两株猪伪狂犬病病毒变异株的分离鉴定及gB、gC和gE基因的分子特征分析
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摘要
为探明近年来河南省猪伪狂犬病病毒(PRV)的遗传变异情况,本研究于2017年采集河南漯河和中牟地区疑似伪狂犬病发病养殖场送检的脑组织病料,通过细胞盲传、噬斑纯化、间接免疫荧光试验、Western blotting和透射电镜技术进行病毒分离鉴定。TCID_(50)法测定分离毒株的病毒滴度、生长曲线,通过小鼠感染试验测定分离毒株对小鼠的致死性。对gB、gC和gE基因进行PCR扩增、测序,并与参考毒株序列进行比对分析。结果显示,对PCR鉴定阳性的病料在PK-15细胞盲传后,两份病料在6代内均出现细胞病变,通过间接免疫荧光试验、噬斑纯化和透射电镜技术,成功分离鉴定了两株PRV,分别命名为HeN-LH株及HeN-YM株。分离毒株在PK-15细胞上的生长曲线显示,HeN-LH和HeN-YM株在感染后36 h病毒滴度分别可达10~(8.35)和10~(6.63) TCID_(50)/mL。用不同浓度的病毒接种小鼠,结果显示,HeN-LH和HeN-YM株LD_(50)分别为10~(2.13)及10~(3.25) TCID_(50)。对gB、gC和gE基因全长扩增测序后构建遗传进化树,结果显示,两株PRV毒株与Bartha、Fa和Ea等经典株的亲缘关系相对较远,而与2011年以来国内不同省份分离的PRV变异株亲缘关系较近。氨基酸序列比对分析显示,与其他变异株相似,gB、gC和gE基因均发生了多个氨基酸的变异,且在特定的位点存在特征性的氨基酸插入和缺失。本研究成功分离鉴定了两株PRV变异株,分离株对小鼠均表现出一定的致病性,本试验结果可为河南省伪狂犬病的防控工作和疫苗株的选择提供科学依据。
To investigate the genetic variation of pseudorabies virus(PRV) in Henan province in recent years,in this study,the virus isolation and identification were performed by cell blind transmission,indirect immunofluorescence,Western blotting,plaque purification and transmission electron microscopy in brain tissue samples from suspected pseudorabies farms in Luohe and Zhongmou in 2017.The viruses titer and growth curve of the isolated strains were determined by 50% tissue culture infective dose(TCID_(50)),and the lethality of the mice was determined by mouse infection test.Futhermore,the gB,gC and gE genes were aligned and analyzed.Two strains of PRV were successfully isolated and identified by indirect immunofluorescence,plaque purification and transmission electron microscopy,named as HeN-LH strain and HeN-YM strain,respectively.The titers of HeN-LH and HeN-YM strains reached 10~(8.35) and 10~(6.63) TCID_(50)/mL at 36 h post infection on PK-15 cells.The lethality of the mice showed that the median lethal dose(LD_(50)) of HeN-LH and HeN-YM strains was 10~(2.13) and 10~(3.25) TCID_(50),respectively.The evolutionary analysis displayed that the two isolates were closer to the PRV variants which were mainly epidemic in China since 2011.Amino acids sequences alignment showed that,similar to other variants,gB,gC and gE genes had multiple amino acid variations,and there were unique amino acid insertions and deletions at specific sites.In short,we successfully isolated and identified two PRV variants,and confirmed the current pseudorabies were mainly caused by the PRV variants,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Henan province.
To investigate the genetic variation of pseudorabies virus(PRV) in Henan province in recent years,in this study,the virus isolation and identification were performed by cell blind transmission,indirect immunofluorescence,Western blotting,plaque purification and transmission electron microscopy in brain tissue samples from suspected pseudorabies farms in Luohe and Zhongmou in 2017.The viruses titer and growth curve of the isolated strains were determined by 50% tissue culture infective dose(TCID_(50)),and the lethality of the mice was determined by mouse infection test.Futhermore,the gB,gC and gE genes were aligned and analyzed.Two strains of PRV were successfully isolated and identified by indirect immunofluorescence,plaque purification and transmission electron microscopy,named as HeN-LH strain and HeN-YM strain,respectively.The titers of HeN-LH and HeN-YM strains reached 10~(8.35) and 10~(6.63) TCID_(50)/mL at 36 h post infection on PK-15 cells.The lethality of the mice showed that the median lethal dose(LD_(50)) of HeN-LH and HeN-YM strains was 10~(2.13) and 10~(3.25) TCID_(50),respectively.The evolutionary analysis displayed that the two isolates were closer to the PRV variants which were mainly epidemic in China since 2011.Amino acids sequences alignment showed that,similar to other variants,gB,gC and gE genes had multiple amino acid variations,and there were unique amino acid insertions and deletions at specific sites.In short,we successfully isolated and identified two PRV variants,and confirmed the current pseudorabies were mainly caused by the PRV variants,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Henan province.
引文
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[13] 王寅彪.猪伪狂犬病野毒感染与疫苗免疫鉴别诊断方法的建立及初步临床应用[D].长春:吉林大学,2015. WANG Y B.Pseudorabies wild virus infection and the establishment of diagnostic methods of vaccination and identification of preliminary clinical application[D].Changchun:Jilin University,2015.(in Chinese)
[14] AN T Q,PENG J M,TIAN Z J,et al.Pseudorabies virus variant in Bartha-K61-vaccinated pigs,China,2012[J].Emerging Infectious Diseases,2013,19(13):1749-1755.
[15] AI J W,WENG S S,CHENG Q,et al.Human endophthalmitis caused by pseudorabies virus infection,China,2017[J].Emerging Infectious Diseases,2018,24(6):1087-1090.
[16] LI A,LU G,QI J,et al.Structural basis of nection-1 recognition by pseudorabies virus glycoproteins D[J].PLoS Pathogens,2017,13(5):e1006314.
[17] 徐国栋.猪体内伪狂犬病毒分布及其神经示踪启示[J].中国动物保健,2017,1:4-7. XU G D.Distribution of pseudorabies virus in pigs and its implications for neural tracing[J].Chinese Animal Health,2017,1:4-7.(in Chinese)
[18] 余文兰,张晓战,黄红亮,等.伪狂犬病病毒Ra株对不同细胞增殖特性及致病性研究[J].中国畜牧兽医,2013,40(11):164-168. YU W L,ZHANG X Z,HUANG H L,et al.Study on proliferative characteristics and pathogenicity of pseudorabies virus Ra strain in different cells[J].China Animal Husbandry & Veterinary Medicine,2013,40(11):164-168.(in Chinese)
[19] YE C,ZHANG Q Z,TIAN Z J,et al.Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence:Evidence for the existence of two major geneotypes[J].Virology,2015,483:32-43.
[20] 余秋颖,常洪涛,陈定文,等.2012-2013年新流行猪伪狂犬病病毒的分离鉴定及其gE、TK、gD基因序列分析[J].中国兽医学报,2014,34(10):1573-1578. YU Q Y,CHANG H T,CHEN D W,et al.Isolation and identification of pseudorabies virus prevailing from 2012-2013 and sequence analysis of gE,TK,gD gene[J]. Chinese Journal of Veterinary Medicine,2014,34(10):1573-1578.(in Chinese)
[21] 伏鹏.猪伪狂犬病流行病学调查及防治[D].郑州:河南农业大学,2016. FU P.Epidemiological investigation and prevention of pseudorabies in pigs[D].Zhengzhou:Henan Agricultural University.(in Chinese)
[22] 黄元,陈晶,赵翠玲,等.伪狂犬病毒变异株的鉴定及伪狂犬病的防控[J].中国畜牧兽医,2016,43(9):2461-2467. HUANG Y,CHEN J,ZHAO C L,et al.Identification of pseudorabies virus variant and control of pseudorabies[J].China Animal Husbandry & Veterinary Medicine,2016,43(9):2461-2467.(in Chinese)
[23] BRITTLE E E,REYNOLDS A E,ENQUIST L W.Two modes pseudorabies virus neuroinvasion and lethality in mice[J].Journal of Virology,2004,78(23):12951-12963.
[24] 刘玉梅,张锐,刁育新,等.猪伪狂犬净化对猪生产性能的影响[J].中国畜牧兽医,2017,44(10):3063-3069. LIU Y M,ZHANG R,DIAO Y X,et al.Effect of pseudorabies purification on the production performance in pig[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):3063-3069.(in Chinese)
[2] 陆承平.兽医微生物学[M].北京:中国农业出版社,2012. LU C P.Veterinary Microbiology[M].Beijing:China Agriculture Press,2012.
[3] POMERANZ L E,REYNOLDS A E,HENGARTNER C J.Molecular biology of pseudorabies virus:Impact on neurovirology and veterinary medicine[J]. Microbiology and Molecular Biology Reviews,2005,69(3):462-500.
[4] HONG W,XIAO S,ZHOU R,et al.Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice,rabbits and piglets[J].Vaccine,2002,20(7):1205-1214.
[5] WHEALY M E,CARD J P,MEADE R P,et al.Effect on brefeldin A on alphaherpes virus membrane protein glycosylation and virus egress[J].Journal of Virology,1991,65(3):1066-1681.
[6] AVTIABILE E,FORGHIERI C,CAMPADELLI-FIUME G.Complex between herpes simplex virus glycoproteins gD,gB and gH detected in cells by complementation of split enhance green fluorescent protein[J].Journal of Virology,2007,81(20):11532-11537.
[7] SUBRAMANIAN R P,GERAGHTY R J.Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D,H,and B[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(8):2903-2908.
[8] HANSSENS F P,NAUWYNCK H J,PENSAERT M B.Involvement of membrane-bound viral glycoproteins in adhesion of pseudorabies virus infect-cells[J].Journal of Virology,1993,67(8):4492-4496.
[9] 阳爱国.伪狂犬病病毒主要毒力基因功能的研究[D].雅安:四川农业大学,2005. YANG A G.Research on the main virulence gene function of pseudorabies virus[D].Ya’an:Sichuan Agricultural University,2005.(in Chinese)
[10] KIT S,LEING W C,GORGENSEN G N,et al.Thymidine kinase isozymes of normal and virus-infected cells[J].Cold Spring Harbor Symposia on Quantitative Biology,1975,39(Pt 2):703-715.
[11] YU X,ZHOU Z,HU D,et al.Pathogenic pseudorabies virus,China,2012[J].Emerging Infectious Diseases,2014,20(1):102-104.
[12] 顾真庆.我国猪伪狂犬病病毒变异株全基因组序列分析及其gE/gI基因缺失灭活疫苗免疫特性[D].南京:南京农业大学,2014. GU Z Q.Whole genome sequence analysis of Chinese pseudorabies virus variant strain and immunological characteristics of inactivated vaccine with gE/gI gene deletion[D].Nanjing:Nanjing Agricultural University,2014.(in Chinese)
[13] 王寅彪.猪伪狂犬病野毒感染与疫苗免疫鉴别诊断方法的建立及初步临床应用[D].长春:吉林大学,2015. WANG Y B.Pseudorabies wild virus infection and the establishment of diagnostic methods of vaccination and identification of preliminary clinical application[D].Changchun:Jilin University,2015.(in Chinese)
[14] AN T Q,PENG J M,TIAN Z J,et al.Pseudorabies virus variant in Bartha-K61-vaccinated pigs,China,2012[J].Emerging Infectious Diseases,2013,19(13):1749-1755.
[15] AI J W,WENG S S,CHENG Q,et al.Human endophthalmitis caused by pseudorabies virus infection,China,2017[J].Emerging Infectious Diseases,2018,24(6):1087-1090.
[16] LI A,LU G,QI J,et al.Structural basis of nection-1 recognition by pseudorabies virus glycoproteins D[J].PLoS Pathogens,2017,13(5):e1006314.
[17] 徐国栋.猪体内伪狂犬病毒分布及其神经示踪启示[J].中国动物保健,2017,1:4-7. XU G D.Distribution of pseudorabies virus in pigs and its implications for neural tracing[J].Chinese Animal Health,2017,1:4-7.(in Chinese)
[18] 余文兰,张晓战,黄红亮,等.伪狂犬病病毒Ra株对不同细胞增殖特性及致病性研究[J].中国畜牧兽医,2013,40(11):164-168. YU W L,ZHANG X Z,HUANG H L,et al.Study on proliferative characteristics and pathogenicity of pseudorabies virus Ra strain in different cells[J].China Animal Husbandry & Veterinary Medicine,2013,40(11):164-168.(in Chinese)
[19] YE C,ZHANG Q Z,TIAN Z J,et al.Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence:Evidence for the existence of two major geneotypes[J].Virology,2015,483:32-43.
[20] 余秋颖,常洪涛,陈定文,等.2012-2013年新流行猪伪狂犬病病毒的分离鉴定及其gE、TK、gD基因序列分析[J].中国兽医学报,2014,34(10):1573-1578. YU Q Y,CHANG H T,CHEN D W,et al.Isolation and identification of pseudorabies virus prevailing from 2012-2013 and sequence analysis of gE,TK,gD gene[J]. Chinese Journal of Veterinary Medicine,2014,34(10):1573-1578.(in Chinese)
[21] 伏鹏.猪伪狂犬病流行病学调查及防治[D].郑州:河南农业大学,2016. FU P.Epidemiological investigation and prevention of pseudorabies in pigs[D].Zhengzhou:Henan Agricultural University.(in Chinese)
[22] 黄元,陈晶,赵翠玲,等.伪狂犬病毒变异株的鉴定及伪狂犬病的防控[J].中国畜牧兽医,2016,43(9):2461-2467. HUANG Y,CHEN J,ZHAO C L,et al.Identification of pseudorabies virus variant and control of pseudorabies[J].China Animal Husbandry & Veterinary Medicine,2016,43(9):2461-2467.(in Chinese)
[23] BRITTLE E E,REYNOLDS A E,ENQUIST L W.Two modes pseudorabies virus neuroinvasion and lethality in mice[J].Journal of Virology,2004,78(23):12951-12963.
[24] 刘玉梅,张锐,刁育新,等.猪伪狂犬净化对猪生产性能的影响[J].中国畜牧兽医,2017,44(10):3063-3069. LIU Y M,ZHANG R,DIAO Y X,et al.Effect of pseudorabies purification on the production performance in pig[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):3063-3069.(in Chinese)