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

In this study, we applied bacterial artificial chromosome (BAC) technology with PRV 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 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, PRV, and PRV) 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 at a 10 TCID dose. Mice immunized with PRV 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 LD of the AH02LA strain, and PRV provided similar protection to that of the control virus PRV. Finally, PRV was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 10 TCID. 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 at a dose of 10 TCID provided complete clinical protection and prevented virus shedding in piglets challenged by 10 TCID of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRV displays great potential as a vaccine candidate.