流感病毒H5N1亚型虎源株的生物学特性及溯源

发布时间：2018-09-07 07:07

【摘要】：高致病性禽流感病毒为甲型流感病毒,主要在禽类中传播。近年来不断有人类感染高致病性禽流感并引起死亡的报导,也有其他物种如猪、犬、猫、虎、豹等感染的报导。该病毒的跨物种传播引起了人们的广泛关注。本试验分离到一株高致病性虎源流感毒株,研究了其生物学特性,并对其来源进行了追溯。1流感病毒H5N1亚型虎源株的分离某动物园饲养的孟加拉国虎突然死亡,荧光PCR鉴定为流感病毒H5N1亚型,用SPF鸡胚从该虎肺脏中分离到一株流感病毒H5N1亚型,命名为A/Tiger/Jiangsu/1/2013。将采集的虎组织病料做病理切片,显示肝脏的中央静脉区有坏死灶,脾内动脉周围淋巴鞘区域的淋巴细胞和边上脾淋巴小结比较明显稀少,肺脏的细支气管内有大量脱落的上皮细胞堵塞管腔。免疫组织化学切片显示,肺泡内有H5N1亚型流感病毒抗原存在,此为孟加拉虎感染H5N1高致病性禽流感病毒提供了组织学证据。2流感病毒H5N1亚型虎源株的核酸序列测定及致病性虎源分离株经SPF鸡胚纯化,并测定其核酸序列。序列经与NCBI数据库比对,发现同源性最高的为2012年一个越南H5N1分离株:A/muscow duck/Vietnam/LBM/227/2012。测定纯化后毒株的EID50,分别以101～106EID50的稀释度接种BALB/c小鼠,并测定出小鼠组织中的病毒滴度,发现在肺组织中病毒滴度最高,用Reed-Muench法计算病毒对小鼠的半数致死量(MLD50),结果显示在第8天时接种不同滴度的所有小鼠均已死亡,提示该毒株对小鼠的致病性极强。3虎源株的溯源样本检测推测饲喂市售的光鸡可能是导致该虎感染的原因,但光鸡来源无从查找。课题协作单位对周边12个地市的活禽交易市场和大型养禽场展开流行病学调查,历时一年,采集样品1168份,样品的种类包括肛拭、咽拭、洗禽污水、养殖饮水、粪便、装禽的笼具和宰禽砧板拭子等。作者进行了荧光PCR检测,甲型流感病毒核酸阳性的样品进而分离和纯化病毒,二代测序技术测定其序列。PCR检测结果显示阳性样品总数为25例,其中常州和镇江阳性样本数量最多,均为6例,阳性率分别为3.8%和2.99%。最少的地区为淮安和泰州,均未能检测到阳性样品;样品总体平均阳性率为1.8%。在所有样品类型中宰杀禽类的砧板阳性检出率最高,为3.53%,其次是笼具表面的拭子,为3.18%,洗禽的污水阳性率排列第三位,为3.09%,检测率最低的是粪便,仅有1例,阳性率为0.62%。25份阳性样品中共分离到流感毒株27株,有两例样品含有混合毒株。27株流感病毒亚型分布为:1株H3N2,1株H5N1,2株H5N6,1株H6N6,3株H5N8亚型,10株H7N9和9株H9N2。所有的分离株用Illumina MiSeq测序系统进行全基因测序,测序结果用于毒株的遗传进化树构建和遗传演化分析。4.虎源株的遗传学特性分析构建H5亚型毒株各基因片段的遗传进化树,分析H5亚型毒株各基因片段的氨基酸序列,发现H5N1亚型虎源株与2014年镇江分离的H5N1亚型禽源株的7个片段在遗传进化树上始终在同一分支上;在各片段氨基酸序列比较上,二者的HA1和HA2断裂位点的氨基酸序列均符合高致病性禽流感毒株的特征;在HA序列上适应哺乳动物受体的突变位点、HA和NA糖基化位点、NA耐药性突变位点、宿主特异性位点及M1、M2、NS1、NS2、NP、PB1、PB2、PA等片段的致病性和耐药性相关的突变位点二者也高度一致,这些分析结果证实,来自镇江的H5N1亚型禽源株与H5N1亚型虎源株存在较高的遗传演化关系,推测二者可能来自同一亲本。在流感病毒H5亚型分离株的进化树图中还发现,虎源株的PB2片段与此次分离的流感病毒H5N8亚型南通株 EV/Nantong/1029/2014(H5N8)和镇江株 EV/Zhenjiang/4342/2014(H5N8)在同一进化支上,而非镇江分离的H5N1亚型禽源株。这一结果提示,虎源株的PB2片段可能来自流感病毒H5N8亚型。5流感病毒H7N9与H9N2亚型毒株与H5亚型毒株内部片段的比较本试验分析的毒株中,H7N9亚型毒株数量最多,占了全部分离株的三分之一以上;其次为H9N2亚型毒株,数量占三分之一。据报道,H7N9亚型流感毒株的内部片段全部来自H9N2亚型。这些毒株与分离鉴定的虎源株出现的时间非常接近,因此有必要分析H7N9亚型、H9N2亚型及其H5亚型毒株基因片段的遗传演化关系。结果显示,H7N9与H9N2亚型毒株的内部6个片段的遗传进化树中,流感病毒不同亚型的片段却在同一分支上,这一现象提示,H7N9亚型毒株的内部片段确实来自H9N2亚型,而且二者的PB2片段与H5亚型毒株的PB2片段也存在一定的遗传演化关系。
[Abstract]:Highly pathogenic avian influenza viruses are mainly spread among birds. In recent years, there have been reports of human infection with highly pathogenic avian influenza and death. There are also reports of other species * such as pigs, dogs, cats, tigers, leopards, etc. the cross species transmission of the virus has attracted wide attention. Isolation of H5N1 subtype influenza virus strain from a zoo of Bangladesh tiger died suddenly. Fluorescent PCR was used to identify the H5N1 subtype influenza virus. A strain of influenza virus H5N1 subtype was isolated from the tiger's lung by SPF chicken embryo and named A/Tiger. / Jiangsu / 1 / 2013. Tiger tissue pathological sections showed necrosis in the central vein of the liver, marked rarity of lymphocytes in the lymphatic sheath around the splenic artery and peripheral splenic lymph nodules, and massive exfoliated epithelial cells in the bronchioles of the lungs. The presence of H5N1 subtype influenza virus antigens provided histological evidence for the infection of Bengal Tigers with H5N1 highly pathogenic avian influenza virus. Nucleic acid sequencing of H5N1 subtype tiger-origin influenza virus strains and purification of pathogenic tiger-origin isolates from SPF chicken embryos were carried out and their nucleic acid sequences were determined. A Vietnamese H5N1 isolate, A/muscow duck/Vietnam/LBM/227/2012, was isolated from Vietnam in 2012. EID50 of the purified strain was determined. BALB/c mice were inoculated with the dilution of 101-106 EID50 respectively. The viral titer in the lung tissue was the highest. Reed-Muench method was used to calculate the half lethal dose of the virus in mice (M LD50) results showed that all mice inoculated with different titers died on the 8th day, suggesting that the virus strain was highly pathogenic to mice. 3 Traceable samples of tiger-origin strain were tested and speculated that the bare chickens fed on the market might be the cause of the tiger infection, but the source of bare chickens could not be found. An epidemiological investigation was carried out in farms and large poultry farms over a period of one year. 1168 samples were collected, including anal swabbing, pharyngeal swabbing, poultry washing wastewater, aquaculture drinking water, feces, poultry cages and slaughter stock swabs. The authors carried out fluorescent PCR detection, isolation and purification of influenza A virus nucleic acid positive samples, and second-generation sequencing. PCR results showed that the total number of positive samples was 25, of which Changzhou and Zhenjiang had the largest number of positive samples, with the positive rates of 3.8% and 2.99% respectively. The smallest areas were Huaian and Taizhou, where no positive samples were detected; the overall average positive rate of the samples was 1.8%. The highest positive rate was found on the cutting board (3.53%), followed by the surface swabs of cages (3.18%), and the third was found in the sewage of poultry washing (3.09%). 1 strain H3N2, 1 strain H5N1, 2 strains H5N6, 1 strain H6N6, 3 strains H5N8 subtypes, 10 strains H7N9 and 9 strains H9N2. All the isolates were sequenced by Illumina MiSeq sequencing system. The sequencing results were used to construct the genetic evolution tree and analyze the genetic evolution of the H5 subtype strain. The amino acid sequence of each gene fragment of H5 subtype strain was analyzed. It was found that seven fragments of H5N1 subtype tiger strain and H5N1 subtype avian strain isolated from Zhenjiang in 2014 were always on the same branch in the genetic evolution tree. The characteristics of susceptible strains; HA and NA glycosylation sites, NA resistance mutation sites, host specific sites and M1, M2, NS1, NS2, NP, PB1, PB2, PA and other fragments of pathogenicity and resistance-related mutation sites are also highly consistent in HA sequence. These results confirmed that the H5N1 subtype from Zhenjiang. The phylogenetic tree of H5 subtype H5 isolates also showed that the PB2 fragment of H5 subtype H5 strain was related to the EV/Nantong/1029/2014 (H5N8) and Zhenjiang EV/Zhenjiang/4342/2014 (H5N8) of Nantong strain H5N8 and H5N8 subtype H5N8. The results suggest that the PB2 fragment of H5N1 subtype strain may come from the internal fragments of H7N9 and H9N2 subtypes of influenza virus H5N8 and H5 subtypes. Among the strains tested, H7N9 subtype was the most abundant, accounting for three of the total isolates. It is reported that the internal fragments of H7N9 subtype influenza virus strain are all from H9N2 subtype. These strains are very close to the origin of tiger strains, so it is necessary to analyze the genetic evolution of H7N9 subtype, H9N2 subtype and H5 subtype. The results showed that the fragments of influenza virus subtype H7N9 and H9N2 were in the same branch among the six internal segments of the evolutionary tree. This phenomenon suggested that the internal fragments of H7N9 and H9N2 subtypes were indeed from H9N2 subtype, and there was a certain genetic evolution relationship between their PB2 fragments and the PB2 fragments of H5 subtype.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】