健康猪体内塞尼卡病毒的分离和鉴定
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摘要
塞尼卡病毒(SVA)属于小核糖RNA病毒科塞尼卡病毒属,可以引起猪的水泡样病变、跛行和新生仔猪突然死亡等。为了解SVA的特性,本研究将从健康猪体内鉴定的SVA阳性样品接种BHK-21细胞分离SVA,采用PCR扩增其VP1基因,并分析其遗传变异特点。结果显示,10份SVA阳性样品中有2份(GXT91和GXT94)能够在BHK-21细胞增殖并产生明显的细胞病变,可达10~(7.6) TCID_(50)/mL,其VP1基因与SVA参考株NC_011349的核酸同源性为90.2%～90.6%,推导氨基酸同源性为96.7%～97.1%,进化树显示新分离的GXT91和GXT94病毒株属于流行分支Ⅱ。本研究为SVA的疫苗研制奠定了基础。
Senecavirus A(SVA) is a single strand positive RNA virus belonging to the Senecavirus genus of family Picornaviridae, which causes porcine idiopathic vesicular lesions on the snout and coronary bands, lameness and acute death of newborn piglets. SVA has been reported in several provinces in China. In order to understand the characteristics of SVA, this paper tried to isolate SVA by inoculating on BHK-21 cell with SVA positive samples by real-time RT-PCR which collected from healthy pigs in slaughterhouse. Then the VP1 gene of SVA was amplified by PCR and analyzed for its genetic variation. The results showed that 2(GXT91 and GXT94) out of 10 positive samples obviously induced cytopathic effect on BHK-21 cells. The TCID_(50) titer of isolate GXT94 reached 10~(7.6)/mL. Further analysis showed that the nucleic acid homology of VP1 gene was 90.2%to 90.6% between SVA isolates and reference strain NC_011349 and the amino acid homology was 96.7% to 97.1%. The isolates of GXT91 and GXT94 were categoried into the second branch in the evolutionary tree of all SVAs. This study laid the foundation for the potential development of SVA vaccine.
Senecavirus A(SVA) is a single strand positive RNA virus belonging to the Senecavirus genus of family Picornaviridae, which causes porcine idiopathic vesicular lesions on the snout and coronary bands, lameness and acute death of newborn piglets. SVA has been reported in several provinces in China. In order to understand the characteristics of SVA, this paper tried to isolate SVA by inoculating on BHK-21 cell with SVA positive samples by real-time RT-PCR which collected from healthy pigs in slaughterhouse. Then the VP1 gene of SVA was amplified by PCR and analyzed for its genetic variation. The results showed that 2(GXT91 and GXT94) out of 10 positive samples obviously induced cytopathic effect on BHK-21 cells. The TCID_(50) titer of isolate GXT94 reached 10~(7.6)/mL. Further analysis showed that the nucleic acid homology of VP1 gene was 90.2%to 90.6% between SVA isolates and reference strain NC_011349 and the amino acid homology was 96.7% to 97.1%. The isolates of GXT91 and GXT94 were categoried into the second branch in the evolutionary tree of all SVAs. This study laid the foundation for the potential development of SVA vaccine.
引文
[1]Hales L M,Knowles N J,Reddy P S,et al.Com plete genome sequence analysis of Seneca Valley virus-001,a novel oncolytic picomavirus[J].J Gen Virol,2008,89(5):1265-1275.
[2]Segalés J,Barcellos D,Alfieri A,et al.Senecavirus A:An emerging pathogen causing vesicular disease and mortality in pigs[J].Vet Pathol,2017,54(1):11-21.
[3]Canning P,Canon A,Bates J L,et al.Neonatal mortality,vesicular lesions and lameness associated with senecavirus A in a USsow farm[J].Transbound Emerg Dis,2016,63(4):373-378.
[4]Montiel N,Buckley A,Guo Bao-qing,et al.Vesicular disease in9-week-old pigs experimentally infected with Senecavirus A[J].Emerg Infect Dis,2016,22(7):1246-1248.
[5]Leme R A,Zotti E,Alcntara B K,et al.Senecavirus A:an emerging vesicular infection in Brazilian pig herds[J].Transbound Emerg Dis,2015,62(6):603-611.
[6]Wu Qi-wen,Zhao Xiao-ya,Chen Yan-shan,et al.Complete genome sequence of Seneca Valley virus CH-01-2015 identified in China[J].Genome Announc,2016,4:e01509-15.
[7]Saeng-chuto K,Rodtian P,Temeeyasen G,et al.The first detection of senecavirus A in pigs in Thailand,2016[J].Transbound Emerg Dis,2018,65(1):285-288.
[8]Sun D,Vannucci F,Knutson T P,Corzo C,et al.Emergence and whole-genome sequence of senecavirus A in Colombia[J].Transbound Emerg Dis,2017,64(5):1346-1349.
[9]Qian Su-hong,Fan Wen-chun,Qian Ping,et al.Isolation and full-genome sequencing of Seneca Valley virus in piglets from China,2016[J].Virol J,2016,13(1):173.
[10]Joshi L R,Fernandes M H,Clement T,et al.Pathogenesis of Senecavirus A infection in finishing pigs[J].J Gen Virol,2016,97(12):3267-3279.
[11]张志,王赛赛,李岚,等.猪全血中多种病原流行病学监测的初步研究[J].中国动物检疫,2012,29(2):46-48.
[12]Leme R A,Alfieri A F,Alfieri A A.Update on Senecavirus infection in pigs[J].Viruses,2017,9(7):E170.
[2]Segalés J,Barcellos D,Alfieri A,et al.Senecavirus A:An emerging pathogen causing vesicular disease and mortality in pigs[J].Vet Pathol,2017,54(1):11-21.
[3]Canning P,Canon A,Bates J L,et al.Neonatal mortality,vesicular lesions and lameness associated with senecavirus A in a USsow farm[J].Transbound Emerg Dis,2016,63(4):373-378.
[4]Montiel N,Buckley A,Guo Bao-qing,et al.Vesicular disease in9-week-old pigs experimentally infected with Senecavirus A[J].Emerg Infect Dis,2016,22(7):1246-1248.
[5]Leme R A,Zotti E,Alcntara B K,et al.Senecavirus A:an emerging vesicular infection in Brazilian pig herds[J].Transbound Emerg Dis,2015,62(6):603-611.
[6]Wu Qi-wen,Zhao Xiao-ya,Chen Yan-shan,et al.Complete genome sequence of Seneca Valley virus CH-01-2015 identified in China[J].Genome Announc,2016,4:e01509-15.
[7]Saeng-chuto K,Rodtian P,Temeeyasen G,et al.The first detection of senecavirus A in pigs in Thailand,2016[J].Transbound Emerg Dis,2018,65(1):285-288.
[8]Sun D,Vannucci F,Knutson T P,Corzo C,et al.Emergence and whole-genome sequence of senecavirus A in Colombia[J].Transbound Emerg Dis,2017,64(5):1346-1349.
[9]Qian Su-hong,Fan Wen-chun,Qian Ping,et al.Isolation and full-genome sequencing of Seneca Valley virus in piglets from China,2016[J].Virol J,2016,13(1):173.
[10]Joshi L R,Fernandes M H,Clement T,et al.Pathogenesis of Senecavirus A infection in finishing pigs[J].J Gen Virol,2016,97(12):3267-3279.
[11]张志,王赛赛,李岚,等.猪全血中多种病原流行病学监测的初步研究[J].中国动物检疫,2012,29(2):46-48.
[12]Leme R A,Alfieri A F,Alfieri A A.Update on Senecavirus infection in pigs[J].Viruses,2017,9(7):E170.