Pierre Delobel

Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen?

1 Foy BD, Kobylinski KC, Chilson Foy JL, et al. Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis 2011; 17: 880–82. 2 Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis 2015; 21: 359–61. 3 Deen GF, Knust B, Broutet N, et al. Ebola RNA persistence in semen of Ebola virus disease survivors—preliminary report. N Engl J Med 2015; published online Oct 14. DOI:10.1056/ NEJMoa1511410. 4 Duff y MR, Chen TH, Hancock WT, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 2009; 360: 2536–43. 5 Roa M. Zika virus outbreak: reproductive health and rights in Latin America. Lancet 2016; published online Feb 12. http:// dx.doi.org/10.1016/S0140-6736(16)00331-7. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen?

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism.

Objective:Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. Design:Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. Methods:HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. Results:The 103 patients had a median CD4+ T lymphocyte count of 268 × 106 cells/l and nadirs of 98 × 106 cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. Conclusion:Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals

Depletion of CD4+ T cells from the gut occurs rapidly during acute HIV-1 infection. This has been linked to systemic inflammation and disease progression as a result of translocation of microbial products from the gut lumen into the bloodstream. Combined antiretroviral therapy (cART) substantially restores CD4+ T cell numbers in peripheral blood, but the gut compartment remains largely depleted of such cells for poorly understood reasons. Here, we show that a lack of recruitment of CD4+ T cells to the gut could be involved in the incomplete mucosal immune reconstitution of cART-treated HIV-infected individuals. We investigated the trafficking of CD4+ T cells expressing the gut-homing receptors CCR9 and integrin α4β7 and found that many of these T cells remained in the circulation rather than repopulating the mucosa of the small intestine. This is likely because expression of the CCR9 ligand CCL25 was lower in the small intestine of HIV-infected individuals. The defective gut homing of CCR9+β7+ CD4+ T cells - a population that we found included most gut-homing Th17 cells, which have a critical role in mucosal immune defense - correlated with high plasma concentrations of markers of mucosal damage, microbial translocation, and systemic T cell activation. Our results thus describe alterations in CD4+ T cell homing to the gut that could prevent efficient mucosal immune reconstitution in HIV-infected individuals despite effective cART.

R5 to X4 switch of the predominant HIV-1 population in cellular reservoirs during effective highly active antiretroviral therapy.

Summary:HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. A switch from CCR5 to CXCR4-using viruses can occur in the natural course of infection and correlates with subsequent disease progression. To investigate whether HIV-1 genetic evolution might lead to changes in virus coreceptor usage during highly active antiretroviral therapy (HAART), a longitudinal genotypic analysis of the virus found in cellular reservoirs was conducted in 32 patients with undetectable viral loads on HAART for 5 years. The genotype of the 3rd variable region of the env gene predicting coreceptor usage was retrospectively determined in the plasma or in peripheral blood mononuclear cells (PBMC) at baseline and then in PBMCs at months 30 and 60 of HAART. There was a switch from R5 to X4 variants in 11 of the 23 patients who harbored a majority virus population of R5 variants at baseline. X4 variants remained predominant in the 9 patients who harbored mainly X4 variants at baseline. The patients harboring predominantly X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority population of R5 variants. These results suggest that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs.

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

Background The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. Methodology/Principal Findings We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14high CD16− and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14high CD16− monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. Conclusions Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART.

Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

Population-Based Sequencing of the V3 Region of env for Predicting the Coreceptor Usage of Human Immunodeficiency Virus Type 1 Quasispecies

ABSTRACT Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.

Zika virus in semen and spermatozoa

The current Zika virus epidemic is a major challenge for the medical and scientific communities for at least two reasons: the severe clinical situation associated with Zika virus infection (neurological complications and adverse fetal outcomes) and the unexpected sexual viral transmission from men to women. These recent findings have shifted the paradigm of arbovirus–host interactions, modifying standard epidemiology and clinical patterns. High infectious Zika virus loads have been detected in semen, but data for viral persistence after symptomatic infections are scarce and even nonexistent for asympto matic ones, with the remaining key issue: how long does semen contain infectious Zika virus? As long as this question is unanswered, the adaptation of preventive measures such as the use of condoms and the abstinence of semen donation will be hampered. Here, we report the longitudinal follow up of Zika virus RNA in the semen of a 32-year-old man returning from French Guyana. At admission, the patient had moderate fever, maculopapular rash, myalgia, and arthralgia and was diagnosed for Zika virus infection upon detection of viral RNA in plasma and urine 2 days after onset of symptoms. The molecular diagnosis for dengue and chikungunya viruses was negative (in-house RT-PCR). The patient was seronegative for HIV, had a normal immunological status, and recovered in few days. Semen (11 samples), blood (ten), and urine (five) were prospectively collected for 141 days after symptom onset. Zika virus RNA was detected on each semen sample and was still positive after 141 days, with viral load decreasing from 8·6 log copies per mL to 3·5 log copies per mL. It was detected until day 37 in both blood (2·5 log copies per mL) and urine (3·7 log copies per mL; fi gure). Such a prolonged RNA Zika virus excretion is quite different from other fl aviviruses, which are rapidly cleared by the immune response and for which the detection of viral nucleic acids is typically limited to a short window after symptom onset. In addition to the present case, we investigated five other symptomatic men for the presence of Zika virus RNA in semen; RNA was still detected 69 days and 115 days after the symptom onset in two patients, but not detected at day 20 in the other three individuals (data not shown). These data suggest that the length of Zika virus excretion varies, probably depending on viral and host characteristics, but long-lasting excretion might be frequent among adults who experienced a symptomatic infection. Such long-lasting excretion of genomic material was recently described in semen from Ebola survivors. The viral persistence in semen is of major concern and could be related to a viral tropism for male sexual cells. Accordingly, we report the immunohistochemical detection of Zika virus in the head of spermatozoa obtained from the fi rst patient (fi gure; appendix). The proportion of infected spermatozoa was estimated at 3·52% (SD 0·71), as assessed by counting Zikavirus-positive sperma tozoa on three smears by fl uorescence micro scopy. The use of both confocal and stimulated emission depletion microscopy (appendix), allowed the unambiguous demonstration that Zika virus antigens are indeed present inside spermatozoa. Additional immuno stainings of sperm from a healthy non-infected control donor for Zika virus antigens or with a mouse IgG2a isotype control confi rmed the specifi city of the staining (data not shown). Spermatozoa do not express the candidate Zika virus entry receptor Axl. Because Sertoli cells highly express Axl and have essential roles in spermatogenesis, we hypothesise that they might be involved in Zika virus transmission to spermatozoa. Appropriately designed studies are needed to discover the modes of spermatozoa infection and length of risk of sexual transmission. Answers to these questions will allow public health authorities to recom mend urgently needed universal safe practices.

Persistence of distinct HIV-1 populations in blood monocytes and naive and memory CD4 T cells during prolonged suppressive HAART.

Objective:Reservoirs of HIV-1 are a major obstacle to virus eradication. There is therefore a need to clearly understand the molecular nature of the virus populations that persist in patients with sustained suppression of plasma viraemia on highly active antiretroviral therapy (HAART). Design:We performed a detailed analysis of the genotypes of HIV-1 quasispecies isolated from highly purified blood cell types taken from three selected patients with sustained undetectable viral loads on HAART for 7 years. Methods:We used polychromatic flow cytometry to sort naive and memory CD4 T cells, CD14 monocytes, and CD56+CD3− natural killer (NK) cells from the total peripheral blood mononuclear cells after 7 years of HAART. Clonal analysis was used to determine coreceptor use and drug-resistance genotypes of HIV-1 quasispecies in the sorted blood cell types. Results:We detected HIV-1 DNA in memory and naive CD4 T cells and in CD14 monocytes, but not in the CD56+CD3− NK cells. Phylogenetic analysis demonstrated that the various blood cells types of two of the three patients harboured genetically distinct HIV-1 quasispecies. Drug-resistance mutations were also distributed differently from one cell type to another. This compartmentalization suggests a minimal virus trafficking between blood cell types during suppressive HAART. Conclusions:We observed a cell-specific compartmentalization of the residual virus populations during prolonged suppressive HAART. The coexistence of numerous HIV-1 quasispecies with different resistance genotypes and coreceptor use in cellular reservoirs may be relevant for future antiretroviral treatment strategies.

Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage

BACKGROUND Clinical trials of CCR5 antagonists have relied on the phenotypic determination of HIV-1 coreceptor usage. Few phenotypic assays are available, with few data on their concordance, and none has been designed to determine tropism from cell-associated HIV-1 DNA. OBJECTIVES To assess the performance of the new Toulouse Tropism Test (TTT) phenotypic assay to characterize HIV-1 tropism using blood plasma and peripheral blood mononuclear cells (PBMC). STUDY DESIGN 434 plasma and 168 PBMC samples were tested with the TTT assay. We determined the correlation between our assay results on plasma samples and those of the commercial Trofile assay. RESULTS The TTT assay determined the tropism of 97% of samples after successful amplification of the env gene. It performed well on both cell samples and plasma samples with various HIV-1 loads and subtypes. It detected 0.5% of minor CXCR4-using variants in the virus population. The TTT and the Trofile assays were >90% concordant for predicting HIV-1 tropism. CONCLUSION We have validated a new recombinant virus phenotypic assay for determining HIV-1 tropism using both plasma and cell samples from patients who are candidates for treatment with CCR5 antagonists.