Jean-Thierry Aubin

Herpesvirus-like DNA sequences in patients with Mediterranean Kaposi's sarcoma

DNA sequences closely related to herpesvirus-like sequences have been found in AIDS-associated Kaposis sarcoma. Using PCR, we found herpesvirus-like DNA sequences in Kaposis lesions and normal adjacent skin in five patients with Mediterranean Kaposis sarcoma. We did not find these sequences in tissues from patients without Kaposis sarcoma. Semi-quantitative PCR revealed many more herpesvirus-like sequences in Kaposis lesions than in unaffected skin. Our results reinforce the hypothesis that an infectious agent closely related to gamma-herpesvirus is implicated in the pathogenesis of Mediterranean and AIDS-associated Kaposis sarcoma.

Frequency of early in utero HIV-1 infection : a blind DNA polymerase chain reaction study on 100 fetal thymuses

Objective: To estimate the prevalence of in utero transmission of HIV‐1 through the second trimester. Material and methods: One hundred consecutive, unselected, intact fetuses, beyond 15 weeks gestational age (mean, 22.4 weeks) were studied. These were obtained following spontaneous intrauterine deaths (n = 4), miscarriages (n = 4), and elective mid‐trimester terminations (n = 92), eight of which were fetuses with malformations from HIV‐1‐positive pregnancies. Coded DNA extracts from the fetal thymuses were tested blindly by polymerase chain reaction in three laboratories using a total of six different primer pairs. Results: Two thymuses tested positive [95% confidence interval (Cl), 0.2‐7]. Results from the three laboratories were consistent in all 100 cases. The two fetuses with HIV in the thymus both tested positive in other organs, demonstrating systemic HIV infection. The first fetus, whose mother had advanced AIDS, had died in utero and had diffuse toxoplasmosis. The second died following extremely premature delivery in a pregnancy complicated by repeated bleeding. HIV infection was observed in none of the 92 fetuses that resulted from elective mid‐trimester terminations (95% Cl, 0‐4). Conclusion: The frequency of early in utero HIV infection appears to be low, compared with transmission rates in infants born to HIV‐1‐infected mothers, suggesting that transmission occurs mostly later in pregnancy and/or at delivery. Specific risk factors may have implications in the occurrence of early as opposed to late transmission. AIDS 1995, 9:359‐366

In vitro sensitivity of human herpesvirus-6 to antiviral drugs

We studied the sensitivity of human herpesvirus-6 (HHV-6) to 4 antiviral drugs known to be effective in the treatment of infections with other human herpesviruses and human immunodeficiency virus. HHV-6 was grown in peripheral blood lymphocytes, and virus multiplication was quantified by evaluation of the cytopathic effect by molecular hybridization and indirect immunofluorescence assay. The 50% and 90% inhibitory concentrations (IC50 and IC90) were determined for each drug. The results obtained by the 3 different quantification techniques were found to correlate, and enabled us to conclude that HHV-6 replication was readily inhibited by foscarnet or ganciclovir. In contrast, inhibition of HHV-6 replication was observed only at high concentrations of acyclovir, and was not detected at the tested concentrations of zidovudine.

Specificity and sensitivity of RHD genotyping methods by PCR‐based DNA amplification

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D‐pos/D‐neg mixtures containing only 0.001% D‐positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false‐negative results were obtained with method II (DIVb variant), method III (DVI and DFR variants) and method IV (DVI variants), but not method I. Weak D (Du) was correctly detected as D‐positive by all methods, but most cases of Rhnull appeared as false‐positives, as they carry normal RH genes that are not phenotypically expressed. Some false‐positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD‐neg but carrying a C‐ or E‐allele, whereas a high incidence of false‐positives was found among non‐Caucasian Rh‐negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.

Selection of the same mutation in the U69 protein kinase gene of human herpesvirus-6 after prolonged exposure to ganciclovir in vitro and in vivo

After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC(50) of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC(50) in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A-->G substitution of the U69 protein kinase (PK) gene resulted in an M(318)V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A(961)V amino acid substitution within the DNA polymerase. The M(318)V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M(460)V and M(460)I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M(318)V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M(318)V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.

Antigenic and genetic differentiation of the two putative types of human herpes virus 6

Ten human herpes virus 6 (HHV-6) strains from different origins were studied using reactivity to monoclonal antibodies and polymerase chain reaction analysis. Using immunofluorescence and neutralization assays, two monoclonal antibodies gave a positive reaction with the ten strains while three others only reacted with a fraction of these strains. This differential reactivity permitted segregation of the ten strains into two non-overlapping antigenic groups, designated as I and II. DNA was amplified from two regions of HHV-6 genome corresponding to the putative large tegument protein (LTP) gene and major capsid protein (MCP) gene, respectively. The restriction analysis of amplified products using HindIII for LTP and HaeII for MCP showed identical patterns among the strains belonging to the same antigenic group while BglII, TaqI and ClaI provided distinct patterns among group II strains. The nucleotide sequence of amplified products was determined and homology was found to be equal to or greater than 99% within each group whereas it was 96% between both groups. The number of amino-acid changes was higher when comparing two strains of different groups than when comparing two strains of the same group. The converging results of antigenic and genetic analyses led us to consider HHV-6 groups I and II as two distinct types of HHV-6 species.

In vivo role of IL-6 on the viral load and on immunological abnormalities of HIV-infected patients.

In vitro experiments have suggested that interleukin (IL)-6 may contribute to human immunodeficiency virus (HIV) burden and to immunological abnormalities in HIV-infected patients. We had the opportunity to directly address this question in vivo through the virological and immunological monitoring of HIV-infected patients treated with an anti-IL-6 monoclonal antibody (mAb) for a lymphoma (ANRS 018 trial). Sixteen courses of anti-IL-6 mAb administration, performed in 11 patients, were studied. All patients were at a late stage of HIV infection. The HIV load and the immunological status were determined at the initiation of each course and at its end, 21 days later. The mAb induced no significant change of HIV load, as evaluated by p24 antigenemia, plasma viremia, and quantification of circulating HIV RNA by reverse transcriptase-polymerase chain reaction and branched DNA techniques. The anti-IL-6 mAb also did not affect CD4+, CD8+, and CD19+ circulating cell counts, nor the serum concentrations of sIL-2R and of sCD8. In contrast, the mAb completely abrogated acute-phase reaction, as demonstrated by the normalization of C-reactive protein and fibrinogen circulating levels (p = 0.013 and p = 0.008, respectively). It increased serum albumin concentration. The latter effect was restricted to patients with a spontaneously low albuminemia (p = 0.01). It decreased B-lymphocyte hyperactivity, as reflected by decreased IgG and IgA serum levels (p = 0.008 and p < 0.001, respectively), and by a decreased production of IgG in vitro (p = 0.017). In contrast, the IgM hyperproduction was not affected by the mAb. Therefore, increased IL-6 production in HIV-infected patients at a late stage of the infection may not stimulate HIV replication in vivo, but it may represent a key mechanism contributing to the metabolic and immunological dysbalance of the disease.

Difference in permissiveness of human fibroblast cells to variants A and B of human herpesvirus-6

Human herpesvirus-6 (HHV6) is a lymphotropic virus genetically related to human cytomegalovirus (CMV) and for which two variants, A and B, have been distinguished. Human CMV is usually cultivated with human fibroblasts (HF). The lack of cell lines useful for HHV6 isolation and propagation led us to investigate whether HHV6 variants A and B could infect HFs as CMV does. Isolates of HHV6 variants A and B were used to infect MRC-5 HFs. HHV6 infection was detected by means of immunoperoxidase assay using three specific monoclonal antibodies. HHV6-specific antigens were detected in 88 and 38% of cases after infection with variants A and B, respectively. The highest number of HHV6-antigen-positive cells was obtained at 4-5 days p.i. The titre of HHV6 stocks was determined in parallel by immunoperoxidase assay on HFs and by observation of cytopathic effect using serial dilutions on peripheral blood mononuclear cells (PBMC). The number of infectious particles inducing the appearance of antigen-positive HF cells was consistently lower than the titre of virus stocks, expressed as TCID50. The amount of HF-associated HHV6 DNA was measured using limiting dilution PCR assay; it was significantly increased during 4-day infection in the case of variant A but not variant B. The yield of virus from infected HFs was demonstrated only for variant A by the serial propagation of virus from HFs to PBMCs and by the increase in cell-free HHV6 DNA in HF culture supernatant. Our results show that HHV6 can reproducibly infect HFs, albeit at a low level, and that HFs are more permissive to variant A than to variant B, as reported previously for PBMCs and human T-cell lines.

Detection of HHV-6 by the polymerase chain reaction

The polymerase chain reaction (PCR) was used to detect human herpes virus 6 (HHV-6) sequences in tissue culture. A pair of primers was synthesized and used to amplify a conserved region of the genome. Amplified products were detected either by visualization of UV illuminated ethidium bromide stained gel or, by hybridization with a specific radiolabeled oligonucleotide. As little as 5 fg of HHV-6 could be detected in infected cells, making this assay suitable for diagnostic purposes.

Human herpesvirus-6 and human herpesvirus-7 in the bone marrow from healthy subjects.

BACKGROUND Human herpesviruses (HHVs) 6 and 7 are recently discovered betaherpesviruses. Although HHV-6 has been associated with disordered hematopoiesis in bone marrow transplant recipients, little information is available on the presence of both viruses in the bone marrow from healthy subjects. METHODS We detected HHV-6 and HHV-7 DNA by means of polymerase chain reaction in bone marrow and peripheral blood samples from 18 healthy subjects who underwent total hip arthroplasty. RESULTS Genomic HHV-6 and HHV-7 DNA were detected in 11% and 67% of the blood samples, respectively, and in 28% and 50% of the bone marrow samples, respectively. CONCLUSIONS Both viruses may be present in the bone marrow without hematopoiesis disorder and can be transmitted through bone marrow infusion. Therefore, the causative role of these two viruses in some bone marrow diseases cannot be inferred simply from the detection of their genome in bone marrow by means of polymerase chain reaction.