Preparation And Characterization Of Cell Adapted Virus Of GPV-CZM-179 Goose Parvovirus

Gosling Plague(Gosling Plague,GP),caused by Goose Parvovirus(GPV)is an acute and lethal infectious disease.Since its high contagiousness,pathogenicity and mortality rate,GP is one of the key infectious diseases that endanger the development of China’s goose industry.Although embryonated goose or duck eggs-adapted attenuated vaccines have been widely applied for immunological prevention and control of GP,there is still a certain range of epidemics of this disease.To develop a safe and efficient cell-adapted attenuated vaccine,the cell-adapted GPV-CZM-179 derived from GPV-CZM-142 was obtained by serial passage on GEF cells.After VP1 and NS1 genes being sequenced and analyzed,safety testing,vaccination-challenge trial and monoclonal antibodies-combined application test were conducted.This study gives reference for the clinical prevention and treatment of GP,and provides a cell-adapted attenuated vaccine candidate for clinical application.1.Preparation and gene analysis of GPV-CZM-179 cell-adapted virusIn this study,cell-adapted goose parvovirus CZM-142 was adapted by serial passage(37 rounds)in GEF to obtain GPV-CZM-179.The TCID50,EID50 and ELD50 of GPV-CZM-165(23th round)and GPV-CZM-179(37th round)cells were determined.The results showed that the virus replicated steadily and maintained good proliferative capacity in the process of passage,with TCIDso,EID50,and ELD50 value of 2.81×107/ml,7.35×104/ml,5×100/ml and 2.57×107/ml,2.81×104/ml,5×100/ml,respectively.At the same time,the sequence analysis of non-structural gene NS1 and structural gene VP1 indicated that the full length of NS1 and VP1 was 1884 and 2199 base pairs respectively and there were 7 and 20 amino acid mutations on the NS1 and VP1 respectively in comparison with GPV-CZM.Whether these mutations are related to the attenuated pathogenicity needs to be explored.2.GPV-CZM-179 animal challenge protection experimentTo evaluate the safety and protective efficacy of GPV-CZM-179 1-day-old goslings were inoculated with GPV-CZM-179 with TCID50 value of 2500,5000,10000 and 15000,respectively.The results showed that the immune doses of 2500TCID50 and 5000TCID50 did not cause death in goslings,while the 10000TCID50 and 15000TCID50 did,which meant it was relatively safe to immunize goslings under 5000TCID50.In the challenge protection experiment,1-day-old goslings were immunized with GPV-CZM-179 at the dose of 500TCID50,1000TCID50,2500TCID50 and 5000TCID50,respectively,six days after which the goslings were infected with highly pathogenic GPV(100LD50).The results showed that the protective efficacy of 500TCID50 was 60%,and the figures for 1000 TCID50,2500TCID50 and 5000TCID50 were 100%indicating that GPV-CZM-179 is a potential celladapted attenuated GP vaccine3.The efficacy of GPV-CZM-179 combined with monoclonal antibody in vivoTo investigate the efficacy of GPV-CZM-179 combined with monoclonal antibody in vivo,four neutralizing antibodies in our laboratory,namely GPV-Mab-2F4,GPV-Mab-2F9,GPVMab-2E11,and GPV-Mab-4D9 were immunized combined with GPV-CZM-179 individually.Meanwhile,control groups corresponding to these antibodies and GPV-CZM-179 were set in parallel.At 6 days post immunization,the goslings were orally challenged with highly pathogenic GPV(GPV-YZGHS20190525)and monitored daily for clinical signs for 21 days.The results showed that the protective efficacy of control groups(GPV-Mab-2F4,GPV-Mab2F9,GPV-Mab-2E11,GPV-Mab-4D9,and GPV-CZM-179)was 60%,80%,100%,60%and 60%,respectively.However,the figure for the group of GPV-Mab-2F4 combined with GPVCZM-179 was 80%,and those of the other three experimental groups were 100%.These results showed that the protective efficacy of GPV-CZM-179 was much higher when it was combined with monoclonal antibody,which provides new ideas for the clinical prevention and treatment of GP.