Genetic and evolutionary characterization of Venezuelan equine encephalitis virus isolates from Argentina

Venezuelan equine encephalitis viruses (VEEV) are emerging pathogens of medical and veterinary importance circulating in America. Argentina is a country free from epizootic VEEV activity, with circulation of enzootic strains belonging to Rio Negro virus (RNV; VEEV subtype VI) and Pixuna virus (PIXV, VEEV subtype IV). In this work, we aim to report the sequencing and phylogenetic analyses of all Argentinean VEE viruses, including 7 strains previously isolated from mosquitoes in 1980, 5 sequences obtained from rodents in 1991 and 11 sequences amplified from mosquitoes between 2003 and 2005. Two genomic regions, corresponding to the non-structural protein 4 (nsP4) and the protein E3/E2 (PE2) genes were analyzed, but only 8 samples could be amplified in the last one (longer and more variable fragment of 702 bp). For both genomic fragments, phylogenetic trees showed the absence of lineages within RNV group, and a close genetic relationship between Argentinean strains and the prototype strain BeAr35645 for PIXV clade. The analysis of nsP4 gene opens the possibility to propose a possible geographic clustering of strains within PIXV group (Argentina and Brazil). Coalescent analysis performed on RNV sequences suggested a common ancestor of 58.3 years (with a 95% highest posterior density [HPD] interval of 16.4-345.7) prior to 1991 and inferred a substitution rate of 9.8×10(-5)substitutions/site/year, slightly lower than other enzootic VEE viruses. These results provide, for the first time, information about genetic features and variability of all VEEVs detected in Argentina, creating a database that will be useful for future detections in our country. This is particularly important for RNV, which has indigenous circulation.