Tick-borne Encephalitis Virus (TBEV) is an emerging flavivirus that causes neurological disorders including viral encephalitis of varying severity. Whilst secondary RNA structures within the 5′ untranslated regions (UTRs) of many flaviviruses determine both virus replication and pathogenic outcomes in humans, these elements have not been systematically investigated for TBEV. In this study, we investigated the role of predicted RNA secondary elements of the first 107 nucleotides (nts) of the viral genome forming the stem-loop A (SLA). Experiments were performed in replicons and infectious TBEV system. This region comprises three distinct structures: 5’ stem 0 (S0), stem-loop 1 (SL1) and stem-loop 2 (SL2). S0 was found to be essential for virus infection as mutations in the lower stem of this region significantly reduced virus replication. Point mutations in SL1 that preserved the Y-shape confirmation delayed viral RNA replication but did not abolish virus infectivity. Deletion of SL2 did not abolish infectivity but had a negligible effect on virus propagation. No correlation was observed between in vitro translation efficiency and virus infectivity, suggesting that the 5’UTR functions independently to virus translation. Together, these findings reveal distinct RNA elements within the 5′UTR that are essential for the stability and replication of viral RNA. We further identify changes in RNA folding that lead to altered TBEV infectivity and pathogenesis.
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