论文部分内容阅读
目的对辽宁省朝阳市蚊虫中分离的一种新病毒进行分子生物学鉴定。方法在辽宁省朝阳市采集蚊虫,用细胞培养方法分离病毒,对引起C6/C36细胞病变(CPE)的病毒进行逆转录-聚合酶链式反应(RT-PCR)检测,并对阳性PCR产物进行测序分析;对病毒编码区全基因进行测序并与GenBank中36株黄病毒参考毒株序列进行比较;用Mega3.1软件、以邻位相连法构建黄病毒属编码区全基因遗传进化树。结果从蚊虫中分离到2株病毒,黄病毒属通用引物RT-PCR检测均为阳性,扩增片段大小986bp,其中有600～750bp核苷酸碱基序列与登革病毒2型、伊桂普病毒、塞皮克病毒和班奇病毒有66%～68%的一致性,2株病毒间碱基序列100%一致,为同一种黄病毒;病毒编码区基因序列全长10308bp,与其他黄病毒比较结果表明,分离株E基因、NS3基因及NS5基因碱基序列与其他黄病毒碱基序列一致性最高分别为59.6%,61.7%和67.0%;编码区全基因遗传进化树分析结果显示,分离株与登革病毒群、日本脑炎病毒群等蚊传黄病毒拥有共同进化祖先,但在系统发生树上处在单独进化分支。结论辽宁省朝阳市分离到的黄病毒毒株不属于现有黄病毒属任何一种群,为黄病毒属的一个新种群,经GenBank分析确认为世界首次发现,并命名为朝阳病毒,收录号为FJ883471。
Objective To identify the molecular biology of a new virus isolated from mosquitoes in Chaoyang City, Liaoning Province. Methods Mosquitoes were harvested in Chaoyang City, Liaoning Province. The virus was isolated by cell culture method. The virus of C6 / C36 cytopathic effect (CPE) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) Sequence analysis of the full-length gene of the coding region of the virus was performed and compared with that of 36 flavivirus reference strains in GenBank. The whole gene phylogenetic tree of the flavivirus coding region was constructed by using the software Mega3.1. Results Two viruses were isolated from mosquitoes, and the flavivirus genotypes were all detected by RT-PCR. The amplified fragment was 986bp in size, including 600 ~ 750bp nucleotide sequence and dengue virus type 2, 66% -68% of the viruses, the same, and the same type of flavivirus. The full length of the coding region of the virus is 10308bp, which is identical to other flaviviruses The results showed that the identities of E gene, NS3 gene and NS5 gene of the isolates were 59.6%, 61.7% and 67.0%, respectively. The results of genetic tree analysis showed that the isolates The strain has a coevolutionary ancestor with mosquito flaviviruses such as the dengue virus group and the Japanese encephalitis virus group, but the phylogenetic tree is in a separate evolutionary branch. Conclusion The flavivirus isolated from Chaoyang in Liaoning Province does not belong to any one of the existing flavivirus groups. It is a new genus of flavivirus. It was identified by GenBank as the first discovery in the world and named as Chaoyang virus, FJ883471.