Construction Of A Recombinant Marek's Disease Virus Expressing The Fusion Protein Of Newcastle Disease Virus Strain ZJ1

Newcastle disease (ND) and Marek's disease (MD) are two important diseases which have made substantial economic costs in poultry industry in recent years. In this study, we devoted to construct a stable polyvalent vaccine against both MD and ND, by using Marek's disease virus (MDV) Rispens CVI988 strain as the vector to express the fusion protein (F) of genotype VII Newcastle disease virus (NDV) which is predominant epidemic in China currently.The sorf1-sorf2 of IRS-US region of MDV Rispens CVI988 genome was used as the insertion site for foreign genes. The recombinant MDV contains the F gene of NDV and lac/smGFP flanked by loxP under the control of the LTR promoter from the reticuloendotheliosis virus (REV). The transfer plasmid pMDV/F/GFP was transfected the chicken embryo fibroblast (CEF) cells infected with MDV Rispens CVI988 strain to generate the recombinant virus rMDV-F/GFP, which inserted the sequence of NDV F and Lac/smGFP genes into the MDV genome by homogeneous recombination. The plaques expressing the GFP were collected by trypsinization and plated on fresh CEF. The cloning process was repeated until all of the plaques stained positively for GFP expression. Cre-based homogeneous recombination was used to delete the Lac/smGFP gene from rMDV-F/GFP to obtain the recombinant virus rMDV-F, expressing fusion protein under the control of REV LTR. According to the results of PCR and IFA, we can draw the conclusion that NDV F gene was cloned into the genome of MDV Rispens CVI988 and stably expressed.We tested the protective efficacy of rMDV-F against NDV and MDV challenges in SPF chickens. For the challenge experiments with NDV, 1-day-old chickens in each group were vaccinated with 3000PFU of the rMDV-F and then challenged with 10LD50 of the NDV strain F48E8 at 3 weeks postvaccination. The chickens were examined for the onset of ND daily, 2 weeks after the challenge. The chickens vaccinated with rMDV-F showed that 83.33% of the chickens were considered protected. In contrast, killed vaccine provided complete protection against ND. Chickens in the challenge control showed high mortality and none of them were protected. For MDV challenge experiments, 1-day-old chickens were vaccinated with 3000 PFU of virus and then challenged with 500 PFU of the vvMDV RB1B strain at 7 days postvaccination. 12 weeks after the challenge, the chickens were examined grossly and histopathologically for the presence of MD lesions in the peripheral nerves, brains, and visceral organs. The vaccination of SPF chickens with rMDV-F result in full protection to chickens against vvMDV just as the Rispens CVI988 strain did, whereas all of the challenge control showed clinical signs. These results showed that the rMDV we constructed induced a immune response against NDV F, furthermore, it can provide complete protection against vvMDV and partial protection against NDV.