A sensitive and specific PCR/Southern blot assay for detection of bovine herpesvirus 4 in calves infected experimentally.

A PCR/Southern blot assay for detection of bovine herpesvirus 4 (BHV-4) in the background of bovine cellular DNA was developed. A BHV-4 specific sequence within the gene coding for the glycoprotein B (gB) was selected for primer sequences to guarantee the specificity of the assay. With a detection limit of six molecules BHV-4 DNA in the background of 1 microg of cellular DNA (equals about 150,000 bovine cells) this PCR/Southern blot assay represents a highly sensitive method for detection of BHV-4 DNA. At low concentrations of BHV-4 genomes, this assay also allows to estimate the copy number of BHV-4: a distinction between fewer than 6, 6-59 and more than 60 BHV-4 genomes/100 microl DNA suspension was possible. Tissue and blood samples of two calves, infected experimentally with BHV-4 were examined for the prevalence of BHV-4 DNA 130 days post infection. Ten days before taking samples, one of the calves was immuno-suppressed with dexamethasone. In both calves, BHV-4 DNA was detected in the leucocyte fraction of the blood, and beyond that in lower quantities in the spleen and the kidney of the immuno-suppressed calf. It is assumed that a latent BHV-4 infection was activated after application of dexamethasone and that the leucocyte fraction of the blood represents one site of latency of BHV-4 in cattle.

Glycoprotein B of bovine herpesvirus type 4: its phylogenetic relationship to gB equivalents of the herpesviruses.

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the gamma-herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.

Prognostic B-cell epitopes on the flagellar protein of Borrelia burgdorferi.

Overlapping decapeptides based on the flagellin sequence of Borrelia burgdorferi B31 (G. S. Gassmann, M. Kramer, U. B. Göbel, and R. Wallich, Nucleic Acids Res. 17:3590, 1989) were used to identify immunologically reactive regions of flagellin. Five serum specimens from patients with late manifestations of Lyme disease and borrelia-specific monoclonal antibody H9724 reacted with an epitope in the central region of the flagellar protein (amino acids 205 to 226), which is heterologous to the amino acid sequences of other bacterial flagellins. This epitope was not recognized by human sera with antibodies to Treponema pallidum, sera of healthy individuals, or sera from patients with stage I or II of Lyme disease.

Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs.

The herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesvirus was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.

Epitopes of the HIV-1-negative factor (nef) reactive with murine monoclonal antibodies and human HIV-1-positive sera.

Murine monoclonal antibodies (MAbs) raised against a recombinant nef protein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HIV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of the nef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinant nef protein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions of nef and cellular proteins.

The antibody response to the HIV-1 specific "out" (vpu) protein: identification of an immunodominant epitope and correlation of antibody detectability to clinical stages.

Overlapping decapeptides based on the sequences of two human immunodeficiency virus type 1 (HIV-1) strains (HXB2 and ELI) were used to identify an immunodominant epitope of the nonstructural protein "out" (vpu) of the human immunodeficiency virus type 1. Of 29 HIV-1 antibody-positive sera, 6 reacted with decapeptides corresponding to the C-terminal amino acid sequence VEMGVEMGHHAPWDVDDL of the "out" (vpu) protein. This oligopeptide was synthesized by the solid phase method and used to develop an enzyme-linked immunosorbent assay (ELISA) for screening of 243 HIV-1-seropositive and 75 HIV-1-seronegative sera. It was found that 26% of the HIV-1 antibody-positive sera were reactive in the "out" (vpu) peptide ELISA, whereas none of the HIV-1-negative sera reacted with the oligopeptide. Correlation of reactivity of sera with the Walter Reed (WR) staging classification demonstrated that individuals classified WR 1 (36%) and WR 2 (42%) were more often reactive than patients classified WR 3-6 (11%).

Epitope mapping of monoclonal antibodies against human immunodeficiency virus type 1 structural proteins by using peptides.

Murine monoclonal antibodies directed against the structural proteins p17 and p24 of human immunodeficiency virus type 1 were investigated in an epitope mapping system. Overlapping peptides consisting of 15 amino acids of the p17 and p24 protein, respectively, were used as competitors in an enzyme-linked immunosorbent assay. Three different immunogenic regions (A, B, and C) could be defined, one on p17 and two on p24. Twenty monoclonal antibodies reacted with the human immunodeficiency virus type 1 peptides of region B, although differences in the reactivity of these antibodies with human immunodeficiency virus type 2 and simian immunodeficiency virus strain mac were detectable. Recognized epitopes were characterized by computer analysis as described by T.P. Hopp and K.R. Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981) and P.Y. Chou and G.D. Fasman (Biochemistry 13:222-245, 1974).