( ) 2014 10 40 5 Journal of Hunan Agricultural University (Natural Sciences), Oct. 2014, 40(5):525 529 DOI:10.13331/j.cnki.jhau.2014.05.013 http://www.hunau.net/qks 育肥猪血清中猪细小病毒 16WS 株的分离与鉴定 ( 410128) * 摘要 214 (PPV) PCR 1 (ST) PPV PPV 15 16 17 18 22 PPV 10% 40% 20% 15.4% 9% 2 14 24 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 S852.65 + 9.2 文献标志码 :A 文章编号 :1007 1032(2014)05 0525 05 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 410128, 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 2012. 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 :2014 07 16 基金项目 (2012NK2001) 作者简介 (1990 ) 18711053351@163.com * xlyu999@126.com
526 ( ) http://www.hunau.net/qks 2014 10 (postweaning mμltisystemic wasting syndrome PMWS) [8] 1966 Mayr [9] PPV Cartwright [10] 1982 [11 12] 214 1 1 1 材料与方法 1.1 材料 1.1.1 病料及细胞 214 2 11 4 10 6 8 9 12 15 10 17 14 25 15 10 16 25 17 10 18 13 20 12 22 11 24 10 (ST) 1.1.2 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 1.2.1 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 63 30 s 72 30 s 30 72 10 min 0.8 % PCR 1.2.2 病毒分离 1 PPV DMEM 1 ml 1 20 1 30 1 40 1 50 1 60 DMEM 1 1 4 24 h ST 50% 0.1 mol/l PBS 2 1 ml ST 37 5% CO 2 1 h 20 μl 2% 37 5% CO 2 3 3 DNA PCR PPV 1.2.3 病毒血凝效价的测定 20 3 3 000 r/min 4min 50 μl [13] 96 V ( 50 μl0.1 mol/l PBS) 50 μl 0.5% 10 s (25 ) 2 h 50 % 1.2.4 PPV 分离株部分基因序列的测定及同源性分析 GenBank DNAStar 1
40 5 16WS 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 55 502 PPV R1 TGCATTATTAACCATCTACTCCAT PPV F2 GTGGAAAAGGCTTACAACAA 692~1 337 bp 55 645 PPV R2 GGTTTTGCCTTTTCAAGTATTA PPV F3 TAGAAATGATGGCTCAAACC 1 220~2 544 bp 55 1 324 PPV R3 GCTGCTGAGAAGTAGAAGTA PPV F4 AAAAGAGCAAGAGGTAAGGG 2 302~3 139 bp 55 837 PPV R4 TGACCAAGGTGTTACCATTT PPV F5 CGCATCAAGACTCATACATC 3 004~4 336 bp 55 1 332 PPV R5 GTCAGCATTGAAATCATCTGTTAG PPV F6 ACAGCACTAAACAATACTGCACCT 4 161~4 538 bp 59 377 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 72 30 s 30 72 10 min 0.8 % PCR PCR GenBank 2 PCR 2 14 24 PPV 15 16 17 18 22 PPV 10% 40% 20% 15.4% 9% 2.2 病毒分离结果 2.2.1 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 AY684866 27a AY684871 BQ EU790641 ZJ EU790642 China AY583318 N EF212027 PPV2010 JN872448 YL JN860197 2 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 2.2.2 分离病毒的 PCR 鉴定 PPV 5 10 1 10 2 10 3 PCR ( 2) PPV 16WS
528 ( ) http://www.hunau.net/qks 2014 10 M 1 2 3 4 98 % [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 ) 2 8 2.4 分离病毒的基因组序列的扩增 5 PCR PCR 0.8% 3 3 M 1 2 3 4 5 6 2 000 bp 1 000 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 4 320 bp DNAStar GenBank 10 2 PPV PPV 214 1 ST PCR 1 PPV 1 1 6 d [15] 214 15 16 17 18 22 10% 40% 20% 15.4% 9% 2~14 24 [16] PPV GenBank 10 PPV PPV PPV 98% 2012 [1] YL 99.1% 27a BQ YL
40 5 16WS 529 参考文献 : [1] YL VP2 [J] 2012 21(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 1997 117(2) 107 118 [4] [J] 2008 30(5) 362 366 [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 1987 15(1/2) 19 29 [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 2002 150(5) 139 143 [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 2011 152(3/4) 284 290 [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 2000 123(4) 302 305 [9] Mayr A Bachmann P A Siegl G et al Characterization of a small porcine DNA virus[j] Archiv frdie gesamte Virusforschung 1968 25(1) 38 51 [10] [M] 2000 [11] [J] 2001 37(1) 22 [12] [J] 1999 21(5) 67 70 [13] HP104 [J] 2013 33(10) 1498 1503 [14] / DNA[J] 2009 37(34) 16771 16772 [15] [M] 2006 [16] PCR [J] 2008(5) 70 72 责任编辑 : 罗维英文编辑 : 罗维