Improved hepatitis delta virus genome characterization by single molecule full‐length genome sequencing combined with VIRiONT pipeline

Hepatitis B virus (HBV) and hepatitis D virus (HDV) coinfection confers a greater risk for accelerated liver disease progression. Full-length characterization of HDV genome is necessary to understand pathogenesis and treatment response. However, owing to its high variability and tight structure, sequencing approaches remain challenging. Herein, we present a workflow to amplify, sequence, and analyze the whole HDV genome in a single fragment. Sequencing was based on the Oxford Nanopore Technologies long-read sequencing followed by a turnkey analysis pipeline (VIRiONT, VIRal in-house ONT sequencing analysis pipeline) that we developed and make available online for free. For the first time, HDV genome was successfully amplified and full-length sequenced in a single fragment, allowing accurate subtyping from 30 clinical samples. High variability of edition, a crucial step in viral life cycle, was found among samples (from 0% to 59%). Additionally, a new subtype of HDV genotype 1 was identified. We provide a complete workflow for assessment of HDV genome at full-length quasispecies resolution overcoming genome assembly issues and helping to identify modifications throughout the whole genome. This will help a better understanding of the impact of genotype/subtype, viral dynamics, and structural variants on HDV pathogenesis and treatment response

Alteration of CD8+ CD45RC[int/neg] regulatory T cells functions in Multiple Sclerosis and correlates with disease severity

Naïl Benallegue, Bryan Nicol, David A. Laplaud and Carole Guillonneau are co-authors.International audienceAutoimmune diseases can develop following pathological activation of autoreactive effector cells and/or, alternatively, after weakening of self-protective regulatory mechanisms. Most of the studies have focused on CD4+ Tregs and the role of CD8+ Tregs in Multiple Sclerosis (MS) remains largely unexplored. We previously reported the suppressive properties of rat and human CD8+CD45RCint/neg Treg cells, expressing Foxp3 and acting through IFNg, TGFb and IL34 cytokines (Guillonneau, JCI, 2007; Bézie, JCI, 2015, Bézie, Front. Immunol., 2018). Thus, their potency of suppression make them strong candidates of disruptive immune tolerance, especially in MS where CD8+ T cells play a major role. Thus, the overreaching goal of this study is to define the role of CD8+ regulatory T cell in MS pathogenesis

ANTI-CD45RC MABS IMMUNOTHERAPY CONTROLS THE DEVELOPPEMENT OF AUTO-IMMUNE SYMPTOMS IN AN APECED RAT MODEL OF AIRE DEFICIENCY

International audienc

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

International audienceViral 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

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

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

Metagenomic Analysis Reveals High Abundance of Torque Teno Mini Virus in the Respiratory Tract of Children with Acute Respiratory Illness

International audienceHuman Anelloviridae is a highly prevalent viral family, including three main genera—Alphatorquevirus (Torque teno virus, TTV), Betatorquevirus (Torque teno mini virus, TTMV), and Gammatorquevirus (Torque teno midi virus, TTMDV). To date, the characterization of Anelloviridae in the respiratory tract of children with acute respiratory infection (ARI) has been poorly reported and mainly focused on TTV. We performed a metagenomic analysis of eight respiratory samples collected from children with an ARI of unknown etiology (eight samples tested negative with a multiplex PCR assay, out of the 39 samples initially selected based on negative routine diagnostic testing). A total of 19 pediatric respiratory samples that tested positive for respiratory syncytial virus (RSV, n = 13) or influenza virus (n = 6) were also sequenced. Anelloviridae reads were detected in 16/27 samples, including 6/8 negative samples, 7/13 RSV samples and 3/6 influenza samples. For samples with a detection of at least one Anelloviridae genus, TTMV represented 87.1 (66.1–99.2)% of Anelloviridae reads, while TTV and TTMDV represented 0.8 (0.0–9.6)% and 0.7 (0.0–7.1)%, respectively (p < 0.001). Our findings highlight a high prevalence of TTMV in respiratory samples of children with an ARI of unknown etiology, as well as in samples with an RSV or influenza infection. Larger studies are needed to explore the role of TTMV in childhood respiratory diseases

Patients With Severe Multiple Sclerosis Exhibit Functionally Altered CD8 + Regulatory T Cells

International audienceBackground and Objectives Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Studies of immune dysfunction in MS have mostly focused on CD4 + Tregs, but the role of CD8 + Tregs remains largely unexplored. We previously evidenced the suppressive properties of rat and human CD8 + CD45RC low/neg Tregs from healthy individuals, expressing Forkhead box P3 (FOXP3) and acting through interferon-gamma (IFN-γ), transforming growth factor beta (TGFβ), and interleukin-34 (IL-34). secretions to regulate immune responses and control diseases such as transplant rejection. To better understand CD8 + CD45RC low/neg Tregs contribution to MS pathology, we further investigated their phenotype, function, and transcriptome in patients with MS. Methods We enrolled adults with relapsing-remitting MS and age-matched and sex-matched healthy volunteers (HVs). CD8 + T cells were segregated based on low or lack of expression of CD45RC. First, the frequency in CSF and blood, phenotype, transcriptome, and function of CD8 + CD45RC low and neg were investigated according to exacerbation status and secondarily, according to clinical severity based on the MS severity score (MSSS) in patients with nonexacerbating MS. We then induced active MOG 35-55 EAE in C57Bl/6 mice and performed adoptive transfer of fresh and expanded CD8 + CD45RC neg Tregs to assess their ability to mitigate neuroinflammation in vivo. Results Thirty-one untreated patients with relapsing-remitting MS were compared with 40 age-matched and sex-matched HVs. We demonstrated no difference of CSF CD8 + CD45RC low and CD8 + CD45RC neg proportions, but blood CD8 + CD45RC low frequency was lower in patients with MS exacerbation when compared with that in HVs. CD8 + CD45RC neg Tregs but not CD8 + CD45RC low showed higher suppressive capacities in vitro in MS patients with exacerbation than in patients without acute inflammatory attack. In vitro functional assays showed a compromised suppression capacity of CD8 + CD45RC low Tregs in patients with nonexacerbating severe MS, defined by the MSSS. We then characterized murine CD8 + CD45RC neg Tregs and demonstrated the potential of CD45RC neg cells to migrate to the CNS and mitigate experimental autoimmune encephalomyelitis in vivo. Discussion Altogether, these results suggest a defect in the number and function of CD8 + CD45RC low Tregs during MS relapse and an association of CD8 + CD45RC low Tregs dysfunction with MS severity. Thus, CD8 + CD45RC low/neg T cells might bring new insights into the pathophysiology and new therapeutic approaches of MS

Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69–V70, France, August to December 2020

International audienceWe report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC