Exploring Genomic, Geographic and Virulence Interactions among Epidemic and Non-Epidemic St. Louis Encephalitis Virus (Flavivirus) Strains

Abstract

St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America. In 2005, an encephalitis outbreak caused by SLEV was reported in Argentina. The reason for the outbreak remains unknown, but may have been related to virological factors, changes in vectors populations, avian amplifying hosts, and/or environmental conditions. The main goal of this study was to characterize the complete genome of epidemic and non-epidemic SLEV strains from Argentina. Seventeen amino acid changes were detected; ten were non-conservative and located in proteins E, NS1, NS3 and NS5. Phylogenetic analysis showed two major clades based on geography: the North America and northern Central America (NAnCA) clade and the South America and southern Central America (SAsCA) clade. Interestingly, the presence of SAsCA genotype V SLEV strains in the NAnCA clade was reported in California, Florida and Texas, overlapping with known bird migration flyways. This work represents the first step in understanding the molecular mechanisms underlying virulence and biological variation among SLEV strains.

Fig 1. Amino acid differences between epidemic (Ep) and non-epidemic (NEp) SLEV strains and the polyprotein conservation profile.

The horizontal bar represents the SLEV polyprotein. (A). Black vertical lines represent residues (in one letter code) that differed between strains studied (CbaAr-4005 (Ep) and 79V-2533 (NEp)). In grey, the non-conservative changes and their respective individual protein positions are highlighted. (B). Polyprotein homology profile for all studied SLEV strains. The light pink areas show the more conserved regions while in the less conserved zones are in blue. (C). Individual protein homology profile for all studied SLEV strains. The Y axis shows the Relative Homology (RH) between 0 and 1 while the X axis shows the residue position. In each protein profile, the zones that show an RH value lower than 0.8 are boxed.

Fig 2. Predicted secondary structures for 5′UTR and 3′UTR of a NEp SLEV strain.

For both ends, the conserved sequences (grey shadowed and boxed) and structures being comparison with DENV2 are shown [16]. The conserved residues in the alignment are shadowed. (A). 5′UTR scheme, where structures of Stem Loop A (SLA), Stem Loop B (SLB), capsid hairpin (cHP), and the 5′ upstream AUG region (5′UAR), and 5′ conserved sequence (5′CS) are shown. (B). 3′UTR scheme, showing the Stem Loop (SL), Short Stem Loop (SSL), the Pseudoknot 1 and 2 (PSK1 and PSK2), Variable Region, the 3′ upstream AUG region (3′UAR) sequences, the 3′ conserved sequence (3′CS), and the repeated conserved sequences 1 and 2 (RCS1 and RCS2).

Fig 3. Correspondence analysis exploring the distribution of 200 non-conservative mutations in 14 SLEV strains through biological features.

Axes CA1 and CA2 account for 84.8% of the total variation observed. HP: high pathogenicity, LP: low pathogenicity, HV: high viremia, LV: low viremia. Source of isolation: Mqt: mosquitoes, Mml: mammals, Brd: birds. Colored dots represent each mutation analyzed distributed by protein.

Fig 4. Spearman’s correlation indices and confidence intervals obtained comparing source of isolation (Mml: mammals, Brd: birds, Mqt: mosquito) and biological behavior (viremia in birds and pathogenicity in mice)in 14 SLEV strains.

Gradient scale colors indicate strength of correlation (positive or negative). *: statistically significant differences.

Fig 5. Phylogenetic analyses performed using the SLEV sequences listed in Table 1. (A): Maximum Likelihood analysis. (B): Bayesian inference analysis. (C) Geographic distribution of analyzed St. Louis encephalitis virus strains and migratory bird flyway.

West Nile virus (Acc. Number: DQ211652), Murray Valley Encephalitis virus (Acc. Number: AF161266) and Japanese Encephalitis virus (Acc. Number: M18370.1) were used as outgroups. Abbreviations for the different geographic origin of strains were used as follow: USA (United States); MEX (Mexico); GTM (Guatemala); ARG (Argentina); BRA (Brazil); PERU (Peru); TRN (Trinidad); PAN (Panamá).