水生动物双RNA病毒的研究进展
2014-03-19葛均青福建省农业科学院生物技术研究所福建福州350003
葛均青, 龚 晖, 陈 超(福建省农业科学院生物技术研究所, 福建 福州 350003)
水生动物双RNA病毒(Aquabirnavirus,ABV)隶属双RNA病毒科Birnaviridae,病毒粒子呈二十面 体球形对称,直径约60nm,无囊膜。传染性胰腺坏死病毒(Infectious Pancreatic Necrosis Virus,IPNV)是ABV的代表种,由Wolf等在1960年首次从河鳟病鱼中分离,可引起多种水产动物爆发急性传染性疾病,其流行范围遍及亚、欧、美各国,导致了水产养殖业的重大经济损失。IPNV也是我国口岸鱼类病害的第一类检疫对象(张奇亚和桂建芳,2008)。ABV的功能基因、诊断检测及其免疫预防等一直是国际上鱼类病毒病研究的热点,Dobos and Roberts(1983)和Bernard and Bremont(1995)曾分别就IPNV的分子生物学的研究作了综述。而我国也开展了包括IPNV、海洋双RNA病毒(Marine Birnavirus,MABV)等的分离、鉴定、基因的表达(林建国,2006;徐海君等,2006;赵丽丽等,2010;胡晓利等,2012;王健楠等,2012)等研究工作。本文结合IPNV及MABV的最新研究进展,对ABV的功能基因、检测及免疫防治等方面的研究进展进行综述。
1 ABV的复制
ABV的基因组以负链RNA为模板进行基因组复制(Cortez-San Martinetal.,2009),编码5种成熟的蛋白,在病毒的侵染过程中,还形成多种蛋白前体;IPNV在感染细胞后6 h,编码蛋白的合成量增加,在 6-9 h达到最高峰,随后减少。IPNV在感染宿主细胞8-10 h后,病毒RNA的合成量达到最大,在感染细胞14 h后,RNA合成量减少。病毒的组装过程中,IPNV首先被包装成病毒前体,通过进一步裂解成为成熟的病毒粒子。ABV需要在低温条件下才能繁殖,这是因为病毒的组装形成与温度有关。如IPNV在28 ℃以下才能在细胞中繁殖。IPNV能够进入哺乳动物细胞,但是不能复制(Orpetveitetal.,2012)。
2 ABV的编码蛋白
ABV的基因组由线性双股、双节段的RNA组成,片段A 编码一个大的多聚蛋白,包括VP2、VP3、VP4、VP5,并通过间隔的ORF编码一个非结构蛋白VP5;片段B编码一个依赖于RNA的RNA聚合酶。
2.1 VP1蛋白
VP1基因编码一个94kD的蛋白,具有RNA聚合酶活性,与病毒RNA转录和基因组复制有关(Grahametal.,2011)。VP1可与病毒基因组通过共价键牢固结合,形成复合体,介导基因组的复制。
2.2 VP2蛋白
VP2是病毒的主要结构蛋白,在与宿主细胞膜受体的互作及进入细胞方面发挥重要作用(Coulibalyetal.,2010)。IPNV的Sp型VP2的Thr217和Ala221决定了IPNV的毒力(Songetal.,2005)。VP2含有与诱导病毒中和抗体有关的抗原决定簇,因此它经常用于不同种或者新分离病毒的疫苗设计、诊断和血清型分类。
2.3 VP3蛋白
VP3是ABV主要的病毒粒子装配者(Pedersenetal.,2007),VP3可与VP1结合且具有强的自我结合能力,且VP3的自我互作功能域在N末端,而与VP1的互作结构域在C末端;另外,VP3可以特异性结合dsRNA,而且C末端在dsRNA的结合能力方面发挥关键作用;动力学分析表明VP1-VP3复合物的存在优先于形成成熟的病毒粒子,这表明其可能在装配过程中发挥重要作用(Baharetal.,2013)。另外,VP3可通过上调Bad的表达及破坏线粒体并激活下游caspase-3介导的细胞凋亡(Chiuetal.,2010)。
2.4 VP4蛋白
VP4是ABV的非结构蛋白(Nonstructural Polypeptide,NS),具蛋白酶活性。在它的作用下,将片段A编码的多聚蛋白NH(2)-pVP2-VP4-VP3-COOH剪切加工为pVP2和VP3,pVP2进一步剪切为核衣壳蛋白VP2。结晶结构分析表明,VP4的裂解位点形成Ser/Lys二联体酰基蛋白酶复合物(Leeetal.,2007)。
2.5 VP5蛋白
VP5是Bcl-2家族的抗凋亡基因,可调控Mcl-1和病毒蛋白的表达,诱导感病细胞产生非典型性细胞凋亡(Hongetal.,2002)。VP5不是病毒在体内复制所必需的,VP5的缺失并不改变病毒的毒力及在宿主上建立持续稳定的侵染(Santietal.,2005)。
3 ABV的侵染机制
ABV具有很广的宿主域,可以侵染多种物种。IPNV可以特异性地与CHSE-214,SHK-1和ASK细胞膜上一个大约220 kDa的膜蛋白结合,然而与非鲑鱼细胞株BF-2细胞膜上的结合的蛋白大小约190 kDa(Orpetveitetal.,2008)。流式细胞试验可以直接检测到IPNV的结合和侵染,在IPNV单独或与IHNV共同感染宿主细胞时,IPNV可以结合88%的细胞,然而IHNV在其它病毒存在时,其结合效率总是较低,但是VHSV的结合不受影响。对宿主进行抗病毒药物的处理,不影响IPNV和VHSV对细胞的结合,但是降低了IHNV的结合。IPNV可以进入通过受体介导的途径进入哺乳动物细胞,但是不能复制(Orpetveitetal.,2012)。当IHNV、VHSV和IPNV同时感染宿主细胞时,其效率明显低于IPNV(de las Herasetal.,2008)。Mx蛋白对IPNV有抗病毒作用,其表达可以明显降低IPNV诱导的细胞凋亡,抑制病毒蛋白的合成(Larsenetal.,2004)。IPNV通过调控大西洋鲑鱼炎症因子的表达建立持续感染(Reyes-Cerpaetal.,2012),而疾病的爆发和死亡率取决于宿主的防御与信号通路的级联调控和病毒基因组特性间的微妙平衡(Skjesoletal.,2011)。
4 ABV的诊断
ABV引起鱼类致病性感染或者潜伏在鱼体内、发病期间流行性传染,最终导致渔业生产的重大损失。ABV的检测方法有多种,而提高灵敏度和去除假阳性是检测方法发展的趋势。免疫学检测是应用较早的IPNV检测方法。但是,IPNV抗体检测常缺乏实际意义,一方面由于曾经感染IPNV的虹鳟血清内的抗体可持续数年,另一方面IPNV携带者不含或只含滴度很低的抗体。此外,正常虹鳟血清中还存在非特异的抗病毒成分,所以大多数情况下难以判断抗体的水平及其意义。近年来,开展的基于RT-PCR的检测方法,为IPNV 的检测和疫情监测提供了可靠依据,如RT-PCR-ELISA(Milneetal.,2006),定量RT-PCR(Bowersetal.,2008;Liuetal.,2008)和RT-LAMP(Solimanetal.,2009)等。
5 ABV的危害
IPNV可感染多种鱼类、牡蛎等淡水和海水水生动物,以进行垂直传播和水平传播。苗期及在环境胁迫条件下容易发病,并引起10%-90%的死亡率(Ronnesethetal.,2013)。病毒感染除可直接导致较高的死亡率外,还可以引起免疫抑制,使得容易感染其它病原(Johansen and Sommer,2001)。染病后存活的鱼类成为无症状的病毒携带者,可将病毒粒子释放到环境中去。
6 ABV的免疫防治
免疫防治是对IPNV进行有效防治的重要措施。IPNV疫苗在控制IPNV的侵染过程中发挥了重要作用(McBeathetal.,2007)。利用杆状病毒表达载体表达的IPNV Sp株的A片段可在昆虫细胞内自我组装形成病毒样颗粒(VLPs);利用浸泡和免疫注射的方法测定了VLP的免疫原性,表明免疫效果与免疫方法和免疫剂量均有关系(Shivappaetal.,2005)。VP2蛋白的糖基化在引起IPNV感染的细胞的免疫反应方面发挥重要作用(Fridholmetal.,2007)。用在细菌、酵母、鱼及哺乳动物细胞中表达的VP2蛋白免疫鲑鱼,都能够产生抗体(Labusetal.,2001)。在酵母中表达的VP2蛋白可以自我组装成约20 nm的亚病毒颗粒,注射纯的VP2亚病毒颗粒(rVP2-SVP)或喂食表达重组蛋白的酵母的虹鳟鱼都可以检测到IPNV抗体;口服或者注射rVP2-SVPs都可以引起鱼的特异性免疫反应,降低IPNV的侵染(Allnuttetal.,2007)。VP2还是开发DNA疫苗的良好候选基因(de Las Herasetal.,2009),用基于VP2基因的DNA疫苗口服免疫鲑鱼,可有效激活鲑鱼的免疫系统,攻毒结果显示鱼的存活率大大提高(de las Herasetal.,2010;Ballesterosetal.,2014)。VP3蛋白也是病毒的主要结构蛋白。用在大肠杆菌中表达的VP2和VP3免疫虹鳟鱼,可诱导鱼体产生抗体,但是免疫VP3鱼体内的抗体水平明显高于免疫VP2鱼体内的抗体水平(Moonetal.,2004)。开发基于IPNV结构蛋白的多联亚单位疫苗是防控IPNV的重要手段(Dharetal.,2010)。研究表明,在病毒装配过程中的病毒前体可激活鱼体的免疫系统(Rivas-Aravenaetal.,2012),而获得性免疫在免疫保护过程中发挥重要作用(Munang'anduetal.,2014)。但有些疫苗虽然能刺激机体免疫反应产生抗体,但并不能产生有效的保护作用,而在试验阶段具保护作用的免疫制剂往往在生产中并不能真正起到保护作用,这就亟待开发疫苗免疫效果的评价体系。
胡晓利,李伟,肇慧君,吴斌.2012.虹鳟鱼传染性胰脏坏死病病毒的分离与鉴定.中国动物检疫,29(3):27-30.
林建国.2006.MABV Y-6 VP2、VP3和VP5基因在昆虫细胞中的表达及VP2、VP3和VP5基因特性分析.浙江大学,博士学位论文.
王健楠,赵丽丽,刘立月,连科迅,李一经,葛俊伟,刘敏.2012.传染性胰腺坏死病病毒分离株VP2基因抗原表位区融合表达及免疫特性的分析.水产学报,36(11):1770-1775.
徐海君,杨章女,林建国,张传溪.2006.海洋双RNA病毒(MABV)vp2e和vp3基因在昆虫细胞中的高效表达.农业生物技术学报,14(4):612-617.
张奇亚,桂建芳.2008.水生病毒学.高等教育出版社.
赵丽丽,刘敏,哈卓,刘巍巍,赵永欣,葛俊伟,乔薪瑗,李一经.2010.传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析.水产学报,34(4):604-610.
Allnutt FC,Bowers RM,Rowe CG,Vakharia VN,LaPatra SE,Dhar AK.2007.Antigenicity of infectious pancreatic necrosis virus VP2 subviral particles expressed in yeast.Vaccine,25(26):4880-4888.
Bahar MW,Sarin LP,Graham SC,Pang J,Bamford DH,Stuart DI,Grimes JM.2013.Structure of a VP1-VP3 complex suggests how birnaviruses package the VP1 polymerase.Journal of Virology,87(6):3229-3236.
Ballesteros NA,Rodriguez Saint-Jean S,Perez-Prieto SI.2014.Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss,Walbaum).Fish Shellfish Immunol,37(2):220-228.
Bernard J,Bremont M.1995.Molecular biology of fish viruses:a review.Veterinary Research,26(5-6):341-351.
Bowers RM,Lapatra SE,Dhar AK.2008.Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling.Journal of Virology Methods,147(2):226-234.
Chiu CL,Wu JL,Her GM,Chou YL,Hong JR.2010.Aquatic birnavirus capsid protein,VP3,induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells.Apoptosis,15(6):653-668.
Cortez-San Martin M,Villanueva RA,Jashes M,Sandino AM.2009.Molecular characterization of IPNV RNA replication intermediates during the viral infective cycle.Virus Research,144(1-2):344-349.
Coulibaly F,Chevalier C,Delmas B,Rey FA.2010.Crystal Structure of an Aquabirnavirus Particle:Insights into Antigenic Diversity and Virulence Determinism.Journal of Virology,84(4):1792-1799.
de Las Heras AI,Perez Prieto SI,Rodriguez Saint-Jean S.2009.In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of the infectious pancreatic necrosis virus.Fish and Shellfish Immunology,27(2):120-129.
de las Heras AI,Rodriguez Saint-Jean S,Perez-Prieto SI.2008.Salmonid fish viruses and cell interactions at early steps of the infective cycle.Journal of Fish Diseases,31(7):535-546.
de las Heras AI,Rodriguez Saint-Jean S,Perez-Prieto SI.2010.Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.Fish and Shellfish Immunology,28(4):562-570.
Dhar AK,Bowers RM,Rowe CG,Allnutt FCT.2010.Expression of a foreign epitope on infectious pancreatic necrosis virus VP2 capsid protein subviral particle (SVP) and immunogenicity in rainbow trout.Antiviral Research,85(3):525-531.
Dobos P,Roberts TE.1983.The molecular biology of infectious pancreatic necrosis virus:a review.Canadian Journal of Microbiology,29(4):377-384.
Fridholm H,Eliasson L,Everitt E.2007.Immunogenicity properties of authentic and heterologously synthesized structural protein VP2 of infectious pancreatic necrosis virus.Viral Immunology,20(4):635-648.
Graham SC,Sarin LP,Bahar MW,Myers RA,Stuart DI,Bamford DH,Grimes JM.2011.The N-terminus of the RNA polymerase from infectious pancreatic necrosis virus is the determinant of genome attachment.PLoS Pathogens,7(6):e1002085.doi:1002010.1001371/journal.ppat.1002085.
Hong JR,Gong HY,Wu JL.2002.IPNV VP5,a novel anti-apoptosis gene of the Bcl-2 family,regulates Mcl-1 and viral protein expression.Virology,295(2):217-229.
Johansen LH,Sommer AI.2001.Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.Dis Aquat Organ,47(2):109-117.
Labus MB,Breeman S,Ellis AE,Smail DA,Kervick M,Melvin WT.2001.Antigenic comparison of a truncated form of VP2 of infectious pancreatic necrosis (IPN) virus expressed in four different cell types.Fish and Shellfish Immunology,11(3):203-216.
Larsen R,Rokenes TP,Robertsen B.2004.Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon Mx1 protein.Journal of Virology,78(15):7938-7944.
Lee J,Feldman AR,Delmas B,Paetzel M.2007.Crystal structure of the VP4 protease from infectious pancreatic necrosis virus reveals the acyl-enzyme complex for an intermolecular self-cleavage reaction.Journal of Biology Chemistry,282(34):24928-24937.
Liu Z,Teng Y,Liu H,Jiang Y,Xie X,Li H,Lv J,Gao L,He J,Shi X,Tian F,Yang J,Xie C.2008.Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.Journal of Virology Methods,149(1):103-109.
McBeath AJ,Snow M,Secombes CJ,Ellis AE,Collet B.2007.Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus.Fish and Shellfish Immunology,22(3):230-241.
Milne SA,Gallacher S,Cash P,Porter AJ.2006.A reliable RT-PCR-ELISA method for the detection of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout.Journal of Virology Methods,132(1-2):92-96.
Moon CH,Do JW,Cha SJ,Bang JD,Park MA,Yoo DJ,Lee JM,Kim HG,Chung DK,Park JW.2004.Comparison of the immunogenicity of recombinant VP2 and VP3 of infectious pancreatic necrosis virus and marine birnavirus.Archive of Virology,149(10):2059-2068.
Munang'andu HM,Mutoloki S,Evensen O.2014.Acquired immunity and vaccination against infectious pancreatic necrosis virus of salmon.Dev Comp Immunol,43(2):184-196.
Orpetveit I,Gjoen T,Sindre H,Dannevig BH.2008.Binding of infectious pancreatic necrosis virus (IPNV) to membrane proteins from different fish cell lines.Archive of Virology,153(3):485-493.
Orpetveit I,Kuntziger T,Sindre H,Rimstad E,Dannevig BH.2012.Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters,but does not replicate in,mammalian cells.Virol J,9:228.
Orpetveit I,Kuntziger T,Sindre H,Rimstad E,Dannevig BH.2012.Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters,but does not replicate in,mammalian cells.Virology Journal,9:228.
Pedersen T,Skjesol A,Jorgensen JB.2007.VP3,a structural protein of infectious pancreatic necrosis virus,interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA.Journal of Virology,81(12):6652-6663.
Reyes-Cerpa S,Reyes-Lopez FE,Toro-Ascuy D,Ibanez J,Maisey K,Sandino AM,Imarai M.2012.IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence.Fish and Shellfish Immunology,32(2):291-300.
Rivas-Aravena A,Cortez-San Martin M,Galaz J,Imarai M,Miranda D,Spencer E,Sandino AM.2012.Evaluation of the immune response against immature viral particles of infectious pancreatic necrosis virus (IPNV):a new model to develop an attenuated vaccine.Vaccine,30(34):5110-5117.
Ronneseth A,Haugland GT,Wergeland HI.2013.Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar L.) leucocytes after vaccination and during the time course of experimental infection.Fish Shellfish Immunol,34(5):1294-1305.
Santi N,Song H,Vakharia VN,Evensen O.2005.Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence.Journal of Virology,79(14):9206-9216.
Shivappa RB,McAllister PE,Edwards GH,Santi N,Evensen O,Vakharia VN.2005.Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system.Developments in Biologicals (Basel),121:165-174.
Skjesol A,Skjaeveland I,Elnaes M,Timmerhaus G,Fredriksen BN,Jorgensen SM,Krasnov A, Jorgensen JB.2011.IPNV with high and low virulence:host immune responses and viral mutations during infection.Virology Journal,8:396.
Soliman H,Midtlyng PJ,El-Matbouli M.2009.Sensitive and rapid detection of infectious pancreatic necrosis virus by reverse transcription loop mediated isothermal amplification.Journal of Virology Methods,158(1-2):77-83.
Song H,Santi N,Evensen O,Vakharia VN.2005.Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation.Journal of Virology,79(16):10289-10299.