Isolation Of Paramyxovirus From Vaccinated Ducks With Newcastle Disease Virus And Development Of Monoclonal Antibody Against DPMV

Duck Paramyxovirus Disease is an acute septic infectious disease that caused by APMV-I.The disease is mainly characterized by diarrhea, neurological symptoms, focal glandulargastric bleeding or ulcers, intestinal mucosal bleeding and necrosis of the pancreas or thespleen.Different day age are susceptible to duck, and the smaller the day age, the higher thesusceptibility, incidence and mortality is higher.Previous to APMV-I have strong resistance,only the performance for the poisonous, even strong poison infection also don’t causedisease.But in recent years, APMV-I to goose, ducks and other waterfowl also produced astrong pathogenic role. Our country’s waterfowl is one of the important disease.Establish and perfect testing means DPMV ducks in the body, to the disease isepidemiological investigation as well as the prevention and control of the key. DPMV eachstrain differences between poisonous force, with normal blood clots inhibit test easily testedout the antigen structure of the differences.Monoclonal antibody can stimulate the hostimmune response to identify produced the antigen of a table, with a single antigen decided tocluster in the specificity and can detect different between DPMV strain the difference ofstructure of an antigen table.Preparation of duck vice sticky virus （DPMV） specificmonoclonal antibody, can for poultry virus nucleic vice sticky analysis, establishing a rapiddetection kit and immune chromatography colloidal gold dipsticks lay the foundation of thedevelopment.Establish ELISA and development colloidal gold immune chromatographydipsticks, facilitate DPMV clinical large number of sample rapid diagnosis and the field testsamples of quick screening.There are three patrs in this study.Part one: A new isolate of DPMV, named as JS0920, was isolated from the vaccinatedducks with Newcastle Disease Virus （NDV） La Sota strain from Jiangsu province in2011.Based on the HA, HI and neutralization test, the isolate was determined to be DPMV. The embryo median lethal dose （ELD50） was10^-5.3. The mean death time （MDT） of the isolate inSPF chicken embryos was47.7h. The intraeerebral pathogenicity index （ICPI） of it in1-day-old SPF chicken was1.875. The intravenous pathogenicity index （IVPI） in6-week-oldchicken was2.7. Non-immunization ducks and SPF chickens were challenged with the isolate（cultures of chick embryo） via intramuscular injection and the results showed thepathogenicity and mortality rates were70%and40%respectively in duck and both were100%in chicken. Sequence analysis of the F gene of the isolate showed that JS0920isolateshared99.2%nucleotide and99.5%amino acid homology to F48E9strain. The peptidecleavage site112R-R-Q-R-R-F117in Fusion protein of the isolate was typical of highpathogenic DPMV. The phylogenetic tree showed JS0920isolate was IX genotype.Part two: Duck paramyxovirus was propagated in chicken embryo and purified bydiscontinuous sucrose density gradient centrifugation. Four hybridomas cell lines secretingMabs against DPMV were established by fusing SP2/0with spleen cells from BALB/c miceimmunized with formaldehyde-killed DPMV, designated as2D7、2F6、3E4and3G2. Theindirect ELISA and IFA showed that the four McAbs could combine with the natural DPMVspecifically, and2D7had neutralizing activity. The Ig isotypes of2D7was IgG1,2F6wasIgG2b, and the others were IgM. Light chain were all the κ. The result of agglutination testshowed that these MAbs did not react with other strains, such as IBV、EDSV-76、IBDV（B87）、AIV（H9N2） and blank chicken embryos allantoic fluid.Part three: Possessed high degree of specificity. In order to detect DPMV, a doubleantibody sandwich ELISA method was established. Used purified monoclonal antibody2F6as the capture antibody and monoclonal antibody2D7as the trace antibody conjugated withthe HPR. Sensitivity of the double antibody sandwich ELISA was the HA was2⁵fordetecting sample.The gold particle in colloidal solution was coupled with purified monoclonalantibody2D7, and then sprayed on the glass fibers. The purified monoclonal antibody2F6served as capture-antibody was coated on the nitrocellulose membrane. Then thesandwich-based gold immunochromatographic （IC） strip for the detection of DPMV wasassembled in regular sequence. The detection results indicated that the IC strip was specific toDPMV and the HA was2⁵for detecting sample. The IC strip is more convenient, rapid andsimple for the clinical diagnosis and field investigation of DPMV infection or contamination.