Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant

Abstract

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE^-/gI^-/TK^-) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE^-/gI^-/TK^- strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 10^6.0 TCID₅₀ of rPRV NY-gE^-/gI^-/TK^- and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE^-/gI^-/TK^- were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3⁺, CD4⁺ and CD8⁺ in rPRV NY-gE^-/gI^-/TK^--immunized mice was higher than that in DMEM-injected mice. After challenge with 10^6.0 TCID₅₀ PRV NY at 42 dpi, all rPRV NY-gE^-/gI^-/TK^--immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE^-/gI^-/TK^- could be a promising next-generation vaccine to control now epidemic PR in China.

Keywords: CRISPR/Cas9 system; Homologous DNA recombination; Immunogenicity; PRV variant; Safety; gE/gI/TK deletion.