臺灣藍舌病病毒感染及病毒株性狀分析

Abstract

藍舌病主要感染畜養及野生的反芻動物，為家畜最重要的動物傳染病之一。我國在2003年曾分離到二株藍舌病病毒，分別命名為BTV2/KM/2003與BTV12/PT/2003。本論文紀錄了對我國藍舌病病毒感染近況的調查以及回溯性研究，並運用綿羊人工感染試驗、病毒核酸序列分析、親緣關係分析、病毒基因轉錄之觀察，對國內的藍舌病病毒株進行深一層的了解。全國性的血清學調查顯示藍舌病病毒感染在國內的牛及山羊族群相當普遍，最早可溯自1989年採集的山羊血清檢出抗體陽性。以BTV2/KM/2003病毒株人工接種於無抗體之柯麗黛綿羊驗並未出現任何臨床症狀及病理變化，顯示該病毒株為弱毒株。前述二個病毒株的基因體已完成核苷酸定序，同時亦推演為各基因產物的胺基酸序列。分析其核苷酸序列，發現此二病毒株除了VP2基因之外極為相似，親緣關係分析亦顯示二者與大陸、印度、印度尼西亞及日本的藍舌病病毒株較為相近。此外，亦利用即時反轉錄聚合酶連鎖反應檢測感染之幼齡倉鼠細胞株及感染綿羊包埋組織內的mRNA含量。在受BTV2/KM/2003病毒株感染的細胞中，NS2、VP4、VP7三種基因的轉錄量最高；然而，在感染綿羊的組織內，三種非結構蛋白基因NS1、NS2、NS3的轉錄量最高。這些研究顯示本土藍舌病病毒分離株的弱毒力與田間未出現任何藍舌病臨床病例相互吻合，而國內存在的病毒株屬於亞洲族系。

Bluetongue (BT) is one of the most important infectious animal diseases, mainly affecting domestic and wild ruminants. Two bluetongue virus (BTV) strains, BTV2/KM/2003 and BTV12/PT/2003, were isolated in 2003. The studies documented in the dissertation aimed to investigate the serological status and chronology of BTV infection in Taiwan, and to characterize the virulence and pathogenicity of Taiwan BTV strains through experimental infection in sheep, determination of sequences, analyses of phylogenetic relationships, and measurement of viral gene transcription. The nationwide serological surveillance revealed that BTV infection is prevalent in cattle and goat populations in Taiwan, and seropositive goat sera existed since 1989. The Corriedale sheep experimentally infected with BTV2/KM/2003 strain demonstrated neither observable clinical signs nor pathological lesions specific to BT, suggesting a low virulence of the Taiwan strain. Full-length nucleotide sequences of the genome of the two virus strains were determined and their amino acid sequences deduced. Nucleotide sequences analyses showed that the two strains were highly similar to each other, except for the VP2 genes, and that the two strains were phylogenetically closely related to strains from China, India, Indonesia, and Japan. Moreover, amounts of mRNAs in the infected baby hamster kidney cell line and in the formalin-fixed paraffin-embedded (FFPE) tissues of the infected sheep were measured by real-time reverse transcription polymerase chain reaction (rRT-PCR). In infected cells, NS2, VP4, and VP7 genes were the most transcribed; whereas in the FFPE tissues of the infected sheep, three nonstructural protein genes, NS1, NS2, and NS3 were the most abundant. To conclude, our studies supported the observation of asymptomatic BTV infection in the field and the virus strains currently circulating in Taiwan might be low virulent and belonged to the Asian lineage.