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Omicron variant is circulating in the presence of a globally acquired immunity unlike the ancestral SARS-CoV-2 isolate. Herein, we investigated the normalized viral load dynamics and viral culture status in 44 fully vaccinated healthcare workers (HCWs) infected with the Omicron BA.1 variant. Viral load dynamics of 38 unvaccinated HCWs infected with the 20A variant during the first pandemic wave was also studied. We then explored the impact of Omicron infection on pre-existing immunity assessing anti-RBD IgG levels, neutralizing antibody titres against 19A, Delta and Omicron isolates, as well as IFN-γ release following cell stimulation with SARS-CoV-2 peptides. We reported that two weeks after diagnosis a greater proportion of HCWs infected with 20A (78.9%, 15/19) than with Omicron BA.1 (44.7%, 17/38; p = 0.02) were still positive by RT-qPCR. We found that Omicron breakthrough infections led to an overall enhancement of vaccine-induced humoral and cellular immunity as soon as a median [interquartile range] of 8 [7-9] days post symptom onset. Among samples with similar high viral loads, non-culturable samples exhibited higher neutralizing antibody titres and anti-RBD IgG levels than culturable samples. Additionally, Omicron infection led to an enhancement of antibodies neutralization capacity against other SARS-CoV-2 isolates. Taken together, the results suggest that Omicron BA.1 vaccine breakthrough infection is associated with a faster viral clearance than that of the ancestral SARS-CoV-2, in addition this new variant leads to a rapid enhancement of the humoral response against multiple SARS-CoV-2 variants, and of the cellular response.

Whole-genome sequencing has become an essential tool for real-time genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide. The handling of raw next-generation sequencing (NGS) data is a major challenge for sequencing laboratories. We developed an easy-to-use web-based application (EPISEQ SARS-CoV-2) to analyse SARS-CoV-2 NGS data generated on common sequencing platforms using a variety of commercially available reagents. This application performs in one click a quality check, a reference-based genome assembly, and the analysis of the generated consensus sequence as to coverage of the reference genome, mutation screening and variant identification according to the up-to-date Nextstrain clade and Pango lineage. In this study, we validated the EPISEQ SARS-CoV-2 pipeline against a reference pipeline and compared the performance of NGS data generated by different sequencing protocols using EPISEQ SARS-CoV-2. We showed a strong agreement in SARS-CoV-2 clade and lineage identification (>99%) and in spike mutation detection (>99%) between EPISEQ SARS-CoV-2 and the reference pipeline. The comparison of several sequencing approaches using EPISEQ SARS-CoV-2 revealed 100% concordance in clade and lineage classification. It also uncovered reagent-related sequencing issues with a potential impact on SARS-CoV-2 mutation reporting. Altogether, EPISEQ SARS-CoV-2 allows an easy, rapid and reliable analysis of raw NGS data to support the sequencing efforts of laboratories with limited bioinformatics capacity and those willing to accelerate genomic surveillance of SARS-CoV-2.

International audience; No abstract available

The sensitivity of SARS-CoV-2 RT-PCR tests developed by Charité (Germany), HKU (Hong-Kong), China CDC (China), US CDC (United-States), and Institut Pasteur, Paris (France) was assessed on SARS-CoV-2 cell culture supernatants and clinical samples. Although all RT-PCR assays performed well for SARS-CoV-2 detection, RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive.

A new member of Anelloviridae, named torque teno mini virus (TTMV)-SH, was recently identified in the serum of three Hodgkin’s lymphoma patients suggesting that TTMV-SH may be associated with this type of hematological malignancy. We investigated by metagenomic analysis the presence of TTMV-SH-related viruses in plasma samples (n = 323) collected from patients with various hematological malignancies (multiple myeloma (MM, n = 256), non-Hodgkin’s lymphoma (NHL, n = 20), acute myeloid leukemia (n = 10)) and from healthy donors (n = 37). TTMV-SH-related strains were identified in 24 samples corresponding to four MM and one NHL patients. Phylogenic analysis revealed that the 24 isolates were close to the TTMV-SH strains previously identified, sharing 79.6–86.7% ORF1 nucleotide sequence identity. These results suggest that TTMV-SH-related viruses might be found in hematological diseases other than Hodgkin’s lymphoma. Due to the high genetic variability within Anelloviridae species, the association between a particular medical condition and a new genotype should be interpreted with caution.

Viral metagenomics next-generation sequencing (mNGS) is increasingly being used to characterize the human virome. The impact of viral nucleic extraction on virome profiling has been poorly studied. Here, we aimed to compare the sensitivity and sample and reagent contamination of three extraction methods used for viral mNGS: two automated platforms (eMAG, MagNA Pure 24, MP24) and the manual QIAamp Viral RNA Mini Kit (QIAamp). Clinical respiratory samples (positive for Respiratory Syncytial Virus or Herpes Simplex Virus), one mock sample (including five viruses isolated from respiratory samples), and a no-template control (NTC) were extracted and processed through an mNGS workflow. QIAamp yielded a lower proportion of viral reads for both clinical and mock samples. The sample cross-contamination was higher when using MP24, with up to 36.09% of the viral reads mapping to mock viruses in the NTC (vs. 1.53% and 1.45% for eMAG and QIAamp, respectively). The highest number of viral reads mapping to bacteriophages in the NTC was found with QIAamp, suggesting reagent contamination. Our results highlight the importance of the extraction method choice for accurate virome characterization.

International audience; Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.

A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175–1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfection of an HERV-W Env expression vector in a panel of cell lines derived from different species resulted in formation of syncytia in primate and pig cells upon interaction with the type D mammalian retrovirus receptor. Moreover, envelope glycoproteins encoded by HERV-W were specifically detected in placenta cells, suggesting that they may play a physiological role during pregnancy and placenta formation.

INTRODUCTION:A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). RESULTS:The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. CONCLUSION:Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

The cellular HERV-W envelope/syncytin-1 protein, encoded by the envelope gene of the ERVWE1 proviral locus is a fusogenic glycoprotein probably involved in the formation of the placental syncytiotrophoblast layer. Syncytin-1-induced in vitro cell-cell fusion is dependent on the interaction with hASCT2. As no receptor binding domain has been clearly defined in the SU of neither the HERV-W Env nor the retroviruses of the same interference group, we designed an in vitro binding assay to evaluate the interaction of the HERV-W envelope with the hASCT2 receptor. Using truncated HERV-W SU subunits, a region consisting of the N-terminal 124 amino acids of the mature SU glycoprotein was determined as the minimal receptor-binding domain. This domain contains several sub-domains which are poorly conserved among retroviruses of this interference group but a region of 18 residus containing the SDGGGX2DX2R conserved motif was proved to be essential for syncytin-1-hASCT2 interaction.

Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation.

International audience; HIV-1 genes are expressed through the complex splicing of a single mRNA precursor leading to three mRNA classes: unspliced, singly-spliced and multiply-spliced. Each class may include several mRNA species specifically encoding one or two HIV-1 proteins. Northern blotting and RT-PCR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classes of viral RNA but does not discriminate between individual RNA species. RT-PCR allows discrimination between different species but does not provide a quantitative analysis. Here, we describe an application of an RNAse mapping assay which gives both quantitative and discriminative HIV-1 RNA detection. A radiolabeled probe overlapping the major splicing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies was synthesized. This probe protects differential sizes of these species, allowing discrimination between them. We investigated the RNA expression pattern in high titer HIV-1 producing cells. The HIV-1-specific probe allowed the detection of multiply-spliced vpr, rev and nef mRNAs, singly-spliced env mRNA and unspliced genomic RNA. With its discriminative and quantitative properties, this application is particularly convenient for the investigation of HIV-1 mRNA expression during the course of HIV-1 infections.HIV-1 genes are expressed through the complex splicing of a single mRNA precursor leading to three mRNA classes: unspliced, singly-spliced and multiply-spliced. Each class may include several mRNA species specifically encoding one or two HIV-1 proteins. Northern blotting and RT-PCR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classes of viral RNA but does not discriminate between individual RNA species. RT-PCR allows discrimination between different species but does not provide a quantitative analysis. Here, we describe an application of an RNAse mapping assay which gives both quantitative and discriminative HIV-1 RNA detection. A radiolabeled probe overlapping the major splicing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies was synthesized. This probe protects differential sizes of these species, allowing discrimination between them. We investigated the RNA expression pattern in high titer HIV-1 producing cells. The HIV-1-specific probe allowed the detection of multiply-spliced vpr, rev and nef mRNAs, singly-spliced env mRNA and unspliced genomic RNA. With its discriminative and quantitative properties, this application is particularly convenient for the investigation of HIV-1 mRNA expression during the course of HIV-1 infections.