魚類神經壞死症病毒持續性感之研究(四)

Abstract

本實驗室所建立的金目鱸腦細胞 株 (Baramundi Brain cell line, BB cell line)是源自於爆發病毒性神經壞 死症之殘活金目鱸(Baramundi)腦組 織的細胞株,已證實有神經壞死症病毒 (Nervous Necrosis Virus, NNV)的持 續性感染。本研究在探討造成NNV 持 續性感染的機制是否與干擾素有關。 以抗NNV之多源抗體治療持續性感染 的BB細胞療後, 其上清液的病毒力價 明顯自103-7 TCID50 ml-1 下降至低於 10 1.5 TCID 50 ml-1，且完全檢測不到 NNV 病毒核酸及外鞘蛋白質的表現， 而成為不再有NNV持續性感染之負對 照組細胞 cured BB (cBB)。干擾素會 誘發細胞產生Mx protein，因此本實 驗以Mx gene 專一核酸引子檢測BB 及cBB 細胞，發現BB 細胞有Mx gene 的表現，而cBB 細胞則沒有Mx gene 的表現，因此干擾素存在於有NNV 持 續性感染的BB 細胞中。此外，以NNV 再度感染的cBB 細胞，以及以 poly I:C 轉染(transfection) 的cBB 細胞則 有Mx gene 的表現，證明NNV 的感 染可以誘導cBB 細胞產生干擾素。在 抗病毒活性測試中，確定BB 細胞上清 液中有干擾素活性的物質，因此，干 擾素的保護效應是造成BB 細胞持續 性感染NNV 的原因。

Nervous necrosis virus (NNV), belonging to betanodavirus of Nodaviridae, has caused mass mortality of many species of cultured fish around the world. A new cell line BB was established from the brain tissue of a surviving barramundi (Lates calcifer) after viral nervous necrosis disease, and was demonstrated to have NNV-persistent infection. The aim of this study was to examine if NNV-persistent infection in the BB cell line was related to interferon (IFN) response. A negative control cell line was obtained by treating with NNV-specific rabbit antiserum for 5 subcultures. Virus titers of the treated BB cells drastically decreased from 103-7 TCID50 ml-1 to levels below 101.5 TCID50 ml-1, and neither NNV RNA nor capsid protein could be detected in the cured BB (cBB) cells. The expression of Mx 3 protein, type I IFN-inducible antiviral protein, was detected in the BB cells but not in the cBB cells; therefore, IFN response existed in the BB cells. In addition, the cBB cells could also express Mx gene through polyinosinic-polycytidylic acid (poly I:C) transfection and NNV infection. The result of antiviral activity assay indicated that IFN-like substances existed in the supernatant of BB cells. In conclusion, IFN protection was responsible for the persistence of NNV infection in BB cells.