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。     

我国猪群H1N1亚型流感病毒流行情况与生物学特性

感染猪群的流感病毒（swine influenza virus,SIV）对养猪业和公共卫生均构成一定威胁。对H1N1亚型SIV监测发现,经典猪H1N1(classical swine H1N1,CS H1N1)、欧亚类禽H1N1(Eurasian avian-like H1N1,EA H1N1)、人源H1N1和2009年大流行H1N1(2009 pandemic H1N1,pdm/09 H1N1)亚型流感病毒均在我国猪群内传播,并且这些SIV相互间不断发生基因重组。一些H1N1亚型SIV会偶发性引起人类感染,对公共卫生存在潜在威胁。此外,H1N1亚型SIV还会引发犬和水貂等动物的感染。经动物模型试验发现,不同H1N1亚型SIV在小鼠、鸡、猪和雪貂中的致病性及传播能力不同,其中一些SIV分离株显示出致死性,需要对其进行生物学特征研究,评估其对人类的潜在风险。SIV导致的猪死亡率不高,因而对其关注较少。但是禽源和哺乳动物源流感病毒可以在猪体内重组为可引起人类大流行的流感病毒毒株,因此应该及时了解SIV的流行情况与生物学特性,并做好相应的控制措施,以减少经济损失,避免具有大流行潜力毒株出现。...     

甲型H1N1流感的特点及其防控

甲型H1N1流感病毒是2009年3月墨西哥出现的一种新型流感病毒。此后不久,这种病毒造成世界范围内的蔓延。论文分析总结了甲型H1N1流感病毒与1918年流感病毒的相似性以及表现出的新特点,归纳了这种病毒的潜在危害性。此外,介绍了甲型H1N1流感的防控策略,主要包括加强猪群监控,流感疫苗和抗流感病毒药物治疗。     

两株H1N1亚型猪流感病毒NS1蛋白抑制Ⅰ型IFN功能比较

本研究分离得到两株猪源H1N1流感病毒,通过进化树分析发现分别是欧亚类禽H1N1猪流感病毒和类鸭源H1N1猪流感病毒,为了研究两株毒株的NS1蛋白对Ⅰ型IFN产生的抑制能力,分别构建了2个毒株的NS1基因的真核表达载体,将NS1真核表达质粒、Ⅰ型IFN报告质粒与干扰素刺激质粒RIG-I共转染293T细胞,利用双荧光素酶报告基因检测Ⅰ型IFN激活水平。结果显示:欧亚类禽H1N1猪流感病毒NS1蛋白对人源细胞干扰素的拮抗作用明显强于类鸭源H1N1猪流感病毒;2种NS1蛋白对RIG-I与TBK-1激活的IFN-I抑制能力存在显著的差别,但是对IRF-3激活的IFN-I抑制能力没有显著差别。本研究结果初步揭示了欧亚类禽与类鸭源H1N1猪流感病毒抑制IFN-I能力的差异,及H1N1禽流感适应猪群的潜在分子机制。     

上海地区猪群中H1N1甲型流感抗体检测

对2009年H1N1甲型流感流行前后的上海地区养殖场户410份猪血清样品,分别采用血凝抑制试验（hemagglutination inhibition,HI）和酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）进行检测H1N1甲型流感病毒和猪流感病毒（Swine in？uenza virus,SIV）。检测结果表明,除2007年外,2008~2010年猪血清中均存在不同水平的HI抗体,阳性率呈显著上升趋势,且抗体水平与猪群饲养周期及饲养密度正相关,而与猪流感病毒的流行无相关性。     

携带H1N1 pdm09基因的重配猪流感病毒分子流行特征研究进展

在2009年甲型H1N1流感(H1N1 pdm09)大流行暴发初期,该病毒就已经成功地潜入到了猪群当中,继而参与到了猪流感病毒(SIV)的进化过程,通过不断地重配和适应,促进了新型SIV变异毒株的产生。目前,世界各地的猪群中普遍流行着携带有H1N1 pdm09基因片段的新型SIV,其重配形式呈现多样化。值得一提的是,由于H1N1 pdm09毒株的内部基因具有广泛的基因相容性,不同亚型、不同谱系的人源流感病毒、禽源流感病毒和本地猪流感病毒更倾向与它们优先发生重组。重配后的子代病毒较亲本毒株容易获得选择优势,能够在猪群中建立稳定的遗传谱系。研究发现H1N1 pdm09部分独特的内部基因片段在其高效的人际间传播过程中发挥着决定性的作用。一旦这类新型SIV成功突破种间屏障,获得适应新的哺乳动物的能力,将会对人类健康造成潜在的威胁。论文综述了携带有H1N1 pdm09基因的新型重配SIV的分子流行情况,特别就H1N1 pdm09内部基因与重组病毒致病性、传播能力等相关的分子基础进行了系统地综述,为防控新型SIV的暴发和保障公共卫生安全提供依据。     

H9N2禽流感病毒与H1N1流感病毒易发生重排

猪源H1N1/2009流感病毒自2009年暴发以来,在世界范围迅速传播。重排是流感病毒进化的主要方式之一,历史上多次流感的流行都是病毒重排的结果。中国农业大学动物医学院刘金华教授课题组研究表明:H9N2禽流感病毒与H1N1流感病毒很容易发生重排,     

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。     

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。     

影响H1N1亚型猪流感病毒致病性与传播性的分子机制研究进展

猪流感(swine influenza,SI)是规模化猪场普遍存在的疾病之一,全球广泛传播的猪流感病毒(swine influenza viruses,SIVs)亚型包括H1N1、H1N2和H3N2,我国以H1N1亚型的流行最为常见。进化上,H1N1亚型SIVs通常分为经典H1N1和欧亚类禽H1N1两大HA分支,分别于1930,1979年被首次分离,且都有直接感染人的病例报道;此后,两者参与重配生成的2009年大流行流感病毒(pdmH1N1/2009)又入侵猪群和人群,严重危害养猪业发展和公共卫生安全。现对致使H1N1 SIVs致病性发生改变或获得跨宿主传播能力的关键氨基酸位点变异及相关机制进行了总结,以期为SI的传播研究和防控提供一定参考。     

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。     

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.     

Pandemic A/H1N1/2009 influenza virus in swine, Cameroon, 2010

Although swine origin A/H1N1/2009 influenza virus (hereafter "pH1N1″) has been detected in swine in 20 countries, there has been no published surveillance of the virus in African livestock. The objective of this study was to assess the circulation of influenza A viruses, including pH1N1 in swine in Cameroon, Central Africa. We collected 108 nasal swabs and 98 sera samples from domestic pigs randomly sampled at 11 herds in villages and farms in Cameroon. pH1N1 was isolated from two swine sampled in northern Cameroon in January 2010. Sera from 28% of these herds were positive for influenza A by competitive ELISA and 92.6% of these swine showed cross reactivity with pandemic A/H1N1/2009 influenza virus isolated from humans. These results provide the first evidence of this virus in the animal population in Africa. In light of the significant role of swine in the ecology of influenza viruses, our results call for greater monitoring and study in Central Africa.     

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.     

中国类禽型H1N1亚型猪流感病毒的发现和遗传分析

采用禽流感病毒通用引物,对2006年发现的1株H1N1亚型的类禽型猪流感病毒的全基因组进行了测序,并进行了遗传学分析。序列分析表明它的8个片段与欧洲的类禽型猪流感病毒A/swine/Ile et Vilaine/1455/99(H1N1)病毒和A/swine/Cotes d'Armor/1488/99(H1N1)病毒的相应基因具有高度的同源性,同源性可达97%~99%,表明类禽型猪流感病毒已在中国出现。其血凝素基因的190E→D和225G→E的突变使得其结合NeuAc-a2,6Gal受体的能力高于NeuAca2,3Gal受体。欧洲的类禽型猪流感病毒可以直接感染人,并且可导致人的肺炎和死亡。中国类禽型猪流感病毒的发现及其的NeuAca2,6Gal受体结合特性使其成为一个潜在可感染人的病毒。     

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。     

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.     

Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.     

Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype

The efficacy of a commercial swine influenza vaccine based on A/New Jersey/8/76 (H1N1) and A/Port Chalmers/1/73 (H3N2) strains was tested against challenge with an H1N2 swine influenza virus. Influenza virus-seronegative pigs were vaccinated twice with the vaccine when they were four and eight weeks old, or with the same vaccine supplemented with an H1N2 component. Control pigs were left unvaccinated. Three weeks after the second vaccination, all the pigs were challenged intratracheally with the swine influenza strain Sw/Gent/7625/99 (H1N2). The commercial vaccine induced cross-reactive antibodies to H1N2, as detected by the virus neutralisation (VN) assay, but VN antibody titres were 18 times lower than in the pigs vaccinated with the H1N2-supplemented vaccine. The challenge produced severe respiratory signs in nine of 10 unvaccinated control pigs, which developed high H1N2 virus titres in the lungs 24 and 72 hours after the challenge. Vaccination with the commercial vaccine resulted in milder respiratory signs, but H1N2 virus replication was not prevented. Mean virus titres in the pigs vaccinated with the commercial vaccine were 1-5 log10 lower than in the controls at 24 hours but no different at 72 hours. In contrast, the H1N2-supplemented vaccine prevented respiratory disease in most pigs. There was a 4-5 log10 reduction in the mean virus titre at 24 hours in the pigs vaccinated with this vaccine, and no detectable virus replication at 72 hours. These data indicate that the commercial swine influenza vaccine did not confer adequate protection against the H1N2 subtype.     

猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立

根据GenBank中H1N1和H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)、神经氨酸酶(neuraminidase,NA)和M基因保守序列,分别设计合成了5对特异性引物,利用RT-PCR技术对SIV的型和亚型进行鉴定。结果表明,该方法的型RT-PCR可以检测出10⁴ EID₅₀病毒量所提取的RNA;H1、H3、N1和N2的亚型RT-PCR均可以检测出10⁴ EID₅₀病毒量所提取的RNA。除每对特异性引物所对应的亚型外,对其他亚型及猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测均为阴性,应用该方法对临床样品进行检测,其结果与病毒分离结果符合率为100%。结果表明,该方法特异性好、敏感性高,有望成为SIV的一种特异、敏感、快速的分型检测方法,为猪流感分子流行病学的调查奠定了良好的基础。