A RNA virus in cells from Culicoides variipennis.

A virus was detected in cells (designated CuVa) cultured from one laboratory colony of the biting midge, Culicoides variipennis. By electron microscopy (30 nm), nonenveloped, icosahedral virions arranged separately and in crystalline matrix arrays were seen in the cytoplasm but not in the nucleus of CuVa cells. Separation by 10% polyacrylamide gel electrophoresis revealed multiple bands of viral-induced double-stranded RNA. Inoculation of this virus onto different cell lines and intracranially into suckling mice revealed no detectable pathology. Immunoperoxidase staining using polyclonal antibody determined that the virus is infectious to toad cells, bovine endothelial cells, bovine kidney cells, mosquito cells, and cells (designated KC) initiated from another laboratory colony of C. variipennis. KC cells infected with this virus were coinfected with bluetongue virus with no decrease in bluetongue virus titer.

Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs.

Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

Limits of detection of bluetongue virus with different assay systems.

The sensitivity of different assay systems for detecting low concentrations of bluetongue virus (BTV) were compared. These assays included blind passage on baby hamster kidney (BHK-21) cells and on cattle pulmonary artery endothelial (CPAE) cells, immunoperoxidase staining of cells on multiwell slides, and cDNA/RNA hybridization of BTV infected cells. Nine serial 10-fold dilutions of a cell culture-adapted BTV serotype 11 were tested (each dilution was treated as a separate sample) in all assays. Visual inspection for cytopathic effects (CPE) during 3 passages in BHK-21 cells detected samples that contained greater than or equal to 3 plaque forming units (PFU)/ml of BTV. Evidence of CPE during 3 passages in CPAE cells detected samples that contained greater than or equal to 0.3 PFU/ml of BTV. A limit of detection (greater than or equal to 0.3 PFU/ml) was obtained faster by immunoperoxidase staining of BTV-inoculated CPAE cells on multiwell slides and incubated for 3 days. The cDNA/RNA hybridizations of CPAE and BHK-21 cells incubated for 2 or 3 days, respectively, with BTV dilution samples detected samples that contained greater than or equal to 30 PFU/ml. Of the assay systems examined, immunoperoxidase staining of CPAE cells on multiwell slides inoculated with cell culture-adapted BTV was the most sensitive and fastest assay for definitive virus identification.

T lymphocyte subset alterations following bluetongue virus infection in sheep and cattle.

To determine potential mechanisms of differential disease expression in ruminants infected with bluetongue virus (BTV), clinically normal, BTV-seronegative, yearling sheep and cattle were infected subcutaneously with a standardized insect-source inoculum of BTV serotype 17 (BTV-17) (three infected and one contact control each) or animal adapted BTV serotype 10 (BTV-10) (three sheep only). BTV was isolated from peripheral blood cell components of infected sheep and cattle and all infected animals showed evidence of seroconversion by 14 days post infection (PI). Sheep infected with both serotypes of BTV developed pyrexia, oral lesions, and leukopenia which were most severe on days 7-8 PI. Analysis of peripheral blood mononuclear leukocytes with specific monoclonal antibodies and flow cytometry revealed panlymphocytopenia on day 7 PI. This response was further characterized by an increase in the CD4/CD8 ratio (greater than 3) resultant from a greater decrease in absolute numbers of circulating SBU-T8(CD8+) ("cytotoxic/suppressor") lymphocytes compared to SBU-T4 (CD4)+ ("helper") lymphocytes. SBU-T19+ lymphocytes were also decreased below baseline values on days 5-14 post infection. On day 14 PI there were increased CD8+ lymphocytes and decreased CD4/CD8 ratios (approximately 0.6) in these sheep. Clinical and hematologic changes in cattle infected with BTV-17 were minimal and consisted of mild pyrexia (rectal temperature 103 degrees F) on day 9 PI in two of three infected animals and mild leukopenia on several days PI in one animal. This leukopenia was the result of a pan T lymphocytopenia with CD4/CD8 ratios in the expected range (1-2). Similar to infected sheep, infected cattle did have a shift (decrease, approximately 0.8) in the peripheral CD4/CD8 ratio associated with an increase in circulating BoT8 (CD8)+ lymphocytes on day 14 post infection. Lymphocytes in the peripheral blood of all sheep and cattle infected with BTV-17 proliferated in vitro in response to purified BTV-17. These results confirm and extend those of previous studies that indicate species differences in the hematologic response to an equivalent BTV infection in domestic ruminants.

Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus.

Cell lines have been developed from 2-day-old embryos of the biting midge, Culicoides variipennis (Diptera: Ceratopogonidae). In North America C. variipennis is the primary insect vector of bluetongue virus (BTV), an orbivirus that causes disease of ruminants. The C. variipennis (CuVa) cells, grown in Schneider's Drosophila medium, consist primarily of a fibroblast-like cell type. CuVa cells are very hardy. They can grow over a wide range of temperature and pH and adapt to growth in minimal essential medium. BTV replicates to high titer (7.5-8.0 log10 50% tissue culture infectious doses/ml) in CuVa cells over a wide range of temperatures (19 degrees to 37 degrees C) without inducing any significant cytopathic effects. The highest BTV titers were obtained in CuVa cells grown at 25 degrees and 32 degrees C. Cells from C. variipennis can be useful for many diverse investigations.

Susceptibilities of 14 cell lines to bluetongue virus infection.

The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations.

Prostaglandin E2 content of regressing and progressing Moloney sarcomas.

Regressing mouse Moloney sarcomas contain macrophages that are activated for tumor cell killing, while those found in progressively growing sarcomas either cannot kill or do so very poorly. Prostaglandin E2 (PGE2) has been shown to down-regulate macrophage activation in vitro. The study described here was designed, therefore, to ascertain and compare the concentrations of PGE2 in regressing and progressing Moloney sarcomas. Tumors were harvested for extraction and analyzed using conditions that minimized artifactual increases in PGE2 levels attributable to de novo synthesis. Concentrations of PGE2 were higher in progressing, compared to regressing, Moloney sarcomas during the early stages of tumor development. At nearly all time points, however, whether the neoplasms were of regressing or progressing type, the estimated concentrations of PGE2 in tumors exceeded the level that completely inhibits macrophage activation for tumor cell killing in vitro, that is, 10(-8) M. These data suggest either that PGE2 is not responsible for down-regulating activation in Moloney sarcomas or that, if PGE2 is responsible for the negative regulation of activation in progressing Moloney sarcomas, there must be something in regressing sarcomas that prevents the hormone from having its inhibitory effect.

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus.

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.