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目的在河南省南阳市采集蚊虫标本,进行西尼罗病毒基因检测,分离流行性乙型脑炎(乙脑)病毒并分析分离株的分子生物学特征。方法 2006年从南阳市采集蚊虫标本,将标本研磨处理后进行巢式PCR检测西尼罗病毒核酸,用细胞培养法对标本进行病毒分离,通过血清学与分子生物学方法鉴定新分离到的乙脑病毒,扩增分离株PrM和E基因区,使用生物学软件进行序列和系统发生分析。结果共采集蚊虫标本6231只,巢式PCR检测西尼罗病毒结果均为阴性。从标本中分离到7株病毒,经鉴定均为乙脑病毒。基因分型显示7株病毒均为基因Ⅰ型,新分离株在E基因之间的核苷酸同源性为98.5%～100%,氨基酸同源性为98.4%～100%。新分离株与P3株和SA14-14-2的E基因核苷酸同源性分别为87.7%～88.2%和87.3%～87.9%。新分离株与P3株相比存在10个共同氨基酸差异位点,但在决定毒力的关键位点不同于SA14-14-2。结论河南省南阳市2006年乙脑病毒流行株为基因Ⅰ型,毒株的毒力基因没有明显改变,所有标本均未检测到西尼罗病毒阳性。
Objective To detect mosquitoes in Nanyang City, Henan Province, and to detect the gene of West Nile virus. The molecular epidemiology of Japanese encephalitis (JE) virus was isolated and analyzed. Methods In 2006, the mosquitoes were collected from Nanyang City. The samples were ground and processed for detection of West Nile virus nucleic acid by nested PCR. The virus was isolated from the samples by cell culture method. The newly isolated B was identified by serological and molecular biological methods Brain viruses were used to amplify the PrM and E gene regions of the isolate using sequence analysis and phylogenetic analysis using biological software. Results A total of 6231 mosquitoes were collected, and the result of nested PCR for detecting West Nile virus was negative. Seven strains of viruses were isolated from specimens and identified as JE virus. The genotypes of all the seven viruses were genotype Ⅰ. The nucleotide sequences of the new isolates were 98.5% -100% and 98.4% -100%. Nucleotide homology of E gene between the new isolate and P3 strain and SA14-14-2 were 87.7% ~ 88.2% and 87.3% ~ 87.9%, respectively. The new isolate has 10 common amino acid difference sites compared with the P3 strain, but differs from SA14-14-2 in determining the virulence of the key sites. Conclusion In 2006, the epidemic strain of Japanese encephalitis was genotype Ⅰ in Nanyang City, Henan Province. There was no significant change in the virulence genes of the strains. None of the specimens were positive for West Nile virus.