中国流行性乙型脑炎病毒分子生物学特性研究

The Molecular Biology of Japanese Encephalitis Viruses Isolated in China

为了从分子水平了解中国流行性乙型脑炎(乙脑)病毒与国外分离株的差异,对1949年以来在中国乙脑主要流行地区分离的19株乙脑病毒的PrM C区及E蛋白基因区的核苷酸序列进行了对比研究,以期了解不同时间不同地区在中国流行的乙脑病毒的分子差异。结果显示:病毒生物学初步鉴定显示,病毒感染BHK细胞后56h所有细胞均发生病变,约50%以上细胞脱落(CPE );3日龄乳鼠接种病毒72h之内死亡;所有病毒均与乙脑病毒(A2株)抗血清发生阳性反应,但反应强度不同,提示所有病毒均为乙脑病毒。病毒PrM C(456～695)区核苷酸序列与各个国家及地区、各个基因型以及各个年代的73株乙脑病毒相应序列,用Woan RuChen建立的方法进行基因分型分析,结果显示,所有19株中国分离的病毒均属于基因型Ⅲ型,与国外相关文献所报道的乙型脑炎病毒中国分离株的基因型相符。以乙型脑炎病毒疫苗株P3株为标准,对各病毒E蛋白500个氨基酸序列进行分析。各毒株核苷酸差异在0和4 2%之间,氨基酸差异在0和4 0%之间。所有核苷酸差异均为碱基的替换,无论核苷酸或氨基酸均无插入或丢失。大多数核苷酸变异在氨基酸编码的第三位,属于沉默突变,不引起氨基酸变化。18株病毒E蛋白活性结构域与疫苗株P3株相比共有247个氨基酸的差异,平均每株病毒有12 35个氨基酸的差异,?

Japanese encephalitis virus(JEV)is a major cause of human encephalitis in Asia-Pacific.Study on molecular character of JEV isolated in China will be useful to understand the pathogenic mechanism of this virus and to evaluate the efficiency of the vaccines,which protect people from this disease.19 JEV strains isolated from different places in China were examined in this study to determine their genotypes and molecular characters.These strains covered most JE epidemic regions in China and their isolation history ranged from 1949 to 1987.Most of these viruses were isolated either from human tissues or from mosquitoes.At first,we examined some of their biological characters and found that all of them caused cytopathogenic effect(CPE)in BHK cells,and were fetal to suckling mice.Serological tests showed that all of these 19 virus stains positively reacted with the antibody of A2 virus strain.In subsequent process,we obtained PreM-E genes of the 19 virus isolates by reverse transcription polymerase chain reaction(RT-PCR)and cloned the fragments into pGEM-T vector.By sequencing these cloned genes,we got a nucleotide sequence about 40 000-base pair in all of them .240 nucleotides of PreM region of all 19 JEV strains were selected and compared them with those of reference strains isolated in different countries from GenBank,to make phylogenetic analysis.The results showed that all of the 19 JEV strains belong to genotype Ⅲ.On the basis of determination of the genotype of these 19 JEV strains,we examined their nucleotide and amino acid sequences in all three active domains of the E gene,which cheifly determine the antigenic property of JE virus,and the vaccine strain(SA14-14-2)was used as control.The results showed that there were 62 amino acid substitutions in all 19 strains of JE virus.These substitutions did not locate at the key sites of three active domains of E protein.So,it suggests that the vaccine strains currently used for preventing JE are able to protect people against JEV.However,in this study,we also found that some JEV srains have many substitutions in key sites of active domains of the E protein.These substitutions are considered important and are worthy to study further.