Study Of Biological Characteristics Of Novel Duck Reovirus And Establishment Of Indirect ELISA Method

Novel duck reovirus disease is an immunosuppressive disease caused by the Novel duck reovirus(NDRV),characterized by hemorrhage/necrosis in the liver and spleen of the ducklings.Since 2017,the disease has outbroken in major commercial duck industry provinces and some regions of China.The disease is geographically spreading and the morbidity and mortality have been rapidly rising,which caused serious economic losses to the waterfowl industry.So far,only a few NDRVs were isolated,and there were few reports on their molecular biological characteristics and pathogenicity.To enrich the database of NDRV strains,nine strains with good purity were successfully isolated from sick ducks by chicken embryo passaging method in this study.The results showed that these nine strains had 100%mortality to chicken embryos and could proliferate well on Vero cells.Except SD19/6202,the other eight strains could induce significant cytopathic effects(CPE)on Chicken embryo fibroblast(CEF)cells.Furthermore,phylogenetic analysis of the?C gene showed that these nine strains and other Chinese NDRVs located in the waterfowl reoviruses genotype 2branches,which could be divided into two different subgroups.SD19/6201 and other seven isolates were located in subgroup?,which were closely related to other 38 Chinese NDRVs,and SD19/6201was the dominant strain of circulating NDRVs in China.SD19/6202,DE150/China and HN5d/China-HN/2013 strains were located in subgroup II,which was significantly different from the existing Chinese NDRVs.The?C amino acid sequences comparison revealed that there were thirteen different amino acid sites between the CEF-adapted virus SD19/6201 and the CEF non-adapted virus SD19/6202.The 1-day-old ducklings infected with SD19/6201 and SD19/6202 showed similar symptoms with ducks infected NDRV naturally.The severe hemorrhage/necrosis and high level of viral loads were observed in spleen of infected ducks.There were no obvious lesions in other tissues and the viral loads were significantly lower than that of spleen.Therefore,spleen hemorrhage/necrosis can be used as an indicator of the morbidity of NDRV-infected ducklings.In order to further understand the molecular characteristics of NDRV,the complete genomic sequences of the two viruses(SD19/6201 and SD19/6202)with obvious differences in replication characteristics were determined.The results showed that these two viruses had typical NDRV genome structure characteristics and belonged to waterfowl reoviruses genotype 2.Subsequently,the proliferation characteristics in vitro of the representative virus strain SD19/6201 were evaluated.The results showed that SD19/6201 can proliferate well in two cell lines including Vero and CEF and poultry embryos(chicken embryos and duck embryos),and was highly lethal to chicken embryos and duck embryos.In addition,we established the infection model of NDRV on 14-day-old and 21-day-old ducklings.The recommended infection dose of NDRV on 14-day-old ducklings and 21-day-old ducklings were 10^5.0 ELD₅₀/0.1 m L and 10^6.0 ELD₅₀/0.1 m L,respectively.and the recommended necropsy time was 7 days after infection,the rate of splenic lesions is 80%(8/10).In summary,the proliferation characteristics of the epidemic NDRV strain were clarified and animal infection models were established in this study,which would provide a theoretical basis for the development of NDRV vaccines and the evaluation of immune effects.The NDRV outer capsid protein?B contains group-specific epitopes that can induce the production of group-specific neutralizing antibodies,it also can be used as a target protein to prepare diagnostic reagents.However,there is no commercial NDRV ELISA kits in our country.Therefore,we developed the NDRV ELISA antibody detection method in this study currently by using?B protein as the coating antigen,which was expressed in prokaryotic cells.This method has no cross-reactivity with positive serums of other waterfowl viruses and has good specificity.More importantly,the NDRV ELISA method developed in this study has higher sensitivity compared with IFA.The coincidence rate between ELISA and IFA methods was 99.40%by detecting 368 clinical sera.The NDRV ELISA antibody detection method could provide an effective technical tool for the epidemiological investigation of NDRV as well as vaccine evaluation.