Table 1.
Cell lines and culture media compositions.
Table 2.
BSQV strains.
Table 3.
ORF sequence comparisons across Aroa serocomplex.
Fig 1.
Phylogenetic relationship of BSQV strains.
Maximum-likelihood tree for the Orthoflavivirus genus based on complete amino acid sequences of eighty-one species across four vector-associated clades (right-most brackets); TB, MB, NK, and IS orthoflaviviruses. Numbers in parentheses at major branch nodes are UFBoot (%); only thresholds over 80% are shown. The scale bar depicts a genetic distance of 0.5 or 50% nucleotide sequence divergence by site. MB subclades of principal vector genera (Aedes, Culex) are indicated by innermost brackets. Consensus sequences of confirmed BSQV (n = 3)(blue arrow) and NAJV (n = 1)(red arrow) isolates. Node labels depict country code/ virus name/ isolate/ year/ NCBI accession. Subclades are listed as: mosquito-borne (MB), tick-borne (TB), insect specific (IS), or no known vector (NKV). The final tree was visualized and edited in FigTree v1.4.4, with supplementary brackets, arrows, and labels (node values, vector clade) added at Biorender.com (https://BioRender.com/5ov1u7j).
Fig 2.
Ultrastructure of BSQV viruses in ultrathin sections.
(A) BeAn 217201 in Vero cells. Replication complex presented by convoluted membranes (CM) and smooth membrane structures (SMS). Bar = 1 um. (B) Fragment of Fig 1A demonstrates immature virions and SMS within the lumen of granular ER. Bar = 0.2 um. (C) BeAn 4116 in a Vero cell. Cluster of immature virions (arrow) in a lumen of expanded granular ER. Bar = 100 nm. (D) BeAn 4116 in a C6/36 cell. Individual immature virions (arrows) within an expanded cistern of granular ER. Bar = 100 nm. (E) BeAn 4073 in a Vero cell. SMS and single immature virions (arrows) within a conglomerate of granular ER cisterns. Bar = 0.2 um. (F) BeAn 4073 in a C6/36 cell. SMS within a tight conglomerate of granular ER cisterns. Bar = 0.5 um.
Fig 3.
Predicted 5’ UTR secondary structure.
Canonical orthoflavivirus RNA structural elements present in predicted folding of BSQV strains. Conformations include two stem loops (SLA, SLB) and the capsid hairpin (cHP) within the 5’ UTR. The capsid protein coding sequence (cds) from the first to second start codon (AUG) was included for software prediction. Nucleotide differences between strains are highlighted in blue. The complementary UAR, DAR, and CS (partial) are color coded to indicate pairing with 3’ UTR motifs in Fig 4. The loop free-energy decomposition (ΔG) values for structure predictions are 49.20 (BeAn 217201) and 45.80 (BeAn 4116/4073). Structure prediction originates from UNAFold Web Server and supplementary graphics added at Biorender.com (https://BioRender.com/qtzxxxr).
Fig 4.
Predicted 3’ UTR secondary structure.
Canonical orthoflavivirus RNA structural elements present in predicted folding include: two stem loops (SL-I, 3’SL), two dumbbells (DB-I, DB-II), and the short hairpin (sHP) across the three domains (DI-III). Nucleotide differences between strains are highlighted in blue. The complementary UAR, DAR, and CS are color coded to indicate pairing with 5’ UTR motifs in Fig 3. The conserved 5’CACAG’3’ pentanucleotide and RCS/CS sequences are outlined with red dashes. The loop free-energy decomposition (ΔG) values for structure predictions are 136.36 (BeAn 217201) and 132.10 (BeAn 4116/4073). Structure prediction originates from UNAFold Web Server and supplementary graphics added at Biorender.com (https://BioRender.com/2kr46w4).
Table 4.
Predicted BSQV Polyprotein Posttranslational Modifications.
Fig 5.
Consensus amino acid sequence across the three BSQV strains. The three nonstructural and seven structural proteins are distinguished by chain color. Polyprotein cleavage sites with the five preceding and five successive residues are defined, with associated proteases distinguished by triangle color. Figure adapted from Plante et al. [83] and created at Biorender.com (https://BioRender.com/jo332fm).
Fig 6.
Kinetic curves of each BSQV strain in mosquito and vertebrate cell lines. Each data point represents the daily mean of three independent replicate wells per BSQV strain per cell line. Dotted lines represent the lower limit of detection (0.09 log10 PFU/mL); data points with no detectable BSQV are represented at the lower limit of detection. BSQV strains are represented by line color, with colored error bands indicating standard error of the mean by dpi.