一株猪伪狂犬病病毒变异株的分离鉴定

doi:10.16431/j.cnki.1671-7236.2018.01.025

摘要/Abstract

摘要：

为了解广东省猪伪狂犬病病毒（pseudorabies virus，PRV）野毒株的基因变异及遗传演化的情况，本试验对广东佛山疑似暴发伪狂犬病的猪场采集的病料（脑、肺脏、扁桃体、肝脏、脾脏）进行了PCR鉴定，初步鉴定为PRV毒株后，将阳性病料接种非洲绿猴肾细胞（Vero），进行毒株传代培养，对分离毒株进行PCR检测及小鼠感染试验，证实该病毒为PRV，并命名为PRV FS-2015株；并对该毒株进行细胞病变观察、病毒TCID₅₀测定、毒株gC和TK基因扩增及序列分析。结果显示，PRV FS-2015株TCID₅₀为10^-7.5/0.1 mL。PRV FS-2015株的gC和TK基因序列与国内外PRV参考毒株进行同源性比对分析发现，其核苷酸序列同源性分别为95.8%~100.0%和99.4%~100.0%，氨基酸同源性分别为92.3%~100.0%和98.7%~100.0%。遗传进化分析表明，PRV FS-2015株与国内近几年分离的PRV变异株GY、ZJ01、HB1201、HN1201、JS2012、BJ/YT和BP属于同一分支，同源性较高，亲缘关系更近；但与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha、Yangsan、Min-A、SS和SL株的同源性较低，基因变异较大，表明PRV FS-2015毒株属于近几年流行的变异株。本研究结果可为广东省伪狂犬病的防控工作和疫苗株的选择提供科学依据。

关键词: 猪伪狂犬病毒; 分离鉴定; 序列分析; gC基因; TK基因; 遗传变异

Abstract:

The purpose of this study was to understand the genetic variation and evolution of pseudorabies virus (PRV) in Guangdong province.In this experiment,the PRV strain was primarily identified by PCR from samples (heart,lung,tonsil,liver and spleen) collected from sick pig in Foshan,Guangdong province,and the positive samples were inoculated Vero cells for subculture. And a strain was identifited as PRV by PCR and mice test,and named as PRV FS-2015.The cytopathic effect of the strain was observed and TCID₅₀ was tested,the whole gene of TK and gC were amplified and the sequence were analyzed. The results showed that the titer of TCID₅₀ of isolated virus in Vero cells was 10^-7.5/0.1 mL.The sequence analysis results showed that the nucleotide identities of the gC and TK genes of PRV FS-2015 strain with other PRV strains in GenBank were 95.8% to 100.0% and 99.4% to 100.0%,respectively,the homology of amino acid sequence were 92.3% to 100.0% and 98.7% to 100.0%,respectively. Phylogenetic tree analysis showed that the PRV FS-2015 stain had closer relationship with GY,ZJ01,HB1201,HN1201,JS2012,BJ/YT and BP stains,while had a distant relationship with Kaplan, Becker,NIA3,Kolchis,Bartha,Yangsan,Min-A,SS and SL stains. The experiment showed that PRV FS-2015 strain belonged to the variant popular in recent years,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Guangdong province.

Key words: pseudorabies virus (PRV); isolation and identification; sequence analysis; gC gene; TK gene; genetic variation

中图分类号:

S852.65

庞旋飞, 练斯南, 李中圣, 万曾培, 白挨泉. 一株猪伪狂犬病病毒变异株的分离鉴定[J]. 《中国畜牧兽医》, 2018, 45(1): 196-205.

PANG Xuanfei, LIAN Sinan, LI Zhongsheng, WAN Zengpei, BAI Aiquan. Isolation and Identification of a Mutant Strain of Pseudorabies Virus[J]. , 2018, 45(1): 196-205.

参考文献

[1] KASTRUKOFF L F,LAU A S,THOMAS E A.The effect of mouse strain on herpes simplex virus type 1 infection of the central nervous system[J].Herpesviridate,2012,3(1):4-8.
[2] FUJISAWA T,YAMAGUCHI Y.Postoperative immunost imulation after complete resection improves survival of patients with stage Ⅰ nonsmall cell lung carcinoma[J].Cancer,1996,78(9):1892-1898.
[3] 袁红,练斯南,张衡,等.新型猪伪狂犬病流行情况及净化措施[J].中国动物检疫,2016,33(3):58-62. YUAN H,LIAN S N,ZHANG H,et al. New epidemic situation and purification measures of pseudorabies[J].China Animal Quarantine,2016,33(3):58-62.(in Chinese)
[4] 张敏.豫南地区猪伪狂犬病野毒感染状况监测和控制措施的研究及运用[D].郑州:河南农业大学,2009. ZHANG M.Study and application of surveillance and control measures of wild pseudorabies virus infection in South Henan province[D].Zhengzhou:Henan Agricultural University,2009. (in Chinese)
[5] 陈溥言.兽医传染病学[M].第5版.北京:中国农业大学出版社,2006. CHEN F Y.Veterinary Infectious Diseases[M].Fifth edition.Beijing:China Agricultural University Press,2006. (in Chinese)
[6] 张青占.变异猪伪狂犬病毒的分离鉴定及生物学特性分析[D].北京:中国农业科学院,2013. ZHANG Q Z.Isolation,identification and biological characteristics of mutant pseudorabies virus[D]. Beijing:Chinese Academy of Agricultural Sciences,2013. (in Chinese)
[7] SUN Y,LUO Y,WANG C H,et al. Control of swine pesudorabies in China:Opportunities and limitations[J].Veterinary Microbiology,2016,183:119-124.
[8] 白挨泉.新型猪伪狂犬病流行情况及净化措施[J].兽医导刊,2016,2:22-24. BAI A Q.The prevalence of pseudorabies in swine model and purification measures[J].Veterinary Ophthalmology,2016,2:22-24. (in Chinese)
[9] 冷依伊,任梅渗,蒙正群,等,猪伪狂犬病病毒致病机理及其变异的研究进展[J].养猪,2016,5:121-126. LENG Y Y,REN M S,MENG Z Q,et al.Research progress of pseudorabies virus pathogenesis and its variation[J].Swine Production,2016,5:121-126.(in Chinese)
[10] 钟承,刘蕾,张乐天,等.伪狂犬病病毒BJ/YT株毒力相关基因gE和TK序列分析[J].中国畜牧兽医,2014, 41(11):73-79. ZHONG C,LIU L,ZHANG L T,et al. Sequence analysis of virulence related genes gE and TK in pseudorabies virus BJ/YT strain[J].Chinese Animal Medicine and Veterinary Medicine,2014,41(11):73-79.(in Chinese)
[11] 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)
[12] 陈超.山东省免疫猪场猪伪狂犬病毒分离鉴定及gE、gC、gD和TK基因的序列分析[D].泰安:山东农业大学,2015. CHEN C.Isolation,identification and sequence analysis of gE,gC,gD and TK genes in pseudorabies pigs of Shandong province[D].Tai'an:Shandong Agricultural University,2015.(in Chinese)
[13] 潘兹书,张楚瑜,丁建华,等.伪狂犬病毒胸苷激酶基因变异引起的病毒致弱作用[J].科学通报,2001,46(16):1364-1367. PAN Z S,ZHANG C Y,DING J H,et al.The attenuated effect of pseudorabies virus gene mutation on gene[J].Chinese Science Bulletin,2001,46(16):1364-1367.(in Chinese)
[14] DARBY G,LARDER B A,INGLIS M M.Evidence that the active centre of herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide[J].Journal of General Virology,1986,67:753-758.
[15] KLEMENS W,THOMAS B,ANDRÉ A,et al.The three dimensional structure of thymidine kinase from Herpes[J]. FEBS Letters,1995,368:289-292.
[16] 郭广君,吕素芳,沈志强,等.山东省分离强毒株猪伪狂犬病病毒SA株TK基因结构与进化分析[J].黑龙江畜牧兽医,2009,8:76-79. GUO G J,LV S F,SHEN Z Q,et al. Structural and evolutionary analysis of TK gene of pseudorabies virus SA strain isolated from Shandong province[J]. Heilongjiang Animal Husbandry and Veterinary Medicine,2009,8:76-79. (in Chinese)
[17] FRERARI M,GUALANDI G L CORRADI A,et al. The response of pigs inoculated with a thymidine Kinase-negative pseudorabies virus to challenge infection with virulent virus[J].Comparative Immunology, Microbiology and Infectious Diseases,2000,23(1):15-26.
[18] 吴德铭.伪狂犬病毒粤A毒株TK基因的扩增、克隆与序列测定[J].华南农业大学学报,2002,23(3):71-73. WU D M. Amplification,cloning and sequencing of TK gene of pseudorabies virus A strain[J].Journal of South China Agricultural University,2002,23(3):71-73. (in Chinese)
[19] HAMPL H,BENPORAI T,EHRLICHER L,et al. Characterization of the envelope proteins of pseudorabies virus[J].Journal of Virology,1984,52:583-590.
[20] 黄海彬,程安春,汪铭书.疱疹病毒糖蛋白的研究进展[J].中国人畜共患病学报,2010,26(2):171-174. HUANG H B,CHENG A C,WANG M S.Research progress of herpes virus glycoproteins[J]. Chinese Journal of Zoonoses,2010,26(2):171-174.(in Chinese)
[21] KARGER A,SCHMIDT J,METTERNLEITER T C. Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D[J].Journal of Virology,1998,72(9):7341-7348.