| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 100 === Japanese encephalitis virus (JEV) is a member of Flavivirus that usually causes encephalitis. The genome of JEV contains a positive sense single-stranded RNA, approximatly 11kb in length. Due to the lack of a proof-reading function and error-repairing ability, the mutation of genomic RNA frequently occurs and accumulates during the process of viral evolution. Recently, RNA recombination was found also involved in viral evolution. Naturally, JEV can infect both mammalian and mosquito cells. However, it was reported that the mutation occurs more frequently in mammalian cells, leading to accelerated evolution of the virus. We have recently observed that RNA recombination may also occur in JEV, actually more frequently in mammalian cells (BHK-21). Furthermore, we have transfected a sequent of 5’3’UTR RNA, followed by being infected with Nakayama virus strain to observe the occurrence RNA recombination. The results suggested a similar trend of RNA recombination that occurs more frequently in mammalian cells than in mosquito cells. In spite, it remains unclear how a differential rate of RNA recombination appeared between the two different cell types. To clarify the phenomenon, we first investigated the virus competition by infecting cells with different ratio of two virus strains (T1P1-S1 and CJN-S1); it turns out it is not related to the occurrence of RNA recombination. We subsequently looked at the viral growth rates, showing that the genome of both virus strains replicates more rapid in mammalian cells compared to that in mosquito cells. When infected , mammalian cells were incubated at lower temperature to reduce viral growth, appearing a higher viral growth rate in mammalian cells. Presumably, it may have contributions in increase of RNA recombination. Finally, we measured the possible role of viral RNA stability in the occurrence of RNA recombination. It seems that the single-stranded RNA degraded quicker in mosquito cells than in mammalian cells. We have tried to inhibit 5’-3’exoribonuclease (Xrn1p) by 3’-phosphoadenosine-5’-phosphate (pAp) in order to recover RNA stability in this study. The results showed that RNA recombination rates increased in BHK-21 cells, however, decreased in C6/36 cells. It implicates that RNA stability may be differentially modulated by endoribonuclease in the two different cell lines. In conclusion, a higher rate of viral replication may elevate the RNA recombination rate, on the other hand, the lower RNA stability may reduce it. Taken together, arboviruses accelerate the speed of viral evolution in mammalian cells, otherwise slow down in mosquito cells.
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