A recombinant E1-deleted porcine adenovirus-3 as an expression vector. (1/13)

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). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B(large) coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B(small) + E1B(large)) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B(large) was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines.  (+info)

Characterization of cis-acting sequences involved in packaging porcine adenovirus type 3. (2/13)

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. To gain comparative insight into adenovirus encapsidation, we examined the packaging domain of porcine adenovirus-3 (PAV-3). Using deletion mutants, we localized the PAV-3 packaging domain to 319 bp (nt 212 to 531), which contains six cis-acting elements. However, this domain does not contain the consensus motifs identified in HAV-5. In addition, consensus motif found in CAV-2 is present only once in PAV-3. Instead, PAV-3 packaging domain appears to contain AT/GC-rich sequences. The packaging motifs of PAV-3, which are functionally redundant but not equivalent, are located at the left end of the genome.  (+info)

Porcine adenovirus type 3 E1 transcriptional control region contains a bifunctional regulatory element. (3/13)

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. This defect was complemented with coinfecting wild-type virus in a mixed infection. The results indicated that the upstream activation sequences (UAS) of E1A overlap the upstream repression sequences (URS) of E1B, although both transcription units are transcribed from different promoters.  (+info)

Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination. (4/13)

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.  (+info)

Viral RNAs detected in virions of porcine adenovirus type 3. (5/13)

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. By reverse transcription (RT) of RNAs isolated from purified PAdV-3 virions, PCR amplification, and DNA sequence analysis of PCR products, we determined the identities of some viral RNAs contained in PAdV-3 virions. The results indicated that the RNAs representing transcripts from E1A, E1B, DNA binding protein (DBP), DNA polymerase (POL), E4 and some of the late genes including pIIIA, pIII, pV, Hexon, 33 K, and fiber were detected from purified PAdV-3 virions. In contrast, we could not detect the RNAs representing transcripts of precursor terminal protein (pTP), 52 kDa, pX, or 100-kDa protein genes in purified virions. Because the transcripts of pIX, IVa2, E3, protease, pVI, pVII, and pVIII overlap with those of other genes in PAdV-3, we could not definitely conclude that RNAs representing these transcripts were packaged in virions although the expected DNA fragments were produced by RT-PCR in the RNAs isolated from purified virions.  (+info)

Porcine adenovirus serotype 3 internalization is independent of CAR and alphavbeta3 or alphavbeta5 integrin. (6/13)

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus-adenovirus receptor (CAR) serves as a primary receptor and alphavbeta3 or alphavbeta5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, alphavbeta3 or alphavbeta5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-alphavbeta3 or anti-alphavbeta5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as alphavbeta3 or alphavbeta5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and alphavbeta3 or alphavbeta5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.  (+info)

A newly identified interaction between IVa2 and pVIII proteins during porcine adenovirus type 3 infection. (7/13)

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. The yeast two-hybrid findings were validated by GST pull-down assays, in vitro binding studies employing cell-free coupled transcription-translation products and in vitro co-immunoprecipitations using protein-specific antibodies. Finally, we demonstrated that IVa2 specifically interacts with pVIII during PAdV-3 infection.  (+info)

Porcine adenovirus type 3 E1B large protein downregulates the induction of IL-8. (8/13)

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). Expression of PAdV-3 E1Blarge inhibited the NF-kappaB dependent transcription of luciferase from IL-8 promoter. Imunofluorescence and electrophoretic mobility shift assays suggested that constitutive expression of PAdV-3 E1Blarge inhibited the nuclear translocation of NF-kappaB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-kappaB to prevent transcription and down regulate proinflammatory cytokine IL-8 production.  (+info)