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Pigs are recognized as important 'mixing vessels' in which influenza viruses can undergo reassortment, but despite the resulting threat to human health there has been a general lack of adequate virus surveillance in pig populations. Malik Peiris and colleagues present a large data set of hundreds of swine influenza isolates and swine sera collected over 12 years of systematic surveillance in southern China, supplemented with samples stretching back 34 years. The data set provides a detailed picture of the evolutionary dynamics of swine influenza in this region. Swine influenza A viruses (SwIV) cause significant economic losses in animal husbandry as well as instances of human disease1 and occasionally give rise to human pandemics2, including that caused by the H1N1/2009 virus3,4. The lack of systematic and longitudinal influenza surveillance in pigs has hampered attempts to reconstruct the origins of this pandemic4. Most existing swine data were derived from opportunistic samples collected from diseased pigs in disparate geographical regions, not from prospective studies in defined locations, hence the evolutionary and transmission dynamics of SwIV are poorly understood. Here we quantify the epidemiological, genetic and antigenic dynamics of SwIV in Hong Kong using a data set of more than 650 SwIV isolates and more than 800 swine sera from 12 years of systematic surveillance in this region, supplemented with data stretching back 34 years. Intercontinental virus movement has led to reassortment and lineage replacement, creating an antigenically and genetically diverse virus population whose dynamics are quantitatively different from those previously observed for human influenza viruses. Our findings indicate that increased antigenic drift is associated with reassortment events and offer insights into the emergence of influenza viruses with epidemic potential in swine and humans.

Besides enormous economic losses to the poultry industry, recent H5N1 highly pathogenic avian influenza viruses (HPAIVs) originating in eastern Asia have posed serious threats to public health. Up to April 17, 2008, 381 human cases had been confirmed with a mortality of more than 60 %. Here, we attempt to identify potential progenitor genes for H5N1 HPAIVs since their first recognition in 1996; most were detected in the Eurasian landmass before 1996. Combinations among these progenitor genes generated at least 21 reassortants (named H5N1 progenitor reassortant, H5N1-PR1–21). H5N1-PR1 includes A/Goose/Guangdong/1/1996(H5N1). Only reassortants H5N1-PR2 and H5N1-PR7 were associated with confirmed human cases: H5N1-PR2 in the Hong Kong H5N1 outbreak in 1997 and H5N1-PR7 in laboratory confirmed human cases since 2003. H5N1-PR7 also contains a majority of the H5N1 viruses causing avian influenza outbreaks in birds, including the first wave of genotype Z, Qinghai-like and Fujian-like virus lineages. Among the 21 reassortants identified, 13 are first reported here. This study illustrates evolutionary patterns of H5N1 HPAIVs, which may be useful toward pandemic preparedness as well as avian influenza prevention and control.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.

For the prediction of future influenza pandemics, global surveillance of avian influenza has been continuing since 1991 and carried out in Russia, Mongolia, China and Japan from 2000 to 2003. Influenza virus isolates of 50 combinations of HA and NA subtypes have been identified and 3 strains selected from each of those are stocked. In addition, 47 other combinations have been generated by standard genetic reassortment procedure in the laboratory. Since we have already shown that influenza viruses have been fully adapted to ducks and cause no disease signs and are in evolutionary stasis in their natural reservoirs, virus isolates from ducks are ideal as vaccine strains. Thus, influenza viruses of 97 combinations of HA and NA subtypes are now available as vaccine strain candidates against emerging pandemic influenza in humans, domestic animals and poultry.

The eminent sinologist and historian, Joseph Needham, writing with his colleague Lu Gwei-Djen in the sixth volume of his mighty tome “Science and Civilization in China,” emphasized the long held Chinese conviction that “the best medicine is preventive medicine,” a conviction that goes back to the Warring States period (475–221 B.C.) when there was a flowering of philosophy and medical understanding. A Han Dynasty (206 B.C.–220 A.D.) writing concludes “A skillful doctor cures illness when there is no sign of disease, and thus the disease never comes” (1, 2). This component of the “Chinese Hippocratic Corpus,” as Needham calls it, holds just as true in today’s modern world and may be simply summed-up in one word: “awareness.” In the case of emerging infectious disease, this means “preparedness,” giving rise to “reaction.” Two thousand or so years onward in the China of today, this conviction is being sorely tested by nature with incursions of respiratory viruses from its southeastern province of Guangdong adjacent to Hong Kong. In 1997, the world was thrown into a state of alarm and high alert arising from the emergence of a potentially new influenza pandemic caused by the transmission of a highly pathogenic H5N1 influenza virus (H5N1/97) of avian origin from chickens to humans in Hong Kong (3–5). On that occasion, a pandemic was only one or two mutational events away. H5N1-like viruses were recognized in chicken and other land-based poultry in Hong Kong in 2001 and 2002 before they could infect humans (6, 7). Probably three or four mutational events from generating a pandemic influenza virus, these more recent incidents upheld the ancient ideal of preventing disease before it appears. In these cases the virus did not “escape” from Hong Kong almost certainly because of its having had sound human and animal disease and virus surveillance systems in place for some time. This led to the recognition of the source of the virus and its removal by the slaughter of poultry in live markets and on farms, or a combination of slaughter and ring vaccination on farms (6–8). Over the years, Hong Kong essentially functioned as an influenza sentinel post for the wider region, stemming from the

In 1997, a high-pathogenicity H5N1 avian influenza virus caused serious disease in both man and poultry in Hong Kong, China. Eighteen human cases of disease were recorded, six of which were fatal. This unique virus was eliminated through total depopulation of all poultry markets and chicken farms in December 1997. Other outbreaks of high-pathogenicity avian influenza (HPAI) caused by H5N1 viruses occurred in poultry in 2001 and 2002. These H5N1 viruses isolated had different internal gene constellations to those isolated in 1997. No new cases of infection or disease in man due to these or other H5N1 viruses have been reported. This paper provides an overview and chronology of the events in Hong Kong relating to avian influenza, covering the period from March 1997 to March 2002.

Retail live poultry markets (LPMs) may act as a reservoir of avian influenza viruses (AIV). In this study we test the hypothesis that a rest day in the LPMs where the stalls are completely emptied of poultry, cleansed, and restocked will reduce the isolation rates of avian influenza viruses. The isolation rate of H9N2 subtype viruses from chicken was significantly lower after the rest day than prior to it, indicating its impact in reducing transmission. In contrast, Newcastle disease virus (NDV) isolation rates appear unaffected by this intervention, possibly reflecting differences in herd immunity or virus transmission dynamics.

Pandemic influenza is a zoonosis. Studies on influenza ecology conducted in Hong Kong since the 1970s in which Hong Kong essentially functioned as an influenza sentinel post indicated that it might be possible, for the first time, to have influenza preparedness at the baseline avian level. This appreciation of influenza ecology facilitated recognition of the H5N1 'bird flu' incident in Hong Kong in 1997 in what was considered to be an incipient pandemic situation, the chicken being the source of virus for humans and, if so, was the first instance where a pandemic may have been averted. The 2001 and 2002 H5N1 incidents demonstrated that it was possible to have an even higher order of baseline preparedness with the recognition in chicken of a range of genotypes of H5N1-like viruses before they had the opportunity to infect humans. Investigations of these incidents revealed a complex ecology involving variously precursor avian H5N1 virus in geese and ducks, and H9N2 and H6N1 viruses in quail, the quail possibly functioning as an avian 'mixing vessel' for key genetic reassortment events for onward transmission of H5N1 viruses highly pathogenic for chicken and humans. These findings highlight the importance of systematic virus surveillance of domestic poultry in recognizing changes in virus occurrence, host range and pathogenicity as signals at the avian level that could presage a pandemic. For example, there is now an increasing prevalence of avian influenza viruses in terrestrial (in contrast to aquatic) poultry. Prior to 1997, no particular virus subtype other than H4N6 would have been considered a candidate for pandemicity and this was based, in the absence of any other data, on its high frequency of occurrence in ducks in southern China. Now, with the isolation of H5N1 and H9N2 viruses from humans supported by genetic, molecular and biological studies on these and other avian isolates, there is credible evidence for the candidacy, in order, of H5N1, H9N2 and H6N1 viruses. These viruses have been made available for the production of diagnostic reagents and exploratory vaccines. The 1997 incident upheld the hypothesis that southern China is an epicentre for the emergence of pandemic influenza viruses. However, the intensification of the poultry (chicken) industry worldwide coupled with the spread of viruses such as the Eurasian lineage of H9N2 suggest that the genesis of a pandemic could take place elsewhere in the world. This re-emphasizes the importance of systematic virus surveillance of poultry globally for international public health and for economic and food concerns. Faced with an incipient pandemic in 1997, Hong Kong brought in international experts to join the investigative effort. Good teamwork at all levels is essential in dealing with the many facets. The threat of a pandemic should not be minimized, nor should governments be lulled into a sense of false security. The media is a powerful channel and has the responsibility and the avenues to convey and influence public perception of events. Close liaison between the media and those on the operational side ensures effective, accurate and timely dissemination of information. This will enhance public confidence in the investigative process and in steps taken for its safety and health.

Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the "bird flu" incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern., published_or_final_version

The H5N1 viruses (H5N1/97) associated with the “bird-flu” incident in the Hong Kong SAR have not been isolated since the slaughter of poultry in December 1997 brought that outbreak to an end. Recent evidence points to this virus as having arisen through a reassortment of a number of precursor avian viruses and a virus related to Goose/Guangdong/1/96 (H5N1) (Gs/Gd/96) was the likely donor of the H5 hemagglutinin. We characterize the Goose/Guangdong/1/96-like viruses isolated from geese and ducks imported into Hong Kong in the year 2000. Antigenically and genetically, these recent H5N1 viruses fall into two groups, one mainly associated with geese, and the other, recently transmitted to ducks. Further, viruses isolated from a goose and a duck in December 2000 have acquired NS, PA, M, and PB2 genes from the aquatic avian influenza gene pool through reassortment. For pandemic preparedness, it is important to monitor whether these reassortant viruses have the capacity for interspecies transmission to terrestrial poultry or mammals.

Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, People's Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs and documents their cocirculation with contemporary human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the porcine H9N2 viruses were closely related to viruses similar to chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants of the latter virus lineage. Phylogenetic analysis suggested that repeated interspecies transmission events had occurred from the avian host to pigs. The Sydney97-like (H3N2) viruses isolated from pigs were related closely to contemporary human H3N2 viruses in all gene segments and had not undergone genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs provides an opportunity for genetic reassortment leading to the emergence of viruses with pandemic potential., published_or_final_version

Background: The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1/97-like) internal genome complex is still present in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Methods: Virus isolation, serological identification and genetic analysis. Results: Two lineages of H9N2 influenza viruses were isolated in the live poultry markets and are represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97) and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97). No reassortment between the two H9N2 virus lineages was detected despite their co-circulation in the poultry markets. The Qa/HK/G1/97-like viruses were isolated most frequently from quail and Dk/HK/Y280/97-like viruses from chicken. Conclusion: The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to H5N1/97-like virus emphasizes the need for continued surveillance of mammals including humans for the H9N2 virus.

Background : Pigs may be an intermediate host for the adaptation of avian influenza viruses to humans. Avian influenza viruses of multiple lineages, including the precursors of the H5N1 causing the avian flu incident in Hong Kong in 1997 (H5N1/97), are now established in geese and land-based poultry in southern China. It is important to examine whether these avian viruses have undergone inter-species transmission to pigs. Methods : Serological and virological surveillance of influenza viruses were carried out in pigs from southern China between 1998 and 2000. Antigenic and molecular characterisation of the virus isolates was done. Results : One hundred and ninety influenza A viruses of subtypes H9N2 ( n =4), H3N2 (A/Sydney/97-like) ( n =13), H3N2 (A/Victoria/75-like) ( n =15), and H1N1 (classical swine-like) ( n =158) were isolated. Seroepidemiology provided independent confirmation of the circulation of each of these viruses. Molecular analysis of all eight gene segments confirmed that the H9N2 viruses belonged to the Duck/HK/Y280/97 lineage. Similarly, the H3N2 Sydney-like viruses were genetically highly homologous to viruses currently circulating in the human population. Conclusions : H9N2 subtype viruses co-circulate with contemporary human A/Sydney/97-like viruses and other porcine H1N1 and H3N2 viruses in pigs in southern China, and the potential for the emergence of viruses of pandemic potential needs to be considered.

The majority of influenza A viruses isolated from wild birds, but not humans, can replicate in the duck intestinal tract. Here we demonstrate that all duck isolates tested universally retain sialidase activities under low pH conditions independent of their neuraminidase (NA) subtypes. In contrast, the sialidase activities of most isolates from humans and pigs practically disappear below pH 4.5, with the exception of four human pandemic viruses isolated in 1957 and 1968. Sequence comparisons among duck, human, and swine N2 NA subtypes indicate that amino acids at positions 153, 253, 307, 329, 344, 347, 356, 368, 390, and 431 may be associated with the low pH stability of duck and human pandemic N2 NAs. This finding suggests that the low pH stability of duck influenza A virus NA may be a critical factor for replication in the intestinal tract through the digestive tract of ducks, and that the properties of NAs are important for understanding the epidemiology of the influenza virus.

This account takes stock of events and involvements, particularly on the avian side of the influenza H5N1 'bird flu' incident in Hong Kong SAR in 1997. It highlights the role of the chicken in the many live poultry markets as the source of the virus for humans. The slaughter of chicken and other poultry across the SAR seemingly averted an influenza pandemic. This perspective from Hong Kong SAR marks the coming-of-age of acceptance of the role of avian hosts as a source of pandemic human influenza viruses and offers the prospect of providing a good baseline for influenza pandemic preparedness in the future. Improved surveillance is the key. This is illustrated through the H9N2 virus which appears to have provided the 'replicating' genes for the H5N1 virus and which has since been isolated in the SAR from poultry, pigs and humans highlighting its propensity for interspecies transmission.

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.

Summary Background Human infection with an avian influenza A virus (subtype H5N1) was reported recently in Hong Kong. We describe the clinical presentation of the first 12 patients and options for rapid viral diagnosis. Methods Case notes of 12 patients with virus-culture-confirmed influenza A H5N1 infection were analysed. The clinical presentation and risk factors associated with severe disease were defined and the results of methods for rapid virus diagnosis were compared. Findings Patients ranged from 1 to 60 years of age. Clinical presentation was that of an influenza-like illness with evidence of pneumonia in seven patients. All seven patients older than 13 years had severe disease (four deaths), whereas children 5 years or younger had mild symptoms with the exception of one who died with Reye's syndrome associated with intake of aspirin. Gastrointestinal manifestations, raised liver enzymes, renal failure unrelated to rhabdomyolysis, and pancytopenia were unusually prominent. Factors associated with severe disease included older age, delay in hospitalisation, lower-respiratory-tract involvement, and a low total peripheral white blood cell count or lymphopenia at admission. An H5-specific reverse-transcription PCR assay (RT-PCR) was useful for rapid detection of virus directly in respiratory specimens. A commercially available enzyme immunoassay was more sensitive than direct immunofluorescence for rapid viral diagnosis. Direct immunofluorescence with an H5-specific monoclonal antibody pool was useful for rapid exclusion of H5-subtype infection. Interpretation Avian Influenza A H5N1 virus causes human influenza-like illness with a high rate of complications in adults admitted to hospital. Rapid H5-subtype-specific laboratory diagnosis can be made by RT-PCR applied directly to clinical specimens.

Summary Background In May, 1997, a 3-year-old boy in Hong Kong was admitted to the hospital and subsequently died from influenza pneumonia, acute respiratory distress syndrome, Reye’s syndrome, multiorgan failure, and disseminated intravascular coagulation. An influenza A H5N1 virus was isolated from a tracheal aspirate of the boy. Preceding this incident, avian influenza outbreaks of high mortality were reported from three chicken farms in Hong Kong, and the virus involved was also found to be of the H5 subtype. Methods We carried out an antigenic and molecular comparison of the influenza A H5N1 virus isolated from the boy with one of the viruses isolated from outbreaks of avian influenza by haemagglutination-inhibition and neuraminidase-inhibition assays and nucleotide sequence analysis. Findings Differences were observed in the antigenic reactivities of the viruses by the haemagglutinationinhibition assay. However, nucleotide sequence analysis of all gene segments revealed that the human virus A/Hong Kong/156/97 was genetically closely related to the avian A/chicken/Hong Kong/258/97. Interpretation Although direct contact between the sick child and affected chickens has not been established, our results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a “mixing vessel”. This event illustrates the importance of intensive global influenza surveillance.

The human influenza pandemics of 1957 and 1968 were caused by reassortant viruses that possessed internal gene segments from avian and human strains. Whether genetic reassortment of human and avian influenza viruses occurs during interpandemic periods and how often humans are infected with such reassortants is not known. To provide this information, we used dot-blot hybridization, partial nucleotide sequencing and subsequent phylogenetic analysis to examine the 6 internal genes of 122 viruses isolated in humans between 1933 and 1992 primarily from Asia, Europe, and the Americas. The internal genes of A/New Jersey/11/76 isolated from a human fatality at Fort Dix, New Jersey in 1976 were found to be of porcine origin. Although none of the geographically and temporally diverse collection of 122 viruses was an avian-human or other reassortant, cognizance was made of the fact that there were two isolates from children from amongst 546 influenza A isolates obtained from The Netherlands from 1989-1994 which were influenza A reassortants containing genes of avian origin, viruses which have infected European pigs since 1983-1985. Thus, genetic reassortment between avian and human influenza strains does occur in the emergence of pandemic and interpandemic influenza A viruses. However, in the interpandemic periods the reassortants have no survival advantage, and the circulating interpandemic influenza viruses in humans do not appear to accumulate avian influenza virus genes.

The nucleotide sequences of the HA1 domain of the H1 hemagglutinin genes of A/duck/Hong Kong/36/76, A/duck/Hong Kong/196/77, A/sw/North Ireland/38, A/sw/Cambridge/39 and A/Yamagata/120/86 viruses were determined, and their evolutionary relationships were compared with those of previously sequenced hemagglutinin (H1) genes from avian, swine and human influenza viruses. A pairwise comparison of the nucleotide sequences revealed that the genes can be segregated into three groups, the avian, swine and human virus groups. With the exception of two swine strains isolated in the 1930s, a high degree of nucleotide sequence homology exists within the group. Two phylogenetic trees constructed from the substitutions at the synonymous site and the third codon position showed that the H1 hemagglutinin genes can be divided into three host-specific lineages. Examination of 21 hemagglutinin genes from the human and swine viruses revealed that two distinct lineages are present in the swine population. The swine strains, sw/North Ireland/38 and sw/Cambridge/39, are clearly on the human lineage, suggesting that they originate from a human A/WSN/33-like variant. However, the classic swine strain, sw/Iowa/15/30, and the contemporary human viruses are not direct descendants of the 1918 human pandemic strain, but did diverge from a common ancestral virus around 1905. Furthermore, previous to this the above mammalian viruses diverged from the lineage containing the avian viruses at about 1880.

Although Southern China has been considered the epicenter of human influenza pandemics, little is known about the genetic composition of influenza viruses in lower mammals or birds in that region. To provide information on the molecular epidemiology of these viruses, we used dot blot hybridization and phylogenetic methods to study the internal genes (PB1, PB2, PA, NP, M, and NS) of 106 avian influenza A viruses isolated from a total of 11,798 domestic ducks, chickens, and geese raised in Southern China including Hong Kong. All 636 genes examined were characteristic of avian influenza viruses; no human or swine influenza genes were detected. Thus, influenza virus reassortants do not appear to be maintained in the domesticated birds of Southeast Asia, eliminating opportunities for further gene reassortment. Phylogenetic analysis showed that the internal genes of these viruses belong to the Eurasian avian lineage, supporting geographical separation of the major avian lineages. The PB1 genes were most similar to A/Singapore/57 (H2N2) and Hong Kong (H3N2) viral genes, supporting an avian origin for the recent human H2N2 and H3N2 pandemic strains. The majority of internal genes from avian influenza viruses in Southern China belong to the Eurasian lineage and are similar to viruses that have recently been transmitted to humans, swine, and horses. This study provides evidence that the transmission of avian influenza viruses and their genes to other species is unidirectional and that the transmission of mammalian influenza virus strains to domestic poultry is probably not a factor in the generation of new pandemic strains.

Influenza viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1999. Phylogenetic analysis of the NP genes of the isolates in Siberia and Japan on their flyway of migration to the south indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virus in Hong Kong. Inactivated vaccine prepared from an avirulent H5N4 virus isolated from a migratory duck in Japan showed enough potency to induce protective immunity against the pathogenic H5N1 virus in mice. To expand surveillance for influenza viruses in migratory and domestic ducks and geese, chickens, quail, and pigs of the world and be informed about what influenza viruses are dominant in the animal reservoirs, we have started the “Programme of Excellence in Influenza”. Influenza virus strains isolated from animal species in the surveillance should be thoroughly characterized, stored, and be provided for the use of diagnosis and vaccine preparation for the control of future pandemics.

Influenza virus surveillance was conducted on wild ducks and shorebirds in Hong Kong at the Mai Po Nature Reserve to determine whether East Asian wild aquatic birds passing through or overwintering in Hong Kong are reservoirs of H5N1 influenza viruses and to establish an epidemiological baseline of influenza virus in wild aquatic birds during the pre-H5N1 endemic era. Three influenza viruses were isolated from 3178 faecal samples collected over three sampling periods from 1988 to 2001 during the southern and northern migration periods. The isolation rates and viruses were, respectively 0.08% (H10N5) in 1988 – 1990, 0.12% (H11N1) in 1998 and 0.09% (H3N8) in 1999 – 2001. Whereas tracheal and intestinal colon explants from representative shorebirds were susceptible to in vitro infection by the H10N5 virus, orally infected shorebirds were apparently not. Genetic analyses indicated that the nucleoprotein, matrix and nonstructural genes of the three viruses were related to those of aquatic bird viruses in Asia, but not to those of the human H5N1 virus. The present study provided epidemiological baseline information for future influenza virus surveillance in wild aquatic birds in southeast China.

Since it was first detected in 1996, the Goose/Guangdong/1/1996 (Gs/GD) H5N1 influenza virus and its reassortants have spread to over 60 countries, with over 20 distinct genetic reassortants previously recognized. However, systematic analysis of their interrelationship and the development of genetic diversity have not been explored. As each of those reassortants was first detected in China, here 318 full-length H5N1 virus genomes isolated from 1996 to 2006 in this region were phylogenetically analyzed. Our findings revealed two major group reassortment events in 2001 and 2002 that were responsible for the generation of the majority of the 44 distinct Gs/GD genotypes identified, excepting those 1997 variants. Genotype replacement and emergence occurred continually, with 34 transient genotypes detected while only 10 variants were persistent. Two major replacements of predominant genotypes were also observed: genotype B replaced by Z in 2002 and then genotype Z replaced by the now predominant genotype V in 2005.

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

An outbreak of highly pathogenic avian influenza caused by multiple genotypes of H5N1 virus occurred in Hong Kong, commencing in January 2002. Infection in local chicken farms was preceded by the detection of virus in multiple retail markets and the main poultry wholesale market. The first case of this disease on a local farm was detected on February 1, 2002. By February 9, 2002, 15 farms were infected, and by late March a total of 22 infected farms had been identified. Three main clusters of infected farms were seen, suggesting multiple incursions of virus, and subsequent limited lateral spread to neighboring firms. Control of this disease has been effected through a combination of quarantine, tightening of biosecurity measures, and depopulation of infected and contact farms. About 950,000 birds have been destroyed. Vaccination using a killed H5 vaccine was introduced in April 2002 to farms in one zone where infection has persisted. None of the viruses isolated contained the internal genes found in the 1997 H5N1 virus.

From February 2000 through September 2001, a limited number of H6N2 influenza viruses were isolated from chickens in California. This report describes the genetic characterization of nine of these H6N2 viruses. All of the viruses analyzed had phylogenetically similar hemagglutinin (HA) and neuraminidase molecules that suggested the viruses shared a recent common ancestor. The analysis of the HA sequence of these viruses with all available H6 viruses from different hosts and locations showed that these genes do not separate into well-defined North American and Eurasian lineages. The neuraminidase genes of the California viruses contain an 18 amino acid deletion, a possible adaptation to growth in chickens. Analysis of the remaining gene segments of the California viruses revealed that three distinct genotypes of H6N2 viruses were present.

The H5N1 virus (H5N1/97) that caused the bird flu incident in Hong Kong in 1997 has not been isolated since the poultry slaughter in late 1997. But the donor of its H5 hemagglutinin gene, Goose/Guangdong/1/96-like (Gs/Gd/96-like) virus, established a distinct lineage and continued to circulate in geese in the area. In 2000, a virus from the Goose/Guangdong/1/96 lineage was isolated for the first time from domestic ducks. Subsequently, it has undergone reassortment, and these novel reassortants now appear to have replaced Gs/Gd/96-like viruses from its reservoir in geese and from ducks. The internal gene constellation is also different from H5N1/97, but these variants have the potential for further reassortment events that may allow the interspecies transmission of the virus.

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known. Civet cats, a raccoon dog, and a ferret badger in an animal market in Gunagdong, China, were infected with a coronavirus identical to the one that causes SARS in humans save for an extra 29-nucleotide sequence. Civet cats, a raccoon dog, and a ferret badger in an animal market in Gunagdong, China, were infected with a coronavirus identical to the one that causes SARS in humans save for an extra 29-nucleotide sequence.

A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus., published_or_final_version

H9 influenza viruses have become endemic in land-based domestic poultry in Asia and have sporadically crossed to pigs and humans. To understand the molecular determinants of their adaptation to land-based birds, we tested the replication and transmission of several 1970s duck H9 viruses in chickens and quail. Quail were more susceptible than chickens to these viruses, and generation of recombinant H9 viruses by reverse genetics showed that changes in the HA gene are sufficient to initiate efficient replication and transmission in quail. Seven amino acid positions on the HA molecule corresponded to adaptation to land-based birds. In quail H9 viruses, the pattern of amino acids at these seven positions is intermediate between those of duck and chicken viruses; this fact may explain the susceptibility of quail to duck H9 viruses. Our findings suggest that quail provide an environment in which the adaptation of influenza viruses from ducks generates novel variants that can cross the species barrier., published_or_final_version

Summary Background In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. Methods We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor α (TNF α) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-α response. Findings The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF α and interferon beta. The concentration of TNF-α protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural ( NS ) gene-segment of H5N1/97 viruses contributed to the increase in TNF α induced by the virus. Interpretation The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF α. This characteristic may contribute to the unusual severity of human H5N1 disease.

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.

The H5N1 influenza virus, which killed humans and poultry in 1997, was a reassortant that possibly arose in one type of domestic poultry present in the live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are still circulating in poultry in southern China, the reassortment event that generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1; Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1 virus, we investigated the continued circulation, host range, and transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused weight loss and death in some mice inoculated with high virus doses. Transmission of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in infection of all birds but limited signs of overt disease. In contrast, oral inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to 50% of infected geese. Transmission from infected geese to chickens occurred only by fecal contact, whereas transmission to quail occurred by either aerosol or fecal spread. This difference is probably explained by the higher susceptibility of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N1/97 virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds, especially quail, supports the wisdom of separating aquatic and land-based poultry in the markets in Hong Kong and the need for continued surveillance in the field and live-bird markets in which different types of poultry are in contact with one another., published_or_final_version

The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple-basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-1ike H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?, published_or_final_version

In order to identify molecular changes associated with the transmission of avian influenza A H5N1 and H9N2 viruses to humans, the internal genes from these viruses were compared to sequences from other avian and human influenza A isolates. Phylogenetically, each of the internal genes of all sixteen of the human H5N1 and both of the H9N2 isolates were closely related to one another and fell into a distinct clade separate from clades formed by the same genes of other avian and human viruses. All six internal genes were most closely related to those of avian isolates circulating in Asia, indicating that reassortment with human strains had not occurred for any of these 18 isolates. Amino acids previously identified as host-specific residues were predominantly avian in the human isolates although most of the proteins also contained residues observed previously only in sequences of human influenza viruses. For the majority of the nonglycoprotein genes, three distinct subgroups could be distinguished on bootstrap analyses of the nucleotide sequences, suggesting multiple introductions of avian virus strains capable of infecting humans. The shared nonglycoprotein gene constellations of the human H5N1 and H9N2 isolates and their detection in avian isolates only since 1997 when the first human infections were detected suggest that this particular gene combination may confer the ability to infect humans and cause disease. J. Med. Virol. 66:107-114, 2002. Published 2002 Wiley-Liss, Inc.

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans., published_or_final_version

In 1997, 18 cases of influenza in Hong Kong (bird flu) caused by a novel H5N1 (chicken) virus resulted in the deaths of six individuals and once again raised the specter of a potentially devastating influenza pandemic. Slaughter of the poultry in the live bird markets removed the source of infection and no further human cases of H5N1 infection have occurred. In March 1999, however, a new pandemic threat appeared when influenza A H9N2 viruses infected two children in Hong Kong. These two virus isolates are similar to an H9N2 virus isolated from a quail in Hong Kong in late 1997. Although differing in their surface hemagglutinin and neuraminidase components, a notable feature of these H9N2 viruses is that the six genes encoding the internal components of the virus are similar to those of the 1997 H5N1 human and avian isolates. This common feature emphasizes the apparent propensity of avian viruses with this genetic complement to infect humans and highlights the potential for the emergence of a novel human pathogen., published_or_final_version

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized., published_or_final_version

The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of “internal” virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.

The origin of the H5N1 influenza viruses that killed six of eighteen infected humans in 1997 and were highly pathogenic in chickens has not been resolved. These H5N1 viruses transmitted directly to humans from infected poultry. In the poultry markets in Hong Kong, both H5N1 and H9N2 influenza viruses were cocirculating, raising the possibility of genetic reassortment. Here we analyze the antigenic and genetic features of H9N2 influenza viruses with different epidemiological backgrounds. The results suggest that the H9N2 influenza viruses of domestic ducks have become established in the domestic poultry of Asia. Phylogenetic and antigenic analyses of the H9N2 viruses isolated from Hong Kong markets suggest three distinct sublineages. Among the chicken H9N2 viruses, six of the gene segments were apparently derived from an earlier chicken H9N2 virus isolated in China, whereas the PB1 and PB2 genes are closely related to those of the H5N1 viruses and a quail H9N2 virus - A/quail/ Hong Kong/G1/97 (Qa/HK/G1/97) - suggesting that many of the 1997 chicken H9 isolates in the markets were reassortants. The similarity of the internal genes of Qa/HK/G1/97 virus to those of the H5N1 influenza viruses suggests that the quail virus may have been the internal gene donor. Our findings indicate that the human and poultry HSN1 influenza viruses in Hong Kong in 1997 were reassortants that obtained internal gene segments from Qa/HK/G1/97. However, we cannot be certain whether the replicate complex of H5N1 originated from Qa/HK/G1/97 or whether the reverse transfer occurred; the available evidence supports the former proposal., published_or_final_version

Influenza A viruses of subtype H9N2 are now considered to be widespread in poultry ([1][1]). They have recently been isolated from pigs originating from southern China ([5][2]) and from two children with influenza-like symptoms in Hong Kong ([2][3]). Crossing the species barrier to mammals

Equine-2 influenza virus A (H3N8) infection occurred among vaccinated thoroughbred horses in Hong Kong during November and December 1992. The outbreak was unique in that it occurred among a large population stabled under intensive conditions. It resulted in the postponement of seven race meetings over a period of 32 days. The outbreak originated after the importation of horses 25 to 32 days before any clinical signs were reported. Vaccination did not prevent 75 per cent of the population from becoming infected, and half the infected horses developed clinical signs. Vaccination did, however, contribute to reducing the morbidity and the severity and duration of the clinical signs.

From immunological and phylogenetic analyses of H3 influenza viruses isolated from pigs and ducks in the People's Republic of China (China), Hong Kong, Taiwan and Japan, between 1968 and 1982, we arrived at the following conclusions. The H3 haemagglutinin and N2 neuraminidase genes from swine isolates can be segregated into four mammalian lineages, including: (i) the earliest human strains; (ii) early swine strains including Hong Kong isolates from 1976–1977; (iii) an intermediate strain between the early swine and recent human strains; and (iv) recent human strains. In this study we found an unusual swine strain (sw/Hong Kong/127/82) belonging to the third lineage which behaved like those of the early swine-like lineage in the haemagglutination inhibition test; but neuraminidase inhibition profiles with monoclonal antibodies indicated that this virus is related to late human strains. On the basis of pairwise comparisons of complete or partial nucleotide sequences the genes encoding the three polymerase proteins (PB2, PB1, PA), the nucleoprotein, the membrane protein and possibly the nonstructural proteins of sw/Hong Kong/127/82 are of the swine H1N1 lineage, whereas genes encoding the two surface glycoproteins belong to the human H3N2 lineage. In contrast, all RNA segments of one swine isolate (sw/Hong Kong/81/78) are similar to those of recent human H3N2 viruses. This study indicated that frequent interspecies infections between human and swine hosts appeared to occur during 1976–82. Although the evolutionary rates of human (0.0122/site/year), swine (0.0127/site/year) and avian (0.0193/site/year) virus genes are similar when based upon synonymous substitutions, nonsynonymous substitutions indicated that viral genes derived from human and swine viruses evolved about three times faster (0.0026–0.0027/site/year) than those of avian viruses (0.0008/site/year). Furthermore, the evolutionary mechanism by which human and swine H3 haemagglutinin genes evolve at a similar rate, based on nonsynonymous substitutions, appeared to be quite different from previous evidence which showed that human H1 haemagglutinin genes evolved three times faster than those of swine viruses. However, comparison of the number of nonsynonymous substitutions in the antigenic sites (A–E) of haemagglutinin molecules demonstrated that swine viruses evolve at a rate that is about one fifth to one tenth that of human viruses, reflecting the conservative nature of the antigenic structure in the former.