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.Venezuelan equine encephalitis virus (VEEV) is a member of the family Togaviridae in the genus Alphavirus (14, 48). The virus is enveloped and includes a nonsegmented, positive-sense RNA genome of approximately 11.5 kb. The 5' two-thirds of the genome encodes four nonstructural proteins (nsP1 to 4) that are involved in viral replication. After virus entry into the cytoplasm of cells, a nonstructural polyprotein is translated and utilized in the production of full-length negative-sense RNA. The negative-sense RNA is used for the generation of genomic RNA as well as a subgenomic mRNA (26S) that is homologous to the 3' one-third of the genome. The subgenomic RNA is translated directly into a structural polyprotein that is proteolytically cleaved into the capsid, E2, and E1 envelope glycoproteins (40).The VEEV antigenic complex of alphaviruses is comprised of six antigenic subtypes (I to VI) (5). Subtype I is comprised of five varieties: AB, C, D, E, and F. Only viruses from subtypes IAB and IC have been implicated in large outbreaks of equine and human encephalitis that have occurred from northern South America to southern Texas (43,47). These viruses cause large outbreaks by e...

Venezuelan equine encephalitis emergence: Enhanced vector infection from a single amino acid substitution in the envelope glycoprotein

“…E2 chimeras were produced by using the restriction enzymes Sse8387I and BlpI (genomic positions 8875 and 9807, respectively) as described in ref. 13. Amplification by using RT-PCR of the CPA201 genome with IE-8875(ϩ) and IE-9821(Ϫ) primers produced an amplicon that was digested and ligated into the digested pIE.AA vector, resulting in a triple-mutant clone (IE.AAE2-E117K,S218N,S407R) that included all three E2 amino acid differences, with respect to strain 68U201 (Table 3).…”

“…To determine the potential role of specific nucleotide and͞or amino acid changes in the emergence of epizootic VEEV subtype IE viruses, the infectious cDNA clone of the enzootic IE strain 68U201 (pIE.AA) (25) was used for mutagenesis to incorporate all E2 amino acid differences between the epizootic VEEV subtype IE strain CPA201 and enzootic strain 68U201 (13). The mutants produced from the 68U201 backbone included all three amino acid differences in all possible combinations (single, double, and triple mutants; Table 3).…”

“…Several strains of VEEV subtype IE were isolated from horse brains and sera and were shown to be closely related to enzootic subtype IE strains isolated in nearby coastal Guatemala from 1968 to 1980 (15). The close genetic relationship of the Mexican equine strains to the Guatemalan enzootic IE viruses suggested that the Venezuelan equine encephalitis (VEE) emergence occurred through a mechanism similar to that involving ID strains, which emerge via host range changes mediated by small numbers of mutations (2,13,16). However, unlike the typical IAB and IC epizootic strains, the subtype IE virus isolates from the Mexican equine epizootics fail to generate high equine viremia (17,18).…”

“…Before 1993, enzootic subtypes, including IE, were not known to have epizootic potential. Genetic analyses have indicated that three different epizootic VEEV subtype IAB and IC genotypes have emerged convergently via similar sets of E2 envelope glycoprotein amino acid substitutions from an enzootic lineage of subtype ID progenitor viruses (12,13).…”

Mosquitos (Diptera: Culicidae) en el caserío de Chingalé, Santander, donde se registró un caso humano de encefalitis equina venezolana

“…Las variantes epizoóticas son oportunistas en la utilización de mosquitos mamófilos para su transmisión, entre los cuales Aedes (Ochlerotatus) taeniorhynchus (Wiedemann, 1821) y Psorophora (Grabhamia) confinnis (Lynch Arribalzaga, 1891) han sido las especies más comprometidas. En cambio, para las variantes enzoóticas, los únicos vectores incriminados son mosquitos de la sección Spissipes del subgénero Culex Melanoconion (1) Por medio de los estudios genéticos se ha demostrado que las cepas epizoóticas han emergido a partir de un linaje enzoótico; el subtipo IC en dos ocasiones independientes a partir del subtipo ID, por sustituciones en los aminoácidos de la glicoproteína E2 de la cubierta del virus (8,9). Además de esto, en la epidemia de México (1993)(1994)(1995)(1996), la cual se presentó en una área completamente deforestada en los estados de Chiapas y Oaxaca, se aisló una variante epizoótica del subtipo IE del cerebro y el suero de caballos enfermos (10); antes de 1993 no se sabía que los subtipos enzoóticos tenían potencial epizoótico por sí mismos (11).…”