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

Pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that causes severe neurological and reproductive disease in swine and can infect a broad range of non-porcine mammals. To compare the phenotypic consequences associated with disruption of different PRV genes within a uniform genetic background, UL21, UL24, UL50, UL56, and gG (US4) were individually disrupted in the PRV JM strain using CRISPR/Cas9. The resulting gene-disrupted viruses were characterized by multi-step growth analysis, plaque formation, and experimental infection in mice. The five viruses displayed distinct and non-parallel phenotypic profiles. PRV JM-ΔUL24, PRV JM-ΔUL50, PRV JM-ΔUL56, and PRV JM-ΔgG showed reduced peak extracellular viral titers, whereas PRV JM-ΔUL21 exhibited only a limited change in viral growth. In contrast, markedly smaller plaques were observed for PRV JM-ΔUL21 and PRV JM-ΔUL56, indicating that extracellular viral yield and plaque phenotype were not necessarily concordant. In mice, PRV JM-ΔUL21 and PRV JM-ΔUL24 showed delayed mortality and 40% survival, whereas PRV JM-ΔUL50 caused delayed but complete mortality and PRV JM-ΔUL56 and PRV JM-ΔgG retained survival patterns similar to parental PRV JM. Thus, attenuation in vivo was not directly predicted by impaired replication or plaque formation in vitro. Among the five gene-disrupted viruses examined, PRV JM-ΔUL21 and PRV JM-ΔUL24 showed the most pronounced attenuation-associated phenotypes in the mouse model, although substantial residual virulence remained. These comparative findings identify UL21 and UL24 as priority loci for further mechanistic evaluation and for assessment in future multi-gene attenuation strategies.