A Simple and Fast Method to Sequence the Full-Length Spike Gene for SARS-CoV-2 Variant Identification from Patient Samples

Since the beginning of the pandemic, a race has been underway to detect SARS-CoV-2 virus infection (PCR screening, serological diagnostic kits), treat patients (drug repurposing, standard care) and develop a vaccine. After almost a year of active circulation worldwide, SARS-CoV-2 variants have appeared in different countries. Those variants include mutations in multiple regions of the genome, particularly in the spike gene. Because this surface protein is a key player in both the spread of the virus and the efficacy of vaccine strategies, the challenge is to efficiently monitor the appearance of spike mutations in the population. The present work describes a procedure based on the widely available Sanger technology to produce a full-length sequence of the spike gene from patient-derived samples

Front Microbiol

Since 2021, 3 variants of concern (VOC) have spread to France, causing successive epidemic waves. To describe the features of Alpha, Delta and Omicron VOC circulation in the Nouvelle-Aquitaine region, France, between February 2021 and February 2022. Data from the three university hospitals (UH) of Nouvelle-Aquitaine were used to describe regional SARS-CoV-2 circulation (RT-PCR positive rates and identified VOC) as well as its consequences (total number of hospitalizations and admissions in intensive care unit). They were analyzed according to the predominant variant and compared with national data. A total of 611,106 SARS-CoV-2 RT-PCR tests were performed in the 3 Nouvelle-Aquitaine UH during the study period. The 37,750 positive samples were analyzed by variant-specific RT-PCR or whole-genome sequencing. In 2021, Alpha VOC was detected from week 5 until week 35. Delta became the most prevalent variant (77.3%) in week 26, reaching 100% in week 35. It was replaced by Omicron, which was initially detected week 48, represented 77% of positive samples in week 52 and was still predominant in February 2022. The RT-PCR positive rates were 4.3, 4.2, and 21.9% during the Alpha, Delta and Omicron waves, respectively. The ratio between intensive care unit admissions and total hospitalizations was lower during the Omicron wave than during the two previous waves due to the Alpha and Delta variants. This study highlighted the need for strong regional cooperation to achieve effective SARS-CoV-2 epidemiological surveillance, in close association with the public health authorities

HIV-1 drug resistance in people on dolutegravir-based antiretroviral therapy:a collaborative cohort analysis

BACKGROUND The widespread use of the integrase strand transfer inhibitor (INSTI) dolutegravir in first-line and second-line antiretroviral therapy (ART) might facilitate emerging resistance. The DTG RESIST study combined data from HIV cohorts to examine patterns of drug resistance mutations (DRMs) and identify risk factors for dolutegravir resistance. METHODS We included cohorts with INSTI resistance data from two collaborations (ART Cohort Collaboration, International epidemiology Databases to Evaluate AIDS in Southern Africa), and the UK Collaborative HIV Cohort. Eight cohorts from Canada, France, Germany, Italy, the Netherlands, Switzerland, South Africa, and the UK contributed data on individuals who were viraemic on dolutegravir-based ART and underwent genotypic resistance testing. Individuals with unknown dolutegravir initiation date were excluded. Resistance levels were categorised using the Stanford algorithm. We identified risk factors for resistance using mixed-effects ordinal logistic regression models. FINDINGS We included 599 people with genotypic resistance testing on dolutegravir-based ART between May 22, 2013, and Dec 20, 2021. Most had HIV-1 subtype B (n=351, 59%), a third had been exposed to first-generation INSTIs (n=193, 32%), 70 (12%) were on dolutegravir dual therapy, and 18 (3%) were on dolutegravir monotherapy. INSTI DRMs were detected in 86 (14%) individuals; 20 (3%) had more than one mutation. Most (n=563, 94%) were susceptible to dolutegravir, seven (1%) had potential low, six (1%) low, 17 (3%) intermediate, and six (1%) high-level dolutegravir resistance. The risk of dolutegravir resistance was higher on dolutegravir monotherapy (adjusted odds ratio [aOR] 34·1, 95% CI 9·93-117) and dolutegravir plus lamivudine dual therapy (aOR 9·21, 2·20-38·6) compared with combination ART, and in the presence of potential low or low (aOR 5·23, 1·32-20·7) or intermediate or high-level (aOR 13·4, 4·55-39·7) nucleoside reverse transcriptase inhibitor (NRTI) resistance. INTERPRETATION Among people with viraemia on dolutegravir-based ART, INSTI DRMs and dolutegravir resistance were rare. NRTI resistance substantially increased the risk of dolutegravir resistance, which is of concern, notably in resource-limited settings. Monitoring is important to prevent resistance at the individual and population level and ensure the long-term sustainability of ART. FUNDING US National Institutes of Health, Swiss National Science Foundation

Impact of the M184V/I Mutation on the Efficacy of Abacavir/Lamivudine/Dolutegravir Therapy in HIV Treatment-Experienced Patients.

Objective The impact of the M184V/I mutation on the virological failure (VF) rate in HIV-positive patients with suppressed viremia switching to an abacavir/lamivudine/dolutegravir regimen has been poorly evaluated. Method This is an observational study from 5 European HIV cohorts among treatment-experienced adults with ≤50 copies/mL of HIV-1 RNA who switched to abacavir/lamivudine/dolutegravir. Primary outcome was the time to first VF (2 consecutive HIV-1 RNA >50 copies/mL or single HIV-1 RNA >50 copies/mL accompanied by change in antiretroviral therapy [ART]). We also analyzed a composite outcome considering the presence of VF and/or virological blips. We report also the results of an inverse probability weighting analysis on a restricted population with a prior history of VF on any ART regimen to calculate statistics standardized to the disparate sampling population. Results We included 1626 patients (median follow-up, 288.5 days; interquartile range, 154-441). Patients with a genotypically documented M184V/I mutation (n = 137) had a lower CD4 nadir and a longer history of antiviral treatment. The incidence of VF was 29.8 cases (11.2-79.4) per 1000 person-years in those with a previously documented M184V/I, and 13.6 cases (8.4-21.8) in patients without documented M184V/I. Propensity score weighting in a restricted population (n = 580) showed that M184V/I was not associated with VF or the composite endpoint (hazard ratio [HR], 1.27; 95% confidence interval [CI], 0.35-4.59 and HR 1.66; 95% CI, 0.81-3.43, respectively). Conclusions In ART-experienced patients switching to an abacavir/lamivudine/dolutegravir treatment, we observed few VFs and found no evidence for an impact of previously-acquired M184V/I mutation on this outcome. Additional analyses are required to demonstrate whether these findings will remain robust during a longer follow-up

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing

Objectives It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. Methods This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. Results Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found. Conclusions Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based AR

Molecular Characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and Impact of T-Cell Epitope Mutations on HLA Recognition (ANRS 12159)

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score). with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area

Sélection et caractérisation d'aptamères inhibiteurs de l'ARN polymérase ARN dépendante du virus de l'hépatite C

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

In vitro analysis of the susceptibility of HIV-1 subtype A and CRF01_AE integrases to raltegravir.

International audienceThe antiviral efficacy of raltegravir (RAL) has been proven against human immunodeficiency virus type 1 (HIV-1) subtypes B and C but remained to be determined against other subtypes. Therefore, the enzymatic activities as well as RAL resistance of HIV-1 subtype A and CRF01_AE integrases (INs) were investigated. Previously published subtype A and CRF01_AE IN sequences from RAL-naïve patients were aligned to generate consensus sequences for both IN subtypes. Subtype A and CRF01_AE INs encoded by these consensus sequences as well as the corresponding enzymes harbouring the N155H resistance mutation were expressed and purified. Enzymatic activities of subtype A and CRF01_AE INs were analysed with regard to typical 3'-end processing (3'-P) and strand transfer (ST) activities both in the presence and absence of RAL and were compared with subtype B IN as well as with the corresponding INs harbouring the N155H resistance mutation. Subtypes B, A and CRF01_AE INs showed similar 3'-P and ST activities. In the presence of RAL, the three wild-type INs exhibited ST activity IC50 values (50% inhibitory concentrations) of 86.3 ± 32.5, 158.3 ± 99.0 and 100.0 ± 65.7 nM, respectively. Analysis of 3'-P activity in the presence of RAL revealed IC(50) > 10 μM for all three enzymes. The three INs harbouring the N155H mutation presented in vitro low but similar resistance levels to RAL. In conclusion, INs from HIV-1 subtypes B, A and CRF01_AE showed similar responses to RAL in vitro, suggesting the potency of this antiretroviral drug to treat HIV-1 subtype A- and CRF01_AE-infected patients

Detection of low-frequency HIV type 1 reverse transcriptase drug resistance mutations by ultradeep sequencing in naive HIV type 1-infected individuals.

International audienceGenotypic resistance testing is recommended to evaluate the susceptibility of HIV to antiretroviral drugs. These tests are based on bulk population sequencing and thus consider only variants representing more than 20% of the viral population, whereas next generation sequencing methods allow detection below this threshold. We aimed to evaluate the potential use of ultradeep pyrosequencing (UDPS) for genotypic resistance testing in clinical routine at the University Hospital of Bordeaux, France. We performed UDPS on reverse transcriptase (RT) from 47 HIV-1 individuals, naive of antiretroviral treatment and for whom genotypic resistance testing was requested for clinical management in 2011-2012. In 8.5% of the patients, only low-frequency variants harboring RT drug resistance mutations were detected raising the question of their clinical significance. Rilpivirine-associated resistance mutations were detected in 19.1% of our population study. To conclude, UDPS could become a routine tool for the evaluation of HIV-infected patients in hospital laboratories