犬與歐亞狗獾SLAM受體比較及犬源細胞Nectin-4受體對犬瘟熱病毒感染的影響

Abstract

犬瘟熱病毒（Canine distemper virus; CDV）為單股負鏈的RNA病毒，其以高傳染性而聞名，此疾病主要影響犬隻的中樞神經、呼吸和消化系統等。CDV可以利用SLAM（Signaling lymphocytic activation molecule）受體感染免疫細胞，也可以利用Nectin-4受體感染上皮細胞。CDV宿主範圍廣，主要以食肉目為主。過去十年，全球發生多次野生動物爆發CDV感染，包含瀕危物種如：衣索比亞狼和東北虎等。在台灣，CDV對本土的食肉動物物種也構成威脅，白鼻心和鼬獾等野生動物近年也陸續出現CDV陽性病例報告。為了瞭解CDV傳播機制，本研究分為三個部分，分別針對Nectin-4、SLAM受體進行研究，並對台灣犬分離株（NTU311）進行序列分析。第一部分，由於Nectin-4受體分佈在上皮細胞和細胞間的連接處，因此使用不同程度的細胞覆蓋率（confluency）進行測試，選用MDCK細胞，並在40%、70%和100%的細胞confluency下感染野外株（NTU311）和疫苗株（Onderstepoort），結果顯示細胞confluency過高時確實會影響Nectin-4的表現，使CDV的傳播受阻，且CDV疫苗株表現出比野外株更好的感染力。第二部分著重在SLAM受體，探討不同物種的SLAM受體對於CDV的感染力，由於無法取得野生動物的外周血單個核細胞（PBMC），因此選擇合成已知且與鼬獾相似物種-歐亞狗獾（Meles meles）的SLAM序列來進行實驗。為了建立可以應用在永生化細胞及初代細胞的表現系統，本研究選擇利用病毒載體的方式將SLAM基因轉導至細胞中。BacMam表現系統能製做出安全的重組昆蟲桿狀病毒，因此本研究製做攜帶歐亞狗獾及狗SLAM受體序列的重組病毒，用此病毒轉導細胞後，使細胞表達不同的SLAM受體，接著進行CDV野外株和疫苗株的感染實驗。結果顯示以化學轉染方式使細胞表達SLAM受體後，能幫助CDV野外株感染BHK細胞；但若使用重組桿狀病毒轉導細胞表達SLAM受體，CDV感染BHK細胞的能力並未增加，推測是重組桿狀病毒力價過低，因此不足以讓基因轉導進細胞中。未來若能取得台灣野生動物的SLAM受體序列並改良所建立的重組桿狀病毒，將其應用在研究CDV於野生動物的傳播機制，將有助保護本土較脆弱甚至瀕危的野生動物。

The canine distemper virus (CDV) is a highly infectious virus with a negative-sense, single-stranded RNA genome.This disease primarily affects the central nervous, respiratory, and digestive systems of dogs. CDV utilizes the Signaling lymphocytic activation molecule（SLAM）receptor to infect immune cells and the Nectin-4 receptor to infect epithelial cells. It has a broad host range, mainly affecting carnivores. Reports from the last decade have documented numerous CDV outbreaks in wildlife globally, including endangered species such as the Amur tiger and the Ethiopian wolf. In Taiwan, CDV threatens native carnivorous species, with recent reports of CDV-positive cases in wild animals like the masked palm civet and the ferret badger. Despite extensive research on CDV in dogs, understanding its transmission mechanism remains crucial. This research is divided into three parts, focusing on the Nectin-4 and SLAM receptors and conducting full-length sequencing of a Taiwanese canine isolate (NTU311). In the first part, due to the distribution of the Nectin-4 receptor in epithelial cells and cell junctions, we tested different degrees of cell confluency using MDCK cells. We infected the cells with the wild strain (NTU311) and the vaccine strain (Onderstepoort) at 40%, 70%, and 100% confluency. Results showed that high cell confluency indeed affects the expression of Nectin-4, impeding CDV transmission. The CDV vaccine strain exhibited better infectivity than the wild strain. The second part focuses on the SLAM receptor, aiming to understand the infectivity of CDV across different species' SLAM receptors. Due to the unavailability of peripheral blood mononuclear cells (PBMCs) from wild animals, we chose the SLAM sequence of the European badger (Meles meles), a species similar to the ferret-badger. To establish an expression system applicable to both immortalized and primary cells, a viral vector was used to transduce cells with the SLAM gene. The BacMam insect expression system was used to create safe recombinant insect baculoviruses carrying the SLAM receptor sequences of the European badger and dog. After transducing cells with this virus, the cells expressed different SLAM receptors and subsequent infection with the CDV wild strain and vaccine strain showed that expressing the SLAM receptor indeed facilitated CDV infection. Results showed that chemically transfecting cells to express the SLAM receptor facilitated CDV infection in BHK cells; however, using recombinant baculovirus to transduce cells did not enhance CDV infection in BHK cells, likely due to the low titer of the recombinant baculovirus, insufficient for effective gene transduction. Future studies should obtain the SLAM receptor sequences of Taiwanese wild animals and improve the recombinant baculovirus system to study CDV transmission mechanisms in wild animals. This would aid in protecting vulnerable and endangered native wildlife.