Progress 01/01/14 to 09/30/18
Outputs
Target Audience:Faculty and students at a veterinary school Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This work has resulted in the completion of a PhD doctoral dissertation entitled "Type 2 Bovine Viral Diarrhea Virus Npro Suppresses Type 1 Interferon Pathway Signaling in Bovine Cells and Augments Bovine Respiratory Syncytial Virus Replication. Abdulrahman Abdulaziz A. Alkheraif, PhD, July 2018 under the supervision of Professor Clayton L. Kelling. How have the results been disseminated to communities of interest?This work has resulted in the preparation and publication of a manuscript entitled "Type 2 BVDV Npro suppresses IFN-1 pathway signaling in bovine cells and augments BRSV replication. A. A. Alkheraif, C. L. Topliff, J. Reddy, C. Massilamany, R. O. Donis, G. Meyers, K. M. Eskridge, C. L. Kelling. Virology 507 (2017); 123-134." What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The bovine respiratory disease complex (BRDC) has a major negative impact on animal well-being and profitability in the U.S. cattle industry. BRDC outbreaks are caused by simultaneous interactions of multiple pathogens in affected animals. Bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) are ubiquitous pathogens and both viruses contribute significantly to causing BRDC. BVDV plays an indirect role in causing BRDC by immunosuppressing the host while BRSV acts directly causing bronchopneumonia. We have characterized the functional role of certain host cell signals influenced by BVDV and BRSV infections that mediate IFN antagonism and enhancement of BRSV replication during co-infection. Specifically, we confirmed the role of type 2 BVDV Npro in the antagonism of IFN-1 in BVDV2-infected cells as well as in BRSV/BVDV-2 co-infected cells. We further determined inhibition of the IRF-3 pathway by BVDV2 Npro in cells co-infected with BRSV/BVDV2-wt resulting in enhanced BRSV replication. This enhancement of BRSV replication in vitro correlates with the in vivo increased pathogenicity of BRSV in calves co-infected with BVDV. In vitro, infection of cells with ncp BVDV having a functional Npro interferes with IFN-1 production, yet in vivo BVDV induces a sustained IFN production in postnatal animals. These findings may contribute to the understanding of the immunosuppressive effects of BVDV and the mechanism(s) involved in the increased pathogenicity of BRSV and other viral infections during co-infection with ncp BVDV, which may lead to a better understanding of the BRDC. Understanding the role of BVDV and up-regulating the host's immune response by use of a mutant BVDV while maintaining antigenicity should add to the development of a safer and more effective BVDV vaccine In response to viral infections, mammalian cells produce type 1 interferon α/β (IFN-1) resulting in the activation of hundreds of genes that transcribe new proteins directed at inhibiting virus replication. IFN induction is mediated by IFN regulatory factors that are transmitted via a pathway dependent upon intracellular receptors located in the cell cytoplasm. These receptors, known as retinoic acid inducible gene (RIG)-like receptors (RLR), are expressed in the cytosol of cells and bind viral RNA. IFN-1 induction cell signals that function via RLR-dependent pathways (including: MAVS, TBK-1, IRF-3 and IRF-7) activate transcription and play key roles among a myriad of adapter molecules in complex pathways that culminate in production of IFN-1. Major Activities/Experiments completed: Using reverse genetics a BVDV2 mutant with a dysfunctional N(pro) due to fusion with enhanced green fluorescence protein (EGFP) was generated from the noncytopathic NY93 infectious clone. The ability of the wild-type and mutant ncpBVDV2 to block the IFN-1 induction by dsRNA was evaluated using a reporter cell line (NCL1-ISRE-Luc-Hygro cells). This is a cell line stably transfected with a plasmid carrying a luciferase gene controlled by the IFN-stimulated response element (ISRE) promoter. The level of IFN production was evaluated by measuring the luciferase activity. Luciferase activity is strongly stimulated when the ISRE-Luc reporter cell line is incubated with bovine IFN-α or dsRNA. We also characterized the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways (including: MAVS, PKR, TBK-1, NF-κB, IRF-3,IRF-7, IFN-β and PIN-1) in IFN antagonism and enhancement of BRSV replication during co-infection. The temporal production of mRNA of IFN-inducing cellular factors post-infection was evaluated by real-time quantitative-RT-PCR assay. Data Collected: We determined that enhancement of BRSV replication by BVDV co-infection was associated with BVDV2 N(pro) (the first protein of the BVDV polyprotein) antagonism of IFN-1 production. Using a mutant virus, (NY93/c EGFP) with a dysfunctional N(pro) gene, we determined that IFN-1 responses in bovine cells infected with NY93/c EGFP were 22 times greater than cells infected with wild-type (wt) NY93/c (functional N(pro)). BRSV RNA levels in cells co-infected with BRSV/BVDV2-wt were 2.5, 2.8, and 2.9 copy number logs significantly greater than in cells infected with BRSV alone or co-infected with BRSV/NY93/c EGFP at 5, 7, and 9 days post-infection, respectively. N(pro) - mediated interference with host defenses plays an important role in BVDV pathogenesis. Using quantitative real-time RT-PCR, we have shown that bovine turbinate cells infected with mutant NY93/c EGFPcompared to wt NY93/c, significantly upregulated the mRNA levels of PKR (P < 0.0001), TBK (P < 0.01), IRF-3 (P < 0.0001),IRF-7 (P < 0.0001) and IFN-β (P < 0.01). No significant differences in MAVS and NF-κB mRNA, as well as the mRNA of the protein that facilitates the IRF-3 degradation, PIN-1, were observed. BRSV-infected bovine turbinate cells expressed significantly up-regulated PKR, IRF-3, IRF-7 and IFN-β mRNAs. Cells co-infected with BRSV and NY93/c EGFP significantly increased the PKR (P < 0.0001), TBK-1 (P < 0.01), IRF-3 (P < 0.0001), IRF-7 ( P < 0.0001) and IFN-β (P < 0.01) mRNA production compared to cells co-infected with BRSV and wt NY93/c. Co-infection of cells with BRSV and wt NY93/c abolished the up-regulation observed with BRSV infection alone, while co-infection with NY93/c EGFP did not. Using a dual-luciferase reporter assay, we have shown that IFN-β luciferase activity in bovine turbinate cells infected with NY93/c EGFP was significantly (P < 0.05) greater than in cells infected with wt NY93/c. The IFN-β luciferase activity in cells co-infected with BRSV and NY93/c EGFP was also significantly (P < 0.05) greater than in cells co-infected with BRSV and wt NY93/c. In contrast, we have shown that wt NY93/c significantly increased NF-κB activity compared to NY93/c EGFP and NF-κB luciferase activity in cells co-infected with BRSV/wt NY93/c was significantly (P < 0.05) greater than cells co-infected with BRSV/NY93/c EGFP. Discussion: Our findings of ncp BVDV2 inhibition of IFN-1 pathway signaling and the enhancement of BRSV replication demonstrates the important role of BVDV2 in single and co-infected cells. Infection with NY93/c EGFP (BVDV2-E having a dysfunctional Npro)induced greater levels of IFN-1 than wt NY93/c (BVDV2-wt), confirming the role of the intact type 2 BVDV Npro in the antagonism of IFN-1 in BVDV2-infected cells. NY93/c EGFP induced mRNA expression of the IRF-3 pathway signals indicating the important inhibition of the IRF-3 pathway by intact BVDV2 Npro. In vitro, wt NY03/c, unlike NY93/c EGFP, decreased mRNA expression of the IRF-3 pathway signals- in cells co-infected with BRSV/BVDV2-wt and enhanced BRSV replication. This enhancement of BRSV replication in vitro correlates with the in vivo increased pathogenicity of BRSV in calves co-infected with BVDV. In vitro, infection of cells with ncp BVDV having a functional Npro interferes with IFN-1 production, yet in vivo BVDV induces a sustained IFN production in postnatal animals. Key Outcomes: These findings contribute to the understanding of the immunosuppressive effects of BVDV and the mechanism(s) involved in the increased pathogenicity of BRSV and other viral infections during co-infection with ncp BVDV, which lead to a better understanding of the BRDC. Understanding the role of BVDV and up-regulating the host's immune response by use of a mutant BVDV while maintaining antigenicity should add to the development of a safer and more effective BVDV vaccine
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Type 2 Bovine Viral Diarrhea Virus Npro Suppresses Type 1 Interferon Pathway Signaling in Bovine Cells and Augments Bovine Respiratory Syncytial Virus Replication
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Scientists at international meeting. Faculty and students at a veterinary college. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One doctoral program is nearing completion. This PhD student is first author on one peer-reviewed scientific journal article and co-author on an additional peer-reviewed scientific journal article. How have the results been disseminated to communities of interest?Publications in peer-reviewed scientific journals. Presentations at the annual meeting of the Conference of Research Workers in Animal Diseases. Presentations at the annual Nebraska Academy of Sciences. University of Nebraska, School of Veterinary Medicine and Biomedical Sciences Seminar Series presentations. What do you plan to do during the next reporting period to accomplish the goals?We will continue working toward development of effective vaccines to contol bovine respiratory disease, which is highly important to the cattle industry. Our work will continue directed toward developing an understanding of the basis of host and pathogen interactions that result in diminished protective immune responses to respiratory tract viral co-infections of cattle. We will continue to characterize the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways in IFN antagonism and enhancement of BRSV replication during co-infection. PKR, TBK-1, IRF-3 and IRF-7 reporter plasmids have been purchased to further evaluate the temporal gene expression of IFN-inducing cellular factors post-infection using the dual-luciferase reporter assay system. In addition, we would like to characterize these responses in additional ruminant cell lines, such as alpaca and bison.
Impacts
What was accomplished under these goals?
The bovine respiratory disease complex (BRDC) has a major negative impact on animal well-being and profitability in the U.S. cattle industry. BRDC outbreaks are caused by simultaneous interactions of multiple pathogens in affected animals. Bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) are ubiquitous pathogens and both viruses contribute significantly to causing BRDC. BVDV plays an indirect role in causing BRDC by immunosuppressing the host while BRSV acts directly causing bronchopneumonia. We determined that co-infection of cattle with BRSV and BVDV leads to more severe disease than that resulting from either virus alone. To elucidate the mechanisms by which BVDV infection enhances the effects of BRSV infection we characterized host responses to BVDV/BRSV co-infections, focusing our work on the host innate immune responses. We have characterized the functional role of certain host cell signals influenced by BVDV and BRSV infections that mediate IFN antagonism and enhancement of BRSV replication during co-infection. Specifically, we confirmed the role of type 2 BVDV Npro in the antagonism of IFN-1 in BVDV2-infected cells as well as in BRSV/BVDV-2 co-infected cells. We further determined inhibition of the IRF-3 pathway by BVDV2 Npro in cells co-infected with BRSV/BVDV2-wt resulting in enhanced BRSV replication. This enhancement of BRSV replication in vitro correlates with the in vivo increased pathogenicity of BRSV in calves co-infected with BVDV. In vitro, infection of cells with ncp BVDV having a functional Npro interferes with IFN-1 production, yet in vivo BVDV induces a sustained IFN production in postnatal animals. These findings may contribute to the understanding of the immunosuppressive effects of BVDV and the mechanism(s) involved in the increased pathogenicity of BRSV and other viral infections during co-infection with ncp BVDV, which may lead to a better understanding of the BRDC. Understanding the role of BVDV and up-regulating the host's immune response by use of a mutant BVDV while maintaining antigenicity should add to the development of a safer and more effective BVDV vaccine. Objectives: The long term goals of our research are to develop new strategies to control BRDC in cattle. Our studies are centered on studying: BVDV virulence determinants and mechanisms of enhanced disease from BVDV/BRSV co-infection. We have undertaken studies to identify and characterize pathogen-specific genes that inhibit immune responses. Specifically, we propose to characterize the functional role of certain host cell signals influenced by BVDV and BRSV infections that mediate IFN antagonism and enhancement of BRSV replication during co-infection. We hypothesized that BVDV2 N(pro) enhances virulence and modulates host cell innate immune responses which augment BRSV virulence during co-infection with BVDV. In response to viral infections, mammalian cells produce type 1 interferon α/β (IFN-1) resulting in the activation of hundreds of genes that transcribe new proteins directed at inhibiting virus replication. IFN induction is mediated by IFN regulatory factors that are transmitted via a pathway dependent upon intracellular receptors located in the cell cytoplasm. These receptors, known as retinoic acid inducible gene (RIG)-like receptors (RLR), are expressed in the cytosol of cells and bind viral RNA. IFN-1 induction cell signals that function via RLR-dependent pathways (including: MAVS, TBK-1, IRF-3 and IRF-7) activate transcription and play key roles among a myriad of adapter molecules in complex pathways that culminate in production of IFN-1. Immediate Objectives: To identify BVDV immunomodulatory proteins and host cell responses which contribute to increased virulence of BRSV during co-infection with BVDV. The effects of BVDV2 N(pro) on IFN-1 cell signaling in IFN antagonism and enhancement of BRSV replication during co-infection will be characterized. Major Activities/Experiments completed: Using reverse genetics a BVDV2 mutant with a dysfunctional N(pro) due to fusion with enhanced green fluorescence protein (EGFP) has been generated from the noncytopathic NY93 infectious clone. The ability of the wild-type and mutant ncpBVDV2 to block the IFN-1 induction by dsRNA was evaluated using a reporter cell line (NCL1-ISRE-Luc-Hygro cells). This cell line is stably transfected with a plasmid that carries a luciferase gene controlled by the IFN-stimulated response element (ISRE) promoter. The level of IFN production was evaluated by measuring the luciferase activity. Luciferase activity is strongly stimulated when the ISRE-Luc reporter cell line is incubated with bovine IFN-α or dsRNA. We characterized the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways (including: MAVS, TBK-1, IRF-3 and IRF-7) in IFN antagonism and enhancement of BRSV replication during co-infection. The temporal production of mRNA of IFN-inducing cellular factors post-infection was evaluated by real-time quantitative-RT-PCR assay. Data Collected: We determined that enhancement of BRSV replication by BVDV co-infection was associated with BVDV2 N(pro) (the first protein of the BVDV polyprotein) antagonism of IFN-1 production. Using a mutant virus, (NY93/c EGFP) with a dysfunctional N(pro) gene, we determined that IFN-1 responses in bovine cells infected with NY93/c EGFP were 22 times greater than cells infected with wild-type (wt) NY93/c (functional N(pro)). Cells infected with BRSV and NY93/c EGFP had BRSV mRNA levels 3 logs lower compared to when BRSV was co-infected with the parental wt NY93. N(pro) - mediated interference with host defenses plays an important role in BVDV pathogenesis. Using a quantitative real-time RT-PCR, we have shown that bovine turbinate cells infected with mutant BVDV2 with dysfunctional N(pro) compared to wild-type BVDV2, significantly upregulated the mRNA levels of PKR, IRF-7, TBK-1, IRF-3 and IFN-β. BRSV-infected bovine turbinate cells expressed significantly up-regulated PKR, IRF-3, IRF-7 and IFN-β mRNAs. Co-infection of cells with BRSV and BVDV2-wt abolished the up-regulation observed with BRSV infection alone, while co-infection with BVDV2 having a dysfunctional N(pro) did not. No significant difference in mRNA levels were observed for MAVS, NF-κB and PIN-1. Using a dual-luciferase reporter assay, we have shown that BVDV2-wt significantly increased NF-κB activity compared to the BVDV2 with the dysfunctional N(pro), while BVDV2 with the dysfunctional N(pro) significantly increased IFN-β activity compared to BVDV2-wt. Discussion: Infection with BVDV2-E having a dysfunctional Npro induced greater levels of IFN-1 than BVDV2-wt, confirming the role of the intact type 2 BVDV Npro in the antagonism of IFN-1 in BVDV2-infected cells. BVDV2-E induced mRNA expression of the IRF-3 pathway signals indicating the important inhibition of the IRF-3 pathway by intact BVDV2 Npro. In vitro, BVDV-wt, unlike BVDV2-E, decreased mRNA expression of the IRF-3 pathway signal in cells co-infected with BRSV/BVDV2-wt and enhanced BRSV replication. This enhancement of BRSV replication in vitro correlates with the in vivo increased pathogenicity of BRSV in calves co-infected with BVDV. In vitro, infection of cells with ncp BVDV having a functional Npro interferes with IFN-1 production, yet in vivo BVDV induces a sustained IFN production in postnatal animals. Key Outcomes: These findings may contribute to the understanding of the immunosuppressive effects of BVDV and the mechanism(s) involved in the increased pathogenicity of BRSV and other viral infections during co-infection with ncp BVDV, which may lead to a better understanding of the BRDC. Understanding the role of BVDV and up-regulating the host's immune response by use of a mutant BVDV while maintaining antigenicity should add to the development of a safer and more effective BVDV vaccine
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Type 2 BVDV Npro suppresses IFN-1 pathway signaling in bovine cells and augments BRSV replication. A. A. Alkheraif, C. L. Topliff, J. Reddy, C. Massilamany, R. O. Donis, G. Meyers, K. M. Eskridge, C. L. Kelling. Virology 507 (2017); 123-134.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Faculty and students of veterinary college as well as to Investigators at an international scientific meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student research training How have the results been disseminated to communities of interest?Seminar presented to university faculty and graduate students as well as presentation at 2016 Conference of Research Workers in Animal Diseases. What do you plan to do during the next reporting period to accomplish the goals?We will confirm the preliminary findings in additional experiments to replicate the initial work by continuing to characterize the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways in IFN antagonism and enhancement of BRSV replication during co-infection. The temporal gene expression of IFN-inducing cellular factors post-infection will be further evaluated using a dual-luciferase reporter assay system.
Impacts
What was accomplished under these goals?
IIa. Impacts Improved vaccines are needed for the control of BVDV infections since currently available BVDV vaccines do not provide protection against systemic viral infections without causing adverse effects. Adverse effects are due to live-attenuated vaccine virus infection of target cells in the host lymphatic organs causing marked depletion of lymphoid cells as well as fetal infection. This research contributes to the understanding the role of BVDV infection on compromising natural defenses during infection. Up-regulating the host's immune response using a mutant BVDV while maintaining antigenicity may contribute to the development of a safer and more effective BVDV vaccine. IIb. Objective #1: To identify BVDV immunomodulatory proteins and host cell responses which are important to increased virulence of BRSV during co-infection with BVDV. The effects of BVDV N(pro) on interferon-1 cell signaling in IFN antagonism and enhancement of BRSV replication during co-infection will be characterized. Using real-time quantitative RT-PCR, we have shown that bovine turbinate cells infected with mutant BVDV2 with dysfunctional N(pro) (with or without BRSV co-infection) compared to wild-type BVDV2 (with or without BRSV co-infection), significantly upregulated mRNA of PKR and IRF-7 two hours post-infection, as well as, the mRNA of TBK-1, IRF-3 and IFN-β starting two days post-infection. No significant difference in MAVS, NF-κB or PIN-1 mRNA was observed. Reporter plasmids for use in a dual-luciferase reporter assay have been developed to further characterize the in vitro temporal production of RLR-dependent pathway gene expression. Preliminary work shows that NF-κB expression is significantly increased with wild-type BVDV2 infection (with or without BRSV co-infection) compared to BVDV2 with dysfunctional N(pro) (with or without BRSV co-infection) or BRSV infection alone starting on day 3 post-infection. Expression of IFN-β was significantly increased in cells infected with BVDV2 with dysfunctional N(pro) (with or without BRSV co-infection) beginning day 1 post-infection and continuing until day 5 post-infection. Understanding the role of BVDV and up-regulating the host's immune response using a mutant BVDV while maintaining antigenicity may contribute to the development of a safer and more effective BVDV vaccine.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
C.L. Topliff, C.A.Kuszinski, W.C.Davis, D.J. Steffen, J.A Schmitz, K.M. Eskridge, B. Charleston, J.N. Henningson, C.L. Kelling. Experimental acute infection of alpacas with BVDV type 1b alters peripheral blood and GALT leukocyte subsets. Journal of Veterinary Diagnostic Investigation
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience will include animal disease worker, diagnostic and extension workers, veterinarians as well as dairy and beef cattle produceers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training and professional development for a graduate student. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will confirm prelimary findings in additional experiments to replicate initial work by continuing tocharacterize the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways (including: MAVS, TBK-1, IRF-3 and IRF-7) in IFN antagonism and enhancement of BRSV replication during co-infection. The temporal production of mRNA and protein of IFN-inducing cellular factors post-infection will be firther evaluated by real-time quantitative-RT-PCR assays and western blot analyses using protein- and phosphoprotein-specific antibodies.
Impacts
What was accomplished under these goals?
We have shown in preliminary work that cells infected with the mutant BVDV2 NY93 with dysfunctional N(pro), synthesized a significantly (p < 0.0001) greater level of IFN compared to the wt BVDV NY93 with a functional N(pro). Further characterization of the in vitro temporal production of mRNA using real-time quantitative-RT-PCR assays suggests that mutant BVDV2 NY93 with dysfunctional N(pro) upregulates the expression and function of IFN-1 cellular signaling components IRF-3, IRF-7, PKR, TBK-1 and MAVS greater than that of the wt BVDV NY93 with a functional N(pro). Upregulation of gene expression by mutant BVDV2NY93 with or without BRSV co-infection was greater than wt BVDV2NY93 with or without BRSV co-infection. Mutant BVDV2 NY93 (with or without BRSV co-infection) upregulation of RLR-dependent pathway genes was observed as early as 2 hours post-infection, depending on the gene, and peaked 5 days post-infection for all signals analyzed. The levels of RLR-dependent pathway gene mRNA expression on day 5 post-infection of mutant BVDV2 NY93 were up regulated [IRF-3 (3.8X), IRF-7 (2.8X), PKR (3X), TBK-1 (2.3X) MAVS (1.7X)] compared to wt BVDV2 NY93. The IFN-inducing gene mRNA levels of mutant BVDVNY93 co-infected with BRSV were up regulated [IRF-3 (4.8X), IRF-7 (4.6X), PKR (4X), TBK-1 (2.8X), MAVS (2.2X)] compared to the corresponding wt BVDV2 NY93 co-infection.BVDV2 N(pro) IFN-1 antagonism and enhancement of BRSV replication correlates with the reduced upregulation of the IFN-1 cellular signaling components IRF-3, IRF-7, PKR, TBK-1 and MAVS. Up regulating the host's immune response using a mutant BVDV while maintaining antigenicity may contribute to the development of a safer and more effective BVDV vaccine.
Publications
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Progress 01/01/14 to 09/30/14
Outputs
Target Audience: The target audience will include animal disease workers, diagnostic and extension workers, veterinariansas well as dairy and beef cattle producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided an opportunity for a PhD graduate student to gain training in molecular biology as well as professional development for two faculty members. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? We are characterizing the effects of BVDV2 N(pro) on the roles of IFN-1 cell signals that function via RLR-dependent pathways (including: MAVS, TBK-1, IRF-3 and IRF-7) in IFN antagonism and enhancement of BRSV replication during co-infection. The temporal production of mRNA and protein of IFN-inducing cellular factors post-infection is being evaluated by real-time quantitative-RT-PCR assays and western blot analyses using protein- and phosphoprotein-specific antibodies.
Impacts
What was accomplished under these goals?
The bovine respiratory disease complex (BRDC) has a major negative impact on animal health, animal well-being and profitability in the U.S. cattle industry. BRDC outbreaks are caused by interactions of multiple pathogens in affected animals such as bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV). Co-infection of cattle with BRSV and BVDV leads to more severe disease than that resulting from either virus alone. Control of BVDV and BRSV infections is achieved by implementing herd health programs which includes optimizing immunity through successful vaccination. There is a need for development of more effective BVDV vaccines because currently available vaccines do not provide complete protection. We will use new insights gained to develop new engineered attenuated-live BVDV vaccines to provide improved protection against BRDC with the goal of enhancing animal health and well-being as well as improving profitability in the cattle industry in Nebraska and throughout the U.S. We have shown that cells infected with the mutant BVDV2 NY93 with dysfunctional N(pro), synthesized a significantly (p < 0.0001) greater level of IFN compared to the wt BVDV NY93 with a functional N(pro). The levels of IFN synthesized by cells infected with dysfunctional BVDV2 NY93 were 22 times greater than the functional wt BVDV2 NY93-infected cells. BRSV RNA levels in cells co-infected with the wt BVDV2 NY93 were significantly (p=0.0008) greater than in cells co-infected with dysfunctional BVDV NY93 at 5, 7 and 9 days post-infection. Preliminary results of real-time quantitative-RT-PCR assays suggest that mutant BVDV2 NY93 with dysfunctional N(pro) up regulates expression and function of IFN-1 cellular signaling components IRF-3, IRF-7 and PKR. While expression of IFN-1 cellular signaling components PIN1, NF KB and TBK-1 were not affected. BVDV2 N(pro) IFN-1 antagonism and enhancement of BRSV replication correlates with the failure to up regulate the IFN-1 cellular signaling components IRF-3, IRF-7 and PKR. This understanding may contribute to development of a safe, effective BVDV vaccine.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Steffen, D.J., C.L. Topliff, J.A Schmitz, J.R. Kammerman, J.N. Henningson, K.M. Eskridge, C.L. Kelling. 2014. Distribution of lymphoid depletion and viral antigen in alpacas experimentally infected with bovine viral diarrhea virus 1. Journal of Veterinary Diagnostic Investigation 26: 35-41.
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