Humoral and cellular responses to influenza vaccination in human recipients naturally tolerant to a kidney allograft.

Rare kidney allograft recipients enjoy unaltered graft function years after interruption of their immunosuppressive treatment. To assess the extent to which this state of 'operational tolerance' (TOL) is specific to the graft and not the result of a global immunodeficiency, we analyzed the response of such patients following influenza vaccination. Hemagglutination inhibition titers and frequency of IFNgamma-secreting T cells were measured before 1 and 3 months after vaccination. The proportion of healthy volunteers (HV) responding to vaccine was significantly higher than that of immunosuppressed (IS) patients. Three 'TOL' patients presented a humoral response similar to that of HV, whereas the two others had a poor response, like the IS recipients. Although the small number of patients does not allow for definitive conclusions to be made, these data suggest that the status of tolerance may be heterogeneous, with some patients with a global immunodeficiency and others with an adapted response to vaccination.

Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir.

Geographic spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compare the sensitivities to oseltamivir of the NAs of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding 50% inhibitory concentrations were determined using a standard in vitro NA inhibition assay. The K(m) for the substrate and the affinity for the inhibitor (K(i)) of NA were determined for a 1997 and a 2005 virus, using an NA inhibition assay on cells transiently expressing the viral enzyme. Our data show that the sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir are about 10-fold higher than those of earlier H5N1 viruses or currently circulating H1N1 viruses. Three-dimensional modeling of the N1 protein predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug-selective pressure may result in significant variations in sensitivity to anti-NA drugs. Although the clinical relevance of a 10-fold increase in the sensitivity of NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly, even in the absence of treatment, underscores the need for continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.

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.

Large-scale screening for human parvovirus B19 DNA by PCR: application to the quality control of plasma for fractionation.

BACKGROUND AND OBJECTIVES: Because human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products, the 'Laboratoire français du Fractionnement et des Biotechnologies' (LFB) implemented PCR screening of plasma pool samples for B19 DNA as part of the quality control of plasma source material. MATERIALS AND METHODS: Plasma pool samples (average of 46.5 donations) were tested for B19 DNA by PCR and by immunological detection of PCR products. The viral DNA content was determined by means of a TaqMantrade mark-based, quantitative PCR. RESULTS: From plasma corresponding to 2-year collections in France, and representing 4.26 million donations from approximately 1.25 million voluntary unpaid donors, the average frequency of positive donations was 1/5,950 and reached 1/1,420 during an epidemic. Levels of B19 DNA in positive pools ranged from <10(2) to 10(11) copies/ml. CONCLUSION: A large-scale PCR plasma screening increased the safety of LFB's wide range of products with respect to B19, a virus particularly resistant to physicochemical inactivation procedures.

Preferential associations of alleles of three distinct genes argue for the existence of two prototype variants of human herpesvirus 7.

We had previously described six distinct alleles of the glycoprotein B (gB) gene of human herpesvirus 7 (HHV-7). The genetic changes corresponding to these alleles did not affect gB gene transcription or translation in in vitro assays. The study of distinct HHV-7-positive human samples showed preferential associations of some gB alleles with some alleles of two other genes, distantly located on the HHV-7 genome, coding for the phosphoprotein p100 (p100) and the major capsid protein (MCP). Two allele combinations, corresponding to 44 and 31% of the samples studied, respectively, were interpreted as the genetic signatures of two major prototype HHV-7 variants.

Definition and distribution analysis of glycoprotein B gene alleles of human herpesvirus 7.

As for other herpesviruses, glycoprotein B (gB) of human herpesvirus 7 (HHV-7) is believed to play a major role in virus infection and as a target of the host immunogenic response. Using nested PCR, we amplified the whole HHV-7 gB gene from 108 human peripheral blood mononuclear cell samples and studied its variability. By means of restriction fragment length polymorphism (RFLP) analysis, three distinct patterns, designated I, II, and III, were defined and detected at frequencies of 93, 5, and 2%, respectively. Determination of the nucleotide sequence allowed us to recognize five critical positions in the gB gene with six specific combinations of point changes at these positions. These combinations were gB alleles A, B, C, D, E, and F. Alleles D and E corresponded to RFLP patterns II and III, respectively, while the other four alleles corresponded to RFLP pattern I. Identical gB alleles were detected in serial samples as well as in paired samples of blood and saliva from the same individuals, except for one case. In contrast, the distribution of gB alleles differed according to the geographical origin of the human samples: C was the most frequent allele in both African and Caribbean samples, whereas F was the most frequent allele in European ones. Although none of the allele-specific nucleotide changes induced any modification at the protein level, the definition of gB alleles provided convenient viral markers for the study of both HHV-7 infections and human population genetics.

Comparison of an amplified enzyme-linked immunosorbent assay with procedures based on molecular biology for assessing human immunodeficiency virus type 1 viral load.

The sensitivity of the enzyme-linked amplified sorbent test (ELAST) was compared with those of other classic enzyme-linked immunosorbent assays (ELISAs), with or without previous acidic immunocomplex dissociation (ICD), in a series of samples at different stages of human immunodeficiency virus type 1 (HIV-1) infection. The limit of viral detection of ELAST was assessed with fresh HIV-1 preparations quantified by reverse transcription-PCR and with the P24 antigen (Ag) Sanofi Pasteur Calibrator containing lyophilized virus. The P24 Ag detection capacity of ELAST was compared with that of NASBA in samples obtained from infected subjects with less than 250 CD4+ cells. The results of the present study show that ELAST was the most sensitive method for detecting P24 Ag compared to classic ELISA and ICD plus ELISA. ELAST was able to detect 0.5 pg of P24 Ag per ml in a whole virus preparation and the equivalent of 330 to 1,000 RNA copies/ml of HIV. The rate of detection of P24 Ag was always higher in subjects with low levels of anti-P24 antibodies. The number of positive results was dramatically enhanced (from 37% to 94% for subjects with <250 CD4+ cells) when the incubation period was prolonged from 1 to 16 h. In a third series of 84 samples (<250 CD4+ cells) tested in parallel, NASBA yielded 83% of the positive results and ELAST yielded 79%. Considering the high sensitivity, low cost, simplicity of equipment (only a plate reader), and possibility for full automation, ELAST appears to be a promising new tool for measuring viral load, especially in areas with few resources, in which the procedures based on molecular biology techniques may be difficult to install.

Selection and characterization of two specific monoclonal antibodies directed against the two variants of human herpesvirus-6.

Monoclonal antibodies (mAbs) specific for human herpesvirus-6 (HHV6) proteins were derived from the splenocytes of mice immunized with HHV6 TAN isolate-infected peripheral blood mononuclear cells. The two mAbs 8C8 and 7C7 reacted by means of immunofluorescence and immunoperoxidase assays with both variant A and variant B isolates giving two different staining patterns. In infected cells, cytoplasmic diffuse staining was observed with mAb 8C8, whereas intense nuclear staining was obtained with mAb 7C7. These different locations of viral target proteins were confirmed by confocal microscopy. The mAb 8C8 reacted with a family of six glycoproteins designated as the gp72 complex in the case of variant A strains and gp63 complex in the case of variant B strains. The endoglycosidases H and F reduced those glycoproteins to a putative precursor molecule of 58 kDa. The mAb 7C7 reacted with 116 and 109 kDa proteins with the two HHV6 variants. These two mAbs did not neutralize virion infectivity in the absence of complement. No cross-reactivity was observed when these mAbs were used in immunoperoxidase assay and immunoblotting against the proteins of human cytomegalovirus or other human herpesviruses. Thus, the two mAbs 8C8 and 7C7 may be valuable tools for the diagnosis and biological investigation of HHV6 infections.

Detection rate and intratumoral virus load of human herpesvirus-8 in immunodeficiency-related B-cell lymphoid malignancies.

Human herpesvirus-8 (HHV-8), associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, has been found in circulating B-cells and might have a causative role in B-cell malignancies associated with immunodeficiency syndromes. We determined the rate of detection and intratumoral virus load of HHV-8 by means of a semiquantitative approach in post-transplant lymphoproliferative diseases (PTLDs), AIDS-related non-Hodgkin's lymphomas (NHLs), including both Burkitt's lymphomas (BLs) and large cell lymphomas (LCLs), as well as in control groups consisting of follicular hyperplasias (FHs) and HIV-negative LCLs. HHV-8 sequences were detected at a similar rate in HIV-negative PTLDs (24%), HIV-negative LCLs (22%) and HIV-negative FHs (17%). The detection rate was significantly higher in HIV-positive BLs (73%), HIV-positive LCLs (67%), and HIV-positive FHs (65%) supporting the view of an epidemiological link between HHV-8 and HIV infections. The viral load was 10(2) genome copies per cell in the single case of primary effusion lymphoma included in the LCL group while it was 10(-3) copy per cell (median value; range: 10(-4)-10(-1)) in all the other HHV-8-positive samples. No significant difference of viral load was found according to HIV status. The virus loads of PTLDs and HIV-positive LCLs were significantly higher than those observed in HIV-positive BLs and FHs, suggesting, to some extent, that the degree of immunodeficiency may influence HHV-8 replication. However, with the exception of the single case of primary effusion lymphoma studied, the low intratumoral load of HHV-8 strongly argues against a direct causative agent of the virus in the occurrence of PTLDs and AIDS-related NHLs.

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 (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) 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.

Detection of human Betaherpesvirinae in saliva and urine from immunocompromised and immunocompetent subjects.

Human cytomegalovirus (HCMV) is a well-known opportunistic agent that reactivates in human immunodeficiency virus (HIV)-seropositive subjects. Human herpesvirus 6 (HHV-6) and HHV-7 were discovered recently and, like HCMV, belong to the Betaherpesvirinae subfamily. We looked for the presence of HCMV, HHV-6, and HHV-7 by PCR with saliva and urine samples from 125 HIV-seropositive patients at different stages of HIV infection and with saliva and urine samples from 29 HIV-seronegative subjects. All three viruses were frequently detected in the saliva (overall rates of detection, 61, 43, and 63% for HCMV, HHV-6, and HHV-7, respectively) with no correlation with the stage of immune deficiency. In contrast, HCMV was detected in urine much more frequently than the two other herpesviruses (overall rates of detection, 37, 2, and 6.5% for HCMV, HHV-6, and HHV-7, respectively) and was associated with immune deficiency. This suggests that these three genetically related viruses differ from each other with regard to replication in the urinary tract.

Use of inverse polymerase chain reaction to characterize a novel human herpesvirus 7 isolate.

Human herpesvirus-7 (HHV-7) is a T-lymphotropic virus detected in peripheral blood mononuclear cells and in saliva, but no reliable link between this agent and a disease has been demonstrated so far. Starting from a 186 bp-fragment described previously, we used an inverse polymerase chain reaction to clone and sequence the adjacent sequences of this known region. A 1062 bp-fragment containing two ORFs was characterized and its sequence was compared with those of two other beta-herpesviruses, human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6). With respect to the first ORF, amino-acid identity was estimated to be 22% between HHV-7 and HCMV, and 47% between HHV-7 and HHV-6. In contrast, only a weak homology between HHV-7 and the two other beta-herpesviruses was demonstrated for the second ORF. The newly characterized 1062 bp-fragment was used to define a novel HHV-7-specific PCR assay. Preliminary data indicate that this region is highly conserved among HHV-7 isolates.

Twin zygosity diagnosis by mailed questionnaire below age twelve months. Le Groupe Romulus.

Parents of a sample of 76 same sexed pairs of twins aged 3 to 9 months completed a mailed similarity questionnaire. It included the Bonnelykke et al.'s questionnaire and a four anthropological variable scale. To improve each of these two methods, three other combined methods were carried out and results were compared with the biological zygosity diagnosis. The Bonnelykke et al.'s classification combined with anthropological scale (method 4) gave only 1.2% misclassified in the whole sample. It is concluded that zygosity diagnosis using this type of procedure to distinguish MZ and DZ pairs would be important not only for epidemiological study but also for pediatricians and parents.

[Human herpesvirus 8]. / L'herpesvirus humain 8.

Human herpesvirus 8 (HHV-8, KSHV) is a novel virus for which fragments of genomic sequence were identified in 1994. This was done by means of a differential amplification technique applied to the DNA of Kaposi's sarcoma lesions and normal tissues obtained from the same individual. The analysis of nucleotide sequence has shown that HHV-8 is closely related to herpesvirus saimiri and Epstein-Barr virus, two members of Gammaherpesvirinae sub-family. The virus has been observed by means of electron microscopy in chronically infected cell lines. Serological studies are still preliminary but seem to demonstrate a low prevalence of HHV-8 infection among the general population at least in Western countries, HHV-8 is mainly detected by means of polymerase chain reaction (PCR) and molecular hybridization. HHV-8 detection in human tissues is strongly associated with three diseases: Kaposi's sarcoma, body-cavity-based lymphomas and Castleman's disease. The demonstration of the causative role of HHV-8 in the occurrence of these three diseases, particularly Kaposi's sarcoma, would have major consequences for their diagnosis and treatment.

Testing for in utero human immunodeficiency virus infection with fetal blood sampling.

OBJECTIVE: Our purpose was to evaluate the potential of midtrimester fetal blood sampling to detect in utero human immunodeficiency virus type 1 infection. STUDY DESIGN: Ultrasonographically guided fetal blood sampling was performed before pregnancy termination in 28 women infected with human immunodeficiency virus type 1. Mean gestational age was 22 weeks (range 15 to 29 weeks). Samples were tested for human immunodeficiency virus with undissociated p24 antigen and deoxyribonucleic acid polymerase chain reaction or viral isolation by cell culture. After terminations fetal thymuses were also tested for human immunodeficiency virus infection by polymerase chain reaction. Polymerase chain reaction was also performed on maternal cells to confirm that the primer pairs used were able to detect human immunodeficiency virus type 1 strains present. RESULTS: All fetal blood samples had negative results by p24 antigen and polymerase chain reaction or virus culture and all fetal thymuses were negative by polymerase chain reaction. CONCLUSION: Early in utero human immunodeficiency virus infection appears infrequent, supporting the hypothesis that mother-to-child transmission predominantly occurs late in pregnancy. Therefore midtrimester prenatal diagnosis is not currently of use to women in making reproductive decisions.

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.

Restricted tissue distribution of extralesional Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS patients with Kaposi's sarcoma.

Specific herpesvirus-like DNA sequences have been found in Kaposi's sarcoma (KS) lesions of AIDS patients, suggesting that a novel gamma herpesvirus, homologous to Epstein-Barr virus and herpesvirus saimiri, could be implicated in the pathogenesis of KS. To better understand the role of this putative etiological agent, named Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), we investigated by the polymerase chain reaction (PCR) the presence of viral DNA sequences in various organs obtained at autopsy from seven AIDS patients with KS and six without KS. For each sample, to exclude positive results due to visceral KS dissemination, the presence of microscopic foci of KS cells was rules out by histology and CD34 immunohistochemistry on serial frozen sections immediately adjacent to those employed for DNA extraction. PCR and nested PCR were performed with primers specific for the HIV-8 330 Bam fragment originally described by Chang et al. (Science 1994;266:1865-1869). As quality control, the extracted DNA was amplified with primers for human beta-globin. All KS legions were HHV-8 positive. In addition, extralesional KSHV DNA sequences were detected in seven of seven lymphoid organs and in five of five prostate glands of KS patients. Normal skin was positive in three of five cases and bone marrow in two of three tested cases, all other tissues being negative by PCR and nested PCR. By contrast, no virus was detected in tissue samples of AIDS cases without KS. The restricted organ distribution here documented argues for a selective tissue tropism of HHV-8 in vivo in AIDS patients and suggests that in the infected host lymphoid organs and the prostate gland may represent privileged sites of viral latency and persistence.