Positively charged amino acid substitutions in the e2 envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus

Abstract

Epidemic-epizootic Venezuelan equine encephalitis (VEE) viruses (VEEV) have emerged repeatedly via convergent evolution from enzootic predecessors. However, previous sequence analyses have failed to identify common sets of nucleotide or amino acid substitutions associated with all emergence events. During 1993 and 1996, VEEV subtype IE epizootics occurred on the Pacific Coast of the states of Chiapas and Oaxaca in southern Mexico. Like other epizootic VEEV strains, when inoculated into guinea pigs and mice, the Mexican isolates were no more virulent than closely related enzootic strains, complicating genetic studies of VEE emergence. Complete genomic sequences of 4 of the Mexican strains were determined and compared to those of closely related enzootic subtype IE isolates from Guatemala. The epizootic viruses were less than 2% different at the nucleotide sequence level, and phylogenetic relationships confirmed that the equine-virulent Mexican strains probably evolved from enzootic progenitors on the Pacific Coast of Mexico or Guatemala. Of 35 amino acids that varied among the Guatemalan and Mexican isolates, only 8 were predicted phylogenetically to have accompanied the phenotypic change. One mutation at position 117 of the E2 envelope glycoprotein, involving replacement of Glu by Lys, resulted in a small-plaque phenotype characteristic of epizootic VEEV strains. Analysis of additional E2 sequences from representative enzootic and epizootic VEEV isolates implicated similar surface charge changes in the emergence of previous South American epizootic phenotypes, indicating that E2 mutations are probably important determinants of the equine-virulent phenotype and of VEE emergence. Maximum-likelihood analysis indicated that one change at E2 position 213 has been influenced by positive selection and convergent evolution of the epizootic phenotype.

FIG. 1.

Guinea pig viremia following infection with enzootic (68U201) and epizootic (CPA201) VEE subtype IE viruses. Guinea pigs were inoculated with 1,000 PFU, and 20 μl of blood was drawn at the postinoculation time points listed. Viremia was determined by plaque assay on Vero cells with a detection limit of 1.7 log PFU/ml. Error bars indicate standard errors. ▴, CPA201; •, 68U201.

FIG. 2.

Plaque diameter analysis of enzootic and epizootic subtype IE VEEVs. All enzootic (MenaII, IE.AA [68U201], and 80U76) and epizootic (CPA201, CPA152, OAX131, and OAX142) viruses were incubated on Vero cells for 3 days under a 0.4% Noble agar overlay. Monolayers were fixed with 20% methanol stained with crystal violet, and plaque diameters (n = 15) were measured.

FIG. 3.

Maximum-parsimony tree generated from complete genomic sequences of enzootic and epizootic VEE subtype I and II viruses. Strains are designated by subtype, location (PA, Panama; GU, Guatemala; MX, Mexico; FL, Florida; TX, Texas; TR, Trinidad; VE, Venezuela; CO, Columbia), year of isolation (last two digits of year only), and strain name. All nodes in the tree have bootstrap values of 100% when nucleotide sequences are analyzed, except for the 68U201-80U76 grouping, with a bootstrap value of 54%. The table shows nucleotide and amino acid changes assigned to the branch representing the emergence of the epizootic subtype IE Mexican epizootic viruses. Trees generated using the neighbor-joining and ML methods had identical topologies.

FIG. 4.

Neighbor-joining tree generated from complete PE2 envelope glycoprotein gene nucleotide sequences of representative VEE subtype I viruses. Strains are designated by subtype, location (PA, Panama; GU, Guatemala; MX, Mexico; FL, Florida; TX, Texas; TR, Trinidad; VE, Venezuela; CO, Columbia; PE, Peru), year of isolation (last two digits of year only), and strain name. Boxes indicate amino acid substitutions in the E2 envelope glycoprotein associated with emergence of epizootic strains from predicted enzootic progenitors (internal nodes) and accompanying attenuation of the TC-83 vaccine strain from its TRD parent strain. Numbers indicate bootstrap values of adjacent nodes. The tree was rooted using an outgroup consisting of all other VEEV subtypes. Trees generated using the maximum-parsimony and ML methods had identical topologies except for some groupings within the IAB clade.