Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells

Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells Andrea Marti, Alexander Nater, Jenny Pego Magalhaes, Noelle Donzé, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9121293/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne Orthoflavivirus that circulates primarily in birds and pigs. Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model. Methods To investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in Aedes albopictus -derived C6/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics. Results For all viruses, serial passaging in MDMs - but not in C6/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaques sizes in porcine endothelial cells. In contrast to C6/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaques phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM. Conclusion This work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed in vitro with other mammalian cells. JEV Orthoflavivirus pigs mosquitos macrophages fitness adaptations Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigures251022.docx DatafileMartietal.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 16 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 14 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9121293","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620145161,"identity":"33e851d2-a07e-476a-829e-65786a32834f","order_by":0,"name":"Andrea Marti","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Marti","suffix":""},{"id":620145162,"identity":"fef4f325-06f8-4c92-bfe7-2de40e19ad6e","order_by":1,"name":"Alexander Nater","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Nater","suffix":""},{"id":620145163,"identity":"f7851d96-2e91-494b-a6bf-80b1bc6d8469","order_by":2,"name":"Jenny Pego Magalhaes","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Jenny","middleName":"Pego","lastName":"Magalhaes","suffix":""},{"id":620145164,"identity":"ee635c40-158b-48ef-ba94-a8691be8a2dc","order_by":3,"name":"Noelle Donzé","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Noelle","middleName":"","lastName":"Donzé","suffix":""},{"id":620145165,"identity":"144300e3-7d76-4945-98e0-190806dd5482","order_by":4,"name":"Markus Gerber","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"Gerber","suffix":""},{"id":620145166,"identity":"5cead0d6-454c-4b39-8958-e084e051fe1e","order_by":5,"name":"Lea Almeida","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Lea","middleName":"","lastName":"Almeida","suffix":""},{"id":620145167,"identity":"7d9efd53-2cdd-42cb-8c6d-aea9f9d0eb8d","order_by":6,"name":"Matthias Liniger","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Liniger","suffix":""},{"id":620145168,"identity":"9f64cfed-cbc6-40b6-bb90-c6ecb0e46000","order_by":7,"name":"Sandra Chenaux","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Chenaux","suffix":""},{"id":620145169,"identity":"5b73726d-dd86-494b-b5e7-c62cada98675","order_by":8,"name":"Nicolas Ruggli","email":"","orcid":"","institution":"Institute of Virology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Ruggli","suffix":""},{"id":620145170,"identity":"87b40a9b-7e3d-4dbd-9f4f-e36818982d6f","order_by":9,"name":"Fadi G. 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Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in \u003cem\u003eAedes albopictus\u003c/em\u003e-derived C6/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFor all viruses, serial passaging in MDMs - but not in C6/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaques sizes in porcine endothelial cells. In contrast to C6/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaques phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed \u003cem\u003ein vitro\u003c/em\u003e with other mammalian cells.\u003c/p\u003e","manuscriptTitle":"Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 17:24:20","doi":"10.21203/rs.3.rs-9121293/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-20T01:33:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57932759119415347372700156043496986156","date":"2026-04-07T08:32:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T07:48:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-16T17:18:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T17:18:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2026-03-14T09:14:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b336b82-b5b6-4612-8b44-c422eb634765","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T17:24:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 17:24:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9121293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9121293","identity":"rs-9121293","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}