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研究生:蓋玉軒
研究生(外文):Yu-Hsuan Kai
論文名稱:石斑魚神經壞死症病毒去活化疫苗之免疫策略與效能
論文名稱(外文):Vaccination Strategies and Efficacies of Inactivated Nervous Necrosis Virus in Groupers
指導教授:齊肖琪齊肖琪引用關係
口試委員:宋延齡周信佑林翰佑邱品文
口試日期:2013-07-25
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:119
中文關鍵詞:神經壞死症病毒石斑魚
外文關鍵詞:NNVgrouper
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神經壞死症病毒(Nervous Necrosis Virus, NNV)造成多種海水養殖魚類在幼苗時期大量死亡。魚苗主要是經由水平及垂直感染的路徑感染NNV,為了降低石斑魚苗感染NNV的機率並提高免疫力,以NNV去活化疫苗浸泡免疫石斑幼苗,或以NNV重組蛋白為疫苗經餌料生物包埋後餵食免疫石斑幼苗,皆證實已有很好的保護效率。然而,石斑幼苗浸泡或餵食免疫後所誘發的免疫反應至今仍有限。因此,本研究以real-time PCR分析石斑幼苗經浸泡與餵食NNV死毒疫苗後的免疫相關基因表現量。結果發現,在早期的免疫反應中,先天性免疫相關基因(IL-1β與Mx)及後天性免疫相關基因(MHC-I, MHC-Ⅱ, CD8, IgT 與 IgM),皆在免疫後第三天及第五天顯著升高。幼苗浸泡免疫與餵食免疫後的一至四週,在內臟組織中可發現細胞免疫相關基因(MHC-I, CD8),體液性免疫相關基因(IgM),以及先天性免疫相關基因(IL-1β and Mx)皆顯著高於對照組魚苗。另外,只有浸泡免疫魚的鰓組織可誘發IgT基因表現,只有餵食免疫魚的腸道組織中可發現IgT表現量上升。因此推測,前人浸泡或餵食免疫石斑幼苗後,在活體攻毒試驗中所引起的保護能力,與細胞免疫相關基因(MHC-I, MHC-Ⅱ及CD8)以及免疫球蛋白基因(IgM, IgT)的上升有關。
為了防止NNV垂直感染石斑幼苗,本研究先以1.35公斤左右的點帶石斑成魚建立NNV死毒疫苗的有效注射免疫條件,再進行種魚的免疫試驗。結果發現,以佐劑混合後肌肉注射NNV抗原免疫劑量109 TCID50 kg-1能誘發最高的中和抗體力價,且17個月後中和抗體力價仍能高於免疫前血清力價的四倍。接著以此免疫條件免疫體重35~60公斤的藍身大石斑種魚,並追蹤免疫後種魚每個月所產魚卵中NNV專一性抗體含量,連續追蹤五個月。結果發現,免疫種魚所產魚卵中的NNV專一性抗體量明顯高於對照組種魚所產魚卵中的抗體量。經由二次PCR檢測連續五個月所採集之魚卵有無感染NNV,結果免疫組種魚在免疫後五
II
個月中所產魚卵皆未測得NNV的感染,但對照組魚卵卻在第五個月採樣中測到NNV的正反應,因此說明,種魚在繁殖季初接種NNV死毒疫苗,可以提高之後五個月繁殖期中產下無NNV帶原魚卵的機率,有助於阻斷垂直感染的效果。
另外,本研究發現孵化後80天的金目鱸對於NNV的感受性明顯低於孵化後14天的幼苗。在實驗中,孵化後80天的金目鱸以NNV攻毒並不會誘發死亡,但可在腦部及肝臟偵測到Mx蛋白以及NNV RNA2的基因表現,證明這些魚已經形成NNV帶原魚。未帶原NNV的金目鱸在RSIV攻毒後其死亡率為100%,而先以NNV攻毒而變成NNV帶原的金目鱸,變得對RSIV具有抵抗能力,在RSIV攻毒後其死亡率為62%。類似的情況也發生在金目鱸腦細胞株的系統中,RSIV在持續性帶原NNV的金目鱸腦細胞株(BB)中複製量明顯低於沒有帶原NNV的細胞(cBB)。測試NNV持續性感染的金目鱸與細胞株對抗RSIV二次感染的潛在原因,發現NNV多源抗體對RSIV不具中和效果,而且NNV的感染也不會影響RSIV的複製,但cBB細胞中干擾素(IFN)反應所誘發的Mx表現卻可抑制RSIV的複製。因此認為,金目鱸因NNV感染所誘發的干擾素反應和Mx的表現,是NNV帶原魚抵抗RSIV感染的最主要原因。

Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured marine fish, especially at the larval and juvenile stages. Fish larvae can be infected with NNV through horizontal and vertical transmission. To prevent the NNV infection of early larvae, bath immunization of inactivated vaccine and oral immunization of recombinant vaccine have been reported to be effective in protection. However, the information of immune response of grouper early larvae after bath or oral immunization is still limited. In this study, grouper larvae were bath or orally immunized with inactivated NNV and the expression levels of immune genes were analyzed by real-time PCR. The early expressions of innate immunity genes (IL-1β, MX) and adaptive immunity genes (MHC-I, MHC-II, CD8, IgT and IgM) of the bath and orally immunized groupers were found to manifest on the 3rd and 5th day post immunization. In the viscera of both immunized fish, the expression levels of cellular immunity marker (MHC-I, CD8), humoral immunity marker (IgM), and innate immunity marker (IL-1β and MX) were all significantly higher than that of the non-immunized fish from one to four weeks post immunization. In addition, only bath immunization induced the elevation of IgT gene expression in the gill; while, only oral immunization raised the IgT gene expression level in the gut. The above data suggested that the previously reported protection against NNV infection through bath
IV
and oral immunization in grouper larvae, are related to the contribution of vaccine-induced elevation of cellular immunity genes (MHC-I, MHC-II and CD8) and immunoglobulin genes (IgM and IgT).
To prevent the vertical transmission of NNV, an effective immunization program was developed and pre-monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35 kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at the dose of 109 TCID50 kg−1, and the enhanced 4-fold neutralization antibody titer could sustain up to 17 months post-vaccination (mpv). The same immunization program was then applied to grouper broodstocks of Epinephelus tukula with body weights among 35-60 kg. The levels of NNV-specific antibodies in the homogenates of the eggs were chased for 5 months. The neutralizing antibody titers found in the eggs from the vaccinated broodfish were higher than that from the non-vaccinated fish. By nested RT-PCR, NNV became detectable in the eggs from the non-vaccinated fish in Month 5, but not in the eggs of the vaccinated fish. It is therefore suggested that vaccination is potentially a practical measure to reduce the risk of vertical transmission of NNV from the grouper broodfish under repeated spawning stress.
We discovered that the pathogenicity of NNV to the 80 days post-hatch (dph) barramundi is lower than that to the 14 dph barramundi. Following NNV challenge,
V
no mortality occurred in the 80 dph barramundi, but NNV RNA2 and barramundi Mx (BMx) gene expression was detectable in the brain and liver. The 80 dph barramundi pre-challenged with NNV became more resistant to the following RSIV challenge (mortality: 62%) compared to the NNV-free barramundi challenged with RSIV (mortality: 100%). A similar phenomenon was revealed in NNV-persistently infected barramundi brain (BB) cell line that RSIV proliferated less progeny virions in BB cells than in NNV-free cured BB (cBB) cells. The potential factors involved in the resistance of the persistently NNV-infected barramundi and BB cells to the secondary RSIV infection were examined in this study. The results indicated that barramundi anti-NNV polyclonal antibodies did not cross-neutralize RSIV, and NNV infection did not interfere with RSIV replication. However, the interferon (IFN) response and BMx gene expression in NNV-infected cBB cells could suppresses the RSIV proliferation. It is therefore suggested that the NNV-induced IFN response and BMx expression are responsible for the resistance against RSIV infection.

Contents
中文摘要 .................................................................................................................... Ⅰ
Abstract .................................................................................................................... Ⅲ
Contents ..................................................................................................................... Ⅵ
Contents of Tables and Figures ................................................................................ XI
Chapter 1 Overview Introduction
1. Viral nervous necrosis ......................................................................................... 1
1.1. Clinical signs of VNN .................................................................................... 1
1.2. Host range of NNV ........................................................................................ 2
2. Fish nodavirus ..................................................................................................... 3
2.1. The characteristics of NNV ......................................................................... 3
2.2. Control of VNN .......................................................................................... 5
3. Fish vaccine ........................................................................................................... 8
3.1. Inactivated vaccines ...................................................................................... 8
3.2. Attenuated vaccines ...................................................................................... 9
3.3. Recombinant vaccine .................................................................................... 9
3.4. DNA vaccines ............................................................................................. 10
3.5. Immunization routs ..................................................................................... 11
4. Inactivated vaccines against betanodavirus in grouper larvae (Epinephelus coioides) ............................................................................................................ 12
VII
5. The purpose of this study .................................................................................... 14
Chapter 2. Innate and adaptive immune gene expression of grouper larvae (Epinephelus coioides) after bath and oral vaccination
1. Introduction .......................................................................................................... 15
2 Materials and Methods ....................................................................................... 17
2.1 Fish and virus .............................................................................................. 17
2.2 Preparation of inactivated NNV vaccine .................................................... 18
2.3 Experimental designs for bath and oral immunization ............................... 18
2.4 Reverse transcription and real-time PCR .................................................... 19
3. Results ................................................................................................................. 20
3.1 Expressions of immune-related genes at 1, 3 and 5 days post immunization .. ............................................................................................................................ 20
3.2 Expression of innate immunity genes 1,2 and 4 weeks post immunization . 21
3.3 Expression of cellular immunity genes 1, 2 and 4 weeks post immunization .. ............................................................................................................................ 21
3.4 Expression of immunoglobulin genes 1,2 and 4 week post immunization .. 22
4. Discussion .......................................................................................................... 23
Chapter 3 Vaccination of grouper broodfish (Epinephelus tukula) reduces the risk of vertical transmission by nervous necrosis virus
VIII
1. Introduction ....................................................................................................... 28
2. Materials and Methods ...................................................................................... 29
2.1. Virus and cell line ........................................................................................ 29
2.2. Fish and rearing condition ........................................................................... 30
2.3. Vaccine preparation ................................................................................... 31
2.4. Vaccination of adult fish and blood sampling ........................................... 31
2.5. Neutralization test for the serum of adult groupers E. coioides ................. 32
2.6. Vaccination of broodfish E. tukula and sampling of eggs ......................... 33
2.7. Sandwich ELISA ....................................................................................... 33
2.8. Nested RT-PCR ........................................................................................... 34
3. Results ................................................................................................................. 35
3.1. Neutralization activity against NNV increased in the serum of vaccinated adult groupers E. coioides ............................................................................ 35
3.2. NNV-specific antibody were detected in the eggs and the hatched fries from vaccinated broodfish E. tukula .................................................................. 36
3.3. NNV was detectable in the eggs from non-vaccinated E. tukula at the end of spawning season .......................................................................................... 37
4. Discussion ............................................................................................................ 38
Chapter 4. Persistently betanodavirus-infected barramundi (Lates calcarifer)
IX
exhibit resistances to red sea bream iridovirus infection
1. Introduction ......................................................................................................... 45
2. Materials and Methods........................................................................................ 47
2.1. Fish, cell lines, viruses, and vaccine ............................................................ 47
2.2. Viral challenge test in barramundi ............................................................... 48
2.3. Reverse transcription and real-time PCR..................................................... 49
2.4. Neutralization assay ..................................................................................... 51
2.5. Infection of NNV and/or RSIV in BB and cBB cells .................................. 53
2.6. Western blot ................................................................................................. 54
2.7. Down-regulation of BMx gene expression by siRNA ................................. 55
3. Results ............................................................................................................ 56
3.1. Pathogenicity of NNV and RSIV to barramundi ......................................... 56
3.2.NNV Existence and BMx gene expression in NNV-challenged barramundi . 57
3.3. Cross-reaction of anti-NNV polyclonal antibodies to RSIV ....................... 59
3.4. Replication of RSIV in cBB and BB cells ................................................... 59
3.5. Influence of NNV and RSIV co-infection on respective viral proliferation .. 60
3.6. Expression of BMx protein in cBB and BB cells after viral infection and poly I:C transfection ............................................................................................ 60
3.7. Antiviral activity of IFN response and BMx against RSIV ......................... 61
X
4. Discussion ............................................................................................................ 62
Chapter 5. Conclusion ........................................................................................... 67
Reference ................................................................................................................... 70
Tables and figures ...................................................................................................... 88
Appendixes .............................................................................................................. 111
Records of Journal Papers ........................................................................................ 117
Records of conference Publication .......................................................................... 118
Published Journal Papers

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