| Bovine viral diarrhea virus(BVDV),also known as bovine viral diarrhea/mucosal disease virus(BVD/MDV),is an important pathogen that endangers animal health all over the world.It can infect cattle,sheep,pig and other animals and caused diseases of respiratory system or reproductive system,resulting in compromised immunity of the infected cattle,susceptible to a secondary pathogen,leading to increased morbidity and mortality.The cattle with persistent infection can spread the virus without any clinical symptoms and this situation brings great difficulties to the disease control and prevention.In order to know the cell culture and genetic characterization of BVDV isolates of beef cattle in Henan province,and to prepare the reagents for immune diagnostic and prevention of BVD,we compared the cell culture and genetic characteristics of cytopathic biotype(CP)BVDV standard strain and BVDV isolates,then layer hens were immunized with immunological antigens of these strains,high-titre IgY have been obtained,which provides material and technical reference for immunological detection methods and clinical treatment of BVD.To begin with,the serum sample which was BVDV positive detected by RT-PCR and added into MDBK cells,one stable isolate of BVDV-MQ03 was obtained,which is a cytopathogenic virus by microscopic examination on cell layer,and CPE were observed after 48 h culturing in MDBK.Monolayer cells show cytopathic effects,such as network structure,cell die and flake off.The evolutionary tree based on 5’-UTR region of MQ03 isolate and reference strains of Pestivirus was established,the result showed that MQ03 locates in the same clade with NY-1 and Osloss,which belongs to BVDV-Ib subtype.The phylogenetic analysis and sequence similarity analysis based on 5’-UTR region also showed that,MQ03 and Osloss has the highest homology of 97.1%,and with that of NY-1 strain was 96.2%,While the homology with other strains are less than 92.0%,MQ03 is identified to be BVDV-Ib subtype.Three BVDV strains,MQ01,MQ03 and Oregon C24V(CP)were cultured in MDBK cells for expanding separately and then the cell cultures were collected for subsequent concentration and purification by PEG and sucrose density gradient centrifugation respectively.And the purified virus were stained negatively by phosphotungstic acid,nearly round enveloped virus particles with size of 30-80 nm in diameter were visualized under electron microscopy.Purified standard virus was using to developing indirect enzyme-linked immune-sorbent assay.The best concentration of the coated antigen concentration was 1μg/mL,and the serum/IgY samples,the second enzyme-labelled antibody working concentration fixed on 1:100and 1:8000 respectively.The optimum concentration of coating buffer is carbonate solution(0.05 mol/L,pH 9.6)and coating condition,working time of serum and enzyme-labelled antibody are all in 37℃,1h.After the parameters of the assay were optimized,the specificity and reproducibility of the method were evaluated too.Except anti-BVDV IgY,antiserum from hen anti-Newcastle disease virus,Egg drop syndrome virus,Avian influenza H5,H9 virus all could not be detected by the method.Intra-assay and inter-assay coefficient of variation of 5 yolk samples contained anti-BVDV IgY were from 3.54% to 7.28%,all less than 10%.After that,Oregon C24 V,MQ01 and MQ03 virus purification were made into immunological antigen and injected into healthy layer hens,and the eggs’ yolk were obtained and treated with water-dilution,ammonium sulfate precipitation.IgY titers were tested by indirect ELISA.The yolk antibodies can be detected at 14 th day after the first immunization.The anitibody titer of Oregon C24 V,MQ01 and MQ03 was1:25600,1: 51200 and 1: 51200 respectively after the third time immunization.The high titer IgY obtained in this study could be developed preparations for BVD prevention and treatment in the future. |
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