Table 1.
Classification and previous reports of characterized viruses in North America. GenBank accession numbers refer to the viral reference sequences used for the assembly of sRNA profiles. The states where each virus was detected are indicated.
Table 2.
List of primers used for viral detection. Primers were designed with primer-BLAST and SnapGene.
Fig 1.
Graphical methods of metagenomics pipeline for virome profiling.
Pools of 3 female mosquitoes per location were used for RNA extraction and sRNA-seq. Raw reads underwent quality checks and were aligned to mosquito and bacterial genomes, then unmapped reads were used for contig assembly. Viral and unknown contigs were used for co-occurrence analyses and contig extension. Extended contigs were analyzed by BLAST for virus identification, and available viral reference sequences were used for virus co-occurrence analyses and sRNA profile design. Figure created in BioRender. Rasgon, J. (2025) https://BioRender.com/o2bfaiw and with RStudio (map figure; libraries ggplot2 and maps).
Fig 2.
(a) Contig classification per sample and ISV distribution. For each library, assembled contigs longer than 200 bp were analyzed by BLAST and classified according to the closest BLAST hit. (b) Heatmap shows the Logâ‚‚ RPKM values for each library, based on mapping to reference genomes of ISVs identified through metagenomics analyses.
Fig 3.
Agarose gel electrophoresis of representative RT-PCR products.
PCR products from each representative sample were gel-extracted and purified for Sanger sequencing. Primer names and expected amplicon sizes are indicated above the lanes, and sample locations of origin are shown below the lanes. A DNA ladder is included for size reference. The full set of RT-PCR screening gels, including positive controls, is provided in S3 Fig. Details for each primer set are provided in Table 2.
Fig 4.
sRNA profiles and coverage plots of the ISVs. All libraries were used to generate these graphs.
sRNA profiles are histograms representing the frequency of mapped read sizes, with colors indicating 5’ nucleotide bias. piRNA profiles are sRNA profiles filtered to include only reads with a 5’ U bias or an A at the 10th nucleotide. Coverage plots were generated using reads of either 21 nt in length, representing siRNAs, or reads of 24–29 nt, representing piRNAs. Colors in the coverage plots indicate whether the reads aligned to the sense (blue) or antisense (red) strand of the reference viral genomes.