{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:21:04Z","timestamp":1768508464397,"version":"3.49.0"},"posted":{"date-parts":[[2024,2,4]]},"group-title":"Molecular Biology","reference-count":66,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,2,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The segmented structure of the Influenza A virus (IAV) genome facilitates reassortment, segment exchange during co-infection. When divergent strains mix across human, agricultural, and wildlife reservoirs novel strains are generated, which has been the source of pandemics. Due to the limited throughput and infection-based assays, IAV reassortment studies has been limited to permissive reassortment. We have developed DE-flowSVP to achieve extremely high throughput, direct profiling of as many as 10\n                  <jats:sup>5<\/jats:sup>\n                  IAV particles in a single-day experiment and enabled quantitative profiling of reassortment propensity between divergent strains for the first time. By profiling reassortants between two naturally circulating low-pathogenicity avian IAVs, we confirmed that molecular incompatibility yields strong preference toward within-strain mixing. Surprisingly, we revealed that two-to-three particle aggregation contributed primarily to genome mixing (75-99%), suggesting that aggregation mediated by sialic acid binding by viral surface proteins provides a secondary pathway to genome mixing while avoiding the co-packaging fitness cost. We showed that genome mixing is sensitively dependent on co-infection timing, relative segment abundances, and viral surface-protein background. DE-flowSVP enables large-scale survey of reassortment potential among the broad diversity of IAV strains informing pandemic strain emergence.\n                <\/jats:p>","DOI":"10.1101\/2024.02.03.578479","type":"posted-content","created":{"date-parts":[[2024,2,4]],"date-time":"2024-02-04T23:30:11Z","timestamp":1707089411000},"source":"Crossref","is-referenced-by-count":0,"title":["Unbiased, Cell-free Profiling of Single Influenza Genomes at High-throughput"],"prefix":"10.64898","author":[{"given":"Thomas W.","family":"Cowell","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wendy","family":"Puryear","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chih-Lin","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruihua","family":"Ding","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan","family":"Runstadler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hee-Sun","family":"Han","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024020800550818000_2024.02.03.578479v1.1","doi-asserted-by":"publisher","DOI":"10.1089\/vim.2004.17.350"},{"key":"2024020800550818000_2024.02.03.578479v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-023-01457-0"},{"key":"2024020800550818000_2024.02.03.578479v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-019-0513-7"},{"key":"2024020800550818000_2024.02.03.578479v1.4","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-020-01479-8"},{"key":"2024020800550818000_2024.02.03.578479v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41572-018-0041-4"},{"key":"2024020800550818000_2024.02.03.578479v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-27838-9"},{"key":"2024020800550818000_2024.02.03.578479v1.7","doi-asserted-by":"crossref","unstructured":"Ganti, K. et al. Avian influenza a viruses reassort and diversify differently in mallards and mammals. Viruses 13, (2021).","DOI":"10.1101\/2021.02.08.430042"},{"key":"2024020800550818000_2024.02.03.578479v1.8","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens12040610"},{"key":"2024020800550818000_2024.02.03.578479v1.9","doi-asserted-by":"crossref","unstructured":"Runstadler, J. A. et al. Field Research Is Essential to Counter Virological Threats. J Virol 97, (2023).","DOI":"10.1128\/jvi.00544-23"},{"key":"2024020800550818000_2024.02.03.578479v1.10","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2022.2155072"},{"key":"2024020800550818000_2024.02.03.578479v1.11","doi-asserted-by":"publisher","DOI":"10.3201\/eid1201.051254"},{"key":"2024020800550818000_2024.02.03.578479v1.12","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa0903810"},{"key":"2024020800550818000_2024.02.03.578479v1.13","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-virology-110615-042203"},{"key":"2024020800550818000_2024.02.03.578479v1.14","doi-asserted-by":"publisher","DOI":"10.3201\/eid2805.220318"},{"key":"2024020800550818000_2024.02.03.578479v1.15","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01445-08"},{"key":"2024020800550818000_2024.02.03.578479v1.16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0908897106"},{"key":"2024020800550818000_2024.02.03.578479v1.17","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-10-1"},{"key":"2024020800550818000_2024.02.03.578479v1.18","doi-asserted-by":"crossref","first-page":"1003421","DOI":"10.1371\/journal.ppat.1003421","article-title":"Influenza Virus Reassortment Occurs with High Frequency in the Absence of Segment Mismatch","volume":"9","year":"2013","journal-title":"PLoS Pathog"},{"key":"2024020800550818000_2024.02.03.578479v1.19","doi-asserted-by":"crossref","first-page":"1003421","DOI":"10.1371\/journal.ppat.1003421","article-title":"Influenza Virus Reassortment Occurs with High Frequency in the Absence of Segment Mismatch","volume":"9","year":"2013","journal-title":"PLoS Pathog"},{"key":"2024020800550818000_2024.02.03.578479v1.20","doi-asserted-by":"crossref","first-page":"113878","DOI":"10.1016\/j.jviromet.2020.113878","article-title":"A method for the unbiased quantification of reassortment in segmented viruses","volume":"280","year":"2020","journal-title":"J Virol Methods"},{"key":"2024020800550818000_2024.02.03.578479v1.21","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1005204"},{"key":"2024020800550818000_2024.02.03.578479v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09078-0"},{"key":"2024020800550818000_2024.02.03.578479v1.23","first-page":"830","article-title":"Seasonal H3N2 and 2009 Pandemic H1N1 Influenza A Viruses Reassort Efficiently but Produce Attenuated Progeny","volume":"91","year":"2017","journal-title":"J Virol"},{"key":"2024020800550818000_2024.02.03.578479v1.24","doi-asserted-by":"crossref","unstructured":"Chen, K. Y. et al. High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing. Proc Natl Acad Sci U S A 120, (2023).","DOI":"10.1073\/pnas.2211098120"},{"key":"2024020800550818000_2024.02.03.578479v1.25","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000012"},{"key":"2024020800550818000_2024.02.03.578479v1.26","doi-asserted-by":"crossref","first-page":"9023","DOI":"10.1093\/nar\/gkac688","article-title":"The influenza A virus genome packaging network - complex, flexible and yet unsolved","volume":"50","year":"2022","journal-title":"Nucleic Acids Res"},{"key":"2024020800550818000_2024.02.03.578479v1.27","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02163-13"},{"key":"2024020800550818000_2024.02.03.578479v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0115-z"},{"key":"2024020800550818000_2024.02.03.578479v1.29","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.05352-11"},{"key":"2024020800550818000_2024.02.03.578479v1.30","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008436"},{"key":"2024020800550818000_2024.02.03.578479v1.31","doi-asserted-by":"crossref","first-page":"e1010865","DOI":"10.1371\/journal.ppat.1010865","article-title":"Beneficial effects of cellular coinfection resolve inefficiency in influenza A virus transcription","volume":"18","year":"2022","journal-title":"PLoS Pathog"},{"key":"2024020800550818000_2024.02.03.578479v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(91)90588-3"},{"key":"2024020800550818000_2024.02.03.578479v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.04.044"},{"key":"2024020800550818000_2024.02.03.578479v1.34","doi-asserted-by":"crossref","unstructured":"Zheng, W. et al. High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome. Science 376, (2022).","DOI":"10.1126\/science.abm1483"},{"key":"2024020800550818000_2024.02.03.578479v1.35","first-page":"1","article-title":"Droplet-based microfluidics","volume":"3","year":"2023","journal-title":"Nature Reviews Methods Primers"},{"key":"2024020800550818000_2024.02.03.578479v1.36","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-016-0009-6"},{"key":"2024020800550818000_2024.02.03.578479v1.37","doi-asserted-by":"publisher","DOI":"10.1021\/ac202578x"},{"key":"2024020800550818000_2024.02.03.578479v1.38","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.0c02378"},{"key":"2024020800550818000_2024.02.03.578479v1.39","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx584"},{"key":"2024020800550818000_2024.02.03.578479v1.40","doi-asserted-by":"crossref","first-page":"39385","DOI":"10.1038\/srep39385","article-title":"Sequence specific sorting of DNA molecules with FACS using 3dPCR","volume":"7","year":"2017","journal-title":"Sci Rep"},{"key":"2024020800550818000_2024.02.03.578479v1.41","doi-asserted-by":"publisher","DOI":"10.1039\/D0LC00261E"},{"key":"2024020800550818000_2024.02.03.578479v1.42","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/978-1-0716-3323-6_12","article-title":"Double Emulsion Flow Cytometry for Rapid Single Genome Detection","volume":"2689","year":"2023","journal-title":"Methods in molecular biology"},{"key":"2024020800550818000_2024.02.03.578479v1.43","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1006888107"},{"key":"2024020800550818000_2024.02.03.578479v1.44","doi-asserted-by":"publisher","DOI":"10.1039\/c004124f"},{"key":"2024020800550818000_2024.02.03.578479v1.45","doi-asserted-by":"crossref","first-page":"20528","DOI":"10.1021\/acsami.1c22692","article-title":"Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis","volume":"14","year":"2022","journal-title":"ACS Appl Mater Interfaces"},{"key":"2024020800550818000_2024.02.03.578479v1.46","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1206069109"},{"key":"2024020800550818000_2024.02.03.578479v1.47","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008671"},{"key":"2024020800550818000_2024.02.03.578479v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(74)90276-1"},{"key":"2024020800550818000_2024.02.03.578479v1.49","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00039"},{"key":"2024020800550818000_2024.02.03.578479v1.50","doi-asserted-by":"crossref","first-page":"e49208","DOI":"10.1371\/journal.pone.0049208","article-title":"Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy","volume":"7","year":"2012","journal-title":"PLoS One"},{"key":"2024020800550818000_2024.02.03.578479v1.51","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1007\/978-1-4939-0758-8_12","article-title":"Avian influenza virus isolation, propagation, and titration in embryonated chicken eggs","volume":"1161","year":"2014","journal-title":"Methods in Molecular Biology"},{"key":"2024020800550818000_2024.02.03.578479v1.52","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-018-1451-y"},{"key":"2024020800550818000_2024.02.03.578479v1.53","doi-asserted-by":"publisher","DOI":"10.1002\/9781118636817.CH5"},{"key":"2024020800550818000_2024.02.03.578479v1.54","doi-asserted-by":"crossref","unstructured":"Johnson, M. P. , Haupt, L. M. & Griffiths, L. R. Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. Nucleic Acids Res 32, (2004).","DOI":"10.1093\/nar\/gnh046"},{"key":"2024020800550818000_2024.02.03.578479v1.55","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1270\/jsbbs.19142","article-title":"Taqman-MGB snp genotyping assay to identify 48 citrus cultivars distributed in the Japanese market","volume":"70","year":"2020","journal-title":"Breed Sci"},{"key":"2024020800550818000_2024.02.03.578479v1.56","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro.2016.46"},{"key":"2024020800550818000_2024.02.03.578479v1.57","doi-asserted-by":"publisher","DOI":"10.3390\/v14020438"},{"key":"2024020800550818000_2024.02.03.578479v1.58","doi-asserted-by":"publisher","DOI":"10.2217\/fvl.13.118"},{"key":"2024020800550818000_2024.02.03.578479v1.59","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.622308"},{"key":"2024020800550818000_2024.02.03.578479v1.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmi.2016.07.007"},{"key":"2024020800550818000_2024.02.03.578479v1.61","doi-asserted-by":"publisher","DOI":"10.1021\/bm049878z"},{"key":"2024020800550818000_2024.02.03.578479v1.62","doi-asserted-by":"crossref","unstructured":"Kaverin, N. V et al. Postreassortment Changes in Influenza A Virus Hemagglutinin Restoring HA-NA Functional Match. Virology (1998).","DOI":"10.1006\/viro.1998.9119"},{"key":"2024020800550818000_2024.02.03.578479v1.63","doi-asserted-by":"publisher","DOI":"10.1016\/j.coviro.2012.07.007"},{"key":"2024020800550818000_2024.02.03.578479v1.64","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1080\/22221751.2019.1581034","article-title":"Neuraminidase activity and specificity of influenza A virus are influenced by haemagglutinin-receptor binding","volume":"8","year":"2019","journal-title":"Emerg Microbes Infect"},{"key":"2024020800550818000_2024.02.03.578479v1.65","doi-asserted-by":"crossref","first-page":"6030","DOI":"10.3390\/ijerph20116030","article-title":"Ecogeographic Drivers of the Spatial Spread of Highly Pathogenic Avian Influenza Outbreaks in Europe and the United States, 2016\u2013Early 2022","volume":"20","year":"2023","journal-title":"Int J Environ Res Public Health"},{"key":"2024020800550818000_2024.02.03.578479v1.66","doi-asserted-by":"crossref","first-page":"84","DOI":"10.3389\/fvets.2018.00084","article-title":"Geographical and historical patterns in the emergences of novel highly pathogenic avian influenza (HPAI) H5 and H7 viruses in poultry","volume":"5","year":"2018","journal-title":"Front Vet Sci"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.02.03.578479","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:54:09Z","timestamp":1768456449000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.02.03.578479"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,4]]},"references-count":66,"URL":"https:\/\/doi.org\/10.1101\/2024.02.03.578479","relation":{},"subject":[],"published":{"date-parts":[[2024,2,4]]},"subtype":"preprint"}}