Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies.

Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.

Identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus 7.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.

Detection of human herpesvirus 6 by reverse transcription-PCR.

The role of human herpesvirus 6 (HHV-6) in disease beyond primary infection remains unclear. We have developed and validated a new reverse transcription-PCR (RT-PCR) assay for HHV-6 that can determine the presence of HHV-6 in clinical specimens and differentiate between latent and replicating virus. Peripheral blood mononuclear cells from 109 children were evaluated for HHV-6 by RT-PCR, DNA PCR, and viral culture. Of these samples, 106 were suitable for analysis. A total of 20 samples were positive for HHV-6 by culture and DNA PCR, of which 19 were positive by RT-PCR (sensitivity, 95%). All 28 samples from children that were negative by viral culture, but positive by DNA PCR, were negative for viral transcripts by our RT-PCR assay. One positive RT-PCR result was observed in 56 samples that were negative by tissue culture and DNA PCR. This indicates a low rate of false-positive results (1.2%) and a specificity of 98.8%. This RT-PCR assay can reliably differentiate between latent and actively replicating HHV-6 and should allow insight into the pathogenesis of this ubiquitous virus.

Use of PCR in detection of Mycobacterium avium complex (MAC) bacteremia: sensitivity of the assay and effect of treatment for MAC infection on concentrations of human immunodeficiency virus in plasma.

We evaluated the sensitivity and specificity of a PCR-based qualitative test for the rapid diagnosis of Mycobacterium avium-M. intracellulare complex (MAC) bacteremia in patients with AIDS disease. Eleven subjects with newly culture-proven MAC bacteremia had the following tests performed at biweekly intervals during the first 8 weeks of therapy: blood culture, Mycobacterium-specific PCR, and quantitative human immunodeficiency virus (HIV) viral-load testing. Mycobacterium genus-specific biotinylated primers were used to amplify a sequence of approximately 582 nucleotides within the 16S rRNA genes of M. avium and M. intracellulare. Detection of the amplified product was performed with an oligonucleotide probe-coated microwell plate combined with an avidin-horseradish peroxidase-tetramethylbenzidine conjugate-substrate system. While not as sensitive as BACTEC culture, PCR detected 17 of 18 specimens which grew >/=40 organisms/ml (94.4% sensitivity) and 9 of 16 specimens which grew

Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe.

Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections.