高致病性猪繁殖与呼吸综合征病毒野毒株持续性感染细胞模型的建立

Establishment of Cell Lines Persistently Infected with the Field High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

旨为建立高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)野毒株持续性感染细胞模型。通过将HP-PRRSV野毒株JX143感染Marc-145细胞,感染的细胞经连续传代35代,建立了稳定的PRRSV持续感染的细胞模型,获得了JX143-Marc-145-P35细胞株。应用RT-PCR、免疫荧光、空斑形态分析等技术跟踪观察了JX143-Marc145细胞株连续传代过程中病毒在细胞内的存在和生物学特性的变化。为了进一步分析病毒持续性感染过程中基因的特征性变化,通过RT-PCR扩增出JX143-Marc145-P35的全基因,并进行测序和序列分析。结果表明,在细胞模型建立的过程中,细胞病变(Cytopathic effect,CPE)的产生经历了从慢到快又到慢的过程,RT-PCR检测和免疫荧光证实JX143-Marc-145细胞中PRRSV持续稳定存在,表现出病毒持续感染的基本特征。该细胞与正常的Marc145细胞相比,细胞周期无显著差异,但JX143-Marc-145-P35的空斑形态与亲本病毒JX143相比略大。通过序列比较分析,JX143-Marc-145-P35v与亲本猪JX143的同源性为99.4%,存在38个非同义突变,其中,31个位于非结构蛋白,7个位于结构蛋白。试验成功建立了稳定的PRRSV持续性感染细胞模型,细胞周期与正常细胞无显著差异,但病毒在持续性感染过程中无论是基因还是表型都发生了变异,且在体外持续性感染建立的过程中,对于氨基酸突变的选择压力很可能施加于病毒的非结构蛋白和主要糖蛋白。该模型的建立对深入研究病毒与细胞间的相互作用,尤其是PRRSV持续性感染的机制具有重要意义。

To establish an in vitro persistent infection cell model of high pathogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV）based on Marc-145 cells.A Marc-145 cell line was infected with a field-isolated HP-PRRSV strain JX143 and then subcultured 35 passages.Hence a steady model of the Marc-145 cells persistently infected by PRRSV was established and the JX143-Marc-145-P35 cell line was obtained.Furthermore,RT-PCR,immunofluorescence assay and plaque assay were used to study the basic viral features of the persistently infected Marc-145 cells.The complete genes of JX143-Marc145-P35v was amplified and sequenced for further analysis the genetic changes involving the persistent infection process of PRRSV.During the process of persistent infection,we observed that the CPE occurs slower,then faster and slower in the end.Both RT-PCR and immunofluorescence assay showed positive results which indicated that PRRSV was quite stable in the JX143-Marc-145-P35 cell line and this cell line had the fundamental characteristics of persistently infected virus.There was no significant difference in their cell cycle between JX143-Marc-145-P35 cell line and the normal Marc-145,but in comparison with the parental virus,larger plaque size was observed.Comparative sequence analysis of the entire genomes of the JX143-Marc-145-P35v and JX143 identified 38 amino acid substitutions,including 31 in the replicase and 7 in the structural proteins.We conclude that persistent infection of the Mrac-145 cell line with PRRSV is established and that multiple genetic and phenotypic variations occur in the virus during persistence.The selective pressure for amino acid change is likely exerted in genes coding for the nonstructural protein and the major viral glycoproteins during establishment of persistence.This cell model can provide a valuable system for studying virus-cell interactions,especially the mechanism of persistent infection.