Generation and characterization of a fowl adenovirus 9 dual-site expression vector.

Fowl adenoviruses (FAdVs) are widely considered as excellent platforms for vaccine development and gene therapy. We improved on our right-end partial TR-2 deleted or a left-end 2.3 kb deleted vectors by developing a single, dual-site delivery vector. We demonstrated that, in addition to ORF11, the right end ORF17 is also dispensable. To further improve the capacity and flexibility of the FAdV-9 based vector system, we generated an infectious recombinant FAdV-9 dual-site expression clone lacking 1.9 kb of the left end and replaced with mCherry under the control of a native promoter, and 3.6 kb of the right-end replaced with an EGFP expression cassette. Five intermediate FAdmid clones were successfully constructed: a) pFAdV-9Δ0-2RED (mCherry replacing the left end 2.2 kb ORF0 to 2); b) pFAdV-9RED (mCherry replacing the left end 1.9 kb ORF1 to 2); c) pFAdV-9Δ17 (deletion of ORF17 and 393 bp downstream untranslated region); d) pFAdV-9GFP (EGFP expression cassette replacing the right end 3.6 kb) and e) pFAdV-9Dual (both mCherry in the left end and the EGFP expression cassette in the right end of our vector). Our novel FAdV-9 dual-site vaccine vector, produced infectious virus and expressed either one or both mCherry and EGFP.

Species determination of fowl adenoviruses based on the 52K gene region.

In the present study, the classification of fowl adenoviruses (FAdVs) based on a part of the 52K gene region was described. A total of 44 FAdV field samples from different countries and sources were detected using a recently developed SYBR Green-based real-time PCR. Amplified products were sequenced, and phylogenetic analyses were conducted on the basis of the 116-bp region. For comparison, the already published sequences of the 52K gene region of aviadenoviruses were used in the analyses. The phylogenetic analysis allowed the grouping of the FAdVs into the established five different FAdV species: Fowl adenovirus A to Fowl adenovirus E. The existence of the species was supported by high bootstrap values (> 70%). This method provides the advantages of quantitation and high sensitivity for FAdV detection in combination with species assignment.

Development and application of quantitative real-time PCR for the rapid detection of hemorrhagic enteritis virus in tissue samples.

Hemorrhagic enteritis virus (HEV) is a type II avian adenovirus that causes intestinal hemorrhages accompanied with immunosuppression in 4-to-12-wk-old turkeys. In the present study, a hexon gene-based, quantitative real-time PCR with TaqMan probe was developed and applied to tissue samples from poultry farms to detect and quantify HEV genome copy numbers. The method was confirmed to be rapid, specific, and sensitive for the detection of HEV. This method is an excellent research and diagnostic tool that can be used to study pathogenesis and to gain insights into different phases of infection on poultry farms and for high-throughput epidemiologic investigations.

[The induction of protective immune response in mice vaccinated by recombinant avian adenovirus CELO expressing glycoprotein G of the rabies virus].

The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive.

[The CELO-ANG recombinant avian adenovirus with human angiogenine gene inducing neovascularization in the anterior tibial muscle of rat]. / Recombinantnyi adenovirus ptits CELO-ANG, nesushchii gen angiogenina cheloveka, indutsiruet neovaskuliarizatsiiu v perednei bol'shebertsovoi myshtse krysy.

The CELO recombinant avian adenovirus carrying the gene coding the human angiogenine (ANG) synthesis was obtained. Expression of the angiogenine gene was shown in the LMH cell culture after infection with the CELO-ANG virus. The ability of CELO recombinant adenoviruses to carry out the delivery and expression of alien genes in muscle cells was demonstrated in experiments with laboratory animals (Wistar line rats). The induced neovascularization in rat muscles after the animals were administered the CELO-ANG viruses was shown.

A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.

The spike peplomer S1 subunit sequence from avian infectious bronchitis virus (IBV) Vic S strain was expressed in a plasmid under the control of the fowl adenovirus (FAV) major late promoter (MLP). Two recombinants were constructed in FAV serotype 8 (FAV 8) by inserting the expression cassette between the SnaBI and XbaI restriction enzyme sites (clone DA3) or between the SpeI sites (clone CA6-20). Expression of the S1 gene in the recombinants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by 20h post-infection. Commercial broiler chickens were orally vaccinated at day 0 or day 6 post-hatch and challenged at day 35 post-hatch. FAV antibody ELISA confirmed that maternal antibody directed against inclusion body hepatitis (serotype 8) had decayed in control birds and that FAV specific serum IgG responses were produced in vaccinated birds at the time of challenge. Further, an S1 specific antibody response was detected prior to challenge. Birds were challenged with either Vic S (serotype B) or N1/62 (serotype C) strains of IBV. The tracheas of challenged birds were analyzed by RT-PCR and re-isolation of virus. In birds vaccinated at day 6, 90-100% protection at the trachea was induced against either homologous or heterologous challenge. The construction of a recombinant FAV expressing S1 of IBV demonstrates the potential of an alternative vaccination strategy against IBV.

Histone deacetylase 1 inactivation by an adenovirus early gene product.

Gam1 is an early gene product of the avian adenovirus CELO and is essential for viral replication. Gam1 has no homology to any known proteins; however, its early expression and nuclear localization suggest that the protein functions to influence transcription in the infected cell. A determinant of eukaryotic gene expression is the acetylation state of chromosomal histones and other nuclear proteins. We find that Gam1 expression increases the level of transcription from a variety of eukaryotic promoters, similar to the effect of treating cells with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA ). We show that Gam1 can effectively inhibit histone deacetylation by HDAC1 and that Gam1 binds to HDAC1 both in vitro and in vivo. A CELO virus lacking Gam1 (CELOdG) is replication defective, but the defect can be overcome by either expressing an interfering HDAC1 mutant or by treating infected cells with TSA. The identification of a viral early gene product having the specific function of binding and inactivating HDAC suggests that deacetylase complexes play an important role in limiting early gene expression from invading viruses.

Defining CAR as a cellular receptor for the avian adenovirus CELO using a genetic analysis of the two viral fibre proteins.

The coxsackievirus and adenovirus receptor (CAR) is a high affinity receptor used by adenoviruses, including adenovirus type 5 (Ad5). The adenovirus fibre molecule bears the high affinity cell binding domain of Ad5, allowing virions to attach to CAR. The avian adenovirus CELO displays two fibre molecules on its capsid and it was logical to expect that the cell binding functions of CELO might also reside in one or both of these fibres. We had previously shown that the cell binding properties of CELO resemble Ad5, suggesting that the two viruses use similar receptors. Experiments with CAR-deficient CHO cells and CHO cells modified to express CAR demonstrated that CELO has CAR-dependent transduction behaviour like Ad5. Mutations were introduced into the CELO genome to disrupt either the long fibre 1 or the short fibre 2. A CELO genome with fibre 2 disrupted did not generate virus, demonstrating that fibre 2 is essential for some stage in virus growth, assembly or spread. However, a CELO genome with disrupted fibre 1 gene produced virus (CELOdF1) that was capable of entering chicken cells, but had lost both the ability to efficiently transduce human cells and the CAR-specific transduction displayed by wild-type CELO. The ability of CELOdF1 to transduce chicken cells suggests that CELOdF1 may still bind, probably via fibre 2, to a receptor expressed on avian but not mammalian cells. CELOdF1 replication was dramatically impaired in chicken embryos, demonstrating that fibre 1 is important for the in vivo biology of CELO.

Characterization of CELO virus proteins that modulate the pRb/E2F pathway.

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.

Genes for fowl adenovirus CELO penton base and core polypeptides.

A 3.5-kilobase DNA fragment of the fowl adenovirus type 1 (CELO), located between map units 31.1 and 39.4 has been determined. The sequence contains the probable CELO equivalents of the IIIa protein, penton base, pVII and pV core protein genes of human adenovirus (HAV). The CELO penton base and major core protein (analog HAV pVII) were found to consist of 514 (56.8 kDa) and 72 amino acids (8.4 kDa), respectively.

Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses.

We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use alpha v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a Leu-Asp-Val (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the alpha 4 beta 1 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of alpha 4 beta 1 integrins in the viral internalization into host cells.

Electron microscopic evidence for infection of splenic dendritic cells by adenovirus in psittacine birds.

The spleens of four psittacine birds suffering from natural type II avian adenoviral infection were studied by histopathological, immunohistochemical and ultrastructural techniques to determine the target cells of this virus in the spleen. Typical adenoviral replication sites were found in lymphocytes and dendritic cells.

VA RNAs from avian and human adenoviruses: dramatic differences in length, sequence, and gene location.

Human adenoviruses encode low-molecular-weight RNAs, so-called VA RNAs, which are transcribed by RNA polymerase III. These RNAs are required for an efficient translation of viral mRNAs late after infection. The genes for the VA RNAs in the genome of CELO virus were mapped and characterized. The results showed a number of surprising differences between CELO virus and human adenovirus type 2 (Ad2). Thus, the CELO virus genome encoded only one VA RNA species, in contrast to human Ad2, which encoded two distinct species. The VA RNA from CELO virus was much shorter than the Ad2 VA RNAs (90 nucleotides compared with 160 nucleotides), and there existed no detectable primary sequence homology between them. The predicted secondary structure of CELO virus VA RNA was, however, similar to that of the Ad2 VA RNAs, implying that the folding rather than the primary sequence was the important feature for biological activity. CELO VA RNA also stimulated translation in a transient expression assay, as did the Ad2 counterparts, albeit with a much lower efficiency. The location of the gene for CELO VA RNA also differed from all previously characterized serotypes, suggesting that the genome organization of avian and human adenoviruses are different. Finally, termination of CELO VA RNA transcription occurred in a TTATT sequence which is unique as a stop signal for RNA polymerase III transcription.

Some biological and physico-chemical properties of egg drop syndrome (EDS) avian adenovirus strain B8/78.

The EDS adenovirus produced either in the allantoic cavity of embryonated duck eggs (A0 virus) or in chicken embryo liver cell cultures (TC0 virus) were comparatively studied by gradient ultracentrifugation in CsCl. The A0 viral particles banded at densities of 1.36 and 1.31 g/ml, whereas, the TC0 viral particle were found at densities of 1.33 and 1.31 g/ml, respectively. The heavy populations contained infectious and hemagglutinating particles if they were not pelleted and sonicated before gradient ultracentrifugation. Pelleting or sonicating the viral particles resulted in loss of hemagglutinating activity of the heavy populations. The light particles were hemagglutinating but not infectious regardless of the previous treatment. The pH- and heat-sensitivity of the B8/78 virus strain was similar to those described for CEL0 (FAV-1) adenovirus (5). The B8/78 virus strain replicated well in tissue cultures of chicken and goose origin causing the formation of intranuclear inclusion bodies in the infected cells.