Generation And Evaluation Of Goose Paramyxovirus Vector Expressing Cap Protein Of Goose-origin Astrovirus As A Bivalent Vaccine

Currently,both Goose Astrovirus（Go Ast V）and Goose Paramyxovirus（GPMV）are widely infectious agents for goslings.There is no vaccine for Go Ast V.Capsid protein can elicit a neutralizing antibody in Human astroviruses（HAst V）.Molecular analysis of the genomic region encoding the capsid protein（ORF2）of goose astrovirus has revealed that contain neutralizing epitopes.Goose-origin NDV is also an infectious.A wide range of NDV strains exists that can be commonly used as vaccine vectors.In the present study,we tried to generate and evaluate of a recombinant GPMV expressing Cap protein of Go Ast V as a candidate vaccine in goslings.The research content mainly includes the following three aspects:（1）Building the reverse genetic platform of GPMV and modify the F gene cleavage site of GPMV/SH12:In this part,the biological features and genotype of wild-type strain GPMV/SH12 which was isolated from a goose farm at Chongming Island,Shanghai,in2012 during clinical diagnosis and treatment were confirmed.The Mean embryo death time（MDT）is 59 hrs and intracerebral pathogenicity index（ICPI）is 1.51.Based on OIE,GPMV/SH12 strain is a virulent strain.The amino acid sequence of cleavage site in F gene is ¹¹²RRQKRF¹¹⁷which belongs to the NDV VII d subtype.Full-length c DNA of GPMV/SH12 was cloned into T-vector and subcloned into the modified vector which modified and include HDZ Rib-T7 terminor sequence;and the F gene cleavage site was also changed into ¹¹²G-R-G-G-G-L¹¹⁷.The plasmid was named p FL/SH.Plasmids p FL/SH and three helper plasmids were cotransfected into Hep-2 cells lines with MAV-T7.The transfection supernant was collected and used to infect chicken embryos for 5 generations continuously.Recombinant strain r GPMV/SH12 was successfully rescused.（2）Determine the best gene expression sites of GPMV/SH12 for expressing the Cap gene of Go Ast V/SH17:In this part,Cap gene of Go Ast V/SH17 strain was analyzed.Cap protein may elicit antibodies to anti-Go Ast V.Based on reverse genetics,to determine the optimal insertion site in the genome of the revised backbone for high level expression,the Cap gene of Go Ast V strain SH17 was inserted into N-P,P-M,M-F,F-HN,HN-L.The levels of Cap transcription were evaluated by q RT-PCR.The levels of Cap translation was evaluated by immunofluorescence assay（IFA）and Western Blot.The insertion of Cap gene into the P-M gene junction resulted in higher level of expression of Cap than when the gene was inserted into N-P.The results of the study also suggested that r NDV/Go Ast V-Cap viruse has potential to be a candidate vaccine.（3）Evaluation the immune efficacy of candidate vaccine preliminarily:r NDV/Go Ast V-Cap was propagation and biological features were also confirmed.Animal experiments were performed.Clinical gross lesions of goslings in nonimmunized groups was showed urate deposition in joint of legs,urate deposition and swelling kidney while goslings were inoculated with r NDV/Go Ast V-Cap showed no apparent signs of disease.Pathological anatomy tests,pathological histology,immunohistochemistry and quantitative RT-PCR were employed to evaluate the animal experiments.The viral load in serum was decreased in treated groups.r NDV/Go Ast V-Cap can induced Go Ast V-Cap specific immune responses and NDV-specific serum antibody.The antibody titer of HI was 3.9±0.8and neutralizing antibody of Go Ast V was 3.55±1.2.It showed that r NDV/Go Ast V-Cap could effectively prevent against both pathogenic of Go Ast V and GPMV challenge.All goslings vaccinated with the r NDV/Go Ast V-Cap in group 5 survived without clinical signs and virus shedding could not be detected after 7 DPC.In conclusion,the results of the study suggested that r NDV/Go Ast V-Cap viruses have potential to be the safe,stable,and effective candidate vaccines.