Development of robust workflow for rescue of coxsackievirus A9 from cDNA clone

Abstract

Human picornaviruses cause mild flu-like illnesses and in some cases, heart or brain infections. Picornaviruses can be detected by specific and sensitive RT-qPCR and genetically typed by amplification of variable genome region for sequencing. However, full-length amplification of picornavirus genomes and virus rescue have not been optimized. Long PCR offers the possibility to amplify full picornaviral clones and to add a T7 promoter into the PCR products. These PCR products can be transfected into cells producing T7 RNA polymerase recognizing T7 promoter. Thus, in vivo transcription of PCR product into virus RNA occurs, which enables direct virus rescue. The virus rescue project with a plasmid cDNA clone of a human picornavirus, coxsackievirus A9 (CVA9), revealed at first that Platinum Super Fi II DNA polymerase had a sensitivity of 100 copies in amplification. The product specificity was good, and PCR reaction setup was also streamlined. Production of DNase-treated in vitro transcribed RNA from PCR products proved to be successful in conjunction with fluorometric quality control assays for RNA. In subsequent 2-step full-length RT-PCR experiments, Induro reverse transcriptase had a sensitivity of 100 000 copies with heterogenous CVA9 RNA samples. Finally, a CVA9 cDNA clone containing the enhanced green fluorescent protein (EGFP) reporter gene insert was used to generate PCR products with a T7 promoter by regulatory primers. T7-BSR cells expressing T7 RNA polymerase were used in virus rescue by transfecting CVA9-EGFP-T7 PCR products into them. EGFP signal indicating viral replication was successfully detected via fluorescence microscopy. However, transfection efficiency was suboptimal. In the end, virus rescue by T7-BSR cells is an efficient technique according to data obtained during the project, but full protocol needs further validation.