Progress 02/01/06 to 01/31/09
Outputs
OUTPUTS: Nine oral presentations or posters were presented as reported in the 2007 CRIS (2006-2007). Pasted below are additional outputs for 2008, which brings the project total outputs to 13 presentations at conferences. Bielefeldt-Ohmann H, Smirnova, N.P., Van Campen, H., Austin, K.J., Ptitsyn, A.A., Hansen, T.R. Up-regulation of the type I interferon (IFN) response and down-regulation of SDF-1/CXCR4 signaling in blood cells from pregnant heifers carrying fetuses infected with bovine viral diarrhea virus (BVDV). Proc. Keystone Symposia "Molecular Evolution as a Driving Force in Infectious Diseases" 2008: 54. Hansen TR, Smirnova N, Campen. V. Maternal and Fetal Response to Fetal Persistent Infection with BVDV. 2008 Animal Protection & Biosecurity PD Workshop 2008; CRAWD Meeting, Chicago, IL: 28-29. Shoemaker ML, Austin, K.J., Van Campen, H., Bielefeldt-Ohmann, H., Rempel, L., Smirnova, N.P., Han, H., Montgomery, D.J., Anthony, R.V., van Olphen, A., Clapper, J.A., Hansen, T.R. Implications for the type I Interferon pathway in intrauterine growth restriction. Society of Gynecological Investigation 2008; Reproductive Sciences, V15, N2 (Supplement): 123A. Smirnova N.P. PAA, Austin K.J., Bielefeldt-Ohmann H., Van Campen H., Hansen T.R Fetal persistent infection with bovine viral diarrhea virus causes down-regulation of T cell receptor and CXCR4 pathways in blood of cows. Journal of Leukocyte Biology 2008; Supplement: Leukocytes: Tissue interactions, Homeostasis, and Host Defense, 41st Annual Meeting of the Society for Leukocyte Biology, Denver, 2008, p. 40. PARTICIPANTS: Thomas R. Hansen, Colorado State University Natalia P. Smirnova, Colorado State University Hana Van Campen, Colorado State University Megan L. Shoemaker, Colorado State University Helle H. Bielefeldt-Ohmann, Colorado State University Alberto L. van Olphen, University of Wyoming Donald L. Montgomery, University of Wyoming Kathleen J. Austin, University of Wyoming Hyungchul Han, Colorado State University TARGET AUDIENCES: Practicing Bovine Veterinarians, bovine practitioners, outreach educators PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Bovine viral diarrhea virus (BVDV) infection occurs in the cattle population worldwide. Non-cytopathic (ncp) BVDV strains cause transient infection (TI) or persistent infection (PI) depending on the host's immune status. Immunocompetent adult animals and fetuses in late gestation resolve the infection. Fetal infection in early gestation results in PI with chronic viremia and life-long viral shedding, ensuring virus perpetuation in the population. Eighteen pregnant heifers, divided into 3 groups, were intranasally inoculated with ncp BVDV2 virus early (day 75) and late (day 175) in gestation, or kept BVDV-naive. Fetuses were retrieved on day 190. Viral infection was confirmed following each inoculation. BVDV RNA was detected in multiple tissues of infected fetuses of both TI and PI groups on day 190 of gestation with significantly higher amounts of viral RNA in PI fetuses when compared to TI fetuses. PI fetuses had lower weight and were smaller based on heart girth and crown rump length. The ponderal index was lower in PI fetuses, indicating significant intrauterine growth retardation. PI fetal femurs had a thickened cortex, leading to a significantly smaller internal diameter and medullar space. Serum from heifers inoculated with ncp BVDV2 in early pregnancy (day 75) showed high antiviral activity by day 7 p.i (day 82 of gestation). Serum from TI heifers and fetuses displayed substantial type I Interferon (IFN-I) antiviral activity on day 15 p.i. (day 190 of pregnancy). Western blot analysis of proteins from fetal spleen presented high amounts of both conjugated and free IFN stimulated gene 15 kDa (ISG15) protein in TI fetuses, while low ISG15 was detected in PI or control fetuses. Affymetrix microarray screen of day 190 maternal blood cell mRNA (n=3 heifers per group) revealed upregulation of IFN-I induced genes (i.e., ISG15) and pathways in TI compared to PI or control heifers. Affymetrix microarray screen of day 160 total white blood cell mRNA from pregnant heifers of PI (n=5) and control heifers (n=4) revealed differential expression of ~1000 genes from numerous pathways. Members of the IFN-I pathway and members of the chemokine family and their receptors, were differentially expressed in blood of heifers with PI fetuses. The two most differentially expressed genes were a chemokine ligand (upregulated) and a chemokine receptor (downregulated), which were confirmed using RT-PCR. It is suggested that PI fetuses lack an adaptive recognition of the viral antigen resulting in an inability to resolve the infection. However, the fetus does display an IFN-I response to the persisting stimulus of the infection, which may have negative consequences for growth and development. Differential maternal blood cell expression of chemokines and their receptors in heifers carrying PI fetuses and altered fetal antiviral responses will be examined further in context of clarifying mechanisms that result in developmental defects caused by the persistence of BVDV in PI fetuses. Also blood cell markers for heifers carrying PI fetuses that were discovered using microarray approaches will be tested for utility in clinical settings.
Publications
- Refereed Publications (2008 manuscripts are listed. Others have already been reported in previous CRIS entries. Note some of these are updates to the electronic publication date)
- Bielefeldt-Ohmann H, Tolnay AE, Reisenhauer CE, Hansen TR, Smirnova N, Van Campen H. Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: viral spread and molecular neuropathology. J Comp Pathol 2008; 138: 72-85.
- Montgomery DL, Van Olphen A, Van Campen H, Hansen TR. The fetal brain in bovine viral diarrhea virus-infected calves: lesions, distribution, and cellular heterogeneity of viral antigen at 190 days gestation. Vet Pathol 2008; 45: 288-296.
- Shoemaker ML, Smirnova, N., Bielefeldt-Ohmann, H., Austin, K.J., van Olphen, A., Clapper, J., Hansen, T.R. Differential expression of the type I interferon pathway during persistent and transient bovine viral diarrhea virus infection. JICR 2008; In press.
- Hansen TR, Smirnova N, Campen. V. Maternal and Fetal Response to Fetal Persistent Infection with BVDV. 2008 Animal Protection & Biosecurity PD Workshop 2008; CRAWD Meeting, Chicago, IL: 28-29.
- Shoemaker ML, Austin, K.J., Van Campen, H., Bielefeldt-Ohmann, H., Rempel, L., Smirnova, N.P., Han, H., Montgomery, D.J., Anthony, R.V., van Olphen, A., Clapper, J.A., Hansen, T.R. Implications for the type I Interferon pathway in intrauterine growth restriction. Society of Gynecological Investigation 2008; Reproductive Sciences, V15, N2 (Supplement): 123A.
- Smirnova N.P., Ptitsyn A.A., Austin K.J., Bielefeldt-Ohmann H., Van Campen H., Hansen T.R. Fetal persistent infection with bovine viral diarrhea virus causes down-regulation of T cell receptor and CXCR4 pathways in blood of cows. Journal of Leukocyte Biology, 2008. Supplement: Leukocytes: Tissue interactions, Homeostasis, and Host Defense. 41st Annual Meeting of the Society for Leukocyte Biology, Denver, 2008, p. 40.
- Patents and Licensing Agreements (In 2009 these were combined as a CIP into one patent application that is under review.International Patent Application PCT/US07/60375, filed January 11, 2007,titled Surrogate Markers for Viral Infections and Other Inflammatory Responses.United States Utility Patent Application, serial number 11/622,124, filed January 11, 2007, titled Surrogate Markers for Viral Infections and Other Inflammatory Responses Provisional Patent leading up to these applications was filed in January, 2006.
- Smirnova NP, Bielefeldt-Ohmann H, Van Campen H, Austin KJ, Han H, Montgomery DL, Shoemaker ML, van Olphen AL, Hansen TR. Acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type I interferon response in pregnant cows and fetuses. Virus Res 2008; 132: 49-58.
- Smirnova NP, Ptitsyn AA, Austin KJ, Bielefeldt-Ohmann H, Van Campen H, Han H, van Olphen A, Hansen TR. Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways. Physiol Genomics 2008, In Press.
- Abstracts or Proceedings Conferences (2008 manuscripts are listed. Others have already been reported in previous CRIS entries)
- Ashley R, Henkes, L., Anthony, R., McBroom, K., Pru, J., Hansen, T. Isg15 is a molecular sentinel that functions to assist mothers in coping with environmental stressors impossed on pregnancy. Society for the Study of Reproduction 2008; 41st Annual Meeting: 186; Abstract 502.
- Bielefeldt-Ohmann H, Smirnova, N.P., Van Campen, H., Austin, K.J., Ptitsyn, A.A., Hansen, T.R. Up-regulation of the type I interferon (IFN) response and down-regulation of SDF-1/CXCR4 signaling in blood cells from pregnant heifers carrying fetuses infected with bovine viral diarrhea virus (BVDV). Proc. Keystone Symposia Molecular Evolution as a Driving Force in Infectious Diseases 2008: 54.
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Progress 02/01/06 to 01/31/07
Outputs
OUTPUTS: The research was presented in the following public meetings and conferences. 1.Hansen T.R., van Olphen A.L., Rempel L.A., Han H., Clapper J.A., Montgomery D.L., Berg B.M., van Campen H. Microarray screening reveals differential gene expression in blood from calves that are persistently infected with bovine viral diarrhea virus. In: Proceedings of the BVDV Special Symposium, Denver, 2006, p. 46. 2.Smirnova N., Van Campen H., Bielefeldt-Ohmann H., Austin K.J., Han H., Montgomery D., van Olphen A.L., Hansen T.R. BVDV induces differential gene expression in blood of pregnant heifers. In: Proceedings of the 87th Annual Meeting of the Conference of Research Workers in Animal Disease 2006, Chicago, Illinois, p. 166. 3.Smirnova N., Van Campen H., Bielefeldt-Ohmann H., Austin K.J., Han H., Montgomery D., van Olphen A.L., Hansen T.R. Analysis of maternal gene expression in pregnant heifers infected with BVDV. In: 3rd Annual Biomedical Sciences Research Retreat. YMCA of the Rockies,
2006, p.15. 4.Shoemaker M., Smirnova N., Van Campen H., Bielefeldt-Ohmann H., Austin K.J., Han H., Montgomery D.J., van Olphen A.L., Hansen T.R. Bovine Viral Diarrhea Virus interaction with the fetal immune system and fetal development. In: 3rd Annual Biomedical Sciences Research Retreat. YMCA of the Rockies, 2006, p.35. 5.Smirnova N., Bielefeldt-Ohmann H., Van Campen H., Austin K.J., Han H., Montgomery D.L., van Olphen A.L., Hansen T.R. Pronounced antiviral response in maternal and fetal ncpBVDV infection is accompanied by upregulation of ISG15. Gordon Research Conferences "Viruses and Cells", Tilton, NH, USA, 2007. 6.Shoemaker M., Smirnova N., Van Campen H., Bielefeldt-Ohmann H, Austin K.J., Han H., Montgomery D.J., van Olphen A.L., Hansen T.R. Maternal viral infection during pregnancy programs fetal development and induces a differential type I Interferon response. In: Biology of Reproduction, Special Issue, 40th Annual Meeting of the Society for the Study of Reproduction, San
Antonio, 2007, p.191. 7.Shoemaker M., Smirnova N., Van Campen H., Bielefeldt-Ohmann H., Austin K.J., Han H., Montgomery D.J., van Olphen A.L., Hansen T.R. Bovine viral diarrhea virus infection during fetal development. In: 8th Annual CVMBS Research Day, Scientific proceedings. CSU, Fort Collins, 2007, p.37. 8.Reisenhauer C.E., Tolnay A.E., Smirnova N., Montgomery D.L., Van Campen H., Bielefeldt-Ohmann H. Transplacental infection of bovine fetuses with non-cytopathic bovine viral diarrhea virus type II: viral spread and brain lesions. In: 8th Annual CVMBS Research Day, Scientific proceedings. CSU, Fort Collins, 2007, p.47. 9.Shoemaker, M.L., Rempel, L.A., Austin, K.J., Smirnova, N.P., Clapper, J.A., Hansen, T.R. Gene Expression in Steers Persistently Infected with Bovine Viral Diarrhea Virus. In: 4th Annual Biomedical Sciences Research Retreat. Pingree Park, CO, 2007, p.29.
PARTICIPANTS: Thomas R. Hansen, PhD, Traubert Professor Director, Animal Reproduction and Biotechnology Laboratory, Director, Division of Physiology, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences Colorado State University. Principle Director of the Project. Natalia Smirnova, PhD, Research Scientist II Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences Colorado State University. Primary scientist in charge of maternal studies and assistance with training the MS student. Megan Shoemaker, MS, Graduate Student Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences Colorado State University. Graduate student in charge of fetal studies. Christina Weiner, DVM, MS graduate Student Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences and Department
of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences Colorado State University. CSU Comparative Medicine Resident completing MS studies on the fetus (i.e., bone marrow). Hana Van Campen, DVM, PhD, Diplomate ACVM Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University. Responsible for in vivo experimental infections, C-sections, necropsies, serum neutralization assay, consultations on the studies. Helle Bielefeldt-Ohmann, DVM, PhD Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Pathologist and consultant on the studies. Alberto L. van Olphen, DVM/PhD Department of Global Health, College of Public Health, University of South Florida. Co-PD on the original grant. Collaborated with the PD to develop the original experimental plan and is currently a consultant. Donald L.
Montgomery, DVM, PhD, Diplomate ACVP Department of Veterinary Sciences, University of Wyoming. Directed all necropsies and completed pathology and histology on fetal brains. Hyungchul Han, PhD, Department of Animal Sciences, Colorado State University. Assisted with all animal care, blood sampling and tissue sampling and real time PCR. Kathleen J. Austin, MS Department of Animal Sciences, University of Wyoming. Assisted with all animal care, blood sampling and tissue sampling and real time PCR. Coordinated necropsies and storage of samples. Andrey A. Ptitsyn, PhD Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University. Provided analysis and interpretation of microarray data.
TARGET AUDIENCES: Target audience is the bovine industry with some relevance to the ovine industry and to viral infections in general.
PROJECT MODIFICATIONS: The primary modification in experimental design was to implement use of the Affymetrix DNA chip in place of the origninally proposed subtractive library. Also, the experimental design is much more extensive in context of what was originally proposed.
Impacts
Bovine viral diarrhea virus (BVDV) infection occurs in the cattle population worldwide. Non-cytopathic (ncp) BVDV strains cause transient infection (TI) or persistent infection (PI) depending on the host's immune status. Immunocompetent adult animals and fetuses in late gestation resolve the infection. Fetal infection in early gestation results in PI with chronic viremia and life-long viral shedding, ensuring virus perpetuation in the population. Eighteen pregnant heifers, divided into 3 groups, were intranasally inoculated with ncp BVDV2 virus early (day 75) and late (day 175) in gestation, or kept BVDV-naive. Fetuses were retrieved on day 190. Viral infection was confirmed following each inoculation. BVDV RNA was detected in multiple tissues of infected fetuses of both TI and PI groups on day 190 of gestation with significantly higher amounts of viral RNA in PI fetuses when compared to TI fetuses. PI fetuses had lower weight and were smaller based on heart girth and
crown rump length. The ponderal index was lower in PI fetuses, indicating significant intrauterine growth retardation. PI fetal femurs had a thickened cortex, leading to a significantly smaller internal diameter and medullar space. Serum from heifers inoculated with ncp BVDV2 in early pregnancy (day 75) showed high antiviral activity by day 7 p.i (day 82 of gestation). Serum from TI heifers and fetuses displayed substantial type I Interferon (IFN-I) antiviral activity on day 15 p.i. (day 190 of pregnancy). Western blot analysis of proteins from fetal spleen presented high amounts of both conjugated and free IFN stimulated gene 15 kDa (ISG15) protein in TI fetuses, while low ISG15 was detected in PI or control fetuses. Affymetrix microarray screen of day 190 maternal blood cell mRNA (n=3 heifers per group) revealed upregulation of IFN-I induced genes (i.e., ISG15) and pathways in TI compared to PI or control heifers. Affymetrix microarray screen of day 160 total white blood cell mRNA
from pregnant heifers of PI (n=5) and control heifers (n=4) revealed differential expression of ~1000 genes from numerous pathways. Members of the IFN-I pathway and members of the chemokine family and their receptors, were differentially expressed in blood of heifers with PI fetuses. The two most differentially expressed genes were a chemokine ligand (upregulated) and a chemokine receptor (downregulated), which were confirmed using RT-PCR. It is suggested that PI fetuses lack an adaptive recognition of the viral antigen resulting in an inability to resolve the infection. However, the fetus does display an IFN-I response to the persisting stimulus of the infection, which may have negative consequences for growth and development. Differential maternal blood cell expression of chemokines and their receptors in heifers carrying PI fetuses and altered fetal antiviral responses will be examined further in context of clarifying mechanisms that result in developmental defects caused by the
persistence of BVDV in PI fetuses. Also blood cell markers for heifers carrying PI fetuses that were discovered using microarray approaches will be tested for utility in clinical settings.
Publications
- Montgomery, D. L., van Olphen, A.L., Van Campen, H., Bielefeldt-Ohmann, H., Hansen, T.R. (2007). The fetal brain in bovine viral diarrhea virus infected calves - lesions, distribution and cellular heterogeneity of viral antigen at 190 days of gestation. Vet Pathol Provisionally accepted.
- Smirnova, N. P., Bielefeldt-Ohmann, H., Van Campen, H., Austin, K.J., Han, H., Montgomery, D.L., Shoemaker, M.L., van Olphen, A.L., Hansen, T.R. (2007). Acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses. Virus Res In Press.
- Hansen T.R., van Olphen A.L., Rempel L.A., Han H., Clapper J.A., Montgomery D.L., Berg B.M., van Campen H. (2006). Microarray screening reveals differential gene expression in blood from calves that are persistently infected with bovine viral diarrhea virus. In: Proceedings of the BVDV Special Symposium, Denver, p. 46.
- Smirnova N., Van Campen H., Bielefeldt-Ohmann H., Austin K.J., Han H., Montgomery D., van Olphen A.L., Hansen T.R. (2006). BVDV induces differential gene expression in blood of pregnant heifers. In: Proceedings of the 87th Annual Meeting of the Conference of Research Workers in Animal Disease, Chicago, Illinois, p. 166.
- Bielefeldt-Ohmann, H., Tolnay, A-E., Reisenhauer, C.E., Hansen, T.R., Smirnova, N., Van Campen, H. (2007). Transplacental infection with non-cytopathic bovine viral diarrhea virus type i and ii: Viral spread and molecular neuropathology. J Comp Pathol In Press.
- Smirnova N., Bielefeldt-Ohmann H., Van Campen H., Austin K.J., Han H., Montgomery D.L., van Olphen A.L., Hansen T.R. (2007). Pronounced antiviral response in maternal and fetal ncpBVDV infection is accompanied by upregulation of ISG15. Gordon Research Conferences, Viruses and Cells, Tilton, NH, USA,
- Shoemaker M., Smirnova N., Van Campen H., Bielefeldt-Ohmann H, Austin K.J., Han H., Montgomery D.J., van Olphen A.L., Hansen T.R. (2007). Maternal viral infection during pregnancy programs fetal development and induces a differential type I Interferon response. In: Biology of Reproduction, Special Issue, 40th Annual Meeting of the Society for the Study of Reproduction, San Antonio, p.191.
- Hansen, T.R., Austin, K.J., van Olphen, A.L., Rempel, L.A. (2007). International Patent Application PCT/US07/60375, filed January 11, 2007,Surrogate Markers for Viral Infections and Other Inflammatory Responses.
- Hansen, T.R., Austin, K.J., van Olphen, A.L., Rempel, L.A.(2007). United States Utility Patent Application, serial number 11/622,124, filed January 11, 2007, Surrogate Markers for Viral Infections and Other Inflammatory Responses
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