Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral testing in flaviviruses
Year: 2026
Type: article
Abstract: As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.
Source: Antiviral Research
Language: English
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $4,160
Funders: European Commission, University of Glasgow, National Research Foundation, Deutsche Forschungsgemeinschaft, Agence Nationale de la Recherche, Institut National de la Santé et de la Recherche Médicale, National Research Foundation of Korea, Université de Strasbourg, Centre National de la Recherche Scientifique, Ministry of Science and ICT, South Korea, HORIZON EUROPE Framework Programme, H2020 Marie Skłodowska-Curie Actions
Awards: G1443766078, G1605585572, G164444715, G1800105685, Par-delà les frontières, l'Université de Strasbourg, G2255650664, G2659019386, G2694206344, Façonner les talents en formation et en recherche à l'Université de Strasbourg, G2980570810, G3000920764, G3357951378, G3730343, G3993934777, G399734812, G4381811279, G4405525097, Integrative Molecular and Cellular Biology, G471331628, G4779185514, RNA-based immunity against flaviviruses in arthropods and mammalian cells, G4941046983, G5296751580, G6136357505, G6276897512, G6364173891, G6389594462, G6803667113, G7240651115, G8886476519
OpenAlex ID: W7206217581