Development Of Novel Detection Methods For Fowl Adenovirus Serotype 4

Since July 2015,severe outbreaks of hepatitis and hydropericardium syndrome(HHS)characterized by a flabby heart with an accumulation of straw-colored fluid in the pericardial sac and a discolored liver with necrotic foci caused by fowl adenovirus serotype 4(FAd V-4)epidemic strains is widespread in Shandong,Henan,Anhui,and Jiangsu provinces.HHS affects mainly 3-to 5-week-old broiler chickens.The disease was characterized by 20% to 60%,up to 80% mortality,causing significant losses to the poultry industry.The results demonstrated that a novel fowl aviadenovirus serotype 4(FAd V-4)was epidemic in China.The comparative analysis with the reported pathogenic and non-pathogenic FAd V-4 strains revealed that the novel Chinese FAd V-4 isolates contain various genomic deletions and multiple distinct amino-acid mutations in their major structural genes.Amino acid mutations and deletions were mainly concentrated on the fiber2 protein.In order to monitor the epidemic situation of FAd V-4 Chinese epidemic strains in chickens objectively and accurately,in this study,new FAd V-4 detection methods were established from the perspective of serology and molecular biology to monitor the flock,to provide methods for the prevention and control of HHS epidemics.1.Rapid detection of fowl adenovirus serotype 4 by recombinase polymerase amplification combined with lateral flow dipstickIn order to establish a rapid,sensitive,and simple method for on-site detection of fowl adenovirus serotype 4(FAd V-4),a recombinase polymerase amplification(RPA)combined with lateral flow dipstick(LFD)was developed using a pair of FAd V-4 hexon-specific primers which were labeled with 6-carboxy-fluorescein(FAM)and Biotin at the 5’-ends,after optimizing concentrations of primers,RPA reaction time and temperature.The RPA-LFD detection could be finished in 15 minutes at a wide temperature range of 25-45 ℃,and the results could be visualized by naked eyes.The established RPA-LFD targeting FAd V-4 hexon gene could detect 100 copies of FAd V-4 DNA,which was 100-fold more sensitive than conventional PCR and showed no cross-reaction with other poultry common pathogens.The developed RPA-LFD is a convenient,sensitive,specific and rapid method for the field diagnosis of FAd V-4,and will provide an efficient method for investigating epidemiology of FAd V-4 infection.2.Development of a Chinese fowl adenovirus serotype 4 fiber2 protein-based indirect ELISA for antibody detectionTo establish a method for fowl adenovirus serotype 4(FAd V-4)epidemiological surveillance,an indirect ELISA was developed using the purified recombinant Chinese FAd V-4 fiber2 protein as coating antigen.The optimal reaction conditions were as follow: amount of coating antigen was 0.25 μg/well,and coating condition was at 37℃ for 1 h then 4℃ overnight;dilution of serum sample was 1:800,and incubated for 2 h;working dilution of second antibody was 1:4 000,incubation time was 1.5 h;TMB incubation condition was 5 min at room temperature.Serum was determined as positive when its S/P≥0.0553 and negative when its S/P<0.0418,respectively.There was no cross-reactivity between FAd V-4 antigen and anti-NDV,-H9/H5 subtype AIV,-IBDV,-IBV and-EDSV serum.,indicating that the method had a good specificity.The maximal intra-and inter-assay coefficient of variation was 5.6% and 5.7%,indicating that the method had a good reproducibility.Fifty serum samples from clinical suspected FAd V-4-infected chicken were detected for FAd V-4-specific antibody,the results showed the coincidence with those of FAd V-4 antigen detection was 100%.This indirect ELISA can be used for surveillance of FAd V-4 infection in chicken flocks.3.Preparation and identification of monoclonal antibodies against hexon protein conserved regionIn this study,four clones of m Abs to clinically relevant FAd V serotype 4,8b and 11 were produced by using whole FAd V-4 virus as immunogen and recombinant FAd V-4 hexon protein representing the consensus region(469aa-775aa)of group I FAd Vs as screening antigen.All m Abs were Ig G2 b sub-isotype,and recognized specifically the putative 100-k Da hexon protein of FAd V-4,8b and 11 in western blot analysis.The results implied that the 469aa-775 aa region of FAd V-4 hexon protein carries FAd V group-specific epitopes.Thus,the recombinant hexon protein and its m Abs can be used to develop methods for detecting clinically relevant FAd V serotype 4,8b and 11,and evaluating antibody responses induced by FAd V vaccine and infections.4.Development of double antibody sandwich ELISA for decetion of fowl adenovirusIn this study,the anti-hexon469-775 aa monoclonal antibody was used as the capture antibody,a double-antibody sandwich ELISA method for detecting fowl adenovirus was established after optimized reacting conditions.The established ELISA method has good specificity and no cross reaction with IBDV,IBV,NDV,H9 subtype AIV,EDSV.The intra-and inter-assay coefficient of variation was less than 10%,indicating that the established ELISA method has good reproducibility.Fifty clinical samples were tested using the established double-antibody sandwich ELISA method,and the results were in accordance with the detection results of the poultry adenovirus PCR pathogen.The results showed that the double-antibody sandwich ELISA method established in this study can be used for the monitoring of clinical infection of fowl adenovirus,and will provide an efficient method for investigating epidemiology of fowl andenovirus infection in China.