我国辽宁、黑龙江两省虫媒病毒调查

发布时间：2018-07-07 18:11

  本文选题：乙型脑炎病毒 + 病毒鉴定　； 参考：《山西医科大学》2005年硕士论文

【摘要】：虫媒病毒是一类由吸血节肢动物叮咬敏感的脊椎动物而传播疾病的病毒。虫媒病毒可引起人畜患病或死亡,造成巨大的经济损失。到目前为止,在我国存在和流行的虫媒病毒只有4种。但我国地域辽阔,气候条件复杂,肯定远远不止这几种。因此,加强虫媒病毒的分离与鉴定的研究是非常迫切和有意义的工作。 2002年7～8月在辽宁、黑龙江两省五市县地区,采集蚊虫标本7900余只,以刺扰伊蚊、背点伊蚊、库蚊、蠓等为主。共处理标本90批。用组织培养法分离到4株病毒,命名为:LN02-102、LN02-104、HLJ02-134、HLJ02-136。其中LN02-102、LN02-104分离自辽宁省东港市采集的凶小库蚊和淡色库蚊,HLJ02-134和HLJ02-136均分离自黑龙江省饶和县五林洞珍宝岛采集的库蠓。 4株病毒在BHK-21细胞病变表现为:圆缩、聚集、脱落等。病变时间在60～72小时左右。这4株病毒感染C6/36细胞病变时间在72小时左右,细胞病变主要表现为:聚集、融合、破碎、脱落等。乳鼠颅内接种病毒后,均能引起乳鼠出现神经系统症状,最终在3天死亡。 应用血清学方法对新分离的病毒鉴定。ELISA检测和免疫荧光试验显示:新分离的4株病毒均与乙型脑炎病毒特异性免疫腹水反应。 新分离的4株病毒Real-Time PCR扩增曲线为S型,Ct值在14～17之间,判定为乙型脑炎病毒。应用RT-PCR方法,利用乙脑病毒特异性引物对这4株病毒的PreM-E区基因分别进行扩增,并对PCR产物进行测序。 对乙型脑炎病毒基因进行分析。利用乙型脑炎病毒PreM区进行基因分型,结果显示:LN02-102、LN02-104株属于基因Ⅰ型,HLJ02-134、HLJ02-136株属于基因Ⅲ型。E蛋白是乙型脑炎病毒的结构蛋白,对病毒株的E基因分析结果显示;4株病毒核苷酸之间同源性为92%～94%,氨基酸同源性为99%～100%。LN02-102株和HLJ02-136株分别疫苗株SA14-14-2株和P3株相比,核苷酸的同源性分别为87.67%、98.33%;氨基酸同源性均为98.20%。LN02-102株与我国上海首次分离到的基因Ⅰ型乙脑病毒SH-53相
[Abstract]:Arboviruses are a class of viruses transmitted by bloodsucking arthropods that bite sensitive vertebrates. Arboviruses can cause human and animal disease or death, causing huge economic losses. So far, there are only 4 species of arboviruses present and prevalent in China. But our country territory is vast, the climatic condition is complex, certainly far more than these kinds. Therefore, it is very urgent and meaningful to study the isolation and identification of insect-borne viruses in Liaoning Province, Heilongjiang Province and five cities and Counties from July to August 2002. More than 7900 mosquito specimens were collected, such as Aedes pipiens, Aedes dorsalis, Culex pipiens, midges and so on. A total of 90 batches of treated specimens were obtained. Four strains of the virus were isolated by tissue culture method and named as: LN02-102LN02-104HL-J02-134HL-J02-136. Among them, LN02-102 LN02-104 was isolated from Culex pipiens, HLJ02-134 and HLJ02-136 collected from Donggang City, Liaoning Province. In BHK-21 cells, the pathological changes of the four strains of the virus were as follows: round contraction, Gather, fall, etc The time of lesion was about 60 ~ 72 hours. The cytopathic time of C6 / 36 virus infection was about 72 hours. The main cytopathic manifestations were aggregation, fusion, fragmentation, shedding and so on. Intracranial inoculation of the virus caused neurological symptoms in the neonatal rats and eventually died in 3 days. Serological methods were used to identify the newly isolated viruses. Elisa and immunofluorescence tests showed that the four newly isolated viruses all reacted with specific immune ascites of Japanese encephalitis virus. Real-time PCR amplification curve of 4 newly isolated viruses was that the S type Ct value was between 14 and 17, and it was identified as Japanese encephalitis virus. The PreM-E region genes of the four strains were amplified by RT-PCR and the PCR products were sequenced. The gene of Japanese encephalitis virus was analyzed. Genotyping of B encephalitis virus PreM region was carried out. The results showed that strain 1: LN02-102 LN02-104 belongs to gene type I, HLJ02-134, HLJ02-136 belongs to gene type 鈪，

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