猪源2009甲型H1N1流感病毒的分离鉴定及其致病性的研究

发布时间：2018-06-17 10:18

本文选题：猪源甲型H1N1流感病毒 + 分离鉴定　；参考：《广西大学》2015年硕士论文

【摘要】：2009年3月新甲型H1N1流感病毒在墨西哥暴发,同年4月第1株2009甲型H1N1流感病毒被分离,之后有越来越多的报道表明2009甲型H1N1流感病毒可以由人向猪传播。研究证明,2009甲型H1N1流感病毒是混合了猪源流感病毒、禽源流感病毒和人源流感病毒三者特征的重组病毒,给养猪业带来巨大经济损失,也对人类的健康构成威胁。因此对猪群中H1N1流感病毒进行流行病学调查和监测具有重要的公共卫生意义。本实验从2013年1月～2014年12月对广西省部分养猪场及发病猪群进行样品采集和病毒的分离,目的为研究该区域猪群中流行的流感病毒亚型、变异规律和发病情况,为猪流感的研究和防控提供参考。实验成果如下：1.从发病猪群分离鉴定得到12株H1N1亚型猪流感病毒,其中1株为2009甲型H1N1流感病毒。本研究从2013年1月～2014年12月采集至广西8个市(地区)发病猪群的猪组织病料共1609份进行病毒分离。经9-11日龄SPF鸡胚分离得到12株猪流感病毒株。运用RT-PCR的方法对12株病毒的HA和NA基因进行扩增和序列测定分析,鉴定12株病毒均为H1N1亚型,其中1株为2009甲型H1N1流感病毒。将这株猪源2009甲型H1N1流感病毒株作为本实验研究的对象,并命名为A/swine/Guangxi/18/2013(H1N1)。2.遗传进化分析表明分离株8个基因片段与其他2009甲型H1N1流感病毒的进化关系一致。对A/swine/Guangxi/18/2013(H1N1)全基因组的8个基因片段进行RT-PCR扩增、克隆及序列测定,运用MegAlign软件进行核苷酸和氨基酸的比较分析,再通过MEGA5.0软件构建遗传进化树。进化分析结果表明分离株与2009甲型H1N1流感毒株处于同一个进化枝,与A/California/04/2009(H1N1)亲缘关系很近,起源于同一祖先。其基因来源情况为：HA、NS和NP基因来源于古典型H1N1猪源谱系,NA和M基因来源于类禽型H1N1猪源谱系,PA和PB2基因来源于禽源谱系,PB1基因来源于H3N2人源谱系。分离株8个基因片段在抗原位点、受体结合位点、糖基化位点以及耐药性位点等多处氨基酸发生了变异,表明其抗原特性、宿主适应性、致病力以及抗药性发生了变化。3.动物实验发现分离株对BALB/c小鼠具有致病性。对BALB/c小鼠的攻毒实验结果显示病毒可感染小鼠,并能在肺脏与鼻甲骨组织中进行有效复制,感染小鼠后引起明显的临床症状,体重迅速下降造成小鼠平均体重最高降幅为13.02%。
[Abstract]:A new influenza A (H1N1) virus broke out in Mexico in March 2009, and the first strain of influenza A (H1N1) virus 2009 was isolated in April of the same year. After that, more and more reports showed that the 2009 A (H1N1) influenza virus could be transmitted from human to pig. It has been proved that the 2009 A (H1N1) influenza virus is a recombinant virus mixed with swine influenza virus, avian influenza virus and human influenza virus, which brings huge economic loss to pig industry and poses a threat to human health. Therefore, epidemiological investigation and surveillance of swine H1N1 influenza virus have important public health significance. From January 2013 to December 2014, samples were collected and virus isolated from some pig farms and infected pigs in Guangxi province. To provide reference for swine flu research and prevention and control. The results of the experiment are as follows: 1. Twelve strains of swine influenza A (H1N1) subtype were isolated and identified from the infected pigs, one of which was A (H1N1) virus. From January 2013 to December 2014, a total of 1609 samples were collected from pigs infected in 8 cities (regions) of Guangxi for virus isolation. Twelve strains of swine flu virus were isolated from 9-11 day old SPF chicken embryo. The HA and na genes of 12 viruses were amplified and sequenced by RT-PCR. All the 12 strains were identified as H1N1 subtypes, one of which was A (H1N1) virus. The swine strain of influenza A (H1N1) 2009 was named as A / swine / 18 / 2013H _ 1N _ 1 / 2. Genetic evolution analysis showed that 8 gene fragments of the isolated strain were consistent with the evolution of other influenza A (H1N1) viruses. Eight gene fragments were amplified by RT-PCR, cloned and sequenced. The nucleotide and amino acids were compared with MegAlign software, and then the genetic evolution tree was constructed by MEGA5.0 software. The results of evolutionary analysis showed that the isolated strain was in the same phylogenetic branch as the influenza A (H1N1) strain 2009, and was closely related to A / California / 04 / 2009 H1 / N1, and originated from the same ancestor. The gene sources were as follows: 1. The gene sources of Ham NS and NP genes were from na and M genes of the classical H1N1 swine lineage. The PA and PB2 genes of the avian H1N1 swine lineage were derived from the avian origin lineage and PB1 gene from the H3N2 human lineage. The amino acids of 8 gene fragments of the isolated strain were mutated at antigenic sites, receptor binding sites, glycosylation sites and drug resistance sites, indicating that the antigenic characteristics, host adaptability, pathogenicity and drug resistance changed .3. Animal experiments showed that the isolated strain was pathogenicity to BALB / c mice. The results of virus attack on BALB / c mice showed that the virus could infect mice and replicate effectively in the lungs and turbinate bone tissues. After infection, the mice caused obvious clinical symptoms, and rapid weight loss resulted in the highest average weight reduction of 13.02% in mice.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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