Comparative Phenotypic Characterization of Five CRISPR/Cas9-Generated Gene-Disrupted Mutants of Pseudorabies Virus JM Strain

Abstract

Simple Summary Pseudorabies virus (PRV) is an important swine pathogen that can also infect a broad range of non-porcine mammals. In this study, five PRV genes, UL21, UL24, UL50, UL56, and gG, were individually disrupted in the same PRV JM genetic background using CRISPR/Cas9, and the resulting viruses were comparatively characterized in vitro and in mice. The five gene-disrupted viruses exhibited distinct phenotypic profiles in viral replication, plaque formation, and survival in mice. Notably, changes in viral growth and plaque phenotype in cultured cells were not necessarily associated with attenuation in vivo. Among the viruses examined, PRV JM-ΔUL21 and PRV JM-ΔUL24 showed comparatively greater attenuation in mice, although substantial residual virulence remained. These findings demonstrate the value of evaluating candidate attenuation-associated loci across multiple phenotypic levels and provide comparative information for prioritizing UL21 and UL24 for further mechanistic investigation and future multi-gene attenuation studies.