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標題: The Host Specificity of Culex flavivirus
Culex flavivirus 之宿主特異性
作者: 賴以勤
Yi-Chin Lai
關鍵字: 日本腦炎
Japanese encephalitis virus
Host Specificity
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摘要: Most members of the Flavivirus genus in the family of Flaviviridae, including Japanese encephalitis virus (JEV), dengue virus (DENV), and West Nile virus (WNV), infect vertebrate and arthropod hosts. Recently, several novel flaviviruses that replicate only in mosquitoes have been isolated and described as insect-specific flaviviruses, including cell fusing agent virus (CFAV), Quanig Binh virus (QBV), and Culex flavivirus (CxFV). In 2010, CxFV was first time detected in Culex tritaeniorhynchus and in Taiwan. In this study, we plans to characterize the host specificity of CxFV and also to identify the possible determinants of CxFV host specificity. The results of immunofluorescence assay and western blotting shown that CxFV replicates in Aedes-derived C6/36, Culex-derived CT and CTR cells, but not in human-derived 293T, monkey-derived Vero, hamster-derived BHK-21, swine-derived PK-15, chicken-derived CER , and Drosophila-derived sf9 cells; and these results indicated the host range of CxFV was mosquito-specific. Because mosquito- and vertebrate-derived cells were cultured in different temperatures, the temperature sensitivity assay was conducted. The results shown the CxFV entry into and replicates in cells at 37℃, and indicated temperature was not the determinant factor for CxFV host specificity. Then, in order to study the determinant step of viral life cycle for CxFV host specificity, several experiments were conducted. The results of viral adsorption assay shown that CxFV binds to C6/36 cells and also to BHK-21 cells. In viral RNA transfection experiment, the results shown the newly synthesized viral proteins and viral positive- and negative-stranded RNA were undetectable. In viral fusion and uncoating processes, the envelope protein was undergoing conformational change among acided-endosome. In summary, in this study, we suggested that the CxFV was a mosquito-specific flavivirus; the restricted host range of CxFV was not determined by the step of receptor binding and viral RNA synthesis, and might be determined by the steps of viral RNA synthesis or viral fusion/uncoating.
黃病毒屬(Flavivirus)家族的大多數成員,包括日本腦炎病毒(Japanese encephalitis virus, JEV)、登革病毒(Dengue virus, DENV)及西尼羅病毒(West Nile virus, WNV),皆可以感染脊椎動物和節肢動物宿主。最近,許多只能在昆蟲體內複製的新興黃病毒被分離出,並命名為昆蟲特異性黃病毒(insect-specific flaviviruses),包括cell fusing agent virus (CFAV)、Quanig Binh virus (QBV)和Culex flavivirus (CxFV)。在2010年,本實驗室從進行日本腦炎病毒監測時,由養豬場所收集三斑家蚊中首次測得Culex flavivirus。因此,本研究計畫分析Culex flavivirus之宿主特異性(host specificity)或宿主範圍(host range),並找出可能的影響因素。根據免疫螢光染色法和西方墨點法之實驗結果,顯示Culex flavivirus可以感染白線斑蚊來源之C6/36、環徑家蚊來源之CT、三斑家蚊來源之CTR細胞株;但卻不能感染人來源之239T、猴子來源之Vero、倉鼠來源之BHK-21、豬來源之PK-15、雞來源之CER以及夜蛾來源之SF9細胞株;這些結果顯示Culex flavivirus的宿主範圍為蚊子特異性(mosquito-specific)。因為蚊子和脊椎動物細胞是在不同的溫度下培養,因此進行溫度敏感性實驗分析,結果顯示在37℃下Culex flavivirus可以進入C6/36細胞並進行複製,所以培養溫度不同不是造成Culex flavivirus宿主特異性的原因。在探討病毒生活史中哪一步驟影響Culex flavivirus宿主特異性的實驗方面,病毒吸附(binding)實驗結果顯示,Culex flavivirus可以結合至C6/36細胞膜上,也可以結合至BHK-21細胞膜上;在病毒RNA轉染(viral RNA transfection)實驗結果方面,將Culex flavivirus病毒RNA直接送入BHK-21細胞中,並未偵測到新合成的病毒蛋白質及正股與負股病毒RNAs;目前因無法直接測量病毒融合(fusion)與脫殼(uncoating)現象,而此兩步驟均與endosome酸化造成病毒套膜蛋白(envelope, E)立體結構改變有關。總結以上結果,Culex flavivirus是蚊子特異性黃病毒(mosquito-specific flavivirus);而病毒生活史中,造成Culex flavivirus宿主特異性(不感染脊椎動物細胞)決定步驟並非在於與接受器的結合或是病毒RNA合成之步驟,推測是病毒蛋白質合成或是病毒進入細胞時融合或脫殼步驟扮演決定性角色。
文章公開時間: 2015-08-28
Appears in Collections:微生物暨公共衛生學研究所



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