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A better understanding of the molecular mechanisms governing flavivirus\u2013host interactions is needed to identify host regulatory pathways involved in infection. This study aimed to investigate how WNV infection remodels the host miRNA\u2013mRNA regulatory landscape. Methods: WNV-induced changes in host miRNA expression in HEK-293 cells were profiled using miRNA-Seq. Transcriptome-wide host gene expression changes in WNV-infected cells were analysed using RNA-Seq. Gene Ontology and pathway enrichment analyses were conducted using DAVID. Integrated miRNA\u2013mRNA network reconstruction was performed using Cytoscape based on the experimentally validated miRNA\u2013mRNA interactions in miRNet database. Results: WNV infection induced global changes in host miRNA expression, with pathogenic NY99 and non-pathogenic Kunjin strains of the virus producing overlapping and strain-specific alterations in the miRNA landscape. Transcriptome analysis showed strong induction of interferon-related responses and activation of NF-\u03baB and MAPK signalling pathways in the infected cells. In contrast, pathways associated with RNA processing, splicing, and proteasomal degradation were downregulated. Integrated miRNA\u2013mRNA network analysis identified miR-197-3p, miR-301b-3p, miR-129-3p, miR-3662, and miR-128-5p as candidate regulatory hubs involved in WNV-induced transcriptome remodelling. These networks suggested that miRNA-mediated regulation may influence antiviral signalling, apoptosis, and RNA metabolism during infection. Conclusions: These findings suggest that WNV infection broadly remodels host miRNA\u2013mRNA regulatory networks and identifies candidate miRNAs that may contribute to the regulation of antiviral and cellular stress responses. These predicted regulatory interactions provide a foundation for future experimental validation.<\/jats:p>","DOI":"10.3390\/epigenomes10020041","type":"journal-article","created":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T08:59:38Z","timestamp":1781773178000},"page":"41","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Remodelling of miRNA Regulatory Landscape During West Nile Virus (WNV) Infection"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7852-2800","authenticated-orcid":false,"given":"Lachlan","family":"De Hayr","sequence":"first","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander A.","family":"Khromykh","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia"},{"name":"Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrii","family":"Slonchak","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia"},{"name":"Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD 4072, Australia"},{"name":"Systems Virology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e0294624","DOI":"10.1128\/mbio.02946-24","article-title":"Emerging mosquito-borne flaviviruses","volume":"15","author":"Nelson","year":"2024","journal-title":"mBio"},{"key":"ref_2","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. 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