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PubMed2006

Reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay for rapid detection of avian influenza virus subtype H9N2.

The performance of a simplified nucleoprotein (NP) and hemagglutinin-subtype-9 (H9) based reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay (RT-PCR-ELISA) for the detection of avian influenza virus (AIV) subtype H9N2 was compared to the standard the virus isolation method and serology testing using hemagglutination (HA) and hemagglutination inhibition (HI) tests. The H9-based RT-PCR-ELISA was 100% sensitive when compared to virus isolation method in detecting H9N2 from experimentally infected specific-pathogen-free (SPF) chickens. The NP- and H9-based RT-PCR-ELISA have a detection limit similar to the virus isolation method in detecting serially diluted tracheal swab samples obtained from chickens inoculated with H9N2. Both RT-PCR-ELISAs were also ten times more sensitive than agarose gel electrophoresis for the detection of PCR products. The result of this study demonstrate that the developed RT-PCR-ELISA is a simple and sensitive assay for the detection of type A influenza virus, particularly AIV subtype H9N2, in chickens.

Animals↗

PubMed2003

[Sequence analysis of NS1 gene of some H9N2 subtype influenza viruses isolated from chickens in China].

The nonstructural protein (NS1) genes of eight H9N2 subtype influenza virus strains isolated from diseased chickens on different farms during 1996 - 2001 were amplified and sequenced. The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were compared. The results showed that the homologies of nucleotide and amino acid of the isolates were 96.5% - 99.5% and 94.5 - 98.6%, respectively. These indicated that NS1 genes of H9N2 influenza viruses isolated in China were well conserved. Comparison of the amino acid sequences of NS1 genes of these isolates with those of H9N2 viruses isolated in Hong Kong of China, Korea and Pakistan demonstrated that nonstructural (NS1) proteins of the eight strains had a deletion of 13 amino acid residues at the carboxy terminus. Phylogenetic analyses showed NS1 genes of these isolates belonged to the same lineage and derived from an early chicken H9 virus isolated in 1994. The NS1 genes of the eight isolates belong to different sublineages from those of the H9N2 viruses isolated in Hong Kong, Korea and Pakistan, suggesting that the geographical distribution plays a significant role in the evolution of the H9N2 subtype influenza viruses.

Amino Acid Sequence↗

PubMed2005

Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland China strain possessing early isolates' genes that have been circulating.

H9N2 subtype influenza virus has become worldwide and prevalent in China. Previous studies illustrated that at least three sublineages had been established in terrestrial poultry of Eurasian avian. In this presentation, eight full-length genes of an H9N2 strain, A/Chicken/Shanghai/F/98 (Ck/SH/F/98) were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes with those of all the available H9N2 strains from the GenBank. The results showed that four genes (HA, NA, M and NS genes) of Ck/SH/F/98 were incorporated into the sublineage represented by the early mainland China strain, Ck/BJ/1/94. However, the other four of RNP genes of Ck/SH/F/98 did not show close relationship with those of the three known sublineages' viruses. Therefore, Ck/SH/F/98 was a natural reassortant between different sublineages. In addition, comparison showed that Ck/SH/F/98 could be a putative precursor of a later isolate from southern China, Dk/ST/1605/01, with at least six genes of both closely related, indicating genes of Ck/SH/F/98 and early isolates had ever been circulating. Further comparison in terms of molecular markers of species specificity of HA1 revealed that DK/ST/1605/01 also resembled Ck/SH/F/98 more than a common earlier duck strain. The results supported the idea of two-way transmission between terrestrial and aquatic birds that emphasized the importance to raise concerns on the natural evolution of all the eight genes of H9N2 avian influenza viruses.

Amino Acid Sequence↗

PubMedDate not supplied

Phylogenetic analysis of the hemagglutinin genes of twenty-six avian influenza viruses of subtype H9N2 isolated from chickens in China during 1996-2001.

The complete coding region of hemagglutinin genes from 26 influenza A viruses of H9N2 subtype isolated from chicken flocks in China during 1996-2001 was amplified and sequenced. Sequence analysis and phylogenetic studies of H9N2 subtype viruses on the basis of data of 26 viruses in this study and 71 selected strains available in the GenBank were conducted. The results revealed that all the mainland China isolates showed high homology (94.19%-100%) and were assigned to a special sublineage in the major Eurasian lineage, in contrast to the high heterogeneity of Hong Kong SAR isolates. All the 29 mainland China isolates and six Hong Kong SAR strains also had the following common characteristics: sharing the same sequence of proteolytic cleavage site with one additional basic amino acid, RSSR, with only two exceptions; having the same amino acid motif of the receptor-binding site, YWTNV/ALY; 23 of 28 isolates bearing seven potential glycosylation sites and the remaining five having six; and sharing characteristic deduced amino acid residues Asn-183 at the receptor-binding site and Ser-130 at the potential glycosylation site. We concluded that the H9N2 subtype influenza viruses circulating in chicken flocks in China since the 1990s and Ck/HK/G9/97-like viruses isolated in Hong Kong SAR should have a common origin, whereas Qu/HK/G1/97-like viruses including human strains isolated in Hong Kong SAR might originate from other places. The available evidence also suggests that the H9N2 viruses of special lineage themselves and factors prone to secondary infections may contribute to the widespread and dominant distribution of viruses of this subtype in chicken flocks in China and other Asian countries.

Animals↗

PubMed2000

Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates.

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.

Animals↗

PubMed2006

Molecular epizootiology of recurrent low pathogenic avian influenza by H9N2 subtype virus in Korea.

The first outbreak of low pathogenic avian influenza (LPAI), H9N2 virus subtype, in 1996 prompted an eradication response, but LPAI returned to Korea in 1999. The relationship between the first and the recurrent viruses is unclear. To determine the molecular epizootiology of recurrent LPAI, we performed phylogenetic analysis with partial nucleotide sequences of four gene segments (HA, NA, NP and PB2) from eight chicken-origin H9N2 viruses. The recurrent H9N2 viruses showed higher nucleotide similarity in haemagglutinin and neuraminidase genes to the 1996 Korean isolates than other Eurasian viruses, and formed a distinct cluster with the early Korean isolates and some isolates from migratory and domestic ducks in Japan and China. Phylogenetic analysis with internal genes showed that some Korean isolates formed a cluster with other subtypes, such as H5N1, H6N1, and H6N2 in China and Taiwan. These results suggest that the recurrent viruses are progeny of the early Korean H9N2 isolates, but further studies are required to explain their phylogenetic relatedness to viruses in China.

Animals↗

PubMed2001

Characterization of hemagglutinin gene of influenza A virus subtype H9N2.

OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed2002

[Recombinant fowlpox virus expressing HA from subtype H9N2 of avian influenza virus and its protective immunity against homologous challenge in chickens].

The hemagglutinin (HA) gene from the AIV, A/Chicken/China/F/1998(H9N2), was amplified with the RT-PCR technique and directionally inserted into transferring vector 1175, resulted in recombinant transferring vector 1175HA. In order to generate recombinant fowlpox virus expressing HA(rFPV-HA), the recombinant transferring vector 1175HA was used to transfect the chicken embryo fibroblasts(CEF) pre-infected with wide type fowlpox virus. Then, by selection of blue plaques on the CEF overlaid with agar containing X-gal, rFPV-HA was obtained and purified. The expression of HA by rFPV-HA was detected in the recombinant virus-infected CEF by indirect immunofluorescence. Experiments on chickens demonstrated that rFPV-HA could induce detectable HI antibodies 7 days post-vaccination and those HI antibodies of relatively high titers could persist 55 days. rFPV-HA also had the same protective efficacies to suppress SPF chickens or commercial broiler chickens with antibodies against FPV from shedding challenged virus from intestine as inactivated vaccine in oil emulsion.

Animals↗

PubMed2006

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel.

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000-2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005-2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.

Animals↗

PubMed2003

[Genetic mutations of heamagglutinin genes of H9N2 subtype influenza A viruses in the field in a five-year period].

Infection in chicken flocks on a farm, where AI outbreak took place in 1998, was monitored in a four-year consecutive period and 22 H9N2 subtype viruses were isolated. The HA gene and antigenicity of 9 strains among isolates were compared with that of isolate of 1998. The results indicated that mutations of HA genes of H9 subtype influenza A viruses were observed during five years. However these mutations of HA genes didn't lead to sufficient antigenic variation to change the cross-protective pattern of these strains. These findings may be fruitful for making further study of genetic mutations of AIVs in the field and improving the control strategies.

Animals↗

PubMed2004

[Discoveries of avian influenza A(H9N2) virus in chickens and men infected by H9N2 virus in Guangzhou area].

OBJECTIVE: To understand the epidemic status of avian influenza A virus in chickens and men in Guangzhou area and to prevent men suffering from avian influenza A (H5N1) virus. METHODS: Etiologic and serological surveys were conducted in chickens and men who were working in the poultry farms and slaughter house. Viruses were isolated with both MDCK cells and embryonated chicken eggs. Hemagglutination inhibition tests were performed by routine method. RESULTS: Anti-H9N2 antibody was found in 12.8% of the chickens and 5.1% of the workers. CONCLUSIONS: Avian influenza virus H9N2 subtype existed in chickens and this subtype of influenza A virus might infect men.

Animals↗

PubMed2002

[Virological and serological surveys for H9N2 subtype of influenza A virus in chickens and men in Shenzhen city].

OBJECTIVE: To understand the distribution of influenza A H9N2 virus in chickens and men in Shenzhen area. METHODS: Virus isolation was performed in embryonated hen s eggs with routine method. The antibody to H9N2 virus was detected with micro-hemagglutination inhibition (HI) test, then the results were checked by using the neutralization assay in MDCK cells. RESULTS: Totally 27 strains of influenza A H9N2 virus were isolated from chickens in farm markets in Shenzhen, whereas no H9N2 virus was isolated from men. Approximately 26% of human sera with the HI titers > or =20 to H9N2 virus were detected. However only 7% of chicken sera with the HI titers > or =20 to H9N2 virus were detected. Meanwhile the HI titer and (MGT) of antibody to H9N2 virus in human sera increased with age. It was also found that there was a close relationship between HI antibody titer to H9N2 virus in human sera and occupation. CONCLUSIONS: The distribution of influenza A H9N2 virus in chicken and men in Shenzhen was rather wide. The human H9N2 virus infection probably derived from chicken H9N2 virus.

Animals↗

PubMed1999

Human infection with influenza H9N2.

We report the clinical features of two cases of human infection with influenza A virus subtype H9N2 in Hong Kong, and show that serum samples from blood donors in Hong Kong had neutralising antibody suggestive of prior infection with influenza H9N2.

Animals↗

PubMed2003

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate.

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.

Adaptation, Physiological↗

PubMed2026

Molecular epidemiological characteristics of H9N2 subtype avian influenza virus in the external environment of western Zhejiang, China, 2014-2025.

OBJECTIVE: To elucidate the epidemiological distribution patterns of avian influenza virus (AIV) in the external environment of western Zhejiang from 2014 to 2025, analyze the molecular epidemiological characteristics of the H9N2 subtype, and assess its public health risks. METHODS: According to the Zhejiang Provincial Surveillance Program for Avian Influenza in Occupationally Exposed Populations and External Environments, real-time RT-PCR was used to detect AIV subtypes in environmental specimens. H9N2-positive samples with cycle threshold values <30 were inoculated into specific pathogen-free (SPF) embryonated chicken eggs for virus isolation, followed by whole-genome sequencing and bioinformatics analysis for phylogenetic and molecular characterization. RESULTS: A total of 7,762 specimens were tested from 2014 to 2025, with an overall positivity rate of 34.64% (2,689/7,762) for AIV. Significant differences in positivity rates were observed in seasons, regions, sampling sites, and specimen types (all p < 0.001). AIV activity peaked in winter and spring, with the highest rates detected in live poultry markets and chopping board swabs. The H9 was the predominant subtype, with co-circulation of multiple subtypes. All 48 H9N2 subtype isolates belonged to the G57 genotype, with the hemagglutinin (HA) and neuraminidase (NA) genes falling into the Y280-like branch, while the internal genes exhibited a mosaic pattern combining G1-like and F/98-like lineages. Molecular characterization analysis revealed multiple mammalian adaptive mutations, involving alterations in receptor-binding sites (T163N, H191N, T197D, T198V, Q234L, Q235M), antigenic epitopes (D280G, N285S), and glycosylation sites (218NRTF, 313NCSK). NA stalk deletion (62-64 aa), along with multiple mutations in the hemadsorption site (E/K368N, D369S/G, D401G/V, N402D, W403L/R, Q432H). Additionally, multiple key amino acid substitutions were also identified in the internal proteins. CONCLUSION: The external environment in western Zhejiang exhibits a high prevalence of AIVs with pronounced spatiotemporal clustering. H9 was the dominant subtype and co-circulated with multiple subtypes, with live poultry markets and slaughterhouses identified as high-risk settings. The H9N2 subtype AIV has accumulated multiple mammalian adaptive mutations, and exhibits genetic linkages across eastern Chinese provinces. These findings collectively underscore the need for an integrated One Health surveillance and early-warning system to reduce the risk of human infections with avian influenza.

Influenza in Birds↗

PubMed2003

Generation and characterization of an H9N2 cold-adapted reassortant as a vaccine candidate.

H9N2 subtype avian influenza viruses have been identified in avian species worldwide, and infections in pigs were confirmed in Hong Kong in 1998. Subsequently, H9N2 viruses were isolated from two children in Hong Kong in 1999, and five human infections were reported from China, raising the possibility that H9N2 viruses pose a potential pandemic threat for humans. These events prompted us to develop a vaccine candidate to protect humans against this subtype of influenza A viruses. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of A/Ann Arbor/6/60 (H2N2)(AA) cold-adapted (ca) virus have been tested extensively in humans and have proved to be attenuated and safe as live virus vaccines. Using classical genetic reassortment, we generated a reassortant that contains the hemagglutinin and neuraminidase genes from A/chicken/Hong Kong/G9/97 (H9N2) and six internal gene segments from the AAca virus. The G9/AAca reassortant virus exhibits the ca phenotype and the temperature-sensitive phenotypes of the AAca virus and was attenuated in mice. The reassortant virus was immunogenic and protected mice from wild-type H9N2 virus challenge. The G9/AAca virus bears the in vitro and in vivo phenotypes specified by the AAca virus and will be evaluated as a potential vaccine candidate in humans.

Acclimatization↗

PubMed2006

Use of strategic vaccination for the control of avian influenza in Pakistan.

The first outbreak of highly pathogenic avian influenza (HPAI) virus caused by subtype H7N3 appeared in Pakistan in 1995. A homologous aqueous-based vaccine prepared from the field isolate employed as ring vaccination around the epicentre of the outbreak helped in controlling the disease. Later, in 1998, an outbreak of avian influenza virus (AIV) subtype H9N2, particularly affecting the broilers and broiler-breeders, was dealt with using the same vaccination approach. However, this time, the virus could not be successfully contained in the affected areas. Later on, low pathogenic avian influenza (LPAI) virus subtype H7N3 re-emerged in the broiler-breeder flocks in different poultry estates of the northern area of the country during 2000. This prompted the introduction of a vaccine strategy, whereby both aqueous and oil emulsion vaccines were routinely employed in and around the affected areas. This helped in the control of disease in this region. However, no AI monitoring and control strategy could be launched on a sustainable basis in these areas. In November 2003, new outbreaks of HPAI subtype H7N3 occurred specifically in commercial layers in the southern part of the country, which were never vaccinated against AI in the past. In many cases, subtype H9N2 was also recovered from the diseased flocks, with or without the presence of subtype H7N3. The disease caused heavy losses in the south. An emergency plan to control this outbreak was developed with the help of FAO in this regard. Under this plan, a nationwide AIV monitoring and vaccination strategy was developed. This paper discusses the role of vaccine type and vaccination schedule in the control of HPAI in this country.

Animals↗