Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease-resistant PrP (PrP-res) of one species induces the in vitro conversion of the normal PrP (PrP-sen) of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents. Here we show that the CWD-associated PrP-res (PrP(CWD)) of cervids readily induces the conversion of recombinant cervid PrP-sen molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrP(CWD)-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD.

First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome.

Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.

Pyruvate metabolism by Anaplasma marginale in cell-free culture.

Partially purified Anaplasma marginale initial bodies were cultivated in a cell-free system in the presence of [3-14C]pyruvate for 24 or 48 h. Experiments showed that a significant portion of the pyruvate supplied to the cultures was incorporated into initial body components. Label incorporation was reduced by 72% in the presence of oxytetracycline. Fractionation and chromatography of the organisms revealed radioactive incorporation as alanine. This is the first report of de novo amino acid synthesis by A. marginale demonstrating that the rickettsia is capable of using pyruvate, an erythrocyte glycolytic product, in its metabolism.

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.

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.