Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties

Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication.