Isolation And Identification Of Novel Duck Reovirus Strain SY And Preparation Of Monoclonal Antibody Against P18 Protein

Novel duck reovirus(NDRV)is a new infectious disease of ducks in recent years,which is characterized by hemorrhage of liver and necrosis of spleen.There is no obvious seasonality of the disease which can be caused by vertical and horizontal transmission,and ducks of all breeds can be infected..Under natural conditions,the mortality caused by the disease is usually low.The morbidity and mortality increase during concurrent infection.It can also cause immunosuppression and growth and development disorders.Since there is no vaccine available for prevention,NDRV disease has now become one of the important infectious diseases that harm waterfowl breeding.Therefore,the development of effective vaccines and reliable diagnostic methods is very important for the prevention and control of NDRV disease.In this study,a strain of NDRV(SY strain)was isolated and identified from the clinical disease of duck tissues.By studying the cell culture characteristics of the SY strain,monoclonal antibodies against the NDRV p18 protein were prepared.This has laid a foundation for the study of NDRV pathogenesis,vaccine and diagnostic reagents.This article includes the following two parts:1.Isolation,identification and culture characteristics of novel duck reovirus strain SYThe main lesions of the diseased duck are liver hemorrhage and spleen necrosis.The liver and spleen tissue are inoculated with SPF chicken embryos for virus isolation.The results showed that all chicken embryos died within 96-120 hours after inoculation,indicating that a virus was successfully isolated from the allantoic fluid of chicken embryos.RCR and RT-PCR were used to detect the pathogen of common duck viral infectious diseases.The results of nucleic acid test proved thatall viruses were negative except for duck reovirus,.The harvested allantoic fluid was inocultated to 3-day-old ducks,symptoms and pathological changes of duck were observed similar to those of natural infections.The above test results indicate that the isolated virus is a novel duck reovirus and is named named as strain SY.To further understand the biological characteristics of strain SY,hemagglutination test and organic solvent sensitivity test were done.It was confirmed that the virus does not agglutinate chicken red blood cells and has no capsule.Electron microscope observation revealed that the virus particles were spherical with a diameter of about 70 nm.In this study,a pair of primers was designed according to the S1 genesequence of NDRV strain 091.Sequencing and phylogenetic tree analysis of S1 gene of strain SY showed that strain SY had the closest genetic relationship with NDRV,with a homology of 95.2%-97.6%.Strain SY was subcultured in Vero cells,and the results showed that it proliferated well on Vero cells and produced obvious cell lesions.This laid the foundation for the research of NDRV vaccine and pathogenic mechanism.2.2.Preparation of p18 monoclonal antibodyEight-week-old BALB / c mices were immunized with purified strain SY).Prepare mouse spleen cells fused with SP2 / 0 myeloma cell line.Screen positive clones by indirect ELISA.A monoclonal antibody hybridoma cell line capable of secreting against p18 protein of NDRV was obtained and named as 1D3.The antibody titer of induced ascites was 106.The indirect immunofluorescence test(IFA)proved that 1D3 reacted specifically with NDRV.Western blot analysis showed that 1D3 can produce specific protein bands with p18 protein.It shows that the specificity and reactogenicity of the monoclonal antibodies screened are excellent.