A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High...

In this study, we applied bacterial artificial chromosome (BAC) technology with PRV^ΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRV^{ΔTK&gE-US3deop-1}, PRV^{ΔTK&gE-US3deop-2}, and PRV^{ΔTK&gE-US3deop-3}) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRV^ΔTK&gEAH02 at a 10^7.0 TCID₅₀ dose. Mice immunized with PRV^{ΔTK&gE-US3deop-1} did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD₅₀ of the AH02LA strain, and PRV^{ΔTK&gE-US3deop-1} provided similar protection to that of the control virus PRV^ΔTK&gEAH02. Finally, PRV^{ΔTK&gE-US3deop-1} was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 10^6.0 TCID₅₀. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRV^{ΔTK&gE-US3deop-1} at a dose of 10^5.0 TCID₅₀ provided complete clinical protection and prevented virus shedding in piglets challenged by 10^6.5 TCID₅₀ of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRV^{ΔTK&gE-US3deop-1} displays great potential as a vaccine candidate.