Identification of the Target Cells and Sequence of Infection during Experimental Infection of Ovine Fetuses with Cache Valley Virus

Abstract

Cache Valley virus-induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the course of infection of cells and tissues with Cache Valley virus. To address these questions, ovine fetuses at 35 days of gestation were inoculated in utero with Cache Valley virus and euthanized at 7, 10, 14, 21, and 28 days postinfection. On postmortem examination, arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphological studies showed necrosis in the central nervous system and skeletal muscle of infected fetuses evaluated after 7 to 14 days postinfection, and hydrocephalus, micromyelia, and muscular loss were observed in infected fetuses after 21 to 28 days postinfection. Using immunohistochemistry and in situ hybridization, intense Cache Valley virus antigen and RNA staining was detected in the brain, spinal cord, skeletal muscle, and, to a lesser degree, in fetal membranes and other tissues of infected fetuses. Viral antigen and RNA staining decreased in targeted and infected tissues with the progression of the infection.

Ovine Fetal Immune Response to Cache Valley Virus Infection

“…Previously, we have demonstrated that fetuses infected with CVV early in gestation (ϳ35 dg) have low viral antigen and RNA signal in tissues around 56 dg and are able to clear the infection before development of an adaptive immune system at 75 dg (15). Similarly, age-based findings have been described in Akabane virus-infected fetuses (9,11,16).…”

“…Viral signal was also markedly reduced or absent in the ganglion, retina, kidney, and heart. CVV targets mainly cells within the CNS and MSS, and viral signal was markedly reduced in the brain and almost completely cleared in the skeletal muscle after 21 to 28 dpi in fetuses infected at 35 dg (15). Similarly, a progressive reduction in viral load, as shown by mean relative amounts of CVV RNA copies, was observed in the brain and SKM in the present study, with significant differences observed between most days of infection in the SKM, and in the brain at between 14 and 28 dpi.…”

“…Immunohistochemistry was performed on 5-m, deparaffinized sections of paraformaldehyde-fixed tissues mounted onto positively charged, silanized slides using an automated system for immunohistochemistry (DakoCytomation Autostainer; Dako, Carpinteria, CA) with minor modifications to a previously described immunohistochemistry protocol (15). Antigen retrieval was performed in a decloaker chamber (Biocare Medical, Concord, CA) with slides soaked in citrate buffer (pH 6.0).…”

“…A group of 15 seronegative, pregnant Rambouillet ewes was housed in BSL2 confinement buildings according to protocols approved by the Institution Animal Care and Use and the Institutional Biosafety Committee. At 35 dg, ewes were inoculated in utero with a 1-ml inoculum containing 10 5 50% tissue culture infectious doses of CVV (infected group) or 1 ml of minimum essential medium (mock-infected/control group), as previously described (15). The viral inoculum was derived from the second passage of an isolate from allantoic membrane from an experimentally infected fetus (3).…”

“…Virus isolation can only be made from infected fetuses in early development, and viral recovery from tissues of term abortions and malformed lambs is uniformly unsuccessful. The virus is cleared from infected tissues within a few weeks after infection (2,15) and before the presumed age of fetal immunocompetency, at approximately 70 to 75 dg (16,17).…”