台灣野生食肉目動物疾病監控調查：鼬獾狂犬病毒鑑定與特性分析首例

Abstract

本研究的目的是調查台灣野生食肉目動物死亡與潛在病因，並針對發現的重要或有趣的疾病進行深入研究。在2011年8月至2015年1月期間共收集51例救傷死亡或路死的食肉目動物屍體，經由詳細解剖與組織病理學檢查、分子與免疫學分析、微生物學及寄生蟲學檢測等進行病因分析。這些案例包括31例台灣鼬獾(TWFBs) (Melogale moschata subaurantiaca)、12例白鼻心(MPCs) (Paguma larvate taivana)、5例麝香貓(SCCs) (Viverricula indica pallida)及3例食蟹獴(CEMs) (Herpestes urva)。人畜共通狂犬病與致死性犬瘟熱分別於4例台灣鼬獾與3例白鼻心被確診。 台灣鼬獾狂犬病的特徵性病理變化為非化膿性腦膜腦脊髓炎、神經節炎及形成典型的細胞質內包涵體- Negri bodies，受影響的中樞神經系統以腦幹病變最為嚴重。此外，大腦皮質部、丘腦與腦幹的神經元與神經氈具有不同程度的海綿狀變性。在非神經組織的代表性病變包括腎上腺壞死與間質淋巴球性唾液腺炎。經由免疫組織化學染色法(IHC)與螢光抗體試驗(FAT)，病毒抗原可在神經組織的神經元細胞與軸突/樹突等區域，以及全身不同組織間的巨噬細胞中被偵測到。結果顯示，腦幹、大腦皮質部、海馬角、丘腦與下視丘是台灣鼬獾狂犬病分子診斷的理想的採樣區。我們利用不同單位提供的2004-2012年間福馬林固定、石蠟包埋的舊有台灣鼬獾腦組織，進行免疫組織化學染色回溯性研究，發現狂犬病陽性案例最早可追溯至2004年。 為了分析台灣鼬獾狂犬病病毒的源起，我們完成三株狂犬病病毒全基因體定序分析，並由公告的幾株狂犬病毒(RABV)核蛋白(N)與醣蛋白(G)序列進行親緣地理學分析顯示，台灣鼬獾狂犬病病毒（RABV-TWFB）來自亞洲譜系並已獨立演化，其近緣病毒株China I (包括中國鼬獾狂犬病病毒株; RABV-CNFB) 和菲律賓狂犬病病毒的分化年代在158-210年前，而台灣鼬獾狂犬病病毒株的最近共祖起源年代約在91-113年前。我們的研究顯示，此次地方性台灣鼬獾狂犬病病毒基因分析的古老親緣結果說明了台灣鼬獾的狂犬病病毒株可能隱晦的潛藏在環境中長期的慢慢傳播，而致一直未被檢出，此病毒與宿主間的交互作用及其潛存的機制值得進一步研究。

The objective of this study was to investigate the causes of death and potential diseases carried by the wild-ranging carnivores in Taiwan. For those interesting and essential diseases, further studies in depths were performed. A total of 51 carcasses from rescued but dead or road-killed carnivores, collected during the period of August 2011 to January 2015, were necropsied for histopathology, molecular and immunological assays, microbiology, and parasitology. The cases included 31 Taiwan ferret badgers (TWFBs) (Melogale moschata subaurantiaca), 12 masked palm civets (MPCs) (Paguma larvate taivana), 5 small Chinese civets (SCCs) (Viverricula indica pallida), and 3 crab-eating mongooses (CEMs) (Herpestes urva). Zoonotic rabies and fatal canine distemper were diagnosed in 4 TWFBs and 3 MPCs, respectively. The characteristic pathological changes of rabid TWFBs were nonsuppurative meningoencephalomyelitis, ganglionitis, and formation of typical intracytoplasmic Negri bodies with brain stem affected the most. Additionally, variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, was observed in the cerebral cortex, thalamus, and brain stem. In the non-nervous tissue, representative lesions included adrenal necrosis and lymphocytic interstitial sialoadenitis. By immunohistochemical (IHC) staining as well as fluorescent antibody test (FAT), positive viral antigens were detected in the perikaryon of the neurons and axonal and/or dendritic processes in the nervous tissue and in the macrophages scattered in various tissues throughout the body. The findings suggest that brain stem, cerebral cortex, hippocampus, thalamus and hypothalamus are ideal sampling regions for molecular diagnosis of RABV in TWFBs. Retrospective study using archived formalin-fixed and paraffin-embedded tissues of TWFBs revealed the earliest IHC-positive rabid TWFB case in 2004. To examine the origin of this viral strain, we sequenced three complete genomes and acquired multiple rabies virus (RABV) nucleoprotein (N) and glycoprotein (G) sequences. Phylogeographic analyses demonstrated that the RABV of TWFB (RABV-TWFB) is a distinct lineage within the Asian group, and has been differentiated from its closest lineages, China I (including Chinese ferret badger isolates; RABV-CNFB) and Philippines, 158-210 years before present. The most recent common ancestor of RABV-TWFB was originated 91-113 years ago. The ancient origin of the endemic RABV-TWFB illustrates that this RABV variant could be cryptically circulated in the environment without being recognized for a long period of time. The underlying mechanism is worthy of further study and may shed light on the complex interaction between RABV and its host.