Isolation And Identification Of ARV And Development Of Inactivated Vaccine

Avian reovirus(ARV)is the pathogen that causes viral arthritis or tenosynovitis in chickens.ARV infection can also lead to stunting syndrome and malabsorption syndrome in infected chickens,causing serious economic losses to the poultry industry in China.There are many ARV genotypes,and the cross-immunity between different genotypes is weak,resulting in unsatisfactory immune effects of existing commercial vaccines.The σC protein encoded by the S1 segment can induce group-specific neutralizing antibodies.The σC gene is the most variable fragment in the ARV genome and is the target gene for epidemiological investigation,genotyping and genetic evolution analysis of ARV strains.In order to better understand the prevalence of ARV in China and develop an efficient vaccine with broad-spectrum protection,two ARV strains were successfully isolated and identified from the arthrosis samples of clinically suspected ARV infected chickens.By sequencing and phylogenetic analysis of the σC gene,it was determined that they belonged to genotype II and genotype V,respectively.The two ARV strains were named as HN01 and HN02.The in vitro replication characteristics and pathogenicity of the isolates HN01 and HN02 were studied.An indirect ELISA method based on different genotypes of ARV σC protein was established.The bivalent whole virus inactivated vaccine and σC subunit vaccines based on ARV epidemic strains were prepared.The immunogenicity and protection efficacy of the two vaccines were preliminarily evaluated in order to provide technical supports for the effective prevention and control of ARV.The research content includes four aspects:1.Isolation and identification of avian reoviruses epidemic strains and genetic evolution analysis of σC genesIn this study,two ARV strains,named HN01 and HN02,were successfully isolated from the arthrosis samples of suspected ARV infected chickens by RT-PCR identification and virus isolation and culturing.The two ARV strains can reproduce highly efficiently on the chicken hepatocytes(LMH),and the cytopathic effect characterized by syncytium can be produced 48 hours after inoculation.The results of sequence determination and genetic evolution analysis of σ C gene showed that HN01 belonged to genotype II and HN02 belonged to genotype V.HN01 was on the same branch with the three Chinese isolates(Gen Bank accession number: MK189465,MK189477,MK189483),and HN02 was on the same branch with a Japanese isolate(Gen Bank accession number: LC605827).The two ARV strains isolated in this study enriched the epidemiological information of ARV infection in China and laid a foundation for ARV genotyping and development of multivalent vaccines of ARV.2.Study on the in vitro replication characteristics and pathogenicity of avian reovirus isolatesIn order to determine the replication dynamics of HN01 and HN02 on LMH cells,HN01 and HN02 were inoculated to LMH cells for TCID50 and one-step growth curve determination.The results showed that HN01 and HN02 had a similar one-step growth curve on LMH cells,the virus titer peaked at 84 h after infection.The virus titer of HN01 was 106.2 TCID50/100μL,and that of HN02 was 106.0 TCID50/100μL.In order to determine the pathogenicity of HN01 and HN02 to SPF chicken embryos and chickens,SPF chicken embryos and chickens were infected with 2×106TCID50 of each virus.The ten-day-old SPF chicken embryos were challenged through the chorioallantoic membrane,and the viral loads were determined by real-time quantitative RT-PCR.The results showed that the mortality of the infected chicken embryos was 40% with HN01 and 80% with HN02 at day 7 post infection.The survival chicken embryos showed poor growth and development of embryos.The results of viral loads showed that the liver and spleen had the highest viral loads.Three-week-old SPF chickens were challenged through footpad injection.After challenge,cloacal swabs were collected to detect viral shedding by RT-q PCR,and the organs were collected for viral loads determination.The results showed that the footpads were swollen after challenge.At day 7 post infection,the mortality was 0,and the duodenum and pancreas showed obvious bleeding.Both groups of chickens in the challenge group showed viral shedding.The viral load in the cecal tonsils and footpads was the highest.This study laid a foundation for in vitro culturing and pathogenicity study of ARV.3.Establishment of indirect ELISA antibody detection methods based on different genotypes of ARV σC proteinsIn order to detect of ARV antibody levels after vaccine immunization and perform seroepidemiological investigation of ARV infection,the indirect ELISA antibody detection methods were established by using purified genotype II and V ARV σC proteins as coating antigen,and HN01 and HN02 positive serum as primary antibody,respectively.The two ELISA antibody detection methods have good specificity,sensitivity and repeatability.In addition,the established ELISA methods were used to detect the cross-reaction of three different genotypes of ARV positive serum(S1133,HN01,HN02).The results showed that the ARV σC protein of genotype II had a strong cross-reaction with the positive serum of genotype V HN02,but a weak cross-reaction with the positive serum of genotype I S1133 vaccine strain,with the OD450 nm value of 1.025,0.866 and 0.469,respectively.The ARV σC protein of genotype V ARV had a strong cross-reaction with the positive serum of genotype II HN01 strain,which was higher than the binding with the isotype antibody,and had a similar level of cross-reaction with the positive serum of genotype I S1133 vaccine strain.The OD450 nm were 1.470,1.058 and 1.042,respectively.The indirect ELISA methods established in this study can be applied to detect a large number of serum samples and obtain a timely understanding of the prevalence of ARV infection in the local area.4.Development of inactivated vaccine based on ARV Chinese epidemic strains and evaluation of immunogenicity and protective efficacyIn order to evaluate the immunogenicity and protective efficacy of vaccines based on different genotypes of ARV epidemic strains,and the cross-protection efficacy of the inactivated vaccine based on the classical strain S1133 of genotype I ARV.Bivalent inactivated vaccine based on HN01 and HN02,genotype II and V ARVσC subunit vaccines and S1133 inactivated vaccine were prepared.The prepared vaccines were used to immunize one-week-old SPF chickens by intramuscular injection in the leg.After immunization,the serum was collected from the subwing vein blood.The ARV-specific antibody level was detected by the established indirect ELISA methods,and the ARV-specific neutralizing antibody titer was detected by the virus neutralization test.The protective efficacy of the vaccines was evaluated after 14 days of immunization.The results showed that the bivalent inactivated vaccine andσC subunit vaccines based on the isolates HN01 and HN02 could produce specific antibodies and neutralizing antibodies against the same genotype strain after immunization,and the S1133 inactivated vaccine could not produce neutralizing antibodies against the ARV epidemic strains after immunization.Both bivalent inactivated vaccine and genotype V ARV σC subunit vaccine can produce complete protection against genotype I,II and V ARV infection,while genotype II ARV σC subunit vaccine can only produce complete protection against genoype II ARV HN01 and genotype I S1133 infection.The immune effect of the inactivated vaccine was better than that of subunit vaccines,and the inactivated vaccine based on genotype I ARV strain S1133 cannot provide complete protection against the infection of genotype II and V ARV epidemic strains.Therefore,the results of this study provided guidance for the screening of ARV vaccine strains and the development of efficient vaccines.