Isolation And Identification Of A Novel Goose Astrovirus And Establishment Of Competivive ELISA For Antibody To Goose Astrovirus

Astrovirus belongs to the Astroviridae family and is a non-enveloped,single-stranded positive-stranded RNA virus,it exists widely in humans and animals and one of the main pathogens that cause diarrhea in infants and young animals.Poultry infection will not only cause diarrhea and poor growth,but also cause serious fatal hepatitis and kidney disease.Since 2017,an infectious disease with gout as the main feature of goslings has appeared in Shandong,Henan,Anhui,Jiangsu and other provinces of China.The disease mainly affects young geese within two weeks of age.The main feature is the deposition of urate.The mortality rate is as high as 50%,and it has caused serious economic losses to the goose industry.In this study,the tissues suspected of being infected with the novel goose astrovirus(Go Ast V)were processed,and then the LMH cells were used to isolate and identify the virus.The virus was confirmed to be a Go Ast V strain by RT-PCR detection and sequencing analysis.The pathogenicity to goslings was confirmed through animal experiments.RT-PCR results showed that the target band of 759 bp was amplified,and the virus inoculated LMH cells showed the characteristics of cell shrinkage and rounding;animal experiments results showed that artificially infected goslings appeared consistent with natural infections.The clinical symptoms and changes of necropsy are mainly manifested as large amounts of urate deposits in various organs and joint cavities.At 3,5,7,9,11 and 13 days post inoculation(dpi),and 5 goslings in each group were randomly necropsied.The kidney,liver,spleen,heart and other tissues were collected and quantified by q PCR was performed,and the results showed that the kidney had the highest viral load on the 7dpi.Use the purified ORF 2 protein in the laboratory to immunize 14-day-old healthy goslings.On the 7th day after the three immunizations,blood will be collected by vein,and the prepared serum will be tested for sterility.At the same time,antibodies to common viruses are detected and the antibody efficacy of the serum is determined.The specificity of the antibody was tested.The result showed that the positive serum was sterile and contained no antibodies to other viruses.The cell neutralization test showed that the prepared serum could specifically react with the Go Ast V.In addition,we resuscitated the frozen hybridoma cells to prepare ascites.The test results showed that the titer of ascites could reach 1:204800.WB and IFA identification results showed that the prepared ascites could specifically react with ORF 2protein,which has immunological activity.In this study,ORF 2 purified protein was used as the coating antigen,and the prepared ascites was used as a competitive antibody.A new competitive ELISA method for the detection of antibodies to Go Ast V was established,and the optimal reaction conditions were optimized.The established competitive ELISA method was used to detect 40 Go Ast V negative sera,and the positive cut-off value was 48% calculated according to the statistical method.The above detection method does not cross-react with AIV,GPV,ARV,DEV virus antibodies,which proves that the method has good specificity;the intra-plate variation coefficient is 5.82-6.75%,and the inter-plate variation coefficient is 3.97-7.70%,indicating that the method has good repeatability.Finally,67 sera were used for competitive ELISA and cell neutralization tests,and the results showed that the coincidence rate was 80%.In summary,this study has finished isolation and identification of a novel goose astrovirus and establishment of competivive ELISA for antibody to goose astrovirus,which can provide technical support for the diagnosis,seroepidemiological investigation and surveillance of the Go Ast V in clinic.