以傳染性胰臟壞死病毒之VP3反意核醣核酸表現抑制本病毒所誘發之細胞凋亡

Abstract

傳染性胰臟壞死病毒（Infectious Pancreatic Necrosis Virus; IPNV）是兩段雙股之RNA病毒，屬於兩段雙股核醣核酸病毒科。在魚類傳染疾病中，是一種分佈廣泛的水生生物病毒的傳染病，在臺灣曾造成多種經濟性魚類的死亡。本實驗室之IPNV-E1S是由病鰻中分離鑑定後，屬於Ab型之水生兩段雙股核醣核酸病毒。 在最近的研究報告中指出，細胞凋亡現像是誘發魚類細胞受IPNV感染後產生病變效應的主要因素，而CHSE-214細胞株可明顯表現出細胞凋亡的外觀及生化之特徵。IPNV所引發之細胞凋亡現像是由何基因所調控，推測是由其VP3伴演了此角色。為了瞭解細胞凋亡現象是否與VP3有關，便利用反意VP3核醣核酸（A2, A4，及A7, 包括nt 501-711, 285-711及1-711）之穩定轉殖CHSE-214細胞作測試。三株轉殖細胞對IPNV所引起之細胞死亡有抑制的功能，由分析結果中可知VP3的表現量受到壓制，而Mcl-1的表現量則下降得較緩慢，及在反意基因表現的初期可使DNA Fragmentation量降低。 由以上的結果推測VP3可受反意RNA的抑制，而VP3可能為IPNV引發之細胞凋亡調控因數之一。

IPNV (Infectious Pancreatic Necrosis Virus) is a double- stranded RNA viruse which has been shown to be a major cause of many contagious and widespread fish diseases. IPNV belongs to Birnaviridae virus family and is also a pathogen of many economically important fishes in Taiwan. The IPNV-E1S (Eel No.1 Spleen) virus strain was isolated from the spleen of diseased Japanese eel (Anguilla japonica) and is an Ab serotype of Aquatic Birnavirus. Recent study has established apoptosis as a major cause for the observed IPNV-induced cytopathic effects in infected fish cells. IPNV-infected CHSE-214 cells displayed all previously known morphological and biochemical hallmarks of apoptosis. At present, the apoptosis-inducing activity of IPNV has not been definitively assigned to any IPNV factors. Nevertheless, there was evidence implicating VP3 as such a factor. To identify the unknown apoptosis-inducing element of IPNV, CHSE-214 cells stably expressing various antisense RNAs (A2, A4, and A7, complement to nt 501-711, 285-711, and 1-711, respectively) against VP3 were established. All antisense RNA expressing cells showed increased resistance to IPNV-induced apoptosis in viral challenge experiments (MOI 1). Further analyses showed that VP3 protein expression was suppressed, Mcl-1 expression was longer sustained, and DNA fragmentation was reduced in these antisense RNA expressing cells during early viral replication cycle. These results suggest that the antisense RNAs could knock down the VP3 expression and VP3 is an apoptosis-inducing factor responsible for the IPNV-induced apoptotic cell death.