猪伪狂犬病毒的分离鉴定及其部分毒力基因序列遗传变异分析

doi:10.16431/j.cnki.1671-7236.2021.05.031

摘要/Abstract

摘要： 为了解吉林省猪伪狂犬病毒（PRV）的遗传变异情况，本研究将采集自吉林省疑似发生猪伪狂犬病的临床样品，通过细胞传代、PCR检测和测序分析进行病毒分离鉴定。TCID₅₀法测定分离毒株的病毒滴度，通过家兔感染试验测定分离毒株对家兔的致病性。对TK、gB、gC、gD和gE基因进行PCR扩增和测序，分析其遗传变异情况。结果表明，临床样品经PK15细胞传代后，有3份样品出现细胞病变，经PCR鉴定和测序分析表明，分离获得3株PRV，分别命名为PRV JL03、JL12和JL15株。通过PK15细胞测定分离毒株的病毒滴度分别为10^6.5、10^6.5和10^7.5TCID₅₀/mL。6只家兔分别接种3株分离毒株（10^3.5TCID₅₀/只）后均表现为体温升高、注射部位奇痒和四肢麻痹等症状，且于病毒接种后3~4 d全部死亡。3株分离病毒TK、gB、gC、gD和gE基因推导氨基酸序列分析结果表明，与参考毒株相比，分离株TK基因未发生氨基酸变异；gB、gC、gD和gE基因部分位点发生氨基酸缺失、插入或突变，其中gE基因在第48和496位分别存在1个天冬氨酸的插入，与国内报道的流行毒株变异特征一致。遗传进化树分析结果表明，3株分离毒株TK、gB、gC、gD和gE基因与国内2012年以后分离的参考毒株JS-2012和ZJ01等的上述基因均表现出较高的同源性，说明毒株间亲缘关系较近，位于同一分支；与国外分离毒株NIA3和Becker等亲缘关系较远，位于不同的分支；而与国内早期流行的毒株SC和Ea等亲缘关系介于二者之间。本研究成功分离鉴定了3株PRV变异株，分离毒株对家兔具有较强的致病性，该结果为吉林省PRV流行病学研究提供了新的数据，并为相关后续研究奠定基础。

关键词: 猪伪狂犬病毒; 分离鉴定; 遗传变异分析

Abstract: To investigate the genetic variation of Porcine pseudorabies virus (PRV) in Jilin province,in this study,the virus isolation and identification were performed by cell passage,PCR and sequencing in clinical samples were collected from pigs with suspected porcine pseudorabies.The viruses titer was determined by TCID₅₀,and the pathogenicity of the isolated strains to rabbits was determined by the rabbit infection test.Futhermore,PCR amplification and sequencing of TK,gB,gC,gD and gE genes were performed to analyze their genetic variation.The results showed that 3 samples of clinical samples had cytopathy after passage by PK15 cells.PCR identification and sequencing analysis showed that 3 strains of PRV were isolated and identified and named as JL03,JL12 and JL15 strain,respectively.The titers of the 3 strains were 10^6.5,10^6.5 and 10^7.5 TCID₅₀/mL on PK15 cells.Six rabbits were inoculated with 3 isolated strains (10^3.5 TCID₅₀ each rabbit),all showed symptoms such as elevated body temperature,itching at the injection site and paralysis of limbs,and all died 3 to 4 days after virus inoculation.The results of the deduced amino acid sequence analysis of the TK,gB,gC,gD and gE genes of the 3 isolated viruses showed that the TK gene had no amino acid variation compared with the reference strains.Amino acid deletions,insertions or mutations occurred in some positions of gB,gC,gD and gE genes.Among them,an aspartic acid insertion in the 48^th and 496^th positions of the gE gene were consistent with the variation characteristics of epidemic strains reported in China.Nucleotide genetic phylogenetic tree analysis showed that the TK,gB,gC,gD and gE genes of the 3 isolates were highly homologous with those of the reference strains JS-2012 and ZJ01 isolated after 2012 in China,and belong to the same branch.They were far from foreign strains NIA3 and Becker, and located in different branches,and the genetic relationship with the early domestic virus strains SC and Ea was between them.In short,we successfully isolated and identified three PRV variants which had strong pathogenicity to rabbits.These results provided new data for the epidemiological study of PRV in Jilin province and lay the foundation for relevant research.

Key words: Porcine pseudorabies virus; isolation and identification; phylogenetic analysis

中图分类号:

S855.3

徐宁, 葛桂阳, 李东丽, 刘艺, 宫庆龙, 麻宝艺, 盛陈艳, 时坤, 李健明, 冷雪, 杜锐. 猪伪狂犬病毒的分离鉴定及其部分毒力基因序列遗传变异分析[J]. 中国畜牧兽医, 2021, 48(5): 1794-1803.

XU Ning, GE Guiyang, LI Dongli, LIU Yi, GONG Qinglong, MA Baoyi, SHENG Chenyan, SHI Kun, LI Jianming, LENG Xue, DU Rui. Isolation and Identification of Porcine Pseudorabies Virus and Genetic Variation Analysis of Partial Virulence Gene[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1794-1803.

参考文献

[1] HSIANG C Y,HO T Y,CHANG T J,et al.Identification of a pseudorabies virus UL12(deoxyri-bonuclease) gene[J].Gene (Amsterdam),1996,177(1-2):100-113.
[2] 童武,李国新,郑浩,等.猪伪狂犬病基因缺失灭活疫苗(JS-2012-△gI/gE株)的制备及免疫效力评价[J].中国动物传染病学报,2016,24(6):12-18. TONG W,LI G X,ZHENG H,et al.Preparation and immune efficacy evaluation of inactivated swine pseudorabies vaccine (JS-2012-△gI/gE strain)[J].Chinese Journal of Animal Infectious Diseases,2016,24(6):12-18.(in Chinese)
[3] CONG X,LEI J L,XIA S L,et al.Pathogenicity and immunogenicity of a gE/gI/TK gene-deleted pseu-dorabies virus variantin susceptible animals[J].Veterinary Microbiology,2016,182:170-177.
[4] POMERANZ L E,REYNOLDS A E,HENGAR-TNER C J.Molecular biology of pseudorabies virus:Impact on neurovirology and veterinary medicine[J].Microbiology & Molecular Biology Reviews,2005,69(3):462-500.
[5] 王继春,曾容愚,DANIEL T,等.猪伪狂犬病活疫苗(Bartha K61株)对变异株的保护效力[J].畜牧与兽医,2015,47(12):1-4. WANG J C,ZENG R Y,DANIEL T,et al.Protection of pseudorabies vaccine (Bartha K61 strain) against Pseudorabies virus variant in pigs[J].Animal Husbandry and Veterinary Medicine,2015,47(12):1-4.(in Chinese)
[6] POL F,DEBLANC C,OGER A,et al.Validation of a commercial real-time PCR kit for specific and sensitive detection of Pseudorabies[J].Journal of Virological Methods,2013,187(2):421-423.
[7] 夏伟,牟迪,唐志芬,等.2016年245个规模猪场7种病毒类疾病病原流行病学调查与2017年防控建议[J].养猪,2017,157(2):93-96. XIA W,MOU D,TANG Z F,et al.Epidemiological survey of pathogens of 7 viral diseases in 245 large-scale pig farms in 2016 and prevention and control recommendations in 2017[J].Swine Production,2017,157(2):93-96.(in Chinese)
[8] 黄艺珠,张渊魁,魏建超,等.猪伪狂犬病毒SD株的分离鉴定及TK基因序列分析[J].畜牧与兽医,2013,45(4):62-67. HUANG Y Z,ZHANG Y K,WEI J C,et al.Isolation and identification of Porcine pseudorabies virus SD strain and TK gene sequence analysis[J].Animal Husbandry and Veterinary Medicine,2013,45(4):62-67.(in Chinese)
[9] 仇华吉,郭宝清,童光志,等.猪繁殖与呼吸道综合征病毒(PRRSV) CH-1a株基因型鉴定[J].中国兽医学报,1998,18(2):118-121. QIU H J,GUO B Q,TONG G Z,et al.Genotyping of Porcine reproductive and respiratory syndrome virus strain CH-1a isolated in China by RT-PCR and indirect immunofluorescence test[J].Chinese Journal of Veterinary Science,1998,18(2):118-121.(in Chinese)
[10] 郭抗抗,邓力,井勇,等.猪瘟病毒强毒株和兔化弱毒疫苗株RT-PCR快速鉴别检测方法的建立[J].西北农林科技大学学报(自然科学版),2010,38(6):13-18. GUO K K,DENG L,JING Y,et al.Development of RT-PCR for detection and differentiation of wild-type and vaccine viruses of classical Swine fever virus[J].Journal of Northwest A&F University (Natural Science Edtion),2010,38(6):13-18.(in Chinese)
[11] 吴淑芳,付瑞,邢瑞昌,等.猪细小病毒PCR检测方法的建立与应用[J].中国生物制品学杂志,2007,20(4):292-295. WU S F,FU R,XING R C,et al.Development of PCR method for determination of Porcine parvovirus[J].Chinese Journal of Biologicals,2007,20(4):292-295.(in Chinese)
[12] 冷雪,崔晓华,黄海龙,等.猪圆环病毒2型吉林地方株ORF2基因的克隆、测序及其在原核细胞中的表达[J].中国兽医学报,2007,27(4):469-472. LENG X,CUI X H,HUANG H L,et al.Cloning,sequencing and expression of ORF2 gene of Porcine circovirus type 2 isolated from Jilin province of JL01 isolate[J].Chinese Journal of Veterinary Science,2007,27(4):469-472.(in Chinese)
[13] MILLER J,ULRICH R.A computer program for Spearman Karber and probit analysis of psychometric function data[J].Behavior Research Methods Instruments and Computers,2004,36(1):11-16.
[14] SZPARA M L,TAFURI Y R,PARSONS L,et al.A wide extent of inter-strain diversity in virulent and vaccine strains of Alphaherpesviruses[J].PLoS Pathogens,2011,7(10):e1002282.
[15] FRANÇOISE P,CÉLINE D,AURÉLIE O,et al.Validation of a commercial Real-time PCR kit for specific and sensitive detection of pseudorabies[J].Journal of Virological Methods,2013,187(2):421-423.
[16] DELVA J L,NAUWYNCK H J,METTERN-LEITER T C,et al.The Attenuated Pseudorabies virus vaccine strain bartha K61:A brief review on the knowledge gathered during 60 years of research[J].Pathogens,2020,9(11):897.
[17] HU R M,ZHOU Q,SONG W B,et al.Novel Pseudorabies virus variant with defects in TK,gE and gI protects growing pigs against lethal challenge[J].Vaccine,2015,33(43):5733-5740.
[18] 陈超,刘存,李海娥,等.山东省免疫猪场猪伪狂犬病病毒分离鉴定及gE毒力基因的序列分析[J].中国兽医学报,2016,36(6):902-907. CHEN C,LIU C,LI H E,et al.Sequence analysis of gE gene among Pseudorabies virus strains isolated from Bartha-K61-vaccinated swine population of Shandong province in 2013 and 2014[J].Chinese Journal of Veterinary Science,2016,36(6):902-907.(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 genotypes[J].Virology,2015,483:32-43.
[20] JU H,YANG D,WANG J,et al.Experimental infection of sheep with Pseudorabies viruses isolated in Shanghai,China,between 2010 and 2012[J].European Journal of Experimental Biology,2017,7(5):201-215.
[21] WANG J,WANG C F,MING S L,et al.Porcine IFITM1 is a host restriction factor that inhibits Pseudorabies virus infection[J].International Journal of Biological Macromolecules,2020,151:1181-1193.
[22] GU Z,HOU C,SUN H,et al.Emergence of highly virulent Pseudorabies virus in Southern China[J].Canadian Journal of Veterinary Research,2015,79(3):221-228.
[23] 傅宏庆,姚志兰,刘轶秋,等.猪伪狂犬病毒YZ株的分离鉴定及遗传进化分析[J].扬州大学学报(农业与生命科学版),2020,41(5):36-41. FU H Q,YAO Z L,LIU Y Q,et al.Solation and genetic evolution analysis of Pseudorabies virus YZ strain[J].Journal of Yangzhou University (Agricul-tural and Life Science Edition),2020,41(5):36-41.(in Chinese)
[24] LU M Q,QIU S Y,ZHANG L L,et al.Pseudorabies virus glycoprotein gE suppresses interferon-β production via CREB-binding protein degradation[J].Virus Research,2020,21(6):902-907.