Negative contrast electron microscopy (NCEM) and direct immune electron microscopy (DIEM) are used in the detection of viruses. Although NCEM provides a simple and rapid method for detecting viruses in clinical specimens, 106 virus particles per milliliter must be present in the original specimen to be detected by transmission electron microscopy. DIEM has been found to be more sensitive than conventional NCEM by forming virus-antibody aggregates which are easily visible by TEM. While a DIEM procedure is utilized for routine diagnostic bovine coronavirus detection in clinical samples submitted to the Wyoming State Veterinary Laboratory, Laramie, WY (WSVL), time-consuming incubations and periodic salt precipitations (Fig. 2) have sometimes delayed the desired rapid turn-around time for TEM virus diagnosis. To help avoid these delays, modifications to the DIEM procedure have been made.

When Culicoides variipennis (Coquillett) ingested a bluetongue virus (BTV)-defibrinated sheep blood suspension, BTV adsorbed to sheep red blood cells (RBCs) within 2 h. The virus had entered RBCs by 6 h and was still seen in RBCs 2 d after ingestion of the blood meal, even though the RBCs had been dehydrated. The peritrophic membrane began to form on day 1, and it contained breaks by day 3. The peritrophic membrane did not prevent infection of the midgut epithelium. Viral replication occurred in the midgut cells; virions matured through the basolateral extracellular membrane into the extracellular spaces between the plasma membrane and the basal lamina. The greatest number of virions was seen on day 3. The virus did not accumulate but rather exited the cells when mature. No cytopathology was observed in virus-infected cells, and midgut cells became vacuolated and sloughed off into the midgut lumen by day 3 in both control and virus-infected cells.

A hallmark of clinical bluetongue virus (BTV) infections in sheep is multisystemic inflammation, which is often most readily noticeable in the oral mucocutaneous tissue of whitefaced breeds of sheep.6J0 Arborvirus infections of the endothelial cells in inflamed cutaneous sites and in visceral organs is do~umented.~ It is thought that infection of endothelial cells, which results in lysis or in direct damage to the microvasculature, is a major pathophysiologic mechanism that accounts for increased vascular permeability and the classic clinical signs of bl~etongue.~ Although perivascular infiltrates of mononuclear cells are described in BTV infection~,~ the phenotype of the infiltrating lymphoid cells has neither been examined, nor has their role in the development of lesions and protection been addressed. Recently, we reported changes in peripheral blood T lymphocytic subsets during acute bluetongue infections in sheep and cattle.4 Of particular interest were the shifts in the OvCD4 and OvCD8 T lymphocytic subsets, which occurred in the peripheral blood of sheep. These occurred at two time periods-approximately l week after infection, at the nadir of the characteristic transient leukopenia, and approximately 2 weeks after infection, early in the convalescent period, when BTV-specific cytotoxic lymphocytes were identified in the thoracic duct lyrn~h.~ Such changes may be a reflection of alterations in lymphoid traffic in sites of viral replication. The purpose of this investigation was to examine the changes in the dynamics of lymphoid populations in inflamed oral mucocutaneous tissue, at time points that corresponded to the shifts observed in the peripheral blood, subsequent to BTV infection in sheep. Each of four BTV-seronegative yearling Columbia x Rambouillet sheep (Nos. 14) were inoculated with approximately 2 to 4 x 10s median chicken embryo intravascular lethal doses (CEIVLD,,) of BTV serotype- 10 (BTV- 10) inoculum.4 Equal amounts ofthe inoculum were injected subcutaneously in each ear and intradermally into bilaterally shaved areas of cervical skin. Two contact sheep (Nos. 5,6) each received an inoculum of an equal volume of uninfected, allogeneic, whole sheep blood. Rectal temperatures, complete blood counts, and occurrence of mouth lesions6 were recorded daily. Two sheep were euthanatized at 7

The cells of eukaryotes are characterized by a filamentous network referred to as the cytoskeleton. It is believed that most animal viruses use the cytoplasmic or nuclear skeletal matrix during at least part of their replication cycle.Transmission electron microscopic studies of thin sections of cells infected with epizootic hemorrhagic disease virus(EHDV), a double-stranded RNA virus belonging to the Reoviridae family, have demonstrated the presence of virus-like particles, virus-specific fibrils and tubules, and viral inclusion bodies. Studies of bluetongue virus (a closely related orbivirus) by Eaton et al. and Hyatt et al. confirmed that these virus-specific structures bind to the cytoskeleton of infected cells, and facilitated study of their viral protein content using monoclonal antibodies in immunogold labeling procedures, This study describes cytoskeletal involvement in the replication of EHDV.The grid-cell-culture technique, preparation of cytoskeletons, and immunolabeling procedure were those described by Hyatt et al. Grids were dehydrated in a graded alcohol series, critical point dried in amyl acetate and CO2, coated with carbon and examined with a Philips LS 410 transmission electron microscope operating at 60 kv.

When an arbovirus enters its arthropod host during a viremic blood meal it quickly reaches the midgut where virus penetration of the host parenchyma and infection of epithelial cells apparently occur. Subsequently, viral particles enter the arthropod's hemolymph and are transported to other tissues which may then become infected. Previous studies have demonstrated that when the arbovirus reaches the salivary glands it increases in titer and persists over long periods, often throughout the life of the vector.It is this persistent presence and associated continual shedding of virus into saliva which is one reason that certain arthropods are extremely efficient biological vectors.Culicoides variipennis(Coquillett) is probably the most economically important species of biting midge in the U.S. due to its involvement in the transmission of bluetongue (BT) disease of sheep, cattle and ruminant wildlife. Although there have been numerous electron microscopic studies of many mosquito-borne viruses in their natural hosts, virtually nothing is known about the replicative cycle of BTV in the salivary glands of its primary vector.

Culicoides variipennis (Coquillett) is probably the most economically important species of biting midge in the U.S. due to its involvement in the transmission of bluetongue (BT) disease of sheep, cattle and ruminant wildlife, and epizootic hemorrhagic disease (EHD) of deer. Proposals have been made to recognize the eastern and western populations of this insect vector as distinct species. Others recommend use of the term “variipennis complex” until such time that the necessary biosystematic studies have been made to determine the genetic nature and/or minute morphological differences within the population structure over the entire geographic range of the species. Increasingly, students of ootaxonomy are relying on scanning electron microscopy (SEM) to assess chorionic features. This study was undertaken to provide comparative chorionic data for the C. variipennis complex.Culicoides variipennis eggs were collected from a laboratory colony maintained in Laramie, Wyoming.