Jacqueline Lecomte

Comparison of the p125 coding region of bovine viral diarrhea viruses

The p125 (p54/p80) coding region of two cytopathic (CP) strains (Oregon and Singer) and two noncytopathic (NCP) strains (NY-1 and Draper) of bovine viral diarrhea virus (BVDV) were amplified by the polymerase chain reaction, cloned and sequenced. The sequence data confirmed that the two CP strains do not possess any insertion or deletion in their p125 gene as observed in many other CP strains. In the p80, which showed a high amino acid sequence homology among all strains, no amino acid substitution should could be found which distinguished these CP strains from the NCP strains NY-1 and SD-1. Many amino acid substitutions were found in p54 but their individual importance in the CP phenotype is not clear since critical domains of p54 have not yet been experimentally defined. The p54 protein is much less conserved than p80, and sequence homology, as well as dendrogram analysis, permitted us to distinguish two genotypic groups of BVDV (Ia and Ib). The mean homology between strains of these two groups was 77.3/80.4% for the nucleic acid/amino acid sequences while it was 88.0/88.8% and 91.6/93.3% within groups Ia and Ib, respectively. Furthermore, we found that the p125 sequence of our NY-1 strain showed only 92% sequence homology with the partial p80 gene reported for NY-1 but 99.8% homology with another partial sequence of the p125 gene of NY-1 reported elsewhere. These observations underscored the difficulty of maintaining a specific BVDV strain, especially the NCP biotype, in cell cultures.

Identification of murine natural killer cell subsets with monoclonal antibodies derived from 129 anti-C57BL/6 immune spleen cells☆

Two hybridomas producing monoclonal antibodies reactive with natural killer cells were selected after fusion of 129 anti-C57BL/6 immune spleen cells with P3X63-Ag8.653 myeloma cells. Treatment of normal or stimulated cells with the 4LO3311 or the 4LO439 mAb and rabbit complement inhibited natural killer and antibody-dependent cellular cytotoxicities, whereas cell lysis mediated by natural cytotoxic cells, cytotoxic T lymphocytes, or activated macrophages was unaffected. Lymphokine-activated killer activity was reduced after complement-mediated treatment of interleukin-2-stimulated spleen cells with the 4LO3311 mAb but not after treatment with the 4LO439 mAb. Similar treatment of spleen cells with either mAb had no effect on the mitogen-induced proliferation of T and B lymphocytes and did not alter the frequency of antibody plaque-forming cells in immune spleen cell suspensions. The 4LO3311 and 4LO439 mAbs thus appear to be specific for NK cells and their progeny. Flow cytometry analysis confirmed that 4LO3311+ and 4LO439+ cells are phenotypically identical to NK-1.1+ cells. The epitope recognized by the 4LO3311 mAb has the same strain distribution as the NK-2.1 alloantigen previously detected with NZB anti-BALB/c antiserum, whereas the 4LO439 mAb appears to identify a new NK cell marker exclusively expressed in mice of C57BL lineage. The relationship of the molecules detected with either the 4LO3311 or the 4LO439 mAb to polymorphic antigens of the Ly series is discussed.

Identification of rubella virus structural proteins by immunoprecipitation

Immunoprecipitation of [3H]amino acid labelled virus with monoclonal or human convalescent rubella sera and subsequent analysis by electrophoresis and fluorography, revealed three structural proteins of rubella virus: VP3: 59,000; VP2: 44,800; and VP1: 33,000.

Further biological, serological and biochemical characterization of North American, European and Southeast Asian strains of bovine herpesvirus 1 compared with other alphaherpesvirinae members

Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.

Comparison of methods for concentration and purification of bovine viral diarrhea virus

The NADL strain of bovine viral diarrhea virus (BVDV) was concentrated by hollow fiber ultrafiltration or polyethylene glycol and purified by centrifugation through sucrose or potassium tartrate gradients. The protein content of polyethylene glycol concentrates was much lower than that of ultrafiltration concentrates. Conversely, recoveries of infectivity were greater using polyethylene glycol (100%) as compared to ultrafiltration (50%). Sucrose or potassium tartrate density gradients appeared comparable in purification of BVD virus. Peak infectivity fractions in both gradients corresponded quite well, having densities of 1.12-1.14 g/cm3, and showed a 150-fold reduction of protein when compared to crude viral supernate. Further examination by negative stain electron microscopy revealed integral pleomorphic, roughly spherical particles in both purified virus preparations. Small knob-like projections could be seen on viral particles.

Rapid titration of bovine, caprine and human RS virus by a micro-immunoperoxidase assay using a monoclonal antibody and a permissive ovine kidney cell line

An indirect immunoperoxidase micro-assay, using a continuous cell line derived from ovine kidney cells (OK) and a previously characterized monoclonal antibody (7C2), specific for an exposed and highly conserved epitope of the fusion protein of different strains of RS virus, was used advantageously to rapidly titrate bovine, caprine and human strains of RSV by either quantal (TCID50) or plaque forming assays. Virus titers, obtained in less than 36 h, were in agreement with those obtained by the conventional plaque assays which required an incubation period of 4 days or more. This assay is also applicable to micro-neutralization of fusion inhibition assays for testing serum or screening monoclonal antibodies.