| Co-Investigator(Kenkyū-buntansha) |
TANAKA Mariko Res. Assoc. Dept. Virol. Inst. Trop. Med., 熱帯医学研究所, 助手 (70197474)
MATSUO Sachiko Res. Assoc. Dept. Virol. Inst. Trop. Med., 熱帯医学研究所, 助手 (80039916)
堀 博之 長崎大学, 熱帯医学研究所, 助手 (30199525)
榊 佳之 九州大学, 遺伝情報実験施設, 教授 (10112327)
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| Budget Amount *help |
¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
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
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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
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