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

由于高死亡率和严重的后遗症，日本脑炎(JE)一直是主要的公共卫生问题之一。虽然日本的乙脑病例在1966年后大幅下降，并自1972年以来一直保持在每年100例以下，但在许多亚洲国家，乙脑仍然是一个严重的公共卫生问题。猪的免疫是乙脑病毒的主要放大因子，在低流行条件下进行免疫接种是为了补充人类免疫。猪免疫弱毒疫苗株ML-17是由强毒株JaOH0566在猴肾细胞培养上反复传代而成。据报道，该菌株与其亲本JaOH0566菌株具有不同的生物学特性，如降低小鼠毒力、不存在猪病毒血症和限制媒介蚊子的生长。为了研究病毒RNA的基因组结构与其致病机制的关系，对这些配对的毒株进行了比较测序。从纯化的病毒粒子w…中提取基因组更多的是因为Poly(A)在3‘端拖尾，并且为两个菌株准备了几乎覆盖整个基因组的互补DNA文库，除了极端的5’端。用双脱氧链终止法对这些基因进行测序，用直接测序的方法对数据进行验证，用引物延伸法测定5‘端序列。比较序列数据显示，ML-17与JaOH0566之间有29个核苷酸14个氨基酸的替换(正义突变率为51%)。大多数突变位于3个突变点(PrM-M、NS4B和NS5蛋白的N端)，其中许多突变是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，第一个突变位于推测的PrM和C蛋白的裂解部位，这表明ML-17株病毒多蛋白的加工发生了改变。发现其他突变改变了M蛋白的二级结构，这可能是ML-17菌株在体外对聚乙二醇处理不稳定的原因。第二个突变部位NS4B蛋白的氨基酸替换被发现影响该蛋白的N-糖基化。NS5蛋白第三个突变点的许多氨基酸替换是ML-17所特有的，可以改变蛋白的二级结构。另一方面，JaOH0566与已测序的JaOArS982株之间存在288个核苷酸和18个氨基酸(6.4%)的替换，因此，自然界中频繁的突变大多是无义突变。较少

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