Changing patterns of h6 influenza viruses in Hong Kong poultry markets.
ABSTRACT: Until 2001, H6N1 influenza viruses in the Hong Kong bird markets were represented by a single stable A/teal/Hong Kong/W312/97-like lineage. Beginning in 2001, despite a reduction in overall prevalence, an increase was observed in the number of H6 viruses isolated from chickens and other hosts. To assess any changes in H6 viruses, we characterized 18 H6 viruses isolated in the Hong Kong bird markets from 2001 to 2003. Experimental data showed that the 2003 H6 viruses had similar infectivity for chickens as did A/teal/HK/W312/97, and they were unable to transmit. Although all hemagglutinin genes were closely related to A/teal/HK/W312/97, 7 isolates were reassortant viruses containing similar gene segments of co-circulating H9N2 or H5N1 viruses. The receptor specificity was different from that of A/teal/Hong Kong/W312/97. Interestingly, similar observations have been documented in H9N2 viruses in Hong Kong. This evolution strongly suggests that some change in the ecology of influenza in the region selected for these changes. Taken together, these findings suggest that the H6 influenza viruses isolated in the Hong Kong markets are not well adapted to chickens and that the likely continued source of these viruses are other "minor" poultry species in which they are undergoing genetic and biologic evolution.
Project description: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?
Project description: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.
Project description: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.
Project description: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.
Project description: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, these 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.
Project description: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 H5N1 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.
Project description: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.
Project description:H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.
Project description: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.
Project description: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.