猪繁殖与呼吸综合征病毒SDZP株与其Marc-145细胞传代株的基因变异分析
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摘要
为了研究PRRSV病毒在Marc-145细胞中长期传代后的基因变异特点,将高致病性PRRSV野毒株SDZP株感染Marc-145细胞,经连续传代120代获得了较为稳定的SDZP-p120传代毒株。通过RT-PCR分段扩增出SDZP亲本株与SDZP-p120株的全长基因,测序分析病毒传代过程中各基因的特征性变化。在传代过程中,病毒增殖速度出现了从慢到快的过程。连续传代后,SDZP-p120株出现了76个碱基的突变,涵盖了绝大部分功能基因,其中出现42个非同义突变,25个位于非结构蛋白,17个位于结构蛋白。囊膜相关结构蛋白的氨基酸突变率明显高于非结构蛋白,且多个囊膜相关结构蛋白的氨基酸突变规律与HuN4、JXA1等其他在Marc-145细胞上传代致弱的毒株具有相似性。研究结果为深入了解病毒与宿主细胞间的相互作用提供了参考数据。
In order to study the gene variation of PRRSV after long-term passages in Marc-145 cells,the highly pathogenic PRRSV isolate SDZP was passaged in Marc-145 cells and a stable strain of SDZP-p120 was obtained after 120 passages.The full-length genome of SDZP parental strain and SDZP-p120 strain were amplified by RT-PCR,and the characteristic changes of each gene during virus passage were analyzed by sequencing.During the passage,virus proliferation rate showed a marked increase.After continuous passage,76 mutations occurred in SDZP-p120 strains,covering most of the functional genes,including 42 non-synonymous mutations,25 of which located in non-structural proteins and 17 sites in structural proteins.The amino acid mutation rate of the envelope-associated structural proteins was significantly higher than that of the non-structural proteins,and the mutation pattern of several envelope-related structural proteins was similar to that of other viruses such as HuN4,JXA1,which were weakened in Marc-145 cells.This study provided a reference for further study of the interaction between PRRSV and host cells.
In order to study the gene variation of PRRSV after long-term passages in Marc-145 cells,the highly pathogenic PRRSV isolate SDZP was passaged in Marc-145 cells and a stable strain of SDZP-p120 was obtained after 120 passages.The full-length genome of SDZP parental strain and SDZP-p120 strain were amplified by RT-PCR,and the characteristic changes of each gene during virus passage were analyzed by sequencing.During the passage,virus proliferation rate showed a marked increase.After continuous passage,76 mutations occurred in SDZP-p120 strains,covering most of the functional genes,including 42 non-synonymous mutations,25 of which located in non-structural proteins and 17 sites in structural proteins.The amino acid mutation rate of the envelope-associated structural proteins was significantly higher than that of the non-structural proteins,and the mutation pattern of several envelope-related structural proteins was similar to that of other viruses such as HuN4,JXA1,which were weakened in Marc-145 cells.This study provided a reference for further study of the interaction between PRRSV and host cells.
引文
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[3] Guo Z,Chen X,Li R,et al.The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in China:a molecular epidemiological perspective[J].Virol J,2018,15(1):2.
[4] 唐娜,管宇,魏凤,等.2006-2012年鲁豫冀地区15株猪繁殖与呼吸综合征病毒的分离鉴定及ORF5/Nsp2基因的遗传变异分析[J].中国畜牧兽医,2013,40(10):26-32.
[5] Tian K.NADC30-like porcine reproductive and respiratory syndrome in China:[J].Open Virol J,2017,11(Suppl-1,M3):59-65.
[6] 刘益民.PRRSV ORF2a和NSP2部分缺失毒株的分离及生长特性的研究[D].黑龙江哈尔滨:东北农业大学,2015.
[7] Ma Z,Yang L,Zhang YJ.Porcine reproductive and respiratory syndrome virus:propagation and quantification[J].Curr Protoc Microbiol,2018,48:15M.1.1-15M.1.14.
[8] Huang L,Zhang Y P,Yu Y L,et al.Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in Marc-145 cells[J].Biochem Biophys Res Commun,2011,414(3):545-550.
[9] Wu J,Peng X,Zhou A,et al.MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151[J].Mol Cell Biochem,2014,394(1-2):275.
[10] Shi C,Liu Y,Ding Y,et al.PRRSV receptors and their roles in virus infection.[J].Arch Microbiol,2015,197(4):503-512.
[11] Ji L,Zhou X,Liang W,et al.Porcine interferon stimulated gene 12a restricts porcine reproductive and respiratory syndrome virus replication in MARC-145 cells[J].Int J Mol Sci,2017,18(8):1613.
[12] Wang H,Bai J,Fan B,et al.The interferon-induced Mx2 inhibits porcine reproductive and respiratory syndrome virus replication[J].J Interferon Cytokine Res,2016,36(2):129-139.
[13] Ge M,Zhang Y,Liu Y,et al.Propagation of field highly pathogenic porcine reproductive and respiratory syndrome virus in MARC-145 cells is promoted by cell apoptosis[J].Virus Res,2016,213:322-331.
[14] Chen Y,He S Y,Sun L.Genetic variation,pathogenicity,and immunogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus strain XH-GD at different passage levels[J].Arch Virol,2015,161(1):77-86.
[15] 张洪亮.PRRSV HuN4-F112与Henan-A11-376株对MARC-145细胞感染性差异的研究[D].北京:中国农业科学院,2016.