Interaction Of Egg Drop Syndrome Virus Penton Protein With Duck Host Proteins GABARAPL1 And RPSA

The egg drop syndrome virus(EDSV)is a stranded DNA virus with non-enveloped icosahedral nucleocapsid of approximately 70–80 nm in diameter.It belongs to genus Atadenovirus under the family Adenoviridae.EDSV was first reported in the Netherlands in 1976,when adenoviruses were isolated from laying chickens The EDSV genome contains 33,213 base pairs.It has also been known as duck adenovirus 1(DAdV-1),duck adenovirus A,adenovirus 127.EDSV primarily causes a sudden severe drop in egg production as well as the production of shell-less,thin-shelled,discolored,or misshapen eggs in apparently healthy laying birds.The hexon,fiber and penton are the main capsid proteins of EDSV.Adenovirus penton base protein has main role in virus internalization into the host cell.The adenovirus penton base can bind to cell membrane intergrins and support the virus to internalize into cell.EDSV penton protein may have same functions with other adenovirus penton protein in viral replication rather than just being a structural protein.Therefore,the objective of this study is to characterize penton,investigate the functions of penton in viral replication and identify the cellular proteins interacting with penton.To find receptors or interaction partners of EDSV penton gene,the yeast two-hybrid(Y2H)screening was carried out.Total of 10 proteins were selected as the potential protein interaction candidates.To determine the positive interactions,co-transformation analysis was used to eliminate the false positives.Among 10 prey proteins,four proteins were selected as the positives.The anas platyrhynchos GABA(A)receptor-associated protein like 1(GABARAPL1),anas platyrhynchos ribosomal protein SA(RPSA),anas platyrhynchos integrin subunit alpha 4(ITGA4)and anas platyrhynchos actin beta(ACTB)were found to be positively interacted with penton in the Y2 H assay.To verify the interaction between penton and selected proteins,the prey and bait proteins were expressed using bacterial and mammalian expression plasmids.Finally the expressed proteins were confirmed by in vitro GST pull-down system.After the confirmation assay,the penton protein of EDSV and both prey candidate proteins(GABARAPL1 and RPSA)have interactions.In diagnosis of avian adenoviruses,the nucleic acid based technologies have been used,such as in vitro amplification of DNA by the PCR method.To detect infection caused by the EDSV,the real-time fluorescence loop-mediated isothermal amplification(RealAmp)is one of the LAMP methods with some advantages as comparison with PCR,including sensitive and specific as well as capable of performing both the amplification and detection by fluorescence in one platform.To detect the EDSV from infected samples(allantoic fluid and cell culture)we utilized RealAmp method directly without viral DNA extraction.RealAmp was successfully employed to detect EDSV fiber gene directly by using diluted samples.RealAmp was amplified viral DNA in short time span of 15–30 minutes.The sensitivity was as low as(26 fg/?l)and specific for EDSV fiber within different DNA viruses.