Tracing the evolutionary and spatial dynamics of the 2022–2023 chikungunya outbreak in Paraguay and its regional spread across the Southern Cone

• The 2022–2023 chikungunya outbreak in Paraguay was driven by a lineage nested within Brazilian sequences. • A lineage introduced around 2021 was associated with sustained local transmission in Paraguay and showed patterns suggestive of southward spread. • The E2:V264A substitution shows no evidence of recurrent independent emergence and is compatible with early or pre-existing fixation. During 2022–2023, chikungunya virus (CHIKV) caused a major outbreak in Paraguay. Previous genomic analyses attributed the epidemic to local circulation since early 2022, but increasing cases in neighboring countries raised questions about its origin and spread. We analyzed 517 CHIKV East/Central/South African (ECSA) genomes from 2014 to 2023, including 49 generated during the 2023 outbreak in Paraguay. Time-scaled phylogenetic and discrete phylogeographic analyses were used to reconstruct viral introductions, regional dissemination, and the evolutionary dynamics of the E2:V264A substitution. The epidemic lineage circulating Paraguay was nested within Brazilian sequences. Three independent Brazil-to-Paraguay introductions were inferred between 2017 and 2022, but only the introduction around 2021 was associated with sustained local transmission and patterns consistent with southward spread into Argentina and Uruguay. Brazil-to-Paraguay was the only route with strong posterior support. The E2:V264A substitution was consistent with being present in the lineage introduced around 2021 or becoming fixed early during expansion, with no evidence of recurrent independent emergence or association with geographic dispersal. The 2022–2023 outbreak is consistent with the expansion of a pre-existing Brazilian lineage introduced prior to sustained local transmission, with phylogeographic patterns suggestive of subsequent dissemination across the Southern Cone.