Establishment And Application Of An ELISA Method For Detection Of Anti-GyH1 Antibody And Antigen Of GyH1

Gyrovirus homsa1(GyH1)is a non-enveloped,icosahedral symmetric,small singlestranded circular DNA virus,a member of the Gyrovirus genus.Originally named Gyrovirus 3(Gy V3),it will be renamed GyH1 in 2021.GyH1 was first found in the feces of children with acute diarrhea.It was subsequently found in marketed chicken,broilers with transmissible viral proventriculitis(TVP),wild migratory birds,and various mammals.In addition,pathogenicity experiments in chickens and mice show that GyH1 is a pathogenic virus and causes aplastic anemia,immunosuppression,and severe multisystem damage.Not only that,reproductive tests confirmed that GyH1 is a direct causative factor for TVP.TVP can cause poor growth and stunting in chickens,seriously affecting the production performance of chickens.However,the infection and prevalence of GyH1 in chickens remains unclear.Based on this,this study aimed to develop indirect ELISA and double-antibody sandwich ELISA for GyH1 to investigate the infection and prevalence of GyH1 in chickens.GyH1 contains three major open reading frames(ORFs)encoding three major viral proteins.VP1 is the major capsid protein of GyH1 and plays a key role in viral assembly,replication and infection.Furthermore,it can induce neutralizing antibodies with the backbone protein VP2.In this study,the antigenicity and hydrophobicity of the VP1 gene were analyzed.The region with high antigenicity of VP1 was selected to construct a prokaryotic expression vector.The VP1 protein was efficiently expressed in this study by optimizing the expression conditions.Similarly,this study used SDS-PAGE and western blot to verify the purification effect and immunogenicity of VP1 protein.The results show that the VP1 protein,with a good purification effect and high immunogenicity,can be used to prepare the next monoclonal antibodies and establish ELISA detection methods.In this study,purified VP1 protein was used as the coating antigen to evaluate the clinical application of indirect ELISA(i-ELISA).Each reaction condition of i-ELISA was gradually optimized to achieve the best reaction effect.The ELISA protocol was as follows: The purified VP1 protein was diluted to 0.9 ?g/m L with 0.05 M phosphate-buffered saline(PBS)and placed at 4°C overnight.The optimal blocking conditions were 5% skimmed milk diluted with Tween 20 in PBS(PBST)and blocked at 37°C for 1 h.Chicken anti-GyH1 positive serum was diluted at 1:640 with PBST and incubated at 37°C for 1 h.The optimal goat antichicken antibody was diluted at 1:5000 and incubated at 37°C for 1 h.The best time for color development is incubation at 37? for 15 min.The cutoff value for i-ELISA was verified to be0.184.The sensitivity and specificity were 93.4% and 98.5%,respectively.The coefficients of variation for repeatability and stability were less than 10%.The above results show that iELISA has high sensitivity,specificity,repeatability,and stability and can be used for Seroepidemiological investigation of GyH1.The purified VP1 protein was fused and emulsified with Freund's adjuvant to immunize mice to obtain an anti-GyH1 monoclonal antibody.After verifications,5 monoclonal antibodies were finally screened from 21 stably expressed hybridoma cell lines.In this study,horseradish peroxidase-labeled monoclonal antibodies were used,and the monoclonal antibody pairing test was performed by checkerboard titration.The results showed that monoclonal antibodies 2F2 and 3H1 could bind stably.Each reaction condition of DASELISA was optimized step by step to establish a DAS-ELISA protocol.The results showed that the optimal concentrations of coating antibody 2F2 and detection antibody 3H1 were 3?g/m L and 6 ?g/m L,respectively.Antigens were diluted 1:4 with PBS and incubated at 37°C for 1 h.The optimal blocking condition is 4% skim milk for 1 h at 37°C.The optimal incubation time for detecting antibodies is 1 h at 37°C.Finally,the optimal time for color development was incubated at 37°C for 15 min.The DAS-ELISA cutoff value was 0.254.The sensitivity and specificity of the DAS-ELISA were 93.14% and 98.67%,respectively.In addition,the reproducibility and stability of the DAS-ELISA were below 10%.The above results indicate that the developed DAS-ELISA has high sensitivity,specificity,stability,and reproducibility.In this study,a seroepidemiological survey was carried out to assess the prevalence of GyH1 infection in chickens using an indirect ELISA developed.The results showed that GyH1 seropositivity ranged from 0.6% to 7.7% in 13 provinces and from 3.1% to 8.1% in eight chicken breeds.The seropositive rate of native chickens was significantly lower than that of foreign species chickens.Similarly,the seropositive rate of broiler breeders and layer breeders by breeder type was 8.6% and 5.3%,respectively.Furthermore,we observed that the seropositive rate of GyH1 was negatively correlated with age.By age,the seropositive rate of GyH1 at 0-30 days,30-60 days,60-180 days,and over 180 days were 13.5%,34.4%,8.3%,and 1%,respectively.To further reveal the prevalence of GyH1,we performed an epidemiological survey of GyH1 in chickens from 15 provinces in China using the developed DAS-ELISA.Epidemiological survey results showed that the positive rate of GyH1 ranged from 3.5% to13.7% in 15 provinces and from 4.5% to 12.3% in 8 different chicken breeds.Likewise,we observed GyH1 positivity rates of 12.6% and 9.6% in broiler breeders and layers,respectively.In addition,age was negatively correlated with the positive rate of GyH1,and the positive rate of GyH1 at 1-14 days,14-35 days,35-98 days,98-189 days,and over 189 days were 14.5%,25.5%,10.8%,8.2%,and 4.6%,respectively.To reveal the regularity of GyH1 virus proliferation and neutralizing antibodies in chickens,we combined the developed i-ELISA and DAS-ELISA to dynamically monitor the different infection stages of 1-day-old SPF chicks infected with GyH1.The results showed that after infection with GyH1,anti-GyH1 neutralizing antibodies could be detected in blood at 21 days after infection,peaked at 35 days after infection,and decreased to negative levels at 49 days after infection.In addition,viremia appeared in the blood 14 days after the GyH1 infection.Virus titers peaked at 21 days post-infection and dropped to negative levels at 35 days.In conclusion,we successfully expressed and purified VP1 protein to develop indirect ELISA and double-antibody sandwich ELISA in this study.Indirect ELISA and doubleantibody sandwich ELISA have been tested with high sensitivity,specificity,stability,and reproducibility and can be used to investigate GyH1 infection and prevalence.The results of seroepidemiological and epidemiological investigations indicate that natural GyH1 infection is widespread in chickens.Different breeds of chickens have different genetic resistance to GyH1 infection.In addition,the age of chickens showed different susceptibility to GyH1 infection,and chicks were more susceptible to GyH1 infection.Interestingly,this study revealed a negative correlation between the increase in neutralizing antibody levels and virus proliferation in GyH1-infected chickens.The results of this study provide a valuable reference for the epidemiology of GyH1.We believe that our research can make positive contributions to the prevention and control of GyH1.