Porcine Adenovirus Serotype Research Articles

High Rates of Detection and Molecular Characterization of Porcine Adenovirus Serotype 5 (Porcine mastadenovirus C) from Diarrheic Pigs.

Since the first report on isolation of porcine adenovirus serotype 5 (PAdV-5, species Porcine mastadenovirus C (PAdV-C)) from pigs with respiratory illness in Japan in 1987, PAdV-5 have been detected in a few fecal samples from healthy pigs and in some environmental samples. To date, only a single PAdV-5 strain (isolate HNF-70 from 1987) has been analyzed for the complete genome. We report here high detection rates of PAdV-5 (25.74%, 26/101 fecal samples) in diarrheic pigs at 3 different farms in the Caribbean country of Dominican Republic. After a long gap, the complete deduced amino acid sequences of the DNA-dependent DNA polymerase (pol) and hexon of two PAdV-5 strains (GES7 and Z11) were determined, revealing >99% sequence identities between PAdV-5 strains (HNF-70, GES7 and Z11) detected in different parts of the world and during different time periods (1987, and 2020–2021). By phylogenetic analysis, the putative hexon and pol of HNF-70, GES7 and Z11 exhibited similar clustering patterns, with the PAdV-5 strains forming a tight cluster near ruminant AdVs, distinct from the species PAdV-A and -B. GES7 and Z11 retained the various conserved features present in the putative pol and major late promoter region of HNF-70. Considering the paucity of data on current epidemiological status and genetic diversity of PAdV in porcine populations, our findings warrant similar studies on PAdV-5 and other PAdVs in clinically ill and healthy pigs. To our knowledge, this is the first report on detection and molecular characterization of PAdV-5 (PAdV-C) from diarrheic pigs.

Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry

Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin-Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized alpha(2,3)-linked and alpha(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.

Comparative analysis of vector biodistribution, persistence and gene expression following intravenous delivery of bovine, porcine and human adenoviral vectors in a mouse model

Nonhuman adenoviruses including bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector gene expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as an alternative or supplement to HAd5 for gene delivery applications.

152. Coxsackievirus-Adenovirus Receptor (CAR) and αvβ3 or αvβ5 Integrin Independent Internalization of Porcine Adenoviral Vectors: Implications in Gene Therapy

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells and circumvents preexisting humoral immunity in humans. Coxsackievirus and adenovirus receptor (CAR) is the primary receptor and |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin is involved as a secondary receptor for various human adenovirus (HAd) subtypes including HAd5. In this study we deduced the role of CAR, |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 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-|[alpha]|v|[beta]|3 or anti-|[alpha]|v|[beta]|5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P<0.05) inhibition in transduction by HAd-GFP was observed in the antibody treated cells as compared to the untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study 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 |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

Porcine adenovirus serotype 3 internalization is independent of CAR and α vβ 3 or α vβ 5 integrin

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 α vβ 3 or α vβ 5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, α vβ 3 or α vβ 5 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-α vβ 3 or anti-α vβ 5 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 α vβ 3 or α vβ 5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and α vβ 3 or α vβ 5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response after the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. E1A-deleted PAd3 vector expressing green fluorescent protein; GFP (PAd-GFP) and E1-deleted HAd5 vector expressing GFP (HAd-GFP) transduced human cell lines with comparable efficiencies. Both of these vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH 3T3 fibroblast cell line significantly better ( P < 0.05) than HAd-GFP. These results suggest that PAd3 vectors would be promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

457. Fibronectin Peptide Reduces the Immune Response to Adenovirus and Enhances Transgene Expression

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response following the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. To compare transduction efficiencies of PAd3 and human adenovirus type 5 (HAd5) vectors, PAd-GFP (E1A-deleted PAd3 vector expressing green fluorescent protein; GFP) and HAd-GFP (E1-deleted HAd5 vector expressing GFP) were generated. PAd-GFP and HAd-GFP transduced human cell lines with comparable efficiencies. Both vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH-3T3 fibroblast cell line significantly better (P<0.05) than HAd-GFP. These results suggest that PAd3 vector system would be a promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

Sequence analysis of porcine adenovirus serotype 5 fibre gene: evidence for recombination.

The nucleic acid and deduced amino acid sequence of the fibre gene of the HNF-61 strain of porcine adenovirus serotype 5 (PAdV-5) was determined and compared to that of the HNF-70 strain of the same serotype (Nagy et al., J Gen Virol 82, 525-529, 2001) and also to adenovirus fibre genes from the genera Mastadenovirus and Atadenovirus. The putative HNF-61 and HNF-70 proteins were similar to each other, with 90% amino acid identity. Conserved amino acid sequences described for mastadenovirus fibre shafts were identified in the shaft regions of both PAdV-5 fibres, except for the so-called TLWT motif. The head regions of the PAdV-5 fibre did not resemble any of the known mastadenovirus fibre heads, but they showed characteristics of the fibre head protein sequences of viruses grouped in the proposed genus Atadenovirus (Benkö et al., Virus Taxonomy, Seventh Report of the International Committee on the Taxonomy of Viruses, Academic Press, New York, San Diego, 2000, pp. 227-238). The findings suggested recombination between viruses of different adenovirus genera.

The complete nucleotide sequence of porcine adenovirus serotype 5.

The complete nucleotide sequence of porcine adenovirus serotype 5 (PAdV-5) has been determined and the putative genomic map was constructed. The size of the genome was found to be 32621 nucleotides. Twenty-eight putative ORFs were identified by their homology to other adenovirus or other virus and eukaryotic genes. Several special protein sequence motifs were identified by their homology to similar protein motifs. The putative promoter regions, polyadenylation and splice sites were predicted and the early and late transcription units were determined. Based on sequence analysis and RNA secondary structure prediction, sequences for virus-associated RNA could not be recognized. Phylogenetic analysis showed that PAdV-5 was more closely related to certain bovine adenoviruses than to other porcine adenoviruses.

Sequence analysis and deletion of porcine adenovirus serotype 5 E3 region

A 3000 basepair (bp) region corresponding to the E3 region, the flanking pVIII and part of the fiber protein genes, of the two prototype strains (HNF-61 and HNF-70) of porcine adenovirus serotype five (PAdV-5) was sequenced. A potential E3 promoter and poly-A signals were identified. The size of the E3 region was 2039 (strain HNF-61) and 2020 bp (strain HNF-70) the largest E3 so far reported among PAdVs. Three open reading frames (ORF2-4) were identified within the E3 region. Based on the predicted amino acid (aa) sequences ORF2 was similar to other adenovirus E3 ORFs, ORF3 showed some similarity to a bovine adenovirus (BAdV-1) ORF. ORF4 was unique to PAdV-5. E3 mRNA transcripts were detected early in infection by Northern blot analysis. Genomic clones of HNF-70 with a 1505 or 1237 bp deletions in the E3 region were constructed to map non-essential regions. After transfection of the DNA into swine testicle cells, virions were recovered for only the shorter 1237 bp deletion. At least 60% of the E3 region was not essential for virus replication, bringing the theoretical vector capacity of a helper independent PAdV-5 to 2.9 kb.

Characterization of early region 4 of porcine adenovirus serotype 5.

To locate the E4 region on the genome of the HNF-70 prototype strain of the porcine adenovirus serotype 5 (PAdV-5), 4.5 kb at the extreme right end was sequenced. This area had 11 open reading frames (ORFs) on the left strand encoding more than 50 amino acids (aa). A Genbank homology search indicated that 8 of these ORFs were unique to PAdV-5. ORF5:253 aa, ORF6:245 aa and ORF11:146 aa showed similarities to known mammalian and avian adenovirus E4 ORFs. The putative E4 promoter sequences were also identified. Two TATA boxes, several GC boxes and two typical CCAAT boxes were located within this region. The presence of several potential poly-adenylation signals and the number of E4 transcripts detected by Northern blot analysis suggested the possibility of a complex mRNA transcription.

Genomic location and nucleotide sequence of a serotype 3 porcine adenovirus hexon gene.

The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein.

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene.

The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.

Nucleotide and amino acid sequence analysis of the porcine adenovirus 23K protein.

The genomic location of the viral encoded protease (23K) of porcine adenovirus serotype 3 (PAV3) was determined and the appropriate fragment cloned and sequenced. An open reading frame (ORF) coding for a polypeptide of 203 amino acids and a calculated molecular weight of 23.3 kDa was found. The ORF was situated in a position similar to that of the human adenovirus 23K, that is, between a putative stop codon for the hexon gene and the polyadenylation signal, AATAAA, for the late region 3. Amino acid sequence alignment of the predicted polypeptide with the sequences of the 23K proteins from other mammalian adenoviruses revealed homology of between 50% and 60% for all except the bovine adenovirus type 7, which displayed appreciable variance from the PAV3 putative 23K with an overall sequence homology of approximately 35%. Conserved cysteine, histidine and proline residues believed to be important in the activity of the 23K protein of human adenoviruses were also present in the PAV3 protein. The genomic location and amino acid sequence of the characterised reading frame suggests that this gene is that of the 23K protein of PAV3.