Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March–April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.     

Genetic characterization of the NS gene indicates co-circulation of two sub-lineages of highly pathogenic avian influenza virus of H5N1 subtype in Northern Europe in 2006

The non-structural (NS) gene of highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI-H5N1) isolated in Baltic Sea area of Sweden in 2006 was studied. The phylogenetic analysis data demonstrated that two distinct sub-lineages of HPAI-H5N1 were circulating during the outbreak in Northern Europe in Spring 2006. Sub-lineage I viruses fell into the same clade as viruses found in Denmark and Germany and formed a sub-clade which also included viruses isolated in the Russian Federation in late 2005. Sub-lineage II viruses formed a sub-clade closely related to European, Middle Eastern and African isolates reported in 2006. Analysis of the inferred amino acid sequences of the NS1 protein showed a deletion of five amino acids at positions 80–84. No viruses represented in this study contained Glu92 in the NS1 and all isolates contained the avian-like ESKV amino acid sequences at the NS1 C-terminal end. Sub-lineage I isolates contained unique substitutions V194I in NS1 and G63E in Nuclear export protein (NEP).     

Genetic characterization of avian influenza viruses isolated in Israel during 2000–2006

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000–2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005–2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.     

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Genetic variation of the hemagglutinin of avian influenza virus H9N2

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.     

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.     

Characterization of H5N1 highly pathogenic avian influenza virus strains isolated from migratory waterfowl in Mongolia on the way back from the southern Asia to their northern territory

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.     

Characterization of a highly pathogenic avian influenza H5N1 virus sublineage in poultry seized at ports of entry into Vietnam

Highly pathogenic avian influenza H5N1 virus was detected in poultry seized at two ports of entry located in Lang Son Province, Vietnam. Sequence analysis of the hemagglutinin (HA) genes from five H5N1 virus isolates and ten PCR amplicons from chicken cloacal samples revealed their close phylogenetic relationship to clade 7 H5N1 HA genes. However, these HA genes exhibited extensive genetic divergence at both the nucleotide and amino acid levels in comparison to previously described clade 7 viruses; e.g., A/chicken/Shanxi/2/2006. In addition, hemagglutination inhibition tests revealed antigenic differences between these and previously isolated H5N1 viruses from Vietnam. These results indicate that viruses with clade 7 HA are evolving rapidly in poultry in Southeast Asia.     

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D¹⁴⁴ mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ408506-DQ408529.     

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.     

北京市首例人感染H9N2禽流感病例的流行病学调查

目的 分析2016年12月北京市朝阳区确诊的l例人感染H9N2禽流感病例流行病学特征.方法 访谈病例发病前后的相关知情人,调查病例发病经过和可能感染来源.采集病例、密切接触者及环境标本,应用实时荧光定量PCR(real-timePCR)方法,检测甲型流感病毒及H9N2禽流感病毒特异的核酸片段.对病例咽拭子标本和l件阳性环境标本中的H9N2禽流感病毒血凝素(Hemagglutinin,HA)基因进行序列测定和分析.结果 病例表现为轻症流感样病例症状,发病前15天曾有活禽市场暴露史.该市场环境标本中H9N2禽流感病毒阳性率为18.2％ (2/11).病例咽拭子标本与市场环境标本中病毒的HA基因高度同源,氨基酸相似度为100％,在进化关系上同属于欧亚系的Y280分支.4名密切接触者在医学观察期内未出现过发热、咳嗽等呼吸道症状.结论 病例感染来源与活禽市场环境暴露有关.加强流感监测以及活禽市场的管理对于防控人感染禽流感疫情具有重要意义.     

Serologic and genetic characterization analyses of a highly pathogenic influenza virus (H5N1) isolated from an infected man in Shenzhen

Highly pathogenic avian influenza (HPAI) H5N1 virus caused a wave of outbreaks in China during 2005--2006, resulting in a total of 20 cases of human infection in 14 provinces of China. On June16, 2006, a case of H5N1 human infection was confirmed in Shenzhen. The virus isolated from the patient, A/Guangdong/2/06, was characterized genetically and the relationship between the tracheal virus load and the antibody titer of the infected man was analyzed. Serological analysis confirmed that the patient's neutralizating antibodies had been generated 2 weeks after the onset of symptoms. The patient's serum antibodies could efficiently neutralize A/Guandong/2/06 infectivity in vitro. Phylogenetic analysis showed that the H5N1 virus of Shenzhen belonged to subclade 2.3.4, which contained viruses that were mainly responsible for the outbreaks in domestic poultry and in the cases of human infection in southern China. Homology and molecular characterization analysis revealed that all the segments of Shenzhen H5N1 virus still belonged to avian segments. Several specific amino acid residue mutations were detected.     

Phylogenetic analysis of low pathogenicity H5N1 and H7N3 influenza A virus isolates recovered from sentinel, free flying, wild mallards at one study site during 2006

From August 2 to October 11, 2006, clusters of low pathogenicity (LP) North American lineage H5N1 and H7N3 avian influenza A viruses (AIV), and other subtypes, were recovered from free-flying, resident, wild mallards used as sentinels at one site. The antigenic subtypes, pathogenicity potential, and Sanger sequencing of the isolates determined the H5N1 and H7N3 isolates were only recovered from samples collected on 8/2/2006 and 9/8/2006, respectively. However, subsequent efforts using next-generation sequencing (NGS) and additional Sanger sequencing found partial H7 segments in other HA-NA virus combinations on 8/2/2006, 9/8/2006 and 10/11/2006. It is well established that over larger geographic areas and years AIVs form transient genomic constellations; this sequential sampling data revealed that over a short period of time the dynamics of AIVs can be active and newer sequencing platforms increase recognition of mixed infections. Both findings provide further insight into the natural history of AIVs in natural reservoirs.     

Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.     

Molecular changes associated with the transmission of avian influenza a H5N1 and H9N2 viruses to humans.

In order to identify molecular changes associated with the transmission of avian influenza A H5N1 and H9N2 viruses to humans, the internal genes from these viruses were compared to sequences from other avian and human influenza A isolates. Phylogenetically, each of the internal genes of all sixteen of the human H5N1 and both of the H9N2 isolates were closely related to one another and fell into a distinct clade separate from clades formed by the same genes of other avian and human viruses. All six internal genes were most closely related to those of avian isolates circulating in Asia, indicating that reassortment with human strains had not occurred for any of these 18 isolates. Amino acids previously identified as host-specific residues were predominantly avian in the human isolates although most of the proteins also contained residues observed previously only in sequences of human influenza viruses. For the majority of the nonglycoprotein genes, three distinct subgroups could be distinguished on bootstrap analyses of the nucleotide sequences, suggesting multiple introductions of avian virus strains capable of infecting humans. The shared nonglycoprotein gene constellations of the human H5N1 and H9N2 isolates and their detection in avian isolates only since 1997 when the first human infections were detected suggest that this particular gene combination may confer the ability to infect humans and cause disease. J. Med. Virol. 66:107-114, 2002. Published 2002 Wiley-Liss, Inc.     

Isolation of a genotypically unique H5N1 influenza virus from duck meat imported into Japan from China

An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed >97% nucleotide identity with their counterparts from recent H5N1 viruses, but that the remaining two genes (PB2 and NP) were derived from other unknown viruses. This duck meat isolate was highly pathogenic to chickens upon intravenous or intranasal inoculation, replicated well in the lungs of mice and spread to the brain, but was not as pathogenic in mice as H5N1 human isolates (with a dose lethal to 50% of mice (MLD50)=5x10(6) 50% egg infectious doses [EID50]). However, viruses isolated from the brain of mice previously infected with the virus were substantially more pathogenic (MLD50=approximately 10(2) EID50) and possessed some amino acid substitutions relative to the original virus. These results show that poultry products contaminated with influenza viruses of high pathogenic potential to mammals are a threat to public health even in countries where the virus is not enzootic and represent a possible source of influenza outbreaks in poultry.     

Full-genome analysis of avian influenza virus H9N2 from Bangladesh reveals internal gene reassortments with two distinct highly pathogenic avian influenza viruses

Low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh, LPAIVs of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in commercial and backyard poultry. The aim of this study was to characterize LPAIVs of subtype H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryos. All eight gene segments were amplified by RT-PCR, cloned, and subjected to full-length sequencing. The sequence data obtained were compared with reference strains available in GenBank. Phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed a close relationship to Indian, Pakistani and Middle Eastern isolates and identified an ancestor relationship to LPAIV H9N2 Quail/HK/G1/1997. The internal genes M and NP belong to lineage G1, whereas NS, PA, PB1 and PB2 belong to the prototype virus A/Chicken/Korea/38349-p96323/96. The internal genes showed high sequence homology to an HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies to recently circulating HPAIV H5N1 from Bangladesh, revealing two independent reassortment events. Examination of the hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor-binding sites indicated a binding preference for human-type receptors. Several mutations in internal proteins are associated with increased virulence and altered host range, while other amino acids were found to be highly conserved among LPAIV H9N2 isolates.     

A/鹅/广东/2/96(H5N1)流感毒株7和8 RNA节段核苷酸序列测定

目的　弄清A鹅广东296(H5N1)毒株RNA7和8核苷酸全序列及它们与AHK15697(H5N1)毒株RNA7和8之间的内在关系,并为今后流感病毒M和NS基因研究打下基础。方法　病毒粒RNA经逆转录合成cDNA,经聚合酶链反应(PCR)扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　A鹅广东296(H5N1)毒株RNA7长度为1027个核苷酸,编码M1(含252个氨基酸)和M2(含97个氨基酸)的蛋白。其RNA8长度为890个核苷酸,编码NS1(含230个氨基酸)和NS2(含121个氨基酸)非结构蛋白。其M1,M2,NS1和NS2蛋白分子上氨基酸序列与AHK15697(H5N1)毒株间同源性分别为976%,928%,657%和769%。结论　A鹅广东296(H5N1)毒株RNA7和8长度分别为1027和890个核苷酸,此两节段RNA均属禽类毒株。AHK15697(H5N1)毒株的RNA7和8不是来自A鹅广东296(H5N1)毒株。     

Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam

Highly pathogenic avian influenza virus H5N1 is endemic in poultry in East and Southeast Asia with disease outbreaks recently spreading to parts of central Asia, Europe and Africa. Continued interspecies transmission to humans has been reported in Vietnam, Thailand, Cambodia, Indonesia and China, causing pandemic concern. Here, we genetically characterize 82 H5N1 viruses isolated from poultry throughout Indonesia and Vietnam and 11 human isolates from southern Vietnam together with sequence data available in public databases to address questions relevant to virus introduction, endemicity and evolution. Phylogenetic analysis shows that all viruses from Indonesia form a distinct sublineage of H5N1 genotype Z viruses suggesting this outbreak likely originated from a single introduction that spread throughout the country during the past two years. Continued virus activities in Indonesia were attributed to transmission via poultry movement within the country rather than through repeated introductions by bird migration. Within Indonesia and Vietnam, H5N1 viruses have evolved over time into geographically distinct groups within each country. Molecular analysis of the H5N1 genotype Z genome shows that only the M2 and PB1-F2 genes were under positive selection, suggesting that these genes might be involved in adaptation of this virus to new hosts following interspecies transmission. At the amino acid level 12 residues were under positive selection in those genotype Z viruses, in the HA and PB1-F2 proteins. Some of these residues were more frequently observed in human isolates than in avian isolates and are related to viral antigenicity and receptor binding. Our study provides insight into the ongoing evolution of H5N1 influenza viruses that are transmitting in diverse avian species and at the interface between avian and human hosts.