猪流行性腹泻病毒云南流行毒株分离与鉴定
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摘要
为有效控制和预防猪流行性腹泻(PED),分离获得猪流行性腹泻病毒(PEDV)云南地方流行毒株,并选取部分经RT-PCR鉴定为阳性的样品,处理后接种在Vero细胞中进行分离培养,细胞培养物盲传至15代后可观察到典型的细胞病变(CPE),对细胞培养物通过RT-qPCR方法检测和间接免疫荧光(IFA)鉴定,并对分离株S基因全序列进行克隆和遗传进化分析。结果表明:获得的细胞培养物均为PEDV阳性,病毒半数细胞感染量(TCID_(50))为10~(-4.85)/0.1mL,将其命名为YNDH-2017毒株。该分离株S基因与参考毒株的同源性为92.8%～98.2%,和国内近年来PEDV流行毒株亲缘关系较近,存在相同的突变位点,而与经典毒株CV777株在两个大的分支上,亲缘关系较远。试验分离获得的YNDH-2017株属于当前国内流行毒株。
The isolated PEDV strain identified as a positive sample by RT-PCR is inoculated into Vero cells for separation culture and the typical cytopathic effect(CPE)of cell cultures is observed after 15 th generation culture. The cell cultures are detected and identified by RT-qPCR and indirect immunofluorescence assay(IFA)and total sequence and genetic evolution of S gene of the isolated strain are cloned and analyzed respectively to effectively control and prevent porcine epidemic diarrhea(PED).Result:The obtained cell culture with PEDV-positive and 10~(-4.85)/0.1 mL of TCID_(50) is named as YNDH-2017 strain.The S gene homology between YNDH-2017 strain and reference strains reaches 92.8%-98.2%.YNDH-2017 strain has a near relationship to PEDV epidemic strains for the past few years in China and there are the same mutation sites between YNDH-2017 strain and other PEDV epidemic strains.YNDH-2017 strain has a distant relationship to typical CV777 strain.The isolated YNDH-2017 strain belongs to current epidemic strain in China.
The isolated PEDV strain identified as a positive sample by RT-PCR is inoculated into Vero cells for separation culture and the typical cytopathic effect(CPE)of cell cultures is observed after 15 th generation culture. The cell cultures are detected and identified by RT-qPCR and indirect immunofluorescence assay(IFA)and total sequence and genetic evolution of S gene of the isolated strain are cloned and analyzed respectively to effectively control and prevent porcine epidemic diarrhea(PED).Result:The obtained cell culture with PEDV-positive and 10~(-4.85)/0.1 mL of TCID_(50) is named as YNDH-2017 strain.The S gene homology between YNDH-2017 strain and reference strains reaches 92.8%-98.2%.YNDH-2017 strain has a near relationship to PEDV epidemic strains for the past few years in China and there are the same mutation sites between YNDH-2017 strain and other PEDV epidemic strains.YNDH-2017 strain has a distant relationship to typical CV777 strain.The isolated YNDH-2017 strain belongs to current epidemic strain in China.
引文
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[3]宣华,邢德坤,王殿瀛,等.应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J].兽医大学学报,1984,4(3):202-208.
[4]张志,李岚,王赛赛,等.猪流行性腹泻新毒株的分离鉴定和致病性研究[J].中国兽医学报,2012,32(10):1423-1428.
[5]孙东波,冯力,时洪艳,等.猪流行性腹泻病毒分子生物学研究进展[J].动物医学进展,2006,27(10):11-14.
[6]孙东波.猪流行性腹泻病毒S蛋白抗原表位鉴定及受体结合域的初步筛选[D].北京:中国农业科学院,2008.
[7]王康,童光志,周艳君等.猪流行性腹泻病毒HeB10/2014株的分离与鉴定[J].中国预防兽医学报,2016,38(4):284-288.
[8]WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus research,2016,226:7-13.
[9]PENSAERT M B,DE Bouck P.A new coronaviruslike particle associated with diarrhea in swine[J].Archives of virology,1978,58(3):243-247.
[10]CHANG S H,BAE J L,KANG T J,et al.Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus[J].Molecules and cells,2002,14(2):295-299.
[11]CHASEY D,CARTWRIGHT S F.Virus-like particles associated with porcine epidemic diarrhoea[J].Research in veterinary science,1978,25(2):255-256.
[12]HOFMANN M,WYLER R.Propagation of the virus of porcine epidemic diarrhea in cell culture[J].Journal of clinical microbiology,1988,26(11):2235-2239.
[13]ZENG Z,LI T T,JIN X,et al.Coexistence of multiple genotypes of porcine epidemic diarrhea virus with novel mutant S genes in the Hubei Province of China in 2016[J].Virologica Sinica,2017,32(4):298-306.
[14]TIAN Y,YU Z,CHENG K,et al.Molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu,China in 2012[J].Viruses,2013,5(8):1991-2004.