Hiding in plain sight: Genomic and phenotypic characterization of mosquito-borne Bussuquara virus

Bussuquara virus (BSQV), an orthoflavivirus discovered in Brazil in 1956, has been detected throughout the Americas in diverse mosquito and vertebrate species, including humans. Critical gaps in BSQV knowledge include its capacity for urban transmission and clinical pathogenesis outcomes, with insufficient historical experimentation to draw genomic or phenotypic comparisons to related orthoflavivirus species. The objective of this study was to conduct morphologic, genomic, phylogenetic, and in vitro viral fitness characterization of BSQV using the four available historical strains. We used next generation sequencing and rapid amplification of cDNA ends to construct consensus genomes, followed by phylogenetic analysis and genome annotation to evaluate orthoflavivirus evolutionary relationships and genome characteristics. Infected mosquito (C6/36) and non-human primate (Vero CCL81) cells were imaged with transmission electron microscopy. Viral replication kinetics were quantified across seventeen cell lines of mosquito, mammal, rodent, avian, non-human primate, and human origin. BSQV morphologic (virion diameter, cytopathic effect) and genomic (size, organization, architecture, sequence motifs) results were in line with canonical orthoflavivirus characteristics. One of the four strains (CoAr 41922) shared greater sequence homology to the Naranjal orthoflavivirus than other BSQV strains and was thus excluded from infection phenotype experiments. All three confirmed BSQV strains replicated robustly in most mosquito and all vertebrate cell lines, causing either minimal (mosquito) or moderate to extreme (vertebrate) cytopathic effects. We conclude that BSQV is a generalist orthoflavivirus with a broad range of susceptible vertebrate and mosquito vectors. Our data build a foundation for pathogenesis and vector competence studies to determine the potential of BSQV to emerge into epizootic and urban transmission cycles.