Construction Of An Infectious Clone Of The Duck Adenovirus Type 1 Genome

Duck adenovirus type 1(DAV1),including egg drop syndrome virus(EDSV) and EDAV-alike fowl adenovirus,is determined to be Atadenovirus.It has the same common antigen with flow adenovirus group 1(CELO).The apathogenetic DAV1 virus is a promising delivery vector in gene pharmacy for poultry,which will avoid the disturbance of immune efficacy produced by inapparent infection of CELO virus, and has the advantage of large range of host,no integration into chromosome and peroral administration.However,non-essential regions for replication of DAV1 virus and its capacity of exogenous genes are not known clearly at present that hold its application back.In order to learn more about its genie construction and function,an attenuated strain of DAV1 QU strain was selected through restriction enzyme analysis of the genome and animal experiment.Then it was cloned as a bacterial artificial chromosome(BAC) by the Cre-loxP site-specific recombination.First,the genome of an Quail-original DAV1 QU strain was digested respectively with BamHⅠ,EcoRⅠ,EcoRⅤ,KpnⅠand Eam1105Ⅰ.The productions were found different from AV127 strain except that of EcoRⅠ.This indicates that the genotype of QU strain is distinguishable from chicken-original DAV1.In order to study the toxicity,chickens at day 20 of life were infected with either QU strain or HS strain.Four days post challenge several serum biochemical indices and hepatic tissue sections were observed.In contrast to healthy chickens,serum biochemical indices and hepatic tissue sections of chickens infected with QU strain displayed no obvious changes.However in the group that received HS strain,a sharp decline of the levels of serum TP,ALB,GLOB,AST,ALP,CHE and AMY was noticed,and necrosis of hepatocytes was detectable by optical microscope.These findings suggest that HS strain mainly leads to losses in liver and pancreas,while QU strain can hardly affect the physiology of chickens.DAV1 QU strain may be a candidate vector for poultry gene engineering vaccine or gene therapy.pAD20 plasmid was constructed in which a amplification from DAV1 QU strain genome was cloned.A green fluorescent protein(GFP) expression cassette flanked by two loxP sites was inserted into the amplified fragment of pAD20 through the BglⅡand SnaBⅠendonuclease,resulting in pADloxH.Following pADloxH cotransfected with QU virus into chick embryo fibroblast(CEF),a recombinant virus Vgfp expressing GFP was constructed by typical targeted homologous recombination.The loxP sites in the Vgfp DNA were then recombined bv cotranstection of the viral DNA with a Cre recombinase expression plasmid pCre,resulting in recombinant virus Vlox with excision of the GFP cassette.A BAC vector,plndigoBAC-5,containing a single loxP site was recombined into the loxP site of Vlox by cotransfection into CEF with pCre.The resulting virus Vbac was plaque-purified by aβ-galactosidase expression assay.Circular viral DNA from Vbac-infected cells was isolated and used to transform E.coli stain DH10B,resulting in a BAC pVbac containing the complete genome of QU strain.The EcoRⅠsite of pVbac was found identical with QU DNA examined by electrophoresis,pVbac was transfected into CEF produced a cytopathic effect which was indistinguishable from that of the parent QU virus.These findings indicate that the BAC clone pVbac containing the QU virus genome is infectious.The propagation of QU virus in bacterial systems will allow for rapid and accurate characterization of QU virus genes.In turn,this will allow for the full utilization of QU virus as a vaccine vector.