean University of China Qingdao (China) Tel. 86 532 2031859, Fax 86 532 2032276, E-Mail wangmin30@hotmail.com

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Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 5977

Avian Influenza H5N1 Virus: An Emerging Global Pandemic


Sunil K. Lala, Vincent T.K. Chowb
Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India; bProgramme in Infectious Diseases, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore
a

Abstract
The specter of avian influenza emerging from Asia and spreading all over the globe is causing deeper concern by the day. As we witness the H5N1 virus evolving and becoming increasingly dangerous, a major pandemic may be unavoidable. The bird flu virus has already claimed more than 140 lives worldwide as of August 2006. Should bird flu spark a global pandemic, several hundred million people could die within a matter of weeks, which is many times the number of deaths due to AIDS so far. This pathogen is completely different from seasonal influenza virus, which kills between 1 and 2 million people worldwide in a typical year. In the worst previous pandemic of 1918, more than 20 million humans died of the Spanish flu. The current bird flu virus has emerged from a pool of animals that have previously never infected humans implying that humans do not have antibodies to combat the infection. This virus also causes severe disease and high fatality within a short time span. The only remaining factor to enable the virus to cause a pandemic is if it acquires the capability of swift transmission among humans through coughing, sneezing or just a simple handshake! The evolving bird flu virus has already crossed the species barrier from chickens to other birds and mammals including pigs. Pigs possess flu virus receptors on their respiratory cells which are similar to human receptors. Thus, pigs serve as an excellent mixing vessel for the virus to exchange genes through genetic reassortment to generate an entirely new viral strain that may be capable of efficient human-to-human transmission. This chapter describes the previous pandemics, influenza virus evolution, its molecular biology, replication, zoonosis and pathogenesis. Issues on pandemic preparedness and important strategies to contain or limit the spread of this virus for the present and future are discussed.
Copyright 2007 S. Karger AG, Basel

Influenza viruses have been known to cause severe respiratory illness killing 250,000500,000 people worldwide annually [1]. Ten influenza pandemics

defined by clinical and epidemiological records have occurred in the past three centuries, with an average of 1 every 33 years [2]. Pandemics are different from epidemics, such that they are defined as a global emerging disease caused by a novel virus or its subtype circulating among the human population. Belonging to the Orthomyxoviridae family, influenza viruses are classified into three major types A, B, C on the basis of two antigens, i.e. nucleoprotein (NP) and matrix (M1) protein. The avian influenza virus belongs to the type A which is the most virulent, and its further subtyping is based on the distinct glycoprotein antigens haemagglutinin (HA) [H1 to H16] and neuraminidase (NA) [N1 to N9]. Influenza A viruses have been isolated from humans and various animals including horses, pigs, sea mammals (e.g. seals and whales), wild waterfowl (e.g. wild ducks) and poultry [3]. Influenza B and C have been isolated from humans, and are known to cause local epidemics. Past influenza A pandemics that afflicted humans during the 20th century apparently arose from the Eurasian avian lineage of viruses [4]. Currently, H5N1 is considered to pose the greatest potential viral threat to humans. Although efficient human-to-human transmission has not yet been confirmed, it is certainly possible that a different subtype may arise by genetic reassortment of H5N1 with other influenza virus strains to generate the next pandemic strain.
Previous Pandemic Outbreaks

Pandemics are global events which arise abruptly, rapidly cause extensive economic damage, killing millions along its path and subsiding eventually as abruptly as they began. During a pandemic, the causative virus achieves dominance over all other circulating influenza viruses in humans and continues to circulate for decades until replaced by another lethal pandemic strain [5]. Antigenic shift caused by genetic reassortment or antigenic drift may give rise to a pandemic strain. The 20th century has witnessed four human influenza pandemics with intervals of 939 years. China is considered the epicenter for pandemic outbreaks of influenza viruses. History indicates that the causative viruses of the influenza pandemics of H2N2 in 1957, H3N2 in 1968, and the re-emergence of H1N1 in 1977 had their origins in China. Recent outbreaks of H5N1 and H9N2 in Hong Kong emphasize the importance of virological surveillance in this region to facilitate early detection of potentially pandemic viruses.
The Spanish Influenza

The H1N1 Spanish influenza (19181919) caused the most devastating pandemic witnessed by the human population, exacting a toll of 100 million lives

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worldwide [1]. This infection had its origin in Europe, and rapidly progressed to parts of Asia and Africa via ships carrying troops from Europe and America. Evidence to link the origin of the 1918 strain to H1N1 swine influenza virus was not forthcoming until 1997 when Taubenberger et al. [6] succeeded in obtaining genetic information of this pandemic strain from a paraffin-embedded lung tissue sample of an infected individual. In addition to the strong sequence similarity to swine influenza A strain, the 1918 HA gene lacked multiple basic amino acids at the cleavage sites suggesting that pigs may have served as an intermediate host for reassortment from an avian influenza strain. Given the speed at which a new pandemic could spread in the modern world, the emergence of a strain as virulent as that of 1918 would be devastating.
The 1957 and 1968 Influenza Pandemics

The 1957 Asian influenza pandemic resulted from the emergence of a reassortant influenza virus in which both the HA and NA segments had been replaced by gene segments closely related to avian strains [79]. The 1957 pandemic strain also acquired a novel N2 subtype replacing N1 of the previous strain. The sequence of the new NA was very closely related to the avian N2 sequences, with only six amino acids differing from the conserved avian sequences [9]. The 1968 pandemic strain H3N2 that circulated in Hong Kong appeared to have had an avian origin as well [10, 11]. This pandemic strain emerged with an avian-derived H3 segment, while retaining the N2 segment derived in 1957. More recently, it was demonstrated that the PB1 gene segment was also replaced in both 1957 and 1968 pandemic strains by a segment of avian derivation. Since PA, PB2, NP, M and NS were preserved from the H1N1 strains of 1918, the hypothesis for the generation of novel pandemic strains became accepted. As in 1918, the epidemic spread resulted in case morbidity, but the mortality rates were much lower. The world was better prepared to cope with the situation in view of the availability of antimicrobial agents and vaccines, and hence the casualties were less compared to the previous pandemic.
Influenza A H5N1 Emergence in Hong Kong, 1997

The first cases of the outbreak appeared in May and November to December 1997 in Hong Kong, where 18 individuals were reported to be infected with H5N1 [12]. The human influenza virus isolates acquired all eight gene segments from Eurasian avian sources, with a preference for binding to the cell surface Neu5Gc 23 sialic acid receptor, a feature typical of avian influenza viruses. Although the outbreak was controlled by the slaughter of poultry stocks (1.5 million), the HA gene continued to circulate in geese in southern China [13].
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Table 1. Human deaths due to bird flu (from 2003 to 23 August 2006) Country Vietnam Indonesia Thailand China Egypt Turkey Azerbaijan Cambodia Iraq Djibouti Total Cases 93 60 24 21 14 12 8 6 2 1 241 Deaths 42 46 16 14 6 4 5 6 2 0 141

From 1997 to 2001, various genotypes of HA remained antigenically homogenous, but a significant change was observed in 2002. The H5N1 strain that was pathogenic only in chickens exhibited high pathogenicity in ducks and aquatic birds, thus implying antigenic change. No further human cases of H5N1 influenza were identified until February 2003, when H5N1 reemerged in a family in Hong Kong. The first patient was a 9-year-old boy who recovered from the infection during hospitalization, but his 33-year-old father and 8-year-old sister succumbed to the infection [14]. Genetic analysis revealed that the causative strain was similar to the antigenically distinct strain that was highly pathogenic for ducks and poultry. Hundreds of millions of poultry animals have been slaughtered due to the unprecedented bird flu epidemic of H5N1 infection in Asia in 2004. The outbreaks were observed in countries such as China, Japan, South Korea, Vietnam, Cambodia, Laos, Thailand, Egypt, Turkey, Iraq, Malaysia, Djibouti and Indonesia. But the virus has taken its greatest toll on human lives in Vietnam, Indonesia, and Thailand followed by China (see table 1 for complete list). The H5N1 strain circulating in Asia is of Z genotype, which is endemic in birds in Southeast Asia and has undergone various antigenic changes and is similar to the strain isolated from Vietnam in 2004 [15]. The intensity of mammalian transmission of H5N1 can be attributed to several factors such as antigenic variation of HA, acquisition of high pathogenicity towards aquatic birds, and the susceptibility to genetic reassortment. These events portend that the potential risk of transmission between humans can eventually lead to the establishment of the H5N1 lineage among humans. The bird flu virus which was initially considered to be endemic in Asia has now spread to Europe and even Africa. Cases of H5N1 infection have been reported from around the globe and the World Health Organization (WHO) now warns that this expanding geographical presence is of great concern as it

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increases the susceptibility of human transmission globally. The outbreaks are attributed to contact and sharing of water sources between domestic poultry and migratory birds known to harbour and excrete the deadly virus.
H5N1 Virology

The influenza A virus genome consists of eight segments of negative-sense single-stranded RNA that encode 10 proteins (HA, NA, M1, M2, NP, PB1, PB2, PA, NS1 and NS2). Under electron microscopy, the viruses are pleomorphic and subsequently appear spherical (approximately 120 nm in diameter). Two distinct types of spikes (approximately 16 nm in length), corresponding to the HA and NA molecules, reside on the surface of the virions. The HA spike appears rod-shaped and protrudes from the envelope as a trimer, while the NA spike is a mushroom-shaped tetramer. These two glycoproteins together with the M2 protein are anchored to the lipid bilayer envelope derived from the plasma membrane of host cells by short sequences of hydrophobic amino acids (transmembrane region). Inside the lipid envelope, eight segments of RNA are associated with nucleoprotein (NP) which resembles a helix. This RNA-NP complex contains the three polymerase proteins (PB1, PB2 and PA) [16]. Haemagglutinin HA is a type I glycoprotein containing an N-terminal ectodomain and a C-terminal anchor which is the major surface antigen of influenza A virus. HA plays a significant role in host cell recognition, attachment and fusion of the viral envelope with the host cell. Encoded by RNA segment 4, HA enables the viral particles to specifically attach to the cell surface receptors containing sialic acid. It is synthesized as a polyprotein precursor (HA0) that is post-translationally cleaved (involving processes such as proteolytic cleavage, glycosylation and fatty acid acylation) into two subunits HA1 and HA2, connected by disulphide linkages. This cleavage is carried out by trypsin-like proteases of the host. After cleavage, HA1 consists of 324 amino acids with variable carbohydrate groups and contains the antigenic determinants, while HA2 has about 222 amino acids with variable carbohydrate and 3 palmitate residues. Due to the error-prone activity of the RNA polymerase, the HA molecule undergoes a high rate of mutation (2 10 3) resulting in the current 16 HA sub-types which are serologically distinct or partially cross-reactive. However, the amino acids that constitute the receptor-binding sites, as well as cysteine and proline residues, are highly conserved [17]. Neuraminidase NA is a type II glycoprotein containing an N-proximal anchor and a C-terminal ectodomain. This second surface antigen of influenza A virus can be

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observed as a tetramer unevenly distributed in the lipid bilayer envelope. Encoded by RNA segment 6, the neuraminidase (sialidase) cleaves terminal sialic acid residues from glycoproteins and glycolipids [18] and hence permits virion entry. This enzyme is also required for elution of newly synthesized virions from infected cells, and aids in the movement of the virus through the mucus of the respiratory tract [19, 20], thus behaving as an essential adjuvant for pathogenicity. The amino-terminal hydrophobic sequence of NA directs transport of the virion to the cell membrane, and prevents viral clumping before the next infectious cycle begins. Similar to HA, NA is prone to mutation and its nine subtypes are not cross-reactive [21]. M1 Protein Influenza virus RNA segment 7 is bicistronic, encoding both M1 and M2 proteins. The M gene encodes two partly overlapping proteins, i.e. a highly conserved 252-amino acid M1 protein and a 97-amino acid M2 protein [22]. The M protein forms an envelope over the nucleocapsid complex. In the infected cell, it is distributed both in the cytoplasm and in the nucleus. It is considered as an important determinant of species specificity, and there is speculation on its role in progeny viral assembly. M2 Protein The bicistronic RNA segment 7 encodes the M2 protein which is derived after splicing from its precursor M1 transcript. The HA and NA glycoprotein spikes together with M2 form tetramers on the infected cell surface forming ionic channels that maintain the pH, preventing the exposure of the viral HA to low intracellular pH to which it is sensitive, thereby facilitating uncoating of the viral nucleoprotein during replication. Despite this preferential association between HA and matrix proteins, the role of the M gene in highly pathogenic influenza virus has not been accorded its due significance. Nucleoprotein The NP is encoded by RNA segment 5, and functions as a structural protein. NP forms a loose association with the RNA of the virion and encapsidates the RNP complex (comprising RNA-NP and the polymerases PB1, PB2 and PA). Besides its structural role, NP is involved in the switching of viral RNA polymerase activity from mRNA synthesis to cRNA synthesis and vRNA synthesis [23, 24]. NP may also play a role in host selection based on its phosphorylation, which is host-dependent. Polymerases (PB1, PB2 and PA) PB2. The polymerase protein constitutes an integral part of the RNP complex, and is encoded by RNA segment 1. Its function includes recognition of the

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5 mRNA cap of the host for use as viral mRNA transcription primers, and cleavage of the cap structures to generate primers for viral mRNA transcription [23, 24]. PB1. Localized in the nuclei of infected cells, PB1 is encoded by RNA segment 2. The basic function of the PB1 protein is in the elongation of viral mRNA primers and vRNA synthesis. PA. The PA polymerase is encoded by RNA segment 3. Also localized in the infected cell nucleus, not much is known about its significance but a possible role as a helix-unwinding protein is suspected. Nonstructural Proteins NS1 and NS2 NS1 and NS2 are encoded by RNA segment 8. NS2 is derived from precursor NS1 by splicing. In the infected cell, NS1 is localized in the nucleus and NS2 in the cytoplasm. NS2 protein is now known to exist in virions [25, 26], and is thought to play a role in the export of RNP from the nucleus [26, 27] through interaction with M1 protein. Based on the evidence that the NS2 protein contains a nuclear export signal and facilitates vRNP export, ONeill et al. [28] have proposed to rename this protein as NEP (viral nuclear export protein). Subsequent studies also confirmed that this protein is essential for vRNP nuclear export [29].

Influenza Virus and Its Replication

Despite their common avian lineage, influenza A viruses exhibit a pattern in host range and replication. This is attributed to the receptor specificity of HA molecules, and how they respond to the presence or absence of certain sialic acid-galactose linkages in the host. Sialic acid is a nine-carbon, acidic amino sugar (5-amino-3,5-dideoxyD-glycero-D-galacto-nonulosonic acid), and is attached to the outermost ends of N-glycan, O-glycans and glycosphingolipids. The 5-carbon position situated at the terminal region of N-glycans, O-glycans and sphingolipids on modification gives an N-acetyl group yielding N-acetylneuraminic acid (Neu5Ac), and on hydroxylation the 5-N-acetyl group gives 5-N-glycolylneuraminic acid (Neu5Gc). The sialic acids can undergo further structural diversities, including substitution of hydroxyl groups with acetyl, methyl, phosphate or sulphate groups, and also by the different linkages from the 2 carbon to the underlying sugar chain to give SA23 Gal - (23) and SA26 Gal - (26). These factors are attributed to the diversity of sialic acid sugar chain receptors among animal species. About 40 different sialic acid receptors have been reported in nature [30, 31]. Krug et al. [32] conducted extensive studies on the replication and expression of influenza viruses. The virion particle infects the target host cell as the HA molecule recognizes and attaches to the specific terminal sialic acid on the cell

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membrane surface. This is followed by endocytosis of the virus. In the acidic environment of the endosome, the virus undergoes a conformational change in the HA, the amino terminus of HA2 is inserted into the membrane of the endosome, fuses the envelope with the endosomal membrane, and the nucleocapsid is released into the cytoplasm of the cell. In the nucleus, the polymerase complex initiates primary transcription to produce necessary proteins for replication. The primary transcription involves what is known as cap snatching [33]. The PB2 polymerase cuts the 5 methylguanosine cap along with thirteen nucleotides of the host RNA which serves as a primer for the transcription of the protein PB1, viral transcriptase followed by the translation of viral proteins, NP and NS1. During the early stage of infection, the host RNA transcription mechanism is inhibited. As the concentration of NP accumulates in the nucleus, a shift in mRNA synthesis to cRNA (positive sense) and vRNA synthesis is observed. The vRNA serves as template for secondary transcription of viral mRNA which in turn results in the synthesis of M1, HA and NA proteins. Accumulation of M1 protein in the nucleus signals the translocation of NP from the nucleus to the cytoplasm. Simultaneously, the HA and NA proteins undergo post-translation modifications (glycosylation, polymerization and acylation), and migrate to the plasma membrane along with the M2 proteins. As the nucleocapsid begins to take form, it gets encased within the shell of M1, initiating the budding process. Finally, the NA cleaves the sialic acid receptors and facilitates the elution of the progeny virions from the host cell. An infectious virion is formed only when the precursor unit of the HA molecule (HA0) is cleaved to produce HA1 and HA2. As the cleaved unit is susceptible to low pH, in birds the cleavage of HA in avian influenza viruses occurs intracellularly, whereas in mammals the HA is cleaved by extracellular proteases of the respiratory tract [34].

Zoonosis

Although certain influenza virus subtypes (e.g. H9) cause mild disease in poultry, the H5 and H7 subtypes are known to cause widespread outbreaks inflicting massive fatalities among domestic poultry. Among animals, wild waterfowl represent the major reservoir for influenza viruses. All influenza viruses in other animal species are believed to be derived from these birds. Migratory birds are considered as carriers, merely serve to carry the virus over great distances, and shed large quantities of virus in their faeces but remain healthy. The excreted virus can survive for several days and withstand low temperatures. Some strains remain infectious for up to 207 days at 17 C, and they remain infectious for longer periods at 4 C. Hence, the virus is acquired by

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poultry animals often via sharing the same water sources with infected wild waterfowl. In poultry, avian influenza virus causes two distinctly different patterns of disease, i.e. one common and mild, while the other is rare but highly lethal. In the mild form, the symptoms vary from reduced egg production to ruffled feathers, and can be detected only by performing corresponding tests. However, the lethal form (often termed chicken Ebola) is highly contagious and can cause 100% mortality within 48 h [35, 36]. Recently, the biology of H5N1 influenza viruses in waterfowl has been changing. The first indication was the death of many domestic and exotic waterfowl in Hong Kongs nature parks in late 2002. These viruses later became dominant in Vietnam and Thailand, and killed aquatic and terrestrial poultry. The H5N1 viruses isolated from humans and poultry in Asia during 20032004 displayed a trend towards decreased pathogenicity in domestic ducks but remained highly pathogenic to chickens and humans. Since 2002, the H5N1 virus has exhibited a difference in its pathogenicity; the virus which predominantly replicated in the intestinal epithelium of ducks is now shed from the upper respiratory tract, and viral shedding in faeces has increased from 10 to 17 days post-infection. There appears to be a trend towards non-pathogenicity to ducks, indicating biological evolution towards equilibrium in their natural host [2]. Despite its spread among land and aquatic birds, the H5N1 virus has also been reported among felines. In October 2004, an outbreak was reported in the biggest zoo in Thailand housing 441 tigers [37]. The tigers were fed with carcasses of chicken which might have been infected. The majority of the infected tigers died from severe respiratory distress and fever. The increasing numbers of sick tigers demonstrated the potential of tiger-to-tiger transmission. H5N1 infection in domestic cats have been reported, and such a scenario may eventually facilitate the transmission of the virus among humans [38, 39]. Pigs Are a Mixing Vessel for Genetic Reassortment Both the 1957 Asian (H2N2) and the 1968 Hong Kong (H3N2) pandemics originated from reassortment of influenza virus. The 1957 pandemic virus acquired three genes (PB1, HA, and NA) from the avian influenza virus gene pool, and retained five other genes from circulating human strains. Influenza virus is capable of considerable genetic reassortment. The lack of proofreading among RNA polymerases contributes to replication errors of the order of 1 in 104 bases. Influenza viruses bind to sialyl sugar chain receptors on the host cell membranes Neu5Ac and Neu5Gc are the two major receptors for influenza A and influenza B virus infections [1]. Avian and human HA differ in their ability to bind to different forms of sialic acid receptors, and this difference acts as a barrier to crossspecies infection. However, pig trachea possesses both Neu5Ac 26 and Neu5Gc 23 sialyl receptors of humans and birds, respectively. Hence, the maintenance of

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virus in pigs and the frequent introduction of different but antigenically related influenza viruses can allow pigs to serve as an intermediate host or mixing vessel for the generation of new reassortant pandemic strains. Mainland China is considered to be the epicenter for all the pandemic influenza strains, given that poultry, swine and humans live in close proximity on farms. Being susceptible to a variety of influenza viruses, pigs should be monitored regularly as part of an early-warning system for detecting viruses with pandemic traits [40, 41].

Evolutionary Pathways

The epidemiological behaviour of influenza in humans is related to the two types of antigenic variation of its envelope glycoproteins, i.e. antigenic drift and antigenic shift. Analysis of virus isolates since 1933 reveals that viruses isolated in the years 19331946, 19471956, 19571967, and from 1968 onwards demonstrated wide variation. It is apparent that pandemics are due to the appearance of new influenza A subtypes against which the population has no immunity. Thus, antigenic shift occurs with the emergence of a novel, potentially pandemic influenza A virus that possesses novel haemagglutinin and/or neuraminidase subtype(s) that have not been present in human viruses for a long time. Based on previous pandemics, antigenic shift is hypothesized to occur due to three main factors, i.e. genetic reassortment, adaptation of animal viruses for replication in humans, and recirculation of existing subtypes. Antigenic drift is a more subtle process in which new strains arise from the accumulation of point mutations in the antibody-binding sites of HA and/or NA glycoproteins. The new strains are antigenic variants, belonging to the same subtype but do not cross-react with antibodies. Variants of epidemiological significance typically have multiple amino acid changes in the haemagglutinin occurring at more than one antigenic site, thus inhibiting the binding of neutralizing antibodies. Antigenic drift is thought to arise through natural mutation, and selection of new strains takes place by antibody pressure in an immune or partially immune population. Biological Evolution of Highly Pathogenic H5N1 Influenza Virus in Asia A series of genetic reassortment events traceable to the precursor of H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 and subsequent avian outbreaks in 2001 and 2002 indicate the rise of the dominant H5N1 genotype Z in chickens and ducks that was responsible for the regional outbreaks in 20022003. In 1997, an H5N1 avian influenza A virus was transmitted directly from chickens to humans in Hong Kong, killing 6 of the 18 people infected [42]. The virus continued to circulate in geese in southern China,

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but was soon replaced by different genotypes that were pathogenic in chickens and not in ducks. As these viruses were eradicated by the slaughter of poultry animals, they soon were replaced by six H5N1 reassortants in 2002, i.e. A, B, C, D, E and X0. From 2002 onwards, eight additional genotypes emerged (V, W, X1, X2, X3, Y, Z and Z0), while A, C, D and E were no longer detected. The viruses exhibited a 20-amino acid deletion in the stalk of NA (position 4968), thus explaining the ability of the virus to infect land-based poultry. Z replaced AE, X and Y to become dominant in land and aquatic poultry, although the genotype is not fully adapted to ducks and may continue to evolve through mutation or reassortment until it becomes well-adapted. Fouchier et al. [43] and Hulse-Post et al. [44] found a trend toward non-pathogenicity to ducks, indicating biological evolution and equilibrium in the natural hosts.

Pathogenesis

Systemic H5N1 infection in birds is characterized by hemorrhage and edema, with virus replication in the endothelium being important in pathogenesis. Factors underlying endotheliotropism include proteolytic activation of haemagglutinin, polarity of virus budding, and tissue-specific expression of virus receptors [45]. Fatal outcome in many patients with H5N1 infection may be attributed to acute respiratory distress syndrome (ARDS), sepsis syndrome, and/or multiorgan failure. H5N1-infected patients who succumb to ARDS and respiratory failure exhibit severe pulmonary injury with diffuse alveolar damage, fibrinous exudate and red cells in alveoli, vascular congestion, hyaline membrane formation, interstitial lymphocytic infiltration, and reactive fibroblast proliferation. Other pathological features include lymphopenia, atypical lymphocytes, reactive histiocytosis, haemophagocytosis, centrilobular hepatic necrosis and acute tubular necrosis. The innate immune responses to H5N1 may contribute to disease pathogenesis since elevated levels of inflammatory mediators were observed among avian influenza patients. These include interleukin-1 , interleukin-6, interleukin-8, interleukin-2 receptor, tumour necrosis factor- , interferon- , interferon- , interferon-inducible protein 10, monokine induced by interferon- and monocyte chemoattractant protein 1. Such responses may be delayed or attenuated by the use of corticosteroids. In patients who recover from infection, specific antibody responses to H5N1 can be detected by the neutralization assay 10 days to 2 weeks following the onset of illness [46]. Genomic and proteomic analyses (infectomics) of H5N1 infection in susceptible target cells can help to elucidate the molecular mechanisms that underpin the pathogenesis of the related disease manifestations. Understanding the

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roles of the individual virulence genes and proteins of the H5N1 virus in causing morbidity and mortality can also lead to the design of new strategies in the treatment and management of avian influenza [47, 48].

Are We Prepared?

The factors responsible for the pathogenesis of influenza are still not completely understood. These include the nature of tissue restriction of proteases, differences in the effect of the innate immune system at different stages of life, and varying susceptibilities to the infection in different populations. Taking these factors into consideration, emphasis must be placed on alternative means for attenuating influenza viruses. Vaccines Currently, influenza vaccine strains are propagated in fertile hens eggs, and inactivated with formaldehyde or -propiolactone. Either the whole virus, detergent-treated split product, HA or NA antigens are utilized. Whole virus vaccines are not preferred due to their adverse side effects in children. Many new developments in vaccine technology have been observed in recent years, including the introduction of subunit influenza vaccine with adjuvant MF59, an emulsion of squalene in water. It increases the antibody response to interpandemic influenza A H3N2 and influenza B viruses. The local reactions are minimal and are well-tolerated among older people [49]. The vaccine is accepted in many of the European countries except in UK. Virosomes, another development in vaccine technology, are phospholipid bilayers enclosing the significant viral surface antigens. Some of their advantages include high rates of antibody production and seroconversion. Virosomes became available in UK from 2002 onwards [50]. Another approach is a live attenuated vaccine which was approved by the US Food and Drug Administration in June 2003. The strategy involves the intranasal delivery of live attenuated strains with genes coding for cold adaptation. The vaccine offers the advantage of imitating a natural infection, but the viral replication is restricted in the nasopharynx and not in the lower airways, thus preventing systemic replication. This vaccine provides a broader immunological response than the inactivated vaccines [51]. Reverse Genetics The development of reverse genetics offers several advantages over the traditional method, and a potential approach for preparing interpandemic vaccines. With plasmid-based reverse genetics, an antigenically new strain

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recommended by the WHO is co-infected along with the laboratory reference strain PR8 in double embryonated hens eggs to produce a high-growth reassortant (HGR) novel vaccine strain. Since there is often an element of unpredictability in the yield of the strain coupled with the restraint of certain strains which cannot be cultivated in eggs, the reverse genetics technique provides an alternative solution. Its advantages include: (a) a direct rational procedure manipulating the negative-sense RNA in contrast to the hit-or-miss traditional method; (b) contamination of wild-type virus derived from a non-validated cell line that may contain other pathogens; (c) the genes such as HA can be manipulated to obtain desired reassortants as previously demonstrated by Schickli et al. [52], Subbarao and Katz [53] and Liu et al. [54]. Nicholson et al. [55] have similarly used reverse genetics to derive a novel H5N1 reassortant virus, NIBRG-14 that utilizes the HA and NA of an avian influenza strain (responsible for several deaths in East Asia in 2004) generated in Vero cells. Nucleic Acid Vaccines DNA vaccines have also proven to be a promising new approach in the development of novel vaccines in the battle against influenza viruses. It evokes a broader immune response, including humoral, cytotoxic and T cell responses. The ease with which the vectors are synthesized and manufactured presents the DNA vaccines as an alternative cost-effective approach for limiting the spread of potential pandemic influenza virus. Limited protection against lethal H5N1 challenge was achieved by DNA vaccination with conserved influenza NP and M. To improve the efficacy of this system, Epstein et al. [56] tested the ability of the recombinant adenovirus (rADV) vector to enhance the potency of vaccination with NP which has 90% homology among influenza A viruses and contains CTL epitopes in mice. Recently, immunization with adenovirusvectored influenza HA vaccine [57] has also shown promising results. Although DNA vaccines using conserved sequences of antigens exhibit protection in animals against infection with several subtypes, further analyses and strategies to improve their efficiency are required. Challenges for Vaccine Development An effective influenza vaccine must be designed to accommodate the potential for viral antigenic change via both antigenic shift and antigenic drift. HA proteins are rapidly subjected to antigenic change compared to NA proteins. Since anti-NA immunity is often suppressed due to antigenic competition of HA, Johansson et al. [58] have demonstrated the strategy of trivalent vaccines which utilizes purified proteins of HA along with both N1 and N2 of NA to reduce the suppression activity. Similar options should be explored to test the immunogenicity of other heterovariant HAs and NAs.

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Vaccines developed by culturing strains in embryonated eggs are being utilized for most interpandemic influenza infections. Such vaccines have limitations, such as contamination with other pathogens, accumulation of endotoxins, and the inability to obtain high yields with certain strains. The major obstacle facing vaccine production of highly pathogenic H5 and H7 is the requirement for a high-level bio-containment facility for handling these viruses. Standardized vaccines that can provide protection for poultry animals appear to be the greatest need. In addition, official approval of reverse genetics technology, cell line-based production, and use of alternative adjuvants need to be focused and expanded. During the initial stages of a pandemic between its detection and the availability of vaccines, vaccine stocks tend to be in short supply, and antiviral drugs will be essential and should be given appropriate emphasis in the control of infection [59]. Anti-Influenza Drugs There are two classes of antiviral drugs active against influenza A. One class of antivirals inhibits the uncoating steps of the virus by blocking the ion channel activity of M2 protein, e.g. amantadine and rimantadine. The other comprises neuraminidase inhibitors, e.g. zanamivir and oseltamivir. The M2 inhibitors amantadine and rimantadine are similar in their activity but differ in the metabolic processing. Amantadine is excreted with little change in renal tubular excretion, but rimantadine is processed in the liver. Viral resistance against these drugs is documented, and hence the neuraminidase inhibitors are preferred as they are also active against influenza B virus. In addition, M2 inhibitors are associated with adverse side effects. Amantadine is known to cause severe neurological disorders, coupled with insomnia, hallucinations and dizziness in elderly people. The genetic basis for resistance is a single nucleotide substitution, resulting in amino acid replacement at positions 26, 27, 30, 31 or 34 in the membrane of M2 [22]. The side effects associated with rimantadine are less severe and infrequent compared to amantadine. The neuraminidase inhibitor zanamivir is a second-generation influenza drug, a sialic acid analogue developed by molecular design with a three-dimensional structure of neuraminidase spikes. Both zanamivir and oseltamivir carboxylate interrupt the replication cycle by preventing viral release and allowing clumping of viral progeny on the surface of the host cell [60]. Zanamivir has poor bio-availability and hence is administered nasally, whereas oseltamivir is taken orally. The side effects associated with zanamivir are negligible except for bronchospasm in asthmatic patients. Oseltamivir is associated with frequent nausea (37%) and vomiting, but these side effects can be avoided if the drug is taken shortly after meals. Bantia et al. [61] have reported a novel neuraminidase inhibitor known as RWJ-270201, a cyclopentane derived by structural design and is effective against both influenza A and B. The drug is active against the virus in mice

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when administered orally for prophylaxis and treatment. Studies have suggested superior activity of this drug compared to zanamivir and oseltamivir carboxylate. Slower dissociation of the compound from the enzyme is suspected. Raising deep concern recently was the disturbing report of oseltamivir resistance associated with the deaths of some patients with avian influenza despite early and aggressive treatment with this antiviral [62, 63]. Rapid Detection Techniques Conventional cell culture methods and serological testing require between 2 days and 2 weeks for detecting the presence of influenza viruses. Such a delay can affect therapeutic decisions and infection control in an emergency situation. Currently, rapid antigen tests are employed such as the Directigen Flu A B or the Binax NOW. Sensitive and rapid RT-PCR techniques which enable the typing and subtyping of influenza strains have been developed. Faster and more reliable than nested RT-PCR, these new multiplex RT-PCR techniques specifically target two different regions of the H5 gene (based on the 2004 Vietnam H5N1 strain). Such techniques also reduce the risk of contamination and are at least three times faster. Uiprasertkul et al. [64] detected viral RNA in lung, intestine and spleen tissues, but found the presence of positive-sense viral RNA in lung and intestine indicating viral replication confined to these sites. They also detected viral replication sites in type II pneumocytes. These pneumocytes are alveolar epithelial cells and progenitors to both type I and II cells. Their findings based on the RTPCR method also imply that sputum and bronchoalveolar lavage samples from the lower respiratory tract may provide a higher sensitivity for viral detection than the upper respiratory tract as is conventionally sampled. Conventional haemagglutination tests are still performed, but provide limited application for the detection of novel subtypes due to the frequent change in HA antigens.
Conclusions The Need for Global Awareness and Surveillance

In the modern world, the emergence of a strain as virulent as that of 1918 would be devastating. The occurrence of an influenza pandemic can cause damage of serious dimensions with grave political, social and economic consequences. Good communication links should be established between various sectors of the community such as clinical, public health and veterinary services. In the event of a pandemic outbreak, emergency decisions have to be made by the health authorities amidst a prevailing atmosphere of uncertainty, thus inspiring the establishment of the Global Outbreak Alert and Response Network (GOARN) in early 2000. Armed with specialized staff and technical resources spanning 120 networks and institutions, their services can be deployed for emergency situations, and on-the-spot assistance in the event of an outbreak.

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Non-medical interventions such as maintaining hygiene and upgrading agricultural practices can potentially reduce the opportunities for transmission. Recommendations have been made by the agricultural authorities including the Food and Agriculture Organization of the United Nations to reduce contact between domestic poultry and wild birds. An example is to eliminate the undesirable practice by many farmers of raising domestic ducks and chickens in ponds, which frequently attracts wild ducks that are often reservoirs for the harmful H5N1 virus. Similarly, other changes in agricultural practices should be implemented, e.g. pigs that are considered as mixing vessels for influenza virus reassortment should be separated from poultry animals and humans in an ecologically controlled manner to prevent future pandemics. Current surveillance should be focused on methods of rapid identification of novel strains in humans and birds, as well as efforts to minimize cross-infection between species. The options available for the control of a pandemic strain are broadened by the introduction of reverse genetics and the development of novel vaccines and antivirals [65, 66]. Proper utilization of antivirals must involve stockpiling in advance of a pandemic threat, which will require coordinated public health planning and political commitment of resources. The flurry of research activities stimulated by the series of outbreaks in 2004 and 2005, including the patterns of evolution, cross-species transmission and origin of the virus must be tracked carefully. Due attention should be given to certain unresolved issues such as the acceptance of genetically modified viruses in humans, the antigen content in vaccines, intellectual property rights and liability matters. Finally, avian influenza is a global problem which may result in catastrophic devastation if it ignites into a pandemic. No single country can handle this all by itself, and concerted efforts in an organized manner across communities and continents need to be established.

Acknowledgements
The authors wish to thank Ms. Mamta Mohandass and Ms. Alisha Lal for help in collecting literature and typing the manuscript.

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Gluck R, Moser C, Metcalfe IC: Influenza virosomes as an efficient system for adjuvanted vaccine delivery. Expert Opin Biol Ther 2004;7:11391145. Betts RF, Douglas RG Jr, Maassab HF, DeBorde DC, Clements ML, Murphy BR: Analysis of virus and host factors in a study of A/Peking/2/79 (H3N2) cold-adapted vaccine recombinant in which vaccine-associated illness occurred in normal volunteers. J Med Virol 1988;26:175183. Schickli JH, Flandorfer A, Nakaya T, Martinez-Sobrido L, Garcia-Sastre A, Palese P: Plasmid-only rescue of influenza A virus vaccine candidates. Philos Trans R Soc Lond [B] 2001;356:19651973. Subbarao K, Katz JM: Influenza vaccines generated by reverse genetics. Curr Top Microbiol Immunol 2004;283:313342. Liu M, Wood JM, Ellis T, Krauss S, Seiler P, Johnson C, Hoffmann E, Humberd J, Hulse D, Zhang Y, Webster RG, Perez DR: Preparation of a standardized, efficacious agricultural H5N3 vaccine by reverse genetics. Virology 2003;314:580590. Nicholson KG, Wood JM, Zambon M: Influenza. Lancet 2003;362:17331745. Epstein SL, Tumpey TM, Misplon JA, Lo CY, Cooper LA, Subbarao K, Renshaw M, Sambhara S, Katz JM: DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice. Emerg Infect Dis 2002;8:796801. Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto A: Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization. J Virol 2006;80:19591964. Johansson BE, Pokorny BA, Tiso VA: Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition. Vaccine 2002;20:16701674. Lipatov AS, Govorkova EA, Webby RJ, Ozaki H, Peiris M, Guan Y, Poon L, Webster RG: Influenza: emergence and control. J Virol 2004;78:8951. Monto AS: Vaccines and antiviral drugs in pandemic preparedness. Emerg Infect Dis 2006;12:5560. Bantia S, Parker CD, Ananth SL, Horn LL, Andries K, Chand P, Kotian PL, Dehghani A, El-Kattan Y, Lin T, Hutchison TL, Montgomery JA, Kellog DL, Babu YS: Comparison of the antiinfluenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir. Antimicrob Agents Chemother 2001;45:11621167. de Jong MD, Thanh TT, Khanh TH, Hien VM, Smith GJD, Chau NV Cam BV, Qui PT, Ha DQ, , Guan Y, Peiris JSM, Hien TT, Farrar J: Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med 2005;353:26672672. Moscona A. Oseltamivir resistance disabling our influenza defenses. N Engl J Med 2005;353: 26332636. Uiprasertkul M, Puthavathana P, Sangsiriwut K, Pooruk P, Srisook K, Peiris M, Nicholls JM, Chokephaibulkit K, Vanprapar N, Auewarakul P: Influenza A H5N1 replication sites in humans. Emerg Infect Dis 2005;11:10361041. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M: Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med 2006;354:13431351. Bresson JL, Perronne C, Launay O, Gerdil C, Saville M, Wood J, Hoschler K, Zambon MC: Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: phase I randomised trial. Lancet 2006;367:16571664.

Sunil K. Lal, PhD Senior Research Scientist, Virology Group International Centre for Genetic Engineering and Biotechnology New Delhi 110067 (India) Tel. 91 11 26189360/61, Fax 91 11 26162316, E-Mail sunillal@icgeb.res.in

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Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 7893

Henipaviruses: New Threats for Southeast Asia and Australia


Joe McCormacka, Greg Smithb
Department of Medicine and Infectious Diseases, University of Queensland and Mater Hospitals, Raymond Terrace, South Brisbane, bPublic Health Virology Unit, Queensland Health Pathology and Scientific Services, Coopers Plains, Australia
a

Abstract
Hendra virus was first encountered in Brisbane, Australia in 1994 and subsequently in other more northerly parts of Queensland. Nipah virus was first encountered in northern Malaysia in 1998 but has subsequently been found in Singapore, Bangladesh, Cambodia, Thailand, Indonesia and India. It has been established that bats, particularly Pteropus species (fruit bats or flying foxes), act as the reservoir for both viruses with horses (Hendra virus) or pigs (Nipah virus) as intermediate hosts and humans as occasional victims. These closely related viruses form a new genus (Henipavirus) of the subfamily Paramyxoviriniae of the family Paramyxoviridiae. The morphology and genomic structure of these viruses have been established and their nucleoprotein, phosphoprotein, C protein, matrix protein and fusion protein have been characterised. Diagnosis of infection with Henipaviruses can be made by serology, molecular testing or culture. Human clinical illness consists of fever, encephalitis and/or pneumonitis; morbidity and mortality rates are high, although the incidence of subclinical infection is uncertain. There is no treatment of proven value but ribavirin may be beneficial; there is no vaccine. Henipaviruses represent a significant threat to the Indian subcontinent, Southeast Asia and Australia.
Copyright 2007 S. Karger AG, Basel

Hendra and Nipah viruses have, as far as we know, become human and animal pathogens only in the last 12 years. Their initial appearances were confined to small areas, were explosive in their onset and were accompanied by considerable debate, drama and excitement in the media. As with many other viruses which have crossed species barriers (e.g. HIV/AIDS, SARS and avian influenza) the circumstances under which such crossing has occurred should be studied and understood so that the likelihood of future similar events can be anticipated and their impact minimised.

Table 1. Taxonomy of the family Paramyxoviridae Family Subfamily Paramyxovirinae Genus Respirovirus Morbillivirus Rubulavirus Henipavirus Avulavirus Pneumovirinae Pneumovirus Metapneumovirus Type species Sendai virus measles virus mumps virus Hendra and Nipah viruses Newcastle disease virus human respiratory syncytial virus avian pneumovirus

Paramyxoviridae

Virology

Taxonomy Hendra (HeV) and Nipah (NiV) virus have been placed into a newly described genus Henipavirus within the virus subfamily Paramyxovirinae and the family Paramyxoviridae [1, 2] (table 1). The Paramyxoviridae belong to the order Mononegavirales which include three additional virus families; Filoviridae, Rhabdoviridae and Bornaviridae. All viruses within the order possess a number of common features and all are believed to share a common ancestral link. Common taxonomic features include a similar genome organisation, a monopartite single-stranded negative sense RNA genome and close sequence similarity in the functional regions of their RNA-dependant RNA polymerase. Other features which are shared by members of the order are: complementarity of the 3 and 5 termini; presumptive single 3 terminal promoter, sequential transcription by interrupted synthesis; replication by synthesis of a complete positive-sense intermediate transcript and maturation by budding [2, 3]. The Paramyxoviridae have been traditionally distinguished from other members of the order by a spherical to pleomorphic morphology and a genome size range of between 15.1 and 15.9 kb. The discovery of HeV and NiV necessitated expansion of this size range to 18.2 kb. It now appears that this expanded size range may need to be revised again to accommodate recently characterised rodent paramyxoviruses [4, 5, Dr Lin-Fa Wang personal communication] a range that would overlap with that traditionally occupied by the filoviruses (19.1 kb). Classification within the family Paramyxoviridae is based on: (1) morphological criteria; (2) genome organization; (3) sequence relatedness, and (4) biological properties of the viral proteins.

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Fig. 1. Electron micrograph of Hendra virus. Courtesy of Howard Prior QDPI&F.

Morphology The virions of both HeV and NiV have been intensively studied at an ultrastructural level [6, 7]. The virions are pleomorphic viruses, 40600 nm in diameter, containing a lipid envelope obtained from the plasma membrane and a helical nucleocapsid (fig. 1). The majority (95%) of HeV virions are doublefringed while NiV is predominately single-fringed. The two different sized surface projections which create the double-fringed appearance of HeV have average lengths of 15 1 and 8 1 nm while those on NiV are slightly longer at 17 1 nm. The nucleocapsid of HeV is approximately 18 1 nm in diameter with a pitch of 5 1 nm [6]. The Henipavirus Genomes HeV and NiV share a similar genome organisation and boast genome sizes of 18,234 [8] and 18,246 nucleotides [9], respectively. Based on these published

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sequences the viruses have a nucleotide homology within the protein coding regions of between 70.8 and 88.5% [8]. The coding region is bounded by a 3 extracistronic leader of approximately 50 nucleotides and a 5 extracistronic trailer of approximately 50 nucleotides, the first 12 nucleotides of which are highly conserved and complementary. The viruses contain six transcription units encoding six major structural proteins in the anti-genome order (3 to 5 ) of nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G) and the RNA-dependent RNA polymerase (L). Each of these transcription units, incorporating conserved gene start and stop signals, are separated by a highly conserved intergenic spacer consisting of a conserved tri-nucleotide junction of 3 -GAA-5 which is shared with the morbilliviruses and respiroviruses. The predicted transcriptional start signal of the HeV and NiV N, P, M and F genes are identical while that of the G gene differs by a single nucleotide (UC). The transcriptional stop signals of the N, P and G genes of the Henipaviruses are also identical but the termination signals of the M and F genes differ by two nucleotide changes each. Nucleoprotein (N) The Paramyxovirus N-protein encapsulates the RNA genome in an RNaseresistant nucleocapsid and possesses specific binding sites which allow it to interact with the P-L polymerase complex during replication and with the M protein during assembly. The N-protein of both HeV and NiV are identical in length (532 aa), 92.1% similar at the amino acid level and have a calculated Mr of 58,481 and 57,993 Da, respectively. Consistent with other paramyxoviruses the Henipavirus N-protein contains two domains; a well conserved N-terminal domain and a highly charged C-terminus which is hypervariable with 52% of the total amino acid variability between HeV and NiV occurring in the last 42 aa of the C-terminal [1, 9, 10]. The C-terminal domain contains most of the proteins phosphorylation and antigenic sites while the N-terminal domain is believed to possess the RNA binding domains and determinants for the helical structure. An invariant, predicted hydrophobic sequence in the middle domain (aa 171383) which is thought to play a part in N:N, N:P and N:L interactions are also present in the Henipaviruses. Phosphoprotein (P) The Paramyxovirus P-protein interacts with the L gene product to produce an RNA dependent RNA polymerase. There is considerable variation in length of the P protein between the different Paramyxovirus genera and at just over 700 aa in length the Henipaviruses P protein is over 100 bp larger than the next largest that of the morbilliviruses (507603 aa). In Henipaviruses it is the largest of three proteins encoded by the P gene and is predicted to be 707 aa in

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length in HeV and 709 aa in length in NiV. The C-terminal domain is crucial to the functioning of the P-protein and contains a domain responsible for the trimerisation of the protein and a domain which interacts with the middle domain of N leading to the formation of the nucleoprotein complex.. The N-terminal portion contains a number of functional domains, includes most of the phosphorylation sites and is rich in acidic residues. The P proteins of HeV and NiV share 67.6% identity at the amino acid level and their respective molecular masses have been calculated to be 78,305 and 78,301 Da although the low pI (4.444.45) of the proteins due to the acidic residues in the C-terminus results in an unusually slow migration in SDS-PAGE gels resulting in an apparent molecular weight of 98 kDa [1]. The P gene of the paramyxoviruses encodes a variable number of additional (accessory) proteins through a complex transcriptional editing mechanism known as pseudotemplating or via the use of alternative internal initiation sites. Both HeV and NiV possess two such proteins V and C [1, 9, 11]. V-Protein The N- and C-terminal domains of the P protein are separated by an AGrich hypervariable region. The V protein is encoded in the P-1 frame through the addition of a single non-templated G residue at this conserved AG-rich editing site (GGGUAAUUUUUCCC) which is identical on the unedited P mRNA of HeV NiV and some morbilliviruses. The addition of the non-templated G , leads to a reading frame shift and a resultant protein which has an N-terminal (402 aa) which is identical to P but a unique cysteine-rich 55 (HeV) or 52 (NiV) amino acid carboxy-terminal which lacks the trimerisation and N-binding sites critical to the functioning of P. The V-protein of HeV is predicted to be 457 aa long and possess a molecular mass of which is predicted to be 50,647 Da but like P migrates much slower in SDS-PAGE gels to produce a band with an apparent Mr of 70,000 Da [1, 11]. C-Protein The Paramyxovirus C proteins are small basic proteins (180204 aa) which are expressed from the P gene as a result of RNA editing and the use of alternate start codons including ACG and GUG. In the case of HeV the proposed AUG initiation codon for C is out of frame with that of P ( 2) and results in a putative C protein of 166 aa in length and a calculated Mr of 19,647 Da [1]. In NiV there are consecutive AUG codons located at 23 and 26 nucleotides downstream of the P protein initiation codon [9]. It is predicted that the first initiator is employed as it would produce a C protein identical in size to that predicted for HeV (166 amino acids in length and a Mr of 19,735 Da). The predicted C proteins of HeV and NiV have 85% homology at the nucleotide level and an identity of 83.2% at

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the amino acid level. There is a hypervariable region between aa 31 and 45 which contains eleven non-conservative amino acid substitutions. The HeV contains a second Open Reading Frame (ORF) that putatively codes for a short basic protein of 65 amino acids but no such ORF is present in NiV [1, 9]. Matrix Protein (M) The Paramyxovirus M protein is a highly basic protein and the most abundant. The protein is believed to form a layer on the inner surface of the cell membrane and serves as an anchor site for the cytoplasmic tails of the two transmembrane proteins the glycoprotein (G) and fusion (F) proteins. This interaction coupled with strong binding to the acidic C-terminal of N is believed to trigger virus budding while interaction with the RNP is thought to terminate viral RNA synthesis. The M genes of HeV and NiV have a nucleotide homology of 77.1% in the ORF but only 40% in the 3 and 5 untranslated regions. The mRNA of NiV is 1359 nt in length and has an ORF of 1059 nt which encodes an M protein of 352 aa with a predicted Mr of 39,928 Da [9]. The M gene of HeV encodes a protein of an identical length with a slightly lower molecular mass of 39,827 [1]. Fusion Protein (F) The Paramyxovirus fusion proteins are type I trans-membrane proteins and one of the two major envelope proteins. The F protein which plays a critical role in virus-cell and cell-cell fusion remains inactive (F0) until cleaved by a host cell enzyme (often furin) which transforms it into a biological active protein consisting of two disulphide-linked subunits (F1 and F2). Paramyxoviruses F protein cleavage/activation sites either have multibasic residues at the cleavage site or just a single basic residue at the cleavage site. Those with multibasic residues are cleaved intracellularly during transport through the trans-Golgi. In both HeV and NiV, the F protein is predicted to be 546 amino acids in length and possess a molecular mass of 59,811 and 60,233 Da, respectively. On SDSPAGE three bands are discernable: F0 at 61 kDa, F1 at 49.5 kDa and F2 at 19 kDa [12]. The HeV and NiV F protein coding regions share 74.2% nucleotide homology and a predicted amino acid identity of 88.1%. The Henipavirus F proteins differ from other paramyxoviruses by possessing a leucine residue in place of a phenylalanine as the first amino acid at the amino terminal of the F1 peptide [9]. The furin cleavage site common to many other members of the family is not present on the Henipaviruses suggesting that another, as yet unidentified host cell enzyme is responsible for F protein cleavage. The ability of the virus to replicate in cell lines that do not produce furin [13] provides phenotypic evidence to support the reliance of Henipaviruses on a non-furin host cell protease.

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Glycoprotein (G) Gene The Paramyxovirus cell attachment protein is a type II transmembrane protein and the major immunodominant determinant. In some genera the protein has both haemagglutinating (HA) and neuraminidase activity (respiroviruses) while in others, (the morbilliviruses) there is an absence of neuraminidase activity. Other genera (henipaviruses and pneumoviruses) lack both HA and neuraminidase activity. The Henipavirus G genes encodes proteins of 602 (NiV) and 604 (HeV) amino acids in length which share 83.35% identity at the amino acid level. The proteins have a calculated Mr of 67,038 Da and 67,192 Da, respectively. Both viruses share 17 identical cysteine residues and 8 N-linked glycosolation sites [9]. The HeV G protein has an apparent MW of 74 kDa on SDS-PAGE gels [12], the variation in size from the 67 kDa predicted is undoubtedly due to glycosolation of at least some of the eight potential sites as HeV has been shown to bind lectin [1, 14]. The Polymerase or L-Gene Product The large gene (L) is the largest, least abundant and most highly conserved of the Paramyxovirus proteins. The RNA-dependent RNA polymerase plays a critical role in RNA synthesis and also serves as a guanyl- and methyltransferase during capping of the viral encoded mRNAs. The Paramyxovirus L protein contains five highly conserved regions located towards the centre of the protein. The Henipavirus L genes each encode a protein of 2244 amino acids in length with calculated molecular masses of 257,280 Da (HeV) and 257,189 Da (NiV), a nucleotide similarity of 74.7% and an amino acid identity of 86.8% [1]. Henipavirus Diagnostics Both HeV and NiV are regarded as biosecurity level 4 agents in most countries and any specific attempt to propagate the virus in cell culture or animal systems from clinical material should be conducted in appropriate containment facilities. Care should also be taken when routinely processing clinical material where either virus is considered in the differential diagnosis but neither is specifically suspected. In such circumstances PC3 facilities and procedures could be used providing rigorous adherence to sound microbiological practices appropriate to that level are observed. There are steps that can be taken to reduce the risk of processing samples for molecular or serological testing. Irradiation of serum or pre-treatment by heat inactivation (56 C for 30 min) followed by diluting the serum sample 1:5 in phosphate-buffered saline containing 0.5% Tween 20 and 0.5% Triton-X100 has been suggested [1]. Although not specifically demonstrated for Henipaviruses, autoclaving has been demonstrated to be effective at

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rendering material non-infectious but still suitable for nucleic acid amplification and this approach may well be a viable alternative for laboratories without appropriate containment facilities [15, 16]. Sample Submission For ante-mortem diagnosis of henipavirus in humans, urine, serum, CSF, nasal and throat swabs should be collected and submitted for molecular testing and/or virus isolation where clinically appropriate. Brain, lung, kidney, spleen, urine and serum are important material to be collected for post-mortem diagnosis. As with any material collected for virus isolation, appropriate aseptic techniques should be used for collection and care taken to maintain a cold chain during transport to the laboratory. Serum, particularly paired acute and convalescent samples, are important for ante-mortem serological diagnosis particularly where the severity of the illness does not warrant more invasive sampling. Virus Isolation Where PC4 facilities are not available virus isolation should not be attempted. Standard virus isolation techniques have proven successful. Briefly, 10% (w/v) tissue suspensions are prepared in cell culture medium, clarified by low-speed centrifugation and the clarified supernatant inoculated onto decanted confluent cell monolayers. Both viruses are capable of infecting a wide variety of cell lines but the most commonly used are Vero E-6 (ATCC C1008) or Vero (ATCC CCL 81) [9, 17]. Following 1 h adsorption at 37 C the inoculated monolayer is refed with culture medium and monitored daily for signs of a cytopathic effect typically large syncytia. Electron microscopy, immunofluorescence or immunoperoxidase staining of acetone- or methanol-fixed slides can provide confirmation of virus recovery [18]. At least three blind passages, each of at least 5 days duration, should be attempted before the isolation attempt is abandoned. Nucleic Acid Detection Reverse transcriptase polymerase chain reaction (PCR) has proven to be a sensitive and specific approach for the diagnosis of both HeV and NiV [19, 20]. The Centre for Disease Control (CDC), Atlanta, has focused on the N gene [20] as a target while the Australian Animal Health Laboratory has focused on the M gene [19]. More recently, the introduction of specific real-time (TaqMan)-based PCR assays for both HeV [21] and NiV [22] has allowed more rapid test results with lower risk of contamination. The high degree of similarity between HeV and NiV combined with the observed sequence stability of both viruses allows the development of henipavirus specific real time PCR assays for epidemiological as well as clinical investigations.

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Serology Enzyme-linked immunosorbent assays (ELISAs) and immunofluorescent antibody assays (IFAs) are the backbone of both human and animal serological investigations. The ELISA assay is particularly well suited for screening large numbers of samples as part of epidemiological investigations or in response to outbreak situations [23]. A variety of different ELISA formats have been described or are under development including IgM capture ELISAs and tests using baculovirus-expressed antigens [17, 24] The IFA test is better suited for screening single specimens or small numbers of specimens. It is quicker to perform and less labour intensive than the ELISA for small numbers of samples. A variation of the IFA, the immune plaque assay, which employs methanol fixed infected cell monolayers in tissue culture plates and peroxidase conjugate for the detection of both HeV and NiV has recently been described [18]. Since both the ELISA and IFA tests rely on inactivated viral antigens they are suited to use outside of secure PC4 environments. When employing tests such as the IFA, it is important that a fourfold rise in antibody titre be demonstrable between acute and convalescent samples. Non-specific reactivity as well as extensive cross-reactivity between HeV and NiV has been reported for the ELISA assays and as a consequence the serum neutralisation tests remain the best confirmatory tests for theses viruses. The test requires live virus and is thus limited to those facilities with PC4 containment laboratories. Several variations have been described [9, 17, 18]. The test is particularly useful for discriminating between HeV and NiV as significant differences between homologous and heterologous titres are observed [9].

Epidemiology

Hendra Virus HeV was named after the suburb in Brisbane, Australia, where the first outbreak occurred. In 1994, two humans and 21 horses were diagnosed with what was known at the time as Equine Morbillivirus infection [25, 26]. Both patients worked at a stable in Hendra, one developed an influenza-like illness from which he recovered, the other had a fulminating septicaemic pneumonia from which he died. Eighteen horses at the stable developed pneumonic illness, 14 of these died. Three other horses at the stable were found to be seropositive with the HeV but were asymptomatic. The second outbreak occurred in Mackay approximately 1,000 km north of Brisbane [26, 27]. A farmer died of a fulminating encephalitic illness in 1995. A year earlier he had aseptic meningitis from which he recovered. At that time two horses on his property had died, one from pneumonia, the other from

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Table 2. Henipavirus Infections Hendra Outbreaks Australia (Brisbane) 1994 Australia (Mackay) 1995 Australia (Cairns) 2000 Australia (Cairns) 2004 fruit bats horses humans fever pneumonitis encephalitis Nipah Malaysia (Nipah) 1998/99 Singapore 1999 India (Siliguri) 2001 Bangladesh (Mahardur) 2001 Bangladesh (Naogaon) 2002 fruit bats pigs humans fever encephalitis pneumonitis

Species involved

Clinical features in humans

Adapted from McCormack [55].

encephalitis and he had assisted at their post-mortems. HeV was found to be the cause of infection and death in the two horses and the farmer as well as being responsible for the farmers earlier aseptic meningitis. In 2000, an isolated case of HeV infection was found in a horse that had died of pneumonia near Cairns in northern Australia [28]. No human cases were found. In 2004, there was a further isolated case of HeV infection in Cairns. A veterinarian suffered from a self-limiting febrile upper respiratory tract infection and had recently carried out a post-mortem on a horse that had died from a respiratory tract infection. In retrospect, the horse was likely to have died as a result of HeV infection but was buried after the post-mortem and no further tissues were available for examination [Jeffrey Hanna, pers. commun.]. In all cases, the diagnosis of HeV infection was made by serological testing or by tissue electron microscopy, culture or molecular testing. In total therefore there have been 4 human and 24 equine cases of HeV infection spread over a 10-year period and covering a 2,000 km distance between Brisbane and Cairns. The features are summarised in table 2. Following the HeV outbreaks in Brisbane and Mackay there were several studies undertaken to determine if other humans or animals were also infected. Two hundred and ninety-six people who had varying levels of contact with the infected humans and horses were tested for evidence of HeV infection and all were sero-negative. These included workplace and domestic contacts and a range of health care workers [29]. Serological tests carried out on potential contacts of the patient in Cairns were also negative [Jeffrey Hanna, pers. commun.].

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The role of horses as a potential reservoir for HeV infection was diminished by failure to find any seropositive equine cases in more than 2,000 tested throughout Queensland. More than 5,000 samples from 40 other animal and bird species were also seronegative [30]. The reservoir role of fruit bats was established when 9% of Pteropus species colonising the east coast of Australia and Papua New Guinea were found to be seropositive [31]. Experimental transmission from bats to horses and between horses has been demonstrated [32]. HeV has been isolated from Pteropus species bats and they seem to be asymptomatic carriers [33]. Nipah Virus NiV is named after the town in northern Malaysia where the virus was first found. Between September 1998 and June 1999 an attack of fever and encephalitis occurred in 265 patients. The causative agent was found to be very similar to HeV There was a strong association with a history of contact with . pigs and the majority of patients were male piggery workers with close and recent exposure [20, 34, 35]. Small numbers of cases of asymptomatic seropositive abattoir workers [36] and military personnel who dealt with pigs involved in the outbreaks [37] were found in Malaysia. At the same time there was a smaller outbreak involving 11 abattoir workers in Singapore who had encephalitis and/or pneumonitis [20, 23]. NiV was also identified as the cause of two outbreaks in two separate provinces in Bangladesh: in Mahardur, 2001 and Naogaon, 2003. A total of 25 cases were found, all had fever and encephalitic features; some also had respiratory involvement [38]. In early 2001, there was an outbreak of fever and encephalitis affecting 66 patients in Siliguri, West Bengal, India. In retrospect, it was demonstrated that the majority of these patients tested were infected with NiV with a strain more , closely related to the Bangladeshi than the Malaysian virus [39]. NiV has been identified in bat species in Malaysia [40, 41], Cambodia [42, 43], Thailand [44] and Indonesia [45]. Infection in bats is thought to be asymptomatic and, as with HeV, these represent the likely reservoir. Pigs act as the intermediate host; transmission between pigs has been demonstrated experimentally [46]. Experimental inoculation of NiV into piglets shows that virus is capable of infecting their lungs and central nervous system [47]; however, most are not unwell. The role of pigs as intermediate hosts was demonstrated in Malaysia by the fact that control of the outbreak was achieved at least in part by destroying more than one million pigs [48]. In the Mahardur (Bangladesh) outbreak, a history of contact with a cow was a significant risk factor but no evidence was found of infection in cows or any other animals [38].

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It has been postulated that the Malaysian outbreak was contributed to by deforestation and drought leading to migration of bats away from their usual forest habitat to fruit orchards that were close to piggeries [49].

Transmission

Hendra Virus The exact mode of transmission of HeV is not known. Human-to-human transmission has not been documented. Horse-to-horse transmission has been documented and demonstrated experimentally [26, 32] and horse-to-human transmission has been assumed [26]. Such transmission has occurred in the cramped conditions of a stable and via post-mortem procedures. It seems likely that transmission occurs by contamination of human hands or in the case of horses, via respiratory tract secretions. Transmission from bats to horses has been well documented but it is not clear how this occurs. It has been suggested that the Australian paralysis tick Ixodes holocyclus may play an intermediary role [50]. Direct transmission from bats to humans has not been demonstrated. In all cases of human HeV infection there has been involvement of a sick or dead horse as an intermediary. Nipah Virus As with HeV the exact mode of NiV infection is not known. There has been one documented episode of human-to-human transmission from a patient to a health care worker [51]. It is thought likely that infection from bats to pigs and humans is by close contact but airborne transmission is also possible. Person-to-person transmission was suspected but not proven in the Bangladeshi outbreak [38]. In the Indian (Siliguri) outbreak many hospital workers were affected but it was not possible to determine if any of these cases represented nosocomial rather than community transmission [39].

Clinical Features

Hendra Virus With only four documented human cases of HeV infection, it is not possible to determine a characteristic pattern. Two of the 4 patients had a self-limiting febrile upper respiratory tract infection. One had a fulminating septicaemia and pneumonia; one had aseptic meningitis followed by acute encephalitis 1 year later. As further human cases immerge a pattern will become more apparent. Of the 24 horses involved 17 died of their illness, 4 were euthanized because of

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their illness, and 3 were asymptomatic. The equine illness consisted predominantly of pneumonitis, but at least 1 horse had encephalitis. Nipah Virus NiV is capable of causing encephalitis and/or pneumonitis. In the Malaysian outbreak, acute encephalitis was the predominant presentation: approximately 40% died, 40% recovered completely, 7.5% had recurrent disease and 3% had late-onset (mean 8.4 months) encephalitis [34, 5254]. The incubation period was less than 2 weeks with fever, headache, giddiness and decreased levels of consciousness in the encephalitic cases [54]. In the Singaporean outbreak 8 of 11 patients presented with encephalitis and 3 with pneumonia [23]. In the Bangladeshi outbreak all patients presented with fever and encephalitic features, some also had respiratory symptoms. In the Indian outbreak all patients presented with fever and encephalitis [39].

Treatment and Vaccines

No antiviral drugs have been used in humans or horses with HeV infection. Ribavirin has been shown in vitro to inhibit HeV RNA synthesis [56]. There was a trend towards a benefit with the use of ribavirin in the NiV-infected patients in the Malaysian outbreak with a 36% reduction in mortality in those who received this drug versus those who did not [57]. More experience of clinical trials is required before any recommendation can be made regarding therapy. Treatment measures are supportive and relate to specific organ involvement. There have been no studies on vaccines for either condition. Their phylogenetic closeness suggests that there may be similarities in their responses to antiviral drugs and vaccines.

Note Added in Proof

A further case of Hendra virus infection has been found in a horse in a rural area 70 km north of Brisbane. The horse suffered from a respiratory tract infection and died. There were no human cases on serological testing of contacts.

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References
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Paton NI, Leo YS, Zaki SR: Outbreak of Nipah virus infection among abattoir workers in Singapore. Lancet 1999;354:12531256. White JR, Narasiman M, Ramlan M, Yeoh S, Daniels PW, Morrisey C, Olsen J, Crameri G, Eaton BT, Jamaluddin A: Establishment and operation of a serological screening capacity within Malaysia for the detection of Nipah virus antibodies in swine. Abstracts of the Australian Society of Microbiology Conference, Cairns, July 2000. Microbiol Austr, 6 Abstract, p 5.5. Selvey LA, Wells RM, McCormack JG, Ansford AJ, Murray K, Rogers RJ, Lavercombe PS, Selleck P, Sheridan JW: Infection of humans and horses by a newly described morbillivirus. Med J Aust 1995;162:642645. Paterson DL, Murray PK, McCormack JG: Zoonotic disease in Australia caused by a novel member of the Paramyxoviridiae. Clin Infect Dis 1998;27:112118. OSullivan JD, Allworth AM, Paterson DL, Snow TM, Boots R, Gleeson LJ, Gould AR, Hyatt AD, Bradfield J: Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet 1997;349:9395. Field HE, Barratt PC, Hughes RJ, Shield J, Sullivan ND: A fatal case of Hendra virus infection in a horse in north Queensland: clinical and epidemiological features. Aust Vet J 2000;78:279280. McCormack JG, Allworth AM, Selvey LA, Selleck PW: Transmissibility from horses to humans of a novel paramyxovirus, equine morbillivirus (EMV). J Infect 1999;38:2223. Ward MP, Black PF, Childs AJ: Negative findings from serological studies of equine morbillivirus in the Queensland horse population. Aust Vet J 1996;74:241243. Young PL, Halpin K, Selleck PW: Serological evidence for the presence in Pteropus bats of the paramyxovirus related to equine morbillivirus. Emerg Infect Dis 1996;2:239240. Williamson MM, Hooper PT, Selleck: Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 1998;76:813818. Halpin K, Young PL, Field HE, Mackenzie JS: Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 2000;81:19271932. Goh KJ, Tan CT, Chew NK: Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. N Engl J Med 2000;342:12291235. Amal NM, Lye MS, Ksiazek TG: Risk factors for Nipah virus transmission, Port Dickson, Negeri Sembilan, Malaysia: results from a hospital-based case-control study. Southeast Asian J Trop Med Public Health 2000;31:301306. Sahani M, Parashar UD, Ali R: Nipah virus infection among abattoir workers in Malaysia, 19981999. Int J Epidemiol 2001;30:10171020. Ali R, Mounts AW, Parashar UD: Nipah virus among military personnel involved in pig culling during an outbreak of encephalitis in Malaysia, 19981999. Emerg Infect Dis 2001;7:759761. Hsu VP, Hossain MJ, Parashar UD, Ali MM, Ksiazek TG, Kuzmin I, Niezgoda M, Rupprecht C, Bresee J, Breiman RF. Nipah virus encephalitis re-emergence, Bangladesh. Emerg Infect Dis 2004;10:20822087. Chadha MS, Comer JA, Lowe L, Rota PA, Rollin PE, Bellini WJ, Ksiazek TG, Mishra AC. Nipah virus-associated encephalitis outbreak, Siliguri, India. Emerg Infect Dis 2006;12:235240. Chua KB, Koh CL, Hooi PS: Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect 2002;4:145151. Yob JM, Field H, Rashdi AM: Nipah virus infection in bats (order Chiroptera) in peninsular Malaysia. Emerg Infect Dis 2001;7:439441. Olson JG, Rupprecht C, Rollin PE: Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambodia. Emerg Infect Dis 2001;7:439441. Reynes JM, Counor D, Ong S, Faure C, Seng V Molia S, Walston J, Georges-Courbot MC, Deubel V , , Sarthou JL: Nipah virus in Lyles flying foxes, Cambodia. Emerg Infect Dis 2005;11:10421047. Wacharapluesadee S, Lumlertdacha B, Boongird K, Wanghongsa S, Chanhome L, Rollin P, Stockton P, Rupprecht CE, Ksiazek TG, Hemachuda T. Bat Nipah virus, Thailand. Emerg Infect Dis 2005;12:19491951. Sendow I, Field HE, Curran J, Darminto, Morrissy C, Meehan G, Buick T, Daniels P. Henipavirus in Pteropus vampyrus bats, Indonesia. Emerg Infect Dis 2006;12:711712.

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Middleton DJ, Westbury HA, Morrissy C: Experimental Nipah virus infection in pigs and cats. J Comp Pathol 2002;126:124136. Weingartl H, Czub S, Copps J, Berhane Y, Middleton D, Mariszal P, Gren J, Smith G, Ganske S, Manning L, Czub M: Invasion of the central nervous system in a porcine host by Nipah virus. J Virol 2005;79:75287534. Lam SK, Chua KB: Nipah virus encephalitis outbreak in Malaysia. Clin Infect Dis 2002;34(suppl 2):S48S51. Chua KB, Chua BH, Wang CW: Anthropogenic deforestation, El Nino and the emergence of Nipah virus in Malaysia. Malaysian J Pathol 2002;24:1521. Barker SC: The Australian paralysis tick may be the missing link in the transmission of Hendra virus from bats to horses to humans. Med Hypothesis 2003;60:481483. Tan CT, Tan KS: Nosocomial transmissibility of Nipah virus. J Infect Dis 2001;184:1367. Chong HT, Kunjapan SR, Thayaparan TT: Nipah encephalitis outbreak in Malaysia, clinical features in patients from Seremban. Can J Neurol Sci 2002;29:8387. Tan CT, Goh KJ, Wong KT: Relapsed and late-onset Nipah encephalitis. Ann Neurol 2002;51:703708. Tan CT, Wong KT: Nipah encephalitis outbreak in Malaysia. Ann Acad Med Singapore 2003;32:112117. McCormack JG: Hendra and Nipah viruses: new zoonotically acquired human pathogens. Respir Care Clin 2005;11:5966. Wright PJ, Crameri G, Eaton BT: RNA synthesis during infection by Hendra virus: an examination by quantitative real-time PCR of RNA accumulation, the effect of ribavirin and the attenuation of transcription. Arch Virol 2005;150:521532. Chong HT, Kamarulzaman A, Tan CT: Treatment of acute Nipah virus encephalitis with ribavirin. Ann Neurol 2001;49:810813.

Joe McCormack Department of Medicine and Infectious Diseases University of Queensland and Mater Hospitals, Raymond Terrace South Brisbane, 4101 (Australia) Tel. 61 738408518, Fax 61 738401548, E-Mail joe.mccormack@mater.org.au

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Lal SK (ed): Emerging Viral Diseases of Southeast Asia. Issues Infect Dis. Basel, Karger, 2007, vol 4, pp 94111

Ross River Virus: An Arthritogenic Alphavirus of Significant Importance in the Asia Pacific
Daniela Tupanceskaa, Ali Zaida, Nestor E. Rullia, Sandra Thomasa, Brett A. Lidburya, Klaus I. Matthaeib, Ruben Ramireza, Suresh Mahalingama
a

Centre for Virology Research, School of Health Sciences, University of Canberra, Gene Targeting Laboratory, The John Curtin School of Medical Research, Australian National University, Canberra, Australia
b

Abstract
Ross River virus is a mosquito transmitted disease endemic in tropical Australia, Papua New Guinea, East Timor, adjacent islands of Indonesia and the Solomon islands. It occurs epidemically in temperate Australia and sporadically in Pacific Islands, such as Fiji, Tonga, Samoa and the Cook Islands. It is the most common arbovirus disease in Australia. The disease occurs mainly in adults, with clinical symptoms rare before puberty. The symptoms are rash, joint pain and general effects such as fatigue, fever and muscle pain, which appear from 3 to 21 days post-infection and can persist for 36 months. In Australia, Ross River virus is responsible for an average of 8,000 cases annually. The long-term effects of Ross River virus disease are thought to be due to the viruss ability to evade the patients immune system. Antibodies produced against the virus may be insufficient to neutralise it and may even improve the ability of the virus to infect host cells (antibody-dependent enhancement). The virus is also capable of persisting for long periods in macrophages and may be reactivated during times of stress. Various human host proteins may also increase Ross River virus infection rates and contribute to disease symptoms.
Copyright 2007 S. Karger AG, Basel

Ross River virus (RRV) belongs to the Togaviridae, a family that consists of RNA viruses, and are primarily arboviruses (arthropod-borne viruses) [1]. The family comprises of two genera, Rubivirus, a genus containing only one recognised
*Ali Zaid and Daniela Tupanceska contributed equally towards the preparation of this book chapter and should be regarded as joint first authors.

Table 1. Alphaviruses and other recognized RNA viruses Virus name Semliki Forest Onyong-nyong Ross River Barmah Forest Chikungunya Sindbis WEE EEE Southern Elephant Seal Family/genus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Togaviridae/Alphavirus Distribution/characteristics Africa, Eurasia; encephalitic Africa; arthralgia, rashes Australia, Oceania; arthritis, rashes Australia, Oceania; arthritis, rashes Africa/Asia; arthralgia, rashes Australia, Oceania; arthritis, rashes North/South America; encephalitis North/South America; encephalitis Antarctic territories, Macquarie Is.

There are currently 26 known alphaviruses which are distributed on all the inhabited continents. RRV is limited to Australia and the neighbouring Pacific Islands. Some alphaviruses arose following a genomic recombination event, while others known as Old World viruses share ancient origins. Most alphaviruses cause diseases in humans, but animals such as horses, marsupials, birds and fish can also be carriers. The most recently identified Alphavirus, Southern Elephant Seal Virus SESV (in bold), was found in tick louse (Lepidophthirus macrorhini) carried by Southern Elephant seals, marine mammals indigenous to marine Antarctic territories.

species, rubella virus, and Alphavirus, the genus which includes 26 recognised members classified antigenically into seven complexes [2, 3] (table 1). These complexes are segregated into New World (American) and Old World (Eurasian-African-Australasian) viruses depending on the nucleotide sequence of their non-structural proteins [4]. An Old World virus, RRV belongs to the genus Alphavirus and is a subtype of Getah virus (GETV) in the Semliki Forest virus (SFV) serological complex. Many members of this genus are important causative agents of disease in humans and other animals [5] (table 1). Most alphaviral infections give rise to transient, debilitating diseases such as arthritis, whilst others may infect the central nervous system, potentially causing encephalitis [6].
Virion and Genomic Properties

Alphaviruses are small spherical, enveloped viruses with a single stranded, positive-sense RNA as the genome [5]. The virion of alphaviruses consists of four major components: the genomic RNA, the nucleocapsid core, the plasma membrane and the glycoprotein shell [7, 8]. The glycoprotein shell consists of 80 spikes with each spike comprising a trimer of heterodimers of two glycosylated envelope proteins: E1, the hemagglutinin (52 kDa) and E2, the neutralising antigen (49 kDa) [1, 9]. The E1 protein

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Glycosylated envelope proteins (Heterodimer spikes)

Nucleocapsid protein 5' NSp1 NSp2 NSp3 NSp4 C E3 E2 6K E1 3'

Non-structural proteins Potassium ion channel protein

Fig. 1. The genome of Ross River virus (prototype T48) is 11851 nucleotides long, and is flanked with a 5 cap and a 3 -poly-A tail. Structural proteins form the rest of the genome, encoding for viral envelope glycoproteins and nucleocapsid proteins. E1 (haemagglutinin) assembles with E2, the neutralising epitope, to form surface heterodimers across the lipid bilayer. E1 also forms heterotrimers, which assemble as hollow-base tripartite heads within the bilayer, and is believed to be involved in membrane fusion. The third glycoprotein, E3, is not involved in virion structure. The 6K protein has been associated with the formation of cationselective ion channels in lipid bilayers. Non-structural proteins are encoded by genes nsP1, nsP2, nsP3 and nsP4, which later assemble as two polyproteins after post-translational cleavage.

forms the core of the trimeric spike, whilst the E2 protein is found primarily on the outer surface. The glycosylated spikes penetrate the host cell-derived plasma membrane and interact directly with the nucleocapsid core. The E1-E2 heterodimers, of which there are 240 in total, form 1:1 associations between E2 and nucleocapsid monomers across the lipid bilayer [1]. The nucleocapsid (40 nm in diameter) has an icosahedral symmetry and is surrounded by a 4.8nm-thick lipid bilayer. Within the core structure are 240 copies of the single nucleocapsid protein, C (32 kDa), and a single copy of the genomic RNA [9]. The genome is approximately 11.7 kb in length, varying marginally between the different alphaviruses [10]. Encoded in the 5 two thirds (7.6 kb) of the genome are the non-structural proteins (nsP14) that are involved in genomic replication and mRNA synthesis, whilst the 3 third (4.1 kb) of the genome encodes the structural proteins of the virus (fig. 1). These consist of the capsid protein, three envelope glycoproteins and an additional 6K protein, which are all translated from the subgenomic mRNA as a single polyprotein in the order C-E3-E26K-E1 [1, 10]. In RRV-infected cells, the 6K proteins have been shown to form cation-selective ion channels in the lipid bilayer, thus increasing the permeability of the cells to monovalent cations and aiding the process of virion budding [10]. It should be noted that the E3 glycoprotein is not incorporated into the virion. The function of E3 and the reasons as to why it

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is not incorporated are unknown [7]. The prototype RRV (T48) genome is an RNA molecule of 11.8 kb, excluding the poly (A) tail, and consists of three regions that are strongly conserved between the alphaviruses. These are believed to play roles in the regulation of viral replication and essentially are composed of a tract of 23 nucleotides situated next to the poly (A) tail at the 3 end, 21 nucleotides at the 3 terminus of the nsP4 gene and 50 nucleotides near the 5 end of the nsP1 gene [1].

Virus Transmission and Propagation

Alphaviruses are largely mosquito-borne viruses, with mosquitoes the primary vectors for transmission and propagation of disease in nature. Propagation of these viruses occurs via a horizontal cycle involving mosquito vectors and vertebrate hosts, i.e. transmission from mosquito to vertebrate to mosquito. The main mosquito vectors are Culex annulirostris in inland areas and Ochlerotatus vigilax, Verrallina funerea and Ochlerotatus camptorhynchus in coastal regions [11]. RRV in particular is only transmitted in placental and marsupial mammals and the virus is maintained in the environment by the marsupials [1, 12]. Serological studies and laboratory investigations have indicated that native marsupials, specifically kangaroos and wallabies, are the primary natural reservoirs or hosts of RRV however many others have also been considered [6]. In addi, tion, RRV transmission from human to mosquito to human, without the involvement of a marsupial, has also been proposed. There is presently little doubt that such a cycle involving only humans and mosquitoes occurs frequently during periods of intense virus activity, e.g. during the summer and autumn months when mosquito vectors are most abundant [1, 13].

Ross River Virus Disease

Discovery of RRV and Viral Activity Disease, thought to be caused by RRV was first reported in 1928 when , an unusual epidemic, resulting in temporary arthritis and rash, occurred in Narrandera and Hay in New South Wales [14, 15]. During the Second World War, epidemics of similar symptoms were described in the tropical regions of Australia and on the islands to the immediate north [13]. It was as a result of these reports, that in 1936 the name epidemic polyarthritis (EPA) was used to describe the disease caused by RRV [1]. In 1956, in the Murray Valley, the first large epidemic was recorded. However, the causative agent was not isolated until 1963 by Doherty and colleagues, from a pool of Aedes vigilax (now called

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Ochlerotatus vigilax) mosquitoes collected near the Ross River in Townsville, North Queensland [1, 16]. Introduction of the virus to several islands of the South Pacific (Fiji, Cook Islands and Samoa) in 1979 resulted in an explosive epidemic, the largest recorded, with more than 50,000 cases of disease reported [17]. In Fiji the polyarthritis outbreak was dramatic and, following the epidemic, up to 90% of residents in some areas were serologically positive for RRV [18]. In American Samoa it was estimated, on the basis of serological tests, that at least 13,500 people were infected. Sera from 393 humans on Tutuila Island showed evidence of infection in 43.8% of the people sampled. In this same island, sera from 100 adults collected before the epidemic had no RRV antibodies, suggesting a recent introduction of the virus [19]. The Cook Islands were the easternmost reach of the epidemic, which affected the majority of the inhabitants of Rarotonga, the most populated island in the group. The virus was isolated from half of the serologically proven infections [20]. Although since 1981 no RRV outbreaks have been reported in the Pacific islands, these regions are not safe from new epidemics. At the beginning of 2004 two Canadian tourists contracted the disease while visiting Fiji. These 2 patients were the first cases notified since 19791981. Both were found serologically positive for RRV and displayed characteristic symptoms such as fatigue, arthralgia and general body pain [21]. Cases of RRV occur throughout the year in some areas, such as northeastern tropical Australia and Papua New Guinea. By contrast, in other areas, mainly Australian coastal regions, disease activity tends to occur in seasonal epidemic outbreaks [22]. Originally endemic in most rural areas, increased virus activity has resulted in an increase in the geographic spread. This has raised considerable concern, as emergence of the virus into major Australian population centres could induce a higher incidence of disease amongst the community [22]. In Australia, on average, RRV is responsible for approximately 8,000 cases of disease annually, directly costing the community an estimated AUD 1,018 per patient [23, 24]. Clinical Manifestations RRV disease, the most common arboviral disease in Australia, is primarily characterized by any or all of three major manifestations; these being rash, rheumatic pain (e.g. arthritis) and constitutional effects such as fatigue, fever and myalgia (muscle pain) [1]. Symptoms become evident from 3 to 21 days (average 9 days) post-infection with arthralgia (joint pain) usually constituting the initial, most prominent and incapacitating symptom of all, commonly affecting the joints of the extremities [13]. Approximately two-thirds of patients are affected by rash, usually lasting 510 days and occurring mainly on the limbs and torso [1, 13]. In addition, one third to one half of patients may experience fever, whilst myalgia and fatigue are reported to occur in over half of

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infected individuals, with fatigue being the most consistent constitutional effect [1, 13]. While viral infection may potentially occur in any individual, clinical disease is very rarely observed in children. The reason for this is not clear but may be associated with differences in immune responses generated between adults and children. The disease has the most prominent incidence in adults of 2050 years of age, particularly in those between the ages of 30 and 40 [25]. No clear gender effect has been established [13]. Although infection by the virus is not fatal, the resulting conditions can be debilitating and may persist in individuals for extended periods [25, 26]. Some controversy is present in the literature regarding the exact duration of disease, with reports of illness lasting from 3 months to several years [24, 27]. The variation is probably due to overestimates of symptom prevalence and duration, especially since established symptoms not solely associated with RRV disease may have been misdiagnosed and included in reported data [2830]. Recently, a study performed by Mylonas et al. [24] demonstrated that although severe at onset, RRV disease usually resolved within 36 months. The researchers also showed that patients who suffered from disease beyond this time point were commonly afflicted with additional conditions. Furthermore, differences observed in the duration and severity of disease may be the result of diversity in the pathogenicity of RRV strains with which the individuals are infected [1]. Currently there is no cure for RRV and treatment is based on empirical antiinflammatory regimes. As reported by Fraser et al. [31], symptomatic relief was achieved by the use of non-steroidal anti-inflammatory drugs. Other treatments, which have, at least to some extent, been found to provide relief, include physical interventions such as massage or physiotherapy, as well as rest [13]. A study by Yu and Aaskov [32] in 1994 reported the inactivation of RRV with binary ethylenimine (BEI) in mice and the ability to protect mice against later challenge with RRV Similarly, an ethnobotanic study of Aboriginal medicinal plants by Semple . et al. [33] showed that RRV infectivity was effectively reduced in vitro by 25% when treated with extracts of fruit, wood and leaf extracts of Pittosporum phylliraeoides var. microcarpa (Pittosporaceae). Developing superior treatment strategies will require better understanding of the immunology and pathology of RRV disease. A candidate RRV vaccine was developed by Aaskov and colleagues [32, 34] and was found to be effective in animal models, however no reports of human clinical trials of a RRV vaccine have been published.

Immunobiology of RRV Infection

RRV has an extensive host range, including marsupials and humans, and has the potential to infect many different cell types within the host [17]. Early

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RRV

Control

Fig. 2. Differences in muscle tissue in uninfected and RRV-infected mice. Stained cells (monocytic cells) are present in high numbers in infected muscle. There is significant destruction of muscle fibres following RRV infection [38] 100.

studies on RRV demonstrated that certain cells and tissues of mice, such as muscle and brain, could readily be infected and facilitated the growth of the virus. However, recently, in vitro studies have identified the association of monocytes and macrophages with human RRV disease [3537]. Both monocytes and macrophages have been identified as the predominant muscleinfiltrating cells at the height of clinical disease [38] (fig. 2). In addition, large mononuclear cell infiltrates, consisting mainly of monocytes and highly activated macrophages, have also been shown in biopsies obtained from rash lesions of infected patients as well as in rheumatic synovial effusions obtained from patients with EPA [39, 40]. Macrophages have been shown to be a primary cellular agent in the development, growth and persistence of RRV and have also been identified as playing a role in the pathology of RRV disease [38, 41]. In addition to the key monocyte/macrophage infiltrate, the primary cellular components identified in the synovial exudates are T lymphocytes, B lymphocytes and a small proportion of natural killer cells, while neutrophils are rarely present [37, 42, 43]. The lack of neutrophils in the synovial fluid of EPA patients distinguishes this condition from other arthritic conditions, such as rheumatoid arthritis. This deficiency is probably due to the absence of immune complexes in the serum of individuals suffering from RRV disease, as discovered by Fraser et al. [44]. Immune complexes attract neutrophils and are commonly involved in the pathogenesis of some viral arthritides [44]. Importantly, the in vivo T cell responses (both CD4 and CD8 ) in RRV disease are significant contributors in the resolution of the infection, where most patients experience a distinct virus-specific T cell proliferative response. In addition, infected

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macrophages can be directly killed via the lytic capabilities of cytotoxic T lymphocytes, which are significant in the elimination of altered cells, such as virusinfected cells [4547]. Several researchers [31, 42, 47] have recognised the crucial role of CD8 T lymphocytes (usually cytotoxic T cells) in the clearance of RRV These cells appear to play a role in determining whether patients . recover rapidly or experience the chronic effects associated with RRV [13]. It has been found that in patients who have experienced rapid recovery from infection, CD8 lymphocytes were predominant in the skin rashes. The virus was swiftly cleared by an RRV-specific CD8 T lymphocyte cell-mediated immune response [31]. The development of chronic arthritis in other patients is probably due to the inability of the cell-mediated immune response to completely clear the infection. Thus, CD4 lymphocytes (helper T cells), as opposed to CD8 lymphocytes, prevail in the serum of these patients and initiate local non-specific immune responses, involving the large influx of mononuclear cells [39, 42, 43].

Viral Evasion Strategies

Innate immunity refers to host defenses that are non-specific and exist prior to exposure to foreign matter [48], whereas adaptive, or acquired immune responses refer to host defenses that are mediated by B and T lymphocytes following exposure to foreign matter [49]. Varying degrees of both innate and adaptive immune responses have been shown to be involved in the hosts responses to RRV infection, as identified in mouse studies [38]. Despite these protective mechanisms established in the host, there are several reports of the ingenious strategies that RRV has implemented in order to evade and manipulate the hosts immune response to infection. Such viral evasion strategies are significant in order for the virus to survive, replicate and establish persistent infection in their host [38, 50]. Viral Latency and Persistence Viral latency refers to the situation where the virus enters a state of dormancy and remains inactive within the body until it is induced by some stimulus, e.g. stress, to regain its infectious activity. This activity by RRV has been demonstrated using immunofluorescence staining of RRV-infected macrophage cells [12]. By employing this strategy, RRV may remain inactive until the immune system is susceptible to infection or has become weakened by some factor, e.g. sickness, before re-inducing its infectious ability. Research by Way et al. [12] has shown that following apparent clearance of RRV infection, relapse may occur either spontaneously or in response to stress. Also reported

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were the presence of viral particles localised within intracellular vesicles during prolonged infection. This may protect the virus, allowing evasion from the hosts immune response and persistence of virus infection during a cycle of latency. The first report of persistent RRV infection was by Eaton and Hapel [51], who performed experimental analyses of RRV in mouse muscle cells and found virus production up to 42 days post-infection. Similar viral persistence has also been reported for RRV infection of synovial fibroblasts [52] and, since then, has also been identified by plaque assay in in vitro-infected cultures of macrophages [41]. In addition, more sensitive molecular approaches, such as reverse transcriptase-polymerase chain reaction (RT-PCR), have detected virus in the synovial tissue of RRV disease patients up to 5 weeks following the onset of disease [43]. A more recent study by Way et al. [12], examining RRV persistence in macrophages, revealed that infection was able to persist for as long as 170 days. Persistent RRV infection has, to date, been reported only in macrophages and not in monocytes and the mechanism responsible for this phenomenon is not entirely known [53]. Despite this uncertainty, it is likely that the chronic infection cycle and disease phases of RRV are due, in part, to viral persistence. For instance, persistent infection of macrophages may be a crucial aspect in facilitating immune evasion, as the activity of key antiviral factors secreted by these cells appears to be altered [12]. Antibody-Dependent Enhancement of Infection As mentioned previously, the hosts adaptive immune responses contribute significantly to protection against infection. Much of this protection is due to specific antiviral antibodies. However, protection from mortality and morbidity associated with viral infections is not always offered by the production of antibodies [38]. It is possible that enhancement of viral infection and pathogenesis may be achieved by the production and presence of sub-neutralising concentrations of antiviral antibodies, an event referred to as antibody-dependent enhancement (ADE) of infection. Since its discovery and implication as an important infection strategy, ADE has become a subject of significant scrutiny by many researchers [26, 54]. ADE was first reported in 1964 by Hawkes, who, whilst examining antiviral antibody neutralisation of several arboviruses, encountered a situation of enhanced viral infectivity [55, 56]. Hawkes and Lafferty [56] suggested that this enhancement of infectivity was an intermediary phase in the pathway leading to viral neutralisation, i.e. corresponding to the early stages of infection that coincides with rising, but subneutralising levels of antibody. Essentially, the mechanism of ADE involves antibody-facilitated virus entry into macrophages and monocytes via Fc- receptors, resulting in an enhanced production of virus [53] (fig. 3). Since the early

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RRV Ross River virus

RRV-ADE Ross River virus

Fig. 3. Electron microscopy of macrophages following infection by Ross River virus. Left: the infection follows the non-ADE pathway; Right: infection following the ADE pathway, clearly showing the enhanced replication of the virus.

reports, ADE has also been documented for several other viruses including dengue virus, human immunodeficiency virus and some alphaviruses (e.g. RRV) [5759]. Infection of cells by RRV occurs via one of two known mechanisms. The first is via a natural receptor, possibly a cell adhesion molecule, which is used by cells to adhere to the extracellular matrix [60]. RRV therefore has the ability to relatively easily infect most adherent cells, including macrophages, the preferred cell for infection. However, this mode of infection is difficult with nonadherent cells, for example, monocytes. The virus consequently requires an alternative route for infection [37]. RRV appears to utilise antibodies to facilitate infection of cells via the ADE pathway [26]. Thus the virus is able to successfully avoid and disable the cells defences, providing it with the opportunity to induce enhanced pathogenesis [37]. The occurrence of ADE for RRV was first reported in 1996 by Linn et al. [37] and has since led to many significant findings. Most intriguing is the ability of RRV to suppress or disrupt the innate antiviral responses of the host via ADE infection pathways [26, 54]. Normally, macrophage cells are able to produce several antiviral proteins, such as tumor necrosis factor (TNF) and inducible nitric oxide synthase (NOS2), which pose a major threat to virus survival. Since it had been shown that the virus grows extensively in macrophages, curiosity was expressed as to the mechanisms involved in providing the virus protection from the antiviral defences of these cells. Interestingly, in vitro studies of RRV infection of macrophages have identified novel aspects in the ADE pathway of infection that allow this to occur [41]. In experiments involving

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lipopolysaccharide (LPS)-induced antiviral activity, it was found that RRV was able to survive and undergo unrestricted replication when the infection of macrophages was facilitated by antibodies [41]. LPS, an oligomer of lipid and carbohydrate, is a large constituent of gram-negative bacterial outer membranes. LPS has the potential to induce innate immunity and the expression of cytokines such as TNF- and type 1 interferons (IFN- / ) and other molecules including nitric oxide [61, 48]. By employing LPS in the experimental analyses, it was possible to increase expression of antiviral proteins and subsequently examine the effects on the production of antiviral factors by macrophages, following ADE-RRV infection. Conditions of considerable antiviral activity induced by LPS do not normally allow the virus to survive when infection occurs via non-ADE pathways [26]. Examination of cytokine levels in these experiments showed that ADE-RRV infection specifically inhibited the production of TNF and NOS2. The decrease in expression of the antiviral proteins prompted further research efforts, which eventually showed that the ADE pathway of infection allowed the virus to compromise antiviral responses at the transcriptional level by targeting interferon regulatory factor-1 and nuclear factor- B; two transcription factors [41] (fig. 4). As nuclear factor- B and interferon regulatory factor-1 are critical in the transcription of TNF and NOS2, respectively, disruption of both of these transcription factors results in the dysregulation of downstream expression of these soluble mediators. Intriguingly, transcription of non-antiviral proteins, which support viral growth, was shown to be unaffected [41]. In addition, ADE-RRV infection also resulted in increased interleukin-10 (IL-10) expression [26]. IL-10 is an immunosuppressive cytokine that is crucial for the down-regulation of inflammatory responses [46]. The increased production of IL-10 after ADE-RRV infection may offer protection to the virus by creating a reduced antiviral environment. Thus, the virus may not only be capable of dysregulating the expression of host antiviral proteins but may also be able to manipulate expression of other proteins to achieve enhanced levels of virus replication [26]. Although observations of antibody-dependent enhancement of infection in vitro have lead to consideration of this mechanism as a significant contributor to the exacerbation of disease, the relevance of ADE in the in vivo situation is not entirely known [56]. ADE infection needs to be carefully considered in the production of suitable vaccines against RRV especially since successful vac, cines are designed to achieve a significant antibody response. Even though no correlation has been found between clinical disease and pre-existing RRV antibodies thus far, the occurrence should not be overlooked in vaccine development. This is especially important for other viruses, which also exhibit ADE of infection that could result in life-threatening conditions, for example, dengue hemorrhagic fever by ADE of dengue virus [59, 26].

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RRV

FcR IL-10 IL-10R IL-10 Viral entry and replication

SOCS3 STAT-1

IFN-

IFN-

R ISRE IFN / TNFIP-10 NO ISRE Early cytokines IRF-1 GAS B

IRF-1 IFNiNOS

NF- B

TLR4 LPS

Fig. 4. Suppression of antiviral cytokines by RRV-ADE-infection in macrophages. LPS binding to Toll-like receptor (TLR4) causes activation of NF- B and IRF-1 which regulates the production of type I IFN. Type I IFNs bind the interferon receptor (IFN- / R), which results in the phosphorylation of STAT-1. STAT-1 in turn promotes transcription of IRF-1 as well as antiviral cytokines (iNOS, IL-10). RRV-ADE-infection of cells results in the suppression of NF- B, IRF-1 and STAT-1, and in the induction of IL-10. IL-10 inhibits signal transduction by IFNs via suppression of kinases that activate STAT-1. GAS IFN- activation sequence; ISRE Interferon-stimulated response element; B kappa B element; SOCS3 suppressor of cytokine signalling.

Roles of Host Proteins in RRV Disease

Various human host proteins may also contribute to increased RRV infection and subsequently to a state of disease. This may be due in part to up- or downregulation of expression of any of these factors [26]. Previously investigated for their dysregulated expression during RRV infection are a group of

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significant host proteins, known as cytokines. Cytokines are low molecular weight glycoproteins or regulatory proteins that are secreted by leukocytes in response to various stimuli. They have crucial functions in many biological activities including the innate and adaptive immune responses, encompassing antiviral responses [48]. By binding to specific receptors on the membrane of target cells, cytokines have the capacity to regulate the duration and intensity of the immune response. This is accomplished by altering gene expression in target cells by triggering signal-transduction pathways. Immune responses are therefore controlled, to some extent, by stimulation or inhibition of the activation, proliferation and/or differentiation of various host cells, as well as by regulation of the secretion of other cytokines by these cells. Monocyte Chemoattractant Protein-1 Mononuclear cell infiltration into inflammatory tissue is mediated by chemotactic cytokines, such as monocyte chemoattractant protein-1 (MCP-1) [62]. Chemotactic cytokines, or chemokines, are a subgroup of cytokines that selectively and specifically mediate several aspects of leukocyte behaviour, including chemotaxis and the activation and regulation of cell adhesion [48]. MCP-1, also known as monocyte chemotactic and activating factor, is secreted by a number of cell types, including fibroblasts, mononuclear leukocytes, monocytes and macrophages, either constitutively or under stimulation [6365]. MCP-1 has been detected in several pathological conditions and is one of the most potent monocyte chemoattractants, as suggested by the vast number of MCP-1 binding sites per cell (with approximately 1,5003,900 MCP-1-binding sites/cell) [66]. MCP-1 contributes to two defence roles in the host, one of cellular immune reactions and responses to acute tissue injury. However, it has also been recognized as a mediator in human disease [67]. For example, MCP-1 is not expressed in normal skin, but expression has been found in human melanomas [68]. In addition, MCP-1 is thought to have a putative role in RRV disease, particularly in association with the establishment of polyarthritis [69]. Recent in vitro studies conducted by Mateo and colleagues [70] provided significant insight into the role of MCP-1 in RRV infection. Upregulated expression of mRNA encoding MCP-1 was found when human synovial fibroblasts and macrophages were exposed to acute RRV infection. Since MCP-1 recruits and activates monocytes and macrophages, this finding is consistent with examinations of synovial exudates obtained from EPA patients, which consist primarily of these cells [42]. Interestingly, persistently infected macrophages (infected for 6 months in vitro) failed to induce expression of mRNA for MCP-1. Again this observation is consistent with the episodic nature of EPA in some patients, where a cycle alternating between periods of no disease and persistent

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infection of nave macrophages, by persistent RRV present in joint cells, occurs [70]. This study therefore implicated MCP-1 as an RRV-induced cytokine, a potentially significant effector protein in macrophage recruitment and activation, associated with EPA [70]. Although the in vivo role of MCP-1 has not been completely defined, the in vitro studies provide reason to consider targeting the production of this chemokine as a therapeutic strategy in the treatment of RRV associated disease [71]. Type I Interferons: Interferon-a and Interferon-b Originally described more than four decades ago as proteins capable of eliciting antiviral activity, the interferons (IFN) have more recently been described as multifunctional proteins synthesized upon activation of the host immune system. Various IFN proteins exist, and belong to one of two IFN families (type I and type II), which are structurally unrelated. Of the many IFNs, the three main human IFNs are IFN- (type I), IFN- (type I) and IFN(type II) [72, 73]. In addition to their significant antiviral roles, the IFNs also possess immunoregulatory and antiproliferative properties and are capable of mediating growth, activation and differentiation of many immune cells [74]. Because of these activities, induction of IFNs, in particular the type I IFNs, is critical in innate immunity and in regulating the effects of cellular immune responses, for example, the activation of natural killer cells and mononuclear phagocytes, which play significant roles in the elimination of pathogens [48]. The mechanisms by which IFN- and IFN- induce antiviral responses involve specific binding of IFN- and IFN- to their respective receptors (IFNand IFN- receptors) [48]. This binding activity activates the signal transducer and activator of transcription-1 pathway, which subsequently results in the transcription of various genes involved in generating resistance to viral replication. Targeting and disrupting the activation of such antiviral pathways has been recognised as an important avoidance strategy for successful viral infection, as considered previously in the case of RRV Recent research has . shown that in ADE infection pathways of macrophages, expression of mRNA for IFN- (amongst other cytokines) was decreased, with levels as low as 10% of those where infection was via non-ADE mechanisms [26]. Interestingly, the diminished levels of IFNs may be related to significantly higher levels of IL-10 expression also found in the RRV-ADE infected macrophage cultures, as IL-10 is a powerful inhibitor of IFN-induced signal transducer and activator of transcription-1 pathway activation [26]. By potentially interrupting the expression of antiviral proteins, such as the type I IFNs, RRV may increase its ability to evade the antiviral defences of the host, thus resulting in chronic disease [41, 26].

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Concluding Remarks

The extensive outbreak of RRV in Pacific Islands in 1979 clearly demonstrates the potential of RRV to spread to other areas. Lessons can certainly be learnt from other alphaviruses such as ONyong nyong (ONNV) and Chinkungunya (CHIKV) viruses that have caused widespread epidemics of debilitating joint pain in Africa and Asia [5]. An outbreak of ONNV in 19591961 began in Uganda and spread over Eastern Africa, affecting over 2 million people [5]. Arboviruses have the potential to spread fast through tropical regions where the vectors are endemic. An example is the spread of CHIKV through south and southeastern Asia in the 1960s, and more recently in the south-west Indian Ocean. CHIKV spread from India, Sri Lanka, Burma, and Thailand to the Philippines and to Indonesia in the 1980s. Thailand and Malaysia also suffered outbreaks in 1995 and 1998, respectively. Therefore, the possibility of RRV spread into neighbouring regions in Southeast Asia cannot be disregarded.

Acknowledgements
We acknowledge funding from the Australian National Health and Medical Research Council (NHMRC) (Grant 303404) and the University of Canberra (Seed funding for establishment of Research Facility 2005). Suresh Mahalingam is an Australian NHMRC R. Douglas Wright Fellow. Daniela Tupanceska is a recipient of the Australian Postgraduate Award.

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Semple SJ, Reynolds GD, OLeary MC, Flower RLP: Screening of Australian medicinal plants for antiviral activity. J Ethnopharmacol 1998;60:163172. Aaskov JG, Williams L, Yu S: A candidate Ross River virus vaccine: preclinical evaluation. Vaccine 1997;15:13961404. Mims CA, Murphy FA, Taylor WP, Marshall ID: Pathogenesis of Ross River virus infection in mice. I. Ependymal infection, cortical thinning and hydrocephalus. J Infect Dis 1973;127:121128. Murphy FA, Taylor WP, Mims CA, Marshall ID: Pathogenesis of Ross River virus infection in mice. II. Muscle, heart and brown fat lesions. J Infect Dis 1973;127:129138. Linn ML, Aaskov JG, Suhrbier A: Antibody-dependent enhancement and persistence in macrophages of an arbovirus associated with arthritis. J Gen Virol 1996;77:407411. Lidbury BA, Simeonovic C, Maxwell GE, Marshall ID, Hapel AJ: Macrophage-induced muscle pathology results in morbidity and mortality for Ross River virus-infected mice. J Infect Dis 2000;181:2734. Clarris BJ, Doherty RL, Fraser JR, French EL, Muirden KD: Epidemic Polyarthritis: a cytological, virological and immunochemical study. Aust NZ J Med 1975;5:450457. Fraser JR, Cunningham AL, Clarris BJ, Aaskov JG, Leach R: Cytology of synovial effusions in epidemic polyarthritis. Aust NZ J Med 1981;11:168173. Lidbury BA, Mahalingam S: Specific ablation of antiviral gene expression in macrophages by antibody-dependent enhancement of Ross River virus infection. J Virol 2000;74:83768381. Fraser JR, Becker GJ: Mononuclear cell types in chronic synovial effusions of Ross River virus disease. Aust NZ J Med 1984;14:505506. Soden M, Vasudevan H, Roberts B, Coelen R, Hamlin G, Vasudevan S, La Brooy J: Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection. Arthritis Rheum 2000;43:365369. Fraser JRE, Cunningham AL, Matthews JD, Riglar A: Immune complexes and Ross River virus disease (epidemic polyarthritis). Rheumatol Int 1988;8:113117. Aaskov JG, Fraser JRE, Dalglish DA: Specific and non-specific immunological changes in epidemic polyarthritis patients. Aust J Exp Biol Med Sci 1981;59:599608. Austyn JM, Wood KJ: Principles of Cellular and Molecular Immunology. Oxford, Oxford University Press, 1993. Linn ML, Mateo L, Gardner J, Suhrbier A: Alphavirus-specific cytotoxic T lymphocytes recognize a cross-reactive epitope from the capsid protein and can eliminate virus from persistently infected macrophages. J Virol 1998;72:51465153. Abbas AK, Lichtman AH, Pober JS: Cellular and Molecular Immunology, ed 4. Philadelphia, Saunders, 2000. Sell S: Immunology, Immunopathology and Immunity, ed 6. New York, ASM Press, 2001. Mahalingam S, Meanger J, Foster PS, Lidbury BA: The viral manipulation of the host cellular and immune environments to enhance viral propagation and survival: a focus on RNA viruses. J Leukoc Biol 2002;72:429439. Eaton BT, Hapel AJ: Persistent noncytolytic togavirus infection of primary mouse muscle cells. Virology 1976;72:266271. Journeaux SF, Brown WG, Aaskov JG: Prolonged infection of human synovial cells with Ross River virus. J Gen Virol 1987;68:31653169. Linn ML, Suhrbier A: Persistence of Ross River virus in macrophages. Arbovirus Res Aust 1997;7:153159. Suhrbier A, Linn ML: Suppression of antiviral responses by antibody-dependent enhancement of macrophage infection. Trends Immunol 2003;24:165168. Hawkes R: Enhancement of infectivity of arbovirueses by specific antisera produced in domestic fowls. Aust J Exp Biol Med Sci 1964;42:465482. Porterfield JS: Antibody-dependent enhancement of viral infectivity. Adv Virus Res 1986;31: 335355. Hawkes RA, Lafferty KJ: The enhancement of virus infectivity by antibody. Virology 1967;33: 250261. Robinson WE, Montefiori DC, Mitchell WM: Antibody-dependent enhancement of human immunodeficiency virus type 1 infection. Lancet 1988;i:790794.

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Morens DM, Halstead SB: Measurement of antibody-dependent infection enhancement of four dengue virus serotypes by monoclonal and polyclonal antibodies. J Gen Virol 1990;71:29092914. White JM: Integrins as viral receptors. Curr Biol 1993;3:596599. Adams DO, Hamilton TA: The cell biology of macrophage activation. Annu Rev Immunol 1984;2:283318. Yoshimura T, Robinson EA, Tanaka S, Appella E, Kuratsu J, Leonard EJ: Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants. J Exp Med 1989;169:14491459. Van Damme J, Decock B, Lenaerts J-P, Conings R, Bertini R, Mantovani A, Billiau A: Identification by sequence analysis of chemotactic factors for monocytes produced by normal and transformed cells stimulated with virus, double-stranded RNA or cytokine. Eur J Immunol 1989;19:23672373. Chang HC, Hsu F, Freeman GJ, Griffin JD, Reinherz EL: Cloning and expression of a -interferoninducible gene in monocytes: a new member of a cytokine gene family. Int Immunol 1989;1: 388397. Colotta F, Borr A, Wang JM, Tattanelli M, Maddalena F, Polentarutti N, Peri G, Mantovani A: Expression of a monocyte chemotactic cytokine by human mononuclear phagocytes. J Immunol 1992;148:760765. Uguccioni M, DApuzzo M, Loetscher M, Dewald B, Baggiolini M: Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 and MIP-1 on human monocytes. Eur J Immunol 1995;25:6468. Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshumura T, Leonard EJ, Witztum JL, Steinberg D: Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci USA 1991;88:52525256. Graves D, Barnhill TR, Galanopoulos T, Antoniades HN: Expression of monocyte chemotactic protein protein-1 (MCP-1) in human melanoma in vivo. Am J Pathol 1992;140:914. Lidbury B, Mahalingam, S: A role for chemokine activity in alphavirus pathogenesis: evidence from the analysis of polyarthritis and myalgia post Ross River virus infection; in Mahalingam S (ed): Chemokines in Viral Infection. Georgetown, Landes/Kluwer, 2004, pp 93107. Mateo L, Linn ML, McColl SR, Cross S, Gardner J, Suhrbier A: An arthrogenic alphavirus induces monocyte chemoattractant protein-1 and interleukin-8. Intervirology 2000;43:5560. Barnes DA, Tse J, Kaufhold M, Owen M, Hesselgesser J, Stroeter R, Horuk R, Perez HD: Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvantinduced arthritis model. J Clin Invest 1998;101:29102919. Weissmann C, Weber H: The interferon genes. Prog Nucl Acid Res Mol Biol 1986;33:251301. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P: Molecular Biology of the Cell, ed 4. Abington, Garland Science, Taylor & Francis Group, 2002. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD: How cells respond to interferons. Annu Rev Biochem 1998;67:227264.

Dr. Suresh Mahalingam School of Health Sciences, University of Canberra Canberra, ACT 2601 (Australia) Tel. 61 02 6201 2368, Fax 61 02 6201 5727 E-Mail suresh.mahalingam@canberra.edu.au

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Emerging and Re-Emerging Infectious Diseases in Our Global Village


David L. Heymann
World Health Organization, Geneva, Switzerland

Abstract
Bacteria, viruses and parasites emerge or re-emerge in human populations as they cross the species barrier from animals to humans, or as they enter human populations through weakened public health systems. Viruses afford the most dramatic examples of emergence and re-emergence. Their potential to adapt to human hosts and the environment with relative ease, and to further evolve in virulence and transmissibility, is facilitated by their rapid reproduction rates and mutation. It is also linked to a host of social, environmental, economic and human behavioural factors that include sub-standard health care procedures, inappropriate use of antimicrobial agents, naturally occurring variations in temperature and rainfall, the modification or destruction of rivers, forests and agricultural land, and the level of investment in public health, especially in surveillance systems that rapidly identify emerging or re-emerging microbes, and mechanisms to ensure a rapid and timely response.
Copyright 2007 S. Karger AG, Basel

The microbial world is complex, dynamic, and constantly evolving. Microbes emerge or re-emerge in human populations as they cross the species barrier from animals to humans, or as they enter human populations through weakened public health systems. Once microbes have crossed the species barrier and successfully infected humans they may be asymptomatic or cause disease; if they cause disease they may maintain their virulence or decrease in virulence with further transmission. Others may not be transmissible from human to human, but rather continue to infect humans as sporadic zoonotic infections; still others, however, transmit easily from human to human, causing major pandemics and/or eventually becoming endemic, adding to the existing endemic human disease burden. Viruses afford the most dramatic examples of these microbial characteristics. Avian influenza A(H5N1), for example, has been a sporadic zoonotic

infection in humans since first identified in human populations in 1997 in Hong Kong. Though as of early September 2006 it still lacks human transmissibility, there is a real but unquantifiable risk for it to obtain the capacity to transmit easily from human to human through reassortment or adaptive mutation, leading to an influenza pandemic. Influenza A(H5N1) has brought the world closer to a major influenza pandemic than at any time since 1968, when the influenza A(H3N2) pandemic emerged in southern Asia as it adapted to human populations, and then spread throughout the world [1]. AIDS, thought to have entered human populations from a non-human primate some time in the early 20th century, escaped detection through weak routine surveillance systems in the late 1970s and early 1980s when humanto-human transmission was being amplified by risky sexual behaviour, and when HIV was causing human disease on the African continent, in island nations of the Caribbean, and in North America [2]. The human monkeypox virus, thought to have a rodent reservoir in the sub-Saharan rain forest, periodically infects humans. First-generation cases are severe, with a case fatality rate that can approach 10%, but with passage through human populations both its virulence decreases and its transmissibility declines [3]. The rabies virus, on the other hand, has remained a zoonotic infection, and though it has recently been transmitted to humans through organ transplantation, including corneas, it has never become endemic in humans [4]. The potential of microbes to adapt to human hosts and the environment with relative ease, and to further evolve in virulence and transmissibility, is facilitated by rapid reproduction rates and mutation. It is also linked to a host of social, environmental, economic and human behavioural factors. Social and behavioural factors include health-care procedures that facilitate the spread of infectious agents among health workers and through them into the general population, and inappropriate use of antimicrobial agents that leads to the evolution of antimicrobial resistance. Environmental factors include naturally occurring variations in temperature and rainfall that impact on breeding sites and biting habits of insect vectors, and the modification or destruction of rivers, forests and agricultural land through economic development that modify animal, vector and human habitats. Other economic factors include the level of investment in public health, especially in surveillance systems that rapidly identify emerging or re-emerging microbes, and mechanisms to ensure a rapid and timely response. More than 40 infectious diseases including bovine spongiform encephalopathy (BSE), hepatitis C, AIDS, and severe acute respiratory syndrome (SARS) have been identified during the past three decades [5, 6]. Some of the infectious agents that cause these diseases were able to adapt immediately to their human hosts when they emerged, while others required a

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period of adaptation in humans. The increase in human mobility and interdependence since the mid-20th century has had a major impact on these and other emerging and re-emerging infections. It facilitates the transfer of the infectious agents that cause emerging and re-emerging infectious diseases from country to country, and from continent to continent in food, animals, insects, or unsuspecting humans. Infections can today spread around the globe and emerge in new geographic areas with amazing ease and speed. Some are transported by the flights of migratory birds. Others, such as disease-carrying mosquitoes, travel in the passenger cabin or luggage hold of jets, to cause tropical infections in temperate countries when they bite airport workers or those who live nearby. And in some instances such as the deliberate release of anthrax spores to cause harm in the United States in 2001 fears continue to mount in some circles that microbial agents will emerge or re-emerge because of their deliberate use to cause harm by terrorists or in warfare [7].

Social and Behavioural Factors that Influence the Emergence and Re-Emergence of Infectious Diseases

The worlds population more than doubled in the second half of the 20th century, accelerating most rapidly in the developing countries of the tropics and sub-tropics, where infectious diseases continued to have a hold [8]. Rural-urban migration resulted in inadequacy of sanitation, crowded living conditions and other basic infrastructures associated with population growth. It thus contributed to the re-emergence of many diseases, such as tuberculosis, cholera, typhoid, and plague, that are transmitted when living conditions and hygiene are sub-standard, and when overcrowding occurs. Cholera emerged and caused epidemics during the 1990s in parts of Latin America where it had previously been quiescent for over 100 years. By the 1980s crowded major urban areas in Africa and South America had experienced repeated emergence of yellow fever epidemics as the yellow fever virus was introduced by mosquitoes from rain forests into new and densely populated urban areas where bednets to protect from mosquito bites were no longer being used [9, 10]. Behaviours such as over- or under-prescribing of antibiotics by health workers and excessive demand for antibiotics by the general population have had a remarkable impact on the selection and survival of resistant microbes, rapidly increasing levels of microbial resistance [11]. Soon after the development of the first antibiotics, warning signs of microbial resilience began to appear. By the end of the 1940s, resistance of hospital strains of staphylococcus to penicillin emerged in the United Kingdom with resistance levels as high as 14%, and by the end of the 1990s levels had risen to 95% or greater [12]. In New York

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City in the 1990s, multidrug-resistant strains of tuberculosis emerged and gained their hold in hospitals, prisons and homeless populations [13]. At the same time, multidrug-resistant tuberculosis emerged in the Russian Federation and more than doubled in less than 7 years, with over 20% of tuberculosis patients in prison settings infected with multidrug-resistant strains [14]. Antimicrobial drugs developed to treat AIDS and other sexually transmitted infections such as gonorrhoea likewise began to lose their efficacy because of the rapid emergence of resistance. The bacterial and viral infections which contribute most to human disease are also those in which emerging resistance is of greatest concern: diarrhoeal diseases such as dysentery; respiratory tract infections, including pneumococcal pneumonia and tuberculosis; sexually transmitted infections such as gonorrhoea, and a host of hospital-acquired infections that are notoriously difficult and expensive to treat. Among the major infectious diseases, the emergence of resistance to drugs commonly used to treat malaria is of particular concern, as is the emerging resistance to tuberculosis and antiretroviral drugs for HIV Most alarming are microbes that have now accumu. lated resistance genes to virtually all currently available antimicrobial drugs, such as Staphylococcus aureus and Salmonella typhi, that now have emerged with the potential to cause infections that are cannot be treated with existing antimicrobial agents. Trends in tourism, with tourists penetrating deep into tropical forests, often without appropriate protection against insect bites and/or vaccination, result in importations of malaria and yellow fever with their emergence in industrialized countries [15]. At the same time, sub-standard infection control procedures by health workers have caused the amplification of transmission in outbreaks of emerging infections such as Ebola to health workers and their contacts in subSaharan Africa, and influenza and SARS to health workers and those with whom they have contact in both developing and industrialized counties [16, 17].

Environmental Factors that Influence Infectious Diseases

Human disturbance and alternation of ecological zones throughout the world has increased the frequency with which microbes, usually confined to animals, cross the species barrier to infect humans. Deforestation disrupts natural habitats of animals, and can force animals, searching for food, into closer contact with humans. Emergence leading to outbreaks of Lassa fever in West Africa and of hantavirus in North America have been linked to such phenomena among rodents [18, 19]. In Latin America, Chagas disease emerged as an important human disease after deforestation caused the insect that transmits the infection to move from its wild natural hosts to involve humans and domestic

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animals in the transmission cycle, eventually transforming the disease into an urban infection that can be now also transmitted by blood transfusion [20]. Climate extremes, whether involving excessive rainfall or drought, can likewise displace animal species and bring them into closer contact with human settlements, or increase vector breeding sites. A 1998 outbreak caused by the re-emergence of Japanese encephalitis in Papua New Guinea has been linked to extensive drought, which led to increased mosquito breeding as rivers dried into stagnant pools [21]. The Japanese encephalitis virus is now widespread in Papua New Guinea and threatening to move farther east. The re-emergence of Rift Valley fever in Eastern Kenya caused an outbreak linked to flooding related to El Nino. Humans and cattle, forced to live in close proximity on islands of dry land surrounded by water, facilitated the transfer of the Rift Valley fever virus from unvaccinated animals to humans by mosquitoes that had increased in numbers because of the abundance of pooledwater breeding sites [22]. Other examples of how insects that carry infectious diseases have emerged by exploiting new opportunities created by environmental degradation include epidemics of dengue and yellow fever that have been fuelled by discarded household appliances, tyres, plastic food containers and jars that have created abundant artificial mosquito breeding sites. The Aedes aegypti mosquito has now emerged and become well established in most, if not all, large African cities, increasing the risk of explosive urban outbreaks of dengue and yellow fever [23]. Similar examples are occurring in Asia where dengue and dengue haemorrhagic fever have caused major outbreaks during 2004 in Indonesia and India [24]. In countries of the former Soviet Union, large amounts of stagnant water, created by ineffective irrigation schemes, provided mosquito breeding sites that permitted the re-emergence of malaria in the most southern states, where a few incidental and probably imported cases in Tajikistan in the early 1990s multiplied to almost 20,000 reported cases in 1998 [25]. Such problems are compounded by the very small number of new cost-effective chemical pesticides, suitable for public health, that have been developed in recent years. Though intensive research has failed to disclose the origins of Marburg and Ebola haemorrhagic fever outbreaks, microbes causing both diseases are also thought to emerge in humans as they encounter animal sources somewhere in the transmission cycle [26]. An outbreak of Ebola haemorrhagic fever in humans in 1995 was linked to a woodsman, who worked deep within the tropical rainforest making charcoal, and who is somehow thought to have become infected with the Ebola virus which he then carried back to his home village and family members, while a Swiss researcher is thought to have become infected with the Ebola virus while doing an autopsy in search of the cause of a major die-out of chimpanzees in a forest reserve in West Africa [27, 28].

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The consequences of the environment and interspecies transmission of microbes are most clearly demonstrated in the case of the influenza virus. It is thought to be only a matter of time until an animal influenza virus circulating in domestic animals undergoes adaptive mutation or recombines with a human influenza virus, and causes the next highly lethal influenza pandemic [29]. Intensive farming practices have placed humans in Asia in close proximity to domestic animals in densely populated areas. In 1997 in the Hong Kong Special Administrative Region of China, crowded conditions and live poultry markets adjacent to residential areas, facilitated the emergence of avian influenza A virus (H5N1), previously thought confined to birds. At least 18 humans were infected and 6 died, raising considerable alarm [30]. Although probable humanto-human transmission of the virus was documented, it was found to be relatively inefficient and uncommon [18, 31]. A re-emergence of this same virus throughout Asia, Africa and the Middle East during the period 20032006 has resulted in increasing numbers of human infections with high case fatality rates in Thailand, Vietnam, Cambodia, China, Indonesia, Iraq, Nigeria, Turkey, Azerbaijan, Djibouti and Egypt. By September 2006, over 241 sporadic human zoonotic infections had occurred, with an approximate case fatality rate of 50%, and the threat of emergence of a global human pandemic continues, either as a result of adaptive mutation or reassortment as the H5N1 virus continues to circulate in non-human mammals and avian populations [32].

Economic Factors that Influence Infectious Diseases

Emerging and re-emerging infections enter a world where public health investment made by governments has decreased during the past 50 years. With the increase in vaccines and the development of antimicrobial agents in the 20th century, as well as major investments in water, sanitation and public health, infectious diseases were well controlled in most industrialized countries. One infectious disease, smallpox, completely disappeared from industrialized countries through routine use of the smallpox vaccine, and then from the rest of the world after a global eradication effort [33, 34]. As infectious diseases in industrialized countries came under control during the 20th century, there was great optimism that they were no longer a health problem. As a result of this optimism, financial resources once used to combat infectious diseases were shifted to other health and disease problems, and there was decreased and insufficient investment during the last quarter of the 20th century to adapt and use newly developed technologies for detection, monitoring and responding to infectious diseases [34].

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In parallel, investment in research and development for new vaccines and antimicrobial drugs for infectious diseases was shifted to non-communicable diseases, i.e. those that still affected industrialized countries and were related to lifestyle, ageing and environmental hazard. The resultant 10/90 research gap, with less than 10% of public and private funds being placed into research for infectious diseases such as tuberculosis, diarrhoeal diseases, malaria and AIDS, created a slowdown in the development of new antimicrobial drugs and vaccines [35]. With decreased investment came the re-emergence of known infectious diseases in industrialized countries, such as tuberculosis and common sexually transmitted infections such as syphilis and gonorrhoea; as well as the new and emerging infectious diseases such as hepatitis C and AIDS that escaped detection until they were firmly implanted in human populations [34, 36]. Developing countries, on the other hand, had never accomplished the same decrease in infectious diseases. In these countries infectious diseases continued to remain an important cause of sickness, disability and death throughout the 20th century because of a lack of access to vaccines and drugs with which to prevent and treat them, and because of weak health systems that failed to consistently reach populations in need. The resources that these countries were able to invest in infectious disease control in the first half of the 20th century, often with the assistance of the colonial powers, decreased as the millennium progressed, with some of the greatest decreases occurring in sub-Saharan Africa [35]. Common infectious diseases such as tuberculosis, that are spread directly from person to person, began to re-emerge and cause significant suffering and death in developing countries because of inequitable distribution of curative drugs within developing-country health systems, and because of the continued lack of development of preventive vaccines [35]. Treatment with partial regimes of tuberculosis drugs because of inconsistent drug supply to developing-country health systems caused a rapid emergence and increase in drug-resistant strains that resulted in higher cost for treatment because of the need to use more expensive second-line tuberculosis drugs [37]. Re-emergence of infections such as diarrhoeal diseases of children, caused by lack of adequate sanitation and safe water, increased as sanitation systems in major metropolitan areas failed with increases in urban populations, and those in rural areas failed to develop. Vaccine-preventable diseases such as measles, for which access to vaccines was likewise not sustained, remained major public health problems. Decreased investment in childhood immunization programmes in Russia and the Ukraine in the early 1990s resulted in re-emergence of diphtheria, with major epidemics in the early 1990s [38]. With decreased financial investment in programmes to control infectioncarrying mosquitoes, a re-emergence of malaria, dengue, yellow fever and chikungunya occurred, and mosquitoes then spread to new geographic areas.

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Following the deterioration of Aedes aegypti control campaigns during the 1970s, dengue re-emerged dramatically, with unprecedented numbers of its haemorrhagic form [39]. Prior to 1970, only nine countries had experienced epidemics of dengue. By 1998, a dengue pandemic occurred in which 1.2 million cases were reported from 56 countries. Since then dengue has continued to emerge in new geographic areas, or to re-emerge where it has occurred before, causing major epidemics. The Asian epidemic of dengue fever that began some time in the early 2000s has resulted in over 60,000 cases and 700 deaths in Indonesia alone [40]. In 2005, the chikungunya virus emerged and spread throughout several southern Pacific islands. A total of 3,100 human infections were reported by a sentinel network on La Runion within the first 6 months of the outbreak, leading to an estimate of over 204,000 human infections by March 2006 (http://www.who.int/csr/don/2006_03_17/en/index.html). Other infectious diseases such as African trypanosomiasis began to re-emerge in the 1980s with a decline of most surveillance and tsetse fly control activities [41]. By the late 1990s, approximately 46% of mortality in lowincome countries was directly caused by infectious diseases that continued to circulate among large urban and rural populations. These low-income countries today represent over 80% of the estimated 14 million deaths caused each year by infectious diseases [35]. Other economic factors also play a role in the spread of emerging and re-emerging infections throughout the world. Globalization, with a phenomenal growth in international travel and trade since the 1950s, has greatly increased the speed with which microbes, incubating in unsuspecting humans, can cross continents and invade new geographic territories. At the same time, microbes living in insects concealed in cargoes or in the luggage holds and cabins of jets, in animals traded internationally, or in improperly or non-processed food and food products can also travel across continents and internationally. As a result, the threat of emergence of epidemic diseases with origins in one country and spread to others has become a real and constant threat. Nothing more clearly demonstrates this global threat than the spread of AIDS in humans during the latter half of the 20th century. Spreading locally and then throughout the world after its emergence, with its transmission amplified by unsafe sexual behaviour, AIDS has had a negative impact on economic development and healthy population growth. In 1999, the lower figure in the world lifeexpectancy range, which had seen a steady increase in previous decades, declined to 33.2 years, just above the 33 years seen in 1949, largely due to the emergence and spread of HIV [42]. In recent years, every continent has experienced an unexpected outbreak of some infectious disease directly related to increased travel and trade, the most recent having been severe acute respiratory syndrome (SARS) [17]. The SARS outbreak rapidly spread around the globe

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through air travel; from an initial exposure to the SARS coronavirus in a Hong Kong hotel, infected persons carried the virus to Viet Nam, Singapore and Canada, and contacts from these countries then carried the virus to other countries including the United States, Germany and Thailand. Health workers were at great risk of infection during this outbreak, and inadvertently served as the entry point of the virus to communities. A worldwide effort that linked clinicians, epidemiologists and virologists in real time for regular discussion and debate stopped the outbreak 7 months after its emergence in human populations. Advances in food production and storage technology, coupled with the globalization of markets, have resulted in a food chain that is unprecedented in its length and complexity, thus creating an efficient vehicle for microbes to spread to new areas and emerge in susceptible hosts. Tracing the origin of all ingredients in a meal has become virtually impossible, constituting an enormous challenge for the control of foodborne diseases [21, 23]. Medical advances in such areas as blood transfusion, organ transplantation and other sophisticated surgical procedures, and the development of intensive care units have likewise opened new opportunities of emergence into human populations for the microbial world, creating ideal conditions for in-hospital transmission of infectious agents to new, atypical hosts [23]. In the late 1990s, infections such as West Nile fever, that is thought, from genetic sequencing, to have arrived in North America through the introduction of a single virus and Rift Valley fever that arrived in the Arabian peninsula in infected livestock have become endemic in animals after emergence in these new geographic areas, adding to the infectious disease burden [4345]. Once established on new continents, emerging or re-emerging infectious diseases have great potential to change the dynamics and epidemiology of local infectious diseases. The universal nature of the microbial threat, with infectious agents, including drug-resistant forms, passing undetected across increasingly porous borders to emerge in new geographic areas, has placed all nations on an equally vulnerable footing. Economic prosperity has produced a world that is interconnected in matters of economics and trade, with the result that health has become both a domestic and a foreign issue [46]. As early as the 14th century, concern was expressed about the international spread of infectious diseases, and quarantine in the city state of Venice was enforced to successfully prevent the entrance and emergence of plague, but this and other successes with quarantine occurred at a time when transport between cities, countries and continents took days or weeks rather than hours. Today such measures cannot prevent the spread of infectious diseases. In fact, they provide a sense of false security that infections can be kept from crossing international borders. In todays globalized and highly mobile world where infectious

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agents travel in food, animals, insects and humans frontiers and quarantine posts alone cannot prevent their spread. The best and only sure defence against the international spread and emergence of infectious disease rests with strong public health measures. These include disease detection systems that take advantage of the latest laboratory technologies and real-time electronic communication systems, and rapid intervention to contain the outbreaks and prevent human sickness and death. As made clear by the recent SARS outbreak, international partners working closely together are a very effective barrier to the international spread of infectious diseases.

The Future of Emerging and Re-Emerging Infectious Diseases

In future years, infectious diseases will continue to emerge and re-emerge, and social, environmental and economic factors, the availability of antimicrobial drugs and vaccines, and the resilience and natural selection and evolution of the microbial world will continue to have an impact on their virulence, transmissibility and impact on human populations. Water and sanitation systems will be challenged as populations continue to move to urban areas in search of work and economic betterment, with continued emergence and re-emergence of intestinal infections and diarrhoeal disease. Behaviour of health workers and the general population will continue to play an important role in the emergence and transmission of infectious diseases, and in the emergence of antimicrobial resistance. Continued lack of vaccines for many of the major infectious diseases will dampen progress in prevention, while continued alterations in temperature and rainfall, and human impact on agricultural lands, forests and rivers will in some instances increase the number of insect vectors and alter the geographic distribution of animal hosts, leading to the emergence of new human infections and/or re-emergence of those that are known. Underlying all these factors is the current acceleration in globalization, increasing the risk that infections that emerge or re-emerge in one country will spread internationally in humans, insects, animals or food. And finally, with continued inequitable distribution of the vaccines, medicines and goods available now to prevent, treat and control infectious diseases, coupled with weak public health delivery systems in developing and low-income countries, a disproportionate burden of human suffering and death from emerging and re-emerging infections diseases will continue to occur in developing countries. To minimize the impact of emerging and re-emerging infectious diseases, increased investment in national pubic health infrastructure is required in order to detect early and rapidly respond to infectious disease outbreaks, as well as

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long-term programmes to modify or remove the factors that facilitate emergence. Likewise, there is a need for an international safety net of global surveillance with a response mechanism should diseases begin to travel internationally. Recently a global partnership the Global Outbreak Alert and Response Network (GOARN) supported by several new mechanisms and a computerdriven tool for real-time gathering of disease intelligence, has been developed to detect and respond to infectious diseases of international importance [47]. Finally, there is a new infectious disease threat that dominates public health thinking and policies in some industrialized countries that of the emergence or re-emergence of a deliberately caused infectious disease outbreak. Following the deliberate dissemination of anthrax spores through the US postal system in 2001, questions concerning the deliberate use of biological or chemical weapons have been raised with great urgency. The prospect of introduction of an infectious disease to non-immune populations that could cause severe illness and death has now become a stark reality. Thus, emerging and re-emerging infectious diseases will continue to threaten human populations into the future. These threats are constantly amplified by social, economic and environmental factors that accelerate the adaptation of bacteria, viruses and parasites for survival in those human populations into which they emerge or re-emerge.

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Levy SB: The Antibiotic Paradox: How Miracle Drugs Are Destroying the Miracle. New York, Plenum Press, 1992. Frieden TF, Fujiwara PI, Washko RM, Hamburg MA: Tuberculosis in New York City turning the tide. N Engl J Med 1995;333:229333. Small MA: A view from the ground: tuberculosis as an example of reemerging infectious disease in the former Soviet Union; in Davis JR, Lederberg J (eds): Emerging Infectious Diseases from the Global to the Local Perspective. Washington, National Academy Press, 2001. World Health Organization: Airport Malaria, 1996. http://www.who.int/docstore/wer/pdf/ 1996/wer7147.pdf Kahn AS, Kweteminga, T, Heymann DL: The reemergence of Ebola haemorrhagic fever: Democratic Republic of the Congo, 1995. J Infect Dis 1999;179(suppl 1):S76S86. Heymann DL, Rodier G: SARS: A global response to an international threat. Brown J World Affairs 2004;X:185197. Birmingham K, Kenyon G: Lassa fever is unheralded problem in West Africa (News). Nat Med 2001;7:878. Update: Hantavirus pulmonary syndrome United States, 1993. MMWR 1993;42:816820. Control of Chagas disease. World Health Organ Tech Rep Ser 1991;811. Mackenzie JS: Emerging diseases in the Australasian region; in Davis JR, Lederberg J (eds): Emerging Infectious Diseases from the Global to the Local Perspective. Washington, National Academy Press, 2001. Outbreak of Rift Valley fever, Yemen, AugustOctober 2000. Wkly Epidemiol Rec 2000;75:392394. Rodier GR, Ryan MJ, Heymann DL: Global epidemiology of infectious diseases; in Strickland GT (ed): Hunters Tropical Medicine and Emerging Infectious Diseases, ed 8. Philadephia, Saunders, 2000. World Health Organization: Dengue Indonesia update 4, 2004. http://www.who.int/csr/don/ 2004_05_11a/en/ Small MA: A view from the ground: tuberculosis as an example of a reemerging infectious disease in the former Soviet Union; in Davis JR, Lederberg J (eds): Emerging Infectious Diseases from the Global to the Local Perspective. Washington, National Academy Press, 2001. Klenk HD, Slenczka W, Feldmann H: Marburg and Ebola viruses; in Webster TG, Granoff A (eds): Encyclopedia of Virology. New York, Academic Press, 1994. Formenty P, Boesch C, Wyers M, Steiner C, Donati F, Dind F, Walker F, Le Guenno B: Ebola virus outbreak among wild chimpanzees living in a rain forest of Cote dIvoire. J Infect Dis 1999;179 (suppl 1):S120S126. Kahn AS, Kweteminga, T, Heymann DL: The reemergence of Ebola haemorrhagic fever, Democratic Republic of the Congo, 1995. J Infect Dis 1999;179(suppl 1):S76S86.9/S120S126. Influenza Pandemic Preparedness Plan: Geneva, World Health Organization, 1999 (WHO/CDS/ CSR/EDC/99.1). Isolation of avian influenza A(H5N1) viruses from humans Hong Kong, MayDecember 1997. MMWR 1997;46:12051207. Class ECJ, Osterhaus ADME, van Beek R, et al: Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 1998;351:472477. Avian Influenza Update: Http://www.who.int/csr/disease/avian_influenza/country/cases_table_ 2004_09_28/en Fenner F, Henderson DA, Jezek Z, Ladnyi ID: Smallpox and Its Eradication. Geneva, World Health Organization, 1988. Lederberg J, Shope RE, Oaks SC Jr (eds): Emerging Infections: Microbial Threats to Health in the United States. Washington, National Academy Press, 1992. World Health Organization: Removing obstacles to Health Development, 1999. http://www. whoint/infectious-disease-report/index-rpt99.html Heymann DL: The fall and rise of infectious diseases. Bioalert: disease knows no borders. Georgetown J Int Affairs 2001;Summer/Fall:714. World Health Organization: Overcoming Antimicrobial Resistance. http://www.who.int/infectiousdisease-report-2000/

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Weinberg J: European responses to emerging infections and their policy implications; in Davis JR, Lederberg J (eds): Emerging Infectious Diseases from the Global to the Local Perspective. Washington, National Academy Press, 2001. Dengue/dengue haemorrhagic fever: situation in 2000. Wkly Epidemiol Rec 2000;75:193196. World Health Organization: Dengue Indonesia update 4, 2004. http://www.who.int/csr/don/ 2004_05_11a/en/ Molyneux DH: Vector-borne infections in the tropics and health policy issues in the twenty-first century. Trans R Soc Trop Med Hyg 2001;95:16. The World Health Report 2000: Health Systems: Improving Performance. Geneva, World Health Organization, 2000. Petersen LR, Toehrig JT: West Nile virus: a reemerging global pathogen. Emerg Infect Dis 2001;7:611614. Outbreak of Rift Valley fever, Saudi Arabia, AugustOctober 2000. Wkly Epidemiol Rec 2000;75: 370371. Outbreak of Rift Valley fever, Yemen, AugustOctober 2000. Wkly Epidemiol Rec 2000;75: 392394. Heymann DL: Evolving infectious disease threats to national and global security; in Chen L, Leaning J, Narasimhani V (eds): Global Health Challenges for Human Security. Cambridge, Harvard University Press, 2003, pp 105124. Heymann DL, Rodier F: Hotspots in a wired world: WHO surveillance of emerging and re-emerging infectious diseases. Lancet Infect Dis 2001;i:345353.

David L. Heymann World Health Organization Avenue Appia 20 CH1211 Geneva 27 (Switzerland) Tel. 41 22 791 2212, Fax 41 22 791 1571, E-Mail heymannd@who.int

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The Role of a Pathology Laboratory in SARS and Other Emerging Infections


John Nichollsa, J.S. Malik Peirisb
Departments of aPathology, and bMicrobiology, The University of Hong Kong, Pok Fu Lam, Hong Kong, SAR, China

Abstract
If there is an outbreak of emerging viral infections in Southeast Asia a pathology laboratory will provide a crucial role to complement the virology laboratory even though the number of suitably trained pathologists in the region is vastly reduced compared to Western countries. A laboratory with limited resources should still be able to perform histological assessment of tissue samples and infected cell lines to determine the nature of the disease. Whilst it is acknowledged that in many regions of Southeast Asia there will be a limit on tissues available after death the use of Trucut samples should be encouraged and submitted tissues should be fixed in alcohol, in addition to formalin to improve the extraction of viral RNA or DNA.
Copyright 2007 S. Karger AG, Basel

If history is going to be an indicator of future trends, emerging infectious diseases (EID) are going to continue to plague our societies and since the 1980s indications have shown that firstly, the Asian regions will be a likely flashpoint, and secondly, the infectious agent will most likely be a virus [1]. There is therefore no doubt that a virology department will play a crucial role in the identification of a new emerging agent, or modification of an existing agent. What role then will a pathology laboratory play? Using SARS as an example, this chapter will deal with the role of a pathology laboratory in an EID outbreak.

The Role of a Pathology Laboratory in SARS

Other chapters in this monograph have dealt with the molecular diagnosis of SARS. What needs to be mentioned was the role of the pathology laboratory in the identification of SARS-CoV as the aetiological agent of SARS. The new

100 nm 40Kx 03 0070 03-04-25

Fig. 1. Electron micrograph of SARS-CoV cultured in FRhK cells from tissue sample of SARS-CoV infection. Numerous viral particles with spikes are present in vesicles 40,000.

agent was identified in the open lung biopsy of one of the index patients [2]. At Hong Kong University there is a close relationship between the microbiology and pathology laboratories so in the index case of SARS the tissue sent for viral study also had a portion submitted for histological examination. This lung biopsy showed the presence of pneumocytes with large nuclei, occasional eosinophilic nucleoli but no evidence of viral inclusions. These histological changes were interpreted as suggestive of viral infection but immunohistochemistry (IHC) for cytomegalovirus, herpes simplex virus, parainfluenza virus, human metapneumovirus and influenza virus were all negative. Electron-microscopic examination eventually demonstrated viral particles similar to those identified in the cell culture from the tissue sample (fig. 1). Later studies on post-mortem material also identified SARS-CoV like particles in pneumocytes and bronchial cells. So, how crucial was the pathology laboratory in the diagnosis of SARS? The electron microscope findings of a coronavirus allowed molecular cloning and

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amplification to target the viral family and reveal that SARS-CoV was a novel strain [3]. Autopsy studies in the early period of the SARS outbreak indicated that the patients were dying of diffuse alveolar damage [4], but owing to the lack of an antibody raised to any other previously known coronavirus reacting with this novel SARS-CoV at the time of the outbreak it was not possible to determine if , the patients were dying with active viral infection or due to overwhelming lung damage. Once specific antibodies became available to SARS-CoV retrospective , studies now indicate that the second scenario is the most likely. During the SARS outbreak, because of the uncertain risk of transmission of the virus to health care personnel, post-mortems were not readily performed in Hong Kong with a total of 299 deaths only 39 post-mortems were performed, 15 involved opening the thoracic cavity compared with 24 Trucut or needle biopsies. In Toronto, with 43 deaths, 20 post-mortems were performed [5]. There is only one reported case of gastrointestinal biopsy material [6] which shows Co-V replication in the epithelium without epithelial necrosis but owing to autolysis of gastrointestinal tissues following death this was not able to be confirmed in larger studies. There were few published reports of the histological changes of SARS during the November to July outbreak [4, 710] and many of the publications studying the pathogenesis were published after the outbreak had ended. One important finding during the outbreak that required autopsy tissue was the fulfillment of Kochs postulates for SARS [11]. The publications after the end of the SARS outbreak have been valuable from a research point of view for determining the time course of disease as well as the cellular tropism of the SARSCoV. Furthermore, the autopsy material from the SARS cases has also been a valuable tissue bank that can be kept for archival purposes.

The State of Pathology in Asia

Pathologists are medically qualified doctors who have undergone a period of post-graduate training, often culminating in an exit exam under the auspices of a professional colleges such as the Royal College of Pathologists of Australasia, The Royal College of Pathologists or the College of American Pathologists. A survey of pathologists in the Asian region carried out in 2002 [Collins RJ, presentation at Amsterdam International Academy of Pathology] indicated that if a benchmark of Australia or USA is used there is a dismal shortage of pathologists in a region where there is (most likely) going to be an outbreak of EID (fig. 2). This shortage is even more acute if the probability that an emerging infection will occur in the rural region than the city is considered [12]. Detailed guidelines have been published for the use of electron microscopy in an outbreak [13] but in

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Russian Federation

ASIA Continent

Kazakhstan Mongolia Kyrgyzstan


NORTH KOREA

ek

ist

an
ta nis n

stan
Afg ha

Tajikistan China Nepal Bhutan


SOUTH KOREA MACAU (CHINA) JAPAN

Pakistan India

Bangladesh

TAIWAN
MYANMAR LAOS

Pacific Ocean

HONG KONG (PRC) VIETNAM


PHILLIPINES

THAILAND

BRUNEI

Sri Lanka Maldives


CAMBODIA

MALAYSIA

SINGAPORE INDONESIA

Indian Ocean

AUSTRALIA

Country

Approximate number of pathologists 2,400 4,000 300 260 3 200 44 2000

Pathologist ratio to population (number/million population 19.2 3 13 37 0.56 3.3 11 105

Japan Peoples Republic of China Taiwan Hong Kong Laos Thailand Singapore Australia and New Zealand

Fig. 2. Survey carried out in 2002 listing the number of pathologists practicing in the depicted Asian region.

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Fig. 3. Trucut biopsy needle that can be used for needle sampling of organs.

reality the number of electron microscope machines in regions susceptible to a viral outbreak appears extremely limited.

Materials for Examination

Many studies indicate a falling autopsy rate over the past 2 decades [14,15] despite their clinical benefit as a tool for quality management [16]. Cultural differences may influence autopsy rates including beliefs about keeping cadavers whole and the religious view on the soul and the body [17]. In Hong Kong, the coronial system is able to override relatives concerns provided certain criteria are met [http://www.judiciary.gov.hk/en/crt_services/pphlt/html/cor.htm#3], but the extent to which this operates in other countries is not clear. Two options to full autopsies were made available that provided useful during the SARS period the first was a limited autopsy where only the lungs were sampled thereby requiring a single, smaller incision than normally performed. The second was the use of a needle para-mortem biopsy obtained with a Trucut needle [18] (fig. 3). In Hong Kong this was used in 24 of the 39 post-mortem cases. Apart from minor puncture wounds there is no disfigurement to the body.

Fixation of Material

For over a century formalin has been the fixative of choice for tissue samples [19]. Fixation of tissue is necessary because as soon as death occurs or tissue is removed from the body in a vital state, it undergoes a process of

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degeneration called autolysis. The fixation of tissue samples in formaldehyde leads to extensive cross-linking of all tissue components and this preserves tissue, preventing autolysis. Recently, the safety of formalin has come under scrutiny where in the European Union it is now classified as a class I carcinogen [20]. Alternatives have been proposed such as Glyo-Fixx, STF-Streck, Omnifix, Histochoice, and Histofix [21], but none of these has met with much approval by pathologists for morphological diagnosis. The main drawback with formalin fixation from a research aspect has been the limited ability to extract DNA and RNA from this fixed tissue. A number of postulated mechanisms for this problem come into play here firstly, the addition of a formaldehyde group to a base in the form of N-methylol (N-CH2OH), the second is the attack of N-methylol on an amino base to form a methylene bridge and the third is the potential deleterious effects of RNAse which are not inactivated by formalin [22, 23]. Indeed, Ding et al. [24] demonstrated that only 0.1% contamination of an extraction reagent by formalin inhibited the ability of the reagent to extract mRNA. The ideal solution for molecular diagnosis of a viral infection, therefore, would not to use any form of fixative as RNA can readily be extracted from fresh tissue using either a combined guanidinium thiocyanate acid-phenol chloroform procedure [26] or a guanidinium thiocyanate-caesium chloride gradient. Commercially available RNA extraction methods are available (RNeasy (Qiagen), Trizol (Invitrogen) and ToTally RNA (Ambion)) but in PCR amplification of fresh samples their success is varied [26] and this may be due to the steps of the individual techniques and the affinity of viral RNA to the spin column. It must be stressed that these solutions do not fix the tissues in the same way as formalin does so will not be useful for morphological analysis or long term storage at room temperature. For long-term storage and transport RNAlater (Ambion) Tissue Protect Tubes provide pre-measured volumes of RNAlater RNA Stabilization Reagent in re-closable tubes for convenient handling and sample storage. The reagent preserves RNA for up to 1 day at 37 C, 7 days at 1825 C, or 4 weeks at 28 C, allowing processing, transportation, storage, and shipping of samples without liquid nitrogen or dry ice. Alternatively, the samples can also be placed at 20 or 80 C for archival storage. Morphological analysis, however, will be very difficult to interpret from tissues placed in this reagent. Fixation in alcohol is superior to formalin for the preservation of RNA but will result in poor morphology [27]. Whilst these authors found that immunohistochemistry was not altered, it is possible that in alcohol fixed tissue some antibodies will not react in alcohol as the antibodies are developed for use in formalin fixed material and small molecules (e.g. peptides) may be solubilized and lost in alcohol-based fixatives [http://www.med.muni.cz/biomedjournal/ pdf/2004/02/63_74.pdf%201.pdf 8 Oct 2005]. In one report, Carnoys fixative has been found to be superior to formalin and ethanol for RNA extraction

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ve

Cell

Cell

ve

ve

B1

B2

L1

L2

R1

R2

ve

710bp 310bp 489bp 404bp 367bp 242bp 190bp 281bp 234bp 194bp 118bp

Using primers with a product size of 250260bp Ladder: BSMSP1 L Positive case (left lung) R Positive case (right lung) M DNA marker Cell Paraffin-embedded cell line

Using primers with a product size of 113bp Ladder: X174 B1 Positive case (brain 1) B2 Positive case (brain 2) L1 Positive case(left lung 1) L2 Positive case (left lung 2) R1 Positive case (right lung 1) R2 Positive case (right lung 2) M DNA ladder

Fig. 4. Comparison of different primers for PCR amplification of formalin fixed, paraffin embedded tissues from fatal human H5N1 case. When a product size of 250260 bp is used no PCR product is present, but when a smaller product size is chosen there is positive amplification from lung tissue.

but this fixative is not widely used in routine practice [28]. Bouins fixative is to be avoided as it causes damage to DNA and RNA [29]. If ethanol fixation is to be used 70% seems the ideal concentration though this should be used for small samples [30]. If one is going to attempt RNA or DNA extraction from formalin-fixed paraffinembedded (FFPE) tissues, a number of factors need to be taken into consideration for successful extraction, especially for RNA. Firstly, the most successful method of total RNA utilizes a proteinase K digestion prior to phenol-chloroform-isoamyl alcohol extraction and carrier precipitation [31]. Both phenol and chloroform denature proteins but leave the RNA and DNA in the aqueous phase. The isoamyl alcohol reduces foaming. Secondly, the RNA that is extracted will be degraded and one should not aim at amplifying fragments larger than 60120 bp [24, 3234] (fig. 4) Thirdly, RNAase from glassware is a potential source of contamination so all reagents must be RNAse free. Trace amounts of DEPC that have been used to eradicate RNAse, however, may be detrimental to RNA by modifying purine residues [23]. It is also recommended that the reverse transcriptase uses primers

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with random hexamers or with a specific antisense primer that will be used in the PCR [23]. Finally a pre-PCR DNA restoration treatment has been proposed that promises to increase the length of amplifiable DNA [34]. There also appears to be progress on the extraction of proteins for protein microarray using novel technologies [http://www.lrz-muenchen.de/ kfbecker/]. In summary, when balancing the multitude of techniques available in a modern laboratory versus the realities of laboratory service in many regions of Asia, we consider that the following protocol is a practical and suitable compromise for obtaining tissue from a region which there are limited resources and facilities which will allow the storage and preservation of this material for analysis by a reference laboratory.

Lung

The area of the lung chosen for Trucut should ideally be corresponding to radiological signs of pneumonia. For negative control, a biopsy from a radiological normal area is also advised. A minimum of 4 cores of tissue should be sampled, 5 if liquid nitrogen is available. Biopsy 1 (fresh): Half is put into viral transport medium, the other half is placed in OCT embedding media and frozen in liquid nitrogen if it is available. Biopsy 2 (fresh): Placed in RNAlater Tissue Protect Tube (Qiagen, #76163) (if available). Biopsy 3 (70% ethanol): Ethanol is superior to formalin for RNA extraction but is inferior for morphology. This fixative can be used if the tissue needs to be transported over distance and liquid nitrogen is not available. (If the same Trucut needle is used for all biopsies fixation in ethanol should be done first as even a small amount of formalin in ethanol will impair RNA extraction.) Biopsy 4, 5 (10% neutral buffered formalin): This will allow good morphology to determine the extent of lung damage, secondary bacterial infection, etc. It will also enable immunohistochemistry to be performed for viral antibodies and co-localization studies to determine the cells infected with the infectious agent. Formalin-fixed tissue can be used for RNA extraction but is less optimal as a fixative in this respect than alcohol. One of the biopsy samples should be processed for paraffin embedding; the other will remain in formalin for future extraction of RNA/DNA or electron microscope examination. If freshly prepared 2.5% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate-HCl buffer pH 7.4) is available a 1- to 2-mm sample from the core can be placed directly into this fixative for electron-microscopic examination.

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Other Organs

Sampling of other organs especially spleen, bone marrow and liver would be important, at least for standard histology, and for detecting the presence of virus (by culture, RT-PCR and viral antigen by immunohistology), and to look for haemophagocytosis or changes in these organs. To avoid possible PCR carryover contamination, a new needle should be used for each organ. This is also important for determining the extent of disease dissemination.

Acknowledgements
Mr. Kevin Fung and Chan Yuk Sing for technical assistance. Supported by RFCID from Government of Hong Kong SAR Number 03040872.

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Dr. John Nicholls Department of Pathology, The University of Hong Kong Pok Fu Lam Hong Kong, SAR (China) Tel. 852 28554883, Fax 852 28725197, E-Mail nicholls@pathology.hku.hk

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The Fight against Emerging Viral Diseases in Asia


S.K. Lam
Department of Medical Microbiology, University of Malaya, Kuala Lumpur, Malaysia

Abstract
Asia has been the epicenter of many emerging diseases in the past decade. Many risk factors have been identified to contribute to this situation, including being economically disadvantaged and lacking in many health amenities. Poor farming practices have been highlighted to be a major risk factor and despite international guidelines and assistance, this situation is unlikely to change in the foreseeable future. The vast movement of peoples, including migrant workers, will continue to make this region vulnerable to the introduction of emerging microbes. The fight against emerging diseases will remain an uphill battle and because of inadequate infrastructure and trained manpower, the situation will worsen. Individual developing countries will not be able to handle outbreaks of emerging diseases and it is suggested that a strong regional effort be formulated. A high biosafety laboratory is needed urgently in the region to allow scientists to work with these highly pathogenic organisms in an appropriate environment and political will and international assistance will be needed to get this going.
Copyright 2007 S. Karger AG, Basel

Despite the impressive advances in the field of antimicrobials and vaccinology in the last two decades, the world is still facing the threat of infectious diseases, with an estimated 15 million deaths globally each year. It is the second most common cause of deaths, accounting for 26% of total deaths, and second only to cardiovascular diseases [1]. Considering that 60% of the worlds population lives in Asia, infectious diseases are probably the most important cause of deaths in developing countries. Among the most important causes of infant deaths in developing countries are microbes causing diarrhoeal and respiratory diseases, many due to viruses. Rotavirus diarrhoea alone is responsible for 800,000 deaths annually and measles, despite the availability of an excellent vaccine, still accounts for

almost a million deaths, much of it in third-world countries. The re-emergence of over 200 cases of poliomyelitis in Indonesia after an absence of a decade is an example of how a vaccine-preventable disease can be re-introduced and reemerged in a developing country despite the inclusion of the vaccine in the expanded programme of immunization [2]. Besides the morbidity and mortality due to known viral infections, there has been an upsurge of emerging and re-emerging viral infections globally, with many of them having their origin in the Asian region. The very first case of avian influenza was reported in Hong Kong in 1997 and resulted in the decimation of millions of poultry stocks and 18 human cases, 6 of whom died [3]. Since then, this highly pathogenic H5N1 influenza virus has infected animals, mainly poultry, in 10 countries, and 4 of them reporting human cases resulting in over 100 deaths. The fear of a pandemic is justified since this virus is known to freely cross species barriers, infecting tigers, leopards, cats, pigs and humans [4]. A pandemic of influenza such as that in 1918 could result in 2530% of the world population being infected and the economic losses of tens of billions of dollars. The severe acute respiratory syndrome (SARS) made its first appearance in Hong Kong in late 2002 and was found to be caused by a new human coronavirus [5]. This SARS coronavirus is believed to have originated in Guangdong, China, and entered Hong Kong from a single individual visiting the colony. Wildlife animals are freely traded legally and illegally and are believed to be the source of human infections, as are at least 75% of newly emergent diseases in the last two decades. Malaysia experienced its first emerging viral disease in 1999 when pig farmers became ill and died from a form of severe viral encephalitis. About 265 cases were reported, with a mortality of nearly 40% [6]. The disease, which was caused by a new emergent paramyxovirus named Nipah virus, was brought to an end by culling of pigs in infected farms. The introduction of Nipah virus into Malaysia by pteropid fruit bats might be due to anthropogenic deforestation and the El Nino climatic effect in the region [7]. Since 1999, Nipah virus has not made a re-appearance in the country but there has been serological evidence that the virus is present in fruit bats in India, Thailand, Cambodia and Bangladesh. Several human outbreaks in Bangladesh between 2004 and 2005 have resulted in mortality as high as 70% and again fruit bats have been implicated in the spread of this virus to humans [8]. Another viral disease that re-emerged in Malaysia was that caused by enterovirus 71 (EV71) during an outbreak of hand-foot-mouth disease in 1997 [9]. EV71 has been responsible for many large fatal outbreaks in the past [10] but rarely in the Asian region. The cause of death among young patients in the Malaysian outbreak was attributed to sudden cardiopulmonary collapse with

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minimal neurological features. The following year, a larger outbreak caused by this virus occurred in Taiwan with at least 80 fatal cases [11]. This EV71 strain has become more virulent and is now endemic in the region.

Asia: The Epicentre for Emerging Diseases

The publication of a report by the US Institute of Medicine (IOM) in 1992 about emerging diseases and the inability to confront them laid the foundation for a global interest and monitoring of emerging diseases. In a follow-up publication by the US IOM in 2003, many risk factors have been attributed to the emergence of infectious diseases and these are documented in a 2003 publication [12]. Some of these risk factors have special significance to Asian region and may account for it acting as an epicenter for emerging diseases. Many countries in the Asian region are economically disadvantaged, with poor access to basic amenities such as clean safe water and good sanitation, and are most vulnerable to infectious diseases. The 90/10 health divide coined by WHO states that 90% of the world is at risk from infectious diseases but that only 10% of resources are spent to fight them is only too true in this region. This is exemplified in the fight against HIV/AIDS where, despite effective medicines being available, the cost prohibits their wide usage in poor developing countries. Globally, there is greater emphasis for the development of drugs to meet the needs of rich countries and neglected diseases such as malaria and tuberculosis do not receive adequate attention. New approaches to drug research should not be driven by economic consideration alone otherwise we will face a major factor for disease re-emergence in Asia. Changes in ecosystems have been identified as a contributing factor in disease emergence. Poor countries in Asia rely on natural resources and land use for agriculture, logging and dam building. This can result in ecological changes and alter the transmission patterns of pathogens. Increased human contact with exotic microorganisms in animal reservoirs and the environment as a result of changing land use patterns can lead to the emergence of new infectious diseases. Increasing populations in developing countries and demographic changes due to rural-urban migration will further erode existing health and social infrastructures. Climatic changes such as El Nino also have an impact on disease burden transmitted by mosquitoes and other arthropod vectors. Dengue has re-emerged as an important cause of epidemics in many countries and Southeast Asia and the Western Pacific regions have reported increasing incidence despite various control measures being implemented. There are four serotypes of dengue virus and each serotype can give rise to mild dengue fever to the more severe

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manifestations of dengue haemorrhagic fever and dengue shock syndrome [13]. The principal mosquito vector, Aedes aegypti, is abundant in urban areas. Despite control measures against this vector, the situation has not improved and large outbreaks are still being reported regularly. This particular species of mosquito is also responsible for a novel outbreak of viral polyarthritis in Malaysia caused by chikungunya virus [14]. The continuous presence of A. aegypti poses a threat of a yellow fever outbreak to the region since the same vector is responsible for its transmission. This possibility becomes real due to increasing tourism and trade between Asian countries and yellow fever endemic countries. In the Southeast Asia region, more developed countries like Singapore and Malaysia have become dependent on migrant workers from less-developed countries such as Indonesia, Bangladesh, Pakistan, India, Thailand, and Myanmar. In Malaysia, over a million workers are currently employed to service various industrial sectors. Many of these workers enter the country to work illegally and are not subjected to medical examination, a mandatory requirement for legal workers prior to entering the country. A pilot study among migrant workers in Malaysia showed that these workers had a high carriage rate for viral hepatitis B, C and E as well as HIV and suffered from sexually trans, mitted diseases due to resistant bacteria pathogens [15]. The spread of HIV in the region can also be attributed to substance abuse such as intravenous drug use and sexual promiscuity. Due to cultural and religious sensitivities, control programmes involving needle exchange, sex education and condom usage are not actively promoted in some Asian countries. Poor farming practices have been identified as a major risk factor for the emergence of new diseases and this is particularly true in Asia. Open mixed farming has contributed to interspecies spread and genetic exchange among viruses. The recommendation by WHO and OIE for close farming in the Asian region cannot be implemented over a short period of time because of the high costs. In addition, backyard farms and free-range domestic animals such as ducks, chickens and pigs abound and prevention of humans inter-mingling with farm animals will require changes in cultural and traditional practices. The keeping of fighting cocks in countries such as Indonesia, Malaysia and Thailand is a flourishing pastime in rural areas and this is proving to be an unusual control problem in the fight against avian influenza. The traditional preference for freshly slaughtered poultry in market places has also posed serious problems in control measures. In addition, exotic meat is a gastronomic delight for Asians, especially among the Chinese, and this has led to the proliferation of wildlife markets and the transmission of viruses such as SARS coronavirus to humans. The use of antibiotics in farm feeds has created many resistant strains of bacteria and the possible use of antivirals such as amantadine to prevent avian influenza in poultry is a cause for concern.

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Uphill Battle against Emerging Diseases in Asia

The WHO has urged member countries to monitor for unusual disease activity and to follow a plan of preparedness for emerging diseases, especially the expected influenza H5N1 pandemic. Since developing countries are unable to adhere to this plan, they have adapted and modified international guidelines based on their limited infrastructure and manpower resources. Weaknesses should be identified and surge capacity improved before such impending pandemics. One of the recommendations in the face of an influenza pandemic is the stockpiling of antivirals and vaccines. Due to economic reasons, such stockpiles are less than adequate in the region and the scarcity of expensive antivirals such as oseltamivir will definitely deprive access to developing countries. Pandemic stockpiling of oseltamivir by rich countries such as Finland, the United Kingdom and USA for 25% of their population in the event of an influenza pandemic may deplete the global supply, placing poor countries at a disadvantage. This is a dilemma faced in Asia where a pandemic influenza is likely to originate. Prepandemic stockpiling of antivirals against influenza has been shown to be cost effective [16] and international agencies such as the WHO is moving ahead with this and will release the drugs where they are needed most. This approach by the WHO is a move in the right direction since containing the outbreak at its source will help developing countries and prevent global spread. The development of a vaccine against H5N1 influenza requires high technology such as reverse genetics and this is beyond the technical capability of many Asian countries. Availability of a pandemic vaccine will again be limited to developed countries initially and the WHO will need to stockpile such vaccines for use in countries where and when the outbreak first occurs. Hopefully commercial production of a pandemic vaccine will increase and become affordable even for the worlds poor. Surveillance of emerging and re-emerging diseases is of paramount importance given the fact that the region is a hotspot of these diseases. This requires continuous resources and funding, which unfortunately are in short supply. The fear of economic trade sanction if the outbreak involves domestic livestock will be a disincentive for reporting and may lead to cover-up and lack of transparency by national authorities. Culling without adequate compensation as has happened in developing countries will lead to illegal trade and smuggling, thus furthering disease spread. A preparedness plan involving infrastructure and manpower development in Asia will also benefit the region in the response to possible bioterrorism

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threat. The hallmark of such a response involves identifying and diagnosing the disease, finding the source and host, containing the outbreak, treating the victims and preventing the spread of the organism. The investment on emerging diseases will therefore strengthen overall surveillance for emerging diseases as well as for bioterrorism. Despite the fact that Asia is a proven epicenter for emerging viral diseases, there is lack of a high biosecurity laboratory (BSL 4) among the countries where recent events happened. This is unlike other regions where a spate of high-tech containment laboratories has mushroomed over the last decade. Some of the latest such laboratories are sited in the USA, United Kingdom, Canada, Sweden and France to compliment existing facilities and there is a notable absence of any such development in Asia. Because of the lack of infrastructure and trained manpower to handle deadly outbreaks, it is therefore the norm for countries in Asia to seek help from international agencies such as the US Centers for Disease Control and Prevention (CDC) either directly or through the WHO. Many of these international agencies have well-equipped laboratories in the region, such as the Institut Pasteur in Vietnam, the Wellcome Trust in India, the US Armed Forces Research Institute of Medical Sciences and CDC in Thailand and the US Naval Medical Research Unit No. 2 in Indonesia. Because of their presence and their willingness to offer assistance, national capacity building for outbreak investigation has not been seen to be urgent, resulting in a dependency syndrome. During a recent ASEAN 3 Expert Group Meeting on Emerging Diseases held in Bangkok, Thailand, a proposal was mooted for the formation of an ASEAN Center for Disease Control (ACDC) to serve the Asian region. It is envisaged that ASEAN countries will form a multinational team of scientists to man the ACDC and to conduct training programmes for outbreak investigations. By so doing, ASEAN will enhance its capability and capacity and lead to the sharing of regional resources. Regional experts in diseases such as dengue, HIV/AIDS, viral hepatitis, SARS, influenza and Nipah would then play a bigger role in outbreak investigations and help to solve their own problems. Presently, regional scientists are unable to conduct research on highly pathogenic agents such as SARS, avian influenza H5N1 and Nipah virus all isolated in the region and have to outsource this to developed countries that have BSL 4 facilities. The setting up of the ACDC with BSL 4 facility is therefore a timely move but it will not materialize without political commitment and funding as well as technical assistance from developed countries. Regional societies such as the Asia Pacific Society for Medical Virology with its 520 members from 39 countries will be able to help by offering advice and expertise in many important viral infectious diseases. ACDC will also provide opportunities for members to conduct research on their own pathogens in a safe environment and at an

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affordable rate. Through such collaboration and sharing of resources, it is envisaged that, in the foreseeable future, Asian countries will be able to throw away their crutches and become self-reliant.

Conclusion

Emerging infectious diseases will continue to pose a threat to global health and Asia is likely to be the epicenter of some of these diseases. It is important to develop and maintain a good surveillance programme in the Asian region so that preventive actions can be taken immediately to nip the problem at source. Resources to counter emerging diseases must be made available in real-time before the problem becomes global due to the convergence of many risk factors encouraging their spread. It will therefore be in the interest of global partners to assist the region in capacity building so that epidemics are curbed rapidly. In this respect, it is seen that international collaboration and partnership is the cornerstone to mount a successful programme. The proposal to set up an ASEAN Center for Disease Control deserves support as this will further enhance and strengthen equal partnerships. Regional experts should be considered to play a frontline role in the on-going battle against emerging diseases.

References
1 2 3 4 Fauci AS, Touchette NA, Folkers GK: Emerging infectious diseases: a 10-year perspective from the National Institute of Allergy and Infectious Diseases EID 2005;11:519525. Poliomyelitis Indonesia: ProMED Digest, 8 August 2005; Vol. 2005; No. 343. Chan PK: Outbreak of avian influenza A (H5N1) in Hong Kong in 1997. Clin Infect Dis 2002;34:S58S64. Keawcgaroen J, Oraveerakul K, Kuiken T, Ron AM, Fouchier Alongkorn Amonsin, Sunchai Payungporn, Suwanna Noppornpanth, Sumitra Wattanodorn, Apiradee Theamboonlers, Rachod Tantilertcharoen, Rattapan Pattanarangsan, Nlin Arya, Parntep Ratanakorn, Albert DME, Osterhaus, Yong Poovorawan: Avian influenza H5N1 in tigers and leopards. EID 2004;10:21892191. Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, Lim W, Nicholls J, Yee WKS, Yan WW, Cheung MT, Cheng VCC, Chan KH, Tsang DNC, Yung RWH, Ng TK, Yuen KY, and members of the SARS study group: Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361:13191325. Kaw Bing Chua, Khean Jin Goh, Kum Thong Wong, Adeeba Kamarulzaman, Patrick Seow Koon Tan, Thomas Ksiazek, Sherif R. Zaki, George Paul, Sai Kit Lam, Chong Tin Tan: Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999;354:12571259. Chua KB, Chua BH, Wang CW: Anthropogenic deforestation, El Nino and the emergence of Nipah virus in Malaysia. Malay J Pathol 2002;24:1521. Vincent P. Hsu, Mohammed Jahangir Hossain, Umesh D. Parashar, Mohammed Monsur Ali, Thomas G. Ksiazek, Ivan Kuzmin, Michael Niezgoda, Charles Rupprecht, Joseph Bresse, Robert F. Breiman: Nipah virus encephalitis re-emergence, Bangladesh. EID 2004;10:20822086. Lum LS, Wong KT, Lam SK, Chua KB, Goh AYT, Lim WL, Ong BB, Paul G, AbuBakar S, Lambert M: Fatal enterovirus 71 encephalomyelitis. J Pediatr 1998;133:795798.

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10

11 12 13 14

15 16

Nagy G, Takatsy S, Kukan E, Mihaly I, Domok I: Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS diseases in Hungary in 1978. Arch Virol 1982;71:217227. Liu CC, Tseng HW, Wang SM, Wang JR, Su IJ: An outbreak of enterovirus 71 infection in Taiwan, 1998: epidemiological and clinical manifestations. J Clin Virol 2000;17:2330. Smolinski MS, Hamburg MA, Lederberg J (eds): Microbial Threats To Health: Emergence, Detection and Response. Washington, National Academies Press, 2003. Lam SK: Review on dengue haemorrhagic fever. Rev Med Microbiol 1995;6:3948. Lam SK, Chua KB, Hooi PS, Rahimah MA, Kumari S, Tharmaratnam M, Chuah SK, Smith DW, Simpson IA: Chikungunya infection: An emerging disease in Malaysia. Southeast Asian J Trop Med Trop Health 2001;32:447451. Ngeow Yun Fong, Ng Kee Peng, Savithiri Devi Puthucheary, Lam Sai Kit: Health problems of foreign workers: microbiological investigations. JUNMEC 2002;1:6769. Balicer RD, Huerta M, Davidovitch N, Grotto I: Cost-benefit of stockpiling drugs for influenza pandemic. EID 2005;11:12801282.

Prof. S.K. Lam 5, Jalan SS20/23 Damansara Utama 47400 Petaling Jaya (Malaysia) Tel. 60 3 7722 1213, Fax 60 3 77259635, E-Mail sklam@nipahvirus.org

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Author Index

Auewarakul, P. 23 Aungtragoolsuk, N. 23 Bai, X. 35 Buranathai, C. 23 Chotpithayasunondh, T. 23 Chow, V.T.K. 59 Fielding, B.C. 1 Heymann, D.L. 112 Kitphati, R. 23

Lal, S.K. VII, 59 Lam, S.K. 136 Lidbury, B.A. 94 Lin, X. 35 Ma, G. 35 Mahalingam, S. 94 Matthaei, K.I. 94 McCormack, J. 78 Nicholls, J. 125

Ramirez, R. 94 Rulli, N.E. 94 Smith, G. 78 Tan, Y.-J. 1 Thomas, S. 94 Tupanceska, D. 94 Wang, M. 35 Zaid, A. 94

Peiris, J.S.M. 125 Puthavathana, P. 23

144

Subject Index

Acquired immunodeficiency syndrome (AIDS) economic factors 119 Malaysia 139 origins 113 Alphavirus, see Ross River virus Amantadine, influenza H5N1 management and resistance 28, 72 Antibiotics farm use 139 prescription behavior impact on disease emergence and re-emergence 114, 115 ASEAN Center for Disease Control (ACDC), establishment 141, 142 Avian influenza, see Influenza H5N1 Biosecurity laboratory, Asia needs 141 Channel catfish virus (CCV) clinical signs 38 culture 38 prevention 39 structure 37, 38 transmission 38, 39 Cholera, epidemiology 114 Climate change, impact on disease emergence and re-emergence 116, 138, 139 C protein, Paramyxovirus 82, 83 Deforestation, impact on disease emergence and re-emergence 115, 116 Diarrhea, economic factors 118

Ebola virus, origins 116 Economic factors, infectious disease influences 117121, 138 Enterovirus 71, epidemiology 137, 138 Environment, impact on disease emergence and re-emergence 115117, 138, 139 Enzyme-linked immunosorbent assay (ELISA) Henipavirus diagnostics 86 severe acute respiratory syndrome diagnostics 6, 7 Farming practices, infectious disease impact 139 Fish channel catfish virus clinical signs 38 culture 38 prevention 39 structure 37, 38 transmission 38, 39 infectious hematopoietic necrosis virus clinical signs 40 genome 39, 40 prevention 40, 41 structure 39 transmission 40 infectious pancreatic necrosis virus clinical signs 36 genome 36 prevention 37 structure 36 transmission 36, 37
145

Fish (continued) infectious salmon anemia virus clinical signs 42 features 41, 42 prevention 42, 43 transmission 42 lymphocystis disease virus clinical signs 44 genome 43, 44 prevention 45 structure 43 transmission 44 Formalin fixation 129, 130 paraffin-embedded tissue nucleic acid extraction 131 F protein, Paramyxovirus 83 G gene, Paramyxovirus 84 Globalization, infectious disease impact 119, 121 Global Outbreak Alert and Response Network (GOARN), functions 122 Hemagglutinin, influenza H5N1 63, 64 Hendra virus clinical features 89, 90 C protein 82, 83 diagnostics reverse transcription-polymerase chain reaction 85 sample handling 84, 85 serology 86 virus isolation 85 epidemiology 8688 F protein 83 genome 80, 81 G gene 84 infection treatment 90 morphology 80 M protein 83 N protein 81 origins 78 P protein 81, 82 RNA polymerase 84 taxonomy 79 transmission 89

Henipaviruses, see Hendra virus; Nipah virus Hepatitis, Malaysia 139 Infectious hematopoietic necrosis virus (IHNV) clinical signs 40 genome 39, 40 prevention 40, 41 structure 39 transmission 40 Infectious hypodermal and hematopoietic necrosis virus (IHHNV) clinical signs 52, 53 epidemiology 53 genome 52 prevention 53, 54 transmission 53 Infectious pancreatic necrosis virus (IPNV) clinical signs 36 genome 36 prevention 37 structure 36 transmission 36, 37 Infectious salmon anemia virus (ISAV) clinical signs 42 features 41, 42 prevention 42, 43 transmission 42 Influenza H5N1 antigenic drift 68 antigenic shift 68 antiviral drugs 28, 72, 73 ducks as pandemic threat 32 epidemiology avian influenza viruses 24 global 63 Thailand outbreaks 2426, 6163 genome 63 genotype evolution 69 origins 26, 27 pandemic preparation 140 pathogenesis 30, 69, 70 proteins hemagglutinin 63, 64 M1 protein 64 M2 protein 64 neuraminidase 64

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NS proteins 65 nucleoprotein 64, 65 polymerases 65 reverse transcription-polymerase chain reaction diagnostics 73 sequencing 26 surveillance 74, 140 Thailand animal infection pig 31, 32, 67, 68 poultry and birds 31, 66, 67 tiger 31, 67 human infection clinical manifestations 29, 30 demographic data of patients 28 source of infection 28, 29, 61, 62 vaccination approaches 70, 71 challenges 72 nucleic acid vaccines 71 reverse genetics 71 virulence 27 Influenza virus classification 60 environment and interspecies transmission 117 epidemiology avian influenza viruses 24 pandemics 5961 influenza A subtypes 23, 24 receptors 65, 66 replication and expression 66 Spanish influenza 60, 61 zoonosis 66, 67 Interferons Ross River virus response 107 types 107 Lung, biopsy 132 Lymphocystis disease virus (LCDV) clinical signs 44 genome 43, 44 prevention 45 structure 43 transmission 44

Marburg virus, origins 116 Monocyte chemoattractant protein-1 (MCP-1), Ross River virus response 106, 107 Mosquito, breeding and disease 116, 139 M protein, Paramyxovirus 83 M1 protein, influenza H5N1 64 M2 protein, influenza H5N1 64 Neuraminidase, influenza H5N1 64 Nipah virus clinical features 90 C protein 82, 83 diagnostics reverse transcription-polymerase chain reaction 85 sample handling 84, 85 serology 86 virus isolation 85 epidemiology 88, 89, 137 F protein 83 genome 80, 81 G gene 84 morphology 80 M protein 83 N protein 81 origins 78 P protein 81, 82 RNA polymerase 84 taxonomy 79 transmission 89 treatment 90 N protein paramyxovirus 81 severe acute respiratory syndrome coronavirus 12, 13 NS genes, influenza H5N1 proteins 65 virulence 27 Nucleoprotein, influenza H5N1 64, 65 Oseltamir, influenza H5N1 management 28 Pathology laboratory Asia status of pathology 127, 129 biopsy 132, 133 fixation of material 129132

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Pathology laboratory (continued) materials for examination 128 severe acute respiratory syndrome role 125127 Polymerase chain reaction, see Reverse transcription-polymerase chain reaction P protein, Paramyxovirus 81, 82 Replicase, severe acute respiratory syndrome coronavirus 911 Reverse transcription-polymerase chain reaction (RT-PCR) fixation considerations 130, 131 Henipavirus diagnostics 85 influenza H5N1 diagnostics 73 severe acute respiratory syndrome diagnostics 8 Ribavirin, Henipavirus management 90 Rimantadine, influenza H5N1 management and resistance 28, 72 RNA polymerase, Paramyxovirus 84 Ross River virus (RRV) antibody-dependent enhancement of infection 102104 clinical manifestations 98, 99 epidemiology 97, 98, 108 genome 96, 97 history of study 97, 98 host proteins in infection interferons 107 monocyte chemoattractant protein-1 106, 107 host range 99, 100 immune response 100, 101 latency and persistence 101, 102 structure 95, 96 taxonomy 94, 95 transmission 97 Seafood, see Fish; Shrimp Severe acute respiratory syndrome (SARS) antiviral drugs 6 collaborations for study 16 coronavirus cultivation in cell culture 46

genome features 8, 9 proteins group-specific proteins and functions 1315 N protein 12, 13 replicase 911 S protein 11, 12 sequencing 2 diagnostics antibody detection 68 biopsy 132, 133 pathology laboratory 125127 RNA detection 8 economic factors 119, 120 epidemiology reservoir 15, 16 Singapore 24, 15 spread 1, 2 gene expression profiles 5 origins 1, 15, 137 Shrimp infectious hypodermal and hematopoietic necrosis virus clinical signs 52, 53 epidemiology 53 genome 52 prevention 53, 54 transmission 53 taura syndrome virus clinical signs 54 epidemiology 54 genome 54 prevention 55 transmission 54, 55 white spots syndrome virus clinical signs 45 genome 49, 50 hosts 45 prevention 50 structure 48 transmission 50 vaccine 50, 51 yellow-head virus clinical signs 51 epidemiology 51 genome 51 prevention 52

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transmission 51, 52 S protein, severe acute respiratory syndrome coronavirus 11, 12 Taura syndrome virus (TSV) clinical signs 54 epidemiology 54 genome 54 prevention 55 transmission 54, 55 Tourism, impact on disease emergence and re-emergence 115 Tuberculosis, economic factors 118 White spots syndrome virus (WSSV) clinical signs 45

genome 49, 50 hosts 45 prevention 50 structure 48 transmission 50 vaccine 50, 51 Yellow-head virus (YHV) clinical signs 51 epidemiology 51 genome 51 prevention 52 transmission 51, 52 Zanamivir, influenza H5N1 management 72, 73

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