Michel Segondy

Human Skin Microbiota: High Diversity of DNA Viruses Identified on the Human Skin by High Throughput Sequencing

The human skin is a complex ecosystem that hosts a heterogeneous flora. Until recently, the diversity of the cutaneous microbiota was mainly investigated for bacteria through culture based assays subsequently confirmed by molecular techniques. There are now many evidences that viruses represent a significant part of the cutaneous flora as demonstrated by the asymptomatic carriage of beta and gamma-human papillomaviruses on the healthy skin. Furthermore, it has been recently suggested that some representatives of the Polyomavirus genus might share a similar feature. In the present study, the cutaneous virome of the surface of the normal-appearing skin from five healthy individuals and one patient with Merkel cell carcinoma was investigated through a high throughput metagenomic sequencing approach in an attempt to provide a thorough description of the cutaneous flora, with a particular focus on its viral component. The results emphasize the high diversity of the viral cutaneous flora with multiple polyomaviruses, papillomaviruses and circoviruses being detected on normal-appearing skin. Moreover, this approach resulted in the identification of new Papillomavirus and Circovirus genomes and confirmed a very low level of genetic diversity within human polyomavirus species. Although viruses are generally considered as pathogen agents, our findings support the existence of a complex viral flora present at the surface of healthy-appearing human skin in various individuals. The dynamics and anatomical variations of this skin virome and its variations according to pathological conditions remain to be further studied. The potential involvement of these viruses, alone or in combination, in skin proliferative disorders and oncogenesis is another crucial issue to be elucidated.

CD4+ T Cell Surface CCR5 Density as a Determining Factor of Virus Load in Persons Infected with Human Immunodeficiency Virus Type 1

The intensity of expression of the chemokine receptor CCR5 is involved in in vitro cell infectability by human immunodeficiency virus (HIV)-1 R5 isolates. Because CCR5 expression varies among individuals, the hypothesis that this expression could determine virus load in HIV-1-infected persons was tested. The mean number of CCR5 molecules per cell was measured on peripheral blood CD4+ T lymphocytes (CCR5 density) from HIV-1-infected, asymptomatic, nontreated adults. There was a strong correlation between HIV RNA plasma level and CCR5 density (P=.009) that was independent of cell activation and was not due to an HIV-induced CCR5 up-regulation. These data are compatible with the hypothesis that CCR5 density is a key factor governing cell infectability and in vivo virus production and explain the protective effect of the Delta32CCR5 deletion, which results in low CCR5 expression. CCR5 density might be of critical predictive value in HIV infection.

Herpes simplex virus and HIV-1: deciphering viral synergy

Recent proof-of-concept randomised controlled trials have shown a causal relation between herpes simplex virus (HSV) type 2 infection and HIV-1 replication in co-infected individuals. We explore the mechanisms that may operate to enhance reciprocal viral replication. Direct interactions could involve HIV-1-related immune deficiency, disruption of mucosal barrier by HSV infection/reactivation, HSV-induced mucosal cell recruitment, transactivation of HIV-1 replication by HSV proteins, and immune modulation by HSV decoys. Indirect interactions might coexist through disturbances of the vaginal flora during HSV shedding and systemic immune activation. In co-infected individuals, suppressive HSV treatment reduces HIV-1 genital and systemic excretion. This finding is a likely result of efficacious prevention of HSV2 reactivations, and perhaps of other herpesviruses. Strategies to control HSV2 and other herpesviruses deserve urgent attention and should become part of the HIV-1 prevention and care package.

Human Bocavirus in French Children

Human bocavirus (HBoV), a new member of the genus Bocavirus in the family Parvoviridae, has been recently associated with respiratory tract infections. We report the epidemiologic and clinical features observed from a 1-year retrospective study of HBoV infection in young children hospitalized with a respiratory tract infection.

Emergence of zidovudine and multidrug-resistance mutations in the HIV-1 reverse transcriptase gene in therapy-naive patients receiving stavudine plus didanosine combination therapy

OBJECTIVE Assessment of genotypic changes in the reverse transcriptase gene of HIV-1 occurring in antiretroviral naive patients treated by stavudine plus didanosine combination therapy. METHODS Sequence analysis (codons 1-230) was performed after amplification of the reverse transcriptase gene from plasma samples collected at baseline and at the end of treatment from 39 previously treatment-naive patients treated for 24-48 weeks. RESULTS At baseline, mutations associated with zidovudine resistance were detected in plasma from two patients: Asp67Asn/Lys219Gln and Leu210Trp. Among the 39 subjects, 18 (46%) developed mutations: one developed the Val75Thr/Ala mutation, four (10%) developed a Gln151Met multidrug-resistance mutation (MDR), associated in one of them with the Phe77Leu and the Phe116Tyr MDR mutations and 14 (36%) developed one or more zidovudine-specific mutations (Met41Leu, Asp67Asn, Lys70Arg, Leu210Trp, Thr215Tyr/Phe). The development of a Met41Leu zidovudine-specific mutation was associated with the development of a Gln151Met mutation in one patient. Other reverse transcriptase mutations known to confer resistance to nucleoside analogues were not detected. At inclusion, there was no statistical difference in HIV-1 load between patients who developed resistance mutations and those who did not. RNA HIV-1 load decrease was higher (P = 0.05) in patients who maintained a wild-type reverse transcriptase genotype (-2.22 log10 copies/ml) than in patients who developed resistance mutations (-1.14 log10 copies/ml). CONCLUSION Stavudine/didanosine combination therapy is associated with emergence of zidovudine-related resistance or MDR mutations in naive patients. These findings should be considered when optimizing salvage therapy for patients who have received a treatment including stavudine/didanosine combination.

Human metapneumovirus infection in young children hospitalized with respiratory tract disease

Background: Human metapneumovirus (hMPV) is a newly recognized pathogen associated with respiratory tract disease (RTD). Objectives: To evaluate the incidence of hMPV infection in children hospitalized with RTD and to analyze the virologic and clinical features of hMPV infection. Study Design: All children younger than 5 years of age hospitalized for RTD were included in this 1-year prospective study. hMPV was detected in nasopharyngeal secretions by reverse transcription polymerase chain reaction. The hMPV F gene amplification products were sequenced, and a phylogenetic tree was constructed. Samples were also tested for other respiratory viruses by both direct immunofluorescence assay and virus culture. Results: hMPV, detected in 50 of 589 (8.5%) children, represented the second leading cause of RTD after respiratory syncytial virus (RSV). Infections with hMPV occurred mainly between December and April. hMPV isolates clustered into the 4 subgroups (A1, A2, B1 and B2) currently recognized; the majority (72%) of hMPV isolates belonged to subgroup A1. Among the 35 children infected with hMPV alone, 23 (65.7%) had bronchiolitis, 5 (14.3%) had pneumonia, 2 (5.7%) had asthma exacerbation and 5 (14.3%) had a limited upper RTD. Fifteen (30%) of the hMPV-infected children were coinfected with RSV. As compared with children infected with hMPV or RSV alone, duration of hospitalization and requirement for supplemental oxygen were increased in the hMPV/RSV-coinfected children. Conclusions: hMPV is a frequent cause of RTD in young children. hMPV/RSV coinfection is frequent and could be more severe than a single hMPV or RSV infection.

Comparative evaluation of three assays for the quantitation of human immunodeficiency virus type 1 RNA in plasma

Reverse transcriptase‐coupled polymerase chain reaction (Amplicor HIV‐1 Monitor), the branched DNA (bDNA) method (Quantiplex HIV‐1 RNA) and the nucleic acid sequence‐based assay (NASBA HIV‐1 RNA QT) were comparatively evaluated for the quantitation of human immunodeficiency virus type 1 (HIV‐1) RNA in plasma. Among 60 plasma specimens from HIV‐1 infected patients, HIV‐1 RNA was detected in 56 by Amplicor (sensitivity, 93.3%), in 41 by bDNA (sensitivity, 68.3%), and in 60 by NASBA (sensitivity, 100%). HIV‐1 RNA was not detected by any of these methods in 34/34 plasma specimens from HIV‐1‐seronegative blood donors (specificity, 100%). The HIV‐1 RNA levels as determined by the different methods were correlated significantly. The frequency of concordant results (log difference <0.50) was 80.4% between Amplicor and NASBA, 77.5% between Amplicor and bDNA, and 58.6% between bDNA and NASBA. After initiation of antiviral therapy, HIV‐1 RNA level variations observed with the three methods were similar. HIV‐1 RNA levels were inversely correlated with the CD4+ T cell counts, whereas no correlation was found with HIV‐1 p24‐antigen levels. When the methods were evaluated for reproducibility, coefficients of variation ranged from 11% to 40% for Amplicor, from 6% to 35% for bDNA, and from 13% to 62% for NASBA. Quantitation of HIV‐1 RNA in culture supernatants from HIV‐1 subtype A to H strains showed that bDNA can be used to quantitate RNA from all HIV‐1 subtypes, whereas Amplicor failed to detect RNA from subtype A strains and NASBA subtype G strains.

Merkel Cell Polyomavirus and Merkel Cell Carcinoma, France

To the Editor: Merkel cell carcinoma (MCC) is a primary cutaneous neuroendocrine tumor. This aggressive skin cancer is uncommon but increasing in frequency. During 1986–2001, incidence rate tripled; average annual increase was 8% (1). MCC shares epidemiologic features with Kaposi sarcoma, a malignant tumor associated with human herpesvirus 8 infection (2). In particular, MCC affects predominantly immunocompromised patients such as organ transplant recipients (3,4), patients with B-cell lymphoid tumors (5), and patients with AIDS (6). This similarity between MCC and Kaposi sarcoma may support the hypothesis of an infectious origin of MCC.

Merkel cell polyomavirus DNA detection in lesional and nonlesional skin from patients with Merkel cell carcinoma or other skin diseases.

Summary Background  A novel polyomavirus, the Merkel cell polyomavirus (MCPyV), has recently been identified in Merkel cell carcinoma (MCC).

Evolution and Taxonomic Classification of Human Papillomavirus 16 (HPV16)-Related Variant Genomes: HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67

Background Human papillomavirus 16 (HPV16) species group (alpha-9) of the Alphapapillomavirus genus contains HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. These HPVs account for 75% of invasive cervical cancers worldwide. Viral variants of these HPVs differ in evolutionary history and pathogenicity. Moreover, a comprehensive nomenclature system for HPV variants is lacking, limiting comparisons between studies. Methods DNA from cervical samples previously characterized for HPV type were obtained from multiple geographic regions to screen for novel variants. The complete 8 kb genomes of 120 variants representing the major and minor lineages of the HPV16-related alpha-9 HPV types were sequenced to capture maximum viral heterogeneity. Viral evolution was characterized by constructing phylogenic trees based on complete genomes using multiple algorithms. Maximal and viral region specific divergence was calculated by global and pairwise alignments. Variant lineages were classified and named using an alphanumeric system; the prototype genome was assigned to the A lineage for all types. Results The range of genome-genome sequence heterogeneity varied from 0.6% for HPV35 to 2.2% for HPV52 and included 1.4% for HPV31, 1.1% for HPV33, 1.7% for HPV58 and 1.1% for HPV67. Nucleotide differences of approximately 1.0% - 10.0% and 0.5%–1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Each gene/region differs in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) /noncoding region 2 (NCR2) >upstream regulatory region (URR)> E6/E7 > E2/L2 > E1/L1. Conclusions These data define maximum viral genomic heterogeneity of HPV16-related alpha-9 HPV variants. The proposed nomenclature system facilitates the comparison of variants across epidemiological studies. Sequence diversity and phylogenies of this clinically important group of HPVs provides the basis for further studies of discrete viral evolution, epidemiology, pathogenesis and preventative/therapeutic interventions.