Isolation And Identification Of An Avian Reovirus And Establishment Of Competitive ELISA Method

Avian reovirus(ARV),belonging to the family Avian reovirus,orthoreovirus genus,mainly causes viral arthritis(Avain viral arthritis)in broiler chickens,the infection rate of broiler chickens can reach 100%,the morbidity rate of 5% to 20%,mortality rate of 1% to3%.It can cause a decrease in carcass quality of broiler chickens and a decrease in egg production rate of laying hens.In recent years,the disease has been on the rise in China,and the ARV variant strain has caused serious economic losses to the poultry industry.Competitive ELISA(C-ELISA)is a commonly used method when quantifying small molecules.In this method,a labeled competitive molecule with the same function as the target is incubated with the test sample,and as the concentration of the target molecule in the sample increases,the competitive molecule that can bind to the solid phase antigen shows a decreasing trend,and the signal of C-ELISA decreases at this time.By detecting the amount of signal interference and observing the degree of interference of the expected signal output to determine the concentration of antibodies in the sample to be tested,the potential secondary antibody cross-reactivity of indirect ELISA can be effectively avoided with high specificity and high sensitivity.In this study,virus was isolated from suspected infected disease material using chicken embryo liver cell isolation method,and the virus was identified by RT-PCR assay,homology comparison,and genetic evolutionary analysis.The inoculation of the disease material grinds with chicken embryo hepatocytes was able to cause cell fusion,which was consistent with the typical cytopathic characteristics.The RT-PCR assay results were consistent with the target bands.Analysis of the homology and genetic variation of the S1 to 4 genes of the isolate revealed that the homology of the S1 gene with the classical strains S1133,1733 and 2048 was only 76.4%,76.5% and 76.5%,respectively;the homology of the S2 gene with the classical strains S1133 and 1733 was 91.6% and 91.5%;the homology of the S3 gene with the classical strains S1133,1733 and 2048 was 88.93%,89.10% and 88.22%;the homology of the S4 gene with the classical strains The homology of S2 gene with classical strains S1133 and 1733 was 91.6% and 91.5%,S3 gene with classical strains S1133,1733 and 2048 was88.93%,89.10% and 88.22%,and S4 gene with classical strains S1133 and 1733 was 80.4%and 80.5%,and all of them were genetically distant from classical strains.All the above data results showed that the strain isolated in this study was an ARV mutant strain,which was named as: ARV-8 strain.Animal regression tests showed that this strain caused swelling of joints,significant exudation from joint cavities,and enlargement of immune organs such as thymus and spleen in chickens,and that broilers were more susceptible than laying hens.The recombinant expression plasmid ?C-pET-32 a constructed in our laboratory was transferred into E.coli BL21(DE3)expressing bacteria to optimize the induced expression of the fusion protein.The protein was purified by nickel column affinity chromatography,and the expression form of the target protein was determined as inclusion bodies by SDS-PAGE electrophoresis.The good specificity and high reactivity of the recombinant protein expression were demonstrated by protein blotting(Western blot)and BCA concentration measurement.SPF chickens were immunized at 14 days of age,and second and third immunizations were performed at two-week intervals.Sera were collected from chickens after third immunization and standard positive sera were prepared.The potency of the positive sera was determined by enzyme-linked immunosorbent assay(ELISA)at 1:8000;the specificity and purity were confirmed by indirect immunofluorescence(IFA),Western blot,and sterility testing.The best encapsulation concentration of protein was determined by the square method as1:400,the best dilution concentration of monoclonal antibody was 1:500;the best dilution concentration of serum was 1:50,the best dilution concentration of sheep anti-mouse Ig G-HRP enzyme-labeled secondary antibody was 1:4000,the closure solution was selected as 5% skim milk powder,the best encapsulation time was 4? overnight,the best closure time was 2h,the best incubation time of serum and monoclonal antibody was 1h,the best incubation time of secondary antibody The method established in this study was initially applied to clinical testing with good results.In conclusion,in this study,a wild strain of ARV variant was isolated and identified from the suspected infected chickens,and prokaryotic expression of its major structural protein ?C,preparation of ARV-positive and negative sera.This has led to the establishment of a competitive ELISA method for the detection of ARV antibodies,which provides technical support for the detection of ARV variant strains,as well as epidemiological investigations and vaccine evaluations.