We performed serologic surveillance for selected arthropod-borne viruses (arboviruses) in bats and opossums in the Lacandona Rainforest, Chiapas, Mexico, in 2023–2024. Sera were collected from 94 bats of at least 15 species and 43 opossums of three species. The sera were assayed by the plaque reduction neutralization test (PRNT) for antibodies to eight orthoflaviviruses (dengue viruses 1–4, St. Louis encephalitis virus, T’Ho virus, West Nile virus, and Zika virus) and one alphavirus (chikungunya virus; CHIKV). Twelve (12.8%) bats and 15 (34.9%) opossums contained orthoflavivirus-specific antibodies. One bat (a Jamaican fruit bat) was seropositive for Zika virus, and 11 bats contained antibodies to an undetermined orthoflavivirus, as did the 15 opossums. All bats and most opossums seropositive for an undetermined orthoflavivirus had low PRNT titers, possibly because they had been infected with another (perhaps unrecognized) orthoflavivirus not included in the PRNTs. Antibodies that neutralized CHIKV were detected in three (7.0%) opossums and none of the bats. The three opossums had low CHIKV PRNT titers, and therefore, another alphavirus may have been responsible for the infections. In summary, we report serologic evidence of arbovirus infections in bats and opossums in Chiapas, Mexico.

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.