吉林省犬细小病毒流行毒株的分离及VP2基因序列分析
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摘要
为了解吉林省宠物医院就诊犬犬细小病毒流行现状及毒株变异情况,从吉林省长春、吉林、辽源、松原、九台地区采集就诊犬肛门拭子73份,采用胶体金快速检测试剂盒检测后,阳性样品15份。选取具有代表性的6份病料使用F81细胞进行病毒的分离,成功分离到6株病毒(暂命名为JL-CC1、JL-CC2、JL-JL、JL-SY、JL-JT、JL-LY),经通用引物鉴定均为犬细小病毒。VP2基因序列分析结果显示,6株CPV分离株之间的核苷酸同源性为99.3%~100%,与近年来国内分离株核苷酸同源性为98.9%~99.9%,与国外分离株核苷酸同源性为98.3%~99.7%。遗传进化树结果表明,6株分离株分属4个分枝:JL-CC1、JL-LY、JL-SY与山东、辽宁分离株同属1个分枝,JL-CC2与黑龙江分离株同属1个分枝,JL-JT与吉林分离株同属1个分枝,JL-JL与2013年吉林、黑龙江分离株同属1个分枝。氨基酸序列比对显示6株分离株JL-CC1、JL-CC2、JL-JT、JL-LY、JL-SY属于New CPV-2a基因亚型,JL-JL株为属于New CPV-2b基因亚型。此外,6株CPV分离株在267、324、377、440位氨基酸发生了变异。结果表明,吉林省CPV以CPV-2a亚型为主,CPV-2b亚型为辅,并存在基因多变现象,可为吉林省CPV的有效防控提供数据支持。
To investigate current prevalence and viariation of canine parvovirus( CPV) among dogs in pet clinics in Jilin province,73 anal swabs of dogs in treatment were collected from Changchun,Jilin,Liaoyuan,Songyuan and Jiutai area,Jilin province. After detection by using colloid gold fast examination reagent kit,6 representative samples from 15 positive samples were added to F81 cell culture,and all 6 isolates( tentatively named JL-CC1,JL-CC2,JL-JL,JL-SY,JL-JT,JL-LY) were successfully isolated and identified as CPV by universal primers. VP2 gene sequence analysis showed that the nucleotide sequence homology among 6 CPV isolates was99. 3% ~ 100%,98. 9% ~ 99. 9% compared to current domestic isolates and 98. 3% ~ 99. 7% compared to abroad isolates. Phylogenetic analysis indicated that the 6 isolates belonged to 4 branches: JL-CC1,JL-LY and JL-SY isolates in same branch with Shangdong and Liaoning isolates; JL-CC2 with Heilongjiang isolate;JL-JT with Jilin isolate; JL-JL with Jilin and Heilongjiang isolates( 2013). Amino acid sequence alignment showed that the 5 CPV isolates JL-CC1、JL-CC2、JL-JT、JL-LY、JL-SY belonged to New CPV-2a gene subtype,while JL-JL belonged to New CPV-2b gene subtype. Besides,there were mutations appeared at the267 th,324th,377 th and 440 th amino acid. The above results indicate that CPV,accompanying with gene variation,is mainly CPV-2a gene subtype,and CPV-2b gene subtype is a supplement in Jilin province,which will provide data support for effective prevention and control of CPV in Jilin province.
To investigate current prevalence and viariation of canine parvovirus( CPV) among dogs in pet clinics in Jilin province,73 anal swabs of dogs in treatment were collected from Changchun,Jilin,Liaoyuan,Songyuan and Jiutai area,Jilin province. After detection by using colloid gold fast examination reagent kit,6 representative samples from 15 positive samples were added to F81 cell culture,and all 6 isolates( tentatively named JL-CC1,JL-CC2,JL-JL,JL-SY,JL-JT,JL-LY) were successfully isolated and identified as CPV by universal primers. VP2 gene sequence analysis showed that the nucleotide sequence homology among 6 CPV isolates was99. 3% ~ 100%,98. 9% ~ 99. 9% compared to current domestic isolates and 98. 3% ~ 99. 7% compared to abroad isolates. Phylogenetic analysis indicated that the 6 isolates belonged to 4 branches: JL-CC1,JL-LY and JL-SY isolates in same branch with Shangdong and Liaoning isolates; JL-CC2 with Heilongjiang isolate;JL-JT with Jilin isolate; JL-JL with Jilin and Heilongjiang isolates( 2013). Amino acid sequence alignment showed that the 5 CPV isolates JL-CC1、JL-CC2、JL-JT、JL-LY、JL-SY belonged to New CPV-2a gene subtype,while JL-JL belonged to New CPV-2b gene subtype. Besides,there were mutations appeared at the267 th,324th,377 th and 440 th amino acid. The above results indicate that CPV,accompanying with gene variation,is mainly CPV-2a gene subtype,and CPV-2b gene subtype is a supplement in Jilin province,which will provide data support for effective prevention and control of CPV in Jilin province.
引文
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[14]Senda M,Parrish C R,Harasawa R,et al.Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines[J].Journal of Clinical Microbiology,1995,33(1):110-113.
[15]丁轲,余祖华,彭春平,等.2011-2013年河南省犬细小病毒分子流行病学调查与分析[J].畜牧兽医学报,2014,45(10):1671-1678.
[2]Goddard A,Leise A L.Canine parvovirus[J].Veterinary Clinics of North America Small Animal Practice,2010,40(6):1041-1053.
[3]梁士哲,魏喜仁,范文光,等.狗传染性肠炎的研究[J].实验动物与比较医学,1982,4:17-20.
[4]孙梅,吴建华,白文军.犬细小病毒研究进展[J].北京农业,2013,36:29-31.
[5]Parrish C R,Have P,Foreyt W J,et al.The global spread and replacement of canine parvovirus strains[J].J General Virology,1988,69(5):1111-1116.
[6]Desario C,Decaro N M,Cavalli A,et al.Canine parvovirus infection:which diagnostic test for virus?[J].Journal of Virological Methods,2005,126(1/2):179-185.
[7]Decaro N,Desario C,Parisi A,et al.Genetic analysis of canine parvovirus type 2c[J].Virology,2009,385(1):5-10.
[8]张仁舟,杨松涛,冯昊,等.中国国内首次检测到犬细小病毒CPV-2c[J].中国病原生物学杂志,2010,(4):246-249.
[9]李廷军.犬细小病毒病的病理学观察[J].动物医学进展,2009,30(8):125-128.
[10]Nicola D,Costantina D,Diane D A,et al.Molecular Epidemiology of Canine Parvovirus,Europe[J].Emerging Infectious Diseases,2007,13(8):1222-1224.
[11]Calderon M G,Mattion N,Bucafusco D,et al.Molecular characterization of canine parvovirus strains in Argentina:detection of the pathogenic variant CPV2c in vaccinated dogs[J].Journal of Virological Methods,2009,159(2):141-145.
[12]Prittie J.Canine parvoviral enteritis:a review of diagnosis,management,and prevention[J].Journal of Veterinary Emergency&Critical Care,2004,14(3):167-176.
[13]Chinchkar S R,Subramanian B M,Rao N H,et al.Analysis of VP2 gene sequences of canine parvovirus isolates in India[J].Archives of Virology,2006,151(9):1881-1887.
[14]Senda M,Parrish C R,Harasawa R,et al.Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines[J].Journal of Clinical Microbiology,1995,33(1):110-113.
[15]丁轲,余祖华,彭春平,等.2011-2013年河南省犬细小病毒分子流行病学调查与分析[J].畜牧兽医学报,2014,45(10):1671-1678.