Use of the bovine leukaemia virus LTR U3 promoter for expressing antisense antiviral RNAs and competitive inhibition of viral infection in cell culture.

Use of viral inducible promoters which can be activated by virus-specific transactivator proteins to drive expression of antisense (as)RNA genes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome. This is the region that is most susceptible to inhibition by asRNA.

Physical mapping and homology studies of egg drop syndrome (EDS-76) adenovirus DNA.

Homologous sequences between EDS-76 adenovirus strain 127 DNA and bovine adenovirus DNA were found by the Southern blotting technique and no homology with CELO virus DNA could be detected. These data suggest a genetic similarity between EDS-76 virus and bovine adenoviruses. The fragments generated form EDS-76 adenovirus DNA by eight restriction endonucleases were physically mapped.

Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81.

A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities.

Characterization of a series of monoclonal antibodies specific for bovine adenovirus serotype BAV3 hexon antigen and their use in an ELISA for detection of adenoviral hexons.

After immunization of mice with purified hexon (the main capsid antigen) of bovine adenovirus serotype BAV3 we have obtained a set of 16 individual hybridoma clones producing MAb's against BAV3 hexon. All MAb's were shown to belong to immunoglobulin G class. Specificity of the most avid MAb marked B3Hx-1 was tested on a panel of representative hexon antigens from 16 adenovirus serotypes of human and animal origin using several immunoassays.