Caution is needed in interpreting HIV transmission chains by ultradeep sequencing.

OBJECTIVES: Molecular epidemiology is applied to various aspects of HIV transmission analyses. With ultradeep sequencing (UDS), in-depth characterization of transmission episodes involving minority variants is permitted. We explored HIV-1 epidemiological linkage and evaluated characteristics of transmission dynamics and transmitted drug resistance (TDR) detection through the added value of UDS. DESIGN: HIV pol gene fragments were sequenced by UDS and Sanger sequencing on samples of 70 HIV-1-infected, treatment-naive recently diagnosed MSM. METHODS: Pairwise genetic distances and maximum likelihood phylogenies were computed. Transmission events were identified as clades with branch support at least 70% and intraclade genetic difference less than 4.5%. TDR mutations were recognized from the TDR consensus list. Transmission directionality, directness and inoculum size were inferred from tree topologies. RESULTS: Both datasets concurred in the identification of seven transmission pairs and one cluster of three patients. With UDS, direction of transmission was inferred in four out of eight chains. Evidence for multiple founder viruses was found in two out of eight chains. No transmission of minority-resistant variants was evidenced. TDR mutations prevalence in protease and reverse transcriptase fragments was 4.3% with Sanger sequencing and 18.6% with UDS. CONCLUSION: Although Sanger sequencing and UDS identified the same transmission chains, UDS provided additional information on founder viruses, direction of transmission and levels of TDR. Nevertheless, topology of clusters was not always consistent across gene fragments, calling for a cautious interpretation of the data. Moreover, unobserved intermediary links cannot be excluded. Phylogenetic analysis use as a forensic technique for HIV transmission investigations is risky

Molecular epidemiology and clinical characteristics of hepatitis B identified through the French mandatory notification system.

BACKGROUND & AIMS: Strains responsible for acute hepatitis B infections (AHB) in France have not been characterized. This study was first designed to analyze the molecular epidemiology of AHB and second to describe the differences between AHB and chronic hepatitis B (CHB) exacerbations. METHODS: This prospective study was based on the French mandatory notification system for AHB. 147 samples corresponding to declared cases were shipped to a central laboratory for classification as AHB or CHB according to the level of anti-HBc IgM and anti-HBc avidity. RESULTS: Based on biological marker values and file examination, 75 cases (59%) were classified as AHB. Independently of the acute or chronic status, genotype A (57%), D (22%) and E (14%) were the most prevalent and no phylogenetic clustering was observed among HBV sequences (n=68). Precore or basal core-promoter variants were not particularly associated with disease severity but were more prevalent in CHB. No antiviral resistant strains or immune-escape HBsAg was observed. HBV viral loads in AHB or CHB were comparable but with opposite distributions. ALT levels reached 10 times the upper normal value in 94% of AHB but only in 24% of CHB. CONCLUSIONS: After rigorous classification, no major difference at the genetic level was found between HBV strains isolated from AHB and CHB. Absence of potentially deleterious variant detection is reassuring. When based upon HBsAg and anti-HBc IgM determination, AHB notification may falsely include more than 40% CHB, leading to an important risk of bias in national surveillance programs of AHB

No difference in HIV-1 integrase resistance between CSF and blood compartments Short title: HIV-1 integrase resistance in compartments

International audienceBackground: Little is known about the HIV-1 integrase resistance in CNS. This study aimed to evaluate integrase resistance in CSF, as a marker of CNS, and compare it to the HIV resistance in plasma. Methods: The HIV integrase was sequenced both in plasma and CSF for 59 HIV-1 patients. The clinical and biological data were collected from clinical routine care. Results: Among the 59 HIV-1 patients, 32 (54.2%) were under antiretroviral (ARV) treatment. The median (IQR) HIV-1 RNA in viremic patients was 5.32 (3.85-5.80) and 3.59 (2.16-4.50) versus 4.79 (3.56-5.25) and 3.80 (2.68-4.33) in CSF log10 copies/mL for ARV naïve and treated patients, respectively. The patients were mainly infected with non-B subtypes (72.2%) with the most prevalent recombinant form CRF02_AG (42.4%). The HIV-1 integrase sequences presented resistance mutations for 9/27 (33.3%) and 8/32 (25.0%) in CSF for ARV naïve (L74I n=3, L74I/M n=1, T97A n=1, E157Q n=4) and treated (L74I n=6, L74M n=1, 1 T97A n=1, 1 N155H n=1) patients, respectively. Integrase resistance mutations in CSF were similar to that in plasma, except for 1/59 patients. Conclusions: This work shows similar integrase resistance profiles in CNS and plasma in a population of HIV-1 viremic patients

Phylogenetic tree obtained from 92 full length sequences.

<p>The evolutionary history was inferred using the Neighbor-Joining method with a bootstrap test (500 replicates); bootstrap values are shown next to the branches. Sequences from 60 acute cases (black dots) and 24 chronic cases (open dot) and 8 reference sequences (grey triangles) were analyzed.</p

General flow chart of the study.

<p>Samples corresponding to notified AHB were classified according to both the values of HBc IgM and avidity index. Each biologically classified coherent case was thoroughly assessed for discrepancies with epidemiological and clinical information. When relevant information was not available physicians were interviewed on an individual basis.</p

General organization of the French AHB registry.

<p>General organization of the French AHB registry.</p

Distribution of HBV-VL (log₁₀ IU/mL) according to disease course.

<p>Black bars represent acute cases and gray bars anti-HBc IgM positive chronic cases and open bars anti-HBc IgM negative chronic cases.</p

Primaquine as a Candidate for HHV-8-Associated Primary Effusion Lymphoma and Kaposi’s Sarcoma Treatment

International audienceHuman Herpesvirus 8 (HHV-8) is associated with three main severe orphan malignancies, Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL), which present few therapeutic options. We identified the antimalarial primaquine diphosphate (PQ) as a promising therapeutic candidate for HHV-8-associated PEL and KS. Indeed, PQ strongly reduced cell viability through caspase-dependent apoptosis, specifically in HHV-8-infected PEL cells. Reactive oxygen species (ROS)- and endoplasmic reticulum (ER) stress-mediated apoptosis signaling pathways were found to be part of the in vitro cytotoxic effect of PQ. Moreover, PQ treatment had a clinically positive effect in a nonobese diabetic (NOD)/SCID xenograft PEL mouse model, showing a reduction in tumor growth and an improvement in survival. Finally, an exploratory proof-of-concept clinical trial in four patients harboring severe KS was conducted, with the main objectives to assess the efficacy, the safety, and the tolerability of PQ, and which demonstrated a positive efficacy on Kaposi’s sarcoma-related lesions and lymphedema

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS

International audienceThe evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV). Recombinant canine adenoviruses expressing the envelope glycoproteins or the Gag protein of a primary strain of FIV were constructed. Three groups of six cats were immunised twice with vectors expressing FIV antigens or with a vector expressing an irrelevant antigen, green fluorescent protein, by intramuscular and subcutaneous routes. Humoral responses were elicited against the transgene product in 6/6, 3/6 and 0/6 cats after immunisation against green fluorescent protein, Gag or the envelope glycoproteins, respectively. Six weeks after the second administration, cats were challenged by the intraperitoneal route with the homologous strain, and viral burden in plasma was followed by quantitative RT-PCR. Immunisation with FIV antigens did not afford protection. Rather, viral RNA was detected at earlier time points in cats immunised against Gag than in cats immunised with a vector expressing an irrelevant antigen. Such immune-mediated enhancement did not appear to have a long-range impact on viral set point or inversion of the CD4(+)/CD8(+) ratio. Thus, in the feline AIDS model pre-existing immunity against a viral antigen exacerbated acute phase infection

Discovery, SAR study and ADME properties of methyl 4-amino-3-cyano-1-(2-benzyloxyphenyl)-1 H -pyrazole-5-carboxylate as an HIV-1 replication inhibitor

International audience(ESI) available: [biology: experimental procedures, full results of the first screening, dose-response curves; chemistry: general procedures, compounds characterization, 1 H and 13 C NMR spectra]. SeeInspired by the antiviral activity of known pyrazole-based HIV inhibitors, we screened our in-house library of pyrazole-based compounds to evaluate their in cellulo activity against HIV-1 replication. Two hits with very similar structures appeared from single and multiple-round infection assays to be non-toxic and active in a dose-dependent manner. Chemical expansion of their series allowed an in-depth and consistent structure–activity-relationship study (SAR) to be built. Further ADME evaluation led to the selection of 4-amino-3-cyano-1-(2-benzyloxyphenyl)-1H-pyrazole-5-carboxylate with an advantageous pharmacokinetic profile. Finally, examination of its mode of action revealed that this compound does not belong to the three main classes of anti-HIV drugs, a feature of prime interest in the context of viral resistance