High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing

Autor: Kuang-Yu Chen, Jayaprakash Karuppusamy, Mary B. O’Neill, Vaitea Opuu, Mathieu Bahin, Sophie Foulon, Pablo Ibanez, Lluis Quintana-Murci, Tatsuhiko Ozawa, Sylvie van der Werf, Philippe Nghe, Nadia Naffakh, Andrew Griffiths, Catherine Isel
Přispěvatelé: Biologie des ARN et virus influenza - RNA Biology of Influenza Virus (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Chimie-Biologie-Innovation (UMR 8231) (CBI), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Génétique Evolutive Humaine - Human Evolutionary Genetics, Max Planck Institute for Mathematics in the Sciences (MPI-MiS), Max-Planck-Gesellschaft, Institut de biologie de l'Ecole Normale Supérieure (IBENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Collège de France - Chaire Génomique humaine et évolution, Collège de France (CdF (institution)), University of Toyama, Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR - laboratoire coordonnateur), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), This work is supported by grants from the Agence Nationale de la Recherche (ANR 18 CE18 0026 01 FLU_REASSORT, ANR-10-LABX-62-IBEID, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31 Institut Pierre-Gilles de Gennes). We also thank the Hospices de Nuits-Saint-Georges for their financial support., We are grateful to Drs. R. Marquet and P. Dumas (Institut de Biologie Moléculaire et Cellulaire, Strasbourg), Dr. G. Simon (ANSES, Ploufragan, France) for insightful discussions. We thank the Genotyping and sequencing core facility at the Institut du Cerveau-ICM (Paris, France) and the Biomics Platform at the Institut Pasteur (Paris, France) for Next Generation Sequencing and S. Behillil and V. Enouf (National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France) for providing the virus samples used in this study., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0031,IPGG_LABEX,Pierre-Gilles de Gennes Institute for microfluidics(2010), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010)
Rok vydání: 2023
Předmět:
Zdroj: Proceedings of the National Academy of Sciences of the United States of America
Proceedings of the National Academy of Sciences of the United States of America, 2023, 120 (6), pp.e2211098120. ⟨10.1073/pnas.2211098120⟩
ISSN: 1091-6490
0027-8424
Popis: International audience; The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.
Databáze: OpenAIRE
Externí odkaz: