{ "abstract": "Bhanja virus (BHAV) is a tick-borne bandavirus (Family Phenuiviridae) with a broad geographic distribution and documented neuroinvasive capacity, yet its molecular biology remains poorly understood. Within the genus Bandavirus, the non-structural protein NSs functions as the primary antagonist of innate immune responses, although mechanistic details for BHAV remain unknown. We established a reverse genetics platform for BHAV by combining virion RNA sequencing with terminal untranslated region mapping, enabling generation of recombinant viruses and systematic investigation of viral determinants. Using this system, we characterized recombinant BHAV replication in mammalian and arthropod cell lines and demonstrated that interferon competence is a critical determinant of viral replication in mammalian cells. Notably, we observed substantial amino acid divergence within the NSs protein across geographically distinct BHAV isolates, despite higher conservation of other viral proteins. To investigate the functional significance of this variation, we generated recombinant viruses expressing heterologous NSs proteins from African and European isolates. Viruses expressing NSs from ibAr2709 or R1819 isolates exhibited enhanced capacity to suppress interferon-beta induction compared to the prototype IG690 strain, correlating with increased viral protein accumulation in interferon-competent cells. Mechanistic studies revealed that BHAV NSs proteins inhibit interferon induction upstream of IRF3, with ibAr2709 and R1819 NSs showing selective inhibition of TBK1 phosphorylation. However, unlike highly pathogenic bandaviruses such as severe fever with thrombocytopenia syndrome virus (SFTSV), BHAV NSs proteins exhibit comparatively weak antagonism of downstream interferon signalling. In vivo studies using interferon alpha/beta receptor-deficient mice demonstrated that NSs sequence variation influences viral replication in splenic tissue without substantially altering disease phenotype. Together, these findings establish BHAV reverse genetics tools for future investigation and reveal how naturally-occurring NSs divergence modulates innate immune antagonism and viral fitness while maintaining an overall attenuated disease phenotype. Author summary: Bhanja virus is a tick-borne virus distributed across Asia, Africa, and Europe that causes neurological disease, yet little is known about how it replicates or evades immunity. We developed the laboratory tools to create recombinant Bhanja viruses and investigate viral functions. We discovered that the virus replicates efficiently in cells lacking interferon responses but is severely restricted in normal immune-competent cells, revealing interferon as a major barrier to viral growth. We observed that Bhanja virus isolates from distinct geographic regions have different versions of a viral protein called NSs that suppresses interferon responses. Isolates from Africa and Europe were better at blocking interferon production than the original Asian strain, correlating with increased viral growth in immune-competent cells. However, unlike related viruses that cause more serious disease such as SFTSV, BHAV NSs proteins only partially antagonise interferon. While this weak antagonism likely contributes to the attenuated phenotype observed in our experimental systems, the natural pathogenesis of Bhanja virus in human hosts and tick vectors remains to be determined. This study provides tools for examining Bhanja virus biology and demonstrates that small viral differences in viral sequence can meaningfully influence evasion of innate immunity and viral fitness without triggering severe disease.", "abstract_shown": true, "affiliations": [ "Medical Research Council–University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom", "CVR Translational Hub, MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom", "Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom", "Departments of Tropical Disease Biology and Vector Biology, Liverpool School of Tropical Medicine, Centre for Neglected Tropical Diseases, Liverpool, United Kingdom" ], "article_type": "Research Article", "arxiv": null, "author_total": 12, "authors": [ { "affs": [ 0 ], "name": "Andrew T. Clarke" }, { "affs": [ 1 ], "name": "Kelsey Davies" }, { "affs": [ 2 ], "name": "Mazigh Fares" }, { "affs": [ 3 ], "name": "Agnieszka M. Szemiel" }, { "name": "Diogo Correa Mendonca" }, { "name": "Karen Kerr" }, { "name": "Vanessa Herder" }, { "name": "Lesley Bell-Sakyi" }, { "name": "Brian J. Willett" }, { "name": "Arvind H. Patel" }, { "name": "Alain Kohl" }, { "name": "Benjamin Brennan" } ], "bibtex": "https://research.draconitic.com/paper/6ac0sbc4t8yqz.bib", "cited_by": 0, "doi": "10.1371/journal.ppat.1014645", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014645", "id": "6ac0sbc4t8yqz", "issns": [ "1553-7374" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014645", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2725|Viral Infections and Vectors" ], [ "plos", "/Biology and life sciences/Biochemistry/Enzymology/Enzymes/Oxidoreductases/Luciferase" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Enzymes/Oxidoreductases/Luciferase" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Interferons" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Recombinant proteins" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Viral pathogens" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral replication" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/DNA construction/Plasmid construction" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Molecular biology assays and analysis techniques/Gene expression and vector techniques/Protein expression" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Sequencing techniques/RNA sequencing" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/Viral pathogens" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Viral pathogens" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/DNA construction/Plasmid construction" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Molecular biology assays and analysis techniques/Gene expression and vector techniques/Protein expression" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Sequencing techniques/RNA sequencing" ] ], "openalex": "W7214175892", "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014645&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-24", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/6ac0sbc4t8yqz.ris", "sources": [ { "first_seen": "2026-10-30T12:17:43Z", "native_id": "10.1371/journal.ppat.1014645", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014645" } ], "subjects": [ { "field": "biochemistry", "origin": "plos:molecular biology", "subfield": "molecular-biology" }, { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" } ], "title": "Functional divergence of the Bhanja virus NSs protein modulates interferon antagonism and viral fitness across divergent lineages", "title_text": "Functional divergence of the Bhanja virus NSs protein modulates interferon antagonism and viral fitness across divergent lineages", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:43Z", "url": "https://research.draconitic.com/paper/6ac0sbc4t8yqz", "venue": "PLOS Pathogens", "versions": [] }