Isolation And Identification Of FAdV-4 Chinese Epidemic Strain Caused Hepatitis And Hydropericardium Syndrome And Development Of Inactivated Vaccine For HHS

Hepatitis and Hydropericardium Syndrome(HHS)is an acute infectious disease in poultry,and impairs the development of the poultry industry.Since June 2015,outbreaks of severe hepatitis-hydropericardium syndrome(HHS)associated with a hypervirulent fowl aviadenovirus 4(FAdV-4)novel genotype infection have emerged in broiler chickens in China,and caused a great loss to the poultry industry in China.So far there is no authentic commercial vaccine for prevention of HHS as well as no objective methods for detection of antibody against FAdV-4 and evaluation of immune efficacy of vaccine.Here,we collected samples from Henan and its neighboring provinces to isolate and identify FAdV-4 strains and then study their biological characteristics.An indirect ELISA was developed by using purified FAdV-4 Chinese variant as coating antigen.The oil emulsion inactivated vaccine was prepared and the immunogenicity and immunological efficacy of the vaccine were studied.The thesis is composed of the following four aspects.1.Isolation and identification of FAdV-4 Chinese epidemic strain and phylogenetic analysisSamples were collected from commercial broiler farms experiencing HHS in five Chinese provinces(Henan,Anhui,Shandong,Shanxi and Jiangsu).The complete genome sequences of the five FAdV-4 isolates were sequenced.The nucleotide and deduced amino-acid sequences were edited,aligned and analyzed with the Meg Align program.It is notable that all the Chinese FAdV-4 strains have a 1966 bp deletion on the right end region of the genome compared with the early isolated pathogenic and non-pathogenic FAdV-4 strains.This deletion resulted in the absence of ORF19,ORF27 and ORF48.Comparisons of the amino acid sequences of the penton base,hexon,fiber 1 and fiber 2 with the corresponding structural proteins from the pathogenic strain and the non-pathogenic strains revealed the presence of various amino acid mutations.Phylogenetic analysis based on the complete genome showed that all Chinese FAdV-4 isolates clustered within FAdV-C together with other FAdV-4 isolates.Notably,the FAdV-C cluster was divided into two major groups.Group 1 consisted of two previously reported nonpathogenic strains,KR5 and ON1.Group 2 comprised all Chinese HHS FAdV-4 strains and the strains from HHS.In summary,our data demonstrated that FAdV-4 with a novel genotype was the predominant serotype involved in the outbreaks of HHS in China.2.Study on the biological characteristics of the FAdV-4 CH/HNJZ/2015 isolateTo further understand the biological properties of the FAdV-4 CH/HNJZ/2015 isolate,its replication characteristics in chicken hepatocellular carcinoma cell line(LMH)were evaluated by using SYBR Green I real-time quantitative PCR and virus titration.The results showed that FAdV-4 grew well in LMH cells and the virus titers at different time points were positively correlated with the viral genome copy number.At 60 h post infection,the cell culture supernatant should be harvested for its contained the maximum amount of mature viral particles.The tissues and organs from SPF chickens challenged with 107TCID50/chicken of the new genotype FAdV-4 intramuscularly were collected and pathological sections were prepared for HE staining and immunohistochemistry.The results showed that all the tissues and organs of the affected chicken exhibited different degrees of damage,and viral antigens could be detected in livers,hearts,spleens,glandular stomachs and small intestines,which prove that the isolate is highly pathogenic for chickens.3.Development and application of a FAdV-4 Chinese variant-based indirect ELISA for antibody detectionIn order to establish a fast,simple and specific assay for investigating the infection status of Chinese fowl adenovirus serotype 4(FAdV-4)variant in chicken flocks and the antibody immune response post-immunization with inactivated whole FAdV-4 vaccine,an indirect ELISA was developed by using purified Chinese FAdV-4 variant as coating antigen.The optimal reaction conditions were as follow: antigen was coated at 0.20 ?g/well and incubated at 37 ?for 1h then 4 ?overnight;working dilution for serum sample and HRP-labelled secondary antibody was 1:800 and 1:2 000,respectively,antibody incubation was carried out at 37 ?for 1.5h,incubation time of TMB substrate was 5 min.Serum sample was determined as positive when its S/P value was?0.073.There were no cross-reactivity between FAdV-4 antigen and anti-NDV,-H9/H5 subtype AIV,-IBDV,-IBV and-EDSV serum,respectively.It indicated that the established ELISA had good specificity.The maximum intra-and inter-assay coefficients of variation of the ELISA were 3.864 % and 3.924 %,respectively,which showed that the ELISA had good reproducibility.Forty serum samples from clinical suspected FAdV-4-infected chickens were detected for FAdV-4 specific antibody.The results showed 100 % coincidence with those of FAdV-4 etiological detection.Six hundred and seventy six sera samples collected from chicken farms in Henan,Shandong,Anhui,Shanxi and Jiangsu provinces were detected for anti-FAdV-4 antibody.The results indicated that positive rate of anti-FAdV-4 antibody in chicken flocks of these areas was 69.3-89.1 %.4.Immunogenicity and protective efficacy of inactivated new genotype fowl adenovirus 4 vaccinesTo effectively prevent and control the spread of HHS in China,FAdV-4 Chinese epidemic strain was propagated in LMH cells and used as antigen to generate oil emulsion inactivated vaccine.7-day-old specific pathogen-free(SPF)chickens were immunized with different doses of inactivated vaccine,and Immunogenicity was evaluated by detecting anti-FAdV-4 specific antibodies and neutralizing antibodies.After two weeks post-immunization,the chickens were challenged with 103TCID50/chicken of the new genotype FAdV-4 intramuscularly,and the protective efficacy was determined by the survival rate.The results showed that all immunized chickens elicited anti-FAdV-4 specific antibodies and virus-neutralizing antibodies.Antibody titers began to increase at two weeks post-immunization,reached its peak at four weeks post-immunization,and lasted for at least 20 weeks.All immunized chickens did not show any HHS clinical symptoms,pathological changes and even death.The study demonstrates that inactivated vaccine prepared from Chinese FAdV-4 epidemic strain can effectively prevent and control infections caused by the novel genotype of FAdV-4.