Safety And Immunogenicity Evaluation Of A GE/gI/TK Gene-deleted Pseudorabies Virus

Pseudorabies(PR), which is caused by pseudorabies virus(PRV), is characterized by fever, unrelieved itchiness, encephalomyelitis, respiratory system and nervous system illness in a variety of domestic and wild animals. Currently, Bartha-K61 vaccine is widely used in the control and eradication of PR worldwide. Since late 2011, however, PR outbreaks occurred in many Bartha-K61-vaccinated pig farms in China.Previously, we isolated the PRV mutant(named as PRV TJ strain), and the pathogenicity of the TJ strain was enhanced compared with the classical virulent SC strain. In order to control the outbreak of PR, we constructed a gE/gI-deleted PRV mutant based on the PRV TJ strain, it can provide rapid and complete protection against the challenge with the PRV TJ strain. Sheep is an ideal model for the efficacy evaluation of PR vaccines. We evaluated the safety of rPRVTJ-delgE/gI in sheep. The results showed that rPRVTJ-delgE/gI induced disease and death in sheep. It revealed that there are some potential biosafety risks in rPRVTJ-delgE/gI.In order to improve the safety of rPRVTJ-delgE/gI, we generated a gE/gI/TK-deleted PRV mutant based on rPRVTJ-delgE/gI using homologous recombination technology and confirmed by PCR and sequencing. The results showed that the plaque formation, growth and size of rPRVTJ-delgE/gI/TK in PK-15 cells was similar to rPRVTJ-delgE/gI, and the growth of rPRVTJ-delgE/gI/TK or rPRVTJ-delgE/gI/TK was attenuated in vitro compared to the parent virus PRV TJ strain.Next, we evaluated the safety and immunogenicity of rPRVTJ-delgE/gI/TK in mice, sheep and pigs. Our results showed that there are no PR-specific clinical signs in mice, sheep and pigs immunized with rPRVTJ-delgE/gI/TK. However, mice and sheep inoculated with rPRVTJ-delgE/gI exhibited severe itching, neurological signs and deaths. It was shown that the safety of rPRVTJ-delgE/gI/TK for susceptible animals(like mice and sheep) and natural host(pigs) was significantly higher than that of rPRVTJ-delgE/gI. Immunogenicity study results showed that PRV specific antibody inoculated with rPRVTJ-delgE/gI/TK in sheep and pigs was similar with rPRVTJ-delgE/gI. We inoculated pigs with 105/104 TCID50 rPRVTJ-delg E/gI/TK, it can provide complete protection against the challenge with the PRV TJ stain, which was consistent with that of rPRVTJ-delgE/gI. It indicates that the deletion of TK gene does not alter the immunogenicity of rPRVTJ-delgE/gI. This suggests that rPRVTJ-delgE/gI/TK has a very high safety and immunogenicity, and might be an attractive vaccine candidate for controlling the currently prevalent PR in China.