广东地区猪群新发塞内卡病毒流行株的分离鉴定及遗传进化分析
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摘要
【目的】检测广东两猪场新发的鼻镜与蹄冠水疱病病原,获得新发塞内卡病毒(Senecavirus A,SVA)流行株,为后续研究提供材料。【方法】应用SVA RT-PCR、荧光PCR等方法对采集的病料进行初步筛查,再将水泡皮进行处理接种于ST细胞、BHK-21细胞上,用SVA特异性引物对细胞上清液进行扩增,测序并分析序列,采用蔗糖浓度梯度方法纯化病毒进行电子显微镜形态观察,并对病毒分离株的TCID50进行测定。【结果】经SVA RT-PCR与荧光PCR方法初步诊断该猪群为SVA感染;细胞接毒培养发现,自第6代起,细胞出现典型CPE,电镜观察可见25 nm左右的病毒颗粒,扩增序列提交NCBI Blastn比对鉴定为SVA;测得毒株SVA CH-GDBL1-2016的TCID50为106.8,毒株SVA CH-GDBL2-2016的TCID50为106.7。【结论】SVA CH-GDBL1-2016和SVA CH-GDBL2-2016可为后续塞内卡病毒病的诊断和疫苗研制提供材料基础。
【Objective】 The pathogen of blistering disease with nasal and coronet in two swine farms in Guangdong province was detected, and new strains of Senecavirus A(SVA) were obtained, which provided materials for subsequent research. 【Method】 RT-PCR and real-time RT-PCR were used to screen the clinical samples. The clinical samples were isolated, treated and cultured with ST cells and BHK-21 cells. The virus was purified by sucrose density gradient centrifugation method and observed by electron microscopy. The specific primers of SVA were designed to amplify the cell supernatant, and sequenced for sequence analysis. The TCID50 of the virus isolated was determined. 【Results】 The swinery were diagnosed as SVA by RT-PCR and real-time RT-PCR. It was found that the cultured cells had CPE from the 6 th generation. The virion observed by electron microscopy was about 25 nm. The amplified sequences were submitted to the NCBI Blastn, which were identified as SVA. The TCID50 result of the strain SVA CH-GDBL1-2016 was 106.8 and the TCID50 result of the strain SVA CH-GDBL2-2016 was 106.7.【Conclusion】 The strains SVA CH-GDBL1-2016 and SVA CH-GDBL2-2016 provide a material basis for the subsequent diagnosis and vaccine development of Senecavirus disease.
【Objective】 The pathogen of blistering disease with nasal and coronet in two swine farms in Guangdong province was detected, and new strains of Senecavirus A(SVA) were obtained, which provided materials for subsequent research. 【Method】 RT-PCR and real-time RT-PCR were used to screen the clinical samples. The clinical samples were isolated, treated and cultured with ST cells and BHK-21 cells. The virus was purified by sucrose density gradient centrifugation method and observed by electron microscopy. The specific primers of SVA were designed to amplify the cell supernatant, and sequenced for sequence analysis. The TCID50 of the virus isolated was determined. 【Results】 The swinery were diagnosed as SVA by RT-PCR and real-time RT-PCR. It was found that the cultured cells had CPE from the 6 th generation. The virion observed by electron microscopy was about 25 nm. The amplified sequences were submitted to the NCBI Blastn, which were identified as SVA. The TCID50 result of the strain SVA CH-GDBL1-2016 was 106.8 and the TCID50 result of the strain SVA CH-GDBL2-2016 was 106.7.【Conclusion】 The strains SVA CH-GDBL1-2016 and SVA CH-GDBL2-2016 provide a material basis for the subsequent diagnosis and vaccine development of Senecavirus disease.
引文
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[13]贺东生，罗天霞，苏丹萍，徐帅飞，熊景峰.规模化猪场暴发塞内加谷病及SVV HN16株的分离鉴定[J].猪业科学，2017,34(10):72-74.HE D S,LUO T X,SU D P,XU S F,XIONG J F.Separation and identification of Senegalosis and SVV HN16 strain in large-scale pig farms[J].Swine Industry Science,2017,34(10):72-74.
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[15]ZHU Z,YANG F,CHEN P,LIU H,CAO W,ZHANG K,LIU X,ZHENGH.Emergence of novel Seneca Valley virus strains in China,2017[J].Transboundary and Emerging Diseases,2017;64:1024-1029.doi:10.1111/tbed.12662.
[16]ZHAO X,WU Q,BAI Y,CHEN G,ZHOU L,WU Z,LI Y,ZHOU W,YANG H,MA J.Phylogenetic and genome analysis of seven senecavirus A isolates in China[J].Transboundary and Emerging Diseases,2017,64(6):2075-2082.doi:10.1111/tbed.12619.
[17]SUN Y,CHENG J,WU R,WU Z,CHEN J,LUO Y,XIE Q,MA J.Phylogenetic and genome analysis of 17 novel Senecavirus A isolates in Guangdong Province,2017[J].Frontiers in Veterinary Science,2018,5:314.doi:10.3389/fvets.2018.00314.
[18]LUO T,XU S,XIONG J,SU D,HE D.Complete genome sequence of Senecavirus A strain SVV HN16 identified China[J].Genome Announcements,2017,5(44):e01168-17.doi:10.1128/genomeA.01168-17.
[19]ZHANG X,XIAO J,BA L,WANG F,GAO D,ZHANG J,PAN C,QI P.Identification and genomic characterization of the emerging Senecavirus A in southeast China,2017[J].Transboundary and Emerging Diseases,2018,65(2):297-302.doi:10.1111/tbed.12750.
[20]黄元，刘涛，陈锦良，王晓虎，陈晶，梁培新，黄武，向华.阳江地区某猪场塞内卡病毒抗原和抗体的检测与分析[J].广东农业科学，2018,45(12):107-111.doi:10.16768/j.issn.1004-874X.2018.12.018.HUANG Y,LIU T,CHEN J L,WANG X H,CHEN J,LIANG P X,HUANG W,XIANG H.Detection and analysis of antigen and antibody of Seneca virus from a pig farm in Yangjiang[J].Guangdong Agricultural Sciences,2018,45(12):107-111.doi:10.16768/j.issn.1004-874X.2018.12.018.
[21]赵晓亚，伍绮文，伍子娴，陈桂华，白杨，马静云.国内首株猪塞内加谷病毒(Seneca Valley virus)的分离鉴定[J].中国预防兽医学报，2016,38(11):839-843.doi:10.3969/j.issn.1008-0425.2016.11.01.ZHAO X Y,WU Q W,WU Z X,CHEN G H,BAI Y,MA J Y.Isolation and identification of the first Seneca Valley virus strain in China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(11).doi:10.3969/j.issn.1008-0425.2016.11.01.
[22]杨彩娟，温肖会，任裕其，冼望强，刘暖华，谢乐新.塞尼卡病毒AVP1基因遗传进化分析[J].安徽农业科学，2017,45(26):106-108,128.doi:10.13989/j.cnki.0517-6611.2017.26.032.YANG C J,WEN X H,REN Y Q,XIE L Q,LIU N H,XIE L X.Phylogenetic analysis of VP1 gene of Senecavirus A[J].Journal of Anhui Agricultural Science,2017,45(26):106-108,128.doi:10.13989/j.cnki.0517-6611.2017.26.032.
[23]SEGALéS J,BARCELLOS D,ALFIERI A,BURROUGH E,MARTHALER D.Senecavirus A[J].Veterinary Pathology,2016,54(1):11-21.doi:10.1177/0300985816653990.
[24]KNOWLES N J,HALES L M,JONES B H,LANDGRAF J G,HOUSE JA,SKELE K L,BURROUGHS K D,HALLENBECK P L.Epidemiology of Seneca Valley virus:Identification and characterization of isolates from pigs in the United States[C]//EUROPIC 2006:XIVth meeting of the European study group on the molecular biology of picornaviruses,Saariselk?,Inari,Finland.European Study Group on the Molecular Biology of Picornaviruses,p G2.2006.
[25]樊晓旭，赵永刚，迟田英，哈登楚日亚，吴晓东，王志亮.塞尼卡谷病毒TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报，2016,38(12):959-962.doi:10.3969/j.issn.1008-0425.FAN X X,ZHAO Y G,CHI T Y,HADENG C R Y,WU X D,WANG ZL.Establishment of a Taq Man real-time PCR assay for detection of Seneca Valley virus[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(12):959-962.doi:doi:10.3969/j.issn.1008-0425.
[26]WU Q,ZHAO Q,BAI X,SUN Y,B,XIE Q,MA J.The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J].Transboundary and Emerging Diseases,2016,64(2017):1633-1640.doi:10.1111/tbed.12557.
[2]BURKE M.Oncolytic Seneca Valley Virus:past perspectives and future directions[J].Oncolytic Virotherapy,2016,5:81-89.doi:10.2147/OV.S96915.
[3]VANNUCCI F A,LINHARES D,BARCELLOS D E S N,LAM H C,COLLINS J,MARTHALER D.Identification and complete genome of Seneca Valley Virus in vesicular fluid and sera of pigs affected with idiopathic vesicular disease,Brazil[J].Transboundary and Emerging Diseases,2015,62(2015):589-593.doi:10.1111/tbed.12410.
[4]FOWLER V L,RANSBURGH R H,POULSEN E G,WADSWORTHJ,KING D P,MIOULET V,KNOWLES N J,WILLIAMSON S,LIU XM,ANDERSON G A,FANG Y,BAI J.Development of a novel realtime RT-PCR assay to detect Seneca Valley Virus-1 associated with emerging cases of vesicular disease in pigs[J].Journal of Virological Methods,2017,239:34-37.doi:10.1016/j.jviromet.2016.10.012.
[5]BRACHT A J,O HEARN E S,FABIAN A W,BARRETTE R W,SAYED A.Real-Time reverse transcription PCR Assay for detection of Senecavirus A in swine vesicular diagnostic specimens[J].PLOSONE,2016,11(1):e146211.doi:10.1371/journal.pone.0146211.
[6]刘健新，查云峰，鲁荣，任旭皎，黎振标，李慧子，张彭涛，宁章勇.猪塞尼卡病毒荧光定量RT-PCR检测方法的建立及初步应用[J].中国兽医科学，2018,1-9.doi.org/10.16656/j.issn.1673-4696.2019.0041.LIU J X,ZHA Y F,LU R,REN X J,LI Z B,LI H Z,ZHANG P T,NING Z Y.Establishment and primary application of real-time RT-PCR for the detection of porcine Senecavirus A[J].Chinese Veterinary Science,2018:1-9.doi.org/10.16656/j.issn.1673-4696.2019.0041.
[7]HAUSE B M,OLIVIA M,DUFF J,HESSE R A.Senecavirus A in pigs,United States,2015[J].Emerging Infectious Diseases,2016,22(7):1323-1325.doi:10.3201/eid2207.151591.
[8]QIAN S,FAN W,QIAN P,LI X.Isolation and full-genome sequencing of Seneca Valley virus in piglets from China,2016[J].Virology Journal,2016,13(1).doi:10.1186/s12985-016-0631-2.
[9]SUN D,VANNUCCI F,KNUTSON T P,CORZO C,MARTHALER D G.Emergence and whole-genome sequence of Senecavirus A in Colombia[J].Transboundary and Emerging Diseases,2017,64(5):1346-1349.doi:10.1111/tbed.12669.
[10]LEME R A,ZOTTI E,ALC?NTARA B K,OLIVEIRA M V,FREITASL A,ALFIERI A F,ALFIERI A A.Senecavirus A:An emerging vesicular infection in brazilian pig herds[J].Transboundary and Emerging Diseases,2015,62:603-611.doi:10.1111/tbed.12430.
[11]SAENG-CHUTO K,RODTIAN P,TEMEEYASEN G,WEGNER M,NILUBOL D.The first detection of Senecavirus A in pigs in Thailand,2016[J].Transboundary and Emerging Diseases,2018,65(1):285-288.doi:10.1111/tbed.12654.
[12]ARZT J,BERTRAM M R,VU L T,PAUSZEK S J,HARTWIG E J,SMOLIGA G R,PALINSKI R,STENFELDT C,FISH I H,HOANG BH,PHUONG N T,HUNG V V,VU P P,DUNG N K,DONG P V,TIENN N,DUNG D H.First detection and genome sequence of Senecavirus A in vietnam[J].Microbiology resource announcements,2019,8(3).doi:10.1128/MRA.01247-18.
[13]贺东生，罗天霞，苏丹萍，徐帅飞，熊景峰.规模化猪场暴发塞内加谷病及SVV HN16株的分离鉴定[J].猪业科学，2017,34(10):72-74.HE D S,LUO T X,SU D P,XU S F,XIONG J F.Separation and identification of Senegalosis and SVV HN16 strain in large-scale pig farms[J].Swine Industry Science,2017,34(10):72-74.
[14]WU Q,ZHAO X,CHEN Y,HE X,ZHANG G,MA J.Complete genome sequence of Seneca Valley virus CH-01-2015[J].Genome Announcements,2019,4(1):e01509-15.doi:10.1128/genomeA.01509-15.
[15]ZHU Z,YANG F,CHEN P,LIU H,CAO W,ZHANG K,LIU X,ZHENGH.Emergence of novel Seneca Valley virus strains in China,2017[J].Transboundary and Emerging Diseases,2017;64:1024-1029.doi:10.1111/tbed.12662.
[16]ZHAO X,WU Q,BAI Y,CHEN G,ZHOU L,WU Z,LI Y,ZHOU W,YANG H,MA J.Phylogenetic and genome analysis of seven senecavirus A isolates in China[J].Transboundary and Emerging Diseases,2017,64(6):2075-2082.doi:10.1111/tbed.12619.
[17]SUN Y,CHENG J,WU R,WU Z,CHEN J,LUO Y,XIE Q,MA J.Phylogenetic and genome analysis of 17 novel Senecavirus A isolates in Guangdong Province,2017[J].Frontiers in Veterinary Science,2018,5:314.doi:10.3389/fvets.2018.00314.
[18]LUO T,XU S,XIONG J,SU D,HE D.Complete genome sequence of Senecavirus A strain SVV HN16 identified China[J].Genome Announcements,2017,5(44):e01168-17.doi:10.1128/genomeA.01168-17.
[19]ZHANG X,XIAO J,BA L,WANG F,GAO D,ZHANG J,PAN C,QI P.Identification and genomic characterization of the emerging Senecavirus A in southeast China,2017[J].Transboundary and Emerging Diseases,2018,65(2):297-302.doi:10.1111/tbed.12750.
[20]黄元，刘涛，陈锦良，王晓虎，陈晶，梁培新，黄武，向华.阳江地区某猪场塞内卡病毒抗原和抗体的检测与分析[J].广东农业科学，2018,45(12):107-111.doi:10.16768/j.issn.1004-874X.2018.12.018.HUANG Y,LIU T,CHEN J L,WANG X H,CHEN J,LIANG P X,HUANG W,XIANG H.Detection and analysis of antigen and antibody of Seneca virus from a pig farm in Yangjiang[J].Guangdong Agricultural Sciences,2018,45(12):107-111.doi:10.16768/j.issn.1004-874X.2018.12.018.
[21]赵晓亚，伍绮文，伍子娴，陈桂华，白杨，马静云.国内首株猪塞内加谷病毒(Seneca Valley virus)的分离鉴定[J].中国预防兽医学报，2016,38(11):839-843.doi:10.3969/j.issn.1008-0425.2016.11.01.ZHAO X Y,WU Q W,WU Z X,CHEN G H,BAI Y,MA J Y.Isolation and identification of the first Seneca Valley virus strain in China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(11).doi:10.3969/j.issn.1008-0425.2016.11.01.
[22]杨彩娟，温肖会，任裕其，冼望强，刘暖华，谢乐新.塞尼卡病毒AVP1基因遗传进化分析[J].安徽农业科学，2017,45(26):106-108,128.doi:10.13989/j.cnki.0517-6611.2017.26.032.YANG C J,WEN X H,REN Y Q,XIE L Q,LIU N H,XIE L X.Phylogenetic analysis of VP1 gene of Senecavirus A[J].Journal of Anhui Agricultural Science,2017,45(26):106-108,128.doi:10.13989/j.cnki.0517-6611.2017.26.032.
[23]SEGALéS J,BARCELLOS D,ALFIERI A,BURROUGH E,MARTHALER D.Senecavirus A[J].Veterinary Pathology,2016,54(1):11-21.doi:10.1177/0300985816653990.
[24]KNOWLES N J,HALES L M,JONES B H,LANDGRAF J G,HOUSE JA,SKELE K L,BURROUGHS K D,HALLENBECK P L.Epidemiology of Seneca Valley virus:Identification and characterization of isolates from pigs in the United States[C]//EUROPIC 2006:XIVth meeting of the European study group on the molecular biology of picornaviruses,Saariselk?,Inari,Finland.European Study Group on the Molecular Biology of Picornaviruses,p G2.2006.
[25]樊晓旭，赵永刚，迟田英，哈登楚日亚，吴晓东，王志亮.塞尼卡谷病毒TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报，2016,38(12):959-962.doi:10.3969/j.issn.1008-0425.FAN X X,ZHAO Y G,CHI T Y,HADENG C R Y,WU X D,WANG ZL.Establishment of a Taq Man real-time PCR assay for detection of Seneca Valley virus[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(12):959-962.doi:doi:10.3969/j.issn.1008-0425.
[26]WU Q,ZHAO Q,BAI X,SUN Y,B,XIE Q,MA J.The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J].Transboundary and Emerging Diseases,2016,64(2017):1633-1640.doi:10.1111/tbed.12557.
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