包含东部马脑炎病毒包膜蛋白重组水疱性口炎病毒的构建及鉴定OA

This study was aimed at constructing a recombinant virus expressing the envelope protein of Eastern equine encephali-tis virus(EEEV),based on vesicular stomatitis virus(VSV).The EEEV envelope protein gene E3E2E1 and the EGFP reporter gene were inserted into the replicating VSV-ΔG backbone to construct the recombinant plasmid pVSV-ΔG-EEEV-E3E2E1-EGFP.This plasmid was co-transfected with helper plasmids(pBS-N/P/G/L)into BHK-21 cells infected with a vaccinia virus carrying the T7 RNA polymerase gene.A single recombinant virus,rVSV-ΔG-EEEV-E3E2E1-EGFP,was obtained through continuous passage of cell supernatants and purification via a plaque assay.Western blotting,immunofluorescence,and transmission electron microscopy analyses confirmed expression of the correct viral proteins after cells infection,as well as a typical bullet-like morphology.Viral growth curve as-says demonstrated replication kinetics largely consistent with those of VSV-EGFP.High-throughput live-cell imaging further revealed that changes in fluorescence intensity exhibited a significant multiplicity of infection(MOI)-dependent pattern.In summary,the success-fully generated rVSV-ΔG-EEEV-E3E2E1-EGFP correctly expressed the EEEV envelope protein while maintaining robust proliferative activity,thereby providing a foundation for the evaluation of EEEV-neutralizing antibodies and research on its infection mechanisms.