2016～2017年中国沿海省市虾类偷死野田村病毒(CMNV)分子流行病学调查
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摘要
偷死野田村病毒(CMNV)引起的病毒性偷死病(VCMD)使中国对虾养殖业遭受了严重的经济损失,为查明CMNV及其变异株的分子流行病学特征,本研究采用逆转录套式PCR (RT-nPCR)、逆转录环介导等温扩增(RT-LAMP)和TaqMan实时荧光定量PCR(TaqMan RT-qPCR) 3种方法,对2016～2017年中国沿海省市CMNV及一种新型野田村病毒——行动障碍野田村病毒(MDNV)的流行、分布和变异情况进行了分析。结果显示,养殖凡纳滨对虾、中国明对虾、日本囊对虾、斑节对虾和罗氏沼虾等种类中均可检测到CMNV阳性;在河北、山东、浙江、福建、广东和海南等省市采集的患病对虾中均存在CMNV。基于RT-nPCR、RT-LAMP和TaqMan RT-qPCR的检测结果显示,2016和2017年样品中CMNV的阳性检出率依次分别为11.8%和7.8%,6.7%和3.9%,17.7%和12.4%;基于上述3种方法检测结果计算得出,2016和2017年样品中CMNV的阳性检出率分别为26.8%和16.3%;基于RT-LAMP的分析显示,2016年样品中MDNV的阳性检出率为9.4%。本研究表明,中国沿海省市养殖虾类中VCMD的流行和危害仍不容忽视,RNA依赖的RNA聚合酶基因不断变异导致前期基于该基因开发的CMNV检测方法可靠性下降,检出假阴性风险升高;同时,发病对虾中出现了新的野田村病毒株系且具有较高的流行率,其传播危害风险值得高度关注。
Covert mortality nodavirus(CMNV), a recently emerged RNA virus, is the pathogen responsible for viral covert mortality disease, which has caused serious losses of shrimp aquaculture in China in recent years. In order to determine the transmission, prevalence, and evolution of CMNV in the coastal provinces and cities in China, national wide survey of the molecular epidemiology of the CMNV and it's variants was conducted, based on analysis of shrimp samples collected from 2016 and 2017 by using the methods of RT-nPCR, RT-LAMP, and TaqMan RT-qPCR in present study. The analyses found the presence of CMNV-positive specimens in the major species of cultured crustaceans, including Litopenaeus vannamei, Marsupenaeus japonicus, Fenneropenaeus chinensis, Penaeus monodon, and Macrobrachium rosenbergii. CMNV-positive specimens appeared in almost all the coastal provinces, such as Hebei, Shandong, Zhejiang, Fujian, Guangdong, and Hainan. The results based on RT-nPCR assays showed that the prevalence rates of CMNV among the collected samples were 11.8%(30/254) and 7.8%(30/387) in 2016 and 2017, respectively. The results based on RT-LAMP assays showed that the prevalence rates of CMNV were 6.7%(17/254) and 3.9%(15/387) in 2016 and 2017, respectively. The results based on TaqMan RT-qPCR assays showed that the prevalence rates of CMNV were 17.7%(45/254) and 12.4%(48/387) in 2016 and 2017, respectively. The total prevalence rates of CMNV were26.8%(68/254) and 16.3%(63/387) in 2016 and 2017, respectively, based on the above mentioned three methods. The results based on RT-LAMP assays showed that the prevalence rate of MDNV was 9.4%(24/254) in 2016. The high prevalence of CMNV in the major shrimp species and in the main farming areas revealed that this virus still threatened shrimp aquaculture in China during 2016～2017. Moreover,mutation of the RNA-dependent RNA polymerase(RdRp) gene of CMNV will raise the risk of appearing of false-negative result in CMNV molecular tests, which is of great concern. Meanwhile, the high prevalence rates of the emerging CMNV variant, MDNV, is a reminder that close attention needs to be paid to the high risk of MDNV transmission widely in farmed crustaceans.
Covert mortality nodavirus(CMNV), a recently emerged RNA virus, is the pathogen responsible for viral covert mortality disease, which has caused serious losses of shrimp aquaculture in China in recent years. In order to determine the transmission, prevalence, and evolution of CMNV in the coastal provinces and cities in China, national wide survey of the molecular epidemiology of the CMNV and it's variants was conducted, based on analysis of shrimp samples collected from 2016 and 2017 by using the methods of RT-nPCR, RT-LAMP, and TaqMan RT-qPCR in present study. The analyses found the presence of CMNV-positive specimens in the major species of cultured crustaceans, including Litopenaeus vannamei, Marsupenaeus japonicus, Fenneropenaeus chinensis, Penaeus monodon, and Macrobrachium rosenbergii. CMNV-positive specimens appeared in almost all the coastal provinces, such as Hebei, Shandong, Zhejiang, Fujian, Guangdong, and Hainan. The results based on RT-nPCR assays showed that the prevalence rates of CMNV among the collected samples were 11.8%(30/254) and 7.8%(30/387) in 2016 and 2017, respectively. The results based on RT-LAMP assays showed that the prevalence rates of CMNV were 6.7%(17/254) and 3.9%(15/387) in 2016 and 2017, respectively. The results based on TaqMan RT-qPCR assays showed that the prevalence rates of CMNV were 17.7%(45/254) and 12.4%(48/387) in 2016 and 2017, respectively. The total prevalence rates of CMNV were26.8%(68/254) and 16.3%(63/387) in 2016 and 2017, respectively, based on the above mentioned three methods. The results based on RT-LAMP assays showed that the prevalence rate of MDNV was 9.4%(24/254) in 2016. The high prevalence of CMNV in the major shrimp species and in the main farming areas revealed that this virus still threatened shrimp aquaculture in China during 2016～2017. Moreover,mutation of the RNA-dependent RNA polymerase(RdRp) gene of CMNV will raise the risk of appearing of false-negative result in CMNV molecular tests, which is of great concern. Meanwhile, the high prevalence rates of the emerging CMNV variant, MDNV, is a reminder that close attention needs to be paid to the high risk of MDNV transmission widely in farmed crustaceans.
引文
Gu SJ.Analysis of causes of the covert mortality disease of Pacific white shrimp and its control strategies.Scientific Fish Farming,2012(8):62-63[顾水江.南美白对虾偷死病发病原因分析及防治对策.科学养鱼,2012(8):62-63]
Liu BB,Yang B,LüXW,et al.Detection and quantification of infectious hypodermal and hematopoietic necrosis virus(IHHNV)and Enterocytozoon hepatopenaei(EHP)of Litopenaeus vannamei by real-time PCR.Progress in Fishery Sciences,2017,38(2):158-166[刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测.渔业科学进展,2017,38(2):158-166]
Liu S,Li JT,Tian Y,et al.Experimental vertical transmission of covert mortality nodavirus in Exopalaemon carinicauda.Journal of General Virology,2017,98(4):652-661
Liu S,Wang XH,Xu TT,et al.Vectors and reservoir hosts of covert mortality nodavirus(CMNV).Journal of Invertebrate Pathology,2018,154:29-36
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Pooljun C,Direkbusarakom S,Chotipuntu P,et al.Development of a TaqMan real-time RT-PCR assay for detection of covert mortality nodavirus(CMNV)in penaeid shrimp.Aquaculture,2016,464:445-450
Song SX,Zhuang SP.Measures for control of“bottom death”of Pacific white shrimp.Fish Science and Technology,2006(6):36-38[宋盛宪,庄世鹏.防治南美白对虾“底死症”的措施.水产科技,2006(6):36-38]
Thitamadee S,Prachumwat A,Srisala J,et al.Review of current disease threats for cultivated penaeid shrimp in Asia.Aquaculture,2016(452):69-87
Xing H.Discussion of the control measures for the“bottom death”(covert mortality disease)of Pacific white shrimp.China Fisheries,2004(4):88-89[邢华.南美白对虾“死底症”(偷死病)的控制措施探讨.中国水产,2004(4):88-89]
Xu ZJ,Ji F.Comprehensive control of the covert mortality disease of Pacific white shrimp.Fishery Guide to be Rich,2009(1):60-61[徐志进,纪富.南美白对虾偷死病的综合防治.渔业致富指南,2009(1):60-61]
Zhang QH.To be cautious of“bottom death”in the intensive farming of Pacific white shrimp.Scientific Fish Farming,2004(10):48-49[章秋虎.南美白对虾高密度养殖谨防“死底症”.科学养鱼,2004(10):48-49]
Zhang QL,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp.Journal of General Virology,2014,95(12):2700-2709
Zhang QL,Liu S,Yang HL,et al.Reverse transcription loopmediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp.Journal of Invertebrate Pathology,2017a,150:130-135
Zhang QL,Xu TT,Wan XY,et al.Prevalence and distribution of covert mortality nodavirus(CMNV)in cultured crustacean.Virus Research,2017b,233(2):113-119
Zhu LL,Zhang QL,Wan XY,et al.Molecular epidemiology of a new yellow head virus strain in China.Progress in Fishery Sciences,2016,37(3):68-77[朱罗罗,张庆利,万晓媛,等.我国一株新型黄头病毒的分子流行病学.渔业科学进展,2016,37(3):68-77]
Liu BB,Yang B,LüXW,et al.Detection and quantification of infectious hypodermal and hematopoietic necrosis virus(IHHNV)and Enterocytozoon hepatopenaei(EHP)of Litopenaeus vannamei by real-time PCR.Progress in Fishery Sciences,2017,38(2):158-166[刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测.渔业科学进展,2017,38(2):158-166]
Liu S,Li JT,Tian Y,et al.Experimental vertical transmission of covert mortality nodavirus in Exopalaemon carinicauda.Journal of General Virology,2017,98(4):652-661
Liu S,Wang XH,Xu TT,et al.Vectors and reservoir hosts of covert mortality nodavirus(CMNV).Journal of Invertebrate Pathology,2018,154:29-36
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Pooljun C,Direkbusarakom S,Chotipuntu P,et al.Development of a TaqMan real-time RT-PCR assay for detection of covert mortality nodavirus(CMNV)in penaeid shrimp.Aquaculture,2016,464:445-450
Song SX,Zhuang SP.Measures for control of“bottom death”of Pacific white shrimp.Fish Science and Technology,2006(6):36-38[宋盛宪,庄世鹏.防治南美白对虾“底死症”的措施.水产科技,2006(6):36-38]
Thitamadee S,Prachumwat A,Srisala J,et al.Review of current disease threats for cultivated penaeid shrimp in Asia.Aquaculture,2016(452):69-87
Xing H.Discussion of the control measures for the“bottom death”(covert mortality disease)of Pacific white shrimp.China Fisheries,2004(4):88-89[邢华.南美白对虾“死底症”(偷死病)的控制措施探讨.中国水产,2004(4):88-89]
Xu ZJ,Ji F.Comprehensive control of the covert mortality disease of Pacific white shrimp.Fishery Guide to be Rich,2009(1):60-61[徐志进,纪富.南美白对虾偷死病的综合防治.渔业致富指南,2009(1):60-61]
Zhang QH.To be cautious of“bottom death”in the intensive farming of Pacific white shrimp.Scientific Fish Farming,2004(10):48-49[章秋虎.南美白对虾高密度养殖谨防“死底症”.科学养鱼,2004(10):48-49]
Zhang QL,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp.Journal of General Virology,2014,95(12):2700-2709
Zhang QL,Liu S,Yang HL,et al.Reverse transcription loopmediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp.Journal of Invertebrate Pathology,2017a,150:130-135
Zhang QL,Xu TT,Wan XY,et al.Prevalence and distribution of covert mortality nodavirus(CMNV)in cultured crustacean.Virus Research,2017b,233(2):113-119
Zhu LL,Zhang QL,Wan XY,et al.Molecular epidemiology of a new yellow head virus strain in China.Progress in Fishery Sciences,2016,37(3):68-77[朱罗罗,张庆利,万晓媛,等.我国一株新型黄头病毒的分子流行病学.渔业科学进展,2016,37(3):68-77]
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