以原位雜交技術應用於九孔鮑皰疹病毒之研究

Abstract

2003年冬天，台灣養殖九孔鮑因鮑魚皰疹病毒（abalone herpesvirus, AbHV）的感染引起大量死亡。組織病理學顯示，AbHV主要感染神經系統，造成神經元的壞死並引發神經節神經炎。本實驗的目的為利用原位雜交技術確認鮑魚皰疹病毒（AbHV）於感染宿主體內的分佈位置，以探討其發病機制。在本實驗中，原位雜交的結果顯示AbHV的陽性訊號主要集中在神經節的神經元及血淋巴細胞，而對照組則無特異性結合，與先前研究資料相符，確認AbHV為一嗜神經病毒。 近年來在台灣懷疑有另一種鮑魚的病毒性疾病。此一未知病毒（AbV2）可利用由皰疹病毒特異的保留區段所設計的引子增幅出特定長度的基因片段，將此一特定長度的基因片段定序，結果顯示該病毒與其他種類的皰疹病毒具有低度的基因相似性。依先前的原位雜交技術流程進行染色，結果顯示AbV2的陽性訊號侷限於血球細胞，為一親血球性的病毒。將AbV2的探針回溯性使用於AbHV陽性的病例中，結果在血球細胞中也可見到陽性訊號，表示在AbHV發病疫情當時，AbV2可能已經存在於台灣的養殖九孔鮑。

Aabalone herpesvirus （AbHV） infection of cultured abalone Haliotis diversicolor supertexta has induced high mortality in Taiwan in 2003. The histopathologic study has revealed AbHV is neurotropic and has caused ganglioneuritis in moribund abalone. In this study, an in situ hybridization protocol was developed to detect AbHV gene distribution within the host to verify its pathogenesis. This study confirmed that the expression pattern of AbHV was restricted to neurons as described previously by light and transmission electron microscopy, and confirmed this is a neurotropic virus. Another abalone virus disease is suspected in Taiwan recently. This undetermined virus （AbV2） can be amplified gene fragment in specific length by primer designed from the conserved region of herpesvirus and further sequencing revealed this virus is belonged to members of the Herpesvirales, however, the identity was low. ISH protocol is followed by aforementioned one. The result revealed AbV2 was restricted to hemocytes and suggested this is a hemocytropic virus. Retrospective study revealed AbV2 may have already existed in the farms before the first outbreak of AbHV.