Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R-S-N-R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.     

Phylogenetic Analysis of Neuraminidase Gene of H9N2 Influenza Viruses Prevalent in Chickens in China during 1995–2002

The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995–2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63–65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.     

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia

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.     

我国猪群中H9N2亚型毒株HA和NA基因特性的研究

目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因，与P^GEM-T Easy Vector4℃过夜连接，重组质粒转化DH-10β细菌，筛选阳性菌落，酶切鉴定，测序。然后，进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第226位上氨基酸为L，这与从人和猪所分离出的H9N2毒株相同，其连接肽属对禽致病的毒株，但它们的序列为R-L-S-R，而不是R-S-S-R；其NA蛋白茎区第62～64位存在掉失，这与A／Shaoguarn/408／98，A／Swine／Hong Kong／9／98及A／Duck／Hong Kong／y280／97(H9N2)毒株相同；HA与NA基因进化树分析表明，两株猪H9N2毒株的HA基因接近于A／Chicken／Hong Kong／G23／97和A／Chicken／Hong Kong／G9／97．而NA基因接近于A／Shaoguan／408／98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换，可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失，造成了它们能直接感染猪。     

Genetic Analysis of the Nonstructural (NS) Genes of H9N2 Chicken Influenza Viruses Isolated in China During 1998–2002

H9N2 subtype avian influenza viruses are widespread in domestic poultry. Genetic analysis indicated that three lineages of H9N2 viruses have been established in Eurasia and only one lineage is present on chicken farms in mainland China. Here, NS1 genes of eight H9N2 chicken influenza viruses, isolated in mainland China during 1998–2002, were completely sequenced and phylogenetically analyzed. By comparison, the homology of the NS1 of the A/chicken/Neimenggu/ZH/02 (Ck/NM/ZH/02) strain had a high identity (93.8%) with that of A/chicken/Korea/323/96 (Ck/Kor/323/96), which is an A/duck/Hong Kong/Y439/97 (Dk/HK/Y439/97)-like virus. NS1 peptides of seven strains possessed 217 amino acids, while that of the strain Ck/NM/ZH/02 coded 230 amino acids. Except for the amino acid at position 225, the additional amino acid sequence (13 AAs) of NS1 of Ck/NM/Zh/02 at the carboxy-terminus is identical with that of Ck/Kor/323/96. Phylogenetic analysis showed that seven of the tested strains belong to the A/duck/Hong Kong/Y280/97 (DK/HK/Y280/97)-like lineage, while the NS1 gene of Ck/NM/Zh/02 belongs to the Dk/HK/Y439/97-like lineage and has a close relationship with that of the Ck/Kor/323/96-like viruses. Therefore, although most of the H9N2 influenza viruses circulating on chicken farms in mainland China belong to the DK/HK/Y280/97-like lineage, the present results indicate that the other two of the three H9N2 lineage viruses also circulate in the chicken population in mainland China.     

Swine infection with H9N2 influenza viruses in China in 2004

In 2004, H9N2 influenza A viruses were isolated from pigs with respiratory syndrome in commercial swine farms in Henan province, China. Antigenic and genetic characterization were performed for seven swine H9N2 influenza viruses. The hemagglutinin antigenicity of swine H9N2 viruses was similar to those of avian H9N2 viruses of A/duck/Hong Kong/Y280/1997 (Dk/HK/Y280/97)-like sublineage prevalent in China. It is noteworthy that the neuraminidase of these isolates had no deletions in the stalk, which was seldom observed in those viruses of Dk/HK/Y280/97-like sublineage. Genetic analysis revealed that all seven isolates had an -R-S-S-R- motif at the HA cleavage site, which was the same as those of Dk/HK/Y280/97-like viruses established in avian population in China. Phylogenetic analyses showed that the seven swine H9N2 viruses were completely derived from avian influenza viruses of Dk/HK/Y280/97-like sublineage. The present results indicated that avian-to-pig interspecies transmission of H9N2 viruses continued to exist in China through 2004; therefore, surveillance of swine influenza should be given a high priority. Yan-Long Cong, Chun-Feng Wang and Chun-Mei Yan have contributed equally to this work. (i) All the authors have agreed to its submission and are responsible for its contents and (ii) all the authors have agreed that Yanlong Cong may act on their behalf regarding any subsequent processing of the paper.     

Epidemiological survey and genetic evolution of H9 subtype influenza viruses in Shanghai, China, from 2006 to 2010

The H9N2 influenza virus is endemic in poultry. We report its occurrence in live-poultry markets, fair-trade markets and poultry farms in the Shanghai region between September 2006 and December 2010. An analysis of partial sequences of the HA, NA, PB1, PB2 and NP genes of eleven distinct H9N2 isolates revealed that all carried an RSSR motif at the cleavage site of HA, diagnostic of low pathogenicity in chickens. A phylogenetic analysis indicated that these isolates are derived from the lineage represented by Duck/HK/Y280/97, but they have evolved a range of reassortments. Their PB1 and NP sequences resembled those of H5N1 strains, indicating a hybrid origin involving both H9 and H5 strains. The HA and NA sequences present in all eleven isolates resembled those of the Duck/HK/Y280/97-like lineage. Infection by H9N2 is commonplace in Shanghai live-poultry markets, allowing the viruses to have evolved rapidly.     

Evolution analysis of the matrix (M) protein genes of 17 H9N2 chicken influenza viruses isolated in northern China during 1998–2008

Matrix (M) protein genes of 17 H9N2 avian influenza viruses (AIVs) isolated from chickens in northern China during the last 10 years were completely sequenced and phylogenetically analyzed. Homology of nucleotide sequences in the M gene of 17 isolates was 92.7–99.9%. Phylogenetic analysis showed that 11 of the tested M genes belong to the A/chicken/HongKong/Y280/97 (Y280)-like lineage, while the other six belong to the A/Quail/HongKong/G1/97 (G1)-like lineage. This is also the first time that a G1-like M gene of a H9N2 virus was detected in chicken flocks in northern China. These newly appearing changes in M genes may be due to reassortment events of AIVs, or they may have come from the H9N2 strains of southern China which surged in northern China after translocation. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S31N in two isolates, which is the molecular characterization of amantadine resistance in AIVs. Results of this study suggest that long-term monitoring should be continued to track the transmission and evolution of H9N2 AIVs in chickens in China.     

H9N2 influenza viruses prevalent in poultry in China are phylogenetically distinct from A/quail/Hong Kongl/G1/97 presumed to be the donor of the internal protein genes of the H5N1 Hong Kong/97 virus

Ten H9N2 influenza virus strains isolated from diseased chickens in different farms in China during 1995 to 1999 were antigenically and genetically characterized. The haemagglutinins of the isolates were not related to those of AlquaillHong KonglG1197 (H9N2) (Qa/HK/G1/97), but were closely related to that of A/chicken/Hong Kong/G9/97 (H9N2) (Ck/HK/G9/97). The neuraminidase of these isolates had a deletion of three aminoacid residues at positions 63 to 65 as compared with those of Ck/HK/G9/97, while that of Qa/HK/G1/97 lackedtwo amino acids at positions 38 and 39. The PB2 genes of the isolates were not related to those of QalHIUG1197 or Ck/HK/G9/97, but showed some relationship to that of A/duck/Hong Kong/Y439/97 (H9N2) (DWHK/Y439197). The PB1 genes of the isolates were not related to those of the three representative strains. The PA,NP, M, and NS genes of the isolates belonged to the same lineage as those of Ck/HK/G9/97, and were distinctfrom those of Qa/HK/G1/97 and Dk/HK/Y439/97. The present results indicate that H9N2 influenza virusesprevalent in chicken populations in China belong genetically to one lineage and are distinct from Qa/HK/G1/97,presumed to be the donor of the internal protein genes of the highly pathogenic H5N1 influenza virus in HongKong in 1997.     

Detection and characterization of new influenza B virus variants in 2002

One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.     

Phylogenetic Analysis of Hemagglutinin and Neuraminidase Genes of H9N2 Viruses Isolated from Migratory Ducks

Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997–2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.     

Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5–99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.     

Characterization of H9N2 influenza viruses isolated from Dongting Lake wetland in 2007

In 2007, a total of eight H9N2 influenza viruses were isolated from the water and fowl feces in Dongting Lake wetland, China. The genomes of the eight viruses were sequenced, and all eight gene segments were subjected to phylogenetic analysis. The results showed that all the isolates belonged to the same genotype, in which the HA, NA and NS gene segments were Chicken/Beijing/94-like; the PB2, PB1, PA and NP gene segments were Chicken/Shanghai/F/98-like; and the M gene was Quail/Hong Kong/G1/97-like. Animal experiments showed low pathogenicity of the selected viruses for chickens, although some chickens died after inoculation. The viruses showed no overt clinical signs in mice, but they could replicate in murine lungs prior to adaptation.     

Genetic Evolution of H9 Subtype Influenza Viruses from Live Poultry Markets in Shanghai,China

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.Avian influenza A viruses of subtype H9N2 were first detected in the United States in 1966 (12). In North America, there are no reports of H9N2 virus-associated disease in chickens to date, although these viruses can be found in wild ducks and have caused a number of outbreaks in turkeys (10, 13, 24, 25). In Asia, H9N2 viruses were detected only in apparently healthy ducks in limited surveillance studies of live poultry markets and farms in Hong Kong from 1975 to 1985. However, in the early 1990s, H9N2 viruses became more prevalent in domestic poultry (26). Reports from Korea, South Asia, Middle Eastern countries, South Africa, and Europe since the late 1990s indicate widespread distribution of H9N2 viruses (1, 4, 14, 19, 20, 23). Previous studies revealed that H9N2 influenza viruses from poultry could occasionally be transmitted from poultry to mammalian species, including humans and pigs (3, 17, 21, 22, 29). The Chicken/Beijing-like (Ck/Bei-like) and G1-like viruses were initially found in specimens from both a human and pigs in the late 1990s and were also observed in 2003 and recently in Hong Kong (5). Genetic analyses demonstrated that the human H9N2 influenza virus isolate from 2003 was a novel reassortant and most likely originated directly from local live poultry markets (3). These recent interspecies transmission events suggest that current H9N2 influenza virus variants are still potentially infectious for humans.During surveillance of live poultry markets in Shanghai from September 2006 to December 2008, the only influenza viruses isolated were H9. It was reported that at least two lineages of H9N2 viruses, represented by A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280) and A/Quail/Hong Kong/G1/97 (Qa/HK/G1), had become established in domestic poultry in Asia (8, 15, 21). Furthermore, human cases of H9N2 virus infection have been reported in Hong Kong and other locations in south China in the late 1990s and early 2000s (3, 9, 17, 22). Follow-up serological surveillance suggests that the incidence of human infections with H9N2 viruses might be higher than previously anticipated (7, 9, 22). The prevalence of H9N2 viruses throughout the world, along with their ability to infect mammals and humans, increases concern about their pandemic potential.Because of the ongoing concern about the transmission of H9N2 viruses to mammals and humans, continued surveillance of H9N2 viruses from live poultry markets is needed.     

Genetic characterization of HA gene of low pathogenic H9N2 influenza viruses isolated in Israel during 2006–2012 periods

H9N2 influenza viruses are isolated in Israel since 2000 and became endemic. From November 2006 to the beginning of 2012, many H9N2 viruses were identified, all belonged to the Asian G1-like lineage represented by A/qu/Hong Kong/G1/97 (H9N2). In the present study, 66 isolates were selected for their hemagglutinin gene characterization. Most H9N2 isolates were distributed between two main groups, identified as the 4th and 5th introductions. The 5th introduction, was represented by a compact cluster containing viruses isolated in 2011–2012; the 4th introduction was subdivided into two subgroups, A and B, each containing at least two clusters, which can be identified as A-1, A-2, B-1, and B2, respectively. Genetic analysis of the deduced HA proteins of viruses, belonging to the 4th and 5th introductions, revealed amino acid variations in 79 out of 542 positions. All isolates had typical low pathogenicity motifs at the hemagglutinin (HA) cleavage site. Most viruses had leucine at position 216 in a receptor binding pocket that enables the virus to bind successfully with the cellular receptors intrinsic to mammals, including humans. It was shown that the differences between the HA proteins of viruses used for vaccine production and local field isolates increased in parallel with the duration and intensity of vaccine use, illustrating the genetic diversity of the H9N2 viruses in Israel.     

H9N2 subtype influenza A viruses in poultry in pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong

Following the outbreak of H5N1 "bird flu" in Hong Kong in 1997, the isolation of H9N2 subtype viruses from patients in southern China and Hong Kong SAR once again raised the spectre of a possible influenza pandemic. H9N2 viruses have recently been responsible for disease in poultry in various parts of the world and preliminary studies of the H9 haemagglutinin (HA) genes of viruses isolated during 1998 and 1999 in Germany, Iran, Pakistan, and Saudi Arabia showed a close relationship to the HA genes of the viruses that infected two children in Hong Kong SAR. Analysis of the complete genome of a Pakistan isolate, A/chicken/Pakistan/2/99, showed that it is closely related in all eight genes (97-99% homology) to the human H9N2 isolates and furthermore that the six genes encoding internal components of the virus are similar to the corresponding genes of the H5N1 viruses that caused 6 (out of 18) fatal cases of human infection. Thus H9N2 viruses similar to those that caused human infections in Hong Kong are circulating more widely in other parts of the world. Whether or not these H9N2 viruses also have features that facilitate avian-to-human transmission is not known. Since avian H9N2 viruses are currently perceived to represent a significant threat to human health it is important to determine whether or not viruses of this subtype circulating in poultry in various parts of the world have the potential to infect people.     

Polygenic virulence factors involved in pathogenesis of 1997 Hong Kong H5N1 influenza viruses in mice

Virulence factors of influenza A (H5N1) viruses collected in 1997 from mammalian hosts were examined using a BALB/c mouse model. Fifteen amino acid (aa) residues in four influenza virus genes which correlated with high- and low-pathogenic phenotypes in mice were identified by analyzing sequence alignments. In addition to these specific residues, the effects of aa residue 627 of the PB2 gene, and the hemagglutinin (HA) and neuraminidase (NA) genes were also investigated using a reverse genetics system established with representative viruses of low (A/Hong Kong/486/97) and high (A/Hong Kong/483/97) pathogenicity for mice. None of 15 aa residues alone had any effect on virulence. The HA and NA genes had a synergistic effect on virulence and the absence of a glycosylation site at aa154 in the HA gene also increased virulence of virus. Multiple genes are involved in virulence of Hong Kong H5N1 influenza A viruses for mice with the presence of lysine at aa627 in the PB2 gene exhibiting a significantly larger effect than the HA and NA genes.     

Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia

Summary.  Influenza A viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1998. Phylogenetic analysis of the NP genes of the isolates in Siberia and those in Hokkaido, Japan on their flyway of migration from Siberia to the south in autumn revealed that they belong to the Eurasian lineage of avian influenza viruses. It is noted that the genes of the isolates in Siberia are closely related to those of H5N1 influenza virus strains isolated from chickens and humans in Hong Kong in 1997 as well as to those of isolates from domestic birds in southern China. The results indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virusin Hong Kong. Vaccine prepared from avirulent A/duck/Hokkaido/4/96 (H5N3) influenza virus was potent enough to protect mice from challenge with lethal dose of the pathogenic H5N1 virus [19]. Intensive surveillance study of aquatic birds especially in Siberia is, therefore, stressed to provide information on the future pandemic influenza virus strains and for vaccine preparation. Received August 24, 1999/Accepted January 7, 2000     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.