Isolation, identification and sequence analysis of complete genome of feline panleukopenia virus in Guangzhou area
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摘要:目的
分析广州地区猫泛白细胞减少症病毒(Feline panleukopenia virus, FPLV)的自然重组、跨宿主传播以及流行变异情况。
方法采集疑似感染猫泛白细胞减少症病毒的猫粪便样品进行细胞分离,并对阳性病料提取基因组DNA,PCR扩增获得病毒的全基因组序列,并与GenBank中相关的参考毒株序列进行遗传进化分析,同时对VP2基因与NS1基因的主要氨基酸位点进行差异分析。
结果成功获得2株猫泛白细胞减少症病毒及其全基因组序列。遗传进化分析显示,广州地区分离的猫泛白细胞减少症病毒FPLV-GZ01、FPLV-GZ02与我国其他分离毒株FPLV-XJ01、FPLV-HRB属同一分支,提示FPLV-GZ01、FPLV-GZ02由FPLV-XJ1进化而来;NS1基因系统进化树显示,FPLV存在与CPV-447重组的可能。主要氨基酸位点分析显示,FPLV在VP2基因中的主要氨基酸位点上的遗传变异比CPV保守;在NS1基因上发现FPLV-GZ01、FPLV-GZ02分别存在不同程度的氨基酸位点突变。
结论广州地区分离的猫泛白细胞减少症病毒仍在不断地重组进化。
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关键词:
- 猫泛白细胞减少症病毒 /
- 细小病毒 /
- 分离鉴定 /
- 全基因组 /
- 遗传分析
Abstract:ObjectiveTo study the natural recombination, cross host transmission and prevalence of feline panleukopenia virus(FPLV)in Guangzhou area.
MethodCells were isolated from stool samples of cats seemingly infected with FPLV, genome DNAs were extracted from positive samples, and complete virus genome sequences were amplified by PCR and were compared with relevant reference sequences from GenBank using genetic evolution analysis. The major amino acid sites were analyzed for differences between VP2 gene and NS1 gene.
ResultTwo FPLV strains were successfully isolated and the complete genome sequences of FPLV were obtained. The genetic evolution analysis indicated that FPLV-GZ01 and FPLV-GZ02 which were isolated from Guangzhou area belonged to the same branch with the other isolated strains FPLV-XJ1 and FPLV-HRB, and implied FPLV-GZ01 and FPLV-GZ02 evolved from FPLV-XJ01. The NS1 gene phylogenetic analysis demonstrated that FPLV might have recombined with CPV-447. Analysis of major amino acid sites indicated that FPLV was more conservative than CPV in the genetic variation of the VP2 gene. FPLV-GZ01 and FPLV-GZ02 had different degrees of mutation in the amino acid sites of the NS1 gene.
ConclusionThe feline panleukopenia viruses isolated from Guangzhou area have been still recombining and evolving.
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表 1 猫泛白细胞减少症病毒全基因分段引物
Table 1 Segmented primers for the complete genome of FPLV
引物名称 引物序列(5′→3′) FPLV-1 F:GAATGATAGGCGGTTTGTGTG R:TCTTCTGCAATTTCTCTGAGC FPLV-2 F:GCAGAAGATAGTGAATGGGTG R:TTGGTTGTGTATGTTCAGGTC FPLV-3 F:TTATGACAACTAATGAAAATA R:TTTTAGTTGGTTTTGTTGGTC FPLV-4 F:TGATACACCAGATCATCCATC R:TATTTGTTTGCCATGTATGTG FPLV-5 F:GAAAATTCTGTGCCAGTACAC R:TACCTTTCCACCAAAAATCTG FPLV-6 F:AAGACTTCATGTAAATGCACC R:AGATTGATACTTATGGTAAGG 表 2 26株细小病毒参考序列
Table 2 Reference sequences of 26 parvovirus strains
毒株 登录号 基因型 年份 来源国 CPV-N M19296 CPV-2 1995 USA CPV-M38 M38245 CPV-2 1996 USA CPV-F261 FJ005261 CPV-2b 2009 Germany CPV-339 AY742933 new CPV-2a 2005 New Zealand CPV-447 AY742934 new CPV-2b 2005 USA CPV-U6 AY742935 new CPV-2a 2005 Germany CPV-395 AY742936 new CPV-2b 2005 USA CPV-EF EF011664 new CPV-2a 2006 China CPV-NJ01 EU310373 new CPV-2a 2008 China CPV-410 EU659119 new CPV-2b 2008 USA CPV-13 EU659118 new CPV-2a 2008 USA CPV-579 EU659116 CPV-2 2008 USA CPV-Y1A D26079 CPV-2a 2008 Japan CPV-LZ2 JQ268284 new CPV-2b 2013 China CPV-UY349 KM457129 CPV-2c 2014 Uruguay CPV-UY135 KM457112 CPV-2c 2014 Uruguay FPLV-M382 M38246 FPLV 1996 USA FPLV-X55 X55115 FPLV 2005 USA FPLV-XJ1 EF988660 FPLV 2007 China FPLV-8b EU659114 FPLV 2008 USA FPLV-367 EU659111 FPLV 2008 USA FPLV-889 EU659113 FPLV 2008 USA FPLV-KA EU659115 FPLV 2008 USA FPLV-464 EU659112 FPLV 2008 USA FPLV-HRB KP280068 FPLV 2015 China FPLV-MG132 KP769859 FPLV 2015 Belgium 表 3 VP2基因中主要的氨基酸位点分析
Table 3 Analysis of major amino acid sites of the VP2 gene
毒株 氨基酸位点 核酸类型 80 87 93 103 300 305 323 426 564 568 FPLV-GZ01 K M K V A D D N N A FPLV FPLV-GZ02 K M K V A D D N N A FPLV FPLV-M382 K M K V A D D N N A FPLV CPV-N R M N A A D N N S G CPV-2 CPV-Y1A R L N A G Y N N S G CPV-2a CPV-NJ01 R L N A G Y N N S G new CPV-2a CPV-F261 R L N A G Y N D S G CPV-2b CPV-410 R L N A G Y N D S G new CPV-2b CPV-UY349 R L N A G Y N E S G CPV-2c 表 4 NS1基因中的部分氨基酸位点分析
Table 4 Analysis of partial amino acid sites of the NS1 gene
毒株 氨基酸位点 核酸类型 248 546 584 FPLV-GZ01 T P A FPLV FPLV-GZ02 T P T FPLV FPLV-M382 T S T FPLV CPV-N I S T CPV-2 CPV-Y1A I S T CPV-2a CPV-NJ01 I S T new CPV-2a CPV-410 I S T new CPV-2b CPV-UY349 I S T CPV-2c -
[1] REED A P, JONES E V, MILLER T J. Nucleotide sequence and genome organization of canine parvovirus[J]. J Virol, 1988, 62(1): 266-276.
[2] AGBANDJE M, MCKENNA R, ROSSMANN M G, et al. Structure determination of feline panleukopenia virus empty particles[J]. Proteins, 1993, 16(2): 155-171. doi: 10.1002/(ISSN)1097-0134
[3] PARRISH C R. Host range relationships and the evolution of canine parvovirus[J]. Vet Microbiol, 1999, 69(1/2): 29-40.
[4] 刘景华.动物病毒学[M].北京:科学出版社, 1985. [5] IKEDA Y, MOCHIZUKI M, NAITO R, et al. Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats[J]. Virology, 2000, 278(1): 13-19. doi: 10.1006/viro.2000.0653
[6] MARTELLA V, DECARO N, ELIA G, et al. Surveillance activity for canine parvovirus in Italy[J]. J Vet Med:Series B, 2005, 52(7/8): 312-315.
[7] DECARO N, ELIA G, DESARIO C, et al. A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus[J]. J Virol Methods, 2006, 136(1/2): 65-70. http://www.academia.edu/14880248/A_minor_groove_binder_probe_real-time_PCR_assay_for_discrimination_between_type_2-based_vaccines_and_field_strains_of_canine_parvovirus
[8] HOELZER K, SHACKELTON L A, PARRISH C R, et al. Phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses[J]. J Gen Virol, 2008, 89(9): 2280-2289. doi: 10.1099/vir.0.2008/002055-0
[9] STEINEL A, MUNSON L, VAN VUUREN M, et al. Genetic characterization of feline parvovirus sequences from various carnivores[J]. J Gen Virol, 2000, 81(2): 345-350. doi: 10.1099/0022-1317-81-2-345
[10] 夏咸柱, 高玉伟, 王化磊, 等.略谈人兽共患病跨种传播与监测及免疫研究中的几个问题[J].中国病毒病杂志, 2012, 2(1): 4-10. http://www.cnki.com.cn/Article/CJFDTOTAL-ZRYX201201003.htm [11] 亢文华, 赵凤龙, 郝霖雨, 等.猫泛白细胞减少症病毒的分离与鉴定[J].中国畜牧兽医, 2008, 35(10): 89-92. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGDX200708052.htm [12] OHSHIMA T, MOCHIZUKI M. Evidence for recombination between feline panleukopenia virus and canine parvovirus type 2[J]. J Vet Med Sci, 2009, 71(4): 403-408. doi: 10.1292/jvms.71.403
[13] LLAMAS-SAIZ A L, AGBANDJE-MCKENNA M, PARKER J S, et al. Structural analysis of a mutation in canine parvovirus which controls antigenicity and host range[J]. Virology, 1996, 225(1): 65-71. doi: 10.1006/viro.1996.0575
[14] TRUYEN U, AGBANDJE M, PARRISH C R. Characterization of the feline host range and a specific epitope of feline panleukopenia virus[J]. Virology, 1994, 200(2): 494-503. doi: 10.1006/viro.1994.1212
[15] KANG B K, SONG D S, LEE C S, et al. Prevalence and genetic characterization of canine parvoviruses in Korea[J]. Virus Genes, 2008, 36(1): 127-133. doi: 10.1007/s11262-007-0189-6