| Pseudorabies virus(PRV) is a member of the alpha herpesvirinae subfamily. PRV genome constitutes approximately 150 K DNA double strand. PRV infection causing pseudorabies is one of the most devastating swine infectious diseases in the swine industry worldwide. In spite of the great efforts on PRV vaccination, pseudorabies re-emerged as one of the top swine epidemic diseases in recent times, most likely due to PRV mutation caused antigenic drift. It is conceivable that this phenomenon is occurring in the entire virus community. The laborious and time-consuming traditional vaccine development strategies, including attenuated vaccines and gene deletion vaccines require many rounds of plaque purification or passages and cannot meet the urgent demand for new vaccines.Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China.1, By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern.2, Next, the recombinant viruses were collected and inoculated to PK15 cells(P1). PK15 cells with fluorescence were then subjected to FACS and plated one cell per well to 96-well plate pre-cultured with PK15 cells. The purity of recombinant virus was validated by PCR amplification.3, Double fluorescent gene excision was achieved through three rounds of plaque purification. Thus, by virtue of these genome-editing tools, the re-emerging PRV HNX was successfully engineered into a PRV-HNX-ΔTK-ΔgE recombinant virus.4, Finally, The safety of this novel vaccine candidate was firstly assessed in mice and piglets. Vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. |
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