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J B Black

Publications and source records attributed to J B Black.

At least 19 recordsLinked to original sources
PubMed1999

Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells.

Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mM) are used, whereas reduction of butyrate concentration to 0.3 mM decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20% of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIPI, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0.3 mM delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate concentration to 0.3 mM, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.

Butyrates↗
PubMed1998

Mapping and serodiagnostic application of a dominant epitope within the human herpesvirus 8 ORF 65-encoded protein.

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi's sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.

AIDS-Related Opportunistic Infections↗
PubMed1998

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia.

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6B's in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.

Adult↗
PubMed1997

Biologic properties of human herpesvirus 7 strain SB.

The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures. The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection. By systematically varying the culture media components, improved culturing conditions were established. Activated lymphocytes were required for virus growth. HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone. Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures. Addition of exogenous IL-2 to the culture media had no effect on viral DNA production. However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media. Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations. However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL. HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines. Nine compounds were tested for antiviral activity against HHV-7 in vitro. Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM. Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production. None of the compounds were cytotoxic at concentrations which inhibited the virus. A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.

Adult↗
PubMed1996

Physical and genetic maps of the human herpesvirus 7 strain SB genome.

Human herpesvirus 7 (HHV-7) is a close relative of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) based on limited biologic and genetic data. In this work we describe physical and genetic maps for HHV-7 strain SB [HHV-7(SB)], which was obtained from the saliva of a healthy adult. The HHV-7(SB) genome length is approximately 144 kb by clamped homogeneous electric field gel electrophoresis and approximately 135 kb by summation of restriction endonuclease fragments. We constructed plasmid clones and PCR amplimers that span the HHV-7 genome, except for the genomic termini, and determined the maps of the restriction endonuclease cleavage sites for BamHI, PstI, and SacI. The HHV-7(SB) genome is composed of a single unique region of approximately 122 kb bounded at each end by a 6 kb direct repeat. Homologs to thirty-five herpesvirus genes were identified. The highest similarity was with the HHV-6 genes, with an average amino acid identity of 50%, followed by the human cytomegalovirus counterpart. The genomic and genetic maps indicated that the HHV-7 and HHV-6 genomes are colinear. There was no sequence variation in a segment of the gene encoding the DNA polymerase-associated factor homolog among six HHV-7 isolates, while the corresponding segment of the HHV-6A and HHV-6B counterparts differed by 4.6%. These data support previous observations that the closest genetic relatives of HHV-7 are betaherpesviruses.

Amino Acid Sequence↗
PubMed1996

Seroconversion to human herpesvirus 6 and human herpesvirus 7 among Brazilian children with clinical diagnoses of measles or rubella.

We collected acute-phase and convalescent-phase serum samples from Brazilian patients who presented with exanthem of unknown origin and evaluated these samples by means of an immunoblot assay for seroconversion to human herpesvirus 6 (HIV-6) or human herpesvirus 7 (HIV-7). Measles or rubella had been clinically diagnosed in all these patients, but their sera were negative for antibodies to both measles virus and rubella virus. Twenty percent of the patients clearly seroconverted to HHV-6 after manifestation of the exanthem, and 8% seroconverted to HHV-7. All seroconversions to HHV-6 occurred in children aged < or = 5 years; a 41% frequency of seroconversion to HHV-6 was noted among children between 3 months and 23 months of age, whereas seroconversions to HHV-7 were detected during infancy and through adulthood. Our data indicate that primary infections due to HHV-6 or HHV-7 can be misdiagnosed as measles or rubella.

Antibodies, Viral↗
PubMed1996

Evaluation of immunoassays for detection of antibodies to human herpesvirus 7.

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.

Adolescent↗
PubMed1995

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

Base Sequence↗
PubMed1993

A strongly immunoreactive virion protein of human herpesvirus 6 variant B strain Z29: identification and characterization of the gene and mapping of a variant-specific monoclonal antibody reactive epitope.

We previously identified a 101-kDa apparent molecular mass polypeptide (101K) as the major immunoreactive virion protein of human herpesvirus 6 variant B strain Z29 [HHV-6B(Z29)] and found that the human immune response to this protein is HHV-6-specific (Yamamoto, M., Black, J. B., Stewart, J. A., Lopez, C., and Pellett, P. E., 1990, J. Clin. Microbiol. 28, 1957-1962). We report here the identification and characterization of the gene encoding 101K. We found 81% amino acid identity between an HHV-6B(Z29) open reading frame (ORF) and its homolog in HHV-6A strain U1102 [HHV-6A(U1102)]. The product of this gene was identified as 101K on the basis of both the reactivity of a 101K-specific monoclonal antibody (MAb C3108-103) with a bacterially expressed portion of the gene and the reactivity of polyclonal rabbit antibodies raised against the bacterially expressed protein with 101K expressed by HHV-6B(Z29)-infected cells. MAb C3108-103 reacted with eight of eight variant B isolates and none of six variant A isolates, indicating that it is a variant-specific MAb. The MAb reactivity was mapped to an eight-amino-acid segment of 101K. HHV-6A(U1102) differs from HHV-6B(Z29) by two amino acids in this region; substitution mapping with synthetic oligopeptides mapped the variant B specificity to Asp723, this explaining the failure of the MAb to react with variant A proteins. A set of transcripts appropriately sized for expression of 101K was identified and precisely mapped. The transcripts originated down-stream from either of two TATA boxes located 139 bp apart in the region 5' to the 101K ORF, with one 5'-species being much more abundant. Two independent polyadenylation sites were identified; the canonical polyadenylation signal located 3' to the 101K ORF was used much more frequently than was the atypical polyadenylation signal located within the 101K ORF. These results suggest a complex regulatory mechanism for this gene.

Amino Acid Sequence↗
PubMed1993

Frequent isolation of human herpesvirus 7 from saliva.

We obtained isolates of human herpesvirus 7 (HHV-7) from 6 of 8 healthy adults by culturing saliva with human umbilical cord blood lymphocytes. These isolates were identified as HHV-7 on the basis of comparisons of restriction endonuclease fragment profiles and hybridization with HHV-7 strain RK DNA. The isolates could be differentiated from HHV-7 strain RK and from each other by their restriction endonuclease fragment profiles. We confirm the finding of frequent isolation of HHV-7 from saliva of healthy adults and report the first dual isolation of human herpesvirus 6 (HHV-6) and HHV-7 from a single saliva specimen. We also describe an in situ hybridization assay that can distinguish between HHV-6 and HHV-7.

Adult↗
PubMed1992

Effects of temperature, potassium concentration, and sugar on human spermatozoa motility: a cell preservation model from reproductive medicine.

Two experiments were performed in vitro with human sperm from a panel of 11 donors. In both experiments, washed sperm from each donor were divided into four equivolume samples, three of which were resuspended in various formulations of Tyrode's solution and the fourth was resuspended in its own seminal plasma. Each of the four samples was then stored at 37 or 3 degrees C for fixed intervals of 0, 2, 6, 12, and 24 h. Motility was assessed at 37 degrees C for all samples at the end of each interval. The results indicate that sperm survival in vitro at 3 degrees C was significantly enhanced by 20 mM K+ in Tyrode's solution relative to Tyrode's with less K+. A metabolizable sugar such as glucose was essential to maintaining sperm viability in K(+)-free media. The addition of raffinose to media containing glucose improved motility of sperm stored at 3 degrees C for 6 h.

Adolescent↗
PubMed1992

Induction of host cell protein synthesis by human herpesvirus 6.

We observed an increase in host cell protein synthesis in human cord blood lymphocytes (CBL) infected with human herpesvirus 6 relative to uninfected cultures. The magnitude of this effect could not be explained by a smaller decrease in cell number in the infected cultures. The induction of host cell protein synthesis by HHV-6 does not appear to be mediated by a stable soluble factor present in the infected cell culture supernatant. When CBL were infected with virus that had been exposed to ultraviolet irradiation (UV) for various intervals, we found that the level of increase in cell number, host protein synthesis, viral DNA and viral antigen was inversely proportional to the length of time of virus exposure to UV. No increase in cell number or host cell protein synthesis was seen in CBL infected in the presence of 50 micrograms/ml phosphonoacetic acid, an inhibitor of HHV-6 DNA replication. These results indicate that components of input virions do not induce the increased protein synthesis and that the induction is dependent on viral DNA replication.

Biological Factors↗
PubMed1990

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.

Antibodies, Monoclonal↗
PubMed1989

Growth properties of human herpesvirus-6 strain Z29.

Experiments performed to optimize the growth conditions of HHV-6(Z29) revealed that the virus grows best in phytohemagglutinin-stimulated umbilical cord blood lymphocytes (CBL) cultured in media containing 32 units/ml interleukin-2 and 0.01 mg/ml hydrocortisone. The titer of maternal antibody in the plasma of the cord blood cells does not affect the ability of the cells to support virus growth. DEAE-dextran and polybrene do not increase virus growth in umbilical cord blood lymphocytes. Phorbol myristate acetate abolishes virus growth. The HHV-6(Z29) growth cycle in CBL was approximately 5 days; capsids were not seen before day 3, and mature virions were not seen before day 5.

Cells, Cultured↗