Specificity of the histone lysine methyltransferases from rat brain chromatin.

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.

Effect of polyamines and cations on the in vitro methylation of histones.

Na+ (0.05-0.15 M) increases both the rate and extent of methylation of chromosomal bound histone H4, while spermidine markedly inhibits this reaction. The effects of spermidine could be mimicked by increasing the concentration of Mg2+ or Ca2+ to 5-10 mM. At the concentrations listed above, these cations have no significant effect on the methylation of free or chromosomal bound histone H3, nor do they affect the rate r extent of methylation of soluble histone H4. Apparently, the accessibility of histone H4 to the methyltransferase is influenced by chromatin structure. Increasing concentrations of Na+ alter the conformation of chromatin (DNA) in such a way as to expose lysing residues in the N-terminal region of histone H4 to the methyltransferase, whereas Mg2+ or spermidine acts in an opposite manner.

A microplate immunofluorescence assay for the detection of monoclonal antibodies against viral antigens in cell culture.

A microplate immunofluorescence assay for the screening of culture fluids from large numbers of hybridoma clones has been developed. This method enables the simultaneous screening of a small volume of culture fluid from initial hybridoma clones on several different viruses or several strains of the same virus, enabling the early isolation and stabilization of the clones with the desired specificity.

Bovine viral diarrhea virus-specific neutralizing antibodies induced by anti-idiotypic antibodies.

Two murine neutralizing monoclonal antibodies (MAbs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.

Quantitation of bovine immunoglobulins in culture fluids by use of sandwich radioimmunoassay with monoclonal antibodies.

Bovine immunoglobulin isotype-specific murine monoclonal antibodies were used in sandwich radioimmunoassays to detect and quantitate bovine IgG1, IgG2, IgM, and IgA in culture fluids. The concentrations of bovine immunoglobulins in unknown samples were extrapolated from standard curves generated with bovine monoclonal immunoglobulins. The lowest detection limits for the bovine immunoglobulin isotypes ranged from 65 to 270 ng/ml.

Bovine monoclonal antibodies specific for bovine herpesvirus-1 glycoprotein gIII.

Spleen cells from a calf immunized with bovine herpesvirus-1 (BHV-1) were fused with the nonsecreting murine cell line SP2/0. Several bovine-murine hybridomas secreting bovine immunoglobulins were stabilized. Of these, 9 hybridomas secreted bovine monoclonal antibodies that specifically bound to BHV-1 in a radioimmunoassay. Two of these monoclonal antibodies reacted specifically with BHV-1 in an indirect fluorescent antibody test and immunoprecipitated a BHV-1 glycoprotein with molecular mass of 97 kilodaltons.

Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1.

A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-113), specific for bovine herpesvirus 1 (BHV-1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (anti-Id) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-113-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotype-matched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentration-dependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV-1 in a cell-binding radioimmunoassay (RIA).

Bovine viral diarrhoea virus-specific bovine monoclonal antibody.

A bovine monoclonal antibody specific for bovine viral diarrhoea virus (BVDV) recognized all the reference strains of BVDV and over 90% of the field isolates tested. It neutralized the virus and precipitated a 56 k-58 k viral protein.

Long-term persistent infection of swine monocytes/macrophages with African swine fever virus.

Long-term persistent infection was established in 100% of pigs (n = 19) experimentally infected with African swine fever virus (ASFV). Viral DNA was detected in peripheral blood mononuclear leukocytes (PBML) at greater than 500 days postinfection by a PCR assay. Infectious virus was not, however, isolated from the same PBML samples. In cell fractionation studies of PBML, monocytes/macrophages were found to harbor viral DNA during the persistent phase of infection. This result indicates that monocytes/macrophages are persistently infected with ASFV and that ASFV-swine monocyte/macrophage interactions can result in either lytic or persistent infection.

Virulent African swine fever virus isolates are neutralized by swine immune serum and by monoclonal antibodies recognizing a 72-kDa viral protein.

Convalescent swine serum to African swine fever virus (ASFV) isolate E75 neutralized the infectivity of virulent ASFV isolates E75, E70, Lisbon 60, Malawi Lil 20/1 and a low passage tissue culture adapted variant of E75, E75CV/V3, by 86-97% in Vero and macrophage cell cultures. A monoclonal antibody, mAb-135D4, recognizing an ASFV protein of 72 kDa also exhibited strong neutralizing activity with these viruses. Unexpectedly, both E75 immune sera and mAb-135D4 failed to neutralize high passage tissue culture adapted ASFV variants including Lisbon 60, Haiti, Dominican Republic I, Dominican Republic II, and Brazil II, even though mAb-135D4 reacted with all viruses. These results suggest that tissue culture adaptation of ASFV isolates may be associated with loss of specific determinants associated with virus neutralization. To our knowledge, this is the first report of an ASFV neutralizing epitope.

Passively transferred African swine fever virus antibodies protect swine against lethal infection.

The role of anti-viral antibodies in homologous protective immunity to a virulent African swine fever virus (ASFV) strain E75 was examined by passive transfer experiments in swine. Eighty-five percent of animals (n = 14) that received anti-ASFV immunoglobulin (Ig) survived challenge infection, while 100% mortality was observed in control group animals (n = 28) that received anti-pseudorabies virus Ig, normal swine Ig, or phosphate-buffered saline. With the exception of a significantly delayed and transient fever response, anti-ASFV Ig group animals remained clinically normal following challenge, whereas control group animals presented with clinical ASF on Day 4 postchallenge. Additionally a significant 3 day delay in onset of viremia and a 10,000-fold reduction in both mean and maximum virus titers were observed for animals given anti-ASFV Ig. These results indicate that anti-ASFV Ig alone will protect swine from lethal infection with virulent ASFV. Further, they support the view that the antibody-mediated protective effect is an early event that effectively delays disease onset.

Characterization of p30, a highly antigenic membrane and secreted protein of African swine fever virus.

We have identified and characterized a 30-kDa phosphoprotein (p30) of African Swine Fever Virus (ASFV) that is synthesized, membrane localized, and released into the culture medium at early times after infection. Sequence analysis of the p30 open reading frame predicts a highly antigenic protein with putative phosphorylation, glycosylation, and membrane attachment sites.