Persistent Bovine Viral Diarrhea Virus Infection in the Bovine Fetus: Morphogenesis of Skeletal Lesions and Innate Immune Response

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Abstract

Introduction. The effects of transplacental viral infections on the developing fetus are dependent upon complex interactions between fetoplacental and maternal immune responses and the stage of fetal development at which the infection occurs. Bovine viral diarrhea virus (BVDV) has the ability to cross the placenta and infect the fetus. Infection early in gestation with noncytopathic (ncp) BVDV leads to persistent infection (PI) due to the development of virus-specific immunotolerance, which results in life-long viremia and detrimental developmental consequences for many organ systems. PI with BVDV has been associated with osteopetrosis, other long bone lesions, and fractures in fetuses and calves. This study was undertaken to characterize the morphogenesis of fetal long bone lesions; determine the effects of PI on biomechanical properties of fetal femora; and delineate the temporal development of the fetal and placental innate immune responses during the establishment of PI.

Materials and Methods. Forty-six BVDV-naïve pregnant Hereford heifers, approximately 18 months old, were inoculated either with non-cytopathic BVDV type 2 containing media or media alone on day 75 of gestation to produce PI and control fetuses, respectively, which were collected via Cesarean section on the following days of gestation: 82 (peak of maternal viremia), 89 (beginning of maternal seroconversion, acute stage of fetal infection), 97 (Maternal seroconversion and aviremia, fetal viremia), 192 (chronic stage of fetal persistent infection), and 245 (chronic stage of fetal persistent infection, near term).

Results. Experiment I: The morphogenesis of fetal skeletal lesions. Radiographic and histomorphometric abnormalities were first detected on day 192, at which age PI fetal long bone metaphyses contained focal densities (4/7 fetuses) and multiple, alternating (cyclic) transverse radiodense bands (3/7 fetuses). Day 245 fetuses were similarly affected. Histomorphometric analysis of proximal tibial metaphyses from day 192 fetuses revealed transverse zones with increased calcified cartilage core (Cg.V/BV, %) and trabecular bone (BV/TV, %) volumes in regions corresponding to radiodense bands (P<0.05). Numbers of tartrate resistant acid phosphatase positive osteoclasts (N.Oc/BS, #/mm²) and bone perimeter occupied by osteoclasts (Oc.S/BS, %) were both decreased (P<0.05). Mineralizing surface (MS/BS, %), a measure of tissue level bone formation activity, was reduced in PI fetuses (P<.05).

Experiment II: The effect of PI on the biomechanical properties and composition of fetal femora. PI fetuses had femora with smaller mid-diaphyseal diameters and lower cortical porosity than controls. There were no differences between PI and Control fetuses in cortical thickness ratio, ash density nor calcium or phosphorous content; however, cortical thickness ratio decreased with fetal age. Although differences in elastic modulus (PI vs Control and day 192 vs day 245) and ultimate stress (day 192 vs day 245) were identified, cortical thickness ratio largely accounted for these observed differences.

Experiment III: Fetal and Placental innate immune response to PI. Fetal viremia was confirmed starting on day 89. Significant up-regulation of mRNA encoding cytosolic dsRNA sensors - RIG-I and MDA5 - was detected on days 82 -192. Detection of viral dsRNA by cytosolic sensors leads to the stimulation of ISGs, which was reflected in significant up-regulation of ISG15 mRNA in fetal blood on days 89, 97, and 192. No difference in IFN-α and IFN-β mRNA concentration was found in fetal blood or caruncular tissue, while a significant increase in both IFN-α and IFN-β mRNA was seen in cotyledons from PI fetuses on day192.

Conclusions. It is concluded that PI with BVDV induces cyclic abnormal trabecular modeling, which is secondary to reduced numbers of osteoclasts. The factors responsible for these temporal changes are unknown but may be related to the time required for osteoclast differentiation from precursor cells. However, fetal infection with BVDV did not significantly impair inherent biomechanical properties of fetal bone but rather resulted in decreased periosteal apposition rates, which manifest as altered cortical geometry. Differences in cortical geometry disproportionally affect the error associated with calculation of stress and strain by classical beam theory equations and, therefore, critical assumptions necessary for use of these equations are not satisfactorily met when comparing samples differing solely in mid-diaphyseal diameter. Furthermore, fetuses respond to early gestational ncp BVDV infection by

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