Isolation, Genome Cloning, Gene Expression Of Avian Adeno-Associated Virus YZ-1 Isolate And Rescuing Of Infectious Virus

Adeno-associated viruses(AAV) are members of dependvirus genus of the family of parvoviridae.AAV virions are approximately 20 to 25 nm in diameter and are composed of VP1,VP2 and VP3 structural proteins that encapsidate a linear 4.7-kb single-stranded DNA of plus or minus polarity.For efficient replication,AAV requires adenovirus or herpes virus as a helper.In the absence of helper virus,however,AAV-2 remains latent within the human cells and integrates its genome into the sequence-specific site AAVS1 on 19q13.3-qter of human chromosome.As gene transfer vectors,AAVs have the advantages of infecting both dividing and nondividing cells,long-term transgene expression and lack of pathogenicity,which make them attractive for use in gene therapy applications.Previous studies have showed that avian adeno-associated virus(AAAV) had the similar genome organizations with its primate counterparts,suggesting that AAAV can be used as the gene transfer vector for gene expression studies in avian cells and for development of recombinant vaccines against avian diseases.In this study,a AAAV isolate was isolated by co-inoculating embryonating eggs with filtered fecal samples from healthy chickens and CELO virus.The complete genome was cloned and characterized by sequence analysis,expression of the viral genes in E.coli and immunological methods,which laid a solid foundation for generation of recombinant AAAV for gene expression studies in avian cells.1.Isolation and identification of AAAVA total of 10 rectum swab samples were obtained from 7-week-old healthy chickens from a local chicken farm.After aseptic treatment,the filtrates were co-inoculated with the type 1 fowl adenovirus(CELO) into 11-day-old embryonated eggs of SPF chickens.Their allantoic fluids were harvested at different time points of post-inoculation and submitted to PCR amplification using primes designed according to the Rep and Cap genes of the reference AAAV VR-865 strain.The results showed that two expected gene fragments of about 480bp and 450bp were detected from the allantoic fluid of the chicken embryos inoculated with one fecal sample.The PCR products were purified by agarose gel electrophoresis for direct sequencing.The sequence analysis showed that the two gene fragments were 97.1%and 94.6%identical to the Rep and Cap genes of the AAAV VR-865 strain.The isolated virus was successfully passed in embryonated SPF eggs based on PCR detection of their egg allantoic fluids.Electron microscopy showed that purified AAAV particles had a diameter of 20 to 25nm.Agarose gel electrophoresis showed that the AAAV genome extracted from the purified particles had an expected size of 4.7kb.These results confirmed the identity of AAAV isolated from healthy chickens.2.Genome cloning and sequencing of AAAVTwo different strategies were used to clone the genome of AAAV YZ-1 isolate.First, based on the abtained rep and cap sequences,one pair of primers were used to amplify the 1.9-kb rep-cap fragment and another pair of primers were used to amplify the 2.1-kb cap-ITR-rep fragment,both of which were subcloned into TA cloning vector for sequence analysis.Second,AAAV particles were isolated from the allantoic fluids of the co-inoculated eggs by ultracentrifugation and the viral DNA was extracted by SDS-proteinase K digestion followed by phenol-chloroform extraction.After annealing and agarose gel separation,the double-stranded genomic DNA was gel-purified and blunt-ended with Taq DNA polymerase.The viral genomic DNA was then cloned into pCR 2.1 vector and six clones with 4.7-kb insert were subjected to sequencing.Sequencing analysis showed that the YZ-1 AAAV genome cloned using two different strategies had a high agreement,which was 4684-nt long and had similar gene organization to other AAVs.The first 119 nucleotides of the 141-nt ITR formed a typical T-shaped palindromic structure and the remaining 22 nucleotides corresponded to the D region of ITR.The putative Rep-binding element(RBE) were tandem repeats of (GAGY)₄ and the putative terminal resolution site(TRS) was TGGCCA.Sequence alignment showed that the YZ-1 AAAV was 95.0%or 92.2%identical to AAAV strain DA-1 or VR-865 at nucleotide sequence level.The Rep78 of the YZ-1 isolate was 99.2%to the DA-1 strain or 95.6%identical to the VR-865 strain at amino acid sequence level.The left Rep78 ORF was 1995nt long,encoding 664 amino acids. The right VP1 ORF was 2217nt long,encoding 738 amino acids,which was 95.3%or 88.6%identical to the DA-1 or VR-865 strain at the amino acid sequence level.The VP3 ORF encoded 535 amino acids with an identity of 94.2%or 91.6%to the DA-1 or VR-865 strain.The whole genome sequences of three AAAV strains were aligned with that of other AAVs and goose parvovirus B strain(GPV-B) and identities ranged from 55.0%to 59.7%to mammalian AAVs were identified with a highest identity of 59.7%to AAV-2 and the lowest identity 55.0%to bovine adeno-associated virus(BAAV).The percent identity between the YZ-1 AAAV and GPV was 52.2%.3.Expression and characterization of AAAV proteinsBoth nonstructural protein Rep78 gene and structural protein VP3 gene were separately amplified by PCR and subcloned into prokaryotic expression vector pET-47b. After transformation into BL21(DE3) E.coli and induction with IPTG,expected proteins of 82 kDa and 56 kDa were revealed by SDS-PAGE,both of which could be recognized by anti-AAAV serum in Western blotting assay.The expressed proteins were eluted from SDS-PAGE gel and injected into BALB/c mice for antiserum production.Indirect immunofluorescence showed that the antisera reacted positively to viral antigens in AAAV-infected CEF cells,but not to CELO virus antigens.Western blotting of AAAV particles showed the three structural proteins VP1,VP2 and VP3 using the VP3-specific antibody as the probe.The rep and cap ORFs of the AAAV YZ-1 isolate was subcloned as a single fragment into the eukaryotic expression vector pcDNA3.0 and transfected into 293T cells. Correct expression of the Rep and Cap proteins were analyzed by IFA using Rep78- or VP3-specific antiserum.Western blotting of the transfected 293T cells detected the three capsid proteins VP1,VP2 and VP3 using VP3-specific antiserum and two nonstructural proteins Rep78 and Rep52 using Rep78-specific antiserum.4.Rescuing of infectious AAAVTo rescue infectious AAAV,the recombinant plasmid pSM525 containing the YZ-1 genome with a 96-nt deletion in the 5' ITR was co-transfected into chicken embryo live (CEL) cells with CELO virus and passed on CEL cells for five passages.A 450-bp cap gene fragment could be amplified by PCR using viral DNA extracted from the cell lysate. After co-transfection of the pSM525 vector into 293T cells with the helper vector pAd12 containing the E2,E4 and VA RNA gene of Ad5,typical AAAV particles of 20-25nm in diameter were revealed by electron microscopy.The rescued virus was passed in 11-day-old SPF embryonated eggs with CELO virus and its infectivity was confirmed by PCR.