2016-2017年江西地区猪繁殖与呼吸综合征病毒分子流行病学调查及ORF5基因变异分析
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摘要
为了解近年来江西地区猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)分子流行病学和其遗传变异情况,本次调查于2016-2017年从江西省各地区规模化猪场采集453份疑似猪繁殖与呼吸综合征(PRRS)的病料,采用RT-PCR方法对所有病料进行检测。结果发现,其中321份病料为PRRSV阳性,阳性率为70.86%,各地区的阳性率在19.15%~84.85%之间。挑选14份阳性样品测序后,经ORF5基因序列分析,江西地区各PRRSV毒株ORF5基因的核苷酸同源性为83%~100%,PRRSV流行毒株与参考毒株的同源性在59.9%~98.5%之间。基于ORF5基因的进化树分析表明,14个测序毒株均为美洲型毒株,其中有4株为基因亚型Ⅰ,即高致病性毒株(HP-PRRSV);3株为基因亚型Ⅱ,即经典毒株;3株为基因亚型Ⅲ,即NADC30-like毒株;4株为新出现的基因亚型Ⅳ。氨基酸序列比对分析表明,基因亚型Ⅰ、Ⅱ、Ⅲ和Ⅳ毒株ORF5基因编码的GP5蛋白氨基酸在3个表位及2个重要的抗原相关区域存在较大变异,其中以NADC30毒株为代表的基因亚型Ⅲ毒株和以GD1404毒株为代表的基因亚型Ⅳ毒株均表现出独有的氨基酸变异,这些变异可能会影响GP5蛋白的免疫原性。本次调查结果表明,2016-2017年江西地区PRRSV流行出现了新形势,美洲型毒株出现了多基因亚型共同存在的局面,以高致病性毒株(HP-PRRSV)为主,NADC30-like毒株和新基因亚型等新毒株的比例较高,同时还存在经典毒株;持续实时监测PRRSV的毒株流行和变异情况,可为临床诊断、药物和疫苗开发及PRRS的科学防控提供依据。
To investigate the genetic variation and molecular epidemiology of porcine reproductive and respiratory syndrome virus(PRRSV)in Jiangxi province in the recent years,a total of 453 tissue samples were collected from porcine reproductive and respiratory syndrome(PRRS)suspected cases in pig farms in different regions of Jiangxi province during 2016 to 2017,and then examined by RT-PCR.Of the 453 clinical samples,321 were identified positive for PRRSV,with a positive rate of 70.86%.The positive rates were varying from 19.15%to 84.85%in different regions.14 positive samples were sequenced,and the nucleotide homologies of ORF5 gene were among 83%to 100%for the strains identified in this study,and 59.9%to 98.5%for reference strains of PRRSV.14 sequenced strains all belonged to North America type(genotype Ⅱ)PRRSV in this study,4 strains belonged to the highly pathogenic PRRSV(HP-PRRSV,subtypeⅠ),3 strains belonged to the classic PRRSV(subtype Ⅱ),3 strains belonged to NADC30-like PRRSV(subtypeⅢ),and 4 strains belonged to the new subtype of PRRSV(subtype Ⅳ).Sequence analysis on deduced amino acid of GP5 coded by ORF5 gene indicated that the significant variations were observed in three epitopes and two important antigenic regions in PRRSVs belonged to the four subtypes(subtypesⅠ,Ⅱ,ⅢandⅣ).Subtype specific variations in subtypesⅢandⅣ were also observed in amino acid of GP5,which might lead to the variation of antigenecity of GP5 protein.The results indicated that there was a new situation of PRRSV strains circulating in Jiangxi province during 2016 to 2017,with multi-subtypes of strains existed and co-infected in pigs.And the most popular subtype was HP-PRRSV,while there were high rates of newly emerged NADC30-like strains and new subtype strains;It was necessary to continuously monitor the epidemiology of PRRSV,which would be benefit for the development of new diagnostic methods,drugs and vaccines for the prevention and control of PRRS.
To investigate the genetic variation and molecular epidemiology of porcine reproductive and respiratory syndrome virus(PRRSV)in Jiangxi province in the recent years,a total of 453 tissue samples were collected from porcine reproductive and respiratory syndrome(PRRS)suspected cases in pig farms in different regions of Jiangxi province during 2016 to 2017,and then examined by RT-PCR.Of the 453 clinical samples,321 were identified positive for PRRSV,with a positive rate of 70.86%.The positive rates were varying from 19.15%to 84.85%in different regions.14 positive samples were sequenced,and the nucleotide homologies of ORF5 gene were among 83%to 100%for the strains identified in this study,and 59.9%to 98.5%for reference strains of PRRSV.14 sequenced strains all belonged to North America type(genotype Ⅱ)PRRSV in this study,4 strains belonged to the highly pathogenic PRRSV(HP-PRRSV,subtypeⅠ),3 strains belonged to the classic PRRSV(subtype Ⅱ),3 strains belonged to NADC30-like PRRSV(subtypeⅢ),and 4 strains belonged to the new subtype of PRRSV(subtype Ⅳ).Sequence analysis on deduced amino acid of GP5 coded by ORF5 gene indicated that the significant variations were observed in three epitopes and two important antigenic regions in PRRSVs belonged to the four subtypes(subtypesⅠ,Ⅱ,ⅢandⅣ).Subtype specific variations in subtypesⅢandⅣ were also observed in amino acid of GP5,which might lead to the variation of antigenecity of GP5 protein.The results indicated that there was a new situation of PRRSV strains circulating in Jiangxi province during 2016 to 2017,with multi-subtypes of strains existed and co-infected in pigs.And the most popular subtype was HP-PRRSV,while there were high rates of newly emerged NADC30-like strains and new subtype strains;It was necessary to continuously monitor the epidemiology of PRRSV,which would be benefit for the development of new diagnostic methods,drugs and vaccines for the prevention and control of PRRS.
引文
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[3] HAN J,ZHOU L,GE X,et al.Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J].Veterinary Microbiology,2017,209:30-47.
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[5]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,2:1-5.GUO B Q,CHEN Z S,LIU W X,et al.Isolation and identification of porcine reproductory and respiratory syndrome(PRRS)virus from aborted fetuses suspected of PRRS[J].Chinese Journal of Preventive Veterinary Medicine,1996,2:1-5.(in Chinese)
[6]杨宗照,方维焕.猪瘟合并猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)感染[J].中国兽医学报,2006,26(3):240-242.YANG Z Z,FANG W H.HCV co-infecting with PRRSV and PCV2[J].Chinese Journal of Veterinary Science,2006,26(3):240-242.(in Chinese)
[7] LI X,BAO H,WANG Y,et al.Widespread of NADC30-like PRRSV in China:Another Pandora’s box for Chinese pig industry as the outbreak of highly pathogenic PRRSV in 2006?[J].Infection Genetics and Evolution,2017,49:12-13.
[8] ZHANG Q,XU X,YOU S,et al.Emerging of two new subgenotypes of porcine reproductive and respiratory syndrome viruses in Southeast China[J].Microbial Pathogenesis,2016,97:27-33.
[9] POPESCU L N,TRIBLE B R,CHEN N,et al.GP5of porcine reproductive and respiratory syndrome virus(PRRSV)as a target for homologous and broadly neutralizing antibodies[J].Veterinary Microbiology,2017,209:90-96.
[10] LUNNEY J K,FANG Y,LADINIG A,et al.Porcine reproductiveandrespiratorysyndromevirus(PRRSV):Pathogenesis and interaction with the immune system[J].Annual Review of Animal Biosciences,2016,4(1):129-154.
[11] LI H,ZHOU E M,LIU C Q,et al.Function of CD163fragments in porcine reproductive and respiratory syndrome virus infection[J].International Journal of Clinical and Experimental Medicine,2015,8(9):15373-15382.
[12]陈希文,汪谦,尹苗,等.猪繁殖与呼吸综合征病毒RT-LAMP快速检测方法的建立[J].中国畜牧兽医,2017,44(6):1630-1636.CHEN X W,WANG Q,YIN M,et al.Establishiment of RT-LAMP for rapid detection of porcine reproduction and respiratory syndrome virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1630-1636.(in Chinese)
[13]施开创,官家明,胡杰,等.同时检测PRRSV美洲型经典株、变异株和TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法的建立及应用[J].中国预防兽医学报,2014,36(6):453-457.SHI K C,GUAN J M,HU J,et al.Establishment and application of a multiplex TaqMan Real-time RT-PCR for differential detection of classical,highly pathogenic and TJM-F92vaccine strains of North American genotype PRRSV[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(6):453-457.(in Chinese)
[14]施开创,许心婷,胡杰,等.PRRSV美洲型经典株、变异株和疫苗株多重RT-PCR鉴别检测方法的建立及应用[J].中国畜牧兽医,2017,44(3):879-887.SHI K C,XU X T,HU J,et al.Establishment and application of a multiplex RT-PCR assay for differential detection of classical,highly pathogenic and vaccine strains of North American genotype PRRSV[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):879-887.(in Chinese)
[15]张帆帆,宋德平,周信荣,等.新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J].中国农业科学,2016,49(7):1408-1416.ZHANG F F,SONG D P,ZHOU X R,et al.Establishment and application of a RT-PCR assay for detection of newly emerged porcine deltacoronavirus[J].Scientia Agricultura Sinica,2016,49(7):1408-1416.(in Chinese)
[16] ZHOU L,YANG B,XU L,et al.Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like[J].Veterinary Microbiology,2017,207:108-116.
[17] LI C,ZHUANG J,WANG J,et al.Outbreak investigation of NADC30-like PRRSV in South-East China[J].Transboundary and Emerging Diseases,2016,63(5):474-479.
[18] SUN Z,WANG J,BAI X,et al.Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus[J].Archives of Virology,2016,161(8):2257-2261.
[19] HAN K,SEO H W,PARK C,et al.Vaccination of sows against type 2porcine reproductive and respiratory syndrome virus(PRRSV)before artificial insemination protects against type 2PRRSV challenge but does not protect against type 1PRRSV challenge in late gestation[J].Veterinary Research,2014,45:12.
[20] CHOI K,PARK C,JEONG J,et al.Comparison of commercial type 1and type 2PRRSV vaccines against heterologous dual challenge[J].Veterinary Record,2016,178(12):291.
[21]蒋新华,陈松昌,万文忠,等.2013年江西省猪病诊断情况总结及分析[J].养猪,2014,3:121-123.JIANG X H,CHEN S C,WAN W Z,et al.Summary and analysis on diagnosis of swine diseases in Jiangxi province in 2013[J].Swine Production,2014,3:121-123.(in Chinese)
[22]徐晓杰,张乔亚,谈晨,等.2015-2016年我国部分地区猪繁殖与呼吸综合征的分子流行病学调查[J].畜牧与兽医,2017,49(6):153-156.XU X J,ZHANG Q Y,TAN C,et al.Molecular epidemiological investigation of porcine reproductive and respiratory syndrome from 2015to 2016in some areas of China[J].Animal Husbandry&Veterinary Medicine,2017,49(6):153-156.(in Chinese)
[23]熊加明.近年来江西部分地区猪瘟、猪繁殖与呼吸综合征和猪伪狂犬病流行病学调查[D].南昌:江西农业大学,2017.XIONG J M.Epidemiological investigation of classical swine fever,porcine reproductive and respiratory syndrome and porcine pseudorabies in parts of Jiangxi province in recent years[D].Nanchang:Jiangxi Agricultural University,2017.(in Chinese)
[24] BROCKMEIER S L,LOVING C L,VORWALD A C,et al.Genomic sequence and virulence comparison of four type 2porcine reproductive and respiratory syndrome virus strains[J].Virus Research,2012,169(1):212-221.
[25]董建国,王瑞,曲哲会,等.2014年~2015年河南省南部地区新近猪繁殖与呼吸综合征病毒流行株ORF5基因的变异分析[J].中国预防兽医学报,2016,38(11):869-873.DONG J G,WANG R,QU Z H,et al.Genetic variations of the ORF5genes of porcine reproductive and respiratory syndrome isolated in Southern Henan regions from 2014to 2015[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(11):869-873.(in Chinese)
[2] NIEDERWERDER M C.Role of the microbiome in swine respiratory disease[J].Veterinary Microbiology,2017,209:97-106.
[3] HAN J,ZHOU L,GE X,et al.Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J].Veterinary Microbiology,2017,209:30-47.
[4] WHITWORTH K M,ROWLAND R R,EWEN C L,et al.Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus[J].Nature Biotechnology,2016,34(1):20-22.
[5]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,2:1-5.GUO B Q,CHEN Z S,LIU W X,et al.Isolation and identification of porcine reproductory and respiratory syndrome(PRRS)virus from aborted fetuses suspected of PRRS[J].Chinese Journal of Preventive Veterinary Medicine,1996,2:1-5.(in Chinese)
[6]杨宗照,方维焕.猪瘟合并猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)感染[J].中国兽医学报,2006,26(3):240-242.YANG Z Z,FANG W H.HCV co-infecting with PRRSV and PCV2[J].Chinese Journal of Veterinary Science,2006,26(3):240-242.(in Chinese)
[7] LI X,BAO H,WANG Y,et al.Widespread of NADC30-like PRRSV in China:Another Pandora’s box for Chinese pig industry as the outbreak of highly pathogenic PRRSV in 2006?[J].Infection Genetics and Evolution,2017,49:12-13.
[8] ZHANG Q,XU X,YOU S,et al.Emerging of two new subgenotypes of porcine reproductive and respiratory syndrome viruses in Southeast China[J].Microbial Pathogenesis,2016,97:27-33.
[9] POPESCU L N,TRIBLE B R,CHEN N,et al.GP5of porcine reproductive and respiratory syndrome virus(PRRSV)as a target for homologous and broadly neutralizing antibodies[J].Veterinary Microbiology,2017,209:90-96.
[10] LUNNEY J K,FANG Y,LADINIG A,et al.Porcine reproductiveandrespiratorysyndromevirus(PRRSV):Pathogenesis and interaction with the immune system[J].Annual Review of Animal Biosciences,2016,4(1):129-154.
[11] LI H,ZHOU E M,LIU C Q,et al.Function of CD163fragments in porcine reproductive and respiratory syndrome virus infection[J].International Journal of Clinical and Experimental Medicine,2015,8(9):15373-15382.
[12]陈希文,汪谦,尹苗,等.猪繁殖与呼吸综合征病毒RT-LAMP快速检测方法的建立[J].中国畜牧兽医,2017,44(6):1630-1636.CHEN X W,WANG Q,YIN M,et al.Establishiment of RT-LAMP for rapid detection of porcine reproduction and respiratory syndrome virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1630-1636.(in Chinese)
[13]施开创,官家明,胡杰,等.同时检测PRRSV美洲型经典株、变异株和TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法的建立及应用[J].中国预防兽医学报,2014,36(6):453-457.SHI K C,GUAN J M,HU J,et al.Establishment and application of a multiplex TaqMan Real-time RT-PCR for differential detection of classical,highly pathogenic and TJM-F92vaccine strains of North American genotype PRRSV[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(6):453-457.(in Chinese)
[14]施开创,许心婷,胡杰,等.PRRSV美洲型经典株、变异株和疫苗株多重RT-PCR鉴别检测方法的建立及应用[J].中国畜牧兽医,2017,44(3):879-887.SHI K C,XU X T,HU J,et al.Establishment and application of a multiplex RT-PCR assay for differential detection of classical,highly pathogenic and vaccine strains of North American genotype PRRSV[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):879-887.(in Chinese)
[15]张帆帆,宋德平,周信荣,等.新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J].中国农业科学,2016,49(7):1408-1416.ZHANG F F,SONG D P,ZHOU X R,et al.Establishment and application of a RT-PCR assay for detection of newly emerged porcine deltacoronavirus[J].Scientia Agricultura Sinica,2016,49(7):1408-1416.(in Chinese)
[16] ZHOU L,YANG B,XU L,et al.Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like[J].Veterinary Microbiology,2017,207:108-116.
[17] LI C,ZHUANG J,WANG J,et al.Outbreak investigation of NADC30-like PRRSV in South-East China[J].Transboundary and Emerging Diseases,2016,63(5):474-479.
[18] SUN Z,WANG J,BAI X,et al.Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus[J].Archives of Virology,2016,161(8):2257-2261.
[19] HAN K,SEO H W,PARK C,et al.Vaccination of sows against type 2porcine reproductive and respiratory syndrome virus(PRRSV)before artificial insemination protects against type 2PRRSV challenge but does not protect against type 1PRRSV challenge in late gestation[J].Veterinary Research,2014,45:12.
[20] CHOI K,PARK C,JEONG J,et al.Comparison of commercial type 1and type 2PRRSV vaccines against heterologous dual challenge[J].Veterinary Record,2016,178(12):291.
[21]蒋新华,陈松昌,万文忠,等.2013年江西省猪病诊断情况总结及分析[J].养猪,2014,3:121-123.JIANG X H,CHEN S C,WAN W Z,et al.Summary and analysis on diagnosis of swine diseases in Jiangxi province in 2013[J].Swine Production,2014,3:121-123.(in Chinese)
[22]徐晓杰,张乔亚,谈晨,等.2015-2016年我国部分地区猪繁殖与呼吸综合征的分子流行病学调查[J].畜牧与兽医,2017,49(6):153-156.XU X J,ZHANG Q Y,TAN C,et al.Molecular epidemiological investigation of porcine reproductive and respiratory syndrome from 2015to 2016in some areas of China[J].Animal Husbandry&Veterinary Medicine,2017,49(6):153-156.(in Chinese)
[23]熊加明.近年来江西部分地区猪瘟、猪繁殖与呼吸综合征和猪伪狂犬病流行病学调查[D].南昌:江西农业大学,2017.XIONG J M.Epidemiological investigation of classical swine fever,porcine reproductive and respiratory syndrome and porcine pseudorabies in parts of Jiangxi province in recent years[D].Nanchang:Jiangxi Agricultural University,2017.(in Chinese)
[24] BROCKMEIER S L,LOVING C L,VORWALD A C,et al.Genomic sequence and virulence comparison of four type 2porcine reproductive and respiratory syndrome virus strains[J].Virus Research,2012,169(1):212-221.
[25]董建国,王瑞,曲哲会,等.2014年~2015年河南省南部地区新近猪繁殖与呼吸综合征病毒流行株ORF5基因的变异分析[J].中国预防兽医学报,2016,38(11):869-873.DONG J G,WANG R,QU Z H,et al.Genetic variations of the ORF5genes of porcine reproductive and respiratory syndrome isolated in Southern Henan regions from 2014to 2015[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(11):869-873.(in Chinese)