STRUCTURAL ANALYSIS OF JAPANESE ENCEPHALITIS VIRUS GENOME RNA IN RELATION TO ITS PATHOGENESIS

日本脑炎（乙脑）因其高死亡率和严重的后遗症而成为主要的公共卫生问题之一。虽然日本的乙脑病例在1966年以后大大减少，并且自1972年以来一直保持在100/年以下，但乙脑仍然是许多亚洲国家的重大公共卫生问题，猪是乙脑病毒的主要扩增者，为了在低免疫力条件下补充人类免疫，已经进行了猪免疫。ML-17是由JaOH 0566强毒株经猴肾细胞反复传代而成的猪用弱毒疫苗株。据报道，该毒株与其亲本JaOH 0566毒株具有不同的生物学特性，如小鼠毒力降低、不存在猪病毒血症和在媒介蚊子中生长受限。为了研究病毒RNA的基因组结构与其致病性的关系，对这些配对的毒株进行了比较测序。从纯化的病毒体中提取基因组RNA， ...更多信息 作为在3'末端加尾的poly（A），并且制备了两种菌株的互补DNA文库，其覆盖了除末端5'末端之外的几乎整个基因组。用双脱氧链终止法测定cDNA序列，用基因组RNA直接测序法确认数据，用引物延伸法测定5 '端序列。比较ML-17和JaOH 0566菌株的序列数据显示29个核苷酸14个氨基酸的替换（有义突变率，51%）。结果表明，在ML-17株中发现了3个突变位点（PrM-M、NS 4 b和NS 5蛋白的N端），其中有许多是ML-17株所特有的，在其他乙脑病毒株中是保守的，在PrM-M区的第一个突变位点，第一个突变发生在PrM和C蛋白的假定切割位点，提示ML-17株病毒多聚蛋白的加工发生了改变。其他突变改变了M蛋白的二级结构，这可以解释ML-17菌株对聚乙二醇处理的体外不稳定性。在第二个突变焦点，NS 4 b蛋白，氨基酸取代，被发现影响该蛋白的N-糖基化。第三个突变点中的许多氨基酸替换是ML-17特异性的，并且可以改变NS 5蛋白的二级结构。另一方面，JaOH 0566与已测序的JaOArS 982株之间有288个核苷酸和18个氨基酸（6.4%）的替换，因此自然界中常见的突变多为无义突变。少

Japanese encephalitis (JE) has been among major public health problems because of high mortality and grave sequelae. Although the number of JE cases greatly decreased after 1966 and has been kept under 100/year since 1972 in Japan, JE is still a great public health problem in many Asian countries.Immunization of swine, a major amplifier of JE virus, has been performed in order to supplement human immunization under low epidemicity conditions. One of the attenuated vaccine strain for swine immunization, ML-17, was derived form virulent JaOH0566 strain by repeated passages in monkey kidney cell cultures. This strain was reported to have different biological characters form those of its parental JaOH0566 strain, such as reduced mouse virulence, absence of swine viremia and limited growth in vector mosquitoes. In order to study genomic structure of viral RNA in relation to its pathogenesis, these paired strains have comparatively been sequenced. Genomic RNA extracted from purified virion w … More as poly(A) tailed at the 3' end, and complementary DNA libraries were prepared for both strains covering almost entire genome except extreme 5' ends. These cDNAs were sequenced by dideoxy-chain termination method and the data were confirmed by direct sequencing genomic RNA and 5'- end sequences were determined by primer extension method. Comparative sequence data showed 29 nucleotide 14 amino acid substitutions between ML-17 and JaOH0566 strains (sense-mutation rate, 51 %). Most of them were found in 3 mutation foci (PrM-M, NS4b, and N-terminal of NS5 proteins), and many of them were unique to ML-17 strain and conserved among other JE virus strains.In the first mutation locus of PrM-M region, the first mutation was at the presumed cleavage site of PrM and C proteins, suggesting altered processing of viral polyprotein of ML-17 strain. Other mutations were found to change secondary structure of M protein, which could explain in vitro instability of ML-17 strain to polyethylene glycol treatment. Amino acid substitution in the second mutation focus, NS4b protein, was found to affect N-glycosylation of this protein. Many amino acid substitutions in the third mutation focus of were ML-17-specific and could change secondary structure of NS5 protein. On the other hand, 288 nucleotide and 18 amino acid (6.4 %) substitutions were found between JaOH0566 and JaOArS982 strain which was previously sequenced.Therefore, frequent mutations in nature appear to be mostly nonsense- mutations. Less