Chimeric Zika Viruses Containing xrRNA1 Elements of Divergent Flaviviruses Retain the Ability to Replicate in Vertebrate and Mosquito Cells and Produce Subgenomic Flaviviral RNA

Abstract

Members of the Flaviviridae encode XRN1-resistant RNA structures (xrRNAs) within their 3′ untranslated regions (UTRs) that stall the cellular 5′→3′ exoribonuclease 1 (XRN1), resulting in the accumulation of subgenomic flavivirus RNAs (sfRNAs). Zika virus (ZIKV) contains two such elements, xrRNA1 and xrRNA2, which generate sfRNA1 and sfRNA2, respectively. A third sfRNA species (sfRNA3) has also been described. Building on evidence that sequences within the orthoflavivirus UTRs modulate host range, we replaced the xrRNA1 element in the ZIKV genome with xrRNA1 or xrRNA1-like elements from nine evolutionarily divergent flaviviruses and assessed the in vitro host ranges of the resulting chimeric viruses to determine whether xrRNA1 contributes to host specificity. We selected mosquito-borne, tick-borne, insect-specific, and vertebrate-specific orthoflaviviruses, together with representative viruses from the Pestivirus, Pegivirus, and Hepacivirus genera. All genetic exchanges yielded chimeric viruses that replicated in both vertebrate (Vero) and mosquito (C6/36) cells. Wild-type ZIKV replicated to substantially higher titers than all chimeric viruses in Vero cells but showed no consistent replication advantage in C6/36 cells. All chimeric viruses produced sfRNA1, whereas detection of sfRNA2 and sfRNA3 varied according to the substituted xrRNA1 sequence and host cell type, indicating that heterologous xrRNA1 substitution can alter downstream sfRNA production in a host cell-dependent manner. In summary, replacement of the ZIKV xrRNA1 element with evolutionarily divergent xrRNA1 or xrRNA1-like sequences did not alter in vitro host range or abolish sfRNA1 production, but substantially reduced viral fitness in vertebrate cells.