辽宁省H1N1亚型猪流感病毒的分离鉴定与遗传演化分析

本研究2012年底从辽宁省某屠宰场猪鼻咽拭子样品中分离到1株流感病毒,经HA—HI试验和RT—PCR鉴定为H1N1亚型猪流感病毒株,命名为A／swine/Liaoning／01／2012（H1N1）,通过对病毒的8个基因片段克隆并测序,并利用分子生物学软件进行遗传演化分析。结果表明,分离株HA基因裂解位点附近的氨基酸序列为IPSIQSRjG,符合低致病力流感病毒的分子特征。全基因组进化树结果表明,分离株的8个基因片段与A／swine／Jiangsu／40／2011（H1N1）株核苷酸同源性最高,分离株处在类禽型H1N1亚型遗传进化分支上;由于类禽型H1N1猪流感病毒具有潜在感染人的潜力,在国外和国内均有感染人的报道,因此,辽宁省首次分离到该型猪流感病毒对全省养猪业和公共卫生安全具有重要意义,值得深入研究。     

一株类禽H1N1亚型猪流感病毒的进化研究

为了解猪流感病毒(SIV)的变异情况,2013年1月份从山西某养殖场采集呈流感症状的猪鼻拭子10份,采用常规方法接种10日龄SPF鸡胚,进行RT-PCR鉴定及全基因序列分析。结果表明:分离到1株类禽H1N1亚型猪流感病毒,命名为A/swine/Shanxi/02/2013(H1N1);全基因序列测定及同源性分析发现,8个基因片段均与2011年江苏地区流行的类禽型H1N1亚型猪流感病毒有较高的同源性;系统遗传演化发现,该病毒株是由2011年类禽型H1N1流感病毒A/swine/Jiangsu/40/2011(H1N1)进化而来的;HA1氨基酸位点差异分析发现,该病毒株与2009年人群中流行的甲型H1N1流感和经典H1N1毒株抗原性差异较大,其HA裂解位点基序为PSIQSRGLF,呈低致病性特征。     

1株欧亚类禽猪流感病毒的分离鉴定及遗传进化分析

【目的】了解广东地区猪流感病毒(Swine influenza virus, SIV)的流行情况并探究其分子生物学特征。【方法】采集广东某猪场疑似猪流感病毒感染猪的鼻拭子和肺脏组织样品进行病毒分离鉴定、遗传进化和关键氨基酸位点分析。【结果】样品经实时荧光定量RT-PCR检测为猪流感病毒核酸阳性;在红细胞凝集试验中,该病毒对鸡红细胞有凝集作用,血凝效价为1∶128;8个基因片段序列结果经BLAST比对和进化树分析显示,HA、NA基因属于欧亚类禽猪流感病毒(H1N1)分支,PA、PB1、PB2、NP和M基因属pdm/09分支,NS基因属于北美三源重组分支,因此,本试验分离株属于G4基因型欧亚类禽猪流感病毒,将其命名为A/swine/Guangdong/CJM2/2022(H1N1)。关键氨基酸位点分析显示,分离株HA蛋白裂解位点序列为PSIQSR/GL,具有典型低致病性流感病毒的分子特征。HA基因在受体结合位点处的190、225、226位氨基酸分别为D、E、Q,表明其既具有结合人型唾液酸受体的潜能又具有结合禽型唾液酸受体的潜能。NA基因关键氨基酸残基均未发生突变,提示分离株对奥司他韦和扎那...     

一株H1N1亚型猪流感病毒的遗传演化分析及在雪貂体内复制和飞沫传播能力评估

本实验室于2016年在天津分离到一株猪流感病毒(SIV),命名为A/swine/Tianjin/312/2016(H1N1)(简称为TJ312株)。为了解该株病毒的生物学特性及遗传演化特征,本研究对经PCR分段扩增该病毒全基因组并测序,采用SeqMan软件拼接成全基因组序列经BLAST比对,得到与该SIV各基因节段序列同源性最高的病毒株,采用mafft分析其各基因节段编码氨基酸序列的分子特征,利用MEGA5.1软件分别构建该病毒8个基因节段的进化树。结果显示,SIV TJ312株各基因节段与2016年分离自天津的H1N1 SIV相应各基因节段的同源性在99%～100%。进化树结果显示该病毒株HA、NA和M基因均来自欧亚类禽H1N1 (EA H1N1) SIV谱系;PB2、PB1、PA和NP基因来自2009/H1N1流感病毒谱系;NS基因来自北美三重配H1N2 SIV谱系。蛋白分子特征分析结果显示,HA蛋白碱性氨基酸裂解位点序列为PSIQSR↓G,符合低致病性流感病毒分子特征;受体结合位点符合人源唾液酸受体(α-2,6唾液酸受体)的分子特性;NA蛋白aa275为H、aa295为N,且该蛋...     

猪流感病毒分离株A/swine/Shanghai/3/2014(H1N1)分子特性研究

采用套式PCR检测方法,结合鸡胚分离鉴定,从20份患呼吸道疾病猪的鼻咽拭子样品中分离到1株流感病毒。经亚型鉴定及全基因组序列分析,证实该分离株为H1N1流感病毒,命名为A/swine/Shanghai/3/2014(H1N1)。遗传进化分析表明该分离株与类禽猪流感病毒(Avian-Like H1N1)相似性最高。蛋白序列分析发现,该分离株具有低致病性流感病毒特征,即其HA蛋白的裂解位点为PSIQSR↓GLFGAI。此外,该基因中有7个潜在的糖基化位点,受体结合位点为108(Y)、148~152(GVTAA)、167(W)、197(H)、204~212(DQQ SLYQNA)和238~243(RDQEGR);其中225~228EQAG显示该分离株具有结合SAα2,6Gal受体的能力,证明该分离株具有感染哺乳动物的能力。同时PB2蛋白的627E、701N及NS蛋白的92D位点均证明了该分离株的低致病性和感染哺乳动物的能力。     

一株欧亚类禽H1N1猪流感病毒分子特征分析

为调查国内猪流感病毒流行和遗传演化状况,将2013年从山东某屠宰场的采集样品接种SPF鸡胚,分离到1株病毒,通过RT-PCR鉴定和全基因测序,并运用生物软件对病毒基因组关键氨基酸位点和遗传演化关系进行分析。结果显示,分离株A/Swine/Shandong/5513/2013(H1N1)为欧亚类禽H1N1猪流感病毒,基因片段未发生重排,与中国大陆近几年分离株类似。HA蛋白受体结合位点具有结合哺乳动物气管上皮细胞特性;HA蛋白抗原位点与欧亚类禽H1N1猪流感代表株A/swine/Hong Kong/1780/2008(H1N1)仅有一处不同,Q196H;裂解位点氨基酸为PSIQSR/GL,PB2蛋白毒力关键氨基酸位点为T271和E627,分离株为典型低致病力毒株。本毒株的分离鉴定为分析中国大陆猪流感流行状况和分子特征提供了数据。     

H1N1亚型猪流感病毒A/Swine/GD/2/12基因特征分析

2012年从广东省某猪场的疑似流感猪群采集鼻拭子样品,接种鸡胚并收集尿囊液,通过血凝、血凝抑制和RT-PCR,鉴定出1株H1N1亚型猪流感病毒,命名为A/Swine/GD/2/12。RT-PCR扩增得到全基因8个片段,与GenBank收录的参考毒株比对并构建进化树,发现本毒株可能是H1N1亚型重组株,其8个片段与我国猪源和北美地区1985—1992年间的猪源、禽源(A/turkey/IA/1992)和人源(A/Maryland/12/1991)流感毒株在同一个大分支上,其中与我国猪源参考株同源性在95.8%以上,与北美地区的H1N1参考株同源性在94%以上。HA受体位点分析表明,本毒株既具备结合Saα2,6Gal型人类流感病毒SA受体的特点,也有结合Saα2,3Gal型禽类流感病毒SA受体的可能。提示本毒株可能是由北美地区猪源、禽源和人源的H1N1亚型流感病毒重排形成的。HA蛋白裂解位点分析、NS和PB2蛋白位点分析表明,本毒株具备低致病性毒株的特点。小鼠致病性试验进一步证实本毒株能够引起小鼠运动减少、食欲欠佳、体重减轻等表现,但不会引起咳嗽和死亡等严重反应。     

一株人源H1N1亚型猪流感病毒的进化分析与生物学特性研究

为了解猪流感病毒(SIV)的变异情况,我们2009年11月从河北某养殖场采集呈流感症状的猪鼻拭子40份,接种10日龄SPF鸡胚,分离到一株猪流感病毒,通过RT-PCR和血凝抑制试验鉴定为H1N1亚型,命名为A/swine/Hebei/15/2009(H1N1),其全基因序列测定及同源性分析发现,8个基因片段均与2000年左右H1N1人流感病毒有较高的同源性。系统遗传演化显示,该病毒分离株是由2000年人源H1N1流感病毒A/Dunedin/2/2000(H1N1)进化而来。抗原性分析显示该株与甲型H1N1流感病毒和经典H1N1病毒株抗原性差异较大。对小鼠致病性试验表明该病毒株可以直接感染小鼠并导致小鼠轻微临床症状和组织病理学变化,但不致死小鼠,表现为低致病性。     

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。     

一株H1N1亚型猪流感病毒全基因组序列分析以及生物学特性研究

本实验室于2016年在江西分离到一株猪流感病毒(SIV),命名为A/swine/Jiangxi/261/2016(H1N1),为进一步弄清该株流感病毒的遗传进化背景和生物学特点,本研究对其进行了全基因组测序,遗传进化分析,BALB/c小鼠致病性试验,抗原性差异试验,受体结合特性试验。测序结果显示该病毒HA碱性裂解位点仅具有一个碱性氨基酸,符合低致病性流感病毒特点。外部基因来自欧亚类禽,内部基因来自2009/H1N1(PB2、PB1、PA、NP),欧亚类禽(M)和Triplereassortant H1N2(NS)。10~6 EID_(50)/50μL剂量可感染小鼠并致死,HA抗原性差异试验结果显示其与欧亚类禽Group1代表株差异较小,受体结合试验结果显示该病毒株主要结合α-2,6唾液酸受体。其传播性和致病性增强机理还有待进一步研究,本实验结果为SIV的检测和防控提供借鉴意义。     

Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.     

欧亚类禽H1N1与古典H1N1亚型猪流感病毒NA基因遗传进化分析

本研究对2011年分离自吉林省猪群的3株流感病毒进行了遗传进化分析。结果表明欧亚类禽H1N1猪流感病毒和古典H1N1猪流感病毒在吉林省猪群中共同流行,因此加强猪流感流行病学调查具有重要意义。     

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.     

猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立

对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。     

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。     

Isolation and phylogenetic analysis of pandemic H1N1/09 influenza virus from swine in Jiangsu province of China

To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.     

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.     

猪流感病毒H1N1亚型天津分离株核蛋白、基质蛋白、非结构蛋白基因克隆与序列分析

从天津地区不同猪场分离到6株H1N1亚型猪源流感病毒（SIV）。根据GenBank发表的H1N1亚型 SIV的核蛋白（NP）、基质蛋白（M）及非结构蛋白（NS）基因序列,分别设计3对引物,将RT-PCR产物克隆至pMD18-T载体,进行测序分析。遗传进化分析结果表明：A/swine/Tianjin/TJ2/2005（H1N1）与A/swine/Tianjin/TJ4/2006（H1N1）的NP、M及NS基因核苷酸序列在遗传进化树中均与A/swine/Guangdong/33/2006（H1N1）位于同一分支上,属于古典型H1N1猪谱系;A/swine/Tianjin/TJ3/2006（H1N1）与A/swine/Tianjin/TJ8/2006（H1N1)的NP、M及NS基因核苷酸在遗传进化树中均与A/Dunedin/2/2000（H1N1）组成一个大分支,可能起源于人谱系;A/swine/Tianjin/TJ6/2009（H1N1）与A/swine/Tianjin/TJ7/2009（H1N1）NP、M及NS基因核苷酸序列在遗传进化树中均与A/swine/Jiangsu/s15/2011（H1N1）位于同一分支上,属于类禽H1N1猪谱系。本试验对6株H1N1亚型SIV的NP、M及NS全基因序列进行分析,在一定程度上揭示了天津地区H1N1亚型SIV的基因进化与流行情况。     

The first detection of influenza in the Finnish pig population: a retrospective study

Background

Swine influenza is an infectious acute respiratory disease of pigs caused by influenza A virus. We investigated the time of entry of swine influenza into the Finnish pig population. We also describe the molecular detection of two types of influenza A (H1N1) viruses in porcine samples submitted in 2009 and 2010.This retrospective study was based on three categories of samples: blood samples collected for disease monitoring from pigs at major slaughterhouses from 2007 to 2009; blood samples from pigs in farms with a special health status taken in 2008 and 2009; and diagnostic blood samples from pigs in farms with clinical signs of respiratory disease in 2008 and 2009. The blood samples were tested for influenza A antibodies with an antibody ELISA. Positive samples were further analyzed for H1N1, H3N2, and H1N2 antibodies with a hemagglutination inhibition test. Diagnostic samples for virus detection were subjected to influenza A M-gene-specific real-time RT-PCR and to pandemic influenza A H1N1-specific real-time RT-PCR. Positive samples were further analyzed with RT-PCRs designed for this purpose, and the PCR products were sequenced and sequences analyzed phylogenetically.

Results

In the blood samples from pigs in special health class farms producing replacement animals and in diagnostic blood samples, the first serologically positive samples originated from the period July–August 2008. In samples collected for disease monitoring, < 0.1%, 0% and 16% were positive for antibodies against influenza A H1N1 in the HI test in 2007, 2008, and 2009, respectively. Swine influenza A virus of avian-like H1N1 was first detected in diagnostic samples in February 2009. In 2009 and 2010, the avian-like H1N1 virus was detected on 12 and two farms, respectively. The pandemic H1N1 virus (A(H1N1)pdm09) was detected on one pig farm in 2009 and on two farms in 2010.

Conclusions

Based on our study, swine influenza of avian-like H1N1 virus was introduced into the Finnish pig population in 2008 and A(H1N1)pdm09 virus in 2009. The source of avian-like H1N1 infection could not be determined. Cases of pandemic H1N1 in pigs coincided with the period when the A(H1N1)pdm09 virus was spread in humans in Finland.