中国几省市猪萨佩罗病毒巢式RT-PCR检测与遗传进化分析
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摘要
为了准确了解猪萨佩罗病毒(Porcine Sapelovirus,PSV)在中国流行情况及遗传进化规律,试验针对PSV 5'-NTR基因利用巢式RT-PCR方法对2015～2017年来自中国部分省市规模化猪场采集的368份样品进行检测,将阳性样品对VP1基因进行克隆、测序、构建进化树。巢式RT～PCR检测结果表明,368份样品中24份样品为阳性,总阳性率为6.52%;8份样品测序成功,序列分析结果表明核苷酸同源性为77.0%～99.7%,推导的氨基酸序列同源性为83.6%～100.0%,遗传进化分析结果显示,中国地区的PSV病毒呈现出遗传多样性,其中3个毒株与德国参考毒株亲缘关系较近,2个毒株与国内外参考毒株的亲缘关系均较远,变异较大。研究丰富了PSV分子流行病学资料,为进一步研究该病毒提供了参考。
In order to clarify the prevalence and genetic evolution of Porcine Saperovirus(PSV)n China,the nested RT-PCR method was used to test PSV 5'-NTR gene of 368 samples which were obtained from large-scale pig farm of several provinces and cities in China from 2015 to 2017.The VP1 of the positive samples were cloned,sequenced and constructed into a phylogenetic tree.Nested RT-PCR results showed that 24 samples were positive,and the total positive rate was 6.52%;8 samples were successfully sequenced,and the sequence analysis showed that the nucleotide homology was 77.0%-99.7%.The amino acid sequence homology was 83.6%-100.0%.The results of genetic evolution analysis showed that the PSV virus in China was genetic diversity.Three of the strains were closely related to the German reference strain,and other two strains had great difference and distant relative relationship from the reference strains in China and abroad.It enriched the epidemiological data of PSV molecules and provided a reference for further study of the virus.
In order to clarify the prevalence and genetic evolution of Porcine Saperovirus(PSV)n China,the nested RT-PCR method was used to test PSV 5'-NTR gene of 368 samples which were obtained from large-scale pig farm of several provinces and cities in China from 2015 to 2017.The VP1 of the positive samples were cloned,sequenced and constructed into a phylogenetic tree.Nested RT-PCR results showed that 24 samples were positive,and the total positive rate was 6.52%;8 samples were successfully sequenced,and the sequence analysis showed that the nucleotide homology was 77.0%-99.7%.The amino acid sequence homology was 83.6%-100.0%.The results of genetic evolution analysis showed that the PSV virus in China was genetic diversity.Three of the strains were closely related to the German reference strain,and other two strains had great difference and distant relative relationship from the reference strains in China and abroad.It enriched the epidemiological data of PSV molecules and provided a reference for further study of the virus.
引文
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[15]刘杉杉,赵亚荣,吕超超,等.检测猪捷申病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的多重RT-PCRPCR方法的建立及应用[C]//中关村全球农业生物技术创新论坛.2011.
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[17] Zell R,Krumbholz A,Henke A,et al.Detection of porcine enteroviruses by nRT-PCR:differentiation of CPE groups I-III with specific primer sets[J].Journal of Virological Methods,2000,88(2):205-218.
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[19] Lan D,Ji W,Yang S,et al.Isolation and characterization of the first Chinese porcine sapelovirus strain[J].Archives of Virology,2011,156(9):1567.
[20] Schock A,Gurrala R,Fuller H,et al.Investigation into an outbreak of encephalomyelitis caused by a neuroinvasive porcine sapelovirus in the United Kingdom[J].Veterinary Microbiology,2014,172(3-4):381-389.
[21]陈俊伟,周庆丰,宋延华,等.2012-2013年华南部分地区猪群中猪萨佩罗病毒分子流行病学调查[C]//中国畜牧兽医学会2014年学术年会论文集.2014.
[22]张露,侯喜林,余丽芸.猪轮状病毒VP7蛋白在E.coli中的表达纯化[J].黑龙江八一农垦大学学报,2018,30(1):12-16.
[23] Sozzi E,Barbieri I,Lavazza A,et al.molecular characterization and phylogenetic analysis of vp1 of porcine enteric picornaviruses isolates in Italy[J].Transboundary&Emerging Diseases,2010,57(6)434-442.
[24]李志强,何德,李翠新.基于兼并引物的PCR扩增特定基因的效果比较[J].湖北民族学院学报:自然科学版,2015(2):170-173.
[25] Tseng C H,Tsai H J.Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus[J].Virus Research,2007,129(2):104-114.
[26]赵婷婷,李晶娇,沈小娟,等.猪萨佩罗病毒研究进展[J].上海交通大学学报:农业科学版,2017(6):62-66.
[2] Belsham G J.Translation initiation on picornavirus RNA[J].Cold Spring Harbor Monograph Archive,2009,31(3):61-63.
[3]张忠信.ICTV第九次报告对病毒分类系统的一些修改[J].病毒学报,2012,28(5):595-599.
[4] Knowles N J,Hovi T,Hyypia咬T,et al.Virus Taxonomy,Ninth Report of the International Committee on Taxonomy of Viruses:Picornaviridae[J].Archives of Virology,1995,140(2):391-392.
[5] Donin D G,De A L R,Alfieri A F,et al.First report of Porcine teschovirus(PTV),Porcine sapelovirus(PSV)and Enterovirus G(EV-G)in Porcine herds of Brazil[J].Tropical Animal Health&Production,2014,46(3):523-528.
[6]兰道亮,吉文汇,王长松,等.华东部分地区猪群中猪萨佩罗病毒分子流行病学调查[J].动物医学进展,2012,33(12):116-121.
[7]范忠良,林宝山,黄偲,等.猪萨佩罗病毒在四川地区猪群中的感染调查[J].四川畜牧兽医,2015(6):29-30.
[8] Buitrago D,Canogómez C,Agüero M,et al.A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.[J].Journal of Veterinary Diagnostic Investigation,2010,22(5):763.
[9] Oberste M S,Maher K,Pallansch M A.Genomic evidence that simian virus 2 and six other simian picornaviruses represent a new genus in Picornaviridae[J].Virology,2003,314(1):283-293.
[10]伍生军,倪婷婷,张守发,等.猪附红细胞体SYBR GreenⅠ荧光定量PCR检测方法的建立[J].延边大学农学学报,2017(3):74-78.
[11] Smith T J,Baker T.Picornaviruses:epitopes,canyons,and pockets[J].Advances in Virus Research,1999,52(1):1-23.
[12] Schock A,Gurrala R,Fuller H,et al. Investigation into an outbreak of encephalomyelitis caused by a neuroinvasive porcine sapelovirus in the United Kingdom[J].Veterinary Microbiology,2014,172(3-4):381-389.
[13]王雪,李海全,徐志文.四川省部分地区公猪精液中蓝耳病分子流行病学调查[J].养猪,2013(5):125-127.
[14]崔迎亚,孟伟杰,唐柯,等.豫北七猪场大肠埃希氏菌的分离及鉴定[J].河南科技学院学报:自然科学版,2016(2):45-48.
[15]刘杉杉,赵亚荣,吕超超,等.检测猪捷申病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的多重RT-PCRPCR方法的建立及应用[C]//中关村全球农业生物技术创新论坛.2011.
[16] Yi J,Liu C.Molecular characterization of porcine circovirus2 isolated from diseased Porcines co-infected with porcine reproductive and respiratory syndrome virus[J].Virology Journal,2010,7(1):1-4.
[17] Zell R,Krumbholz A,Henke A,et al.Detection of porcine enteroviruses by nRT-PCR:differentiation of CPE groups I-III with specific primer sets[J].Journal of Virological Methods,2000,88(2):205-218.
[18] Sozzi E,Barbieri I,Lavazza A,et al.Molecular characterization and phylogenetic analysis of VP1 of porcine enteric picornaviruses isolates in Italy[J].Transboundary&Emerging Diseases,2010,57(6):434-42.
[19] Lan D,Ji W,Yang S,et al.Isolation and characterization of the first Chinese porcine sapelovirus strain[J].Archives of Virology,2011,156(9):1567.
[20] Schock A,Gurrala R,Fuller H,et al.Investigation into an outbreak of encephalomyelitis caused by a neuroinvasive porcine sapelovirus in the United Kingdom[J].Veterinary Microbiology,2014,172(3-4):381-389.
[21]陈俊伟,周庆丰,宋延华,等.2012-2013年华南部分地区猪群中猪萨佩罗病毒分子流行病学调查[C]//中国畜牧兽医学会2014年学术年会论文集.2014.
[22]张露,侯喜林,余丽芸.猪轮状病毒VP7蛋白在E.coli中的表达纯化[J].黑龙江八一农垦大学学报,2018,30(1):12-16.
[23] Sozzi E,Barbieri I,Lavazza A,et al.molecular characterization and phylogenetic analysis of vp1 of porcine enteric picornaviruses isolates in Italy[J].Transboundary&Emerging Diseases,2010,57(6)434-442.
[24]李志强,何德,李翠新.基于兼并引物的PCR扩增特定基因的效果比较[J].湖北民族学院学报:自然科学版,2015(2):170-173.
[25] Tseng C H,Tsai H J.Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus[J].Virus Research,2007,129(2):104-114.
[26]赵婷婷,李晶娇,沈小娟,等.猪萨佩罗病毒研究进展[J].上海交通大学学报:农业科学版,2017(6):62-66.