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 PRVATK/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 ((PRVATK & gE-US3deop-1, PRVATK & gE-US3deop-2, and PRVATK & 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 PRVATK & gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVATK & 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 PRVATK & gE-US3deop-1 provided similar protection to that of the control virus PRVATK & gEAH02. Finally, PRVATK & gE-US3deop-1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 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 PRVATK & gE-US3deop-1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVATK & gE-US3deop-1 displays great potential as a vaccine candidate.

Automated summary

In this study, bacterial artificial chromosome (BAC) technology was used to replace segments of the US3 gene with codon-deoptimized fragments. Three recombinant viruses were obtained and shown to have similar growth kinetics to the parental strain. Immunization with one of the recombinant viruses showed decreased virus load and attenuated pathological changes in mice. In piglets, immunization with the recombinant virus provided clinical protection and prevented virus shedding after challenge.