Construction and verification of an infectious cDNA clone of encephalomyocarditis virus from pigs.

Abstract

In this study, a novel Encephalomyocarditis virus (EMCV) reverse genetic operating system was developed utilizing CMV promoters, enabling EMCV genome expression under the transcriptional control of the CMV immediate early promoter and BGH polyA transcriptional-termination signal. The full-length cDNA of EMCV BJC3 was ligated to the pRK5 vector, incorporating the CMV eukaryotic promoter sequence, resulting in the construction of recombinant plasmid EMCV (pEMCV). Subsequently, the recombinant plasmid was transfected into BHK-21 cells to generate the rescue virus. Further investigation of the neuropathogenicity and histopathology of the rescue virus was conducted using a mouse infection model. The findings demonstrated that the proliferation dynamics of the virus verified that the rescue virus and the wild-type virus exhibited comparable proliferative traits in BHK-21 cells, and whole genome sequencing confirmed the genetic stability of the rescue virus. Although the neuropathogenicity of the rescue virus in mice was less than that of the wild-type parent virus, it retained robust proliferative capacity in vivo. The study indicates that CMV promoter-based EMCV infectious clones can efficiently and stably rescue the virus, preserving similar in vivo and in vitro biological characteristics as the wild-type parent viruses. The development of the infectious cDNA clone offers a genetic platform for studying gene function and facilitating the rational design of vaccines.