一株鹌鹑源禽流感病毒A/quail/Jilin/7/2015(H9N2)的遗传进化分析和致病性研究

为探究从死亡鹌鹑肺脏分离鉴定出的1株H9N2亚型禽流感病毒(AIV)A/quail/Jilin/7/2015(简称QA/JL/7/15)在流感病毒生态分布中的进化关系,本研究对QA/JL/7/15株进行了全基因序列测定,并结合Gen Bank中登录的代表性H9N2亚型流感病毒的基因组序列及其它参考株序列进行了遗传进化分析。结果显示,QA/JL/7/15为一株重组病毒,血凝素基因(HA)来自A/chicken/Jiangsu/1/00类病毒株;神经氨酸酶(NA)来自A/chicken/Guangxi/KMIII/1999类病毒株;PB2基因来自A/duck/Shantou/163/2004类病毒株;PB1、PA、NP和NS基因来自A/chicken/Shanghai/F/98类病毒株;M基因来自A/Quail/Hong Kong/G1/97类病毒株。HA基因与近年中国湖北分离株A/chicken/Hubei/R386/2012(H9)的核苷酸序列同源性最高(98.5%),HA裂解位点推导氨基酸序列为"-RSSR-",为典型低致病性AIV的特征序列,动物实验也显示该病毒株对鸡和小鼠呈现低致病性。本研究首次证实H9N2重组病毒株在我国鹌鹑中出现,为AI防控提供了实验依据。     

3株H9N2亚型禽流感病毒HA基因变异分析

对近10年来在同一地区分离到的3株H9N2亚型禽流感病毒血凝素(HA)基因进行扩增和序列分析,探讨H9N2亚型流感病毒变异情况.3个毒株的HA基因全长1 683bp,编码560个氨基酸;其裂解位点均为R-S-S-R,属于低致病力毒株;推导的HA基因氨基酸序列均含7个相同的潜在糖基化位点,受体结合位点为禽源性流感病毒特异性序列,左侧臂氨基酸均为NGQQG,右侧臂均为GTSKA.3个分离株与参考株核苷酸与氨基酸同源率均较高,仅有一些非关键位点突变.它们均属欧亚种系,与A/Duck/Hong Kong/Y280/97代表株亲源关系较近.研究结果从分子水平上证明H9N2亚型禽流感病毒近年来未发生较大变异.     

2011～2014年广西H9N2亚型禽流感病毒HA基因遗传变异分析

为了解H9N2亚型禽流感病毒广西分离株的遗传变异情况,采用RT-PCR技术扩增了10株从2011~2014年广西不同地区分离的H9N2亚型AIV的HA基因片段,并对所得序列进行同源性和遗传进化分析。结果显示,10个毒株的HA基因全长1 683 bp,编码560个氨基酸;10株分离株的裂解位点均为PSRSSR↓GLF,为典型低致病性禽流感病毒的特征序列。10株分离株234位氨基酸均为L,具有与哺乳动物唾液酸α-2,6受体结合的特征。分离株与参考毒株HA基因的核苷酸与氨基酸同源性均较高,分别为79.3%~98.7%和85.0%~98.6%,均属欧亚种系,其中与A/Duck/Hong Kong/Y280/97亲源关系较近。研究结果从分子水平上证明H9N2亚型禽流感病毒近年来未发生较大变异。     

2011～2014年安徽省发病鸡群H9亚型禽流感病毒HA基因的遗传进化分析

为了解安徽省发病鸡群H9N2亚型禽流感病毒(Avian infl uenza virus,AIV)的变异情况,本研究对2011~2014年安徽省发病鸡群的368份组织样品进行AIV的鸡胚分离和RT-PCR检测,分离鉴定到17株H9N2 AIV。对这17株进行HA基因测序及系统进化分析,结果表明:17株HA基因核苷酸序列的相似性为88.5%~99.6%,属于A/Duck/Hong Kong/Y280/1997病毒亚系。氨基酸比对分析显示,裂解位点326~329位氨基酸为典型的低致病性AIV特征性序列。与A/Chicken/Shanghai/F/98疫苗株相比,17株HA蛋白受体结合位点的226位均为亮氨酸(L),呈现了人流感受体结合特性;5株200位的点突变(T→I),导致了200位潜在糖基化位点的缺失。可见安徽省分离的H9N2 AIV大部分毒株基因序列已发生变异,目前使用的疫苗能否为禽群提供足够的保护力还需要作进一步的研究。     

H9N2亚型猪流感病毒A／Swine／Shandong／1／02分离株的基因序列分析

从有肺炎症状的病猪中分离到1株H9N2亚型猪流感病毒（SIV）A／Swine／Shandong／1／02，对其进行了全病毒基因序列分析。结果表明，该毒株8个基因片段的核苷酸序列均来自禽流感病毒（AIV），与我国目前家禽中流行的H9N2亚型AIV毒株具有很高的同源性，与A／Duck／Hong Kong／Y280／97（H9N2）的同源性为94．1％～98．9％，与A／Chicken／Beijing／1／94（H9N2）的同源性为94．5％～98．2％；推导的其血凝素（HA）裂解位点处的氨基酸序列为P—A-R—S-S-R，完全符合H9亚型AIV欧亚分支中的类A／Chicken／Beijing／1／94亚分支的特征；基因分析结果表明，该分离株的所有基因片段均来源于H9N2亚型AIV，它可直接感染猪，并导致发病，但它并未在猪体内发生重组。     

14株H3亚型禽流感病毒全基因组序列分析

为了解2016年至2017年在我国部分省市活禽市场的家禽体内分离得到的14株H3亚型禽流感病毒的流行情况和遗传特点。采用RT-PCR技术对这14株H3亚型禽流感毒株的全基因进行扩增,并对所得序列进行遗传演化分析。14株分离株中有5株H3N8,7株H3N2,2株H3N3。结果表明,14株毒株中AIV的HA蛋白的裂解位点氨基酸序列均为PE-KQTR↓GLF,只有1个碱性氨基酸,符合低致病性禽流感的分子特征。HA基因的受体结合位点为226(Q)、228(G)具有典型的禽源特征。遗传进化分析表明,除了毒株A/Duck/Fujian/F1142/2016(H3N8)的NS基因属于北美分支外,其余毒株全部基因均属于欧亚分支。通过对H3亚型AIV的遗传进化关系和流行情况进行分析,以期对H3亚型AIV的进化研究提供一些参考依据。     

鸭流感病毒4/2004-H6N2亚型毒株的血凝素基因全序列克隆与分析

2004初从正常鸭群中分离到一株鸭源禽流感病毒，命名为A／Duck／HN／4／2004(H6N2)。经对血凝素基因(HA)序列分析发现HA基因全长为1744bp，共编码566个氨基酸，在裂解位点仅含一个碱性氨基酸-精氨酸(R)，符合LPAIV的标准。将所得基因序列与已发表的同一亚型参考序列分析表明，与H6亚型流感HA基因同源性为89．2％-97．1％，经分子遗传演化分析表明本次分离株与香港分离株A／Duck／Hong Kong／3600／99(H6N2)、A／Duck／Hong Kong／3600／99(H6N2)最近。     

鸭源流感病毒分离株表面蛋白基因的克隆及序列分析

从湖南分离得到一株H5N1亚型禽流感病毒(AIV),首先设计合成两对HA和NA基因特异性引物,采用两步法RT-PCR,对鸭流感病毒株 A/Duck/HunanWugang/2/2004(H5N1)(简称DK/HNWG/2/04)的表面蛋白基因进行序列测定,并与国内外已经发表的H5N1亚型毒株表面蛋白基因进行序列分析和比较.结果表明,HA和NA基因全长分别约为1.7 kb和1.4 kb,分离株与14个参考毒株HA基因的同源性为95.9%～98.0%,NA基因的同源性为87.0%～98.4%.根据HA基因核苷酸序列推导HA裂解位点氨基酸,发现分离株的裂解位点包含多个碱性氨基酸,符合高致病性禽流感的特征.     

H9N2亚型禽流感病毒西藏分离株的全基因组遗传进化分析

根据GenBank登陆的H9N2亚型AIV全基因组序列,设计了8对引物,运用RT-PCR的方法获取了3株H9N2亚型AIV西藏分离株A/Chiken/Tibet/S1/2009、A/Duck/Tibet/S2/2009和A/Chiken/Tibet/S4/2009的8个基因序列,并对所得序列进行同源性及遗传进化分析.结果显示,分离毒株的各基因片段均有完整的开放阅读框,HA基因裂解位点处均为RSSR/G,符合低致病性禽流感的特征;3株西藏分离毒株之间同源性为98%～99%,可能起源于同一种系;各分离毒的HA、NS和NA基因与欧亚分支A/Chiken/Beijing/1/94分支中的A/Duck/HongKong/Y280/97遗传距离最近,而M、NP、PA、PB1和PB2基因与欧亚分支中另一分支A/Quail/HongKong/G1/97群系遗传关系近.由此可见3株分离株的各基因所属分支不具有统一性,说明本研究所分离的3株H9N2亚型AIV西藏分离毒株可能是来源于不同亚系AIV的毒株在感染同一种宿主时发生基因重排的产物.     

4株H3N2亚型猪源流感病毒HA基因的序列测定与特性分析

从广东省不同猪场分离到4株H3N2亚型猪源流感病毒A/Swine/Guangdong/01/2004、A/Swine/Guang-dong/02/2004、A/Swine/Guangdong/03/2004、A/Swine/Guangdong/04/2004.根据GenBank公布的H3N2亚型猪源流感病毒的HA基因序列,设计1对引物,运用RT-PCR方法扩增四株病毒的HA基因,并进行测序和分析.同源性分析和遗传进化分析表明本实验的4株H3N2亚型SIV HA基因核苷酸序列同源性为99.8%～99.9%,在遗传进化树中均位于同一分支上.与参考毒株的比较分析表明,4个毒株与WHO推荐的2001-2004年北半球H3N2亚型流感疫苗株A/Moscow/10/99 HA基因的核苷酸序列同源性最高为99.4%～99.5%,4个毒株与A/Moscow/10/99 HA基因在遗传进化树中位于同一个小分支上.氨基酸序列比较发现,4个毒株HA基因裂解位点处的氨基酸序列均为PEKQTR↓G,4个毒株推导的氨基酸序列中均有11个糖基化位点,4个毒株HA蛋白226位受体结合位点(RBS)处氨基酸均为异亮氨酸(Ⅰ).4个毒株HA基因的氨基酸序列、受体结合位点以及糖基化位点均与A/Moscow/10/99相应的氨基酸序列一致.本试验的4株H3N2亚型猪源流感病毒的HA基因属于以A/Moscow/10/99为代表的近代类人H3N2亚型流感病毒,在一定程度上揭示了广东省H3N2亚型猪流感病毒HA基因进化与流行情况.     

福建省鸭禽流感病毒分离株（A/Duck/Fujian/FQ107/2007〔H9N2〕）全基因测序及遗传进化分析

为了解一株可引起产蛋鸭产蛋异常的H9N2亚型禽流感病毒A/Duck/Fujian/FQ107/2007（H9N2）（以下简称Dk/FQ107/07）分离株的分子特性及其遗传进化地位,运用RT-PCR方法对其基因组进行扩增,克隆至pMD18-T载体后测序。结果显示,Dk/FQ107/07病毒株的血凝素（haemagglutini,HA）蛋白裂解位点的氨基酸组成为-PARSSR↓GLF-,其静脉接种指数（intravenous pathogenicity index,IVPI）为0.04,符合低致病性禽流感病毒特征;Dk/FQ107/07株HA基因与A/chicken/Shantou/5269/2005（H9N2）同源性最高,为98.9%,和我国首次哺乳动物流感病毒分离株A/Swine/HongKong/9/98（H9N2）有较近的遗传进化关系,三者均属于经典的H9N2/Y280群系;神经氨酸酶（neuraminidase,NA）基因与中国大陆首次分离株A/chicken/Beijing/1/1994（H9N2）相比,在63、64、65位点上缺失3个氨基酸（T、E、I）;核蛋白（nucleoprotein,NP）基因与高致病性鸭源禽流感分离株A/Duck/Fujian/1734/05（H5N1）和A/Duck/Fujian/9713/2005（H5N1）在同一遗传进化分支上,而从聚合酶（polymerase PA,PA）基因的遗传进化分析发现其基因属于H9N2/Y439群系。由此可见,Dk/FQ107/07可能是由不同禽流感病毒基因亚群间发生自然重排的产物。     

上海市三株H9N2鸭禽流感病毒全基因遗传进化分析

为了解上海市鸭群中H9N2亚型禽流感病毒（Avian influenza virus,AIV）的遗传变异特征,以及与疫苗株A/Chicken/Shan dong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离,对2007年和2009年分离自上海市鸭气管和泄殖腔样品采用荧光RT-PCR检测,将H9亚型禽流感病毒核酸阳性样品处理后,经鸡胚尿囊腔接种分离病毒,HI进一步确定血凝素（haemagglutin,HA）亚型,随后进行了全基因测序,并结合GenBank中的相关序列进行遗传进化分析。结果表明：3株分离毒株为H9N2亚型鸭禽流感病毒,HA蛋白裂解位点的氨基酸组成为PARSSRGLF,符合低致病性禽流感病毒特征,均属于经典的H9N2 Ck/Bei群系;NA基因均属于Y280系;NP、PA基因和A/Goose/Guangdong/1/1996（H5亚型）归为一群;PB2和M基因属于Qa/HK/G1/97系;NS基因仍为Ck/Bei系;2007年的分离株和2009年的分离株在PB1基因上分属不同亚群。3株病毒的HA1基因与疫苗株A/Chicken/Shandong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离均大于7%。由此可见,3株鸭H9N2亚型毒株可能是由不同禽流感病毒基因亚群间发生自然重排的产物,现有疫苗对分离株的保护性需要进一步评估。     

H9N2亚型禽流感病毒血凝素和神经氨酸酶基因的遗传分析

H9N2亚型禽流感病毒自1994年在中国首次发现以来,一直在家禽中流行,其导致的产蛋下降和发病死亡给养禽业发展带来严重危害。以前的研究发现中国的H9N2亚型禽流感病毒在进化过程中形成多个基因型,其表面抗原蛋白血凝素基因(HA)可被划分为以A/chicken/Beijing/1/94、A/quail/Hong Kong/G1/97(G1)和A/chicken/Heilongjiang/35/01等为代表的3个亚群,神经氨酸酶基因(NA)可被划分为以A/chicken/Beijing/1/94、A/quail/Hong Kong/G1/97(G1)和A/chicken/Hong Kong/G9/97(G9)等为代表的3个亚群。其中类G1病毒的HA基因只在香港分离株中出现。本研究对我国2003年～2004年从禽类中分离的H9N2亚型禽流感病毒血凝素(HA)和神经氨酸酶(NA)基因进行测定和遗传演化分析,结果表明其中11株病毒的HA基因属于CK/BJ/1/94群系,NA基因属于CK/BJ/1/94或DK/HK/G9/97群系,并首次发现两株病毒含有类G1病毒HA和NA基因,而且这些类G1病毒具有不同的抗原性以及人流感病毒的受体结合位点。本研究结果提示应对H9N2病毒的防治及其公共卫生意义予以高度重视。     

近年来中国H9亚型禽流感分离株谱系分析

从GenBank中下载所有来自中国（含港、澳、台）的H9亚型禽流感病毒血凝素基因885条核苷酸序列（长度≥900bp）,用MEGA5.0软件进行谱系分析。结果表明我国近年来H9亚型禽流感病毒以第h9.4.2.5分支为主（代表株为A/chicken/Guangxi/55/2005）,而不是WHO新近报告所列出的4株病毒（A/Quail/HongKong/G1/97、A/chicken/HongKong/G9/97、A/duck/HongKong/Y280/97、A/HongKong/33982/2009）所代表的分支。此分析结果对于研制针对这一病毒感染的疫苗有重要指导意义。     

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.     

两株H6N2亚型鸡源禽流感病毒的分离鉴定

本研究利用血凝抑制试验(HI)、反转录-聚合酶链式反应(RT-PCR)、基因测序等方法,对广东省某活禽交易市场进行流行病学调查时获得的两株非H5、H9亚型禽流感病毒65株和C7株进行了亚型鉴定。结果表明这两株病毒均具有血凝活性,且能被抗H6亚型禽流感病毒标准阳性血清特异性抑制。用针对禽流感病毒的M基因、H6亚型禽流感病毒HA基因、N2亚型禽流感病毒NA基因特异性鉴定引物对65株和C7株进行RT-PCR扩增,分别获得特异性目的片段。测序及BLAST分析表明两株分离株与H6N2亚型禽流感广东分离株的HA基因和NA基因核苷酸序列相似性均高达95%以上。将该两分离株鉴定为H6N2亚型禽流感病毒,并命名为A/Chicken/Guangdong/65/2009、A/Chicken/Guangdong/C7/2009。     

Isolation and genetic characterization of avian origin H9N2 influenza viruses from pigs in China

As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the adaptation of avian influenza viruses to humans. In April 2006, a disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) occurred in several pig farms and subsequently overwhelmed almost half of China with more than 2,000,000 cases of pig infection. Here we report a case in which four swine H9N2 influenza viruses were isolated from pigs infected by highly pathogenic PRRSVs in Guangxi province in China. All the eight gene segments of the four swine H9N2 viruses are highly homologous to A/Pigeon/Nanchang/2-0461/00 (H9N2) or A/Wild Duck/Nanchang/2-0480/00 (H9N2). Phylogenetic analyses of eight genes show that the swine H9N2 influenza viruses are of avian origin and may be the descendants of A/Duck/Hong Kong/Y280/97-like viruses. Molecular analysis of the HA gene indicates that our H9N2 isolates might have high-affinity binding to the alpha2,6-NeuAcGal receptor found in human cells. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially after the emergence of highly pathogenic PRRSVs in pigs in China.     

Comparative analysis of receptor-binding specificity and pathogenicity in natural reassortant and non-reassortant H3N2 swine influenza virus

Genetic reassortment between human and avian influenza viruses can create pandemic viruses. Influenza surveillance of pigs in Jilin Province, in China during 2007–2008 revealed that there were two distinguishable genotypes: a human-like H3N2 genotype and a double-reassortant genotype derived from the human H3N2 and avian H5 viruses. In this study, viral infection potential, replication kinetics, and pathogenicity were compared. The solid-phase binding assay demonstrated that both viruses prominently maintained a preference for the human-type receptor and the reassortant A/swine/Jilin/37/2008 (Sw/JL/37/08) showed relatively higher binding affinities than the non-reassortant A/swine/Jilin/19/2007 (Sw/JL/19/07). Replication kinetics showed that Sw/JL/37/08 had higher replicability in MDCK cells than Sw/JL/19/07. The mouse experiments clearly revealed that Sw/JL/37/08 had higher virulence than Sw/JL/19/07 as measured by more significant body weight loss, higher viral lung load, delayed viral clearance from lungs, and more severe pulmonary lesions. Sequence analysis indicated that the absence of glycosylation sites at residue 126 of HA and 93 of NA, as well as the characteristic NS1 C-terminal PL residues of ESEV may account for the increased replication and pathogenicity of Sw/JL/37/08. These results may imply that human may have infection risk by the reassortant swine influenza virus and emphasize the necessity for enhanced viral surveillance strategies, which monitor reassortment events in nature to reduce the public health threat posed by influenza viruses with the potential for human-to-human transmission currently circulating in pig populations.     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

2株H3N8亚型禽流感病毒HA和NA基因遗传变异分析

采用RT-PCR技术扩增了2株H3N8亚型流感毒株A/duck/Guangxi/69/2009和A/chicken/Guangxi/2117/2010的HA和NA基因,并与GenBank中收录的其他毒株序列进行比较分析和遗传进化分析.结果表明,分离株的HA与NA基因全长分别为1 733 bp、1 432 bp.A/du...