石斑魚神經壞死病毒的分離與定性

Abstract

近年來南台灣石斑養殖孵化場魚苗屢有病毒性神經壞死症（Viral nervous necrosis disease，NNV disease）疫情發生，造成極高的死亡率。為瞭解病毒基本特性以提供未來防治方法之參考，本篇報告利用本實驗室新建立的石斑魚鰭細胞株（GF-1 cell line）來分離罹患VNN症石斑魚苗體中的病原體，並做特性的分析，包括：以光學及電子顯微鏡做病理觀察，用PCR檢定病毒核酸，用螢光免疫染色法檢測病毒在感染細胞中出現的時間，並探討溫度與酸鹼值因數對此病毒在細胞株與活體中感染力之影響。 將石斑病魚分離出來的病毒，利用GF-1細胞增殖、純化並萃取核酸，以條紋?神經壞死症病毒（Striped Jack Nervous Necrosis Virus, SJNNV）鞘蛋白質基因的兩組核酸引子對，（F1，R3）及（F2，R3），進行PCR檢測，結果可得到兩個引子對的目標核酸片段T2及T4，故證實病原體是屬於魚類結病毒（Fish nodavirus），並命名為GNNV（Grouper nervous necrosis virus）。純化的GNNV在電子顯微鏡觀察下，為不具外套膜，直徑20-25nm之圓形至20面體顆粒；經核酸萃取與電泳分析，測到兩段RNA，分子量分別是1.02×103kDa及0.5×103kDa；純化GNNV在SDS-PAGE電泳分析下，可得到兩條主要蛋白質，分子量分別是為43kDa及41kDa。GNNV在GF-1細胞株及活體中的複製能力受溫度影響，最適複製溫度範圍是28 - 32℃，低溫則會降低病毒的複製量並延緩活體感染魚發病的時間。GNNV在強鹼的環境（pH10、pH12）下感染力受到的抑制最大。比對臺灣養殖石斑分離出來的GNNV與日本養殖石斑魚分離出來的RGNNV的T2核酸序列，相似度高達99％，顯示GNNV與日本分離之RGNNV屬於同一類海水魚NNV。本實驗結果除了建立GNNV的物化特性資料外，更證實了GF-1細胞無論在分離或增殖GNNV方面都有很好的效率。由GF-1細胞所複製出的GNNV，還能保有在活體內時的特性。

Mass mortalities caused by nervous necrosis virus (NNV) of hatchery-reared grouper larvae and juveniles have been found in southern Taiwan. In the present study, a cell line derived from the fin tissue of a grouper hybrid (Einephelus malabarius × Epinephalus akaara) and nominated as GF-1 cell line, is used for isolation and characterization grouper nervous necrosis virus (GNNV). The purified virus was examined by PCR using striped jack nervous necrosis virus (SJNNV) RNA2 specific primer sets (F1, R3) or (F2, R3), and the target fragments T2 and T4 were detected in the PCR products. Viruses after replication in GF-1 cells were purified and observed under electron microscope and follow by analysis by using electrophoretic technique. The purified virus has spherical to icosahedral morphology with diameter of 20-25 nm, and the buoyant density of the virus is 1.34 g/mL. The protein profile of purified virus revealed two major proteins with molecular weight of 43 kDa and 41 kDa, respectively. Viral genome extracted from the purified virus showed two separate RNAs with molecular weights of 1.02 × 103 kDa and 0.5 × 103 kDa, respectively. The results of in vitro and in vivo effect of temperature on GNNV infection indicated that the suitable temperature range for viral replication is 28-32℃, and lower temperature could decrease viral replication rate in vitro and delay the outbreak of mass mortality in vivo. GNNV was inactivated under a culture medium of pH 10 or pH 12. The T2 sequence similarity between GNNV and grouper virus RGNNV was about 99%. It is therefore suggested that these two viruses should be classified in the same group. All the properties of GNNV in vitro suggested that GNNV replicated in GF-1 cells sustained most similarities of GNNV in vivo, GF-1 cell line is recommended as an important tool for the studying NNV.