10株H9N2亚型禽流感病毒血凝素基因的序列分析

为了解中国目前H9N2亚型禽流感病毒(avian influenza virus,AIV)血凝素(HA)基因的遗传变异情况,对中国不同地区分离的10株H9N2亚型AIV的HA基因进行扩增、克隆和测序,并对所获得的HA全序列进行同源性和遗传进化分析。结果表明,10个分离株的裂解位点均为RSSR↓GLF,符合低致病性AIV的分子特征;10个分离株有7~9个潜在糖基化位点,由于基因突变有些HA基因出现了新的糖基化位点;与参考株相比,发现了4个抗原表位的突变,这些表位的突变可能引起病毒致病性的改变;受体结合位点除198位有变异外,其他位点均较保守;6株病毒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;10个分离株HA基因与国内疫苗株的核苷酸及氨基酸序列同源性分别为90.4%~99.2%和92.2%~98.7%;10个分离株同属于欧亚谱系中的A/duck/Hong Kong/Y280/97群,但差异显著,为此本试验又将其分为4个不同的亚群。人工感染排毒试验结果表明,BJ15和NJ17分离株在鸡体内具有较强的复制能力,排毒周期较长且排毒量也较大,而S145N的漂变导致在145-147位氨基酸多出1个糖基化位点NGT,可能是分离株复制能力增强的原因。     

Classic fowl plague--a review

Highly pathogenic avian influenza (HPAI) represents a severe form of generalized avian influenza which is characterized by a rapid and severe course of disease and a very high mortality. All poultry species are susceptible. Turkeys and chickens are most vulnerable. There are no pathognomonic symptoms or specific pathological alterations. The disease is caused by avian influenza virus strains of the subtypes H5 or H7. These viruses arise spontaneously from apathogenic progenitors by insertional mutation in the HA gene. Until recently, outbreaks of HPAI were rare events, however, they have been found to cause increasing losses over the past few years. Since 2003, a widespread occurrence of HPAI has been registered in southeast Asia, and some countries are endemically infected with HPAIV strain H5N1. In six countries this virus has also caused fatal human infections. This has sparked fears that this agent may be the progenitor of a new pandemic influenza virus. During summer 2005 the disease has slowly spread westward. Isolated outbreaks have been reported from Kazakhstan, Russia, Romania, Turkey, Croatia and Ukraine. Migratory birds have been tentatively accused for spreading the infection along their flyways.     

一株绿鹭源H9N2亚型禽流感病毒全基因组序列分析

为了解野生水禽绿鹭(Butorides striata)中分离到的1株H9N2亚型禽流感病毒(A/striated heron/Yunnan/2018)的生物学特性;对其进行全基因组序列扩增、测序、进化分析;序列分析显示:该分离株HA、NA基因位于Y280-like分支、PB2、M基因位于G1-like分支、PA、PB1、NP、NS基因位于F98-like分支,分别与H9、H7、H10等多种亚型的AIV同源性较高,该分离株不同基因片段来源较复杂。HA裂解位点氨基酸序列为333PSRSSR↓GL340,符合低致病性禽流感病毒(LPAIV)氨基酸序列特征;S145N突变增加了一个糖基化位点,提示该位点出现可能会使毒株致病性提高,免疫原性发生改变;HA受体结合位点发生Q234L突变,表现出人流感病毒受体结合特性;NA基因出现第63—65位氨基酸缺失,M1发生N30D,T215A突变,M2发生S31N的突变,PB2、PB1、NS、NP、PA关键位点未发生变化,分析结果提示当前分离株已出现耐药性、致病性增强的变化。本研究表明该分离株呈现遗传演化的多样性及基因重组的复杂性,因此加强对野生水禽类禽流感病毒的监测和研究具有重要的公共卫生意义。     

鹅源H5N1亚型禽流感病毒株HA基因的克隆与序列分析

本研究对1株鹅源H5N1亚型禽流感病毒血凝素（HA）基因进行了扩增、克隆及序列测定。结果表明：所扩增的片段长1707bp，包含完整的开放阅读框架，编码568个氨基酸。该毒株裂解位点的氨基酸序列为RRRKKR，具有高致病性的分子特征；该毒株有6个潜在糖基化位点；受体结合位点的氨基酸分别为YWIHELY，其左侧壁氨基酸为SGVSSA，右侧壁为NGQSGR。该毒株与国内外一些参考毒株的HA基因序列比较，核苷酸同源率为76.8％～98．1％，氨基酸同源率为85．7％～97．4％，属于欧亚种系。     

南京市首例甲型H1N1(2009)流感病毒分离及其血凝素基因遗传变异分析

对南京市首例甲型H1N1(2009)病毒进行细胞分离,获得一株具有较高血凝活性的病毒,命名为A/Nanjing/1/2009。在全基因组测序的基础上,对分离株的血凝素基因(haemagglutinin,HA)的遗传特征进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Nanjing/1/2009HA蛋白的有5个氨基酸发生了突变,其中一个位于Ca抗原位点208位氨基酸(R→K),这一突变虽然还不会影响抗原性的改变,但预示了新甲型H1N1(2009)抗原漂移的启动。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1病毒的特点。与禽H1病毒相比,分离株HA蛋白受体结合位点上的190(E→D)和225(G→D)位点发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究对南京市早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。     

猪流感病毒广东株分离鉴定及遗传进化分析

本研究从广东省某猪场采集37份疑似猪流感症状的猪鼻拭子样品,接种于9日龄SPF鸡胚并收集尿囊液,通过血凝试验、血凝抑制试验和RT-PCR鉴定,分离得到一株猪流感病毒,经RT-PCR分别扩增8个基因片段,进行基因测序及序列分析,与GenBank收录的参考毒株比对并构建进化树。结果显示,分离毒株为H1N1亚型猪流感病毒,将其命名为A/swine/Guangdong/2/2018（H1N1）。遗传进化分析显示,分离株8个片段的核酸序列与A/swine/Guangdong/L3/2009（H1N1）对应序列的同源性均达99%以上,与经典型H1N1亚型猪流感病毒处于同一分支。分离毒株HA的裂解位点为PSIQSR↓GL,符合低致病性流感病毒分子特征。HA基因受体位点为190D、225G和226Q,表明本毒株既可以结合SAα-2,6-Gal型人类流感病毒SA受体,也有结合SAα-2,3-Gal型禽类流感病毒SA受体的可能,在28、40、104、304、498、557位氨基酸处有6个潜在糖基化位点;NA蛋白在50、58、63、68、98、146、235位氨基酸处有6个潜在糖基化位点,NA蛋白氨基酸序列活性中心位点为119E、199D、223I、275H、293R、295N,氨基酸分析位点未出现突变,表明本分离株对神经氨酸酶抑制剂类药物的敏感性较高,但在M2蛋白中,31位氨基酸由敏感型的（S）突变为抗药的（N）,提示可能对金刚烷胺类药物产生耐药性。开展猪流感病毒分离鉴定与遗传进化分析将为广东地区的猪流感流行和变异情况提供重要信息。     

H5亚型鸭源流感病毒HA基因序列生物信息学分析

A型流感病毒（AIV）引起的禽类禽流行性感冒（avian influenza,AI）或相关疾病,被国际兽疫局定为甲类传染病。AIV中最容易突变的基因是血凝素（HA）基因,具有亚型和株的特异性,是区分病毒亚型的依据之一。本研究从GenBank中下载了所有209条鸭源H5禽流感病毒HA基因的全序列,利用生物信息学软件构建进化树研究序列的聚类特点;比较不同年份毒株的HA基因的受体结合位点氨基酸,找出受体结合位点氨基酸的变异规律;比较不同年份分离的鸭源H5N1序列的HA1和HA2蛋白的剪切位点,找出各年份毒株的剪切位点的特点;比较不同年份分离序列的潜在糖基化位点,统计各年份潜在糖基化位点的数量以及序列变化。期望找到H5亚型鸭源流感病毒的变异规律和变异方向,为鸭源流感的防控起到积极的作用。     

2株云南H9N2亚型禽流感病毒HA和NA基因序列分析

在2019年1月-2019年6月对云南出现呼吸道疫病的57个鸡场进行H9亚型禽流感检测的基础上,选取石林和楚雄2个H9亚型禽流感阳性样品进行病毒分离。从分离的H9N2亚型禽流感病毒感染鸡胚尿囊液中提取总RNA,采用特异性引物经反转录PCR分别扩增HA和NA基因,PCR产物纯化后进行测序。序列比对及系统发育分析结果表明,云南2株H9N2毒株HA基因核苷酸序列同源性为94.2%,NA基因核苷酸序列同源性为93.6%,系统进化分析表明云南H9N2亚型禽流感病毒HA和NA基因均属于欧亚谱系中的类ADKHKY28097分支（Y280-like）,ACKYN12019和ACKYN72019 HA基因之间的同源性为94.3%,与参考毒株ACKJX2448的同源性最高,为95.6%~98.5%,与中国流行的H9N2代表株和疫苗株同源性较低。HA蛋白333-340位裂解位点为PSRSSR↓GLF,具有低致病性禽流感病毒分子特征,受体结合位点均发生E198T和Q234L的突变,具有人样受体结合特征,在29、141、298、305、313、492位氨基酸有6个糖基化位点。ACKYN12019和ACKYN72019 NA基因同源性为93.6%,与Y280-like代表毒株的同源性分别为97.1%~97.5%和93.7%~94.6%,NA蛋白缺失63、64、65位氨基酸,在44、69、86、146、200、234位氨基酸处存在6个潜在的糖基化位点,NA蛋白红细胞结合（HB）位点分析发现,368-369、399-403、432位氨基酸处存在变异。研究结果显示,H9N2亚型禽流感病毒一直处于不断的变异之中,故应加强其监测与防控。     

Full sequence analysis of hemagglutinin and neuraminidase genes and proteins of highly pathogenic avian influenza H5N1 virus detected in Iran, 2015

Over the last two decades, the highly pathogenic avian influenza H5N1 virus has gained a lot of attention due to its zoonotic and mutative nature. Iran is among the countries significantly affected by the virus as it hosts migratory birds during seasonal migration. In this study, the molecular characterizations of hemagglutinin (HA) and neuraminidase (NA) genes and proteins of H5N1 strain A/chicken/Iran/8/2015 detected in backyard poultry, Mazandaran province, were investigated. Phylogenetic analysis classified this virus as a member of subclade 2.3.2.1c, with the cleavage site motif of “PQRERRRK-R/GLF”. HA carried a few mutations altering affinity to mammalian cells; however, the virus was categorized as avian. NA protein had the 20-amino acid deletion at aa position 49–69 similar to those isolated since 2000. Mutations of H253Y and H274Y contributing to antiviral resistance were present in NA. From this analysis, it can be concluded that the wild migratory birds flying from Western Asia to Eastern Africa are probably the main carriers of seasonal H5N1 in the country.

     

禽流感病毒A/Afri.Star/Eng-Q/983/79/(H7N1)血凝素基因的全序列分析

对反转录－聚合酶链反应扩增克隆的Ｈ７亚型禽流感病毒（ＡＩＶ）Ａ／Ａｆｒｉ．Ｓｔａｒ／Ｅｎｇ－Ｑ／９８３／７９／（Ｈ７Ｎ１）的血凝素（ＨＡ）基因进行了核苷酸序列分析。结果,克隆的ＨＡ基因共１７０７ｂｐ,包括完整的阅读框架;同源性分析表明该毒株起源于欧亚群系;ＨＡ裂解位点只有一个碱性氨基酸,显示典型的低致病力特征。Ｈ７亚型ＡＩＶＨＡ基因的克隆成功为分子诊断和基因工程疫苗的研制奠定了基础。     

Improvement of a rapid diagnosis kit to detect either influenza A or B virus infections

To improve the sensitivity of a kit, ESPLINE INFLUENZA A&B for rapid diagnosis of influenza by detecting influenza A or B virus specific nucleoproteins (NP), the ESPLINE INFLUENZA A&B-N was developed by using newly established monoclonal antibodies (MAbs) to the respective NP molecule. MAbs FVA2-11 and FrB1-03 recognize the epitope on the amino acid region 59-130aa of the NP molecule of influenza A virus, and that on the region 72-191aa of the NP of influenza B virus, respectively. The new kit detected influenza A and B virus antigens with a detection limit of 10(2.0)-10 (2.7) pfu/test, which is 4-1000 times higher than that of the original kit. Importantly, this kit detected each of influenza A viruses of the known hemagglutinin (HA) subtypes (H1-H15) including the H5N1 viruses recently isolated from human and avian sources in Asia. The kit also detected all of the 15 representative influenza B virus strains tested. The ESPLINE INFLUENZA A&B-N is thus a rapid and highly sensitive and specific kit for the diagnosis of either influenza A or B virus infections.     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

H5亚型禽流感病毒多肽疫苗的初步研制

本试验利用生物软件对H5亚型禽流感病毒A/CK/GD/178/04的血凝素进行抗原特性分析,结合关于禽流感病毒血凝素抗原位点的文献报道,选定97～212位氨基酸和369～529位氨基酸两个区域作为多肽表位候选区域.利用本实验室自主研发的原核表达载体pBT载体,串联表达HA的两个区域,经SDS-PAGE和Western Blotting分析,证明重组产物得到表达,Ni2+柱纯化重组蛋白后,对2周龄SPF鸡进行免疫,经HI试验检测该重组蛋白可以刺激机体产生HI抗体.本研究为H5亚型AIV多肽疫苗的进一步研制奠定了基础.     

Genetic and pathobiologic characterization of H3N2 canine influenza viruses isolated in the Jiangsu Province of China in 2009-2010

The newly emerging canine influenza virus (CIV) causes considerable concerns for both veterinary and public health. During 2009-2010, six strains of H3N2 influenza virus were isolated from dogs in Jiangsu Province, China. Sequence and phylogenetic analysis of eight gene segments revealed that the six viruses were most similar to a recent canine-derived subtype H3N2 influenza virus isolated in cats from South Korea, which originated from avian strain. By comparing the deduced amino acid sequences of the hemagglutinin 1 (HA1) and neuraminidase (NA) genes of the six Jiangsu isolates against the most similar avian strains, we found that all isolates had several common mutations at the receptor-binding sites, potential glycosylation sites and cleavage site in HA1, and antigenic sites in both the HA1 and NA segments. Significantly, a unique two amino acid insertion in the NA stalk was found. Experimental infection of BALB/c mice revealed that viral RNA could be detected in the major rodent organs, such as brain, heart, spleen, kidney, liver and intestine, as well as the lung. All the sampled organs from infected mice showed significant lesions and viral antigen staining. This study highlights the potential of domesticated animals to become a reservoir for influenza virus and the need for surveillance programs to detect cross-species transmission.     

2011年－2014年我国部分省区 H9N2亚型禽流感病毒HA 基因序列分析

本研究于2011年－2014年在我国部分省区鸡群中鉴定出49株 H9N2亚型禽流感病毒,并对所有毒株的 HA 基因进行克隆、测序及序列分析。结果表明,49个毒株的 HA 基因开放阅读框全长均为1683 bp,编码560个氨基酸。所有分离株均属于以 HK/Y280/97株为代表的 H9．4．2谱系,并明显分成2个亚分支(H9．4．2．5和 H9．4．2．6)。分离株 HA 基因核苷酸同源性在87．1％~100％之间,与疫苗株 SH/F/98株、GD/SS/94株和 SD/6/96株核苷酸同源性在89．4％~92．5％之间。对 HA 基因的推导氨基酸序列分析表明,所有分离株裂解位点附近没有连续的碱性氨基酸插入,符合低致病力毒株特征,受体结合位点为PWTN?LY 形式,受体结合位点左沿为 NGLM/QGL 形式,右沿均为 GTSKA 形式。在49个分离株中共发现10个潜在糖基化位点,但只有6个糖基化位点保守。研究表明,近年来 H9N2亚型禽流感在我国多个地区流行,2013年以后流行毒株趋势以 H9．4．2．5为主,但病毒基因仍在不断发生变异,因此需要继续加强对H9N2亚型禽流感分子流行病学的监控。     

禽流感病毒A/Chicken/Yangzhou/N/2005(H9N2)HA基因的克隆及序列分析

根据GenBank公开发表的H9N2型禽流感病毒血凝素(HA)基因序列设计1对引物,用RT-PCR方法成功扩增出H9N2型禽流感病毒分离株A/Chicken/Yangzhou/N/2005(H9N2)HA基因,凝胶电泳结果显示,扩增产物为1.7 kb的单一条带,与预期结果相符.将PCR扩增片段连接到pCR2.1-T载体上,重组质粒酶切鉴定正确.序列测定分析结果显示,所获序列与GenBank中收录的H9N2亚型分离株核苷酸同源性最高达99%,与原型毒株A/Furkey/Wisconsin/1/1966(H9N2)氨基酸同源性为89%.对HA裂解位点附近的氨基酸序列分析表明,此毒株为低致病力毒株.     

13株H9N2亚型禽流感病毒HA基因变异分析

为了从分子水平上掌握我国H9亚型禽流感的病原变异情况和流行规律,本研究汇集近年来从我国部分省市养殖场分离的13株H9N2亚型禽流感毒株,采用RT-PCR技术对其HA基因进行扩增、克隆和测序,并对所得全序列进行同源性和遗传进化分析。结果显示,13株病毒的HA基因在遗传进化树中均属于欧亚分支中的类Y280-like亚分支,与A/DK/HK/Y280/97的HA基因核苷酸序列同源性为92.2%～97.6%,与中国最早的分离株A/Chicken/Beijing/1/94(简称BJ94)相距较远,初步说明H9亚型禽流感病毒随着流行时间而发生了遗传分化。推测的HA糖基化位点的氨基酸序列12株病毒均与上述亚系相似,但有一株病毒由于一个核苷酸的变异,缺失了HA上218～220位的一个潜在的糖基化位点。     

两广地区2011--2012年H9N2亚型禽流感病毒的HA基因进化分析

为探究两广地区H9N2亚型禽流感病毒（avianinfluenzavirus,AIV）的变异情况及分子流行规律,于2011-2012年从该地区发病鸡群中共分离到16株H9N2亚型A1V,并对分离株HA基因进行测序与进化分析。结果表明,分离株HA基因开放阅读框全长均为1683bp,编码560个氨基酸;HA基因核苷酸同源性为88．7％～99．6％,编码氨基酸同源性为91．8％～99．5％。本试验分离毒株与国内疫苗株（GD-SS、SH—F和SD-6）的核苷酸同源性在90．1％～92．6％之间,推导的氨基酸序列同源性在91．6％～94．8％之间。进化分析显示分离株可分为Group1和Group2两个亚分支,与疫苗株均属于欧亚谱系的Y280分支,但亲缘关系较远。分离株HA蛋白裂解位点附近序列有3种形式：PARSSR＋GLF、PSRSSR＋GLF和PARLSR0GLF,均无连续碱性氨基酸的插A,符合低致病性AIv的特征。本试验发现分离株GD4、GX2在HA1的127、295位分别增加一个潜在的糖基化位点;除分离株GD5和GD6外,其余分离株在HAl的216位发生Q216L氨基酸突变,表明其存在感染人的可能性。     

不同源性H5亚型禽流感病毒株HA基因序列分析

本研究对1株鹅源和一株鸽源的H5N1亚型禽流感病毒血凝素（HA）基因进行扩增、克隆和序列分析。结果表明，所扩增的片段长均为1707bp，包含完整的开放阅读框架，编码568个氨基酸；该毒株有7个潜在糖基化位点；在HA裂解位点附近有6个碱性氨基酸序列插入。两毒株间核苷酸与氨基酸的同源性均为96．7％。该毒株与参考毒株的序列比较分析结果表明：核苷酸同源率分别为95．3％～99％；氨基酸同源率分别为95．1％～99.3％。     

H5亚型禽流感疫苗变异候选株的构建

本文就2008年在江苏某鸡场分离到的一株发生抗原变异的H5N1亚型禽流感病毒A/chicken/Huadong/4/2008(wt-DT株)开展如下研究:利用反向遗传技术,删除wt-DT株病毒血凝素(HA)基因多碱性氨基酸序列,使之成为低致病特征,再与wt-DT株的神经氨酸酶(NA)基因组合,结合鸡胚高产病毒PR8株的6个内部片段骨架,构建针对此种变异H5亚型禽流感的疫苗株,结果成功拯救出重组病毒rH5N1/PR8.病毒在鸡胚和MDCK细胞上均具有较好的繁殖能力,相对于wt-DT株,rH5N1/PR8在MDCK细胞上的繁殖能力明显提高.rH5N1/PR8对SPF鸡和鸡胚无致病性,在鸡体内的免疫保护效果良好,符合疫苗候选株的标准.