犬细小病毒河南株的分离鉴定及全基因组序列分析
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摘要
为了研究河南省当前犬细小病毒(Canine parvovirus,CPV)的流行情况,收集经CPV胶体金试纸检测阳性的病犬血便病料,对其处理后进行特异性PCR扩增,然后将粪便处理物经滤器过滤后接种于F81细胞,进行细胞盲传并观察细胞的病变情况,同时对病毒进行理化性质及血凝试验等检测,最后对病毒的全基因组进行测序。结果表明,该病毒经特异性PCR扩增初步鉴定为CPV,接种于F81细胞盲传2代后,出现明显细胞病变。全基因组测序显示,该病毒分离株基因组全长5 052 bp,其中包含U型发卡结构188 bp,Y型发卡结构120 bp。此外,其ORF1全长2 007 bp,能够编码非结构蛋白NS1,ORF2全长2 257 bp,能够编码结构蛋白VP1、VP2。经VP2氨基酸序列分析,鉴定其为New CPV-2b亚型,命名为CPV/HN1,该病毒分离株TCID_(50)为10~(-4.85)/0.1 mL,血凝效价为1∶256。与国内的17株参考毒株全基因组序列核苷酸同源性为96.4%～99.9%,其中与北京分离毒株同源性为99.9%。
In order to study the current epidemic situation of canine parvovirus(CPV) in Henan province,we collected the stool of dogs which was positive after testing by CPV colloidal gold test paper,and amplified two genes by specific PCR after treatment. Then the fecal treatment content was filtered through a filter and inoculated in F81 cells,and blind transmission of F81 cells was performed to observe whether there was obvious cytopathic effect.Meanwhile,the physical and chemical properties and hemagglutination tests of the virus were carried out,and finally the whole genome of the virus was sequenced.The results showed that the virus was identified as CPV by specific PCR amplification.After being inoculated in F81 cells for two generations of blind transmission,the cytopathic effect appeared.The whole genome sequencing showed that the genome of the virus isolate was 5 052 bp in length,including 188 bp U-type hairpin structure and 120 bp Y-type hairpin structure.ORF1 was 2 007 bp and encoded non-structural protein NS1,ORF2 was 2 257 bp and encoded structural protein VP1 and structural protein VP2.After the VP2 amino acid sequence analysis,we identified it as New CPV-2 b subtype and named CPV/HN1.The TCID_(50) of this virus isolate strain was 10~(-4.85)/0.1 mL and its coagulation titre was 1∶256.Besides,it showed 96.4%—99.9% nucleotide sequence identity with 17 domestic reference virus strains and its homology with the virus strain isolated from Beijing was up to 99.9%.
In order to study the current epidemic situation of canine parvovirus(CPV) in Henan province,we collected the stool of dogs which was positive after testing by CPV colloidal gold test paper,and amplified two genes by specific PCR after treatment. Then the fecal treatment content was filtered through a filter and inoculated in F81 cells,and blind transmission of F81 cells was performed to observe whether there was obvious cytopathic effect.Meanwhile,the physical and chemical properties and hemagglutination tests of the virus were carried out,and finally the whole genome of the virus was sequenced.The results showed that the virus was identified as CPV by specific PCR amplification.After being inoculated in F81 cells for two generations of blind transmission,the cytopathic effect appeared.The whole genome sequencing showed that the genome of the virus isolate was 5 052 bp in length,including 188 bp U-type hairpin structure and 120 bp Y-type hairpin structure.ORF1 was 2 007 bp and encoded non-structural protein NS1,ORF2 was 2 257 bp and encoded structural protein VP1 and structural protein VP2.After the VP2 amino acid sequence analysis,we identified it as New CPV-2 b subtype and named CPV/HN1.The TCID_(50) of this virus isolate strain was 10~(-4.85)/0.1 mL and its coagulation titre was 1∶256.Besides,it showed 96.4%—99.9% nucleotide sequence identity with 17 domestic reference virus strains and its homology with the virus strain isolated from Beijing was up to 99.9%.
引文
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[2] 徐汉坤,金淮,郭宝发,等.某犬群爆发犬病毒性肠炎的流行病学和临床病理学观察[J].畜牧与兽医,1983(5):5-7.
[3] 陈强,嵇辛勤,段志强,等.贵阳地区犬细小病毒VP2基因的克隆及遗传进化分析[J].中国畜牧兽医,2017,44(4):1061-1068.
[4] 吴植,曹斌,贺生中,等.犬细小病毒VP2基因编码主要抗原表位区的核酸免疫及其免疫原性研究[J].中国预防兽医学报,2015,37(6):473-476.
[5] 王斌,徐绮嫔,郭慧琛,等.犬细小病毒病毒样颗粒研究进展[J].动物医学进展,2016,37(3):81-85.
[6] 张传鹏,张航,高全新,等.犬细小病毒SH15株的分离鉴定与全基因组分析[J].中国兽医科学,2017,47(2):165-171.
[7] 王建科,刘辉哲,林鹏,等.犬细小病毒新2b型分离株的分离鉴定[J].中国兽医学报,2016,36(5):734-738.
[8] 李晓楠,唐青海,李羽,等.1株猪传染性胃肠炎病毒的分离鉴定及其生物学特性研究[J].河南农业科学,2017,46(8):131-136.
[9] BARRETT P N,MEYER H,WACHTEL I,et al.Determination of the inactivation kinetics of hepatitis A virus in human plasma products using a simple TCID50 assay[J].Journal of Medical Virology,2015,49(1):1-6.
[10] 马爱民,李倩,王超,等.血凝-血凝抑制试验在快速诊断犬细小病毒性肠炎中的应用[J].中国兽医学报,2015,35(6):858-861.
[11] GODDARD A,LEISEWITZ A L.Canine parvovirus[J].Vet Clin North Am Small Anim Pract,2010,40(6):1041-1053.
[12] PINTO L D,STRECK A F,GON?ALVES K R,et al.Typing of canine parvovirus strains circulating in Brazil between 2008 and 2010[J].Virus Research,2012,165(1):29-33.
[13] DECARO N,DESARIO C,ELIA G,et al.Occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration:A clinical and laboratory diagnostic dilemma[J].Vaccine,2007,25(7):1161-1166.
[14] HOELZER K,PARRISH C R.The emergence of parvoviruses of carni-vores[J].Vet Res,2010,41(6):39-46.
[15] HUALEI W,HONGLI J,QIAN L,et al.Isolation and se- quence analysis of the complete NS1 and VP2 genes of canine parvovirus from domestic dogs in 2013 and 2014 in China[J].Arch Virol,2016,161(2):385-393.
[16] SHACKELTON L A,PARRISH C R,TRUYEN U,et al.High rate of viral evolution associated with the emergence of carnivore parvovirus[J].Proc Natl Acad Sci USA,2005,102(2):379-384.
[17] KESTLER J,NEEB B,STRUYF S,et al.Cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses[J].Human Gene Therapy,1999,10(10):1619-1632.
[18] 于永乐,王吉贵,郗冀,等.犬细小病毒北京分离株的鉴定及其全基因组序列分析[J].中国兽医学报,2017,37(6):1023-1029.