魚類結病毒持續性感染之研究

Abstract

病毒性神經壞死症（Viral nervous necrosis disease, VNN disease）是目前重要的海水養殖魚類疾病，致病原為神經壞死病毒（nervous necrosis virus, NNV），分類上屬結病毒科（Nodaviridae）之魚類結病毒屬（piscine nodavirus）。本研究中對於持續性感染進行了活體及體外細胞株的實驗。NNV急性感染後的倖存魚可形成持續性感染的狀態。持續性感染石斑魚可以藉著糞便釋放具感染性的病毒至環境中，可能成為水準傳染的傳染源。結病毒以低量分佈在持續性感染石斑魚多種的組織或器官中，包括腦、眼、心臟、肝臟、泳鰾、小腸等，沒有專一的組織趨性，個體間的分佈也不一致。對NNV持續性感染的石斑魚進行NNV再攻毒可以引發病癥及死亡，病毒的分佈在攻毒後會由部份內臟擴及全身組織器官，而後在腦及眼大量複製，同時會出現體色變深及游泳行為異常等病癥，接著發生死亡，這是帶原魚發病完整的病程。 本研究中建立了由天然持續感染結病毒之金目鱸（barramundi）的腦細胞株，命名為BB細胞株，可用來模擬活體持續性感染的情形，所以是個有價值的體外研究系統。RT-PCR的結果證明BB細胞株中有NNV存在。BB細胞株40至70代上清液的力價在102至106TCID50/0.1ml之間波動。免疫染色結果指出細胞族群中只有少數細胞可偵測到NNV抗原。溫度轉移實驗結果指出溫度突變株並非造成持續性感染主因。來自BB細胞株的結病毒（BBNNV）與急性發病石斑魚結病毒（GNNV）鞘蛋白分子量並無差異，但在鞘蛋白基因核酸序列比對中，BBNNV與其他魚類結病毒分離株有兩個核?酸的差異，影響了相對位置胺基酸抗原性的差別，以抗GNNV單株抗體分析BBNNV也發現抗原性有所不同，這項特性可能和持續性感染的形成有關。

Viral nervous necrosis disease, VNN disease, is one of the most important marine diseases affecting finfish. Nervous necrosis virus, NNV, which belongs to Nodaviridae is the causative agent of VNN disease. This study was focused on persistent infection with NNV. The results suggested that groupers (Epinephelus sp.) surviving VNN outbreak could be persistently infected by nodavirus. Infectious NNV particles were released via feces by these carrier groupers. Carrier groupers are therefore a possible reservoir of NNV. NNV was located in various tissues, including nervous tissues and many visceral organs. Death could be induced among carrier groupers by superinfection with NNV. Progression of VNN disease was described. NNV persistently infected cell line (BB cell line) was established. BB cell line can release low levels of NNV detectable at each subculture. Experiments were performed to characterize the nature of persistent infection in BB cell line, and the possible mechanisms of maintenance of persistent infection were discussed. Infectious virus was detected in BB cell culture supernatant, and the titers of different subcultures fluctuated between 102-106TCID50/0.1 ml. NNV coat protein gene was detected in BB cell line by RT-PCR. NNV antigens could be found in small portion of BB cells by immunostaining using GNNV-specific antiserum. Viral persistence in BB cells was not affected by shifting incubation temperature, so temperature and temperature sensitive virus mutants are probably not involved. BBNNV, isolated from BB cell line, had unique variation in coat protein gene when compared with other strains of piscine nodavirus. The difference of antigenicity of BBNNV from GNNV was shown by ELISA using GNNV-specific monoclonal antibodies. This characteristic was a candidate for further investigation of persistent infection.