兩種抗菌肽抗神經壞死症病毒活性測試及機制探討

Abstract

神經壞死病毒（nervous necrosis virus, NNV）屬於野田病毒科，魚類野田病毒屬，造成全球海水養殖魚苗的高死亡率及重大損失。抗菌蛋白（antimicrobial peptide, AMP）為先天性免疫中的一員，由白血球和消化道、生殖泌尿道的表皮和黏膜細胞分泌，多數是帶正電的小分子（1-5 kDa）。不同物種的AMP具有不同抗細菌、真菌、病毒的機制和免疫調節的能力。本研究首先測試出八種AMP在石斑魚鰭細胞株（grouper fin-1, GF-1）中的抗NNV效果，將AMP以不同溫度及方式處理NNV，然後測AMP中和病毒的效力。結果顯示， tilapia hepcidin 1-5 (TH1-5) 和cyclic shrimp anti-lipopolysaccharide factor (cSALF)，在感染細胞前先和NNV作用，可大幅降低病毒對細胞的感染力。以Western blot檢測TH 1-5和cSALF分別和NNV作用後再感染細胞的結果，發現AMP處理過的NNV對細胞的吸附量遠低於沒有 AMP處理過的NNV。將NNV/AMP混合物離心後，以western blot檢測上清液與沉澱物，發現絕大部分的NNV 鞘蛋白在沉澱物層；以穿透式電子顯微鏡觀察TH1-5和cSALF分別處理後的NNV，結果發現處理組的病毒顆粒聚集成堆，對照組的病毒則呈分散狀態；以奈米懸臂樑測試這兩種AMP的抗病毒機制，證實是透過凝集病毒顆粒來阻擋NNV吸附寄主細胞。以八種AMP處理金目鱸腦細胞株（cBB），並以RT-PCR檢測 Mx的表現，結果TH1-5和cSALF無法誘導Mx表現，但grouper epinecidin-1處理組則有些微Mx表現，顯示grouper epinecidin-1可能透過先天性免疫干擾素的誘導作用來抵抗NNV。

Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. In this study, the antiviral activities of eight antimicrobial peptides (AMPs) against grouper nervous necrosis virus (GNNV) were screened in a fish cell line GF-1. Two of the eight AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able found to agglutinate purified GNNV particles into clumps, which were further reconfirmed to be viral proteins by TEM observation, western blot and nanomechanical microcantilever (MCL) biosensor. GNNV was premixed with AMP (TH 1-5 or cSALF) or deionized distilled water for 1 h before infection of GF-1 cells respectively, and viral capsid protein in the infected cells was examined by western blot at 1 h post infection. The levels of capsid protein in GNNV-AMP-infected cells were much lower than that in GNNV-H2O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infect the cells. Pretreatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression, suggesting that they failed to induce innate immune response of host cells. However, grouper epinecidin-1 could induce the minor expression of Mx in treated cBB cells, indicating the potential its application in grouper.