Continuing evolution of H9N2 influenza viruses in Southeastern China

H9N2 influenza viruses are panzootic in domestic poultry in Eurasia and since 1999 have caused transient infections in humans and pigs. To investigate the zoonotic potential of H9N2 viruses, we studied the evolution of the viruses in live-poultry markets in Hong Kong in 2003. H9N2 was the most prevalent influenza virus subtype in the live-poultry markets between 2001 and 2003. Antigenic and phylogenetic analysis of hemagglutinin (HA) showed that all of the 19 isolates found except one belonged to the lineage represented by A/Duck/Hong Kong/Y280/97 (H9N2). The exception was A/Guinea fowl/NT184/03 (H9N2), whose HA is most closely related to that of the human isolate A/Guangzhou/333/99 (H9N2), a virus belonging to the A/Chicken/Beijing/1/94-like (H9N2) lineage. At least six different genotypes were recognized. The majority of the viruses had nonstructural (and HA) genes derived from the A/Duck/Hong Kong/Y280/97-like virus lineage but had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses isolated in 2001. Viruses of all six genotypes of H9N2 found were able to replicate in chickens and mice without adaptation. The infected chickens showed no signs of disease, but representatives of two viral genotypes were lethal to mice. Three genotypes of virus replicated in the respiratory tracts of swine, which shed virus for at least 5 days. These results show an increasing genetic and biologic diversity of H9N2 viruses in Hong Kong and support their potential role as pandemic influenza agents.     

The continued pandemic threat posed by avian influenza viruses in Hong Kong

In 1997, a highly pathogenic avian H5N1 influenza virus was transmitted directly from live commercial poultry to humans in Hong Kong. Of the 18 people infected, six died. The molecular basis for the high virulence of this virus in mice was found to involve an amino acid change in the PB2 protein. To eliminate the source of the pathogenic virus, all birds in the Hong Kong markets were slaughtered. In 1999, another avian influenza virus of H9N2 subtype was transmitted to two children in Hong Kong. In 2000-2002, H5N1 avian viruses reappeared in the poultry markets of Hong Kong, although they have not infected humans. Continued circulation of H5N1 and other avian viruses in Hong Kong raises the possibility of future human influenza outbreaks. Moreover, the acquisition of properties of human viruses by the avian viruses currently circulating in southeast China might result in a pandemic.     

Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.     

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.     

The Surface Glycoproteins of H5 Influenza Viruses Isolated from Humans, Chickens, and Wild Aquatic Birds Have Distinguishable Properties

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.     

Characterization of a human H5N1 influenza A virus isolated in 2003

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.     

Molecular Evolution of H6 Influenza Viruses from Poultry in Southeastern China: Prevalence of H6N1 Influenza Viruses Possessing Seven A/Hong Kong/156/97 (H5N1)-Like Genes in Poultry

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-like 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?     

Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat

Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.     

Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans

Since the outbreak in humans of an H5N1 avian influenza virus in Hong Kong in 1997, poultry entering the live-bird markets of Hong Kong have been closely monitored for infection with avian influenza. In March 1999, this monitoring system detected geese that were serologically positive for H5N1 avian influenza virus, but the birds were marketed before they could be sampled for virus. However, viral isolates were obtained by swabbing the cages that housed the geese. These samples, known collectively as A/Environment/Hong Kong/437/99 (A/Env/HK/437/99), contained four viral isolates, which were compared to the 1997 H5N1 Hong Kong isolates. Analysis of A/Env/HK/437/99 viruses revealed that the four isolates are nearly identical genetically and are most closely related to A/Goose/Guangdong/1/96. These isolates and the 1997 H5N1 Hong Kong viruses encode common hemagglutinin (H5) genes that have identical hemagglutinin cleavage sites. Thus, the pathogenicity of the A/Env/HK/437/99 viruses was compared in chickens and in mice to evaluate the potential for disease outbreaks in poultry and humans. The A/Env/HK/437/99 isolates were highly pathogenic in chickens but caused a longer mean death time and had altered cell tropism compared to A/Hong Kong/156/97 (A/HK/156/97). Like A/HK/156/97, the A/Env/HK/437/99 viruses replicated in mice and remained localized to the respiratory tract. However, the A/Env/HK/437/99 isolates caused only mild pathological lesions in these tissues and no clinical signs of disease or death. As a measure of the immune response to these viruses, transforming growth factor beta levels were determined in the serum of infected mice and showed elevated levels for the A/Env/HK/437/99 viruses compared to the A/HK/156/97 viruses. This study is the first to characterize the A/Env/HK/437/99 viruses in both avian and mammalian species, evaluating the H5 gene from the 1997 Hong Kong H5N1 isolates in a different genetic background. Our findings reveal that at least one of the avian influenza virus genes encoded by the 1997 H5N1 Hong Kong viruses continues to circulate in mainland China and that this gene is important for pathogenesis in chickens but is not the sole determinant of pathogenicity in mice. There is evidence that H9N2 viruses, which have internal genes in common with the 1997 H5N1 Hong Kong isolates, are still circulating in Hong Kong and China as well, providing a heterogeneous gene pool for viral reassortment. The implications of these findings for the potential for human disease are discussed.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A (H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses (compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.     

Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus

Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.     

Genome Sequence of a Novel Reassortant H3N2 Avian Influenza Virus in Southern China

The distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a potential threat to both human and avian health. We report a complete genome sequence of a novel reassortant H3N2 avian influenza virus. Phylogenetic analysis showed that HA and NA showed the highest sequence homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2). However, the internal genes had the highest sequence homologies with those of H6 and H7 subtypes. The data provide further evidence of the existence of a natural reassortant H3N2 strain in southern China.     

Pathogenesis of avian influenza A (H5N1) viruses in ferrets

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.     

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.     

Establishment of influenza A virus (H6N1) in minor poultry species in southern China

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of those viruses had a common source. Continuing surveillance in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants in 1999; however, the further development of H6N1 viruses has not been investigated since 2001. Here we report influenza virus surveillance data collected in southern China from 2000 to 2005 that show that H6N1 viruses have become established and endemic in minor poultry species and replicate mainly in the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had W312-like hemagglutinin and neuraminidase genes. However, reassortment of internal genes between different subtype virus lineages, including H5N1, H9N2, and other avian viruses, generated multiple novel H6N1 genotypes in different types of poultry. These novel H6N1/N2 viruses are double, triple, or even quadruple reassortants. Reassortment between a W312-like H6N1 virus and an A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest that W312-like viruses may not be the precursors of HK/97 virus but reassortants from an HK/97-like virus and another unidentified H6 subtype virus. These results provide further evidence of the pivotal role of the live poultry market system of southern China in generating increased genetic diversity in influenza viruses in this region.     

A review on human influenza A H5N1 infections in Hong Kong

Avian influenza A H5N1 remains the most threatening virus that may cause another devastating pandemic in the foreseeable future. In 1997, Hong Kong was the first place to detect human infections due to this virus originated from birds. The experience and lessons learnt provide important information for controlling further outbreaks caused by avian influenza viruses.     

H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China

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.