台灣石斑魚虹彩病毒基因型分析、病毒分離特性與疫苗效力之評估

Genetic analysis, characterization and evaluation the efficacy of vaccination for grouper iridovirus isolates in Taiwan

黃淑敏(Sue-Min Huang)

指導教授 ： 張紹光

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2019年)

https://doi.org/10.6342/NTU201900537

摘要

石斑魚(Epinephelus spp)是東南亞國家水產養殖業的重要經濟魚類之一。依據聯合國糧食及農業組織之估計，到2020年，點帶石斑魚的市場需求規模可能達到10萬噸。雖然石斑魚養殖產業在亞洲地區不斷地增長，但產業常因稚魚階段易受到病毒性疾病的感染，進而限制了整體之成長；其中以虹彩病毒(grouper iridovirus, GIV)和神經壞死病毒(viral nervous necrosis,VNN) 對台灣石斑魚產業造成之經濟損失最為慘重。為了解決前述病毒感染的問題，本研究即是以台灣重要經濟魚類之虹彩病毒株的基因型別之親緣分析為基礎，進而建立新的細胞株用於分離和增殖石斑魚虹彩病毒，最後以新發展的虹彩和神經壞死病毒雙價不活化疫苗，實際應用於種苗場以降低垂直感染的風險。

病毒親源分析中，比較23個台灣虹彩病毒分離株(2001~2009年)之病毒外膜蛋白(major capsid protein, MCP)和腺苷三磷酸酶(adenosine triphosphatase, ATPase)的開放閱讀框的基因型別與其分群。研究結果發現，臺灣之重要經濟魚種同時具有兩種病毒屬之感染:其中蛙病毒屬(Ranavirus)，主要感染海水魚；而巨大細胞病毒屬(Megalocystivirus)，則可同時感染海水及淡水魚。另基於MCP胺基酸序列分析得知，臺灣流行之虹彩病毒株主要為傳染性脾臟和腎臟壞死病毒(infectious spleen and kidney necrosis virus, ISKNV)、嘉魶虹彩病毒(sea bream iridovirus, RSIV)、比目魚虹彩病毒(flounder iridovirus)和石斑虹彩病毒(GIV)等4種主要基因型別，前3種病毒株屬於巨大細胞病毒屬；而GIV屬於蛙病毒屬。此外，分析自田野收集之虹彩病毒MCP基因中核苷酸之同義突變率(Synonymous substitution rate; Ks)和非同義突變率(Non-synonymous substitution rate; Ka)的比率，顯示了大多數核苷酸的變異是屬於同義突變。計算病毒MCP基因中非同義突變率與同義突變率的比值(Ka/Ks)，結果皆小於1，顯示病毒處於陰性選擇。此結果表明，虹彩病毒在野外海水和淡水環境之魚類宿主中，病毒的演化速度是緩慢的。

為分離目前台灣田間流行之虹彩病毒株，從點帶石斑魚(Epinephelus coioides)的脾臟組織開發出一種稱為GS-1(grouper spleen-1, GS-1)的新細胞株。此細胞株適合生長在20℃〜32℃的溫度範圍內。形態上，GS-1細胞呈現紡錘狀樣細胞，並能夠以抗细胞角质蛋白(cytokeratin)、波形蛋白(vimentin)、纖維連線蛋白(fibronectin)及肌間線蛋白(desmin)之單源抗體進行免疫分型法(immunophenotyping)確認，顯示GS-1細胞源自於纖維母細胞(fibroblast)。GIV與ISKNV於GS-1細胞的複製能力分別可達到108.4與105.2 TCID50/mL之病毒產量。電子顯微鏡觀察之超薄切片顯示，於GIV感染於GS-1細胞的細胞質中，可發現許多二十面體且直徑介於180〜220 nm的病毒顆粒存在，其大小較ISKNV(160〜200 nm)為大。以負染色觀察GIV成熟病毒顆粒具有三層外膜包覆結構，外膜平均厚度約為(27 ± 3 nm) ; ISKNV則具有二層外膜包覆結構。動物接種攻毒試驗顯示，GIV於水溫25℃的毒力高於30℃; ISKNV則於水溫30℃下的毒力高於25℃。綜合前述之研究結果顯示，GS-1細胞株為分離及比較這兩屬的石斑虹彩病毒之有效工具之一。

為評估以疫苗為控制病毒性疾病之防疫效益，將雙價NNV和GIV不活化疫苗應用於41尾之點帶石斑魚(Epinephelus coioides)種魚，以評估疫苗之有效性。以即時定量聚合酶連鎖反應(Real-time quantitative PCR)分析，在種魚接種疫苗前，NNV和GIV於卵子之樣本中病毒核酸檢出率為10.5% (2/19)，其中卵子之樣本經定量分析分別攜帶1780 copies的GIV病毒DNA，此結果顯示種魚的卵子也可能是垂直傳染之途徑。在接種疫苗後1個月，以血清中和抗體試驗，分別檢測並分析NNV和GIV之血清中和抗體力價之分布，其結果顯示已有超過50%之種魚血清中NNV中和抗體位於高力價(1：1810〜1：5120倍)及45%之種魚血清中NNV中和抗體位於中力價(1：452〜1：1280倍)；相較於GIV之血清中和抗體力價分布，計有50% (10/20)的血清中和抗體力價位於1：57倍至1：320倍及40% (8/20)位於1：452倍至1：1280倍。結果顯示在單次免疫注射後，NNV比GIV更容易誘發高力價之血清中和抗體。評估各試驗組於疫苗使用前後，其體內病毒核酸檢出量之變化發現，其中2尾種魚(晶片號碼: 45266724與146237372)於免疫前被檢出分別攜帶有GIV (1780 copies/µL)與NNV (97.1 copies/µL)之病毒核酸，經免疫後1個月，2尾種魚之卵子樣本就檢測不出GIV與NNV病毒核酸之存在。綜合上述之評估結果顯示，種魚經免疫注射可提高種魚體內具有保護力之特異性中和抗體，清除體卵細胞表面或生殖液的病毒量，降低垂直感染之可能性。

關鍵字

石斑魚虹彩病毒 ； 病毒性神經壞死病毒 ； 死毒雙價疫苗 ； 魚類細胞株 ； 石斑魚 ； 病毒株親緣分析

並列摘要

Groupers are economically important fish species of the aquaculture industry in many southeast Asian countries. The Food and Agriculture Organization estimates that the market demand of orange-spotted grouper may reach 100,000 tons in 2020. Although the culture of groupers continues to grow in Asia, its development is constrained by the limited availability of fingerlings, mainly because commercial fish farming has been severely hit by epidemics associated with viruses. Iridoviruses and nervous necrosis virus (NNV) species severely impacted the grouper industry and caused heavy economic losses in Taiwan. The purpose of the studies included in this thesis were to investigate the genetic relationships of iridoviruses gathered from various fish species in Taiwan, to establish a cell line for the isolation and propagation of grouper iridovirus (GIV) and infectious spleen and kidney necrosis virus (ISKNV), and to evaluate an inactivated bivalent vaccine applied to the broodfish in an injection vaccination program to reduce the risk of vertical transmission to the eggs.
In the phylogenetic analysis, we compared the open reading frames encoding the viral major capsid protein (MCP) and adenosine triphosphatase (ATPase) from the 23 Taiwanese isolates of iridoviruses that were collected from 2001 to 2009 and determined their relationships. Our results indicated that the iridoviruses from the Taiwan aquaculture fishes could be classified into two groups: Prior to 2005, the viruses were closely related to members of the genus Ranavirus, and after 2005, they were similar to members of the genus Megalocytivirus. Based on the analysis of the MCP amino acid sequences, Taiwanese iridovirus isolates were divided into four major genotypes related to ISKNV, sea bream iridovirus (RSIV), flounder iridovirus (FLIV) and GIV. Pairwise comparisons of the MCP genes showed that the Ranavirus-like isolates caused epidemics in economically important species from major production regions and major in cultured marine fish, while the Megalocytivirus-like isolates mostly affected both in marine and freshwater fish. In addition, the distribution of synonymous and non-synonymous changes in the MCP gene revealed that the iridoviruses were evolving slowly, and most of the variations were synonymous mutations. The Ka/Ks values were lower than 1, and hence, the viruses were under negative selection.
A new continuous cell line, designated as GS-1, from the spleen tissue of the marine grouper, Epinephelus coioides was developed in this study. Morphologically, the GS-1 cell line is a spindle-shaped cell, which was confirmed by immunophenotyping with cytokeratin, vimentin, fibronectin, and desmin antibodies. The optimal temperature for GIV and ISKNV proliferation in GS-1 cells was 25°C. The highest titer of GIV and ISKNV was 108.4 TCID50/mL and 105.2 TCID50/mL respectively. The electron micrographs showed that the mean diameter of the GIV virions was 180–220 nm, which was larger than that of the ISKNV virions (160–200 nm). The negatively stained GIV particles possessed an envelope structure that was assembled by the three-layered structure with an inner electron-dense core surrounded by a lighter coat (mean diameter, 27 ± 3 nm). The highest GIV- and ISKNV-induced mortality of groupers occurred at 25°C and 30°C, respectively. In summary, the GS-1 cell line is a valuable tool for isolating and investigating fish ranaviruses and megalocytiviruses in the same host system.
Forty-one orange-spotted grouper (Epinephelus coioides) broodfish were selected to ‎evaluate the effectiveness of an NNV and GIV inactivated bivalent vaccine. Real-time quantitative PCR analysis showed that an NNV and GIV detection rate of 10.5% (2/19) was found in the broodfish egg specimens, which carried approximately 1,780 copies of GIV viral DNA before vaccination. These results indicating that vertical transmission of GIV may exist in broodfish. One month after vaccination, the anti-NNV serum antibody titer increased to high titer level (1:1810 to 1:5120) in more than 50% of the fish, and to moderate titer level (1:452 to 1:1,280) in 45% of the fish, which were higher than the increases seen for the anti-GIV serum antibody titer, with 50% (10/20) in a titer of 1:57 to 1:320 and 40% (8/20) in a titer of 1:52 to 1:1,280. These results showed that NNV is a highly antigenic virus and can effectively induce neutralizing antibodies better than GIV. In this study, the viral copy numbers of NNV and GIV were 97.1 and 1,780 copies per μg of the egg specimens from two broodfish with ID number 146237372 and 45266724 before vaccination, respectively. However, viral genome was undetectable in the egg samples collected at one month post bivalent vaccine immunization. It is suggested that the neutralization antibodies induced by the bivalent vaccine was helpful to eliminate the viruses on the eGroupers are economically important fish species of the aquaculture industry in many southeast Asian countries. The Food and Agriculture Organization estimates that the market demand of orange-spotted grouper may reach 100,000 tons in 2020. Although the culture of groupers continues to grow in Asia, its development is constrained by the limited availability of fingerlings, mainly because commercial fish farming has been severely hit by epidemics associated with viruses. Iridoviruses and nervous necrosis virus (NNV) species severely impacted the grouper industry and caused heavy economic losses in Taiwan. The purpose of the studies included in this thesis were to investigate the genetic relationships of iridoviruses gathered from various fish species in Taiwan, to establish a cell line for the isolation and propagation of grouper iridovirus (GIV) and infectious spleen and kidney necrosis virus (ISKNV), and to evaluate an inactivated bivalent vaccine applied to the broodfish in an injection vaccination program to reduce the risk of vertical transmission to the eggs.
In the phylogenetic analysis, we compared the open reading frames encoding the viral major capsid protein (MCP) and adenosine triphosphatase (ATPase) from the 23 Taiwanese isolates of iridoviruses that were collected from 2001 to 2009 and determined their relationships. Our results indicated that the iridoviruses from the Taiwan aquaculture fishes could be classified into two groups: Prior to 2005, the viruses were closely related to members of the genus Ranavirus, and after 2005, they were similar to members of the genus Megalocytivirus. Based on the analysis of the MCP amino acid sequences, Taiwanese iridovirus isolates were divided into four major genotypes related to ISKNV, sea bream iridovirus (RSIV), flounder iridovirus (FLIV) and GIV. Pairwise comparisons of the MCP genes showed that the Ranavirus-like isolates caused epidemics in economically important species from major production regions and major in cultured marine fish, while the Megalocytivirus-like isolates mostly affected both in marine and freshwater fish. In addition, the distribution of synonymous and non-synonymous changes in the MCP gene revealed that the iridoviruses were evolving slowly, and most of the variations were synonymous mutations. The Ka/Ks values were lower than 1, and hence, the viruses were under negative selection.
A new continuous cell line, designated as GS-1, from the spleen tissue of the marine grouper, Epinephelus coioides was developed in this study. Morphologically, the GS-1 cell line is a spindle-shaped cell, which was confirmed by immunophenotyping with cytokeratin, vimentin, fibronectin, and desmin antibodies. The optimal temperature for GIV and ISKNV proliferation in GS-1 cells was 25°C. The highest titer of GIV and ISKNV was 108.4 TCID50/mL and 105.2 TCID50/mL respectively. The electron micrographs showed that the mean diameter of the GIV virions was 180–220 nm, which was larger than that of the ISKNV virions (160–200 nm). The negatively stained GIV particles possessed an envelope structure that was assembled by the three-layered structure with an inner electron-dense core surrounded by a lighter coat (mean diameter, 27 ± 3 nm). The highest GIV- and ISKNV-induced mortality of groupers occurred at 25°C and 30°C, respectively. In summary, the GS-1 cell line is a valuable tool for isolating and investigating fish ranaviruses and megalocytiviruses in the same host system.
Forty-one orange-spotted grouper (Epinephelus coioides) broodfish were selected to ‎evaluate the effectiveness of an NNV and GIV inactivated bivalent vaccine. Real-time quantitative PCR analysis showed that an NNV and GIV detection rate of 10.5% (2/19) was found in the broodfish egg specimens, which carried approximately 1,780 copies of GIV viral DNA before vaccination. These results indicating that vertical transmission of GIV may exist in broodfish. One month after vaccination, the anti-NNV serum antibody titer increased to high titer level (1:1810 to 1:5120) in more than 50% of the fish, and to moderate titer level (1:452 to 1:1,280) in 45% of the fish, which were higher than the increases seen for the anti-GIV serum antibody titer, with 50% (10/20) in a titer of 1:57 to 1:320 and 40% (8/20) in a titer of 1:52 to 1:1,280. These results showed that NNV is a highly antigenic virus and can effectively induce neutralizing antibodies better than GIV. In this study, the viral copy numbers of NNV and GIV were 97.1 and 1,780 copies per μg of the egg specimens from two broodfish with ID number 146237372 and 45266724 before vaccination, respectively. However, viral genome was undetectable in the egg samples collected at one month post bivalent vaccine immunization. It is suggested that the neutralization antibodies induced by the bivalent vaccine was helpful to eliminate the viruses on the eggs, and protect the fish from vertical infection of both viruses. This chapter, thus, demonstrates that the specific GIV and NNV antibodies induced by vaccination can reduce the risk of vertical transmission of NNV and GIV in grouper broodfish.

並列關鍵字

Grouper iridovirus ； Nervous necrosis virus ； Inactived bivalent vaccine ； Fish cell line ； Epinephelus coioides ； Phylogenetic analysis

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