一株欧洲类禽H1N1猪流感病毒的分离鉴定与反向遗传系统的建立

猪流感是猪常见的呼吸道传染病,临床以高热、呼吸困难、咳嗽和衰竭、迅速康复或死亡为特征。猪流感不仅给养猪业造成巨大损失,也严重威胁着人类健康。本研究从发病猪场中分离到1株H1N1亚型猪流感病毒,序列分析结果显示,分离毒株属于欧洲类禽猪流感H1N1亚型病毒。将分离毒株分别接种到MDCK与ST细胞,观察病毒的生长特性,结果显示分离的猪流感病毒在ST细胞中复制能力较强。采用RT-PCR技术分别扩增8个基因片段,克隆到流感病毒反向遗传系统,成功拯救出猪流感病毒毒株,测序结果显示拯救的猪流感病毒与亲本毒序列一致。本研究成功分离的猪流感病毒,以及建立的反向遗传技术为研究欧洲类禽猪流感病毒跨种传播的机制以及研发新型猪流感疫苗株奠定了基础。     

1株禽源猪流感H6N6病毒的反向遗传系统的建立

为建立禽源猪流感病毒A/swine/Guangdong/K6/2010(H6N6)的反向遗传系统,本试验构建了包含有GDK6毒株基因组的8个重组质粒,转染293T细胞后成功拯救出病毒r GDK6。救获病毒与亲本病毒拥有相同基因序列,其抗原性、经HI试验、TCID50试验和小鼠攻毒试验表明,救获病毒的生物学特性与对小鼠的致病性与亲本病毒一致。禽源猪流感H6N6病毒的反向遗传系统的成功建立为进一步研究H6亚型流感病毒的分子致病机理与病毒基因功能及新型疫苗创制提供了技术平台。     

一株类禽型H1N1亚型猪流感病毒的反向遗传系统的建立

为建立H1N1亚型猪流感病毒A/swine/Jiangsu/40/2011(JS40)的反向遗传系统,本研究分别构建了JS40株8个基因节段的重组质粒,经转染293T和MDCK混合细胞,拯救出病毒R-JS40。序列测定结果表明,救获病毒与亲本病毒的核苷酸序列一致,无氨基酸变异,可以稳定传代;抗原性未发生变化;对小鼠的致病性结果显示R-JS40与JS40对小鼠的组织嗜性以及在肺脏中复制的病毒滴度基本一致。以上结果表明R-JS40保持了亲本病毒JS40的生物学特性,该病毒反向遗传操作系统的建立,为进一步开展病毒的致病分子基础以及新型疫苗的研制提供有效的技术平台。     

中国流行的2009甲型H1N1猪源流感病毒HA和NA基因的分子和遗传特征

目前流行的甲型H1N1流感病毒是一个复杂的基因重配病毒。对病毒的分子生物学研究,尤其是病毒囊膜蛋白血凝素（haemagglutini,HA）基因和神经氨酸酶（neuraminidase,NA）基因的研究,为控制和预防H1N1流感病毒具有重要的意义。本研究对中国流行的2009甲型H1N1猪源流感病毒的HA和NA基因与疫苗株A/California/07/2009（H1N1）,以及不同国家和地区的病毒株进行核苷酸和氨基酸序列分析。从NCBI的GenBank数据库下载所需要毒株的序列,采用Lasergene 6.0软件包中的EditSeq和MegAlign进行序列分析,进化树分析采用MEGA4.1软件。进化分析表明,中国流行的2009 H1N1流感病毒与疫苗株的核苷酸同源率分别在98.8%~99.7%和98.6%~99.6%之间;裂解位点处为I/VPSIQSR↓G,不具备高致病性流感病毒的特征;有1株NA抗性病毒。尽管与疫苗株相比,中国流行株2009甲型H1N1猪源流感病毒的HA和NA基因有部分突变,但这些突变并不是重要的。本研究首次详细分析了中国流行的2009甲型H1N1猪源流感病毒株与疫苗株的HA和NA基因的分子特征,对实时监测流感病毒HA和NA基因的变化具有重要意义。     

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。     

两株H1N1亚型重组流感病毒的猪致病性研究

选取2种不同宿主来源的新型甲型重组H1N1亚型流感病毒(犬流感病毒(A/canine/Guangxi/QZ5/2013)(简称CIV-QZ5)和猪流感病毒(A/swine/Guangxi/QZ5/2014)(简称SIV-QZ5),以猪为动物模型,分别以106 PFU/mL病毒剂量和气管攻毒的方式感染3周龄仔猪,从而探究新型甲型重组犬流感病毒对猪的致病性。试验发现CIV-QZ5及SIV-QZ5均能有效感染仔猪,攻毒仔猪均出现发热(≥39.5℃)、流鼻涕、食欲减退、活动减少等流感症状,其中CIV组出现1头仔猪(A3)死亡。通过对肺脏灌洗液进行病毒滴定,发现CIV组在攻毒后3d和5d在肺脏的复制能力较SIV组强,最高达到3.12log10PFU/mL。病理剖检发现,肺脏出现不同程度的实变。肺脏病理切片发现两组均出现不同病变,其中以死亡仔猪(A3)最为明显,主要以支气管上皮细胞坏死脱落,肺泡壁增厚,肺泡腔以及支气管内有大量的弥漫性浸润的单核细胞为主要特征。结果表明,猪源甲型重组CIV不仅能引发猪出现明显流感症状,而且能有效地在肺脏复制,为了解潜在的猪-犬跨宿主传播机制奠定了基础。     

猪源新型甲型H1N1流感病毒的分离鉴定及遗传进化特点

本研究旨在了解猪源新型甲型H1N1流感病毒山东分离株的遗传进化特点。对山东地区出现的疑似H1N1流感病死猪进行病料采样,然后进行病毒的分离鉴定,并对分离病毒株(A/swine/Shandong/07/2011)的HA、NA、PB2、PB1、PA、NP、NS和M基因进行遗传进化分析。结果显示,该株病毒8个片段的核酸序列与A/H1N1(2009)对应序列的相似性都大于99%,并且该毒株HA蛋白的裂解位点和优先识别唾液酸α-2,6受体的位点与A/H1N1(2009)也高度一致,分别为PSIQSR↓GLFGAI和190D、225D。但是,与A/H1N1(2009)毒株的HA蛋白相比,受体结合位点处出现了重要的突变(Q226R)。该研究结果为进一步研究猪源新型甲型H1N1流感病毒的分子进化提供了重要信息。     

甲型H1N1流感病毒株的分离鉴定以及PR8株的相关基因对重组病毒增殖能力的影响

为分离流感病毒,本研究将疑似甲型流感患者的咽喉拭子接种SPF鸡胚尿囊腔,经检测收获的鸡胚尿囊液具有血凝活性.采用流感病毒通用引物进行PCR扩增出8个基因节段,经测序与GenBank中登录的序列比对后确定该病毒株为2009年爆发的甲型H1N1流感病毒,并命名为A/Harbin/LM/2009 (H1N1),简称LM株.由于LM分离株在鸡胚中增殖能力比较弱,为提高其在鸡胚中的增殖能力,我们利用反向遗传技术将LM株的HA和NA基因与高增殖特性的人流感病毒细胞高产株PR8的另外6个节段进行“6+2”重配,获得拯救病毒,但该重配病毒血凝价并未显著提高.为进一步鉴定影响病毒增殖能力的基因,本研究将LM株所有节段以“7+1”的方式逐一与PR8的7个片段搭配构成完整的流感病毒基因组,分别得到8个重配病毒.结果显示分离株的PB1、PA、HA基因显著降低了重配病毒在鸡胚细胞中的增殖能力.     

H1N1猪流感病毒环介导等温扩增快速检测方法的建立

目的:建立H1N1猪流感病毒环介导等温扩增(LAMP)快速检测方法。方法:从GenBank中获得H1N1猪流感病毒血凝素(HA)、神经氨酸酶(NA)基因序列,应用DNAStar软件MegAlign程序分析其序列,利用Primer ExplorerV4软件在序列保守区域设计LAMP引物,即外引物和内引物,同时以H1N1猪流感病毒的cDNA作为阳性模板,对试验中的几个反应条件进行优化。结果:LAMP检测方法对H1N1猪流感病毒的灵敏度达到4～6个拷贝,其引物对于H9亚型禽流感病毒、猪瘟病毒和猪圆环病毒均无非特异性扩增,表现出良好的特异性。结论:建立的H1N1猪流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测猪流感病毒提供了新方法和新思路。     

Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

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.     

Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

H5N1亚型人禽流感病毒A/Anhui/2/2005株反向遗传操作系统的建立

为建立H5N1亚型人源禽流感病毒A/Anhui/2/2005(W-AH05)反向遗传操作系统,本研究构建了W-AH05的8重组质粒,拯救出人源禽流感病毒R-A/Anhui/2/2005(R-AH05)。全基因序列测定结果表明,救获病毒R-AH05与野生病毒W-AH05的核苷酸序列完全一致;动物试验证实,R-AH05保持了W-AH05的对BALB/c小鼠呈高致病力的特性,其MLD50分别为1.5log10EID50和1.2log10EID50。R-AH0与W-AH05分别以106EID50剂量鼻腔感染BALB/c小鼠,病毒在小鼠体内的分布情况及各个脏器中的病毒滴度基本相同。由此可见,R-AH05保持了W-AH05的生物学特性,从而为进一步研究人源禽流感病毒的致病机理及跨宿主传播机制等提供了操作平台。     

H6N6亚型禽流感病毒反向遗传操作系统的建立

为建立H6N6亚型禽流感病-毒(AIV) A/Chicken/GuangDong/S1311/10 (W-CKGD)反向遗传操作系统,本研究构建了W-CKGD的8个重组质粒,拯救出AIV A/Chicken/GuangDong/S1311/10 (R-CKGD).全基因序列测定结果表明,救获病毒R-CKGD与野生病毒W-CKGD的核苷酸序列完全相同.R-CKGD与W-CKGD具有相同的受体结合特性和对BALB/c小鼠均呈低致病性,以106 EID50剂量鼻腔感染BALB/c小鼠,病毒仅在小鼠的呼吸道复制,可以引起小鼠体重下降但均不造成死亡.实验结果表明,R-CKGD与W-CKGD保持了一致的生物学特性,从而为进一步研究H6亚型AIV的致病机理及跨宿主传播机制等提供了良好的平台.     

Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic influenza virus is superior to commercially available swine influenza vaccines

In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.     

Phylogenetic evolution of swine-origin human influenza virus: a pandemic H1N1 2009

The knowledge of the genome constellation in pandemic influenza A virus H1N1 2009 from different countries and different hosts is valuable for monitoring and understanding of the evolution and migration of these strains. The complete genome sequences of selected worldwide distributed influenza A viruses are publicly available and there have been few longitudinal genome studies of human, avian and swine influenza A viruses. All possible to download SIV sequences of influenza A viruses available at GISAID Platform (Global Initiative on Sharing Avian Influenza Data) were analyzed firstly through the web servers of the Influenza Virus Resource in NCBI. Phylogenetic study of circulating human pandemic H1N1 virus indicated that the new variant possesses a distinctive evolutionary trait. There is no one way the pandemic H1N1 have acquired new genes from other distinguishable viruses circulating recently in local human, pig or domestic poultry populations from various geographic regions. The extensive genetic diversity among whole segments present in pandemic H1N1 genome suggests that multiple introduction of virus have taken place during the period 1999-2009. The initial interspecies transmission could have occurred in the long-range past and after it the reassortants steps lead to three lineages: classical SIV prevalent in the North America, avian-like SIV in Europe and avian-like related SIV in Asia. This analysis contributes to the evidence that pigs are not the only hosts playing the role of "mixing vessel", as it was suggested for many years.     

利用反向遗传技术拯救H9N2重组病毒

为研制高效特异的H9N2亚型AI疫苗,选取我国具有代表性的毒株A/chicken/Shanghai/10/01(H9N2)(简称SH10),以12质粒系统为基础,利用反向遗传操作技术构建了1株表面基因由SH10提供、内部基因由12质粒系统提供的重组病毒SH/PR8,为新型疫苗的研制提供了新的毒株。