本文選題：乙型腦炎病毒 + 病毒鑒定�。� 參考：《山西醫(yī)科大學》2005年碩士論文

【摘要】：蟲媒病毒是一類由吸血節(jié)肢動物叮咬敏感的脊椎動物而傳播疾病的病毒。蟲媒病毒可引起人畜患病或死亡,造成巨大的經(jīng)濟損失。到目前為止,在我國存在和流行的蟲媒病毒只有4種。但我國地域遼闊,氣候條件復雜,肯定遠遠不止這幾種。因此,加強蟲媒病毒的分離與鑒定的研究是非常迫切和有意義的工作。 2002年7～8月在遼寧、黑龍江兩省五市縣地區(qū),采集蚊蟲標本7900余只,以刺擾伊蚊、背點伊蚊、庫蚊、蠓等為主。共處理標本90批。用組織培養(yǎng)法分離到4株病毒,命名為:LN02-102、LN02-104、HLJ02-134、HLJ02-136。其中LN02-102、LN02-104分離自遼寧省東港市采集的兇小庫蚊和淡色庫蚊,HLJ02-134和HLJ02-136均分離自黑龍江省饒和縣五林洞珍寶島采集的庫蠓。 4株病毒在BHK-21細胞病變表現(xiàn)為:圓縮、聚集、脫落等。病變時間在60～72小時左右。這4株病毒感染C6/36細胞病變時間在72小時左右,細胞病變主要表現(xiàn)為:聚集、融合、破碎、脫落等。乳鼠顱內(nèi)接種病毒后,均能引起乳鼠出現(xiàn)神經(jīng)系統(tǒng)癥狀,最終在3天死亡。 應用血清學方法對新分離的病毒鑒定。ELISA檢測和免疫熒光試驗顯示:新分離的4株病毒均與乙型腦炎病毒特異性免疫腹水反應。 新分離的4株病毒Real-Time PCR擴增曲線為S型,Ct值在14～17之間,判定為乙型腦炎病毒。應用RT-PCR方法,利用乙腦病毒特異性引物對這4株病毒的PreM-E區(qū)基因分別進行擴增,并對PCR產(chǎn)物進行測序。 對乙型腦炎病毒基因進行分析。利用乙型腦炎病毒PreM區(qū)進行基因分型,結果顯示:LN02-102、LN02-104株屬于基因Ⅰ型,HLJ02-134、HLJ02-136株屬于基因Ⅲ型。E蛋白是乙型腦炎病毒的結構蛋白,對病毒株的E基因分析結果顯示;4株病毒核苷酸之間同源性為92%～94%,氨基酸同源性為99%～100%。LN02-102株和HLJ02-136株分別疫苗株SA14-14-2株和P3株相比,核苷酸的同源性分別為87.67%、98.33%;氨基酸同源性均為98.20%。LN02-102株與我國上海首次分離到的基因Ⅰ型乙腦病毒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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