Recombinant bovine adenovirus-3 co-expressing bovine respiratory syncytial virus glycoprotein G and truncated glycoprotein gD of bovine herpesvirus-1 induce immune responses in cotton rats.

One of the impediments in the development of safe and cost effective vaccines for veterinary use has been the availability of appropriate delivery vehicle. We have chosen to develop and use bovine adenovirus (BAdV)-3 as vaccine delivery vector in cattle. Here, we describe the construction of recombinant E3 deleted BAdV-3 vectors expressing single vaccine antigen (BAV360; bovine respiratory syncytial virus G) or two vaccine antigens (BAV851; bovine herpesvirus-1gDt and bovine respiratory syncytial virus G).

Bovine adenovirus-3 E1A coding region contain cis-acting DNA packaging motifs.

To elucidate further the regulation of E1 gene transcription and viral DNA packaging, we constructed and analyzed mutant BAdV-3s in which the deletion of sequences between left ITR and E1A ATG codon was combined with the functional blocking of E1A gene expression by introducing deletion mutations into E1A open reading frame (ORF). The results suggest that E1A coding region contains cis-acting packaging motifs for efficient encapsidation of BAdV-3 DNA into preformed empty capsids. In addition, E1A is not required for the transcription of E1B.

Porcine adenovirus type 3 E1B large protein downregulates the induction of IL-8.

Replication-defective (E1-E3 deleted) adenovirus vector based gene delivery results in the induction of cytokines including IL-8, which may contribute to the development of inflammatory immune responses. Like other adenoviruses, E1 + E3 deleted porcine adenovirus (PAdV) 3 induces the production of IL-8 in infected cells. In contrast, no IL-8 production could be detected in cells infected with wild-type or mutant PAdV-3s containing deletion in E1A + E3 (PAV211) or E1Bsmall + E3 (PAV212).

E1A promoter of bovine adenovirus type 3.

Conserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR.

A newly identified interaction between IVa2 and pVIII proteins during porcine adenovirus type 3 infection.

The adenovirus IVa2 is an intermediate viral gene product that appears to perform multiple essential roles in viral infection. Using IVa2 as bait in the yeast two-hybrid system, we screened selected open reading frames (ORFs) of porcine adenovirus (PAdV)-3 for potential interaction with IVa2. Interestingly, pVIII showed specific interaction with IVa2.

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3.

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication.

cis-Acting packaging motifs of porcine adenovirus type 3.

The cis-acting packaging domain is required for selective encapsidation of adenovirus DNA into preformed empty capsids late in the viral life cycle. Earlier, it was demonstrated that the cis-acting packaging domain of porcine adenovirus type (PAdV)-3 is located between nucleotide position (nt) 212 and 531 at the left end genome which contains six AT/GC rich motifs. Removal of packaging domain from left end to the right end of the genome produced a viable mutant virus suggesting that the identified cis-acting packaging domain represents the DNA sequences required for selective packaging of PAdV-3 DNA, whose position and orientation appear to be flexible.

Viral RNAs detected in virions of porcine adenovirus type 3.

It has been demonstrated that cellular and viral RNAs were packaged in the virions of human cytomegalovirus (CMV) and herpes simplex virus 1 (HSV 1), members of the Herpesviridae family, both of which are enveloped double-stranded DNA viruses. Here, we provide evidence suggesting that RNAs are packaged in the virions of porcine adenovirus type 3 (PAdV-3), which is a member of the Adenoviridae family, a non-enveloped double-stranded DNA virus. The RNAs packaged in PAdV-3 virions were enriched in the size range of 300-1000 bases long.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX.

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins.

Porcine adenovirus type 3 E1 transcriptional control region contains a bifunctional regulatory element.

We identified a bifunctional regulatory element located between nt 374 and 431 upstream of TATA box of porcine adenovirus (PAV) 3 E1A promoter. Deletion of the element dramatically reduced the steady-state level of E1A mRNA, but increased that of E1B, which lies immediately downstream of E1A. The mutant virus displayed defective replication at early times of infection, but replicated nearly as efficiently as wild-type PAV-3 at late times of infection.

Altered tropism of recombinant bovine adenovirus type-3 expressing chimeric fiber.

Recombinant bovine adenovirus-3 (BAV-3) has been used as a gene delivery vector for vaccination of calves. However, its usefulness as a vector for non-bovine species is limited due to poor transduction efficiency. To develop BAV-3 based vector for non-bovine species, we determined the feasibility of making targeted BAV-3 vector by modifying its natural tropism.

Identification of cis-acting sequences required for selective packaging of bovine adenovirus type 3 DNA.

The assembly of adenovirus particles is a multistep process, in which viral genomic DNA is selected and subsequently inserted into preformed empty capsids. The selective encapsidation of the adenovirus genome is directed by cis-acting packaging motifs, termed A repeats due to their AT-rich character in DNA sequence. A repeats are usually located at the left end of the viral genome.

Characterization of cis-acting sequences involved in packaging porcine adenovirus type 3.

Encapsidation of adenovirus DNA involves specific interactions between cis-acting genomic DNA sequences and trans-acting proteins. The cis-acting packaging domain located near the left inverted terminal repeat is composed of a series of redundant but not functionally equivalent motifs. Such motifs are made up of the consensus sequence 5'-TTTGN(8)CG-3' and 5'-TTTG/A-3' in human adenovirus 5 (HAV-5) and canine adenovirus-2 (CAV-2), respectively.

A recombinant E1-deleted porcine adenovirus-3 as an expression vector.

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B(large) coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B(large) of PAV-3 and also complemented PAV214 (E1A+E1B(small) deleted).

Genetic organization and sequence analysis of pVIII, fiber and early region 4 of bovine adenovirus type 7.

The DNA sequence of 8,810 nucleotides at the right end of bovine adenovirus type 7 (BAV7) genome was determined and compared with similar regions of other adenoviruses. This genomic region of BAV7 consists of sequences encoding partial 33K, pVIII, fiber, putative early region 4 (E4) proteins and other unassigned proteins. However, BAV7 E3 region is not present in the expected location between pVIII and fiber as BAV7 intergenic region between pVIII and fiber genes is only 183 nucleotides.