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INVESTIGATOR: Webb, B. T.

PERFORMING INSTITUTION:
NORTH DAKOTA STATE UNIV
FARGO, NORTH DAKOTA 58105

IN UTERO INFECTION WITH BOVINE VIRAL DIARRHEA VIRUS IMPAIRS FETAL BONE DEVELOPMENT

NON-TECHNICAL SUMMARY: Bovine Viral Diarrhea Virus (BVDV) causes a wide range of clinical manifestations in cattle, which include infertility, abortion, stillbirth, birth of weak calves and suppression of the immune system, which secondarily leads to infection by other disease agents. BVDV, which infects cattle populations throughout the USA and world, causes significant economic losses to the cattle industry making it arguably the most important viral disease of cattle. The research examines transplacental infection with BVDV, which leads to persistent infection (PI) of the fetus and life-long shedding of virus. The latter is responsible for maintaining the virus in cattle populations. PI negatively impacts the developing fetus often leading to a myriad of developmental abnormalities including abnormal long bone development, which is the main focus of this research. Abnormalities of the long bones caused by PI with BVDV are characterized by an excessive accumulation of bone, known as osteopetrosis which results from impaired bone resorption. Resorption of bone is mediated by bone resorbing cells known as osteoclasts, which share a common precursor cell with an important type of white blood cell called monocytes or macrophages. The research tests the hypotheses that long bone abnormalities in PI fetuses are caused by decreased bone resorption due to impaired differentiation or development of osteoclast from precursor cells. The studies will address gaps in knowledge concerning the mechanisms by which PI with BVDV impacts osteoclast and immune cell precursor development. Summarily, the information gained from these studies has promise to impact the knowledge base of how this economically important virus causes disease.

OBJECTIVES: The objectives of the research are to define the exact character of the long bone lesions and the cellular and molecular mechanisms underlying lesion development in fetuses persistently infected (PI) with Bovine Viral Diarrhea Virus. Specifically, the research aims to: 1) quantitatively determine the relative contributions of formation and resorption processes in the development of bone lesions on Days 175 to 185 of gestation and 2) systematically evaluate the effect of PI on osteoclast differentiation, precursor cell proliferation, and osteoclast function in vitro.

APPROACH: The methods employed to test the research hypotheses include light microscopy with histomorphometric and standard cellular and molecular biological approaches. Aim 1 entails the use of intravital flurochrome labeling of fetal bone via maternal administration coupled with standard histomorphometry to measure dynamic bone processes, namely bone formation. Aim 2 will utilize a cell culture system, in which a cytokine modulated osteoclast differentiation, colorimetric cell proliferation and pit resorption assays have been standardized. The use of RT-PCR and western blot approaches will compliment these assays for the purpose of identifying alterations in candidate pathways with know relevance to the processes under study. Statistical analysis will employ a Student's t-test or nonparametric alternative following the results of a q/q plot to assess for normality.

PROGRESS: 2013/02 TO 2014/05
Target Audience: Animal disease researchers and veterinarians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing additional to report. How have the results been disseminated to communities of interest? The results have been disseminated in presentations and publications detailed in this reportand previous progress reports. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2013/02 TO 2014/05
What was accomplished under these goals? Aim one and two have been completed. The findings of aim one are detailed in previous reports (also see COLV-2010-05138). In summary persistent infection with BVDV inhibits of bone resorption and bone formation rate. The observed reduction in bone resorption corresponds to reduced numbers of osteoclasts. In aim two the effects of PI fetal serumon peripheral blood mononuclear cell derived osteoclast precursor proliferation, differentiation and resorptive capacity were evaluated. Consistent differences in permissible precursor proliferation, differentiation, fusion and resorptive capacity were not observed. This suggests the processes resulting in the observed inhibitory effects of persistent BVDV infection on bone resorption and osteoclast numbers involve interactions in the bone marrow microenvironment or manifest through alteration of specific precursor cells that cannot be easily replicated in vitro. It further suggest that the inhibitor effects are not the sole result of soluble factors present in PI fetal serum as previous hypothesized.

PUBLICATIONS (not previously reported): 2013/02 TO 2014/05
Type: Journal Articles Status: Published Year Published: 2014 Citation: Smirnova, N.P., Webb, B.T., McGill, J.L., Schaut, R.G., Bielefeldt-Ohmann, H., Van Campen, H., Sacco, R.E., Hansen, T. R. (2014) Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus. Virus Research 183:21, 183-195.

PROGRESS: 2013/02/01 TO 2014/01/31
Target Audience: Researchers and veterinarians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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? Efforts during the remaining period of this award will be focused on analysis of data obtained from experiments completed in aim two.

IMPACT: 2013/02/01 TO 2014/01/31
What was accomplished under these goals? The goal of this project was to evaluate the effects of chronic in utero viral infection on the developing skeleton using maternal infection with bovine viral diarrhea virus to generate persistently infected fetuses. Aim one quantitatively determined the relative contributions of formation and resorption processes in the development of bone lesions on Days 175 to 185 of gestation in persistently infected fetuses. Aim two evaluated the effect of persistent BVDV infection on osteoclast differentiation, precursor cell proliferation, and osteoclast function in vitro. The objectives of aim one and a majority of those for aim two were completed at Colorado State University. Accomplishments and outputs are detailed under project COLV-2010-05138.The PD moved from Colorado State University to North Dakota State University in February 2013 and transferred the remaining funds. To follow up on the findings from aim one, bone composition, bone geometry and biomechanical properties were evaluated. In short, significant differences in bone composition and geometry were observed in persistently infected fetuses but measureable alterations in biomechical properties were not identified. The results of these experiments are detailed in the publication cited in the current report.With respect to aim two, in vitro experimentsto evaluate osteoclast differentiation and resorption have been completed using isolated peripheral blood monocytes that were differentiated into osteoclasts in the presence of serum obtained from persistently infected and control fetuses.

PUBLICATIONS: 2013/02/01 TO 2014/01/31
Type: Journal Articles Status: Published Year Published: 2013 Citation: Webb BT, McGilvray KC, Smirnova NP, Hansen TR, Norrdin RW. Effects of in utero pestivirus infection on bovine fetal bone geometry, biomechanical properties and composition. Vet J. 2013;198:376-381.