Objective: To study the genetic diversity of Culex theileri flavivirus and the spread of this virus among Spain, Portugal and Turkey. Methods: A database consisting of 55 sequences of the NS5/3'UTR region of Culex theileri flavivirus group downloaded from GenBank were aligned and manual edited with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using MEGA7. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. Results: The phylogenetic tree showed three clusters. Myanmar sequences clusterd together with Turkish sequences, Spain and Portugal strains grouped together and two Turkish sequences grouped separately. Selective pressure analysis showed a moderate percentage of sites (22.5%) under pervasive negative selection and only 1% under pervasive positive selection. The sites subject to selective pressure in CTFV RdRp NS5 fragments have been located onto the predicted three-dimensional structure. Conclusions: Phylogenetic and evolutionary analysis can be an important tool for understanding the evolutionary impact of the probable contemporary existence between non-pathogenic and pathogenic flaviviruses among these vectors.

Objective: To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling. Methods: Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied. Results: Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity. Conclusion: Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.