Characterization of cis-acting sequences involved in packaging porcine adenovirus type 311Published as VIDO Journal article no. 340.

AbstractEncapsidation 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′-TTTGN8CG-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

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

AbstractReplication-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 E1Blarge coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1Blarge of PAV-3 and also complemented PAV214 (E1A+E1Bsmall 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 E1Blarge coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1Bsmall + E1Blarge) 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 E1Blarge 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

Porcine adenovirus type 3 E1Blarge 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). Expression of PAdV-3 E1Blarge inhibited the NF-κB 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-κB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-κB to prevent transcription and down regulate proinflammatory cytokine IL-8 production

Pathogenesis and Immunogenicity of Bovine Adenovirus Type 3 in Cotton Rats (Sigmodon hispidus)

AbstractIntranasal inoculation of cotton rats (Sigmodon hispidus) with 108 PFU of bovine adenovirus type 3 (BAd3) resulted in limited virus replication in the lung and trachea. Histopathological changes in the lungs were characterized by necrosis and hyperplasia of bronchiolar epithelium, eosinophilic intranuclear inclusions, pneumocyte type II hyperplasia in the alveoli, and mild peribronchiolar and perivascular lymphocytic infiltration. Immunohistochemically, viral antigens were observed more frequently in bronchiolar epithelial cells than in alveolar cells in cotton rat lung sections stained using a rabbit anti-BAd3 serum. Bronchiolar epithelial changes, intranuclear inclusion bodies, type II pneumocyte proliferation, peribronchiolar infiltration, and immunohistological staining were maximum at Day 3 or Day 4 postinoculation, whereas perivascular infiltration was first observed at Day 8 postinoculation. In addition to the histological study of the pathogenesis of BAd3 infection, we monitored the BAd3-specific immune response in cotton rats. Anti-BAd3 IgG and virus neutralizing antibodies were detected in sera, whereas anti-BAd3 IgA antibodies were found in the sera, lung, and nasal washes. Our results suggest that the cotton rat can serve as a useful small-animal model for investigating the pathogenesis of BAd3 infection, as well as immune responses to BAd3 recombinant virus vaccines

Leucine residues in conserved region of 33K protein of bovine adenovirus – 3 are important for binding to major late promoter and activation of late gene expression

AbstractThe L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37kDa in BAdV-3 infected cells and one protein of 42kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP

Transcription Mapping and Characterization of 284R and 121R Proteins Produced from Early Region 3 of Bovine Adenovirus Type 3

AbstractWe established the transcription map of early region (E) 3 of bovine adenovirus 3 (BAV-3) by Northern blot, S1 nuclease protection assays, cDNA sequencing, and RT-PCR analysis. Five major classes of mRNAs were identified, which shared the 3′ ends. Four classes of mRNAs transcribed from the E3 promoter also shared the 5′ end, while one major class of mRNA transcribed from the major late promoter contained a tripartite leader sequence at the 5′ end. These five transcripts have the potential to encode four proteins, namely 284R, 121R, 86R, and 82R. To identify the proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding 284R or 121R protein. Serum against 284R immunoprecipitated protein of 26–32 kDa in in vitro translated and transcribed mRNA and three proteins of 48, 67, and 125 kDa from BAV-3-infected cells. Western blots and enzymatic digestions confirmed that the 284R protein is a glycoprotein, which contains only N-linked oligosaccharides, both high mannose (48 kDa) and complex types (67 kDa). Serum against 121R immunoprecipitated a protein of 14.5 kDa from in vitro translated and transcribed mRNA and BAV-3-infected cells. Although 121R protein shows limited sequence similarity to a 14.7-kDa protein of human adenovirus 5, the 284R protein appears to be unique to BAV-3. Since proteins encoded by the E3 region appear to influence adenovirus pathogenesis, the 284R protein may contribute to the unique pathogenic properties of BAV-3

Meme marketing: How marketers can drive better engagement using viral memes?

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Construction and Characterization of E3-Deleted Bovine Adenovirus Type 3 Expressing Full-Length and Truncated Form of Bovine Herpesvirus Type 1 Glycoprotein gD

AbstractUsing the homologous recombination machinery ofE. coli,a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3.E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replicationin vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3.E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals

Identification and Characterization of a Bovine Herpesvirus-1 (BHV-1) Glycoprotein gL Which Is Required for Proper Antigenicity, Processing, and Transport of BHV-1 Glycoprotein gH

AbstractDNA sequence analysis of the bovine herpesvirus-1 (BHV-1) genome revealed the presence of an open reading frame named UL1 which exhibited limited homology to glycoprotein gL of herpes simplex virus-1 (S. K. Khattar, S. van Drunen Littel-van den Hurk, L. A. Babiuk, and S. K. Tikoo,Virology213, 28–37). To identify the BHV-1 UL1 protein, rabbit antisera were prepared against two synthetic peptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides immunoprecipitated a 16- to 17-kDa protein fromin vitrotranslatedin vitrotranscribed mRNA, BHV-1-infected MDBK cells, and purified virions. Enzymatic deglycosylation and lectin binding assays confirmed that the BHV-1 UL1 protein contains only O-linked oligosaccharides and was named glycoprotein gL. Sera against UL22 protein immunoprecipitated a protein of 108 kDa from BHV-1-infected MDBK cells and purified virions, which was modified only by N-linked oligosaccharides and was named glycoprotein gH. Glycoprotein gL expressed by recombinant vaccinia virus was properly processed and secreted into the medium. In contrast glycoprotein gH expressed by recombinant vaccinia virus was found to be retained in the rough endoplasmic reticulum. However, gH coexpressed with gL by recombinant vaccinia viruses was properly processed and transported to the cell surface, suggesting that complex formation between gH and gL is necessary for the proper processing and transport of gH but not gL. In addition gH–gL complex formation is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane