Publications by authors named "Suresh K Tikoo"

61 Publications

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles.

Sci Rep 2021 Apr 27;11(1):9028. Epub 2021 Apr 27.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, S7N 5B4, Canada.

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host's metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.
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http://dx.doi.org/10.1038/s41598-021-88386-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079682PMC
April 2021

Adenovirus Core Proteins: Structure and Function.

Viruses 2021 02 28;13(3). Epub 2021 Feb 28.

Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac). University of Saskatchewan, Saskatoon, SK S7N5E3, Canada.

Adenoviruses have served as a model for investigating viral-cell interactions and discovering different cellular processes, such as RNA splicing and DNA replication. In addition, the development and evaluation of adenoviruses as the viral vectors for vaccination and gene therapy has led to detailed investigations about adenovirus biology, including the structure and function of the adenovirus encoded proteins. While the determination of the structure and function of the viral capsid proteins in adenovirus biology has been the subject of numerous reports, the last few years have seen increased interest in elucidating the structure and function of the adenovirus core proteins. Here, we provide a review of research about the structure and function of the adenovirus core proteins in adenovirus biology.
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http://dx.doi.org/10.3390/v13030388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998265PMC
February 2021

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V.

Front Microbiol 2020 6;11:579593. Epub 2021 Jan 6.

VIDO-InterVac, University of Saskatchewan, Saskatoon, SK, Canada.

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81-120, 190-210, and 380-389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21-50 and 380-389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380-389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.
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http://dx.doi.org/10.3389/fmicb.2020.579593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815533PMC
January 2021

Bovine Adenovirus-3 Tropism for Bovine Leukocyte Sub-Populations.

Viruses 2020 12 12;12(12). Epub 2020 Dec 12.

VIDO-InterVac., 120 Veterinary Road, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada.

A number of characteristics including lack of virulence and the ability to grow to high titers, have made bovine adenovirus-3 (BAdV-3) a vector of choice for further development as a vaccine-delivery vehicle for cattle. Despite the importance of blood leukocytes, including dendritic cells (DC), in the induction of protective immune responses, little is known about the interaction between BAdV-3 and bovine blood leukocytes. Here, we demonstrate that compared to other leukocytes, bovine blood monocytes and neutrophils are significantly transduced by BAdV404a (BAdV-3, expressing enhanced yellow green fluorescent protein [EYFP]) at a MOI of 1-5 without a significant difference in the mean fluorescence of EYFP expression. Moreover, though expression of some BAdV-3-specific proteins was observed, no progeny virions were detected in the transduced monocytes or neutrophils. Interestingly, addition of the "RGD" motif at the C-terminus of BAdV-3 minor capsid protein pIX (BAV888) enhanced the ability of the virus to enter the monocytes without altering the tropism of BAdV-3. The increased uptake of BAV888 by monocytes was associated with a significant increase in viral genome copies and the abundance of EYFP and BAdV-3 19K transcripts compared to BAdV404a-transduced monocytes. Our results suggest that BAdV-3 efficiently transduces monocytes and neutrophils in the absence of viral replication. Moreover, RGD-modified capsid significantly increases vector uptake without affecting the initial interaction with monocytes.
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http://dx.doi.org/10.3390/v12121431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763465PMC
December 2020

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV.

Virology 2020 07 11;546:25-37. Epub 2020 Apr 11.

VIDO-InterVac, University of Saskatchewan, SK, Canada; Veterinary Microbiology, WCVM, University of Saskatchewan, Saskatoon, SK, Canada; Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address:

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.
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http://dx.doi.org/10.1016/j.virol.2020.04.006DOI Listing
July 2020

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against .

J Immunol Res 2020 13;2020:2704728. Epub 2020 Jan 13.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as and sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 g of CpG-ODN or saline ( = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs ( = 3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4 and CD8 T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 g and 50 g of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of (1 × 10 or 1 × 10 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 g and 50 g of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.
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http://dx.doi.org/10.1155/2020/2704728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201825PMC
March 2021

The dynamics of molecular evolution of emerging avian reoviruses through accumulation of point mutations and genetic re-assortment.

Virus Evol 2020 Jan 10;6(1):veaa025. Epub 2020 May 10.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4, Canada.

In the last decade, the emergence of variant strains of avian reovirus (ARV) has caused enormous economic impact in the poultry industry across Canada and USA. ARVs are non-enveloped viruses with ten segments of double-stranded RNA genome. So far, only six genotyping cluster groups are identified worldwide based on sequence analysis of the σC protein encoded by the S1 segment. In this study, we performed deep next generation whole-genome sequencing and analysis of twelve purified ARVs isolated from Saskatchewan, Canada. The viruses represent different genotyping cluster. A genome-wide sequence divergence of up to 25 per cent was observed between the virus isolates with a comparable and contrasting evolutionary history. The proportion of synonymous single-nucleotide variations (sSNVs) was higher than the non-synonymous (ns) SNVs across all the genomic segments. Genomic segment S1 was the most variable as compared with the other genes followed by segment M2. Evidence of positive episodic/diversifying selection was observed at different codon positions in the σC protein sequence, which is the genetic marker for the classification of ARV genotypes. In addition, the N-terminus of σC protein had a persuasive diversifying selection, which was not detected in other genomic segments. We identified only four ARV genotypes based on the most variable σC gene sequence. However, a different pattern of phylogenetic clustering was observed with concatenated whole-genome sequences. Together with the accumulation of point mutations, multiple re-assortment events appeared as mechanisms of ARV evolution. For the first time, we determined the mean rate of molecular evolution of ARVs, which was computed as 2.3 × 10 substitution/site/year. In addition, widespread geographic intermixing of ARVs was observed between Canada and USA, and between different countries of the world. In conclusion, the study provides a comprehensive analysis of the complete genome of different genotyping clusters of ARVs including their molecular rate of evolution and spatial distribution. The new findings in this study can be utilized for the development of effective vaccines and other control strategies against ARV-induced arthritis/tenosynovitis in the poultry industry worldwide.
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http://dx.doi.org/10.1093/ve/veaa025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211400PMC
January 2020

Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections.

Sci Rep 2019 01 23;9(1):341. Epub 2019 Jan 23.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3-4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4 and CD8 T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4 and CD8 T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.
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http://dx.doi.org/10.1038/s41598-018-36588-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344490PMC
January 2019

US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Bovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the -Golgi apparatus but not in the -Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔU47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection. Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the -Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.
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http://dx.doi.org/10.1128/JVI.02151-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401447PMC
March 2019

Generation of infectious clone of bovine adenovirus type I expressing a visible marker gene.

J Virol Methods 2018 11 31;261:139-146. Epub 2018 Aug 31.

College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China; Wuhan BioCom Pioneers Co. Ltd., China. Electronic address:

Background And Objective: Bovine adenovirus type 3 (BAdV3) has been widely used as a vector for vaccine research and development, whereas BAdV1 biology and BAdV1-based vectored vaccine have been less frequently reported. We aimed to construct an infectious BAdV1 clone and explore the functions of BAdV1 genes.

Methods: First, the infectious clone of pUCBAdV1 containing the full-length BAdV1 DNA and the recombinant plasmid pUCBAV1-EYFP expressing the marker gene EYFP were constructed. Then, the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued. The stability of the exogenous EYFP gene was analyzed by continuous passage, PCR, and western blotting. Finally, the virus neutralization titer of the rescued viruses was evaluated.

Results: The infectious clones of pUCBAdV1 and pUCBAV1-EYFP were constructed and the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued successfully. Moreover, the results showed that the EYFP gene could be expressed continuously. In addition, the replication of rBAdV1-EYFP was less efficient than that of the wild-type virus wtBAdV1 in vitro, while the efficacy of BAdV101 replication was almost the same as that of wtBAdV1. Furthermore, the neutralization test showed that the neutralization titer of rBAdV1-EYFP was consistent with that of wtBAdV1.

Conclusion: To our knowledge, the infectious genome of pUCBAV1-EYFP expressing a visible marker gene EYFP was constructed for the first time, and the finding forms a basis for the development of BAdV1-based efficient vectored vaccine.
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http://dx.doi.org/10.1016/j.jviromet.2018.08.020DOI Listing
November 2018

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.

J Virol 2018 10 26;92(20). Epub 2018 Sep 26.

VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

The adenovirus E3 region encodes proteins that are not essential for viral replication The porcine adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.7 expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.7 suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.7 by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions. Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of porcine adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an adenovirus E3-encoded protein is an essential structural protein.
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http://dx.doi.org/10.1128/JVI.00680-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158433PMC
October 2018

Domains of bovine adenovirus-3 protein 22K involved in interacting with viral protein 52K and cellular importins α-5/α-7.

Virology 2018 09 24;522:209-219. Epub 2018 Jul 24.

VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3; Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3; Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3. Electronic address:

The L6 region of bovine adenovirus-3 (BAdV-3) encodes unspliced and spliced proteins named 22K and 33K, respectively. Earlier, anti-22K sera detected two proteins of 42- and 37-kDa in infected cells and 42-kDa protein in transfected cells. Here, we demonstrate that 22K protein localizes to the nucleus of BAdV-3 infected or transfected cells. Analysis of mutant 22K proteins suggested that amino acids 231-250 of non-conserved C-terminus of 22K are required for nuclear localization. The nuclear import of 22K appears to utilize multiple importin (α-5 and α-7) of importin α/β nuclear import pathway. Mutational analysis of 22K identified four basic residues RRRK, which apparently are essential for the nuclear localization of 22K. Our results suggest that the nuclear localization of 22K appear essential for virus replication and production of progeny BAdV-3. Furthermore, we demonstrate that N-terminus amino acid 35-65 conserved in 22K and 33K interact with 52K protein in BAdV-3 infected cells.
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http://dx.doi.org/10.1016/j.virol.2018.07.015DOI Listing
September 2018

Bovine adenovirus-3 protein VIII associates with eukaryotic initiation factor-6 during infection.

Cell Microbiol 2018 08 17;20(8):e12842. Epub 2018 Apr 17.

VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein-protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2-hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S-transferase-pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus-3 (BAdV-3) infected cells. The C-terminus amino acids 147 to 174 of protein VIII and N-terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV-3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV-3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.
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http://dx.doi.org/10.1111/cmi.12842DOI Listing
August 2018

Broad spectrum protection of broiler chickens against inclusion body hepatitis by immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine.

Res Vet Sci 2018 Jun 5;118:262-269. Epub 2018 Mar 5.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada. Electronic address:

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was considered a secondary disease in broiler chickens associated with immunosuppression. However, we previously reported the occurrence of IBH as a primary disease in the broiler chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the objectives of this study were to develop an immunization strategy in broiler breeders using live FAdV 11-1047 and FAdV8a-TR59 to confer homologous and heterologous protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1 × 10 TCID/bird) and FAdV11-1047 (1 × 10 TCID/bird), FAdV8a-TR59 (1 × 10 TCID/bird) and FAdV11-1047 (1 × 10 TCID/bird) or FAdV8b (1 × 10 TCID/bird) transferred substantial levels of neutralizing antibodies to their progeny. The efficacy of maternal antibodies was studied by challenging 14-day old broiler chickens with 1 × 10 TCID of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were significantly protected against all serotypes of FAdV (P < 0.05). Comingling of unvaccinated broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59 and FAdV11-1047.
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http://dx.doi.org/10.1016/j.rvsc.2018.03.003DOI Listing
June 2018

Intrapulmonary Delivery of CpG-ODN Microdroplets Provides Protection Against Escherichia coli Septicemia in Neonatal Broiler Chickens.

Avian Dis 2017 12;61(4):503-511

A Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine phosphodiester guanine (CpG) motifs (CpG-ODN) are effective immunostimulatory agents against a variety of viral, bacterial, and protozoan diseases in different animals including poultry. We have recently demonstrated that in ovo injection of CpG-ODN confers protection in neonatal chickens against bacterial septicemias. The objective of this study was to investigate the effectiveness of needle-free intrapulmonary (IPL) delivery of CpG-ODN microdroplets against Escherichia coli infection in neonatal chicks. In the present study, we used 880 chicks in total keeping 40 chicks per group. Chicks were delivered CpG-ODN or saline by IPL at the day 1 of hatch. Three days later, chicks were challenged with two doses (1 × 10 CFU, n = 20 or 1 × 10 CFU, n = 20) of E. coli. Chicks treated with CpG-ODN by the IPL route had significantly lower clinical signs and bacterial load compared to the group treated with saline ( P < 0.05). CpG-ODN-treated groups were significantly protected against E. coli septicemia. We observed dose- and exposure time-dependent immunoprotective effects of IPL CpG-ODN in chicks. We found that IPL delivery of CpG-ODN can induce protective immunity as early as 6 hr that remains effective at least until day 5 post-treatment. Moreover, there were no adverse effects of IPL delivery of CpG-ODN on growth or mortality up to 42 days of age. Based on these findings, it can be suggested that CpG-ODN delivery by IPL route can be a promising alternative to antibiotics for inducing protective immunity in chicks during the critical first week of neonatal life.
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http://dx.doi.org/10.1637/11684-060617-Reg.1DOI Listing
December 2017

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH).

Vaccine 2018 01 29;36(5):744-750. Epub 2017 Dec 29.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada. Electronic address:

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 10 TCID of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 10TCID of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log. Approximately 26 ± 7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 10 TCID/bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98-100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH.
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http://dx.doi.org/10.1016/j.vaccine.2017.12.047DOI Listing
January 2018

Induction of PrP-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease.

Prion 2017 Sep;11(5):368-380

a Vaccine and Infectious Disease Organization, University of Saskatchewan , Saskatoon , Saskatchewan , Canada.

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrP into PrP. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrP-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrP-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.
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http://dx.doi.org/10.1080/19336896.2017.1367083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639826PMC
September 2017

Phenotypic, genotypic and antigenic characterization of emerging avian reoviruses isolated from clinical cases of arthritis in broilers in Saskatchewan, Canada.

Sci Rep 2017 06 15;7(1):3565. Epub 2017 Jun 15.

Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

In recent years, emerging strains of pathogenic arthrogenic avian reovirus (ARV) have become a challenge to the chicken industry across USA and Canada causing significant economic impact. In this study, we characterized emerging variant ARV strains and examined their genetic and antigenic relationship with reference strains. We isolated 37 emerging variant ARV strains from tendons of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in Saskatchewan, Canada. Viral characterization using immunocytochemistry, gold-immunolabeling and electron microscopy revealed distinct features characteristic of ARV. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the viral Sigma C gene revealed genetic heterogeneity between the field isolates. On phylogenetic analyses, the Sigma C amino acid sequences of the isolates were clustered into four distinct genotypic groups. These ARV field strains were genetically diverse and quite distant from the vaccine and vaccine related field strains. Antibodies produced against a commercial Reo 2177 vaccine did not neutralize these variants. Moreover, structure based analysis of the Sigma C protein revealed significant antigenic variability between the cluster groups and the vaccine strains. To the best of our knowledge, this is the first report on the genetic, phenotypic and antigenic characterization of emerging ARVs in Canada.
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http://dx.doi.org/10.1038/s41598-017-02743-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472580PMC
June 2017

Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b.

Vaccine 2017 05 7;35(20):2716-2722. Epub 2017 Apr 7.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Electronic address:

Inclusion body hepatitis (IBH) is an economically important diseases in broiler chicken industry. Several serotypes of fowl adenovirus (FAdV) can cause IBH, among them, serotype FAdV-8b is associated with the majority of the IBH cases in Canada. Here, we evaluated FAdV-8b virus-like particles (VLPs) and recombinant FAdV-8b fiber proteins (expressed in E. coli) as potential broiler-breeder vaccines against IBH. For assessing the immunogenicity of vaccines, we investigated both humoral and cellular immunity. The humoral immune response was evaluated by determining total IgY and virus-neutralizing antibody in serum at 14, 28, 35 and 60days post-immunization (dpi). We examined cellular immunity using flow cytometry by determining CD4:CD8 ratio change in peripheral blood after the booster vaccination. The protective effect of vaccines was tested by challenging 14day-old progeny (n=30/group) carrying maternal antibodies (MtAb) by challenging with virulent FAdV-8b virus (1×10 TCID, FAdV-8b-SK). Although total IgY levels were comparable in all groups, the neutralizing antibody response in broiler-breeders at 35 and 60 dpi was significantly (p<0.05) higher those vaccinated with FAdV-8b VLPs followed by FAdV-8b fiber compared to fiber-knob. Moreover, vaccines comprised of FAdV-8b VLPs and FAdV-8b fiber rather than FAdV-8b fiber-knob efficiently elicited the cell-mediated immune response as evidenced by a statistically significant (p<0.05) CD8 T-cell proliferative response in broiler-breeders four days after the booster vaccination. Unlike FAdV-8b fiber-knob, FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders were able to transfer a substantial amount (28.4±9%) of MtAb to their progeny. Challenge revealed that MtAb provided 100% and 82.7% protection in progeny hatched from FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders, respectively. Collectively, our data suggest that FAdV-8b subunit vaccine-induced MtAb efficiently protected progeny against clinical IBH and broiler-breeder vaccination with subunit vaccines is a potential approach to protect against IBH.
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http://dx.doi.org/10.1016/j.vaccine.2017.03.075DOI Listing
May 2017

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine.

Vaccine 2017 02 12;35(6):882-888. Epub 2017 Jan 12.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada. Electronic address:

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×10 EID) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8 T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.
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http://dx.doi.org/10.1016/j.vaccine.2017.01.005DOI Listing
February 2017

Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) and avian beta defensin as adjuvants in chickens.

Vaccine 2017 02 10;35(6):981-986. Epub 2017 Jan 10.

Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, SK S7N 5E3, Canada. Electronic address:

Inclusion body hepatitis (IBH) is one of the major viral infections causing substantial economic loss to the global poultry industry. The disease is characterized by a sudden onset of mortality (2-30%) and high morbidity (60-70%). IBH is caused by a number of serotypes of fowl adenovirus with substantially low levels of serotype cross protection. Thus far, there is no effective and safe vaccine commercially available in the North America for the control of IBH in chickens. Poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) is a high molecular weight, biodegradable water soluble polymer that has been well characterized as a safe and effective adjuvant for a number of experimental veterinary vaccines. Similarly, host defence peptides, including β-defensins, have also been shown to exhibit strong adjuvant potential. In this study, we evaluated the adjuvant activity of PCEP and avian beta defensin (ABD) in a vaccine formulation containing inactivated fowl adenovirus (FAdV) serotype 8b administered in ovo. Our data showed that a combination of PCEP and inactivated virus is capable of inducing a robust and long lasting antibody response. Moreover, significant enhancement of IFN-γ, IFN-α, IL-12(p40) and IL-6 gene expression under the influence of PCEP suggests that as an in ovo adjuvant PCEP has the ability to activate a substantial balanced immune response in chickens. To our knowledge, these are the first studies in which PCEP and ABD have been characterized as adjuvants for the development of an in ovo poultry vaccine. It is expected that these preliminary studies will be helpful in the development of safer and more effective in ovo vaccine against IBH and other infectious diseases affecting chickens.
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http://dx.doi.org/10.1016/j.vaccine.2016.12.023DOI Listing
February 2017

Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap.

Front Microbiol 2016 27;7:2119. Epub 2016 Dec 27.

Vaccine and Infectious Disease Organization - International Vaccine Centre, University of Saskatchewan, Saskatoon, SKCanada; Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SKCanada; Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, SKCanada.

Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport and induction of interferon production. This study was conducted to investigate the function of bovine adenovirus (BAdV)-3 pVIII during virus infection. Here, we provided evidence regarding involvement of DDX3 in cap dependent cellular mRNA translation and demonstrated that targeting of DDX3 by adenovirus protein VIII interfered with cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacted with DDX3 in transfected and BAdV-3 infected cells. pVIII inhibited capped mRNA translation and by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E, eIF4G, and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in cap binding complex. The total amount of eIFs appeared similar in uninfected or infected cells as BAdV-3 did not appear to degrade eIFs. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E, or PABP. These data indicate that interaction of pVIII with DDX3 interferes with the binding of eIF3, eIF4E and PABP to the 5' Cap. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA possibly by interfering with the recruitment of eIFs to the capped cellular mRNA.
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http://dx.doi.org/10.3389/fmicb.2016.02119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5186766PMC
December 2016

Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada.

Res Vet Sci 2016 Oct 4;108:54-9. Epub 2016 Aug 4.

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, SK, Canada. Electronic address:

Antibiotic-free and safe animal products are most desirable among consumers. However, ensuring safe poultry products is a challenging task when the chicken immune system is compromised. Infectious bursal disease virus (IBDV) causes immunosuppression and predisposes chickens to secondary infections. Breeder vaccination against IBDV is routinely practiced for producing chicks with maternally-derived antibody (MAb) to prevent infection in newly hatched chicks. The majority of IBDV circulating in Canadian farms are variant strains (vIBDV). Whether circulating vIBDV strains are immunosuppressive in chicks or are amenable to current vaccine regimens has not previously been tested through challenge studies. In this study, one-day-old broiler chicks (n=240) carrying MAb were obtained from broiler breeders vaccinated with commercial IBDV vaccines. In the first set of experiments (n=40/group), at six days post-hatch, one group was challenged with a Canadian field isolate, vIBDV (strain-SK09) (3×10(3) EID50). The second and the third groups (controls) were inoculated with non-immunosuppressive IBDV D-78 (10×10(3) TCID50) and saline, respectively. Histopathological examination on days 14 and 30 post-challenge revealed that despite the high level of MAb, vIBDV (SK09) caused severe bursal damage in chicks. Another set of experiments with treatment groups as above, demonstrated that pre-exposure of chicks with vIBDV (SK09) caused immunosuppression resulting in significantly higher mortality and disease severity in chicks challenged with a virulent strain of Escherichia coli (E. coli). Our data provide evidence that IBDV strains circulating in Canada are immunosuppressive, not amenable to current anti-IBDV vaccination strategy, and a potential threat to antibiotic-free chicken farming.
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http://dx.doi.org/10.1016/j.rvsc.2016.08.002DOI Listing
October 2016

Bovine adenovirus type 3 virions cannot be rescued after full-length viral genome transfection in the absence of detectable polypeptide IX.

J Vet Sci 2017 Jun;18(2):217-227

College of Veterinary Medicine, North-west A&F University, Yangling 712100, China.

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.
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http://dx.doi.org/10.4142/jvs.2017.18.2.217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489469PMC
June 2017

Deletion of pV affects integrity of capsid causing defect in the infectivity of bovine adenovirus-3.

J Gen Virol 2016 10 11;97(10):2657-2667. Epub 2016 Aug 11.

Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada.

Members of the genus Mastadenovirus including bovine adenovirus 3 (BAdV-3) encode a genus-specific unique protein named pV. The pV encoded by BAdV-3 is a protein of 423 aa showing 40.9 % identity to pV of human adenovirus 2. Here, we report the construction and analysis of recombinant BAdV-3 (BAV.dV) containing deletion of pV. The BAV.dV could only be isolated in CRL.pV cells expressing pV, suggesting that pV appears essential for the infection of BAdV-3. Analysis of BAV.dV suggested that despite affecting some late gene expression in virus-infected cells, there was no significant difference in the incorporation of viral proteins in the mature virions. Moreover, analysis of mature virions revealed degraded capsids leading to change in morphology and infectivity of BAV.dV. Furthermore, analysis of the genome sequence of different clones of BAV.dV passaged in different cell lines revealed no mutations in core proteins pVII and pX\Mu suggesting that the replication defect may not be rescued. Our results suggest that pV is required for proper viral assembly of BAdV-3 as lack of pV produces aberrant capsids. Moreover, altered capsids lead to the production of non-infectious BAV.dV virions.
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http://dx.doi.org/10.1099/jgv.0.000570DOI Listing
October 2016

Investigation of the cause of geographic disparities in IDEXX ELISA sensitivity in serum samples from Mycobacterium bovis-infected cattle.

Sci Rep 2016 Mar 7;6:22763. Epub 2016 Mar 7.

Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada.

Accurately identifying Mycobacterium bovis-infected cattle is critical for bovine tuberculosis prevention and control. One method for identifying infected cattle is an ELISA developed by IDEXX laboratories, which detects antibodies to two M. bovis proteins, MPB70 and MPB83. The assay's sensitivity varies by geographic region, with sensitivities of 77%, 45%, and 9% in bovine serum samples from the United Kingdom (n = 126), the United States (n = 146), and Mexico (n = 128), respectively. We hypothesized that geographically-biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may explain these differing sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries. For each gene, a single, common sequence was identified in most genomes of the M. bovis strains collected in all three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA-positive genomes and three of the five ELISA-negative genomes contained the most common sequence of all four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain the geographic disparities in IDEXX ELISA sensitivity.
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http://dx.doi.org/10.1038/srep22763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780098PMC
March 2016

DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation.

J Virol 2016 Jan 20;90(7):3661-75. Epub 2016 Jan 20.

Vaccine and Infectious Disease Organization-International Vaccine Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Unlabelled: DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation.

Importance: DDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.
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http://dx.doi.org/10.1128/JVI.03010-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794679PMC
January 2016

Proteomic analysis of purified turkey adenovirus 3 virions.

Vet Res 2015 Jul 9;46:79. Epub 2015 Jul 9.

Vaccine and Infectious Disease Organization -International Vaccine Center (VIDO- InterVac1), University of Saskatchewan, Saskatoon, S7N 5E3, SK, Canada.

Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.
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http://dx.doi.org/10.1186/s13567-015-0214-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497381PMC
July 2015

Porcine retinal cell line VIDO R1 and Chlamydia suis to modelize ocular chlamydiosis.

Vet Immunol Immunopathol 2015 Aug 14;166(3-4):95-107. Epub 2015 Jun 14.

LUNAM Université, Oniris, Nantes-Atlantic College of Veterinary Medicine and Food Sciences and Engineering, UMR BioEpAR, F-44307 Nantes, France; INRA, UMR1300 Biology, Epidemiology and Risk Analysis in Animal Health, CS 40706, F-44307 Nantes, France. Electronic address:

Human ocular Chlamydia trachomatis infections can lead to trachoma, the major cause of infectious blindness worldwide. Trachoma control strategies are very helpful but logistically challenging, and a trachoma vaccine is needed but not available. Pigs are a valuable large animal model for various immunological questions and could facilitate the study of human ocular chlamydial infections. In addition, a recent study identified the zoonotic potential of Chlamydia suis, the natural pathogen of pigs. In terms of the One Health Initiative, understanding the host-pathogen-interactions and finding a vaccine for porcine chlamydia infections would also benefit human health. Thus, we infected the porcine retinal cell line VIDO R1 with C. suis and analyzed the chlamydial life cycle and the innate immune response of the infected cells. Our results indicate that C. suis completes its life cycle in VIDO R1 cells within 48 h, comparable to C. trachomatis in humans. C. suis infection of VIDO R1 cells led to increased levels of various innate immune mediators like pathogen recognition receptors, cytokines and chemokines including IL6, TNFα, and MMP9, also most relevant in human C. trachomatis infections. These results illustrate the first steps in the host-pathogen-interactions of ocular C. suis infections in pigs and show their similarity to C. trachomatis infections in humans, justifying further testing of pigs as an animal model for human trachoma.
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http://dx.doi.org/10.1016/j.vetimm.2015.06.003DOI Listing
August 2015

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication.

J Gen Virol 2015 Sep 1;96(9):2749-2763. Epub 2015 Jun 1.

Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3 Canada.

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/β transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.
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http://dx.doi.org/10.1099/vir.0.000205DOI Listing
September 2015