Search

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.

The cellular immune response of cattle to virulent and avirulent (inactivated) bluetongue virus (BTV) was studied. Each of three calves received three vaccinations (sensitizations) with binary ethyleneimine (BEI)-inactivated BTV, 3 weeks apart. The sensitized animals were challenged with BTV-11 strain UC8 3 weeks after the last vaccination. BTV-seropositive and BTV-seronegative calves were used as controls. The animals were bled weekly for virus isolation and for evidence of cell-mediated immunity (CMI) as determined by the lymphocyte stimulation test (LST). Peripheral blood mononuclear leukocytes (PBM L) cultures were induced with purified BTV antigen; the phytomitogens phytohemagglutinin (PHA), Concanavalin A (ConA) and pokeweed (PKW) mitogen, and combinations of phytomitogens and BTV antigen. LST data were analysed by ANOVA and reported as counts per minute (CPM) and stimulation index (SI). Following BTV challenge exposure, significant SI to mitogens were found in PBML cultures for all animals. BTV antigen induced a weak CMI response. There was evidence of perturbations in lymphocyte response as characterised by a sharp decrease in lymphocyte response to mitogens following combined BTV-antigen and mitogen PBML induction. The SI diminished in PBML cultures after a 4 day incubation period, except for ConA. These results provide evidence that the cell-mediated immune response could be affected by BTV and that inhibitory mediators might play an important role in the pathogenesis of BTV in cattle.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.

Antiviral responses of bovine fetuses to bluetongue virus (BTV) infection were determined. Fetuses inoculated with BTV at 125 days of gestation apparently had cleared all virus by the time of birth; however, the mechanism responsible for virus clearance was not determined. Interferon production and virus clearance were temporally unrelated. Cell-mediated responses to BTV antigens were not detected by the lymphocyte stimulation test using lymphocytes from infected fetuses or cows. Although virus clearance and fetal production of high titers of neutralizing antibody did coincide, it was considered unlikely that antibody alone was responsible for virus clearance. As congenital BTV infection did not lead to specific immunologic tolerance or postnatal persistance of virus, such animals would not be expected to have a major role in the dissemination of BTV.

Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance.