魚類神經壞死病毒之分子演化與分子流行病學探討

Molecular phylogeny and epidemiology of Nervous Necrosis Virus

蔡昆展(Kun-Chan Tsai)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/動物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01155

摘要

魚類結病毒（Betanodavirus）是造成全球海水養殖魚苗罹患神經壞死症之病原體，根據鞘蛋白質基因序列可分成Red-spotted Grouper Nervous Necrosis Virus（RGNNV）、Striped Jack（SJ）NNV、Barfin Flounder（BF）NNV，以及Tiger Puffer（TP）NNV四種基因型，其中RGNNV基因型寄主範圍及分佈地區最廣。本研究擬針對台灣周邊海域野生魚，以RT-PCR進行帶原NNV的篩檢，並利用分子演化學相關軟體分析該病毒分子流行病學的特徵。比對野生魚NNV分離株的鞘蛋白基因序列，發現台灣周邊海域野生魚帶原SJNNV及RGNNV，分佈地區以台灣北部海域為主，其中有4目12科的魚類首次發現為NNV寄主。根據基因交流的分析結果顯示，台灣周邊海域的RGNNV族群會經由水平傳播的方式影響養殖魚，再加上兩病毒族群均受到強烈正向天擇力量的影響，顯示未來可能有新變種病毒出現。根據病毒序列變異分佈的計算結果，台灣與中國目前是太平洋西岸最主要的NNV傳播中心。過去十年，台灣養殖魚的NNV分離株皆只有RGNNV基因型，不曾在爆發疫情的養殖魚中發現SJNNV，但在本研究中，台灣沿海野生魚帶原SJNNV的機率卻大於RGNNV數量。將台灣沿海帶原魚的SJNNV鞘蛋白質基因序列，和日本及歐洲養殖發病魚分離出的SJNNV序列進行比對，結果台灣的SJNNV自成一枝序群，日本和歐洲合成另一枝序群，顯示造成這兩群的不是地理隔絕的因素，反而可能是帶原魚與發病魚的差異；此外，台灣野生帶原魚的SJNNV枝序群不受天擇力量的影響，但日本和歐洲養殖發病魚的SJNNV枝序群卻受負向天擇影響，顯示決定SJNNV病原性有無的遺傳機制是非常保守的。

關鍵字

NNV ； RGNNV ； SJNNV ； 分子演化 ； 野生帶原魚 ； 正向天擇 ； 自然儲所

並列摘要

Betanodaviruses, the causative agents of viral nervous necrosis disease, are classified into 4 genotypes, including red-spotted (RG), striped jack (SJ), barfin flounder (BF) and tiger puffer (TP) nervous necrosis virus (NNV). The aim of this study is to screen NNV in the wild fish collected from different aquatic area around Taiwan, and investigated the molecular phylogeny and epidemiology of those NNV isolates. Nested RT- PCR was applied for the screening of T4 segment of coat protein gene of NNV. Both RGNNV and SJNNV were detected in the wild fish in Taiwan, and the fish of 12 species of 4 orders were first reported to be NNV host. Strong positive selection was observed in RGNNV populations from both wild-field and cultured fish, indicating that the environment preferred to keep the mutations of RGNNV. Unequal gene flow, from trash fish to the seashore fish and then to cultured fish, was monitored in RGNNV populations. It is therefore suggested that the carrier fish or sea water played a role of RGNNV reservoir and might transmit the virus from sea area to land pond. The phylogenetic tree of SJNNV revealed that all isolates from carrier fish of Taiwan were clustered into one lineage, and all isolates from diseased fish in Japan and Europe were clustered into another lineage, indicating that the distinction was not due to geographic barrier. Negative evolution was revealed in SJNNV population of cultured fish in Japan and Europe, and neutral evolution was found in SJNNV from wild-field carrier fish around Taiwan, indicating that mutation of SJNNV would reduce viral pathogenecity on cultured fish, but would not affect the fitness in wild carrier fish. It is therefore suggested the SJNNV population would be less harm than RGNNV on aquatic industry.

並列關鍵字

NNV ； RGNNV ； SJNNV ； molecular evolution ； wild carrier fish ； positive selection ； natural reservoir

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