A gE/gI/US9-triple deletion inactivated vaccine provides complete protection against emerging PRV variant infection

Since 2011, pseudorabies virus (PRV) variant strains have emerged in China, against which conventional vaccines confer inadequate protection, resulting in substantial economic losses. Consequently, there is an urgent need to develop a novel vaccine capable of effectively addressing this challenge. Here, the homology-directed repair (HDR)-CRISPR/Cas9 technology was employed to specifically target and knockout the gI, gE, and US9 genes, which were further developed into inactivated vaccines. Subsequently, these three recombinant viruses, along with a previously constructed gE/gI/US9/US2 deleted strain, were systematically evaluated. In vitro analysis revealed that all mutants exhibited viral titers and growth kinetics comparable to the parental strain but formed smaller plaques. No genetic reversion was detected after 20 passages, confirming favorable genetic stability. In animal studies, the gE/gI/US9 triple-gene deletion mutant induced stronger gB-specific and neutralizing antibodies, enhanced Th1 cytokine secretion (IFN-γ, TNF-α), and promoted lymphocyte proliferation, without altering Th2 cytokines (IL-6, IL-4). Additionally, all three inactivated virus-immunized groups achieved complete protective efficacy, surpassing the 80% protection rate observed with a commercial inactivated vaccine. Notably, the triple-gene deletion group experienced less weight loss, had a lower brain and lung viral load following the challenge. In conclusion, this study identified the triple-gene deletion strain rPRV ΔgE/gI/US9 as a highly promising inactivated vaccine candidate, offering a novel strategy for PRV prevention and control.