Abstract
Japanese encephalitis virus (JEV) is a mosquito-borne virus that belongs to the family Flaviviridae, genus Flavivirus. JEV causes fatal disease in human infection case and leads half of the survivors to neural system disrupted and psychoneurosis. The attenuated variant CH2195LA and the non-attenuated variant CH2195SA of Japanese encephalitis virus were both isolated from wild-type Taiwanese mosquito isolates CH2195. The attenuated variant CH2195LA was sensitive to mAb E3.3 neutralization and the non-attenuated variant CH2195SA was resistant to mAb E3.3 neutralization. The purpose of this research is to analyze the complete nucleotide sequence genotype and cell-line multiplication pattern phenotype of the attenuated variant CH2195LA of Japanese encephalitis virus. In the genotype study, we determined the complete nucleotide sequences of the two variants and found amino acid changes between them. Then we used complete nucleotide sequences phylogenetic analysis to compare the evolutionary relationship of the attenuated variant CH2195LA and other 14 JEV strains. Our study found there were 9 amino acid changes between the complete sequences of the two variants, including E-85, E-306, E-331, E-387, NS2A-215, NS3-350, NS4B-196, NS4B-197, and NS4B-198. There were 12 amino acid changes between CH2195LA and CH2195LA-SM4, including M-73, M-80, E-161, E-170, E-276, NS2A-136, NS2A-215, NS3-346, NS4A-128, NS4B-196, NS4B-197, and NS4B-198. The phylogenetic analysis indicated the attenuated variant CH2195LA is closely related to the non-attenuated variant CH2195SA and JaOArS982, and is distantly related to SA14-14-2, SA14-2-8, and RP-2ms of the attenuated JEV strains.The multiplication patterns of the two variants were investigated in eight different cell lines. The virus replication rate and maximum infectivity of the attenuated variant CH2195LA in Vero cell multiplication pattern were both higher than those of the non-attenuated variant CH2195SA. The multiplication patterns for CH2195LA compared with CH2195SA in other seven cell lines were different from Vero cell. We further determined the relative fitness of CH2195LA verse CH2195SA by using direct competitive mixture of both variants to measure the relative fitness change in three different cell lines passages, including Vero, C6/36, and THP-1. Relative fitness vector plot indicated the attenuated variant CH2195LA showed increased viral fitness in Vero cell line passages, but viral fitness of the mouse brain passage sample CH2195LA-SM4 was lower than the attenuated variant CH2195LA. The multiplication patterns and relative fitness results indicated that the attenuated variant CH2195LA was unique to culture in Vero cell lines, but it changed after mouse brain passage. The results suggested that the genome of the attenuated variant CH2195LA was not stable after mouse brain passaged. We can raise its gemome stability by continuous passaging the attenuated variant CH2195LA in Vero cell line. The results of this research were important for realizing the attenuation mechanism of the attenuated variant CH2195LA and developing attenuated JEV vaccines.英文摘要……………………………………………………….3第一章 緒論………………………………………………….5第二章 材料與方法……………………………….………………22第三章 結果……………………………………….………………36第四章 討論……………………………………….………………46第五章 結論………………………………………………..58圖表…………………………………………………………60參考文獻……………………………………………………….…..81