Live Poultry Trade in Southern China Provinces and HPAIV H5N1 Infection in Humans and Poultry: The Role of Chinese New Year Festivities

Background

The number of outbreaks of highly pathogenic avian influenza virus of the H5N1 subtype (HPAIV H5N1) over the past 5 years has been drastically reduced in China but sporadic infections in poultry and humans are still occurring. In this study, we aimed to investigate seasonal patterns in the association between the movement of live poultry originating from southern China and HPAIV H5N1 infection history in humans and poultry in China.

Methodology/Principal Findings

During January to April 2010, longitudinal questionnaire surveys were carried out monthly in four wholesale live bird markets (LBMs) in Hunan and Guangxi provinces of South China. Using social network analysis, we found an increase in the number of observed links and degree centrality between LBMs and poultry sources in February and March compared to the months of January and April. The association of some live poultry traders (LPT’s) with a limited set of counties (within the catchment area of LBMs) in the months of February and March may support HPAIV H5N1 transmission and contribute to perpetuating HPAIV H5N1 virus circulation among certain groups of counties. The connectivity among counties experiencing human infection was significantly higher compared to counties without human infection for the months of January, March and April. Conversely, counties with poultry infections were found to be significantly less connected than counties without poultry infection for the month of February.

Conclusions/Significance

Our results show that temporal variation in live poultry trade in Southern China around the Chinese New Year festivities is associated with higher HPAIV H5N1 infection risk in humans and poultry. This study has shown that capturing the dynamic nature of poultry trade networks in Southern China improves our ability to explain the spatiotemporal dissemination in avian influenza viruses in China.     

A Cross-Sectional Study of Avian Influenza in One District of Guangzhou, 2013

Since Feb, 2013, more than 100 human beings had been infected with novel H7N9 avian influenza virus. As of May 2013, several H7N9 viruses had been found in retail live bird markets (LBMs) in Guangdong province of southern China where several human cases were confirmed later. However, the real avian influenza virus infection status especially H7N9 in Guangzhou remains unclear. Therefore, a cross-sectional study of avian influenza in commercial poultry farms, the wholesale LBM and retail LBMs in one district of Guangzhou was conducted from October to November, 2013. A total of 1505 cloacal and environmental samples from 52 commercial poultry farms, 1 wholesale LBM and 18 retail LBMs were collected and detected using real-time RT-PCR for type A, H7, H7N9 and H9 subtype avian influenza virus, respectively. Of all the flocks randomly sampled, 6 farms, 12 vendors of the wholesale LBM and 18 retail LBMs were type A avian influenza virus positive with 0, 3 and 11 positive for H9, respectively. The pooled prevalence and individual prevalence of type A avian influenza virus were 33.9% and 7.9% which for H9 subtype was 7.6% and 1.6%, respectively. None was H7 and H7N9 subtype virus positive. Different prevalence and prevalence ratio were found in different poultry species with partridges having the highest prevalence for both type A and H9 subtype avian influenza virus. Our results suggest that LBM may have a higher risk for sustaining and transmission of avian influenza virus than commercial poultry farms. The present study also indicates that different species may play different roles in the evolution and transmission of avian influenza virus. Therefore, risk-based surveillance and management measures should be conducted in future in this area.     

Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City,China

Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response.     

Complete Genome Sequence Analysis of an H6N1 Avian Influenza Virus Isolated from Guangxi Pockmark Ducks

We report here the complete genomic sequence of a novel H6N1 avian influenza virus strain, A/Duck/Guangxi/GXd-5/2010(H6N1), isolated from pockmark ducks in Guangxi Province, Southern China. All of the 8 gene segments of A/Duck/Guangxi/GXd-5/2010(H6N1) are attributed to the Eurasian lineage; the amino acid motif of the cleavage site between HA1 and HA2 was P-Q-I-E-T-R-G. These are typical characteristics of the low-pathogenicity avian influenza virus. This study will help to understand the epidemiology and molecular characteristics of avian influenza virus in ducks.     

2012-2015年广东省活禽市场外环境禽流感病毒污染状况研究

研究广东省活禽市场外环境禽流感病毒污染状况并及时发现人流感发病潜在的危险因素,为人流感防治提供科学参考依据。应用传染病技术监测平台信息管理系统数据,采用描述性流行病学方法分析各种亚型病毒感染的流行病学特征,研究2012-2015年广东省活禽市场外环境禽流感病毒污染。共采集检测广东省21个地市级样本33079份,FluA 总阳性率为24．23％,H5、H7和 H9型高致病性禽流感病毒阳性率分别为3．70％、3．89％和13．53％;除2012年阳性率呈现季节性增加外,其他年份 FluA 核酸检测阳性率均在冬春季出现一个高峰。不同部位或地点采集的标本中,宰杀或摆放禽肉案板表面阳性率最高（FluA39．49％,H58．41％,H77．41％,H923．84％）,而采集的粪便标本阳性率最低（FluA14．99％,H51．73％、H72．38％、和 H97．23％）;所采集的标本所对应的相关动物种类中,鸡（64．08％）、鸭（55．84％）和鸟类（51．92％）的禽流感病毒阳性率都达到50％以上,H5、H7和 H9在各禽类中均可以检出。同时发现,在环境中检出 H7亚型多的地区分布与其相应地区 H7N9感染的病例数呈显著相关性,（r ＝0．689,P ＜0．05）;对2322份样本进行 H6亚型核酸检测,总阳性率为2．58％,并选取 H5、H6和 H9亚型标本153份进行 N 亚型检测,检测出 H5N1、H5N2、H5N6、H6N2和 H9N2等多种亚型。2012-2015年广东省21个地市活禽市场均存在 HA 亚型（H5、H7、H9和 H6）和 NA 亚型（N1、N2、N6）等多种亚型的污染,污染程度呈现季节性分布,不同样本类型和禽类其禽流感病毒分状况不同,H7亚型的污染严重程度与 H7N9的病例感染数呈正相关性。     

Molecular Surveillance of Low Pathogenic Avian Influenza Viruses in Wild Birds across the United States: Inferences from the Hemagglutinin Gene

A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV) in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV). We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA) diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event), as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.     

2017–2019年我国南方地区高致病性H5N6亚型禽流感病毒血凝素蛋白分子特征分析

【背景】自2014年以来,H5N6禽流感病毒在我国家禽和活禽市场持续进化,成为人类和动物健康的重大威胁。【目的】对2017–2019年中国南方地区93株高致病性H5N6禽流感病毒的HA基因进行分子进化分析。【方法】接种9–11日龄鸡胚分离核酸检测阳性的H5N6标本,运用下一代测序平台对病毒分离物进行全基因组测序,从NCBI和GISAID数据库下载参考序列,利用BLAST、MEGA6.1及Clustal X等软件进行序列分析。【结果】2017–2019年,从189份江苏省H5亚型禽类/环境标本和1名H5N6患者咽拭子标本中共分离到43株病毒,完成了33株H5N6病毒的全基因组测序。下载网上同时期中国其他地区流行的H5N6毒株序列,对总计93株H5N6病毒的HA基因进行分子进化分析。93株H5N6病毒中有78株属于Clade 2.3.4.4h,9株病毒属于Clade 2.3.4.4e,4株H5N6病毒属于Clade 2.3.4.4b,1株属于Clade 2.3.4.4f,1株属于Clade 2.3.4.4g。所有93株病毒HA蛋白的裂解位点含有多个碱性氨基酸,表明它们都属于高致病性禽流感病...     

1株H6N6亚型禽流感病毒分子遗传特性分析

本研究采用无特定病原体(specific pathogen free,SPF)鸡胚,从某活禽市场环境中分离出1株H6N6亚型禽流感病毒(A/environment/Zhenjiang/zj18/2013,en/zj18)。通过二代测序技术进行全基因组测序,通过BLASTn 进行同源性检索,并采用MEGA5.0软件构建系统发生树。基因进化树分析表明,分离株en/zj18的所有8个基因节段(PB2、PB1、PA、HA、NP、NA、M和NS)均与近年来中国华东地区流行的H6N6亚型禽流感病毒的相应基因位于同一进化分支,与参考株的核苷酸同源性达96.7%～99.6%。分离株en/zj18的HA蛋白裂解位点为PQIETR↓GL,是低致病性禽流感病毒的分子特征。HA蛋白上关键受体结合位点190和228位(按H3亚型的HA蛋白序列排序)氨基酸分别是E和G,理论上更易与α2,3-半乳糖苷唾液酸受体结合。结果提示,需加强活禽市场禽流感病毒的持续监测,从而为有效应对禽流感病毒对公共卫生的持续威胁提供科学依据。     

Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×10² vs. 2.3×10⁴ vs. 8.7×10⁴; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.     

Cocirculation of Three Hemagglutinin and Two Neuraminidase Subtypes of Avian Influenza Viruses in Huzhou,China, April 2013: Implication for the Origin of the Novel H7N9 Virus

We detected three avian influenza hemagglutinin (HA) subtypes (H7, H9, and H5) and two neuraminidase (NA) subtypes (N9 and N2), as well as H7N9-related H9N9 reassortant intermediates, cocirculating among poultry in Huzhou, China, during April 2013. The results of our study reveal not only that Huzhou is one of the geographic origins of the novel H7N9 virus but also that cocirculation poses a potential threat to humans in the future.     

Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.     

Genetics,Receptor Binding Property,and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.     

Continued Evolution of H5N1 Influenza Viruses in Wild Birds,Domestic Poultry,and Humans in China from 2004 to 2009

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.The highly pathogenic H5N1 influenza viruses that emerged over a decade ago in southern China have evolved into over 10 distinct phylogenetic clades based on their hemagglutinin (HA) genes. The viruses have spread to over 63 countries and to multiple mammalian species, including humans, resulting in 498 cases of infection and 294 deaths by 6 May 2010 according to the World Health Organization (WHO) (http://www.who.int). To date, none of the different H5N1 clades has acquired the ability to consistently transmit among mammalian species. The currently circulating H5N1 viruses are unique in that they continue to circulate in avian species. All previous highly pathogenic H5 and H7 viruses have naturally “burned out” or were stamped out because of their high pathogenicity in domestic poultry. While there is growing complacency about the potential of H5N1 “bird flu” to attain consistent transmissibility in humans and develop pandemicity, it is worth remembering that we have no knowledge of the time that it took the 1918 Spanish, the 1957 Asian, the 1968 Hong Kong, and the 2009 North American pandemics to develop their pandemic potentials. We may therefore currently be witnessing in real time the evolution of an H5N1 pandemic influenza virus.H5N1 avian influenza viruses (AIVs) were first detected in sick geese in Guangdong province in 1996, and both nonpathogenic and highly pathogenic (HP) H5N1 viruses were described (18). In 1997, H5N1 reassortant viruses that derived the HA gene from A/goose/Guangdong/1/96 (GS/GD/1/96)-like viruses and the other genes from H6N1 and/or H9N2 viruses caused lethal outbreaks in poultry and humans in Hong Kong (6, 7). Since then, long-term active surveillance of influenza viruses in domestic poultry has been performed, and multiple subtypes of influenza viruses have been detected in chickens and ducks in China (16, 19, 37). H5N1 influenza viruses have been repeatedly detected in apparently healthy ducks in southern China since 1999 (4, 13) and were also detected in pigs in Fujian province in 2001 and 2003 (39).Since the beginning of 2004, there have been significant outbreaks of H5N1 avian influenza virus infection involving multiple poultry farm flocks in more than 20 provinces in China (2). H5N1 viruses resulted in the deaths of millions of domestic poultry, including chickens, ducks, and geese, as the result of infection or of culling and the deaths of thousands of wild birds (5, 20). Thirty-eight human cases of HP H5N1 infection with 25 fatalities have been associated with direct exposure to infected poultry (WHO; http://www.who.int). Since 2004, the vaccination of domestic poultry has been used for the control of HP H5N1 influenza virus in China. While this strategy has been effective at reducing the incidence of HP H5N1 in poultry and at markedly reducing the number of human cases, it is impossible to vaccinate every single bird due to the enormous poultry population. Outbreaks of H5N1 influenza virus still continue to occur in poultry although at a reduced frequency.A previous study by Smith et al. reported that a “Fujian-like” H5N1 influenza virus emerged in late 2005 and predominated in poultry in southern China (26). Those authors suggested that vaccination may have facilitated the selection of the “Fujian-like” sublineage. Here, we analyzed 51 representative H5N1 viruses that were isolated from wild birds, domestic poultry, and humans from 2004 to 2009 in China and described their genetic evolution and antigenicity profiles. Our results indicate that H5N1 influenza viruses in southern China, including the “Fujian-like” viruses, are complicated reassortants, which could be well protected against by GS/GD/1/96 virus-based vaccines. We documented the emergence of the latest variant of H5N1 (A/chicken/Shanxi/2/06 [CK/SX/2/06]) that broke through existing poultry vaccines. We show that this variant is less pathogenic in mice and ducks than the earlier strains and propose that the variant was not selected by the use of vaccines.     

人感染H7亚型禽流感事件

本研究综述了自1959年以来国内外发生的人感染H7亚型禽流感事件。大多数是在家禽爆发禽流感期间,农场工人在处置感染鸡群过程中被暴露而感染;也有曾接触活禽或曾到过活禽市场而感染;有经禽流感病毒致病的哺乳动物（海豹）感染于人或实验室感染（事故）所致。引起人感染的H7亚型中已知有H7N2、H7N3、H7N7以及2013年在中国发现的新的致病亚型H7N9。H7N2、H7N3、H7N7感染以结膜炎为主,大多为轻症;而H7N9感染以严重的呼吸道感染为特征,表现为重症肺炎,呼吸窘迫综合症,病死率高达33.6%。     

近年来华东地区家鸭中禽流感病毒的亚型分布

[目的]为了研究近年来华东地区家鸭中禽流感病毒的亚型分布情况.[方法]对2002-2006年分离自华东地区家鸭的180株禽流感病毒的HA亚型和其中88株禽流感病毒的NA亚型分别进行了测定.[结果]近年来华东地区家鸭中至少存在9种HA亚型和6种NA亚型组成的H1N1,H3N1,H3N2,H3N8,H4N6,H5N1,H5N2,H6N2,H6N8,H8N4,H9N2,H10N3,H11N2共13种亚型的禽流感病毒.[结论]华东地区家鸭中有多种亚型的禽流感病毒分布,应加强家鸭禽流感的监测和防制工作.     

Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States

The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.     

Complete genomic sequence of a novel natural recombinant H6N2 influenza virus from chickens in Guangdong, Southern China

Here, we reported the complete genome sequence of a novel H6N2 avian influenza virus (AIV) isolated from chicken in Guangdong, Southern China, in 2011 which was a natural recombinant virus between the H6N2 and H5N1 subtypes. It will help to understand the epidemiology and molecular characteristics of H6N2 influenza virus in Southern China.     

Live bird markets of Bangladesh: H9N2 viruses and the near absence of highly pathogenic H5N1 influenza

Avian influenza surveillance in Bangladesh has been passive, relying on poultry farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, the results of an active surveillance effort focusing on the live-bird markets are presented. Prevalence of influenza infection in the birds of the live bird markets is 23.0%, which is similar to that in poultry markets in other countries. Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also observed. The highly pathogenic H5N1-subtype virus was observed at extremely low prevalence in the surveillance samples (0.08%), and we suggest that the current risk of infection for humans in the retail poultry markets in Bangladesh is negligible. However, the high prevalence of the H9 subtype and its potential for interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and attenuation of host morbidity, highlight the importance of active surveillance of the poultry markets.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

水禽流感病毒分离株A/Duck/Yangzhou/233/02(H6N2)膜蛋白基因遗传进化分析

采用常规的血清学收验和特异性RT-PCR方法对华东地区家养水禽中流感病毒的带毒状况进行两年多的监测,分离鉴定出多株H6亚型禽流感病毒。对其中的一株A/Duck/Yangzhou/233/02(H6N2)(简称DkYZ23302)(H6N2)的表面膜蛋白基因进行了序列测定,并与GenBank中收录的其它序列进行了比较,遗传进化结果表明DkYZ23302的血凝素基因(HA)与近年香港分离的鸭源毒株DkHK346199(H6N1)、中国台湾鸡源毒株CkTaiwanna398的亲缘关系最近;而神经氨酸酶基因(NA)遗传进化分析结果表明DkYZ23302(H6N2)的NA基因起源于禽源H9N2亚型流感病毒,这可能是不同亚型禽流感病毒在水禽体内发生基因重配的结果。DkYZ23302(H6N2)的HA推导的氨基酸剪切位点序列为P-Q-I-E-T-R-D,为典型低致病性禽流感病毒的特征序列,与对SPF鸡的致病力试验相吻合。