Isolation And Identification Of A Novel Duck Reovirus And Screening Of Oil Emulsion Inactivated Vaccine

Novel duck reovirus?NDRV?disease is characterized by tumefaction,hemorrhage and necrosis of spleen.The morbidity and mortality are relatively high since 2017,which caused severe economic losses.In this study,NDRV was isolated and identified from clinically suspected samples,duck infection model constructed,and an oil emulsion inactivated vaccine prepared,which provides technical support for the prevention and control of NDRV.30 suspected disease samples Shandong,Anhui and other places from 2017 to 2019 were detected by RT-PCR,among which 10 samples were positive.SPF embryonic body and liver cells were inoculated with virus,after which the chicken embryo showed obvious symptoms of bleeding,and the syncytial lesions were produced on liver cells.The strains were then sequenced for homology comparison analysis and genetic evolution tree.The homology comparison results showed that the homology of the 10 isolated strains were between 97.4%and 99.4%.The homology of the isolated strains and classical strain ranged from 94.6%to 96.5%,indicating that NDRV had a significant variation compared to DRV.The genetic phylogenetic tree results showed that the isolated strains were located in the same branch as the waterfowl reovirus,indicating that the genetic relationship between NDRV and waterfowl reovirus were relatively closed.Through homology comparison and genetic evolution tree analysis of the isolated strains,the SDHC strain was most significantly different from DRV in homology.Based on the analysis of pathogenicity and immunogenicity,the SDYC strain was selected as a vaccine strain.One-day-old ducklings were inoculated through the leg muscles to perform animal regression experiment.The results showed that after the inoculation of 0.2 mL(TCD₅₀0 of 10^-7.2)virus solution,ducklings appeared clinical symptoms such as depression and loss of appetite,and autopsies such as bleeding and necrosis of spleen,necrosis of liver and other characteristic pathological changes.After inoculated with the virus solution of SDYC strain,chicken embryonic hepatocytes were collected in 48-72 hours after virus infection.In this study,rotary flask culture was used for cell culture and virus proliferation.This method can rapidly amplify a amount of antigen solution.Formaldehyde was used to inactivate the antigen solution and then mixed with Tween-80 at a ratio of 94:6 as the vaccine aqueous phase.The NDRV oil emulsion inactivated vaccine was prepared by emulsification with the oil-water ratio of 3:2.The physical and chemical characteristics,the optimal immunization dose,immune protection effect,and changes in antibody levels in the serum of ducklings after immunization of the vaccine were tested.The results showed that the optimal inactivation condition was inactivation of 0.2%formaldehyde solution at 37°C for 24 h.The physical and chemical properties,and the safety test of the vaccine showed that the vaccine was water-in-oil type with stable physical properties,and without microorganisms such as bacteria and mycoplasma,which could be stored at 4°C for 12 months.The neck of 5-day-old ducklings injecting with1.0 mL of the inactivated vaccine showed no local inflammatory reaction and systemic adverse reactions,indicating that the vaccine is safe.The results of the immunoprotective test showed that the optimal dose of the vaccine was 0.3 mL/feather.The ELISA OD450 value reaching 0.508 and the neutralizing antibody level reaching 24 represents that the vaccine can effectively defend against NDRV infection.In the challenge protection test,most of the samples in the non-immune group were positive,and the samples within 21 days were positive,indicating that the ducklings in the non-immune group had long-term poisoning and detoxification.Some positive samples were also detected in 0.1mL immune group.Only one positive sample was detected in the 0.3mL¹.0mL immune group,and the protection rate was above 96%.In summary,the NDRV oil emulsion inactivated vaccine prepared in this study has good immune protection effect and can effectively resist NDRV infection.