Avian Influenza Virus Subtype H5N1 Research Articles

Using Risk Assessment as Part of a Systems Approach to the Control and Prevention of HPAIV H5N1

Since its emergence in China in 1996, highly pathogenic avian influenza virus subtype H5N1 has spread across Asia, Africa, and Europe. Countries had to promptly implement control and prevention measures. Numerous research and capacity building initiatives were conducted in the affected regions to improve the capacity of national animal health services to support the development of risk-based mitigation strategies. This paper reviews and discusses risk assessments initiated in several South-East Asian and African countries under one of these projects. Despite important data gaps, the risk assessment results improved the ability of policy makers to design appropriate risk management policies. Disease risk was strongly influenced by various human behavioral factors. The ongoing circulation of HPAIV H5N1 in several Asian countries and in Egypt, despite major disease control efforts, supports the need for an interdisciplinary approach to development of tailored risk management policies, in accordance with the EcoHealth paradigm and the broad concept of risk governance. In particular, active stakeholders engagement and integration of economic and social studies into the policy making process are needed to optimize compliance and sustainable behavioral changes, thereby increasing the effectiveness of mitigation strategies.

Supplementation of oil-based inactivated H9N2 vaccine with M2e antigen enhances resistance against heterologous H9N2 avian influenza virus infection

Avian influenza virus (AIV) subtype H9N2 has been evolving rapidly and vaccine escape variants have been reported to cause circulation of infections and economic losses. In the present study, we developed and evaluated ectodomain of the AIV matrix 2 (M2e) protein as a supplementing antigen for oil-based inactivated H9N2 vaccine to increase resistance against vaccine escape variants. AIV H9N2 M2e antigen was expressed in Escherichia coli and supplemented to inactivated H9N2 oil emulsion vaccine. Specific pathogen-free chickens received a single injection of inactivated H9N2 oil emulsion vaccines with or without M2e supplementation. At three weeks post vaccination, hemagglutination inhibition tests and enzyme-linked immunosorbent assays were performed to determine serological immune responses. Challenge study using a vaccine escape H9N2 variant was performed to evaluate the efficacy of M2e supplementation. M2e antigen supplemented in oil emulsion vaccine was highly immunogenic, and a single M2e-supplemented vaccination reduced challenge virus replication and shedding more effectively than non-supplemented vaccination.

Innate immune response gene expression profiles in specific pathogen-free chickens infected with avian influenza virus subtype H9N2

In poultry species, H9N2 low pathogenic avian influenza (LPAI) virus infections frequently lead to low to moderate mortality rates and morbidity characterized by depression, respiratory disease symptoms, and decreases in egg production. Since current knowledge on the avian immune response to H9N2 infection is limited, we used microarray analysis to examine global changes in immune-related gene expression induced by H9N2 LPAI infection in specific pathogen-free chickens. The expression profiles of approximately 800 genes, including those that influence cell differentiation, transcription, transport, immune responses, and signal transduction, were altered by H9N2 infection. A complete chicken genome microarray analysis identified a strong innate antiviral host response after infection in spleens. In particular, a significant number of immune response genes, including interferon genes and related immune response genes, were upregulated following H9N2 infection. The increased transcription of 2′-5′-oligoadenylate synthetase and myxovirus resistance genes was confirmed by real-time RT-PCR. These results suggest that the host response generated against H9N2 infection may contribute to protective effects by manipulating innate immune responses.

Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model.

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 107.2 TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV.

Effect of infectious bursal disease virus on pathogenicity of avian influenza virus subtype H9N2 in broiler chicks

In this experiment, pathogenesis of H9N2 avian influenza virus (AIV), experimentally infected with infectious bursal disease virus (IBDV) in broiler chicks was examined. Three groups of twenty were randomly selected. Day old chickens in group 1, were infected by 103 CID50 of IBDV intrabursaly, and in thirty days of age groups 1 and 2 were challenged with 106 EID50 H9N2, intranasaly-intraoculary. Chickens in group 3 remained as control (uninfected with neither IBDV or AIV). Tracheal and cloacal swabs, and tissue samples, were collected at 3, 7, and 11 days postinoculation (PI). Serum samples examined for antibodies against avian influenza virus (AIV) by hemagglutination inhibition test (HI). IBD caused lower H9N2 antibody level. IBDV infected chickens (g1) shed AI virus for a longer period than AIV infected birds (g2), from both trachea and cloac. IBDV was related with AIV in brain and liver. Isolation of AIV from trachea, conjunctiva, bursa and lung in IBDV infected group (1), prolonged till 11 days PI. Our study provides evidence that a previous history of IBDV infection in chickens may cause them to be more susceptible to H9N2 low pathogenic avian influenza (LPAI) virus infection and may alter its tissue tropism. Key words: Infectious bursal disease, avian influenza, virus shedding, broiler chicks.

Survey on ELISA Based on anti Influenza A NS1 Antibodies to Differentiate the Infected and Vaccinated Poultries

Background: The Vaccination programs to control avian influenza (AI) infection in poultry, have limitations due to the difficulty in differentiating the vaccinated and naturally infected birds AI vaccination would bring greater global acceptance if a reliable test, which could clearly distinguish the naturally infected and vaccinated-only animals (DIVA), was available. Since the nonstructural protein (NS1) is expressed in influenza infected cells, and it is not presented as a virion, it could be a proper candidate for DIVA differential diagnostic test. Objectives: Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. The use of AI vaccination in poultry would have greater worldwide acceptance if a reliable test were available that clearly discriminated between naturally infected and vaccinated-only animals (DIVA). Because the nonstructural protein (NS1) is expressed in influenza virus–infected cells, and it is not packaged in the virion, it is an attractive candidate for a DIVA differential diagnostic test. Materials and Methods: A total of 300 day-old broiler chicks (Ross 308) divided into three equal groups (1 to 3) .The chicks in group 1 were immunized with killed AIV H9N2. The chicks in group 2 were infected with AI virus subtype H9N2. The chicks in group 3 were kept as controls and did not receive any vaccined or lived virus. Chicks sera were collected at day 42 usingrNS1-ELISA and Commercial ELISA kit. Results: Designed ELISA test for detection of antibody against influenza NS1 could experimentally distinguish the chicks infected with AIV and chicks immunized with killed influenza viruswith93.3% sensitivity and 100 ℅ specificity. 3 weeks after infection or vaccination, sera from all two treated groups were positively tested using commercial ELISA kit (IDEXX). In contrast, by NS1-ELISA, only infected groups sera were tested and the result was positive, and all sera samples from the vaccinated group were NS1-antibody titer were evaluated and the result were negative. Conclusions:: It was concluded that antibodies against AIV NS1 protein was only detected in the sera of chickens experimentally infected with AIV, not in the sera of chickens immunized with inactivated vaccine.

Characterization of recombinant H9N2 influenza viruses isolated from wild ducks in China

Wild birds are considered to be the natural reservoirs for avian influenza A viruses (AIV). During active influenza surveillance in Poyang Lake of southeast China, we isolated and characterized 11 H9N2 viruses from two species of wild ducks. Phylogenetic analysis showed that the 11 isolates were almost identical with 99.3-100% nucleotide homology in their entire genome, and they all closely related in whole eight genes (95.6-99.4% homology) to human H9N2 isolates (HK/33982/2009) and clustered in the same sublineage. The isolates belonged to triple reassortant H9N2 genotype viruses containing Ck/Bei-like NA genes, Y439-like PA genes and six other G1-like genes. We also found that the subtype of virus replicated efficiently in the lungs and tracheas of BALB/c mice and caused mortality in 20-40% of infected groups after 3-6 days, which indicates that the subtype of virus is capable of establishing lethal mammalian infections. However, whether or not the virus has features transmittable from wild ducks to humans is not known. This study showed H9N2 subtype avian influenza virus for the first time in wild birds, and suggests that wild birds may carry the virus along migratory routes, highlighting the need for continued surveillance of wild birds.

Identification of Gene Resistance to Avian Influenza Virus (Mx Gene) among Wild Waterbirds

The Mx gene is an antiviral gene used to determine the resistance or the susceptibility to different types of viruses, including the Avian Influenza (AI) virus subtype H5N1. The AI virus subtype H5N1 infection in chickens causes Mx gene polymorphism. The Mx + gene shows resistant to the AI virus subtype H5N1, whereas the Mx gene shows signs of susceptible. The objective of this research was to detect the Mx gene in wild aquatic birds using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method with the primer pairs F2 and NE-R2/R and the RsaI restriction enzyme. DNA samples were obtained from eight species of wild waterbirds with positive and negative exposure to the AI virus subtype H5N1. DNA amplification results showed that the Mx gene in wild aquatic birds is found in a 100 bp fragment, which is the same as the Mx gene found in chickens. However, unlike chickens, the Mx gene in wild aquatic birds did not show any polymorphism. This study proves that Mx- based resistance to AI virus subtype H5N1 in different in wild birds than in chickens.

Experimental Assessment of Houseflies as Vectors in Avian Influenza Subtype H5N1 Transmission in Chickens

In this study, laboratory-reared houseflies were experimentally exposed to the high pathogenicity avian influenza virus (HPAI) subtype H5N1 virus to evaluate the houseflies as vectors in HPAI-H5N1 virus transmission in chickens. One hundred and fifty houseflies (Musca domestica L.) were equally allocated into three groups. Groups 2 and 3 were exposed to the HPAI-H5N1 virus by allowing the flies to consume food containing the virus for 15 min, while the flies in group 1 were allowed to consume H5N1-free food and would serve as a negative control group. Group 2 flies were euthanatized immediately after H5N1 exposure, while group 3 were held at room temperature for 24 hr and euthanatized. The houseflies in the transmission of the HPAI-H5N1 virus were examined by challenging three groups of housefly homogenates into layer chickens via the oral drop. Morbidity and mortality were observed for 14 days, and virus shedding monitored via oropharyngeal swabs (OS) and cloacal swabs (CS), which were collected daily and determined by real-time reverse transcription-PCR and virus titration. Experimental challenge showed that all the chickens of groups 2 and 3 died within 7 days of inoculation. The OS had higher concentrations of virus than CS. Moreover, the chickens of group 2 had higher concentrations of virus shedding than the chickens of group 3. Immunohistochemistry detected the nucleoprotein of the type A influenza virus in all tissue samples collected, including the trachea, duodenum, pancreas, and brain. In summary, this study demonstrates that houseflies could serve as vectors in HPAI-H5N1 virus transmission in chickens under experimental conditions.

Rapid detection of the avian influenza virus H5N1 subtype in Egypt

Influenza A virus continue to cause widespread morbidity and mortality. The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Egypt is threatening poultry and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, polyclonal antibodies (PAbs) were developed against the H5N1 avian influenza virus (AIV) and implemented an enzyme-linked immunosorbent assay (ELISA) to detect the H5 viral antigen. A group of rabbits was immunized with viral vaccine (H5N1) therefore, purifiedantibodies from rabbit serum, which secrete immunoglobulin G (IgG) was served as the detector antibody after conjugation with horse radish peroxidase and fluorescent isothiocyanate (FITC). The reactivity of the obtained peroxidase and fluorescent conjugated PAb of influenza virus revealed that they are specifically recognized H5N1 virus antigen. Specimens containing AIV subtypes collected from different Governments in Egypt yielded specific and strong signals with hemagglutination test. The detection limit of ELISA using the prepared peroxidae conjugated PAbs was 1:100000, while using fluorescent conjugated PAb was 1:10000. Reconstituted clinicalsamples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in ELISA, and the results were confirmed using reference virus antigens. This investigation enhanced the usage of these PAbs in the surveillance and diagnosis of H5N1 AIV in Egypt. Key words: Avian influenza virus, H5N1, fluorescent antibody enzyme-linked immunosorbent assay (ELISA) technique, polyclonal antibodies.

Interventions for avian influenza A (H5N1) risk management in live bird market networks

Highly pathogenic avian influenza virus subtype H5N1 is endemic in Asia, with live bird trade as a major disease transmission pathway. A cross-sectional survey was undertaken in northern Vietnam to investigate the structure of the live bird market (LBM) contact network and the implications for virus spread. Based on the movements of traders between LBMs, weighted and directed networks were constructed and used for social network analysis and individual-based modeling. Most LBMs were connected to one another, suggesting that the LBM network may support large-scale disease spread. Because of cross-border trade, it also may promote transboundary virus circulation. However, opportunities for disease control do exist. The implementation of thorough, daily disinfection of the market environment as well as of traders' vehicles and equipment in only a small number of hubs can disconnect the network dramatically, preventing disease spread. These targeted interventions would be an effective alternative to the current policy of a complete ban of LBMs in some areas. Some LBMs that have been banned still are very active, and they likely have a substantial impact on disease dynamics, exhibiting the highest levels of susceptibility and infectiousness. The number of trader visits to markets, information that can be collected quickly and easily, may be used to identify LBMs suitable for implementing interventions. This would not require prior knowledge of the force of infection, for which laboratory-confirmed surveillance would be necessary. These findings are of particular relevance for policy development in resource-scarce settings.

Epidemiological and risk analysis of the H7N9 subtype influenza outbreak in China at its early stage.

Dozens of human cases infected with H7N9 subtype avian influenza virus (AIV) have been confirmed in China since March, 2013. Distribution data of sexes, ages, professions and regions of the cases were analyzed in this report. The results showed that the elderly cases, especially the male elderly, were significantly more than expected, which is different from human cases of H5N1 avian influenza and human cases of the pandemic H1N1 influenza. The outbreak was rated as a Grade III (severe) outbreak, and it would evolve into a Grade IV (very severe) outbreak soon, using a method reported previously. The H7N9 AIV will probably circulate in humans, birds and pigs for years. Moreover, with the driving force of natural selection, the virus will probably evolve into highly pathogenic AIV in birds, and into a deadly pandemic influenza virus in humans. Therefore, the H7N9 outbreak has been assumed severe, and it is likely to become very or extremely severe in the future, highlighting the emergent need of forceful scientific measures to eliminate any infected animal flocks. We also described two possible mild scenarios of the future evolution of the outbreak.

Preparation and characterization the polyclonal antibody of the nonstructural protein of human highly pathogenic H5N1 avian influenza viruses

Of this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency. Construct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 (DE3), induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity. NS1 fusion protein was highly expressed in a purity of greater than 90%, with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80 000, and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein. NS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus detection kit.

Genetic and biological characteristics of recent Korean isolates of avian influenza virus subtype H9N2

Genetic and biological characteristics of recent Korean isolates of avian influenza virus subtype H9N2

Social Network Analysis for Assessment of Avian Influenza Spread and Trading Patterns of Backyard Chickens in Nakhon Pathom, Suphan Buri and Ratchaburi, Thailand

The aim of this study is to explain the social networks of the backyard chicken in Ratchaburi, Suphan Buri and Nakhon Pathom Provinces. In this study, we designed the nodes as groups of persons or places involved in activities relating to backyard chickens. The ties are all activities related to the nodes. The study applied a partial network approach to assess the spreading pattern of avian influenza. From 557 questionnaires collected from the nodes, the researchers found that the degree (the numbers of ties that a node has) and closeness (the distance from one node to the others) centralities of Nakhon Pathom were significantly higher than those of the others (P<0.001). The results show that compared with the remaining areas, this area is more quickly connected to many links. If the avian influenza virus subtype H5N1 was released into the network, the disease would spread throughout this province more rapidly than in Ratchaburi and Suphan Buri. The betweenness centrality in each of these provinces showed no differences (P>0.05). In this study, the nodes that play an important role in all networks are farmers who raise consumable chicken, farmers who raise both consumable chicken and fighting cocks, farmers' households that connect with dominant nodes, and the owners and observers of fighting cocks at arenas and training fields. In this study, we did not find cut points or blocks in the network. Moreover, we detected a random network in all provinces. Thus, connectivity between the nodes covers long or short distances, with less predictable behaviour. Finally, this study suggests that activities between the important nodes must receive special attention for disease control during future disease outbreaks.

Pinon shell polysaccharide enhances immunity against H9N2 avian influenza virus in chickens

This study investigated the potential of pinon shell polysaccharide (PSP) to enhance immunity against H9N2 subtype avian influenza virus (H9N2 AIV) in chickens. The effect of PSP treatment and H9N2 virus infection after PSP pretreatment were investigated by [3-(4, 5-dimethylthiazol-2-yl)-2, 3-diphenyl tetrazolium bromide] assay and analysis of major histocompatibility complex and cytokine mRNA expression. The in vivo effects on lymphocytes and serum antibody titers were also investigated. The IL-6 expression and antibody titers were enhanced in the first week after PSP treatment. These data indicate that prophylactic PSP administration reduces H9N2 AIV replication and promotes early humoral immune responses in young birds.

Complete Genome Sequence of a Novel H9N2 Subtype Influenza Virus FJG9 Strain in China Reveals a Natural Reassortant Event

A/chicken/FJ/G9/09 (FJ/G9) is an H9N2 subtype avian influenza virus (H9N2 AIV) strain causing high morbidity that was isolated from broilers in Fujian Province of China in 2009. FJ/G9 has been used as the vaccine strain against H9N2 AIV infection in Fujian Province of China. Here, we report the complete genome sequence of FJ/G9 with natural six-way reassortment, which is the most complex genotype strain in China and even in the world so far. The present findings will aid in understanding the complexity and diversity of H9N2 subtype avian influenza virus.

Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China

To investigate the risk of H5N1 subtype avian influenza virus (AIV) transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of haemagglutinin (HA) genes of H5N1 AIV in the environment were analyzed. One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis (SRH) while 160 environment samples (from sewage, birds stools, feathers and smearing samples of poultry cages) in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% (26/102), 50.0% (9/18) and 25.4% (17/67) respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% (50/160), and the positive rate of townships poultry markets was 37.3% (31/83), which were both higher than those from the inner cities poultry markets (24.7%, 19/77). H5 subtype AIV positive rate was different in the tested specimens, with ranking of positive rates were sewage (50.0%, 24/48), feathers (44.5%, 4/9), birds stools (29.8%, 14/47) and smearing samples of poultry cages (14.3%, 8/56), with statistically significant differences (P < 0.01). Four H5N1 HA genes TA cloning were successfully constructed and identified as Eurasian branch, similar to viruses isolated in mainland China and Hong Kong in the same group, according to genetic analysis. Sequence data of the four HA genes showed the same feature of high pathogenicity, compared to the H5N1 AIV from mainland China of human origin. The receptor specificities were still with avian influenza origin (QSG) and the connecting peptide between HA1 and HA2 possessing the polybasic motif (RERRRKK or RERRGKK). One of the reasons for H5N1 antibody positive rate of 25.5% among poultry markets workers was that there were large numbers of H5N1 subtype AIV detected in the environment of poultry markets and HA genes of H5N1 subtype AIV in the poultry markets environment carried molecular characteristics of highly pathogenic which could increase the risk for H5N1 subtype AIV transmission in the environment of poultry markets.

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first reported in Myanmar in 2006. In this study, we have characterized 6 HPAI (H5N1) viruses recovered from 2007-2010 as well as three additional available nucleotide sequences representing Myanmar AI outbreaks. Phylogenetic analysis showed that the Myanmar viruses belong to HPAI (H5N1) clades 7, 2.3.2 and 2.3.4. The result suggested that the HPAI (H5N1) viruses recovered from Myanmar had been introduced into the country by multiple introductions. Genetic analysis of the viruses confirmed the HPAI characteristics of the viruses.