Genomic Characterization and Seroprevalence Studies on Alphaviruses in Uruguay

Abstract

Alphaviruses (Togaviridae) are arboviruses frequently associated with emerging infectious diseases. In this study, we aimed to investigate the presence of alphaviruses in Uruguay by detecting the viral genome in mosquitoes and neutralizing antibodies in equines. A total of 3,575 mosquitoes were analyzed for alphavirus genome detection. Serologic studies were performed on 425 horse sera by plaque reduction neutralization test (PRNT₈₀) against Venezuelan equine encephalitis virus (VEEV) subtype IAB, Pixuna virus (PIXV), Rio Negro virus (RNV), western equine encephalitis virus (WEEV), and Madariaga virus (MADV). Mosquitoes belonging to six genera were captured and 82.9% were identified as Culex pipiens. Two Cx. pipiens pools collected in Fray Bentos and Las Toscas localities were alphavirus positive, and phylogenetic analyses showed that the sequences grouped into two different clusters: the lineage I of eastern equine encephalitis virus and RNV (VEEV complex), respectively. Plaque reduction neutralization test assays showed antibodies against strains of the VEEV complex, MADV, and WEEV. Rio Negro virus was the most geographically widespread virus, showing higher seroprevalences (up to 20%). Seroprevalences against VEEV IAB ranged between 4.6% and 13%; antibodies against PIXV, WEEV, and MADV were less frequent (3-4%). In conclusion, RNV exhibited the highest seroprevalence in horses, a wide geographical distribution, and viral genome was detected in Cx. pipiens mosquitoes. Madariaga virus had a low seroprevalence in equines, but an epizootic lineage typical of North America was detected in Cx. pipiens mosquitoes. Taken together, our results show that alphaviruses are present in Uruguay with variable occurrence and geographical distribution being a potential threat for human and equine health.

Figure 1.

Mosquito trapping sites. Mosquito traps were placed in nine of the 19 departments of Uruguay between 2006 and 2014. The map was built with the free software QMapShack v1.9.1 and edited in Microsoft PowerPoint v2016. This figure appears in color at www.ajtmh.org.

Figure 2.

Bayesian phylogenetic analysis of alphavirus detected in Uruguay. Partial nsP4 genome sequences from mosquito samples were analyzed in comparison with sequences of the VEEV and EEEV complex. The analysis was conducted under the general time reversible + gamma + proportion invariant (GTR + Γ + I) model of nucleotide substitution. Two runs of four chains each (one cold and three heated, temperature 0.20) were run for three million generations; trees were sampled every 100 generations. Convergence was assessed by using the average standard deviation in partition frequency values across independent analyses with a threshold value of 0.01; burn-in was set to 25%. Supports above nodes are the posterior probabilities and below are depicted the approximate likelihood ratio test supports from maximum likelihood analysis. GenBank accession numbers: CpFB: MG009260 and CpLT: MG009261. CABV = Cabassou virus; EEEV = eastern equine encephalitis virus; MADV = Madariaga virus; MAYV = Mayaro virus; MDPV = Mosso das Pedras virus; MUCV = Mucambo virus; PIXV = Pixuna virus; RNV = Rio Negro virus; and VEEV = Venezuelan equine encephalitis virus.