Investigating the emergence potential of Bussuquara virus, a neglected mosquito-borne orthoflavivirus from the neotropics

The orthoflavivirus Bussuquara (BSQV) was discovered in Brazil in 1956, with sporadic virus isolations or antibody detections 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. Comprehensive field and experimental studies are urgently needed to assess risk factors for emergence.

Consensus RNA sequences of all available BSQV strains were obtained through next-generation sequencing. Phylogenetic analysis and genome annotation were performed to assess orthoflavivirus evolutionary relationships and genome characteristics for each strain. BSQV-infected cells (mosquito, non-human primate) were imaged with transmission electron microscopy. Replication kinetics studies were performed with BSQV infection (MOI=0.01) across a panel of mosquito, mammal, rodent, avian, non-human primate, and human cell lines. Vector competence was assessed in peri-domestic and domestic Aedes and Culex mosquito colonies from Asia, Africa, and South and North America.

We described the initial morphologic, genomic, phylogenetic, and replication relationships of the four BSQV strains available for use. Morphologic (virion diameter, cytopathic effect) and genomic (size, organization, architecture) results were in line with canonical orthoflavivirus characteristics. One of four strains shared greater sequence homology to the related Naranjal virus. Kinetic studies demonstrated robust replications in most mosquito and all vertebrate cell lines. Moderate-to=extreme cytopathic effects were prominent in vertebrate cells, with minimal cytopathy observed in mosquito cells. Several mosquito colonies, particularly Culex, appear refractory to BSQV, while others demonstrated transmission potential with infectious virus present in saliva.

BSQV poses a threat to humans as a generalist arbovirus with a broad host range of susceptible vertebrate and mosquito vectors, with capacity for BSQV transmission across urban and rural transmission cycles.