2.7. Phylogenetic Analysis and Genome Characterization

The partial cox1 sequence dataset generated for blood-meal source identification and the viral RdRp fragment dataset were imported into Geneious Prime [45]. Multiple sequence alignments were performed for each using the MAFFT v. 1.4.0 add-on in Geneious Prime operated under the standard ‘Pairwise/Multiple Align’ option [45,46]. The alignment included the generated sequences in the present study and reference sequences retrieved from GenBank, including CuaCua virus (CuCuV) (GenBank accession no: {"type":"entrez-nucleotide","attrs":{"text":"KX245154","term_id":"1069777798"}}KX245154) reported in Mansonia sp. in Mozambique in 2014 and other flaviviruses. Phylogenetic relationships of the identified viruses and related ISFs were inferred using the maximum likelihood approach in PhyML v. 2.2.4, under the general time reversible (GTR) model of sequence evolution [47]. Branch support was assessed over 1000 bootstrap replicates. The complete coding sequence generated from NGS was annotated in Geneious Prime using InterProScan [45,48] and compared with other mosquito-specific flavivirus genomes.