Publications by authors named "Martine Valette"

44 Publications

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.

Euro Surveill 2021 01;26(3)

The members of the COVID-Diagnosis HCL Study Group are listed below.

We 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.
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http://dx.doi.org/10.2807/1560-7917.ES.2021.26.3.2100008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848679PMC
January 2021

Estimation of influenza-attributable burden in primary care from season 2014/2015 to 2018/2019, France.

Eur J Clin Microbiol Infect Dis 2021 Jan 20. Epub 2021 Jan 20.

Sorbonne Université, INSERM, Institut Pierre Louis d'épidémiologie et de Santé publique (IPLESP UMRS 1136), Paris, France.

Influenza viruses cause seasonal epidemics whose intensity varies according to the circulating virus type and subtype. We aim to estimate influenza-like illness (ILI) incidence attributable to influenza viruses in France from October 2014 to May 2019. Physicians participating in the French Sentinelles network reported the number of patients with ILI seen in consultation and performed nasopharyngeal swabs in a sample of these patients. The swabs were tested by RT-PCR for the presence of influenza viruses. These clinical and virological data were combined to estimate ILI incidence attributable to influenza viruses by subtypes and age groups. Influenza incidence rates over seasons ranged from 1.9 (95% CI, 1.9; 2.0) to 3.4% (95% CI, 3.2; 3.6) of the population. Each season, more than half of ILI cases were attributable to influenza. Children under 15 years were the most affected, with influenza incidence rates ranging from 3.0 (95% CI, 2.8;3.3) to 5.7% (95% CI, 5.3;6.1). Co-circulation of several (sub)types of influenza viruses was observed each year, except in 2016/2017 where A(H3N2) viruses accounted for 98.0% of the influenza cases. Weekly ILI incidences attributable to each influenza virus (sub)type were mostly synchronized with ILI incidence, except in 2014/2015 and 2017/2018, where incidence attributable to type B viruses peaked few weeks later. The burden of medically attended influenza among patients with ILI is significant in France, varying considerably across years and age groups. These results show the importance of influenza surveillance in primary care combining clinical and virological data.
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http://dx.doi.org/10.1007/s10096-021-04161-1DOI Listing
January 2021

Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France.

Emerg Microbes Infect 2021 Dec;10(1):167-177

CIRI, Centre International de Recherche en Infectiologie, Team VirPatH, Lyon, Fracne.

During routine molecular surveillance of SARS-CoV-2 performed at the National Reference Center of Respiratory Viruses (Lyon, France) ( = 229 sequences collected February-April 2020), two frameshifting deletions were detected in the open reading frame 6, at the same position (27267). While a 26-nucleotide deletion variant (D26) was only found in one nasopharyngeal sample in March 2020, the 34-nucleotide deletion (D34) was found within a single geriatric hospital unit in 5/9 patients and one health care worker in April 2020. Phylogeny analysis strongly suggested a nosocomial transmission of D34, with potential fecal transmission, as also identified in a stool sample. No difference in disease severity was observed between patients hospitalized in the geriatric unit infected with WT or D34. D26 and D34 characterization revealed comparable replication kinetics with the wild-type (WT), but differential host immune responses. While interferon-stimulated genes were similarly upregulated after infection with WT and ORF6 variants, the latter specifically induced overexpression of 9 genes coding for inflammatory cytokines in the NF-kB pathway, including , , and , for which high plasma levels have been associated with severe COVID-19. Our findings emphasize the need to monitor the occurrence of ORF6 deletions and assess their impact on the host immune response.
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http://dx.doi.org/10.1080/22221751.2021.1872351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850418PMC
December 2021

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation.

Virus Evol 2020 Jul 5;6(2):veaa075. Epub 2020 Oct 5.

Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, Lyon cedex 4, France.

Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next-Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty-four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialised kits including a hybridisation capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP), and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples ( = 8), with mNGS and RVOP producing the most complete genomes. For mid viral loads (Ct 20-25), amplicon-based enrichment methods led to genome coverage >99 per cent for all samples while 1/8 sample sequenced with RVOP and 2/8 samples sequenced with mNGS had a genome coverage below 99 per cent. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in three samples was observed in CleanPlex the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34nt-deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a loss of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer-annealing or probe-hybridizing regions and would require further validation using unbiased metagenomic sequencing.
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http://dx.doi.org/10.1093/ve/veaa075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665770PMC
July 2020

Salivary and Nasal Detection of the SARS-CoV-2 Virus After Antiviral Mouthrinses (BBCovid): A structured summary of a study protocol for a randomised controlled trial.

Trials 2020 Nov 2;21(1):906. Epub 2020 Nov 2.

University of Lyon, University Claude Bernard Lyon 1, Laboratory "Systemic Health Care", EA4129, 69008, Lyon, France.

Objectives: - To describe the evolution of the SARS-CoV-2 salivary viral load of patients infected with Covid-19, performing 7 days of tri-daily mouthwashes with and without antivirals. - To compare the evolution of the SARS-CoV-2 nasal and salivary viral load according to the presence or absence of antivirals in the mouthwash.

Trial Design: This is a multi-center, randomised controlled trial (RCT) with two parallel arms (1:1 ratio).

Participants: Inclusion criteria - Age: 18-85 years old - Clinical diagnosis of Covid-19 infection - Clinical signs have been present for less than 8 days - Virological confirmation - Understanding and acceptance of the trial - Written agreement to participate in the trial Exclusion criteria - Pregnancy, breastfeeding, inability to comply with protocol, lack of written agreement - Patients using mouthwash on a regular basis (more than once a week) - Patient at risk of infectious endocarditis - Patients unable to answer questions - Uncooperative patient The clinical trial is being conducted with the collaboration of three French hospital centers: Hospital Center Emile Roux (Le Puy en Velay, France), Clinic of the Protestant Infirmary (Lyon, France) and Intercommunal Hospital Center (Mont de Marsan, France).

Intervention And Comparator: Eligible participants will be allocated to one of the two study groups. Intervention group: patients perform a tri-daily mouthwash with mouthwash containing antivirals (β-cyclodextrin and Citrox®) for a period of 7 days.

Control Group: patients perform a tri-daily mouthwash with a placebo mouthwash for a period of 7 days.

Main Outcomes: Primary Outcome Measures: Change from Baseline amount of SARS-CoV-2 in salivary samples at 4 and 9 hours, 1, 2, 3, 4, 5 and 6 days. Real-time PCR assays are performed to assess salivary SARS-CoV 2 viral load.

Secondary Outcome Measures: Change from Baseline amount of SARS-CoV-2 virus in nasal samples at 6 days. Real-time PCR assays are performed to assess nasal SARS-CoV-2 viral load.

Randomisation: Participants meeting all eligibility requirements are allocated to one of the two study arms (mouthwash with β-cyclodextrin and Citrox® or mouthwash without β-cyclodextrin and Citrox®) in a 1:1 ratio using simple randomisation with computer generated random numbers.

Blinding (masking): Participants, doctors and nurses caring for participants, laboratory technicians and investigators assessing the outcomes will be blinded to group assignment.

Numbers To Be Randomised (sample Size): Both the intervention and control groups will be composed of 103 participants, so the study will include a total of 206 participants.

Trial Status: The current protocol version is 6, August 4, 2020. Recruitment began on April 6, 2020 and is anticipated to be complete by April 5, 2021. As of October 2, 2020, forty-two participants have been included.

Trial Registration: This trial was registered on 20 April 2020 at www.clinicaltrials.gov with the number NCT04352959 .

Full Protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol." The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2)."
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http://dx.doi.org/10.1186/s13063-020-04846-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604647PMC
November 2020

Analytical Performances of the Panther Fusion System for the Detection of Respiratory Viruses in the French National Reference Centre of Lyon, France.

Microorganisms 2020 Sep 7;8(9). Epub 2020 Sep 7.

Laboratoire de Virologie, Hospices Civils de Lyon, Institut des Agents Infectieux, Centre National de Référence des virus des infections respiratoires (dont la Grippe), Hôpital de la Croix-Rousse, 103 grande rue de la Croix-Rousse, CEDEX 04, 69317 Lyon, France.

Respiratory infection are mainly caused by viral pathogens. During the 2017-2018 epidemic season, Panther Fusion Respiratory kits (Influenza virus A&B (FluA&B), respiratory syncytial virus (RSV), adenovirus (ADV), metapneumovirus (MPV), rhinovirus (RV), parainfluenzae virus (PIV), were compared to the Respiratory MultiWells System r-gene. Respiratory clinical specimens were tested retrospectively ( = 268) and prospectively ( = 463). Analytical performances were determined (sensitivity -Sep-, specificity -Spe- and κ) considering concordances of ≥2 molecular testing specific to each viral target (discrepant results were verified at the National Reference Centres for Enteroviruses or Respiratory viruses, Lyon, France). After retrospective (and prospective) testing, Sep, Spe, and κ were 100% (97.7%), 100% (99%) and 100% (94%) for FluA: 100% (95.5%), 100% (99.3%) and 100% (94%) for FluB, and 100% (88.5%), 100% (98.7%) and 100% (89%) for RSV; 82.1% (41.7%), 100% (99.5%) and 86% (54%) for ADV; 94.7% (73.7%), 96.1% (98.0%) and 91% (65%) for MPV; 96.1% (94.6%), 90.2% (98.5%) and 86% (91%) for HRV; and 90% (72.7%), 100% (99.3%) and 91% (72%), respectively, for PIV. Analytical performances were above 85% for all viruses except for ADV, MPV and PIV, confirming the analytical performance of the Panther Fusion system, a high throughput system with reduced turn-around-time, when compared to non-automated systems.
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http://dx.doi.org/10.3390/microorganisms8091371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563737PMC
September 2020

Incidence of asymptomatic and symptomatic influenza among healthcare workers: a multicenter prospective cohort study.

Clin Infect Dis 2020 Aug 4. Epub 2020 Aug 4.

Service d'Hygiène, Epidémiologie, Infectiovigilance et Prévention, Hospices Civils de Lyon, Lyon.

Background: Influenza is an important cause of viral hospital-acquired infection involving patients, healthcare workers (HCW), and visitors. The frequency of asymptomatic influenza among HCW with possible subsequent transmission is poorly described. The objective is to determine the cumulative incidence of asymptomatic, pauci-symptomatic and symptomatic influenza among HCW.

Method: A multicenter prospective cohort study was done in 5 French university hospitals, including 289 HCW during the 2016-2017 influenza season. HCW had 3 physical examinations (Time [T] 0, before epidemic onset; T.1, before epidemic peak; T.2, T.3 after epidemic peak). A blood sample was taken each time for influenza serology and a nasal swab was collected at T1 and T2 for influenza detection by PCR. Positive influenza was defined as either a positive influenza PCR, and/or virus-specific seroconversion against influenza A, the only circulating virus, with no vaccination record during follow-up. Symptoms were self-reported daily between T1 and T2. Cumulative incidence of influenza was stratified by clinical presentation per 100 HCW.

Findings: Of the 289 HCW included, 278 (96%) completed the entire follow-up. Overall, 62 HCW had evidence of influenza of whom 46·8% were asymptomatic, 41·9% were pauci-symptomatic, and 11·3% were symptomatic. Cumulative influenza incidence was 22·3% (95%CI:17·4%-27·2%). Cumulative incidence of asymptomatic influenza was 5·8% (95%CI: 3·3%-9·2%), 13·7% (95%CI:9·9%-18·2%) for pauci-symptomatic influenza, and 2·9% (95%CI:1·3%-5·5%) for symptomatic influenza.

Interpretation: Asymptomatic and pauci-symptomatic influenza were frequent among HCW, representing 47% and 42% of the influenza burden respectively. These findings highlight the importance of systematic implementation of infection control measures among HCW regardless of respiratory symptoms from preventing nosocomial transmission of influenza.

Trial Registration: clinicaltrials.gov identifier: NCT02868658.
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http://dx.doi.org/10.1093/cid/ciaa1109DOI Listing
August 2020

Clinical and virological data of the first cases of COVID-19 in Europe: a case series.

Lancet Infect Dis 2020 06 27;20(6):697-706. Epub 2020 Mar 27.

Department of Infectious and Tropical Diseases, Assistance Publique-Hôpitaux de Paris, Bichat-Claude Bernard University Hospital, Paris, France; Infections Antimicrobials Modelling Evolution (IAME) UMR 1137, University of Paris, Paris, France. Electronic address:

Background: On Dec 31, 2019, China reported a cluster of cases of pneumonia in people at Wuhan, Hubei Province. The responsible pathogen is a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the relevant features of the first cases in Europe of confirmed infection, named coronavirus disease 2019 (COVID-19), with the first patient diagnosed with the disease on Jan 24, 2020.

Methods: In this case series, we followed five patients admitted to Bichat-Claude Bernard University Hospital (Paris, France) and Pellegrin University Hospital (Bordeaux, France) and diagnosed with COVID-19 by semi-quantitative RT-PCR on nasopharyngeal swabs. We assessed patterns of clinical disease and viral load from different samples (nasopharyngeal and blood, urine, and stool samples), which were obtained once daily for 3 days from hospital admission, and once every 2 or 3 days until patient discharge. All samples were refrigerated and shipped to laboratories in the National Reference Center for Respiratory Viruses (The Institut Pasteur, Paris, and Hospices Civils de Lyon, Lyon, France), where RNA extraction, real-time RT-PCR, and virus isolation and titration procedures were done.

Findings: The patients were three men (aged 31 years, 48 years, and 80 years) and two women (aged 30 years and 46 years), all of Chinese origin, who had travelled to France from China around mid-January, 2020. Three different clinical evolutions are described: (1) two paucisymptomatic women diagnosed within a day of exhibiting symptoms, with high nasopharyngeal titres of SARS-CoV-2 within the first 24 h of the illness onset (5·2 and 7·4 log copies per 1000 cells, respectively) and viral RNA detection in stools; (2) a two-step disease progression in two young men, with a secondary worsening around 10 days after disease onset despite a decreasing viral load in nasopharyngeal samples; and (3) an 80-year-old man with a rapid evolution towards multiple organ failure and a persistent high viral load in lower and upper respiratory tract with systemic virus dissemination and virus detection in plasma. The 80-year-old patient died on day 14 of illness (Feb 14, 2020); all other patients had recovered and been discharged by Feb 19, 2020.

Interpretation: We illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies.

Funding: REACTing (Research & Action Emerging Infectious Diseases).
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http://dx.doi.org/10.1016/S1473-3099(20)30200-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156120PMC
June 2020

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020.

Euro Surveill 2020 03;25(9)

These authors have contributed equally to the manuscript.

In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.9.2000178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164PMC
March 2020

Impact of Pregnancy on Intra-Host Genetic Diversity of Influenza A Viruses in Hospitalised Women: A Retrospective Cohort Study.

J Clin Med 2019 Dec 25;9(1). Epub 2019 Dec 25.

Virpath, INSERM U1111, CNRS UMR5308, International Center for Infectiology Research, ENS Lyon, Claude Bernard Lyon 1 University, 69008 Lyon, France.

The authors wish to make the following corrections to this paper [...].
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http://dx.doi.org/10.3390/jcm9010058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019403PMC
December 2019

Impact of Pregnancy on Intra-Host Genetic Diversity of Influenza A Viruses in Hospitalised Women: A Retrospective Cohort Study.

J Clin Med 2019 Nov 14;8(11). Epub 2019 Nov 14.

Virpath, INSERM U1111, CNRS UMR5308, International Center for Infectiology Research, ENS Lyon, Claude Bernard Lyon 1 University, 69008 Lyon, France.

Characterising dynamics of Influenza A Viruses (IAV) within-host evolution is an active field of research which may lead to a better understanding of viral pathogenesis. Using a pregnant mouse model, a study has recently suggested that immune modulation during pregnancy could promote the emergence of IAV quasispecies with increased virulence. Herein, we assess the clinical relevance of these findings in humans. We studied IAV intra-host diversity (ihD) in pregnant ( = 36) and non-pregnant ( = 23) women hospitalized in Lyon for IAV infection (01/2015-05/2018). Whole IAV genomes present in nasopharyngeal samples were sequenced in duplicate to analyze reproducible intra-host single nucleotide variants (ihSNV). Counts, relative frequencies and locations of ihSNV were used as indicators of ihD. The median ihSNV/kb counts per segment were between 0 and 1.3. There was >81% ihSNV at relative frequencies between 1-5% for H1N1 and >51% for H3N2 IAV. No significant difference was noted between pregnant and non-pregnant women when considering all or only non-synonymous ihSNV. Seven convergent non-synonymous ihSNV were found; none were significantly associated with pregnancy. These results suggest that modulation of the immune system during pregnancy in humans does not impact IAV ihD, in contrast to mice.
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http://dx.doi.org/10.3390/jcm8111974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912736PMC
November 2019

Web-based analysis of adherence to influenza vaccination among French healthcare workers.

J Clin Virol 2019 07 28;116:29-33. Epub 2019 Apr 28.

Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Centre National de Référence des virus des infections respiratoires (dont la Grippe), HCL, Hôpital de la Croix-Rousse, 103 grande rue de la Croix-Rousse, 69317, Lyon Cedex 04, France; Virpath, CIRI, Université de Lyon, CIRI, Inserm U1111 CNRS UMR5308, ENS, UCBL, Faculté de Médecine Lyon Est, 7-11 rue Guillaume Paradin, 69372, Lyon Cedex 08, France. Electronic address:

Background: Influenza is recognized as a public health threat. However, vaccine hesitancy and poor vaccine uptake have been seen in French healthcare workers (HCWs). As a result, authorities have considered implementing mandatory influenza vaccination for HCWs.

Objectives: This study aimed to describe factors associated with influenza vaccine adherence or non-adherence in French HCWs and to collect their perception about mandatory influenza vaccination.

Study Design: In February 2017, during the influenza season, a standardized questionnaire was sent electronically to the professional email addresses of French HCWs. Analyses were performed having collected 3000 responses.

Results: Between February 1 and 16, 2017, a 14-question survey was sent to HCWs professional email addresses. After a two week period had elaspsed, 3000 answers were collected for analysis. Overall, 45.7% of responders reported they had received influenza vaccination in 2016-2017, with statistical differences relating to professional status, age and practice. In addition, 92.2% reported caring for at-risk patients and 62.9% had a community-based practice. Finally, accepting mandatory influenza vaccination was statistically associated with higher age, a higher socio-professional category, and seasonal influenza vaccine uptake, but not with management of at-risk patients.

Conclusions: Electronically submitted questionnaires are a rapid and easy tool that can be used to describe factors associated with influenza vaccine uptake in HCWs. In our study, differences in receiving influenza vaccination related to age, practice and professional categories, and provided an insight into potential adherence to mandatory influenza vaccination in HCWs. If repeated, these surveys may also monitor the evolution of vaccine uptake by professional categories.
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http://dx.doi.org/10.1016/j.jcv.2019.04.008DOI Listing
July 2019

Residual Activatability of Circulating Tfh17 Predicts Humoral Response to Thymodependent Antigens in Patients on Therapeutic Immunosuppression.

Front Immunol 2018 5;9:3178. Epub 2019 Feb 5.

French National Institute of Health and Medical Research (Inserm) Unit 1111, Lyon, France.

The generation of antibodies against protein antigens (such as donor-specific HLA molecules) requires that T follicular helper cells (Tfh) provide help to B cells. Immunosuppressive (IS) armamentarium prevents T cell activation, yet a significant proportion of renal transplant patients develop donor-specific antibodies (DSA), which suggests that IS drugs do not efficiently block T follicular helper cells. To test this hypothesis, the number of circulating Tfh, their polarization profile, and ability to up-regulate (i) the co-stimulatory molecules CD40L and ICOS, and (ii) the activation marker CD25, following stimulation in presence of IS drugs, were compared between 36 renal transplant patients (6-72 months post transplantation) and nine healthy controls. IS drugs reduced the number of Tfh1 and 2 but had little impact on Tfh17, which was the dominant subset in transplant patients. Although, IS drugs decreased activation-induced expression of co-stimulatory molecules by Tfh, the impact was highly variable between individuals. Furthermore, 20% of transplant patients displayed normal expression of CD25 on Tfh following stimulation (i.e., "residual activatability"). To test whether residual activatability of Tfh correlates with antibody response against thymo-dependent antigens we took advantage of the 2015 influenza vaccination campaign, which provided a normalized setting for antigenic stimulation. In line with our hypothesis, responders to influenza vaccine exhibited significantly higher percentage of CD25-expressing Tfh17 after stimulation. A results that was confirmed retrospectively in nine transplanted patients at the time of first DSA detection. We concluded that "residual activatability" of Tfh17 might be used as a non-invasive biomarker to identify transplant patients at higher risk to develop DSA under immunosuppression. If validated in larger studies, this assay might help optimizing the prevention of DSA through personalized adaptation of immunosuppressive regimen.
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http://dx.doi.org/10.3389/fimmu.2018.03178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370621PMC
October 2019

Whole Genome Sequencing of A(H3N2) Influenza Viruses Reveals Variants Associated with Severity during the 2016⁻2017 Season.

Viruses 2019 01 28;11(2). Epub 2019 Jan 28.

Virpath, CIRI, Univ Lyon, Inserm U1111 CNRS UMR5308, ENS, UCBL, Faculté de Médecine Lyon Est, 69372 Lyon, France.

Influenza viruses cause a remarkable disease burden and significant morbidity and mortality worldwide, and these impacts vary between seasons. To understand the mechanisms associated with these differences, a comprehensive approach is needed to characterize the impact of influenza genomic traits on the burden of disease. During 2016⁻2017, a year with severe A(H3N2), we sequenced 176 A(H3N2) influenza genomes using next generation sequencing (NGS) for routine surveillance of circulating influenza viruses collected via the French national influenza community-based surveillance network or from patients hospitalized in the intensive care units of the University Hospitals of Lyon, France. Taking into account confounding factors, sequencing and clinical data were used to identify genomic variants and quasispecies associated with influenza severity or vaccine failure. Several amino acid substitutions significantly associated with clinical traits were found, including NA V263I and NS1 K196E which were associated with severity and co-occurred only in viruses from the 3c.2a1 clade. Additionally, we observed that intra-host diversity as a whole and on a specific set of gene segments increased with severity. These results support the use of whole genome sequencing as a tool for the identification of genetic traits associated with severe influenza in the context of influenza surveillance.
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http://dx.doi.org/10.3390/v11020108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410005PMC
January 2019

Clinical management and viral genomic diversity analysis of a child's influenza A(H1N1)pdm09 infection in the context of a severe combined immunodeficiency.

Antiviral Res 2018 12 11;160:1-9. Epub 2018 Oct 11.

Hospices Civils de Lyon, Centre National de Référence des virus des infections respiratoires, Laboratoire de Virologie, Institut des Agents Infectieux, Groupement Hospitalier Nord, F-69317, Lyon Cedex 04, France; Univ Lyon, CIRI, Inserm U1111, CNRS UMR5308, ENS, UCBL1, équipe Virpath, Faculté de Médecine Lyon Est, 7-11 Rue Guillaume Paradin, F-69372, Lyon Cedex 08, France. Electronic address:

Introduction: A child with severe combined immunodeficiency (SCID) had an influenza A(H1N1)pdm09 infection with viral excretion longer than 6 months, during 2013-2014 influenza season, despite cord blood transplantation and antiviral treatments.

Methods: Conventional real-time RT-PCR methods were used to estimate viral load and to detect the presence of the common N1 neuraminidase (NA) H275Y substitution responsible for oseltamivir resistance. Next-generation sequencing (NGS) of influenza viruses was performed retrospectively to characterize viral quasispecies in specimens.

Results: The patient was first treated with oral oseltamivir, leading to detection of low-levels of NA-H275Y substitution. Concomitant cord blood cell transplantation, intravenous administration of zanamivir and immunoglobulins led to an increase in white blood cells and influenza viral load decrease. A viral rebound occurred as soon as the antiviral treatment was discontinued. Eventually, influenza viral load was negated with immune reconstitution. NGS found influenza quasispecies harboring NA-E119A substitution (10.3%). Moreover, NGS showed that viral genomic diversity evolved under antiviral treatment and immune status.

Conclusions: Conventional virological techniques were sufficient for influenza infection follow-up but NGS performances allowed characterization of viral variants evolution in this specific case of prolonged influenza virus infection. New and efficient treatments against influenza in immunocompromised patients are needed.
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http://dx.doi.org/10.1016/j.antiviral.2018.10.009DOI Listing
December 2018

The NS Segment of H1N1pdm09 Enhances H5N1 Pathogenicity in a Mouse Model of Influenza Virus Infections.

Viruses 2018 09 17;10(9). Epub 2018 Sep 17.

Laboratoire de Virologie et Pathologies Humaines Virpath, CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Inserm U1111, CNRS UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, 69372 CEDEX 08 Lyon, France.

In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that a reassortment event may lead to highly pathogenic influenza strains. H1N1pdm09 and H5N1 are able to infect the same target cells of the lower respiratory tract. To investigate the capacity of the emergence of reassortant viruses, we characterized viruses obtained from the co-infection of cells with H5N1 (A/Turkey/13/2006) and H1N1pdm09 (A/Lyon/969/2009 H1N1). In our analysis, all the screened reassortants possessed the PB2, HA, and NP segments from H5N1 and acquired one or two of the H1N1pdm09 segments. Moreover, the in vivo infections showed that the acquisition of the NS segment from H1N1pdm09 increased the virulence of H5N1 in mice. We conclude, therefore, that reassortment can occur between these two viruses, even if this process has never been detected in nature.
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http://dx.doi.org/10.3390/v10090504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164720PMC
September 2018

[Viral genomic diversity: Characterization and consequences on respiratory infections virulence].

Virologie (Montrouge) 2018 06;22(3):173-182

Centre hospitalier universitaire de Lyon, Laboratoire de virologie, Institut des agents infectieux (IAI) de Lyon, Centre national de référence des virus respiratoires France Sud, Centre de biologie et de pathologie Nord, Groupement hospitalier Nord F-69317, Lyon cedex 04, France Université Claude Bernard Lyon 1, Faculté de médecine Lyon Est, CIRI, ENS, Inserm U1111 CNRS UMR5308, équipe Virpath, F-69372, Lyon cedex 08, France.

During host infection, viral replication generates multiple subpopulations. Studies of viral diversity using high-throughput sequencing technologies provide a better understanding of the therapeutic effects as well as of the viral pathogenesis. This technical evolution led to an impressive number of studies analyzing this viral characteristic. In this review, we will discuss the principles of the evaluation of viral diversity, before summarizing the main physiological consequences for respiratory viruses. To date, although no study clearly established its role in pathogenesis of severe forms, viral diversification can be alternately a formidable virulence advantage or deleterious to the virus, resulting in its extinction (error-threshold). Because of these differences, it is important to study it in the context of respiratory virus infection, such as Influenza, respiratory syncitial virus (RVS) or rhinovirus. The precise understanding of this property allows us to consider multiple clinical applications, i.e. therapeutic or preventive.
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http://dx.doi.org/10.1684/vir.2018.0738DOI Listing
June 2018

Early estimates of 2016/17 seasonal influenza vaccine effectiveness in primary care in France.

J Clin Virol 2017 10 9;95:1-4. Epub 2017 Aug 9.

EA7310, Université de Corse, Inserm, France.

Background: The ongoing 2016/17 influenza epidemic in France is characterized by the circulation of A(H3N2) viruses, known to cause more severe illness among at risk populations.

Objectives: The purpose of our study was to provide early influenza vaccine effectiveness (IVE) estimates for the ongoing influenza epidemic in France and compare these estimates over the six post-pandemic IVE.

Study Design: We used clinical and virological data collected in primary care by the French Sentinelles network. IVE in preventing influenza infection was estimated by the test-negative design method. The screening method was used to estimate IVE in preventing medically-attended influenza-like illness among target groups (<65year with chronic diseases and ≥65 years) since 2010/11 influenza epidemic.

Results: Early IVE estimates in primary care against influenza A(H3N2) were 48% (95% confidence interval (CI): 22-66) overall and 39% (95% CI: -17 to 69) among elderly (aged 65 and older). In comparison to the last six epidemics, 2016/17 early IVE in preventing influenza-like illness among target groups showed VE estimates higher to those reported during the 2011/12 and 2014/15 epidemics.

Conclusions: The moderate 2016/17 IVE estimates were higher than those estimated during influenza A(H3N2) epidemics with vaccine mismatch.
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http://dx.doi.org/10.1016/j.jcv.2017.08.002DOI Listing
October 2017

Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis.

Antiviral Res 2017 Sep 3;145:160-167. Epub 2017 Aug 3.

Hospices Civils de Lyon, Centre National de Référence des virus Influenzae France Sud, Laboratoire de Virologie, Institut des Agents Infectieux, Groupement Hospitalier Nord, F-69317, Lyon Cedex 04, France; Univ Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, CIRI, Inserm U1111, CNRS UMR5308, équipe Virpath, F-69372, Lyon Cedex 08, France. Electronic address:

Introduction: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV.

Methods: RT-qPCR was performed using the ABI7500 platform. RT-ddPCR was carried out using the QX200 ddPCR platform. We strengthened our results by a NGS assay (Ion PGM™ sequencer). Discrimination performance and sensitivity of the RT-ddPCR assay were evaluated using mixes of wild type (WT) and mutated H275Y-NA-coding segments.

Results: The performance of RT-ddPCR was better than RT-qPCR, using NGS assay as a gold standard. RT-ddPCR was able to detect 0.28% oseltamivir-resistant IV in a WT IV population and 0.55% WT IV in an oseltamivir-resistant IV population. For the first patient, the H275Y-NA substitution was selected by oseltamivir treatment and reached about 50% of the IV population before dropping to less than 2% after treatment discontinuation which was under the lower limit of quantification by RT-qPCR and RT-ddPCR (<2%) after treatment stop. Then, five days after oseltamivir was re-introduced, the H275Y-NA substitution rose up to 100%. For the second patient, the H275Y-NA substitution reached about 30% two days after oseltamivir discontinuation.

Conclusion: RT-ddPCR demonstrated better performances than classical RT-qPCR to estimate oseltamivir-resistant IV proportions. This technique could be used to detect earlier emergence of H275Y-NA substitution.
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http://dx.doi.org/10.1016/j.antiviral.2017.07.021DOI Listing
September 2017

Performance of influenza case definitions for influenza community surveillance: based on the French influenza surveillance network GROG, 2009-2014.

Euro Surveill 2017 Apr;22(14)

Univ Lyon, Virpath, CIRI, INSERM U1111, CNRS, ENS, Université Claude Bernard Lyon 1, Lyon, France.

International case definitions recommended by the Centers for Disease Control and Prevention (CDC), the European Centre for Disease Prevention and Control (ECDC), and the World Health Organization (WHO) are commonly used for influenza surveillance. We evaluated clinical factors associated with the laboratory-confirmed diagnosis of influenza and the performance of these influenza case definitions by using a complete dataset of 14,994 patients with acute respiratory infection (ARI) from whom a specimen was collected between August 2009 and April 2014 by the Groupes Régionaux d'Observation de la Grippe (GROG), a French national influenza surveillance network. Cough and fever ≥ 39 °C most accurately predicted an influenza infection in all age groups. Several other symptoms were associated with an increased risk of influenza (headache, weakness, myalgia, coryza) or decreased risk (adenopathy, pharyngitis, shortness of breath, otitis/otalgia, bronchitis/ bronchiolitis), but not throughout all age groups. The WHO case definition for influenza-like illness (ILI) had the highest specificity with 21.4%, while the ECDC ILI case definition had the highest sensitivity with 96.1%. The diagnosis among children younger than 5 years remains challenging. The study compared the performance of clinical influenza definitions based on outpatient surveillance and will contribute to improving the comparability of data shared at international level.
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http://dx.doi.org/10.2807/1560-7917.ES.2017.22.14.30504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388124PMC
April 2017

A major impact of the influenza seasonal epidemic on intensive care units, Réunion, April to August 2016.

Euro Surveill 2016 Nov;21(47)

Santé publique France, French national public health agency, Regional unit (Cire) Océan Indien, Réunion, France.

The 2016 seasonal influenza in Réunion in the southern hemisphere, was dominated by influenza A(H1N1)pdm09 (possibly genogroup 6B.1). An estimated 100,500 patients with acute respiratory infection (ARI) consulted a physician (cumulative attack rate 11.9%). Sixty-six laboratory-confirmed cases (65.7/100,000 ARI consultations) were hospitalised in an intensive care unit, the highest number since 2009. Impact on intensive care units was major. Correlation between severe cases was 0.83 between Réunion and France and good for 2009 to 2015.
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http://dx.doi.org/10.2807/1560-7917.ES.2016.21.47.30405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291144PMC
November 2016

Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9).

J Antimicrob Chemother 2016 11 17;71(11):3036-3045. Epub 2016 Jul 17.

Univ Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, CIRI Inserm U1111, équipe Virpath, F-69008, Lyon, France

Objectives: While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ± I222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness.

Methods: Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ± I222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, K of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined.

Results: NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA.

Conclusions: NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued.
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http://dx.doi.org/10.1093/jac/dkw275DOI Listing
November 2016

Factors associated with poor outcomes among adults hospitalized for influenza in France: A three-year prospective multicenter study.

J Clin Virol 2016 06 12;79:68-73. Epub 2016 Apr 12.

Inserm, CIC 1417, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Department of Infectious Diseases, CIC Cochin Pasteur, Cochin Broca Hôtel-Dieu hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France; Inserm, F-CRIN, Innovative Clinical Research Network in Vaccinology (I-REIVAC), Paris, France. Electronic address:

Background: Influenza is an important cause of serious illness and death, particularly in elderly and high-risk groups.

Objectives: Aim of this study was to identify factors associated with poor outcomes among adults hospitalized in France for laboratory-confirmed seasonal influenza.

Study Design: Patients hospitalized for influenza were identified in a prospective, multicenter study carried out in French hospitals during three consecutive influenza seasons (2012-2015). Influenza virus infection was confirmed by reverse transcription polymerase chain reaction. Sociodemographic and clinical variables were compared according to the virus type and subtype. Risk factors for complications, intensive care unit (ICU) admission and death were analyzed by backward stepwise logistic regression.

Results: The study population consisted of 566 patients, of whom 56% were older than 65 years and 82% had underlying chronic illnesses. Type A influenza viruses infected 422 patients (75%), including subtype H3N2 in 239 patients (57%). The prior vaccine coverage rate was 38%. Complications occurred in 255 patients (45%), consisting mainly of pneumonia (n=143, 30%) and respiratory failure (n=116, 20%). Eighty-three patients (15%) were admitted to an ICU, and the in-hospital mortality rate was 4% (n=21). Sixty-six patients (12%) received oseltamivir. Age over 65 years was the only identified risk factor for complications. Risk factors for ICU admission were an absence of vaccination, no oseltamivir administration before admission, pre-existing chronic respiratory disease, and current smoking. Age over 65 years and ICU admission were risk factors for death.

Conclusions: Older individuals and patients with underlying conditions are most at risk of influenza complications. Vaccination and early oseltamivir administration, both of which are recommended for these patients, appear to reduce ICU admissions.
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http://dx.doi.org/10.1016/j.jcv.2016.04.005DOI Listing
June 2016

Striking Similarities in the Presentation and Duration of Illness of Influenza A and B in the Community: A Study Based on Sentinel Surveillance Networks in France and Turkey, 2010-2012.

PLoS One 2015 1;10(10):e0139431. Epub 2015 Oct 1.

Royal College of General Practitioners, Surveillance and Research Unit, Birmingham, United Kingdom.

Influenza B represents a high proportion of influenza cases in some seasons (even over 50%). The Influenza B study in General Practice (IBGP) is a multicenter study providing information about the clinical, demographic and socio-economic characteristics of patients affected by lab-confirmed influenza A or B. Influenza B patients and age-matched influenza A patients were recruited within the sentinel surveillance networks of France and Turkey in 2010-11 and 2011-12 seasons. Data were collected for each patient at the swab test day, after 9±2 days and, if not recovered, after 28±5 days. It was related to patient's characteristics, symptoms at presentation, vaccination status, prescriptions of antibiotics and antivirals, duration of illness, follow-up consultations in general practice or emergency room. We performed descriptive analyses and developed a multiple regression model to investigate the effect of patients and disease characteristics on the duration of illness. Overall, 774 influenza cases were included in the study: 419 influenza B cases (209 in France and 210 in Turkey) and 355 influenza A cases (205 in France and 150 in Turkey). There were no differences between influenza A and B patients in terms of clinical presentation and number of consultations with a practitioner; however, the use of antivirals was higher among influenza B patients in both countries. The average (median) reported duration of illness in the age groups 0-14 years, 15-64 years and 65+ years was 7.4 (6), 8.7 (8) and 10.5 (9) days in France, and 6.3 (6), 8.2 (7) and 9.2 (6) days in Turkey; it increased with age but did not differ by virus type; increased duration of illness was associated with antibiotics prescription. In conclusion, our findings show that influenza B infection appears not to be milder disease than influenza A infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139431PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591015PMC
June 2016

Early estimates of 2014/15 seasonal influenza vaccine effectiveness in preventing influenza-like illness in general practice using the screening method in France.

Hum Vaccin Immunother 2015 ;11(7):1621-5

a INSERM, UMR_S 1136; Institut Pierre Louis d'Epidémiologie et de Santé Publique ; Paris , France.

The ongoing influenza epidemic is characterized by intense activity with most influenza infections due to the A (H3N2) viruses. Using the screening method, mid-season vaccine effectiveness (VE) in preventing influenza-like illness in primary care was estimated to 32% (95% CI; 23 to 40) among risk groups and was 11% (95% CI; -4 to 23) among the elderly (≥ 65 y). The VE in ≥ 65 y was the lowest estimate regarding the 4 previous seasonal influenza epidemics.
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http://dx.doi.org/10.1080/21645515.2015.1046661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514301PMC
May 2016

Functional balance between the hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 HA D222 variants.

PLoS One 2014 13;9(8):e104009. Epub 2014 Aug 13.

Université de Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, Laboratoire de Virologie et Pathologie Humaine, EA 4610, Lyon, France.

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104009PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131921PMC
April 2015

Ultrastructural fingerprints of avian influenza A (H7N9) virus in infected human lung cells.

Virology 2014 May 28;456-457:39-42. Epub 2014 Mar 28.

Virologie et Pathologie Humaine VirPath, EA 4610, Université Claude Bernard Lyon 1-Hospices Civils de Lyon, Lyon, France; VirNext, Faculté de Médecine RTH Laennec, Université Lyon 1, Lyon, France. Electronic address:

In this study, we investigated the ultrastructural modifications induced by influenza A (H7N9) virus in human lung epithelial cells. One particular characteristic of H7N9 viral infection is the formation of numerous M1-associated striated tubular structures within the nucleus and the cytoplasm, which have only previously been observed for a limited number of influenza A viruses, notably the 2009 pandemic (H1N1) virus.
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http://dx.doi.org/10.1016/j.virol.2014.03.013DOI Listing
May 2014

A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.

J Infect Dis 2014 Oct 3;210(8):1260-9. Epub 2014 May 3.

Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.

Unlabelled: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.

Methods: Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed.

Results: The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir.

Conclusions: Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.
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http://dx.doi.org/10.1093/infdis/jiu244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176448PMC
October 2014

Incidence of H1N1 2009 virus infection through the analysis of paired plasma specimens among blood donors, France.

PLoS One 2012 22;7(3):e33056. Epub 2012 Mar 22.

Institut de Veille Sanitaire, St Maurice, France.

Background: Knowledge of the age-specific prevalence of seroprotection and incidence of seroconversion infection is necessary to complement clinical surveillance data and statistical models. It provides the basis for estimating the future impact of influenza A (H1N1pdm09) and implementing appropriate prevention and response strategies.

Methods: Using a cross-sectional design, two-stage stratified sampling and paired plasma samples, we estimated the age-specific prevalence of a protective level of H1N1pdm09 antibodies in the French adult population before and after the 2009/10 pandemic, and the proportion of those susceptible that seroconverted due to infection, from a single sample of 1,936 blood donors aged 20-70 years in mainland France in June 2010. Samples with a haemagglutination inhibition (HI) titre ≥1∶40 were considered seropositive, and seroconversion due to infection was defined as a 4-fold increase in titre in the absence of H1N1pdm09 vaccination or pre-pandemic seropositivity.

Results: Out of the 1,936 donors, 1,708 were included in the analysis. Seroprevalence before the pandemic was 6.7% (95% CI 5.0, 8.9) with no significant differences by age-group (p = 0.3). Seroprevalence afterwards was 23.0% (95% CI 17.7, 29.3) with 20-29 year olds having a higher level than older groups (p<0.001). Seroconversion due to infection was 12.2% (95% CI 6.9, 20.5). Younger age-group, vaccination against H1N1 and being seropositive before the pandemic were strongly associated with post-pandemic seropositivity.

Conclusions: Before the 2009/2010 winter influenza season, only 6.7% of the French mainland population aged 20-70 had a level of antibodies usually considered protective. During the first pandemic wave, 12.2% of the population seroconverted due to infection and the seroprevalence after the wave rose to 23%, either due to prepandemic seropositivity, infection or vaccination. This relatively low latter figure contributed to an extension of target groups for influenza vaccination for the 2010/2011 season.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033056PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310844PMC
August 2012

H1N1 influenza A virus neuraminidase modulates infectivity in mice.

Antiviral Res 2012 Mar 1;93(3):374-80. Epub 2012 Feb 1.

Université de Lyon, F-69000 Lyon, France.

In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.
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http://dx.doi.org/10.1016/j.antiviral.2012.01.008DOI Listing
March 2012