山东省一例山羊伪狂犬病的病原分离鉴定
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摘要
2017年4月,山东省某羊场饲养的山羊发生疑似伪狂犬病疫情。为确诊引发疫情的病原,采集死亡山羊组织样品,匀浆处理后接种家兔,并进行病原分离、PCR鉴定、效价测定、电镜观察及主要毒力基因测序分析。结果显示:家兔接种病料上清液后出现奇痒、麻痹,最后死亡;Marc145细胞接种病例样品后,产生变亮、变大、破裂,呈"包涵体"样等PRV特征性细胞病变,细胞分离物伪狂犬病病毒(PRV)g E PCR检测结果为阳性,病毒效价可达10-7.50 TCID50/0.1 m L,电镜观察可见病毒粒子呈圆形,有囊膜,直径约150 nm,将其命名为SDPD-17株。测序发现该毒株g E糖蛋白48、497位各插入了1个天冬氨酸,有12个氨基酸位点发生点突变;g C蛋白氨基酸序列64～70位出现了7个氨基酸(AASTPAA)插入;TK基因无碱基插入或缺失。分离鉴定结果证实,该病例由PRV野毒感染所致。
In April 2017,a suspected outbreak of pseudorabies occurred in a goat farm in Shandong Province. Then the dead goat tissue samples suspected of being infected with pseudorabies virus(PRV)were collected,homogenized,and inoculated into rabbits,and the virus was isolated and identified by PCR,the viral titer was determined,the cytopathic effect(CPE)in Marc145 cells was observed by electron microscope,and main virulence genes were sequenced and analyzed. According to the results,the rabbits showed itching,paralyzed and then died after inoculated with the tissue supernatant;after inoculated with viral samples,the Marc145 cells appeared characteristic cytopathic lesions,such as becoming brighter and larger,breaking up and showing appearance of"inclusion body",the PCR result of ampli?cation of PRV gE gene was positive;and the viral titer in cellular upernatant could reach 10-7.50 TCID50/0.1 mL;under the electron microscope,the virus particles were round,enveloped and the diameter was about 150 nm,the isolate was named as PRV SDPD-17 strain. Gene sequencing revealed that each of the 48 th and497 th amino acid in gE glycoprotein was inserted with an aspartic acid,and a total of 12 amino acid sites were pointmutated;the amino acid region(from 64 to 70)of gC protein had 7 amino acid insertion(AASTPAA);besides,the TK gene had no base insertion or deletion. In conclusion,the death of goat was caused by wild PRV infection.
In April 2017,a suspected outbreak of pseudorabies occurred in a goat farm in Shandong Province. Then the dead goat tissue samples suspected of being infected with pseudorabies virus(PRV)were collected,homogenized,and inoculated into rabbits,and the virus was isolated and identified by PCR,the viral titer was determined,the cytopathic effect(CPE)in Marc145 cells was observed by electron microscope,and main virulence genes were sequenced and analyzed. According to the results,the rabbits showed itching,paralyzed and then died after inoculated with the tissue supernatant;after inoculated with viral samples,the Marc145 cells appeared characteristic cytopathic lesions,such as becoming brighter and larger,breaking up and showing appearance of"inclusion body",the PCR result of ampli?cation of PRV gE gene was positive;and the viral titer in cellular upernatant could reach 10-7.50 TCID50/0.1 mL;under the electron microscope,the virus particles were round,enveloped and the diameter was about 150 nm,the isolate was named as PRV SDPD-17 strain. Gene sequencing revealed that each of the 48 th and497 th amino acid in gE glycoprotein was inserted with an aspartic acid,and a total of 12 amino acid sites were pointmutated;the amino acid region(from 64 to 70)of gC protein had 7 amino acid insertion(AASTPAA);besides,the TK gene had no base insertion or deletion. In conclusion,the death of goat was caused by wild PRV infection.
引文
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[14]孙海凤,董静,姜辰龙,等.江苏省猪伪狂犬病病毒强毒株的部分基因进化分析与致病性研究[J].中国预防兽医学报,2018,40(4):283-287.
[15]朱玲云,张正学,郑龙龙,等.山羊源伪狂犬病病毒的分离与鉴定[J].动物医学进展,2016,37(12):113-117.
[16]钟承,张乐天,王巨实,等.犬源伪狂犬病毒BJ/RD株的分离鉴定及gE基因分析[J].中国动物传染病学报,2013,21(5):1-6.
[17]WANG Y B,QIAO S L,LI X W,et al.Molecular epidemiology of outbreak-associated pseudorabies virus(PRV)strains in central China[J].Virus genes,2015,50(3):401-409.
[2]CRAMER S D,CAMPBELL G A,NJAA B L,et al.Pseudorabies virus infection in Oklahoma hunting dogs[J].Journal of veterinary diagnostic investigation,2011,23(5):915-923.
[3]向柯宇,潘慧,吉艺宽,等.猪伪狂犬病病毒新流行株主要糖蛋白的分子特征分析[J].华南农业大学学报,2016,37(3):23-28.
[4]张乐天,陆梓杰,吕艳丽.犬伪狂犬病病毒致病机理研究进展[J].动物医学进展,2013,34(6):145-149.
[5]SCH?NIGER S,KLOSE K,WERNER H,et al.Nonsuppurative encephalitis in a dog[J].Veterinary pathology,2012,49(4):731-734.
[6]高英.猪伪狂犬病病毒在不同细胞中增殖情况的比较[J].畜牧兽医科技信息,2015(7):96.
[7]彭金美,安同庆,赵鸿远,等.猪伪狂犬病病毒新流行株的分离鉴定及抗原差异性分析[J].中国预防兽医学报,2013,35(1):1-4.
[8]仇华吉.伪狂犬病病毒变异株的致病性、分子特征及基因缺失疫苗的研制[J].兽医导刊,2016(21):21.
[9]王娟,杭学果.猪伪狂犬病病毒引起哺乳仔猪腹泻的治疗[J].畜禽业,2012(5):73-74.
[10]芮萍,刘曜综,马增军,等.貂源伪狂犬病病毒的分离鉴定及gE基因分子特征[J].畜牧兽医学报,2015,46(7):1268-1272.
[11]中国政府网.实验动物管理条例[EB/OL].(2017-03-01)[2019-03-10].http://www.gov.cn/gongbao/content/2017/content_5219114.htm.
[12]国家市场监督管理总局,国家标准化管理委员会.伪狂犬病诊断技术:GB/T 18641-2018[S].北京:中国标准出版社,2018.
[13]郭容利,王继春,茅爱华,等.猪伪狂犬病病毒流行株gE基因的遗传变异分析及其对仔猪致病性的初步研究[J].农业科学与技术(英文版),2015(5):926-930.
[14]孙海凤,董静,姜辰龙,等.江苏省猪伪狂犬病病毒强毒株的部分基因进化分析与致病性研究[J].中国预防兽医学报,2018,40(4):283-287.
[15]朱玲云,张正学,郑龙龙,等.山羊源伪狂犬病病毒的分离与鉴定[J].动物医学进展,2016,37(12):113-117.
[16]钟承,张乐天,王巨实,等.犬源伪狂犬病毒BJ/RD株的分离鉴定及gE基因分析[J].中国动物传染病学报,2013,21(5):1-6.
[17]WANG Y B,QIAO S L,LI X W,et al.Molecular epidemiology of outbreak-associated pseudorabies virus(PRV)strains in central China[J].Virus genes,2015,50(3):401-409.
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