Comparative study of three rescue strategies of Coxsackievirus A10
10.13200/j.cnki.cjb.004445
- VernacularTitle:3种柯萨奇病毒A10型拯救策略的比较
- Author:
PEI Jie
- Publication Type:Journal Article
- Keywords:
Coxsackievirus A10(CV-A10);
Transfection;
Virus rescue
- From:
Chinese Journal of Biologicals
2025;38(03):338-343
- CountryChina
- Language:Chinese
-
Abstract:
Objective To compare three rescue strategies of Coxsackievirus(CV)-A10 in order to lay a foundation for selecting appropriate rescue strategies for the research of enterovirus-related strains.Methods The plasmid pBR322-CVA10 was constructed by cloning the genome of CV-A10 strain into pBR322 vector,with T7 promoter introduced before the5'UTR,polyA tail and Not I restriction site inserted after the 3'UTR of the viral genome.The plasmid pBR322-CV-A10-HdvRZ was constructed by further introducing the sequence of hepatitis delta virus ribozyme(HdvRZ) after poly A tail in the plasmid pBR322-CV-A10.The three virus rescue strategies involved:transfection of Vero cells with full-length RNA of viral genome;co-transfection of Vero cells with linearized viral genomic cDNA and T7 RNA polymerase expression plasmid pCAGGS-T7;co-transfection of Vero cells with pBR322-CV-A10-HdvRZ and pCAGGS-T7 plasmids.After the rescue viruses obtained by the three strategies were amplified to the third generation in Vero cells,the gene fragments of about 1 500 bp were amplified by RT-PCR and sequenced respectively.The expression of viral structural protein VP1 was detected by Western blot.The virus rescued was inoculated into Vero cells,and the growth kinetics of the virus was analyzed.Results All three strategies successfully rescued the CV-A10 virus.The growth characteristics of the rescued viruses in Vero cells were consistent across all strategies,and the genome sequence was consistent with that of the parent strain.All three strategies realized the expression of virus structural protein VP1 in cells.The CV-A10 viruses rescued by the three strategies had the same proliferation characteristics in Vero cells.Conclusion All three strategies can successfully rescue the CV-A10virus.The preparation steps of transfecting Vero cells with full-length RNA of virus genome are complicated,and the time required for virus production after transfection is short,which is suitable for rapid rescue of specific strains.Co-transfection of Vero cells with two plasmids is the simplest operation,but it takes a long time for virus production after transfection,which is applicable for the rescue of a series of recombinant viruses in basic research.The above strategies provide flexible and efficient avenues for the rescue of CV-A10 virus.
