Isolation, Identification And Biological Characterization Of Fowl Adenovirus Group Ⅰ And The Construction Of Recombinant Fowl Adenovirus

Adenovirus was found in 1949, which is tendency to infect epithelial cell. Adenoviridae is divided into two genuses, the Mastadenoviridae and the Aviadenoviridae in the past. However, Atadenoviridae and Siadenoviridae were added to this family in the ICTV 8th report, 2005. Duck adenovirus A is classified as members of Atadenoviridae while HEV as Siadenoviridae, an intermediate between the Mastadenoviridae and the Aviadenoviridae because both encode sialidase in the right structure of the genome. It would be worth notice that EDSV is called Duck adenovirus A and Turkey adenovirus A is designated as new species after this report. Fowl adenoviruses are divided into three groups by differences in serotype, most of which only replicate in birds and don't cause clinical symptoms or only have low pathogenicity. But most of them will influence the health of birds if there are other inducing factors or they superinfect with other pathogens while some fowl adenoviruses themselves are pathogenic. There are some reports about the prevalence or variation of fowl adenovirus (FAV) in foreign countries, but little in China.With the development of molecular biology, many viral vectors are being used for gene therapy, vaccination or transfer. Differently from retrovirus, adenovirus does not depend on host cell division for its replication, and its chromosome rarely integrates into the cell genome, remaining episomal in most cases. Adenoviral vectors have a broad spectrum of cell infectivity that includes virtually all post-mitotic and mitotic cells, and also can be produced in high titers. Therefore, it is convenient to use adenoviral vectors for gene therapy or transfer by many routs. It has been found that birds were reinfected with the same strain after 8 week, eliciting a secondary response neutralizing and precipitating antibodies; virus excretion also occurred despite of humoral antibody. So, if the virus can be constructed into a vector to express foreign protein, it would induce strong immunity against foreign protein in chickens for a long time. Up to now, there is some preliminary research on FAV vector in foreign countries, while there was little research progress in China. The CMV promoter is used more popularly than the major late promoter of FAV in the construct of recombinant virus, but which one is more efficient to express the foreign gene is unknown.In this study, fowl adenoviruses (FAV) distributions in flocks of chickens, ducks and geese were investigated by PCR. The results show that FAV was extensively popular in chickens, geese and ducks, with the positive rate of 51.4%, 51.7%, and 17.3%, respectively. Three positive samples were selected separately from chickens, ducks and geese, and then the open reading frame 8 (ORF8) and the right terminal of viral genome (ITR), were amplified, sequenced and analyzed. All nine isolates were 94.7%-99.9% homologous to FAVI when compared with that of CELOV, FAVI-JS, but shared low nucleotide sequence homology to FAV-9 and EDSV. They all converged into the same lineage with CELOV, FAVI-JS, while the viruses from group III shared the other one. The sequence of the ORF8 from all the above fowl adenoviruses shared very high homology to each other, which is in accordance with the genomic structural feature of adenovirus. From these results, we can presume that the fowl adenoviruses isolates belong to FAV group I.In order to investigate the possibility of FAV as live vaccine vector, the biological characterization of FAVs isolates were further studied with technology of electron microscope, virus morphology, cytopathogenic effect (CPE) and experiments in ovo and in vivo. The results of these experiments showed that the virus particles are spherical, non-enveloped, and about 70-80nm in diameter and couldn't agglutinate the erythrocytes of chickens. The virus couldn't cause CPE in chicken embryo fibroblast cells (CEFs), but cause typical CPE of FAV in chicken embryonic kidney cells (CEKs), which became round with stronger light refraction. No gross lesions and growth retardation was observed in chicken embryo inoculated with the isolates, which are not completely in conformity with the characterization of CELOV (Chicken embryo lethal orphan virus).To evaluate the safety of isolated viruses, SPF chickens were inoculated with the FAVI-G24-0605 at 1-day-old by subcutaneous injection or oral way. The weight of body and some immune organs were measured when a couple of chicken was euthanized at 10d, 20d, and 30d post infection (p.i). The results showed that the inoculated chickens were all healthy with no gross lesions during the animal experiment and there were no serious effects on the body weight of SPF chickens, although the body weight of chickens was lower than the control at 10d p.i and became normal at 20d and 30d p.i. The same changes also occurred in the weight of major immune organs of chickens inoculated. Most of tissue samples had no histopathological changes except some minor lesions at the early stage. These results indicate that the FAVI-G24-0605 strain may be a good candidate for recombinant FAV.The non-essential region for replication of FAVI-JS was determined and the plasmid pFACMV3.1, a universal transfer vector for gene transferred to FAVI-JS, has been constructed by our studying team. On the basis of this research, FAVI-JS was selected from all the fowl adenovirus isolated in our lab for recombination. The major late promoter (MLP) of FAVI-JS, which belongs to group I avian adenovirus, were amplified by PCR and the sequence of MLP has high homology (99%) with that of CELOV. Then the MLP, together with a fragment from pFACMV3.1, containing the R fragment and the large part of L fragment, were cloned into pcDNA3.1/zeo (+). The above large fragment, digested with another two restrict enzymes, was subcloned to pFACMV3.1 to produce pFAMLP3.1, an universal transfer vector controlled by the MLP. By the same way, the ORF of eGFP gene was cloned into pFAMLP3.1 to construct pFAMLP-eGFP as a transfer vector with eGFP controlled by the MLP.CEK was transfected with pFAMLP-eGFP plasmid. Expression of eGFP was found at 48h post transfection. Then CEK infected with wt-FAVI-JS was transfected with pFAMLP-eGFP to produce recombinant virus. The transfected cells and supernatant was freezed, thawed and centrifuged after 3-4d. And the diluted supernatant was re-infected CEK. The expression of eGFP was observed for at least 3 passages. Thus the recombinant fowl adenovirus rFAMLP-eGFP was successfully constructed and provided the basis for study the influence of the promoter on the immune efficiency of FAV.