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1 ( ) Journal of Hunan Agricultural University (Natural Sciences), Oct. 2014, 40(5): DOI: /j.cnki.jhau 育肥猪血清中猪细小病毒 16WS 株的分离与鉴定 ( ) * 摘要 214 (PPV) PCR 1 (ST) PPV PPV PPV 10% 40% 20% 15.4% 9% PPV PPV ST 5 (CPE) 2 8 PPV GenBank PPV 98% 2012 [1] YL 99.1% PPV PPV 关键词中图分类号 S 文献标志码 :A 文章编号 : (2014) Isolation and identification of porcine parvovirus strain 16WS in serum of fattening pigs WU Hai-chao, HU Cheng-cai, LIU Chong-ling, LI Run-cheng, YU Xing-long * (College of Veterinary Medicine, Hunan Agriculture University, Changsha , China) Abstract: We collected 214 sera samples from a farm which its growing-finishing pigs with poor health conditions and detected porcine parvovirus (PPV) by PCR. A sample contained PPV identified by PCR was inoculated into ST cells to isolate PPV and the isolate was sequenced and we described partial of its biological characteristics. We found that there were no sera contain PPV detected by PCR from pigs aged from 2 to 14 weeks and 24 week, however, PPV can be detected from serum of pigs aged from 15 to 18 and 22 weeks with positive rate 10%, 40%, 20%, 15.4% and 9%, respectively. The isolate was serially passaged in ST cells and the ST showed stable cytopathic effects when the isolate passaged to the fifth generation. Hemagglutination titer of the isolate was 2 8 by the hemagglutination assay. We also found that the partial genome of the isolate share more than 98% identity with PPV genomes found in GenBank, especially as high as 99.1% with the strain YL which was isolated by Shi [1] in Therefore, we demonstrated that the isolate is PPV and nursing pigs in late stage as well as fattening pigs in early stage have a high PPV rate in this farm. Key words: fattening pigs; porcine parvovirus(ppv); serum; isolation; identification [2] [3] [4 5] [6] (porcine circovirus, PCV) (porcine pseudorabies, PRV) (Torque torque sus virus, TTSuV) [7] 收稿日期 : 基金项目 (2012NK2001) 作者简介 (1990 ) @163.com * xlyu999@126.com

2 526 ( ) (postweaning mμltisystemic wasting syndrome PMWS) [8] 1966 Mayr [9] PPV Cartwright [10] 1982 [11 12] 材料与方法 1.1 材料病料及细胞 (ST) 主要试剂 DNA OUT PCR MIX 3.0 DMEM(high glucose) 0.25 % Trypsin EDTA HyClone Gibco (80 ) (100 ) DM2000 Plus DNA marker 1.2 方法病原检测 GenBank DNAStar PPVfp1 5' AGCGAGCCAACAACAC CAACTTT 3' PPVrp1 5' TCT CGGCGATCTTCTTACCTCTG 3' 63 DNA OUT DNA PCR 20 μl PCR MIX 9 μl 10 pmol/l PPVfp1 PPVrp1 0.5 μl DNA 1 μl 20 μl 95 5 min 95 5 min s s min 0.8 % PCR 病毒分离 1 PPV DMEM 1 ml DMEM h ST 50% 0.1 mol/l PBS 2 1 ml ST 37 5% CO 2 1 h 20 μl 2% 37 5% CO DNA PCR PPV 病毒血凝效价的测定 r/min 4min 50 μl [13] 96 V ( 50 μl0.1 mol/l PBS) 50 μl 0.5% 10 s (25 ) 2 h 50 % PPV 分离株部分基因序列的测定及同源性分析 GenBank DNAStar 1

3 WS 527 表 1 猪细小病毒基因组扩增引物 Table 1 Sequencing primers for PPV (5' 3') / /bp PPV F1 ACTCTCAGCTACTGCAGCAT 273~775 bp PPV R1 TGCATTATTAACCATCTACTCCAT PPV F2 GTGGAAAAGGCTTACAACAA 692~1 337 bp PPV R2 GGTTTTGCCTTTTCAAGTATTA PPV F3 TAGAAATGATGGCTCAAACC 1 220~2 544 bp PPV R3 GCTGCTGAGAAGTAGAAGTA PPV F4 AAAAGAGCAAGAGGTAAGGG 2 302~3 139 bp PPV R4 TGACCAAGGTGTTACCATTT PPV F5 CGCATCAAGACTCATACATC 3 004~4 336 bp PPV R5 GTCAGCATTGAAATCATCTGTTAG PPV F6 ACAGCACTAAACAATACTGCACCT 4 161~4 538 bp PPV R6 CTTGGTATAAGTTGTGAATATTCTG [14] 5 DNA 1 PCR 50 μl PCR PCR MIX 23.5 μl 1 μl DNA 1 μl PCR 95 5 min 95 5 min 55 /59 30 s s min 0.8 % PCR PCR GenBank 2 PCR PPV PPV 10% 40% 20% 15.4% 9% 2.2 病毒分离结果细胞病变观察 ST 5 ( 1) 表 2 毒株信息 Table 2 Information of isolates NADL 2 NCOO1718 Kresse U44978 Challenge AY a AY BQ EU ZJ EU China AY N EF PPV2010 JN YL JN 结果 2.1 病原检测结果 214 A B A B 图 1 分离株感染 ST 细胞产生细胞病变 Fig1 Cytopathogenic effect induced by isolate in ST cell 分离病毒的 PCR 鉴定 PPV PCR ( 2) PPV 16WS

4 528 ( ) M % [1] YL 99.1% 3 讨论 500 bp M DM2000 Plus DNA marker 1~3 5 4 图 2 接毒细胞的 PCR 检测结果 Fig 2 Cells inoculated with ppv detected by PCR 2.3 分离病毒对豚鼠红细胞的血凝效价测定 PPV 5 ( PCR ) 分离病毒的基因组序列的扩增 5 PCR PCR 0.8% 3 3 M bp bp 500 bp 250 bp M DM2000 Plus DNA marker 1~6 PPV F1 PPV R1 PPV F2 PPV R2 PPV F3 PPV R3 PPV F4 PPV R4 PPV F5 PPV R5 PPV F6 PPV R6 图 3 测序引物 PCR 结果 Fig 3 Amplification of PPV by sequencing primers 2.5 分离病毒的基因组测序分析 PCR bp DNAStar GenBank 10 2 PPV PPV ST PCR 1 PPV d [15] % 40% 20% 15.4% 9% 2~14 24 [16] PPV GenBank 10 PPV PPV PPV 98% 2012 [1] YL 99.1% 27a BQ YL

5 WS 529 参考文献 : [1] YL VP2 [J] (6) 6 12 [2] [M] 2008 [3] Bolt D M Hani H Muller E et al Non-suppurative myocarditis in piglets associated with porcine parvovirus infection[j] J Comp Pathol (2) [4] [J] (5) [5] Choi C S Molitor T W Joo H S et al Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses[j]. Vet Microbiol (1/2) [6] Drolet R D'Allaire S Larochelle R et al Infectious agents identified in pigs with multifocal interstitial nephritis at slaughter[j] The Veterinary Record (5) [7] Nieto D Aramouni M Grau-Roma L et al Dynamics of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) DNA loads in serum of healthy and postweaning multisystemic wasting syndrome (PMWS) affected pigs[j] Veterinary Microbiology (3/4) [8] Choi C Chae C Distribution of porcine parvovirus in porcine circovirus 2 infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization[j] Journal of Comparative Pathology (4) [9] Mayr A Bachmann P A Siegl G et al Characterization of a small porcine DNA virus[j] Archiv frdie gesamte Virusforschung (1) [10] [M] 2000 [11] [J] (1) 22 [12] [J] (5) [13] HP104 [J] (10) [14] / DNA[J] (34) [15] [M] 2006 [16] PCR [J] 2008(5) 责任编辑 : 罗维英文编辑 : 罗维

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<4D6963726F736F667420576F7264202D20B2CCA3BA4542B2A1B6BE52746120496747BFB9CCE5D3EBB1C7D1CAB0A9B7D6C6DAB5C4B9D8CFB52E646F63> EB 病毒 Rta/IgG 抗体与鼻咽癌分期的关系蔡永林, 郑裕明, 成积儒, 李军, 莫永坤, 钟青燕 ( 广西梧州市红十字会医院, 梧州市肿瘤防治研究所, 广西梧州 543002) 摘要 : 目的探讨 EB 病毒 Rta/IgG 抗体与鼻咽癌临床分期的关系方法用酶