{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T17:05:58Z","timestamp":1771002358569,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T00:00:00Z","timestamp":1770940800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003710","name":"Ministry of Health & Welfare","doi-asserted-by":"publisher","award":["HI23C0710"],"award-info":[{"award-number":["HI23C0710"]}],"id":[{"id":"10.13039\/501100003710","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011705","name":"Ministry of Oceans and Fisheries, Republic of Korea","doi-asserted-by":"publisher","award":["20210466"],"award-info":[{"award-number":["20210466"]}],"id":[{"id":"10.13039\/501100011705","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) co-circulate widely and cause significant morbidity, yet effective broad-spectrum antivirals are limited. This study evaluated the antiviral efficacy, cytotoxicity, and host transcriptional responses to the nucleic acid\u2013hydrolyzing antibody fragment 3D8 scFv in mono- and co-infection models. RNA sequencing of A549 cells treated with 3D8 scFv revealed a dose-dependent activation of the MAPK\u2013HSP70 stress response, with minimal transcriptomic disruption at antiviral concentrations. Comparative transcriptomic analysis identified distinct host signatures for ZIKV and DENV2, and machine learning classifiers accurately distinguished infection states (AUC &gt; 0.95). In Vero E6 cells, prophylactic treatment with 3D8 scFv significantly reduced viral RNA, protein expression, and infectious particle production for both viruses, including during co-infection. Optimized post-entry treatment also demonstrated antiviral activity. Cytotoxicity assays confirmed good tolerability at effective concentrations. These findings indicate that 3D8 scFv inhibits viral replication through early cleavage of viral nucleic acids while inducing a limited protective stress response, supporting its development as a broad-spectrum antiviral candidate.<\/jats:p>","DOI":"10.3390\/ijms27041806","type":"journal-article","created":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T16:09:32Z","timestamp":1770998972000},"page":"1806","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Antiviral Efficacy, Cytotoxicity, Transcriptomics, and Discriminatory Function of 3D8 scFv Against Dengue and Zika Viruses"],"prefix":"10.3390","volume":"27","author":[{"given":"Muhammad Salman","family":"Akram","sequence":"first","affiliation":[{"name":"Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-8808-0179","authenticated-orcid":false,"given":"Chengmin","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rimsha","family":"Riaz","sequence":"additional","affiliation":[{"name":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad 38000, Pakistan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Quynh Xuan Thi","family":"Luong","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Ton Duc Thang University, Ho Chi Minh City 70000, Vietnam"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Muhammad Faizan","family":"Khurram","sequence":"additional","affiliation":[{"name":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad 38000, Pakistan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"SeonHyeon","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0842-1769","authenticated-orcid":false,"given":"Ramadhani Qurrota","family":"Ayun","sequence":"additional","affiliation":[{"name":"Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min-Jeong","family":"Kim","sequence":"additional","affiliation":[{"name":"Ecological Risk Research Department, Korea Institute of Ocean Science & Technology, Geoje 53201, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6090-507X","authenticated-orcid":false,"given":"TaekKyun","family":"Lee","sequence":"additional","affiliation":[{"name":"Ecological Risk Research Department, Korea Institute of Ocean Science & Technology, Geoje 53201, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9964-0176","authenticated-orcid":false,"given":"Sukchan","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Badia, R., Garcia-Vidal, E., and Ballana, E. (2022). Viral-Host Dependency Factors as Therapeutic Targets to Overcome Antiviral Drug-Resistance: A Focus on Innate Immune Modulation. Front. Virol., 2.","DOI":"10.3389\/fviro.2022.935933"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"eabg9478","DOI":"10.1126\/scitranslmed.abg9478","article-title":"Dengue and Zika virus infections in children elicit cross-reactive protective and enhancing antibodies that persist long term","volume":"13","author":"Katzelnick","year":"2021","journal-title":"Sci. Transl. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The global distribution and burden of dengue","volume":"496","author":"Bhatt","year":"2013","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Krauer, F., Riesen, M., Reveiz, L., Oladapo, O.T., Mart\u00ednez-Vega, R., Porgo, T.V., Haefliger, A., Broutet, N.J., Low, N., and WHO Zika Causality Working Group (2017). Zika Virus Infection as a Cause of Congenital Brain Abnormalities and Guillain\u2013Barr\u00e9 Syndrome: Systematic Review. PLoS Med., 14.","DOI":"10.1371\/journal.pmed.1002203"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1038\/nrd.2017.33","article-title":"Broad-spectrum agents for flaviviral infections: Dengue, Zika and beyond","volume":"16","author":"Boldescu","year":"2017","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"23696","DOI":"10.1038\/s41598-021-03211-0","article-title":"High resolution proteomics of Aedes aegypti salivary glands infected with either dengue, Zika or chikungunya viruses identify new virus specific and broad antiviral factors","volume":"11","author":"Chowdhury","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"10731","DOI":"10.1038\/s41598-025-95346-7","article-title":"Effects of the fatty acid synthase inhibitors triclosan and lapatinib on dengue virus and Zika virus infection","volume":"15","author":"Sornprasert","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1038\/s41564-020-0714-0","article-title":"The continued threat of emerging flaviviruses","volume":"5","author":"Pierson","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1186\/s12917-020-02462-9","article-title":"The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens","volume":"16","author":"Byun","year":"2020","journal-title":"BMC Vet. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1985","DOI":"10.1007\/s00018-009-9179-2","article-title":"A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity","volume":"66","author":"Jang","year":"2009","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1016\/j.bbrc.2010.04.032","article-title":"An RNA-hydrolyzing recombinant antibody exhibits an antiviral activity against classical swine fever virus","volume":"395","author":"Jun","year":"2010","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"773","DOI":"10.14348\/molcells.2015.0073","article-title":"Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL\/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity","volume":"38","author":"Lee","year":"2015","journal-title":"Mol. Cells"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Lee, Y., Hoang, P.T., Kim, D., Ayun, R.Q., Luong, Q.X.T., Na, K., Kim, T., Oh, Y., Kim, W.-K., and Lee, S. (2022). A Therapeutically Active Minibody Exhibits an Antiviral Activity in Oseltamivir-Resistant Influenza-Infected Mice via Direct Hydrolysis of Viral RNAs. Viruses, 14.","DOI":"10.3390\/v14051105"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Lee, G., Budhathoki, S., Lee, G.-Y., Oh, K.-J., Ham, Y.K., Kim, Y.-J., Lim, Y.R., Hoang, P.T., Lee, Y., and Lim, S.-W. (2021). Broad-spectrum antiviral activity of 3d8, a nucleic acid-hydrolyzing single-chain variable fragment (Scfv), targeting SARS-CoV-2 and multiple coronaviruses in vitro. Viruses, 13.","DOI":"10.1101\/2020.11.25.398909"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"8472","DOI":"10.1038\/s41598-024-52810-0","article-title":"An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models","volume":"14","author":"Luong","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2793","DOI":"10.1007\/s00253-014-6257-7","article-title":"Development of Lactobacillus paracasei harboring nucleic acid-hydrolyzing 3D8 scFv as a preventive probiotic against murine norovirus infection","volume":"99","author":"Hoang","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5938","DOI":"10.1038\/s41598-017-05270-8","article-title":"Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission","volume":"7","author":"Byun","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"113706","DOI":"10.1016\/j.celrep.2024.113706","article-title":"Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans","volume":"43","author":"Ratnasiri","year":"2024","journal-title":"Cell Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s13073-023-01216-0","article-title":"A machine learning classifier using 33 host immune response mRNAs accurately distinguishes viral and non-viral acute respiratory illnesses in nasal swab samples","volume":"15","author":"Pandya","year":"2023","journal-title":"Genome Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1007\/s00018-004-4464-6","article-title":"Hsp70 chaperones: Cellular functions and molecular mechanism","volume":"62","author":"Mayer","year":"2005","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1016\/j.yrtph.2018.02.012","article-title":"Survival of Escherichia coli harboring nucleic acid-hydrolyzing 3D8 scFv during RNA virus infection","volume":"94","author":"Park","year":"2018","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4462","DOI":"10.1074\/jbc.M111.311522","article-title":"Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains","volume":"287","author":"Hutt","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"105516","DOI":"10.1016\/j.antiviral.2022.105516","article-title":"Flavivirus proteases: The viral Achilles heel to prevent future pandemics","volume":"210","author":"Teramoto","year":"2023","journal-title":"Antivir. Res."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Fiorucci, D., Meaccini, M., Poli, G., Stincarelli, M.A., Vagaggini, C., Giannecchini, S., Sutto-Ortiz, P., Canard, B., Decroly, E., and Dreassi, E. (2024). Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25042437"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e202400039","DOI":"10.1002\/ansa.202400039","article-title":"Prodigiosin Demonstrates Promising Antiviral Activity Against Dengue Virus and Zika Virus in In-silico Study","volume":"5","author":"Rahman","year":"2024","journal-title":"Anal. Sci. Adv."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"de Castro-Amarante, M.F., Pereira, S.S., Pereira, L.R., Santos, L.S., Venceslau-Carvalho, A.A., Martins, E.G., Balan, A., and Ferreira, L.C.d.S. (2023). The Anti-Dengue Virus Peptide DV2 Inhibits Zika Virus Both In Vitro and In Vivo. Viruses, 15.","DOI":"10.3390\/v15040839"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Roozitalab, A., Zhang, J., Zhang, C., Tang, Q., and Zhao, R.Y. (2025). The Evolving Role of Zika Virus Envelope Protein in Viral Entry and Pathogenesis. Viruses, 17.","DOI":"10.3390\/v17060817"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"BSR20240134","DOI":"10.1042\/BSR20240134","article-title":"Dengue virus: Pathogenesis and potential for small molecule inhibitors","volume":"44","author":"Chauhan","year":"2024","journal-title":"Biosci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"105517","DOI":"10.1016\/j.antiviral.2022.105517","article-title":"Therapeutics for flaviviral infections","volume":"210","author":"Bifani","year":"2023","journal-title":"Antivir. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1038\/s41577-023-00858-w","article-title":"Antiviral neutralizing antibodies: From in vitro to in vivo activity","volume":"23","author":"Burton","year":"2023","journal-title":"Nat. Rev. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e0004022","DOI":"10.1128\/cmr.00040-22","article-title":"Antiviral Approaches against Influenza Virus","volume":"6","author":"Kumari","year":"2023","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Hoang, P.T., Luong, Q.X.T., Ayun, R.Q., Lee, Y., Oh, K.-J., Kim, T., Lee, T.-K., and Lee, S. (2024). A synergistic therapy against influenza virus A\/H1N1\/PR8 by a HA1 specific neutralizing single-domain VL and an RNA hydrolyzing scFv. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1355599"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"40687","DOI":"10.1038\/s41598-025-24248-5","article-title":"A data-driven machine learning framework to predict side effects of AstraZeneca and sinopharm COVID-19 vaccines","volume":"15","author":"Eterafi","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"100396","DOI":"10.1016\/j.patter.2021.100396","article-title":"Machine learning and network medicine approaches for drug repositioning for COVID-19","volume":"3","author":"Torres","year":"2022","journal-title":"Patterns"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1108","DOI":"10.1016\/j.cell.2015.10.046","article-title":"Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection","volume":"163","author":"Taguwa","year":"2015","journal-title":"Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.antiviral.2016.03.017","article-title":"An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target","volume":"130","author":"Howe","year":"2016","journal-title":"Antivir. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"109377","DOI":"10.1016\/j.vetmic.2022.109377","article-title":"Heat shock protein 70 (HSP70) plays important role in tembusu virus infection","volume":"267","author":"Dai","year":"2022","journal-title":"Vet. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1038\/s41587-019-0201-4","article-title":"Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype","volume":"37","author":"Kim","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"Ritchie","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Wickham, H. (2016). Data Analysis, Springer International Publishing.","DOI":"10.1007\/978-3-319-24277-4_9"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc. Ser. B (Methodol.)"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"15545","DOI":"10.1073\/pnas.0506580102","article-title":"Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles","volume":"102","author":"Subramanian","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Chen, T., and Guestrin, C. (2016, January 13\u201317). XGBoost: A scalable tree boosting system. Proceedings of the ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, San Francisco, CA, USA.","DOI":"10.1145\/2939672.2939785"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1806\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T16:10:00Z","timestamp":1770999000000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1806"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,13]]},"references-count":45,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ijms27041806"],"URL":"https:\/\/doi.org\/10.3390\/ijms27041806","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,13]]}}}