乙脑病毒持续感染株preM区序列分析

Significance and preM Sequence Analysis of Different Mutant Japanese Encephalitis Virus Strains in Persistently Infected KN73 Cells

为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒。利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较。preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸)。对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同。认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关。

The significance and preM sequence analysis of two different mutant strains of Japanese encephalitis virus during persistent infection was studied. Two wild strains of Japanese encephalitis viruses （JaGAr-01 and Nakayama） were used to infect a human hepatoma cell line. Persistent infection was established after the cells were subcultured several times. Mutant viruses in persistently infected cells were collected by freezing and thawing of the cells. Viral titers were examined by plaque assys in BHK cells. The preM coding region of four Japanese encephalitis strains, two wild-type and two mutant viruses from infected cells were amplified by RT-PCR. The PCR products were sequenced and the preM sequences of the four strains were compared. The results showed that there was a single aminot acid mutation （E9 Leu→Arg） in the JaGAr-01 persistently infecting mutant in comparison to wild-type JaGAr-01. Two amino acid replacements （E9 Leu→Arg and El3 Val→Ile） were noted in the persistently infecting Nakayama and its wild-type strains. The amino acid sequences of the mutant strains JaGAr-01 and Nakayama were completely the same. We inferred here that genotypic variation existed in preM region of all mutant viruses and that genotypic variation of protein encoded by preM region may play a role in persistent infections and may contribute to the maintenance of the characters of Japanese encephalitis virus and replication.