猪圆环病毒2型的分离鉴定及其全基因组序列分析
|
摘要
为分离鉴定流行于河南省的猪圆环病毒2型(PCV2),对河南省不同地区猪场的11份疑似断奶仔猪多系统衰竭综合征(PMWS)猪的病料进行分离鉴定,得到PCV2毒株。通过PCR检测,11份临床疑似病料中,7份确诊为PCV2阳性病料,阳性率为63.6%。挑选出阳性值较高的毒株命名为zmd-20161022,通过增殖培养筛选出优良毒株,并对其进行单层过氧化物酶试验(IPMA)鉴定,分析免疫原性;应用PCR对该病毒全基因组进行特异性扩增,经测序后对该病毒的同源性和系统进化进行分析。结果显示,该毒株序列全长1 767 bp,同源性分析发现与国内3株PCV2毒株(AY686763、HM641752、HM038034)的同源性为95.2%~98.3%,与基因登录号为HM038030.1的同源性高达98.6%。系统进化树分析发现,zmd-20161022毒株与基因登录号为HM038030.1的亲缘关系很近,被鉴定为PCV2b-1c型,属于PCV2b的一种亚型。
In order to isolate and identify porcine circovirus type 2,11 clinical samples from weanling pigs suspected of PCV2 infection,which is endemic to pig farms in Henan province,were tested and identified.The 7 out of 11 clinical samples were diagnosed as PCV2 positive by PCR,and the positive rate was63. 6%. The strain with the highest positive value was selected and named as zmd-20161022,which was further tested by IPMA to analyze the immunogenicity,and the complete genome was amplified by PCR and sequenced for homology and phylogenetic analysis. The full length of zmd-20161022 was 1 767 bp,95. 2% —98. 3% homology with three PCV2 strains( AY686763,HM641752,HM038034) from China,and 98. 6% homology with the PCV2 strain( the gene accession number HM038030. 1) in Gen Bank.Phylogenetic analysis showed that the zmd-20161022 strain belonged to PCV2 b-1 c,a subtype of PCV2 b,which was closely related to the gene accession number HM038030. 1.
In order to isolate and identify porcine circovirus type 2,11 clinical samples from weanling pigs suspected of PCV2 infection,which is endemic to pig farms in Henan province,were tested and identified.The 7 out of 11 clinical samples were diagnosed as PCV2 positive by PCR,and the positive rate was63. 6%. The strain with the highest positive value was selected and named as zmd-20161022,which was further tested by IPMA to analyze the immunogenicity,and the complete genome was amplified by PCR and sequenced for homology and phylogenetic analysis. The full length of zmd-20161022 was 1 767 bp,95. 2% —98. 3% homology with three PCV2 strains( AY686763,HM641752,HM038034) from China,and 98. 6% homology with the PCV2 strain( the gene accession number HM038030. 1) in Gen Bank.Phylogenetic analysis showed that the zmd-20161022 strain belonged to PCV2 b-1 c,a subtype of PCV2 b,which was closely related to the gene accession number HM038030. 1.
引文
[1]Ge X,Wang F,Guo X,et al.Porcine circovirus type 2 and its associated diseases in China[J].Virus Res,2012,164(1/2):100-106.
[2]Guo L J,Lu Y H,Wei Y W,et al.Porcine circovirus type2(PCV2):Genetic variation and newly emerging genotypes in China[J].Virol J,2010,7(1):273.
[3]郭抗抗,张彦明,张红,等.猪圆环病毒2型分离株优势基因型分析与检测[J].中国预防兽医学报,2010,32(10):808-811.
[4]Kweon C H,Nguyen L T K,Yoo M S,et al.Differential recognition of the ORF2 region in a complete Genome sequence of porcine circovirus type 2(PCV2)isolated from boar bone marrow in Korea[J].Gene,2015,569(2):308-312.
[5]吴瑗,章红兵,王鑫炎,等.2007—2012年浙江省及周边地区猪圆环病毒2型(PCV2)分子流行病学调查[J].农业生物技术学报,2013,21(4):456-463.
[6]Zhang D,He K,Wen L,et al.Genetic and phylogenetic analysis of a new porcine circovirus type 2(PCV2)strain in China[J].Arch Virol,2015,160(12):3149-3151.
[7]Kekarainen T,Mccullough K,Fort M,et al.Immune responses and vaccine-induced immunity against porcine circovirus type 2[J].Veterinary Immunology and Immunopathology,2010,136(3):185-193.
[8]郎洪武,陈晓春,吴华伟,等.猪圆环病毒病及其生物制品学的研究进展[J].中国兽医科学,2012,42(5):545-550.
[9]Takahagi Y,Nishiyama Y,Toki S,et al.Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis[J].The Journal of Veterinary Medical Science,2008,70(6):603-609.
[10]Davies B,Wang X,Dvorak C M,et al.Diagnostic phylogenetics reveals a new porcine circovirus 2 cluster[J].Virus Res,2016,217:32-37.
[11]董林,王艳萍,唐娜,等.猪圆环病毒2型基因型鉴别PCR检测方法的建立[J].中国兽医科学,2013(10):1073-1078.
[12]Yuan W,Li J,Zuo Y,et al.Complete genome sequence of porcine circovirus type 2 strain HB-MC1[J].Genome Announc,2014,2(6):e00987-14.
[13]Shi J,Xu S,Fu F,et al.Phylogenetic analysis of 32 porcine circovirus type 2 isolates from Shandong,China[J].Arch Virol,2015,160(2):465-468.
[14]李鹏,郭军庆,金前跃,等.猪圆环病毒2型荧光定量PCR检测方法的建立[J].华北农学报,2014,29(2):66-70.
[15]Alarcon P,Velasova M,Werling D,et al.Assessment and quantification of post-weaning multi-systemic wasting syndrome severity at farm level[J].Preventive Veterinary Medicine,2011,98(1):19-28.
[2]Guo L J,Lu Y H,Wei Y W,et al.Porcine circovirus type2(PCV2):Genetic variation and newly emerging genotypes in China[J].Virol J,2010,7(1):273.
[3]郭抗抗,张彦明,张红,等.猪圆环病毒2型分离株优势基因型分析与检测[J].中国预防兽医学报,2010,32(10):808-811.
[4]Kweon C H,Nguyen L T K,Yoo M S,et al.Differential recognition of the ORF2 region in a complete Genome sequence of porcine circovirus type 2(PCV2)isolated from boar bone marrow in Korea[J].Gene,2015,569(2):308-312.
[5]吴瑗,章红兵,王鑫炎,等.2007—2012年浙江省及周边地区猪圆环病毒2型(PCV2)分子流行病学调查[J].农业生物技术学报,2013,21(4):456-463.
[6]Zhang D,He K,Wen L,et al.Genetic and phylogenetic analysis of a new porcine circovirus type 2(PCV2)strain in China[J].Arch Virol,2015,160(12):3149-3151.
[7]Kekarainen T,Mccullough K,Fort M,et al.Immune responses and vaccine-induced immunity against porcine circovirus type 2[J].Veterinary Immunology and Immunopathology,2010,136(3):185-193.
[8]郎洪武,陈晓春,吴华伟,等.猪圆环病毒病及其生物制品学的研究进展[J].中国兽医科学,2012,42(5):545-550.
[9]Takahagi Y,Nishiyama Y,Toki S,et al.Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis[J].The Journal of Veterinary Medical Science,2008,70(6):603-609.
[10]Davies B,Wang X,Dvorak C M,et al.Diagnostic phylogenetics reveals a new porcine circovirus 2 cluster[J].Virus Res,2016,217:32-37.
[11]董林,王艳萍,唐娜,等.猪圆环病毒2型基因型鉴别PCR检测方法的建立[J].中国兽医科学,2013(10):1073-1078.
[12]Yuan W,Li J,Zuo Y,et al.Complete genome sequence of porcine circovirus type 2 strain HB-MC1[J].Genome Announc,2014,2(6):e00987-14.
[13]Shi J,Xu S,Fu F,et al.Phylogenetic analysis of 32 porcine circovirus type 2 isolates from Shandong,China[J].Arch Virol,2015,160(2):465-468.
[14]李鹏,郭军庆,金前跃,等.猪圆环病毒2型荧光定量PCR检测方法的建立[J].华北农学报,2014,29(2):66-70.
[15]Alarcon P,Velasova M,Werling D,et al.Assessment and quantification of post-weaning multi-systemic wasting syndrome severity at farm level[J].Preventive Veterinary Medicine,2011,98(1):19-28.