Isolation And Identification Of Serum Type 4 Avian Adenovirus And Establishment Of Indirect ELISA Detection Assay

Avian adenovirus is a member of the adenovirus family.It is widely distributed worldwide.The main manifestation of inclusion body hepatitis and pericardial effusion syndrome is acute infection.Since 2013,Hydropericardium-hepatitis Syndrome has been widely popular in Shandong,Hebei,Shanxi,Henan,Jiangsu and other provinces in China,which has caused tremendous economic losses to the poultry industry in China.In this study,a suspected case of pericardial effusion hepatitis syndrome was collected from a chicken farm in Tai’an,Shandong Province,and the virus was isolated from SPF chicken embryo.A pair of specific primer sequences were designed according to the Hexon gene sequence of FAdV-4 published in GenBank,and the virus PCR was identified as positive.The isolated virus was sera type IV avian adenovirus.The isolated virus was concentrated as the antigen of the agar diffusion test as well as the the antigen of specific positive sera.It was found that there was a clear precipitation line between the antigen hole and the pore of the serum.The typical clinical symptoms of the disease were replicated by animal regression test,and the liver was made as pathological section and observed by electron microscopy.The typical hepatocyte inclusion body,the nucleus swelling and denaturation,and the intracellular were full of virus particles.The hemagglutination test showed that the virus had no hemagglutination activity.The PCR amplification product was connected with the expression vector pET28 a and transformed into the receptive cell.The recombinant protein was well expressed by IPTG,and the weight of expression was proved to be heavy by SDS-PAGE electrophoresis.Histones mainly exist in the form of inclusion bodies in precipitation.After purification,the protein concentration was 4.1 mg/m L.Western-blot analysis showed that the purified recombinant protein had good reactivity and could be used as an antigen of indirect ELISA.Using Hexon recombinant protein as inclusion antigen,an indirect ELISA detection method was established for detecting FAdV-4 antibody.The experimental results showed that ELISA detection method has good specificity,repeatability and sensitivity.A total of 100 clinical samples were detected by this method,and the positive rate was 25%.The above results show that this study has established a Hexon protein indirect ELISA method for the detection of FAdV-4 antibody,which provides a technical reference for the prevention,diagnosis and treatment of this disease.