Abstract:

Marek's disease (MD), caused by Marek's disease virus (MDV), is one of the most important immunosuppressive and neoplastic diseases. It can be prevented and controlled with MD vaccines, but the virulence of epidemic MDV strains persistently increases under the highly immune pressure of widely used MD vaccines. Recent studies have found that the classical MD vaccines are no longer able to provide ideal immune protection for the current prevalent hypervirulent MDV (HV-MDV) variant, which implies the urgent need to develop a new generation of highly effective MD vaccines. Presently, we have used the newly isolated HV-MDV variant HNSQ01 as the parental virus, first passaged on CEF monolayers in vitro, to generate a meq gene-edited and knocked out mutant of SQ01Δmeq utilizing the CRISPR/Cas9-based gene editing technology. A series of experiments and analysis have demonstrated a stable MD vaccine candidate strain of SQ01Δmeq has been successfully generated. Furthermore, the pathogenicity analysis was performed with 1-day-old specific-pathogen-free (SPF) chickens for virus challenge experiments. During the 77-days animal experiments, the parental HNSQ01 had caused serious atrophy of host immune organs and inhibited the growth of virus-challenged birds, together with the 100% MD morbidity, 100% mortality and 80% gross tumor occurrence. However, SQ01Δmeq had not caused the immunosuppression or inhibited the growth of virus-challenged birds, together with the 0% rates of MD morbidity, mortality and tumor occurrence. Our data indicate that the generation of SQ01Δmeq provides an important basis for the future research and development of novel efficient genetically engineered MD vaccines.

Key words: Marek's disease, MDV, meq gene, CRISPR/Cas9, Gene editing, gene deletion, novel vaccine