DORAVIR: a French national survey of people with HIV-1 treated with an antiretroviral regimen including doravirine.

BACKGROUND: Doravirine is the latest NNRTI to be approved for the treatment of HIV-1 and has a different resistance profile from first-generation NNRTIs. Our aim was to investigate the virological efficacy of antiretroviral treatment including doravirine in people living with HIV-1 (PLWHIV), the factors associated with virological failure (VF) and those associated with the emergence of reverse transcriptase (RT) mutations in the case of VF. METHODS: A retrospective national survey of PLWHIV who were either naive or experienced on antiretroviral treatment including doravirine was conducted. VF was defined as two consecutive plasma viral loads (VLs) of ≥50 copies/mL or one VL of ≥200 copies/mL. Genotypic resistance tests were interpreted using the Stanford (v9.4.1) and ANRS (v33) algorithms. RESULTS: Of the 589 PLWHIV treated with a doravirine-containing regimen, 8.5% were naive and 91.5% had prior antiretroviral experience; 56.9% were infected with HIV-1 B subtype. Overall, 88.3% and 85.1% of participants were virologically controlled at Month (M)3 and M6 of doravirine treatment, respectively. In multivariable analysis, CRF02_AG subtype, higher zenith plasma HIV-1 RNA VL, doravirine initiation in the context of failure and baseline V179D mutation presence were associated with VF. Among 88 PLWHIV who experienced virological failure at M6, 15.9% had a median of 2 (IQR 1-3) HIV RT mutations. In multivariable analysis, the only factor associated with the occurrence of mutations was a genotypic sensitivity score that was not fully sensitive. CONCLUSIONS: This study is one of the largest to characterize the virological efficacy of doravirine-containing regimens in clinical practice and to identify factors associated with VF or emergence of resistance mutations that should be considered in clinical management.

Chronic persistent HHV-6B infection after sulfasalazine-induced DRESS with demonstration of HHV-6 encoded small noncoding RNAs (sncRNAs) in Crohn's-like colitis: Case report.

A sulfasalazine-induced DRESS (Drug Reactivation with Eosinophilia and Systemic Symptoms) was complicated by a Crohn's-like colitis. We demonstrated HHV-6 reactivation with presence of HHV-6 DNA and small noncoding RNA in colonic lesions. This observation confirms the major role of HHV-6 reactivation in DRESS manifestations and the importance of looking for HHV-6 reactivation in DRESS.

Donor-to-recipient transmission and reactivation in a kidney transplant recipient of an inherited chromosomally integrated HHV-6A: Evidence and outcomes.

Human herpesvirus (HHV)-6A can be inherited and chromosomally integrated (iciHHV-6A), and donor-to-recipient transmission has been reported in solid organ transplant. However, when HHV-6A reactivation happens after transplant, the source of HHV-6A is often not evident and its pathogenicity remains unclear. Here, we present an exhaustive case of donor-to-recipient transmission and reactivation of iciHHV-6A through kidney transplant. The absence of HHV-6A genome from the nails of the recipient excluded a recipient-related iciHHV-6A. Viral loads > 7 log10 copies/106 cells in donor blood samples and similarities of U38, U39, U69, and U100 viral genes between donor, recipient, and previously published iciHHV-6A strains are proof of donor-related transmission. Detection of noncoding HHV-6 snc-RNA14 using fluorescence in situ hybridization analysis and immunofluorescence staining of HHV-6A gp82/gp105 late proteins on kidney biopsies showed evidence of reactivation in the transplanted kidney. Because HHV-6A reactivation can be life threatening in immunocompromised patients, we provide several tools to help during the complete screening and diagnosis.

Evaluation of liver failure in a pediatric transplant recipient of a liver allograft with inherited chromosomally integrated HHV-6B.

BACKGROUND: Active infections of human herpesvirus 6B (HHV-6B) are frequent in immunocompromised recipients after transplantation. Nevertheless, they need to be distinguished from latent inherited chromosomally integrated genomes (iciHHV-6) present in about 1% of the population to avoid unnecessary administration of toxic antivirals. METHODS: A 5-year-old child presented with acute liver allograft rejection associated with HHV-6 DNA in plasma, which led to an unfavorable outcome. We investigated the possibility of HHV-6 infection derived from an iciHHV-6 present in the donor's liver using molecular and histopathology studies in various tissues, including quantification of HHV-6 DNA, genotyping, sequencing for antiviral resistance genes, relative quantification of viral transcripts, and detection of gB and gH viral proteins. RESULTS: The presence of iciHHV-6B was evidenced in the donor with signs of reactivation in the gallbladder and transplanted liver (detection of HHV-6B mRNA and late proteins). This localized expression could have played a role in liver rejection. Low viral loads in the recipient's plasma, with identical partial U39 sequences, were in favor of viral DNA released from the transplanted liver rather than a systemic infection. CONCLUSIONS: Determination of iciHHV-6 status before transplantation should be considered to guide clinical decisions, such as antiviral prophylaxis, viral load monitoring, and antiviral therapy.

Phenotypic and Functional Differences between Human Herpesvirus 6- and Human Cytomegalovirus-Specific T Cells.

Human herpesvirus 6 (HHV-6) infects >90% of the population and establishes a latent infection with asymptomatic episodes of reactivation. However, HHV-6 reactivation is associated with morbidity and sometimes mortality in immunocompromised patients. To date, control of the virus in healthy virus carriers and the failure to control it in patients with disease remain poorly understood. In particular, knowledge of HHV-6-specific T-cell responses is limited. Here, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses from peripheral blood mononuclear cells (PBMCs) of healthy donors. We studied the phenotype of effector HHV-6-specific T cells ex vivo, as well as of induced specific suppressive regulatory CD4+ T cells in vitro poststimulation, in comparison to human cytomegalovirus (HCMV) responses. Compared to that for HCMV, we show that ex vivo T-cell reactivity in peripheral blood is detectable but at very low frequency, both for HHV-6A and -6B viruses. Interestingly, the phenotype of the specific T cells also differs between the viruses. HHV-6A- and HHV-6B-specific CD4+ T lymphocytes are less differentiated than HCMV-specific T cells. Furthermore, we show a higher frequency of HHV-6-specific suppressive regulatory T cells (eTregs) than HCMV-specific eTregs in coinfected individuals. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection.IMPORTANCE T cells are central to an effective defense against persistent viral infections that can be related to human cytomegalovirus (HCMV) or human herpesvirus 6 (HHV-6). However, knowledge of HHV-6-specific T-cell responses is limited. In order to deepen our knowledge of T-cell responses to HHV-6, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses directly ex vivo from healthy coinfected blood donors. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection. Our findings may encourage immunomonitoring of patients with viral replication episodes to follow the emergence of effector versus regulatory T cells.

Vaginal Neoplasia Induced by an Unusual Papillomavirus Subtype in a Woman with Inherited Chromosomally Integrated Human Herpesvirus Type 6A.

We describe here a case of high-grade vaginal squamous lesion in a 54-year-old woman with a papillomaviruses (HPV) genital infection that developed from a cervical low-grade squamous intraepithelial lesion (SIL) to a high-grade SIL (H-SIL) on cytological examination. A colposcopy exam led to the detection of suspect vaginal lesions with granulomatous infiltrations, which were classified as a Vaginal Intra-Epithelial Neoplasia grade 2 after pathologists' analyses. After a laser vaginal surgery and a loop excision of the transformation zone, the analyses of the anatomical pieces using a near-complete HPV screening panel revealed an HPV-4 infection that was not detected before in cervical smears. This HPV-infection is associated with a high human herpesvirus type 6A (HHV-6A) viral load in the same anatomical piece. The presence of an inherited chromosomally integrated HHV-6A (iciHHV-6A) was proved in this patient by real-time polymerase chain reaction on hair follicles and nail. This case suggests reconsidering both the benign nature of low-grade lesions in the female genital tract and the well-known "good" prognosis of low-risk HPV infection, especially when iciHHV-6A is diagnosed. This clinical course insists on the benefits of the multiplex panel use or global sequencing in order to optimize biological testing sensitivity, and so enhance clinical management of infection-induced neoplasia.

Human Herpesviruses 6A, 6B, and 7.

Human roseoloviruses include three different species, human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), genetically related to human cytomegalovirus. They exhibit a wide cell tropism in vivo and, like other herpesviruses, induce a lifelong latent infection in humans. In about 1% of the general population, HHV-6 DNA is covalently integrated into the subtelomeric region of cell chromosomes (ciHHV-6). Many active infections, corresponding to primary infections, reactivations, or exogenous reinfections, are asymptomatic. They also may cause serious diseases, particularly in immunocompromised individuals, including hematopoietic stem-cell transplant (HSCT) and solid-organ transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients. This opportunistic pathogenic role is formally established for HHV-6 infection and less clear for HHV-7. It mainly concerns the central-nervous system, bone marrow, lungs, gastrointestinal tract, skin, and liver. As the best example, HHV-6 causes both exanthema subitum, a benign disease associated with primary infection, and severe encephalitis associated with virus reactivations in HSCT recipients. Diagnosis using serologic and direct antigen-detection methods currently exhibits limitations. The most prominent technique is the quantification of viral DNA in blood, other body fluids, and organs by means of real-time polymerase-chain reaction (PCR). The antiviral compounds ganciclovir, foscarnet, and cidofovir are effective against active infections, but there is currently no consensus regarding the indications of treatment or specifics of drug administration. Numerous questions about HHV-6A, HHV-6B, HHV-7 are still pending, concerning in particular clinical impact and therapeutic options in immunocompromised patients.

[Diagnosis and practice of virological monitoring of infections by the human herpesviruses 6A and 6B]. / La pratique du diagnotic et du suivi virologiques des infections par les herpesvirus humains 6A et 6B.

The diagnosis of the infection by the human herpesviruses 6A and 6B (HHV-6A, HHV-6B) is based on a direct and an indirect approaches. Serological methods are mainly used to ask primary infection diagnosis and carry out epidemiological studies. However, limitations are numerous with, in particular, the existence of cross-reactivity with other herpesviruses, and the inability to differentiate the two kinds of HHV-6. Initially based on virus isolation in cell culture, direct diagnosis evolved with the development of gene amplification methods that provide sensitivity and specificity, and allow viral quantitation in many biological systems and the identification of present species. Its main current indications are the identification of active infection, the identification of the integrated form of HHV-6 (iciHHV-6, inherited chromosomally integrated HHV-6) and the monitoring of the effectiveness of antiviral treatment.

HHV-6 infection after allogeneic hematopoietic stem cell transplantation: From chromosomal integration to viral co-infections and T-cell reconstitution patterns.

OBJECTIVES: Human herpes virus 6 (HHV-6) can reactivate after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be associated with significant clinical manifestations. METHODS: Case control study of HHV-6 infections after allo-HSCT. Chromosomal integration (ciHHV-6) for viral loads ≥ 5.5-log10 copies/mL was investigated. Viral co-infections, T-cell recovery, risk factors and outcome were compared in HHV-6- and non-HHV-6-infected patients. Antiviral treatment strategies were reviewed. RESULTS: Among 366 adult allo-HSCT recipients, 75 HHV-6 infections occurred. Three (4%) recipients were ciHHV-6. HHV-6 infections were associated with CMV (p = 0.05; sdHR 1.73, CI 0.99-3.02) and/or BKV infections (p < 0.0001; sdHR 4.63, CI 2.04-10.53) but not EBV reactivation (p = 0.34). A slower CD8+ T-cells recovery was observed until 6 months after allo-HSCT in the HHV-6-infected group (p < 0.001), independently of acute and/or chronic graft-versus-host disease. The overall probability of survival after allo-HSCT was diminished for active HHV-6-infected patients (p = 0.0326). Cord blood unit recipients had a higher risk of developing HHV-6 infection compared to bone marrow recipients (p = 0.0007; sdHR 3.82, CI 1.76-8.27). Anti-HHV-6 treatment achieved complete response in only 2/3 of the cases. CONCLUSIONS: In this series of allo-HSCT recipients, 4% were ciHHV-6, active HHV-6 infection was likely associated with CMV and BKV co-reactivations, delayed CD8+ T-cell recovery and poorer outcome.

Où en est la recherche sur les antiviraux ?

Research for novel antivirals: where are we going? Antiviral drugs have become a critical component of anti-infective treatments, as illustrated by the development of antiretrovirals and antiviral drugs directed against hepatitis B and C viruses. Several other molecules are used in clinical practice against herpesviruses, adenoviruses, poxviruses, papillomaviruses and influenza viruses. Current antivirals often target viral enzymes involved in the replication of viral genomes. They also target the early stages of binding and intracellular penetration of viral particles and the late steps leading to their assembly, maturation and release. Most nucleoside analogs such as acyclovir and nucleotide analogs such as cidofovir, require phosphorylation prior to inhibit the activity of DNA or RNA polymerases through mechanisms of competition and optionally termination. Foscarnet, a pyrophosphate analog, makes this inhibition directly without any modification. Antiretrovirals also include protease, integrase and entry inhibitors while the neuraminidase inhibitors have proven to be effective against influenza viruses. Research for novel drugs aims at increasing the number and specificity of antivirals to overcome the current limitations of antiviral chemotherapy which include the inability to eradicate latent viral infections, emergence of resistance, adverse effects related to cell toxicity and cost. It is essential that economic imperatives do not block or distort the expected progresses in this particularly innovative field of contemporary medicine.

Où en est la recherche sur les antiviraux ? Les antiviraux sont devenus une composante essentielle des traitements anti-infectieux, comme cela est illustré par le développement des antirétroviraux et des antiviraux dirigés contre les virus des hépatites B et C. Plusieurs autres molécules sont utilisées en pratique clinique contre les herpèsvirus, adénovirus, poxvirus, papillomavirus et virus grippaux. Les antiviraux actuels ciblent fréquemment des enzymes virales impliquées dans la réplication des génomes viraux. Ils ciblent aussi les étapes précoces de fixation et de pénétration intracellulaire des particules virales, ainsi que les étapes tardives conduisant à leur assemblage, leur maturation et leur libération. La plupart des analogues nucléosidiques tels que l'aciclovir, et les analogues nucléotidiques tels que le cidofovir, nécessitent une phosphorylation préalable pour inhiber, par un mécanisme de compétition et éventuellement de terminaison, l'activité d'une ADN ou d'une ARN polymérase. Le foscarnet, analogue de pyrophosphate, exerce cette inhibition directement sans modification. Les antirétroviraux incluent également des inhibiteurs de protéase, d'intégrase et d'entrée du virus, alors que les inhibiteurs de la neuraminidase ont montré leur efficacité contre les virus grippaux. La recherche sur les antiviraux vise à augmenter leur nombre et leur spécifi- cité d'action pour dépasser les limitations actuelles de la chimiothérapie antivirale que sont l'impossibilité à éradiquer les infections virales latentes, l'émergence de la résistance, les effets indésirables liés à la relative toxicité cellulaire et le coût. Il est essentiel que les impératifs économiques ne viennent pas bloquer ou biaiser les progrès attendus dans un domaine particulièrement innovant de la médecine contemporaine.

Laboratory and clinical aspects of human herpesvirus 6 infections.

Human herpesvirus 6 (HHV-6) is a widespread betaherpesvirus which is genetically related to human cytomegalovirus (HCMV) and now encompasses two different species: HHV-6A and HHV-6B. HHV-6 exhibits a wide cell tropism in vivo and, like other herpesviruses, induces a lifelong latent infection in humans. As a noticeable difference with respect to other human herpesviruses, genomic HHV-6 DNA is covalently integrated into the subtelomeric region of cell chromosomes (ciHHV-6) in about 1% of the general population. Although it is infrequent, this may be a confounding factor for the diagnosis of active viral infection. The diagnosis of HHV-6 infection is performed by both serologic and direct methods. The most prominent technique is the quantification of viral DNA in blood, other body fluids, and organs by means of real-time PCR. Many active HHV-6 infections, corresponding to primary infections, reactivations, or exogenous reinfections, are asymptomatic. However, the virus may be the cause of serious diseases, particularly in immunocompromised individuals. As emblematic examples of HHV-6 pathogenicity, exanthema subitum, a benign disease of infancy, is associated with primary infection, whereas further virus reactivations can induce severe encephalitis cases, particularly in hematopoietic stem cell transplant recipients. Generally speaking, the formal demonstration of the causative role of HHV-6 in many acute and chronic human diseases is difficult due to the ubiquitous nature of the virus, chronicity of infection, existence of two distinct species, and limitations of current investigational tools. The antiviral compounds ganciclovir, foscarnet, and cidofovir are effective against active HHV-6 infections, but the indications for treatment, as well as the conditions of drug administration, are not formally approved to date. There are still numerous pending questions about HHV-6 which should stimulate future research works on the pathophysiology, diagnosis, and therapy of this remarkable human virus.

Impact of HIV-1 infection on herpes simplex virus type 2 genetic variability among co-infected individuals.

Herpes simplex virus type 2 (HSV-2) is the most common cause of genital ulcer disease worldwide. While the contribution of HSV-2 to acquisition and course of human immunodeficiency virus (HIV) infection has been well described, less attention has been paid to the impact of HIV infection on the variability and the pathophysiology of HSV-2 infection. The goal of the present study was to characterize genotypically and phenotypically HSV-2 strains isolated from 12 patients infected by HIV-1 and from 12 HIV-negative patients. Replication capacity analyses were carried out in Vero cells and full-length nucleotide sequences were determined for glycoproteins B (gB), D (gD), G (gG), thymidine kinase (TK), and DNA polymerase (POL) HSV-2 genes. Sequence alignments and phylogenetic trees were performed. No significant differences were found in terms of replication capacity. The interstrain nucleotide identities of the 3 glycoprotein genes (gB, gC, and gG) ranged from 99.5% to 100% among the 24 HSV-2 strains. The phylogenetic analysis showed no clustering of HSV-2 strains when correlating to the HIV status of the patients. A lower variability was observed for the functional proteins TK and DNA polymerase (98.9% to 100% identity). Genetic analysis of TK evidenced mutations related to acyclovir-resistance in two HSV-2 strains. No specific differences regarding replication capacity and gene sequence were found when comparing HSV-2 strains isolated from patients infected with HIV-1 and HIV-negative patients, suggesting that the virological properties of HSV-2 infection are not influenced by HIV-1 infection among co-infected patients.

Impact of novel mutations of herpes simplex virus 1 and 2 thymidine kinases on acyclovir phosphorylation activity.

The acyclic analogue of guanosine acyclovir (ACV) constitutes the first-line drug for the treatment of herpes simplex virus (HSV) infections. ACV activation requires primophosphorylation by virus-encoded HSV thymidine kinase (TK). In 95% of cases, HSV resistance to ACV is associated with mutations located in TK. The aim of this work was to address the question of the potential involvement of novel HSV-1 and HSV-2 TK mutations in reduced susceptibility to ACV using a novel nonradioactive method, based on luminescent quantitation of ADP, for the evaluation of in vitro phosphorylation activity of TK. All recombinant TKs tested exhibited significantly lower ACV phosphorylation activities in comparison with those of reference KOS or gHSV-2 TKs (p<0.015), therefore indicating that amino acid changes Y53D, L170P, R176W, A207P (HSV-1) and S66P, A72S, I101S, M183I (HSV-2) were likely to be involved in HSV resistance to ACV.

Quantitation of human herpesvirus-6A, -6B and -7 DNAs in whole blood, mononuclear and polymorphonuclear cell fractions from healthy blood donors.

BACKGROUND: Diagnosis of human herpesvirus-6A (HHV-6A), -6B (HHV-6B) or -7 (HHV-7) infections is often based on the measure of viral load in blood. OBJECTIVES: The aim of this study was to define usual values of HHV-6A, HHV-6B and HHV-7 loads in blood fractions (whole blood [WB], mononuclear cells [PBMCs], polymorphonuclear leukocytes [PMNLs]) of blood donors. STUDY DESIGN: HHV-6A, HHV-6B and -7 DNAs were quantitated using real-time PCR assays in WB, PBMCs and PMNLs separated on Ficoll or dextran gradients, respectively, for 200 blood donors. Viral loads were expressed as the number of viral genomic copies per million cells (Cop/M) for all fractions, and also per milliliter for WB. RESULTS: HHV-6B DNA was rarely detected in WB (8%), PBMCs (16.5%), and PMNLs (10.5%), HHV-6A was never detected, whereas HHV-7 DNA was often present in WB (51.5%), PBMCs (62%) and PMNLs (51.5%). Median loads were low with 81 Cop/M in WB, 62 Cop/M in PBMCs and 34.5 Cop/M in PMNLs for HHV-6B, and 129 Cop/M in WB, 225 Cop/M in PBMCs and 62 Cop/M in PMNLs for HHV-7. Viral load expression per million cells and per mL were equivalent. One subject had chromosomally integrated HHV-6 with high viral loads ranging from 2.23×10(6) to 3.21×10(6) Cop/M in all compartments and plasma. CONCLUSIONS: These results allow to propose viral load in WB as a sensitive and suitable marker, with values for healthy subjects at approximately 100 Cop/M for both viruses. The prevalence of chromosomally integrated HHV-6 was 0.5%.

HHV-6 et antiviraux : le sixième herpèsvirus humain fait aussi de la résistance.

The human herpesvirus 6 (HHV-6) is an opportunistic agent like the genetically related human cytomegalovirus (HCMV). After primary infection usually occurring in childhood, it may reactivate during immunosuppression, leading to symptoms of varying severity, such as encephalitis. If no antiviral regimen has been formally approved yet, the anti-HCMV drugs, ganciclovir (GCV), cidofovir (CDV) and foscarnet (PFA), are also active in vitro and in vivo against HHV-6. However, prolonged treatment may lead to the selection of mutants resistant to these drugs. In recent years, resistant clinical or laboratory HHV-6 strains have emerged and have been studied. The involvement of various viral genomic mutations in the resistance has been addressed by different genetic, functional or structural approaches, enabling to understand the underlying molecular mechanisms. Until now, the evidence of the role of a mutation in GCV resistance has only been obtained for the M318V substitution of the viral phosphotransferase, enzyme involved in metabolism of GCV, by means of phenotype restoration tests. This technique remains to be developed to study mutations in the gene of the final target of antiviral drugs, the viral DNA polymerase.

Conservation of HHV-6 DNA polymerase processivity factor sequence and predicted structure suggests it as a target for antiviral development.

The replication of human herpesvirus-6 (HHV-6) DNA is catalyzed by the viral DNA polymerase pU38 and the processivity factor pU27 which stabilizes the enzyme on the DNA template. The genetic polymorphism of pU27 among 46 clinical strains of HHV-6 variant A or B and four strains resistant to antivirals was investigated. Overall, 28 amino acid changes (7.6%) and a two-amino acid deletion were identified among the 368 residues of pU27, when using the U1102 (variant A) sequence as the reference. Eleven amino acid changes (3.0%) specifically differentiated both variants. The median intravariant amino acid variability was 1.2% and 0.3% for A and B, respectively. Except for a single change, the pU27 sequence of multi-drug resistant HHV-6 strains was also conserved. Structural models of pU27 for variants A and B were derived from that of the human cytomegalovirus homologue pUL44, and showed either identical or very similar residues in the regions interacting with viral DNA polymerase and viral DNA molecule. As pU27 is both highly conserved and essential for viral replication, it might constitute an interesting target for antiviral chemotherapy.