(PDF) TITLE: Evolutionary Patterns of Eastern Equine Encephalitis Virus in North versus South America Suggest Ecological Differe

JOURNAL OF VIROLOGY, Jan. 2010, p. 1014–1025 Vol. 84, No. 2 0022-538X/10/$12.00 doi:10.1128/JVI.01586-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved. Evolutionary Patterns of Eastern Equine Encephalitis Virus in North versus South America Suggest Ecological Differences and Taxonomic Revision䌤† Nicole C. Arrigo, A. Paige Adams, and Scott C. Weaver* Department of Pathology and WHO Collaborating Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609 Received 30 July 2009/Accepted 28 October 2009 The eastern equine encephalitis (EEE) complex consists of four distinct genetic lineages: one that circulates in North America (NA EEEV) and the Caribbean and three that circulate in Central and South America (SA EEEV). Differences in their geographic, pathogenic, and epidemiologic profiles prompted evaluation of their genetic diversity and evolutionary histories. The structural polyprotein open reading frames of all available SA EEEV and recent NA EEEV isolates were sequenced and used in evolutionary and phylogenetic analyses. The nucleotide substitution rate per year for SA EEEV (1.2 ⴛ 10ⴚ4) was lower and more consistent than that for NA EEEV (2.7 ⴛ 10ⴚ4), which exhibited considerable rate variation among constituent clades. Estimates of time since divergence varied widely depending upon the sequences used, with NA and SA EEEV diverging ca. 922 to 4,856 years ago and the two main SA EEEV lineages diverging ca. 577 to 2,927 years ago. The single, monophyletic NA EEEV lineage exhibited mainly temporally associated relationships and was highly conserved throughout its geographic range. In contrast, SA EEEV comprised three divergent lineages, two consisting of highly conserved geographic groupings that completely lacked temporal associations. A phylogenetic compar- ison of SA EEEV and Venezuelan equine encephalitis viruses (VEEV) demonstrated similar genetic and evolutionary patterns, consistent with the well-documented use of mammalian reservoir hosts by VEEV. Our results emphasize the evolutionary and genetic divergences between members of the NA and SA EEEV lineages, consistent with major differences in pathogenicity and ecology, and propose that NA and SA EEEV be reclassified as distinct species in the EEE complex. Eastern equine encephalitis virus (EEEV) is an important served throughout its geographic and temporal spectra. Mul- veterinary and human pathogen belonging to one of seven tiple robust analyses have demonstrated less than 2% antigenic complexes in the Alphavirus genus, family Togaviri- nucleotide sequence divergence among NA EEEV strains dae (32). Isolated throughout the Americas, EEEV is classified isolated between 1933 and 2007 (5, 7, 64, 68, 69). An overall as the only species in the eastern equine encephalitis (EEE) temporal trend of genetic conservation is also maintained, complex (9, 10), which was originally divided into North and with newer isolates differing most from ancestral strains at Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. South American varieties based on antigenic properties (11). the base of the North American clade (7, 64). In contrast, However, additional antigenic and phylogenetic analyses have SA EEEV is highly divergent both between and among the refined its classification to include four subtypes that corre- three lineages/subtypes. Although less robust than previous spond to four major genetic lineages (I to IV) (7, 55). North NA EEEV phylogenetic analyses, those of SA EEEV show American EEEV (NA EEEV) strains and most strains from a tendency for geographic clustering of isolates rather than the Caribbean comprise subtype/lineage I, while subtypes/lin- temporal relationships (7). Differing patterns of genetic conser- eages II to IV include South and Central American EEEV (SA vation between NA and SA EEEV may be the result of differ- EEEV) strains. The EEEV genome consists of a nonseg- ences in their ecology and adaptation to different mosquito and mented, single-stranded, positive-sense RNA of approximately vertebrate hosts (65). 11.7 kb, which includes a 5⬘ cap and a 3⬘ poly(A) tail. The 5⬘ Transmission of NA EEEV occurs in an enzootic cycle in- end of the genome encodes four nonstructural proteins (nsP1 volving the ornithophilic mosquito vector Culiseta melanura to -4), while a subgenomic RNA (26S) is encoded by the 3⬘ end and passerine birds in hardwood swamp habitats (32, 43). The and ultimately produces three main structural proteins: capsid broad geographic distribution and distinctly ornithophagic be- and envelope glycoproteins E1 and E2 (46). havior of Cs. melanura result in a close relationship between Despite considerable nucleotide sequence divergence be- NA EEEV and avian vertebrate hosts, which is one proposed tween NA and SA EEEV lineages, NA EEEV is highly con- mechanism for its highly conserved genetic nature. Infected birds provide for efficient geographic dispersal and the mixing * Corresponding author. Mailing address: 301 University Blvd., De- of strains with distant origins. While genetic drift tends to have partment of Pathology, University of Texas Medical Branch, less impact on large, panmictic populations, competition and Galveston, TX 77555-0609. Phone: (409) 747-0758. Fax: (409) 747- natural selection may periodically constrain genetic diversity in 2415. E-mail:

. † Supplemental material for this article may be found at http://jvi the NA EEEV population, resulting in the antigenic and ge- .asm.org/. netic conservation observed (64, 66). Transmission of NA 䌤 Published ahead of print on 4 November 2009. EEEV by bridge vectors probably does not impact viral evo- 1014 VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1015 lution; however, it does result in sporadic outbreaks of severe MATERIALS AND METHODS disease in humans, equids, and other domestic animals, includ- Virus preparation, RNA extraction, and reverse transcription-PCR (RT- ing game birds, swine, and dogs that are considered dead-end PCR). Table 1 includes a list of all EEEV strains included in this study, which were either from our collection or kindly provided by Robert Tesh (UTMB) from hosts (22, 23, 43, 50). the World Reference Center for Emerging Viruses and Arboviruses. RNA was Although they are associated with equine disease, SA strains extracted using a QIAamp viral RNA extraction kit (Qiagen Inc., Valencia, CA), of EEEV are not clearly associated with human disease (4, 17, according to the manufacturer’s protocol. The cDNA synthesis and PCR ampli- 18, 40). This lack of human pathogenicity has limited research fication reactions were conducted simultaneously using a Titan one-tube RT- PCR kit (Roche Diagnostics Corp., Indianapolis, IN), according to the manu- to expand our epidemiologic and ecologic understanding of SA facturer’s protocol. The complete structural polyprotein ORFs of all EEEV strains. EEEV isolations from Culex (Melanoconion) spp. in strains were amplified by producing three overlapping fragments (primer se- the Spissipes section (Culex pedroi in South America and Culex quences available upon request). SA EEEV strain GU68 required the use of taeniopus in Central America) suggest that they are the pri- additional strain-specific primers to fill gaps, and random hexamer primers were used to produce cDNA, followed by PCR in two-step RT-PCRs for strains BR75, mary enzootic, and potentially epizootic, vectors (28, 33, 53, BR76, BR77, PE75, and GU68. The PCR amplifications included 35 cycles, with 58). Movement of these vectors beyond their tropical forest annealing temperatures set to 3 to 5°C below the lowest melting temperature of habitat is typically limited (29), which may influence the focal- each primer pair, and a 1-min extension step per kb of genome amplified. ity of transmission. However, these species are relatively cath- DNA extraction, purification, and sequencing. PCR amplicons were extracted using agarose gel electrophoresis and purified using the QIAquick PCR purifi- olic in their feeding behavior, which broadens the potential cation kit (Qiagen). DNA sequencing was performed using the BigDye Termi- transmission cycles used by SA EEEV. Greater vector diversity nator version 3.1 cycle sequencing kit (Roche) and an Applied Biosystems 3100 in tropical regions may also contribute to genetic diversity genetic analyzer (Foster City, CA). Independent sequencing reactions used both among the SA EEEV lineages, although vector competence the forward and reverse amplification primers (3.2 pmol) and multiple internal sequencing primers. data are limited. Genetic and phylogenetic analysis. Nucleotide sequences were aligned using The vertebrate ecology of SA EEEV is not well described, ClustalW (48) in the MacVector 9.0 software package (MacVector, Inc.). The with serological associations including wild birds, ground-dwelling final sequence alignments were manually adjusted according to the translated rodents, marsupials, and reptiles (12, 17, 31, 45, 56, 57, 58). ORF alignment. Pairwise comparisons were performed using MacVector; phy- logenetic analyses were performed with multiple methods using the PAUP* The observed genetic divergence and geographic clustering of version 4.0b10 (47) and BEAST version 1.4.7 (20) software packages, and boot- the SA EEEV phylogeny could reflect the use of ground- strap resampling was performed with 1,000 replicates (25). The heuristic search dwelling mammals as primary hosts for enzootic transmission algorithm was used in maximum parsimony (MP) analyses, and the neighbor- (43, 65). With limited mobility, these vector and vertebrate joining (NJ) distance matrix algorithm was used with Hasegawa-Kishono-Yano, 85, Kimura 3, and general time-reversible (GTR) substitution models. Maximum species may restrict the distribution of SA EEEV to geograph- likelihood (ML) analyses were performed with the heuristic search method using ically defined regions, thus limiting competition among distant the GTR plus gamma distribution plus a proportion of invariant sites strains and allowing for the independent evolution of genetic (GTR⫹G⫹I) model, as recommended by Modeltest 3.7 (35), and refined with lineages (65). Geographically delineated transmission foci may multiple iterations of parameter estimates. The resultant ML substitution model parameters were also applied to NJ analyses for additional validation and boot- also be more susceptible to the impacts of genetic drift, thus strapping. BEAST was used to implement a Bayesian Markov chain Monte Carlo constraining genetic diversity locally. Venezuelan equine en- (MCMC) method using the codon-based SRD06 nucleotide substitution model cephalitis viruses (VEEV), which also utilize Culex (Melano- (44). Further details of the Bayesian analysis are provided below. As the most conion) sp. vectors and small mammals as primary vertebrate closely related alphavirus, VEEV was used as an outgroup to root some EEEV Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. trees. hosts (15, 42, 51, 52, 59, 67), exhibit a similar genetic pattern of Coalescent analysis. The BEAST software package was used to conduct independent evolution and multiple, cocirculating subtypes in Bayesian evolutionary analyses, including phylogenetic and coalescent analyses, Central and South America (60). However, a robust compar- from data sets compiled using the BEAUti interface. BEAST analyses produce ison of the evolutionary patterns between SA EEEV and rooted phylogenetic trees that incorporate a time scale based on rates of evolu- tion estimated for each tree branch or group of related sequences. Rates of VEEV has not been conducted. evolution were independently estimated as substitutions per nucleotide site per Elucidation of patterns of enzootic transmission and dis- year (s/n/y), assuming both the relaxed and strict molecular clock models. Ap- persal of zoonotic, arboviral pathogens is critical for under- propriate single or variable rates were then used to estimate divergence times standing and predicting the risk to human health. Therefore, (i.e., time since most recent common ancestor [TMRCA]) of the EEEV complex and of individual lineages. When available, dates of isolation for each strain were we studied the evolutionary progression of the EEE complex provided to the month; otherwise, they were designated as midway through the to clarify the extent of divergence between NA and SA EEEV. calendar year. All analyses were initially run with the relaxed molecular clock Because previous analyses of SA EEEV were either limited in model using the uncorrelated lognormal distribution (UCLD) (19) to account for their geographic scope or utilized only partial, concatenated rate heterogeneity among lineages and indicate the degree to which the data fits a clock-like model of evolution. If unable to reject a clock-like evolution (as sequences, conclusions regarding the genetic relationships of measured by the UCLD standard deviation [UCLD.STDEV] and coefficient of members within and among EEEV lineages were limited. In variation parameters), the analyses were then conducted under the strict molec- addition, previous analyses utilized linear regression and were ular clock model to further refine the rate of evolution and divergence dates. based on few representatives of a single SA EEEV lineage. The Bayesian skyline coalescent model (21) was used in all strict and relaxed molecular clock analyses. The SRD06 model parameters were applied because Here we exploited contemporary techniques to sequence and they have been shown to impose a reasonable balance of prior information to fit analyze the structural protein open reading frames (ORFs) of coding nucleotide data (44). This model links first and second codon positions all available SA EEEV and additional NA EEEV isolates and but allows the third position to differ in the rate of nucleotide substitution, the phylogenetically compared SA EEEV and VEEV. Our results transition/transversion (Ti/Tv) ratio, and gamma-distributed rate heterogeneity. Convergence was monitored using the Tracer version 1.4 (http://beast.bio.ed.ac support evolutionary and ecological diversity between NA and .uk/Tracer) software program, and the MCMC algorithm was run for a number SA EEEV and suggest that NA and SA lineages be considered of generations sufficient to obtain estimated sample size (ESS) values of at least independent species in the EEE complex. 200 for each parameter in the model. At least two independent runs were 1016 ARRIGO ET AL. J. VIROL. TABLE 1. EEEV strains used in phylogenetic and coalescent analyses Date isolated GenBank Abbreviation Strain Location of isolation Sourceb Passage historyc Reference (yr or mo–yr)a accession no. VA33 Ten Broeck Virginia, USA 9–1933 Horse sm12, v1 68 U01558 MA38 M 463 Massachusetts, USA 9–1938 Human Unknown 7 AF159550 LA47 Decuir Louisiana, USA 1947 Human p1 63 U01552 LA50 Arth167 Louisiana, USA 1950 Cs. melanura gp2, ch2 7 AF159551 NJ60 New Jersey 60 New Jersey, USA 10–1959 Cs. melanura p6, sm1 63 U01554 MA77 ME77132 Massachusetts, USA 8–1977 Cs. melanura m1, C6/36-1 68 U01555 WI80 WiAn-5000 Wisconsin, USA 1980 Horse de2, sm1, v1 68 U01559 FL82 82V-2137 Florida, USA 1982 Mosquito sm1, v1 68 U01034 MS83 MS-4789 Mississippi, USA 9–1983 Human rd2, sm3 7 AF159552 MD85 215-85 Maryland, USA 9–1985 Cs. melanura BHK1 68 U01556 CT90 Williams Connecticut, USA 10–1990 Horse v1 68 U01557 MD90A 3067-90 Maryland, USA 10–1990 Cs. melanura Unpassaged 64 U01553 FL91 FL91-4679 Florida, USA 6–1991 Ae. albopictus sm1, v3, BHK2 34 AY705241 GA91 PorEEE Georgia, USA 1991 Pig Unknown 7 AF159557 TX91 VR1-7164 Texas, USA 10–1991 Horse sm1 7 AF159553 FL93-939 FL93-939 Florida, USA 5–1993 Culex spp. v1 1 EF151502 FL93-969 FL93-969 Florida, USA 5–1993 Cs. melanura v1 GU001911 FL93-1637 FL93-1637 Florida, USA 7–1993 Cx. erraticus v1 GU001912 TX95 PV5-2547 C Texas, USA 11–1995 An. crucians sm1 7 AF159555 FL96 FL96-14834 Florida, USA 8–1996 Bird v1 7 AF159556 MX97 97-1076 Mexico, USA 10–1996 Horse v1 7 AF159558 GA97 GA97 Georgia, USA 8–1997 Human v2 34 AY705240 GA01 DES189-01 Georgia, USA 7–2001 Bird v1 GU001913 TX03 TX1634 Texas, USA 7–2003 Bird v1 GU001914 MA06 MA06 Massachusetts, USA 9–2006 Seal v1 GU108612 TN08 TN08 Tennessee, USA 2008 Horse v1 GU001921 AR36 ArgLL Argentina 1936 Horse p3 GU001915 AR38 ArgB Argentina 1938 Horse p5 GU001916 BR56 BeAn-5122 Brazil 7–1956 Monkey sm2 7 AF159559 AR59 ArgM Argentina 1959 Horse p5 GU001917 TR59 24443 Trinidad 5–1959 Cx. nigripalpus sm7, BHK1, v1 GU001918 BG60 25714 Guyana 8–1960 Horse ?, sm1, v1 GU001919 BR60 BeAr 18205 Brazil 1960 Horse v1, sm1 GU001920 PA62 900188 Panama 1962 Horse sm2, v1 GU001922 BR65 BeAr 81828 Brazil 1965 Cx. taeniopus v1, sm2 GU001923 BR67 BeAr 126650 Brazil 1967 Mansonia spp. v1, sm3 GU001924 GU68 68U231 Guatemala 1968 Sentinel hamster sm1, v1 GU001925 Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. PE70 77U1104 Peru 1970 Sentinel hamster v1 GU001926 EC74 75V1496 Ecuador 1974 Culex (Melanoconion) v2, sm2, BHK1 GU001927 spp. BR75 BeAr 300851 Brazil 4–1975 Cx. taeniopus v1, sm? GU001928 PE75 75U40 Peru 4–1975 Sentinel hamster sm1, CEC1, BHK1 GU001929 VE76 El Delirio Venezuela 1976 Horse sm7 GU001930 BR76 76V25343 Brazil 3–1976 Culex (Melanoconion) sm1, BHK1, v1 GU001931 spp. BR77 77U1 Brazil 3–1977 Hamster v1 GU001932 BR78 BeAr 348998 Brazil 1978 Ae. fulvus v2, sm? GU001933 VE80 IVICPan57151 Venezuela 1980 Sentinel hamster sm1, v2 GU001934 BR83 BeAn416361 Brazil 1983 Bird v1 GU001935 PA84 903836 Panama 1984 Cx. ocossa v2 GU001936 BR85 BeAr436087 Brazil 1985 Culex spp. sm1, v1 7 AF159561 PA86 435731 Panama 1986 Horse v2 7 AF159560 CO92 C49 Colombia 10–1992 Sentinel hamster v1 GU001937 PE-0.0155-96 0.0155 Peru 8–1996 Cx. pedroi v1 28 DQ241304 PE-3.0815-96 3.0815 Peru 12–1996 Cx. pedroi v1 28 DQ241303 PE-16.0050-98 16.0050 Peru 9–1998 Cx. pedroi v3 GU001938 PE-18.0140-99 18.0140 Peru 2–1999 Cx. pedroi v3 GU001939 PE-18.0172-99 18.0172 Peru 1999 Cx. pedroi v3 GU001940 a Month of isolation provided if available. b Mosquito species listed in italics. Ae., Aedes; An., Anopheles; Cs., Culiseta; Cx., Culex. c sm, suckling mouse; v, Vero cell culture; p, unknown passage source; gp, guinea pig; ch, chicken embryo; m, mosquito; C6/36, C6/36 Aedes albopictus cell culture; dec, duck embryo cell culture; rd, human embryonal rhabdomyosarcoma cell culture; BHK, baby hamster kidney cell culture; CEC, chick embryo cell culture; ?, unknown passage source or number. VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1017 ness to VEEV (Table 2). The NA and SA EEEV lineages consistently showed 23 to 24% nucleotide and 9 to 11% amino acid sequence divergence. The SA EEEV were only slightly more conserved than the overall EEE complex, with 17 to 21% nucleotide divergence between the two main lineages (II and III) but only 3 to 5% amino acid divergence, indicating a high proportion of synonymous nucleotide changes. Greater diver- gence was observed between SA EEEV lineage IV and the other two SA lineages, particularly at the amino acid sequence level. The degree of genetic divergence within each EEEV lineage varied greatly. NA EEEV lineage I was highly conserved, with less than 3% nucleotide divergence throughout its temporal and geographic range. The independent clades comprising SA EEEV lineage II differed from one another by approximately 5% and from the basal isolate (GU68) by 11 to 12%. SA EEEV lineage III was more highly conserved, with only 4 to 5% sequence divergence among strains. Consistent with previous alphavirus intercomplex comparisons (37), all EEEV lineages, and each of their members, differed from subtype I VEEV by 41 to 43% in both nucleotides and amino acids. NA EEEV. The temporally dominated evolution and mono- phyletic nature of the NA EEEV lineage were robustly sup- ported by MP and Bayesian analyses, which placed the older isolates (1933 to 1977) at the base of clade, followed by sub- sequent divergence into 2 distinct, cocirculating groups in the 1970s (Fig. 2B). However, the use of some NJ and ML models resulted in either the placement of MD90/FL93-939 isolates FIG. 1. Map showing the geographic distribution of EEEV lineages basal to the NA lineage or the paraphyletic codivergence of I to IV. Symbols represent locations of isolation for virus strains used those isolates from the older isolates. While this arrangement in this study. supports the early cocirculation of two monophyletic groups in North America prior to 1970, low bootstrap values and the lack of basal resolution (polytomies) with these methods limited performed for each data set. While chain length varied for each analysis con- confidence in this theory. Similar inconsistencies in NA EEEV ducted, they generally consisted of 10,000,000 to 50,000,000 generations, with topology were encountered in earlier analyses (64). However, parameters sampled and logged every 1,000 generations. Maximum clade cred- ibility trees were generated (with 10% burn-in) to display median node heights the limited sequence data and lack of early sequences led to Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. using TreeAnnotator version 1.4.7 and visualized using FigTree version 1.2.2 the conclusion that NA EEEV evolves as a single lineage. Our (http://tree.bio.ed.ac.uk/software/figtree). robust MP and Bayesian phylogenies validated these previous assumptions. The basal inconsistencies we observed may re- flect the inherent limitations of various phylogenetic methods RESULTS to resolve relationships among very highly conserved sequences. Genetic and phylogenetic analyses of the EEE complex. The Although the placement of the MD90/FL93-939 group was complete structural polyprotein ORF of approximately 3.7 kb inconsistent, the divergence of the NA EEEV lineage into was sequenced for 25 SA EEEV strains and 4 NA EEEV additional monophyletic groups after 1970 was robustly sup- strains. These new sequences were combined with all homol- ported in all analyses (Fig. 2B). Previously termed group A and ogous EEEV sequences available from GenBank for a data set group B by Weaver et al. in 1994 (64), the sympatric cocircu- comprising 29 SA EEEV and 22 NA EEEV strains (Table 1; lation of these two groups was further validated by our distinct Fig. 1). The monophyletic nature of the EEE complex within phylogenetic placement of two newly sequenced group A Flor- the Alphavirus genus and the presence of four major EEEV ida 1993 strains, FL93-969 and FL93-1637, from the group B lineages were validated using all phylogenetic methods (Fig. FL93-939 strain. FL93-969 and FL93-939 were isolated from 2A). Consistent with previous findings (7), lineage I included two different mosquito species that were collected simulta- isolates from North America, lineages II and III included iso- neously from the same county (30). lates from Central and South America, and lineage IV con- A temporally dominated pattern of NA EEEV evolution was tained a single strain from Brazil. The inclusion of longer and also evident in the terminal groupings of our most recent additional sequences in our analysis further supported the sis- isolates, GA01, TX03, MA06, and TN08 (Fig. 2B). The group- ter grouping of SA EEEV lineages II and III and the polyphy- ing of all recent isolates from Georgia, Tennessee, and Florida letic nature of all three Central/South American clades. supported regional EEEV evolution, with only occasional geo- Pairwise comparisons of both nucleotide and amino acid graphic dispersal (5, 7, 64, 69). While other regional clusters sequences were used to determine the genetic relatedness (TX91/MX97/TX95, GL91/FL96, and MD85/CT90) also sup- among members of the EEE complex as well as their related- ported regionally confined transmission, their persistence ap- 1018 ARRIGO ET AL. J. VIROL. FIG. 2. Phylogenetic and coalescent analyses of EEEV isolates using Bayesian methods, with the complete structural polyprotein open reading frames. (A) Phylogenetic tree of NA and SA EEEV. Bayesian posterior probability (PP) values and maximum parsimony (MP) bootstrap values are noted for all major nodes of lineage divergence (PP/MP values). Within each SA EEEV lineage, values for PP/MP are shown only if either value is less than or equal to 0.90 (PP) or 90 (MP) for the adjacent node. Boxes represent the time since most recent common ancestor (TMRCA) in years for respective nodes, estimated using BEAST analysis. TMRCAs within the gray and white boxes were estimated with data sets including all EEEV and all SA EEEV lineages, respectively. TMRCAs within the lined box adjacent to the basal node were estimated with a data set including all SA EEEV lineages and a single representative of the NA EEEV lineage (TX03). Scale bar shows a genetic distance of 5% nucleotide Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. sequence divergence. (B) Magnified version of NA EEEV phylogeny. Values for PP/MP are shown only if either value is greater than or equal to 0.90 (PP) or 90 (MP) for the adjacent node. Asterisks indicate a polytomy in MP bootstrap analysis. Scale bar shows a genetic distance of 0.3% nucleotide sequence divergence. peared to be limited, and their topological placement generally SA EEEV. The phylogeny of SA EEEV was stable regardless followed a temporal trend. However, the basal relationship of of the methods and models used and demonstrated an evolu- a Massachusetts isolate (MA06) to the most terminal Southern tionary pattern very different from that of NA EEEV. Multiple grouping also emphasized the wide geographic dispersal and highly divergent lineages of SA EEEV have coevolved and temporal conservation of NA EEEV. continue to cocirculate in overlapping geographic regions (Fig. TABLE 2. Nucleotide and amino acid sequence divergence among EEEV and VEEVa % Sequence divergence fromb: Data set NA EEEV lineage I SA EEEV lineage II SA EEEV lineage III SA EEEV lineage IV VEEVc NA EEEV lineage I 22.8–23.9 22.5–23.5 22.7–23.0 41.1–42.2 SA EEEV lineage II 8.9–10.5 16.5–18.0 20.7–21.2 41.6–42.5 SA EEEV lineage III 8.2–9.7 3.3–4.6 19.3–19.9 41.6–43.2 SA EEEV lineage IV 10.2–11.2 7.8–8.9 6.9–7.6 41.3–42.4 VEEVc 42.1–43.0 41.6–42.8 41.3–42.2 41.3–42.2 a Values in the upper diagonal indicate nucleotide sequence divergence; values in the lower diagonal indicate amino acid sequence divergence. b All members of each EEEV lineage were compared and are represented by ranges of percent sequence divergence. c VEEV includes representatives of subtypes IAB, IC, ID, and IE. VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1019 1). A temporal trend of evolution was lacking, and multiple grouping that included Panama/Colombia/Ecuador isolates geographic clusters were evident within both of the main SA and the Mexican/Guatemala VEEV IE grouping were compa- EEEV lineages (Fig. 2A). The inclusion of longer, contiguous rable in their geographic dimensions, their collection times genomic sequences provided the robust support that had been spanned 30 to 40 years, and they maintained similar levels of lacking for previously recognized clades (7, 28), and the addi- genetic conservation at approximately 98 to 99%. The lineage tion of more recent isolates revealed newly recognized geo- II and III Peruvian and lineage III Argentinean EEEV clusters graphic groupings that also lacked a temporal association. were spatially more focal but equally conserved, which corre- Despite its limited representation, lineage II consisted of mul- sponded in geographic and collection time span to the VEEV tiple genetically divergent SA clades. Brazilian (BR65/BR67) subtype ID Venezuelan and VEEV subtype IE Mexican (MX63/ and Peruvian (PE70/PE3.0815-96/PE18.0172-99/PE18.0140-99) MX08) and Guatemalan clusters. Although isolated decades groups exhibited a high degree of localized genetic conserva- apart, the viruses within each group differed in nucleotide tion, particularly exemplified by the isolates collected in the sequences by less than 2%. Amazon basin of Peru over a span of 30 years. Although Despite the well-established role of rodent hosts with limited lineage III was more highly conserved overall, it was more mobility in the transmission of enzootic VEEV (2, 62), exam- extensive in its geographic scope and contained numerous geo- ples of closely related viruses with distant geographic origin graphically based groupings. One such northern South/Central were also observed in the VEEV phylogeny (e.g., VEEV sub- American cluster included isolates from Panama, Colombia, type ID PA61/PE98). Although fewer sequences are available and Ecuador, with a collection time span from 1962 to 1992. for other subtypes of the VEE complex, the recent phylogeny Argentinean isolates collected from 1936 to 1959 also formed (6) of VEE complex subtype IIIA (Mucambo virus) generally a robust grouping on the most terminal branches of the lin- agreed with those observed with SA EEEV and VEEV sub- eage, further emphasizing the lack of widespread EEEV dis- types ID and IE. persal in SA. Finally, a Peruvian clade (PE75/PE16.0050-98/ Rates of EEEV evolution. The rates of evolution of the EEE PE0.0155-96) similar to that in lineage II further supported the complex, NA EEEV lineage I, and SA EEEV lineages II to genetic conservation among isolates from the same geographic IV were independently analyzed under the relaxed molecular area over the same period of time. Most interesting was the clock model of evolution (Table 3). We observed a high degree apparent cocirculation and persistence of subtypes II and III of rate heterogeneity in all 3 data sets, which signified that for multiple decades. these data sets were best modeled with the relaxed molecular Interestingly, some of the highly conserved geographic SA clock; therefore, the use of a strict molecular clock model of EEEV clades were closely related to geographically distant evolution was rejected. Mean substitution rates (UCLD.mean) isolates. For example, the Peruvian isolates of lineage II grouped were 2.1 ⫻ 10⫺4 s/n/y for the entire EEE complex, 2.7 ⫻ 10⫺4 with a distant Brazilian isolate (BR56), and those from Argen- s/n/y for the NA EEEV lineage, and 1.2 ⫻ 10⫺4 s/n/y for SA tina consistently grouped with BR83 in lineage III. While long- EEEV lineages II to IV. term geographic groupings could indicate maintenance by ver- Branch rate variation within the SA EEEV data set was not tebrate hosts with limited mobility, these distant relationships surprising because it included diverse SA EEEV lineages. could represent historical introductions, perhaps via alterna- Therefore, lineages II and III were individually analyzed using tive vector or vertebrate hosts. Sampling bias is also inherent in the relaxed clock model to determine the degree of intraclade Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. these analyses, as the majority of SA EEEV isolates originated variation. UCLD.STDEV parameter estimates abutting zero from equine epizootics, structured arbovirus surveillance, or indicated that a strict molecular clock could not be rejected for focused scientific research studies. EEEV circulation in sparsely SA EEEV lineages II and III. A strict clock model applied to inhabited tropical regions may go undetected, resulting in an the analysis of each lineage (Table 4) yielded a median substi- incomplete representation of the SA EEEV phylogeny. tution rate (clock rate) of lineage II (1.5 ⫻ 10⫺4 s/n/y) that was VEEV phylogenetic comparison. Because VEEV ecology, approximately 1.5 times higher than that of lineage III (1.0 ⫻ and especially reservoir host use, is better understood in SA, 10⫺4 s/n/y). Both the strict and relaxed clock models yielded the phylogenetic patterns of enzootic VEEV subtypes ID and similar rates of nucleotide substitution for each SA EEEV IE were compared to those of SA EEEV. VEEV subtypes IAB lineage, further supporting the robustness of the groupings and IC utilize fundamentally different epizootic cycles of lim- within these lineages and their clock-like evolution (20). ited duration and were therefore not considered. To provide an There was considerable rate variation estimated with the accurate comparison of the topologies and scales of divergence, relaxed molecular clock model analysis of NA EEEV; there- the phylogeny in Fig. 3 was generated using the structural fore, clades were analyzed individually via the relaxed and polyprotein ORFs of both VEE and EEE complex viruses (see strict clock models. Based on NA EEEV phylogenetic analyses Table S1 in the supplemental material). Representative mem- conducted in this study and those of previous studies (64), the bers of all VEE subtypes and two NA EEEV representatives individual groups analyzed consisted of the following: (i) all (VA33 and MA06) were included in the tree for context and to strains isolated prior to 1977, designated “pre-1977”; (ii) all provide an accurate topology of the VEE and EEE complexes. strains isolated after 1977, designated “post-1977”; and (iii) Similar evolutionary patterns were observed between SA post-1977 strains minus MD90 and FL93-939, termed “group EEEV and VEEV subtypes ID and IE, which overlap both B,” which corresponds with that of Weaver et al. in 1994 (64). geographically and temporally. Many geographic clusters of Because only 2 isolates from group A were included in the SA EEEV and VEEV subtype ID/IE isolates were analogous present study, substitution rates were not estimated for these in their spatial and temporal scales and their degree of genetic isolates. conservation (Fig. 3). For example, the SA EEEV lineage III The pre-1977 group was unable to efficiently reach conver- 1020 ARRIGO ET AL. J. VIROL. Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. FIG. 3. Phylogenetic tree including SA EEEV and VEEV generated with Bayesian methods using the complete structural polyprotein open reading frames. Geographic clusters with less than 2% nucleotide sequence divergence are shaded in gray for comparison between SA EEEV and VEEV. The brackets and captions refer to the strains included in each geographic cluster and their collection time span. NA EEEV is represented by two isolates to denote the phylogenetic placement and temporal span of lineage I. Representatives of all VEE subtypes are included to provide an accurate topology of the VEE complex. Numbers refer to percent nucleotide identity among members of clades defined by the adjacent node. VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1021 TABLE 3. Summary of coalescent analysis parameters estimated using the relaxed molecular clock modela Nucleotide substitution rateb (⫻10⫺4) Intraclade rate variationc Molecular clock Data set Median Lower 95% HDP Upper 95% HDP Median Lower 95% HDPd Upper 95% HDP model fit EEE complex 2.1 1.7 2.6 0.4 0.3 0.5 Relaxed NA EEEV lineage I 2.7 1.9 3.7 0.6 0.3 0.8 Relaxed SA EEEV lineages II-IV 1.2 0.6 1.8 0.2 0.1 0.4 Relaxed SA EEEV lineage II 1.8 0.2 4.6 0.2 0.0 0.6 Cannot reject strict SA EEEV lineage III 1.1 0.5 1.8 0.2 0.0 0.4 Cannot reject strict a Parameters estimated using the uncorrelated lognormal relaxed molecular clock (UCLD) model in BEAST. HPD, highest posterior density intervals. b Nucleotide substitution rate is measured by the UCLD.mean parameter and is the mean result of the branch substitution rates, with units of substitution/nucleotide site/year. c Measured by the UCLD.STDEV parameter, which is used to determine if the data set rejects or cannot reject a strict molecular clock. d Lower 95% HPD values for UCLD.STDEV parameter abutting zero indicate that data cannot reject a strict molecular clock; if not abutting zero, a relaxed clock model is most appropriate. Values of zero reflect rounding to the nearest tenth. gence for all parameters using the relaxed clock model, sug- SA EEEV lineages 1,307 (868 to 1,794) years ago (701 AD), gesting a poor fit of this model to the data. Alternatively, followed by the divergence of lineages II and III 878 (577 to convergence was quickly reached using the strict clock model, 1,239) years ago (1129 AD). However, the relaxed model anal- with a median substitution rate estimate of 9.4 ⫻ 10⫺5 s/n/y. ysis that included only SA EEEV lineages produced much The post-1977 and group B data sets ultimately reached con- earlier TMRCA estimates of 2,166 (1,057 to 4,020) years since vergence using the relaxed clock model; however, the strict the divergence of lineage IV (158 BC) and 1,617 (836 to 2,926) clock model resulted in more efficient convergence and substi- years since the divergence of lineages II and III (391 AD). An tution rates similar to those with the relaxed model. The evo- additional analysis including all SA EEEV lineages and a sin- lutionary rate estimate of 2.2 ⫻ 10⫺4 s/n/y in the post-1977 gle representative of the predominate NA EEEV clade (TX03) isolates was more than twice that of the pre-1977 group, sup- was performed in order to generate a TMRCA for the basal porting previous observations of an increase in evolutionary divergence of NA and SA EEEV that corresponded to those of rate following the divergence of NA EEEV into two distinct, the SA EEEV analysis. This analysis resulted in TMRCAs for cocirculating clades in the 1970s (64). Differentially higher all internal nodes that were similar to those generated by the passage histories in these two groups may have slightly im- SA EEEV strains only. In addition, the estimate for NA and pacted these estimated evolutionary rates. However, many of SA EEEV divergence was much earlier, 2,866 years (1,689 to the oldest EEEV isolates had very low passage histories (e.g., 4,856 years; ca. 878 BC), than that generated from the entire 1 to 4 for LA47 and LA50), and extensive passage of EEEV is EEE complex data set. Although the confidence intervals broadly accompanied by relatively few mutations (61), suggesting that overlapped, these wide differences in TMRCAs and the cor- any effect on evolutionary rate estimates was minimal. Inter- responding dates of divergence highlight the variation ob- estingly, the rate for group B isolates (1.8 ⫻ 10⫺4 s/n/y) was tained with the different models and data sets used for coales- lower than that of the post-1977 group, i.e., when MD90 and cent analyses and the imprecision of the estimates based on Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. FL93-939 (group A) were removed, which implies that MD90 rate variation among virus lineages. and FL93-939 evolved at a higher rate than those isolates in group B. DISCUSSION Times of divergence. The times since most recent common ancestor (TMRCAs) were estimated using the model that best Geographic, pathogenic, and epidemiologic differences be- fit the corresponding data (Fig. 2A). Using the relaxed model tween NA and SA EEEV have prompted exploration of their and the entire EEE complex, NA and SA EEEV last shared a genetic diversity and evolutionary history. However, a lack of common ancestor 1,598 (922 to 2,370) years ago, or around the corresponding sequence data had previously limited a robust year 410 AD of the Gregorian calendar. The same analysis comparison. By expanding the length and number of available estimated a divergence of lineage IV (BR85) from the other EEEV sequences, we produced an equal platform upon which to compare and contrast the evolutionary patterns of NA and SA EEEV and to compare SA EEEV to the closely related TABLE 4. Rates of nucleotide substitution estimated using the VEEV. Our results emphasized the differences between NA strict molecular clock model and SA EEEV and provided insights into the extent that this Nucleotide substitution rate (⫻10⫺4)a divergence likely reflects extant transmission dynamics. Data set EEEV evolution. To explore the evolutionary history of the Lower 95% Upper 95% Median EEE complex, a Bayesian coalescent analysis was performed. HDP HDP Depending upon the data set used, median estimates of when NA EEEV pre-1977 0.9 0.4 1.6 NA and SA EEEV last shared a common ancestor were ap- NA EEEV post-1977 2.2 1.5 2.9 NA EEEV group B 1.8 1.1 2.6 proximately 1,600 and 2,300 years ago, with ranges stretching SA EEEV lineage II 1.5 0.5 2.5 much earlier than previously estimated. Data dominated by SA SA EEEV lineage III 1.0 0.6 1.5 EEEV produced an earlier range of TMRCAs (1,689 to 4,856 a Measured by the clock rate parameter, with units of substitutions/nucleotide years ago) due to the slower evolutionary rate estimated for site/year. these lineages (1.2 ⫻ 10⫺4 s/n/y), while those dominated by the 1022 ARRIGO ET AL. J. VIROL. entire EEE complex or just the NA EEEV yielded more recent mine its molecular epidemiologic patterns. Similar patterns are TMRCAs (922 to 2,370 years ago) based on their higher evo- observed with other New World alphaviruses, e.g., western lutionary rate estimates (2.1 ⫻ 10⫺4 and 2.7 ⫻ 10⫺4 s/n/y, equine encephalitis virus (WEEV), which also uses avian ver- respectively). While it is unclear why analysis of the entire EEE tebrate hosts throughout its North and South American trans- complex was influenced more by NA EEEV than by SA mission range (27, 38, 45), and Highlands J virus that circulates EEEV, the variation in evolutionary rates among EEEV lin- in eastern North America in a manner indistinguishable from eages limits the precision of estimates for divergence events. EEEV (13). Alternatively, arboviruses that utilize less mobile The stability and uniformity of the slower evolutionary rates of mammalian hosts tend to share a molecular epidemiologic pat- the SA EEEV lineages, as well as their concordance with tern more similar to that observed for SA EEEV. Ground-dwell- estimates of other alphaviruses (66), support the earlier esti- ing mammals, such as rodents and marsupials, lack the ability mates of key divergence events. to physically disperse acutely infecting viruses. Theoretically, The consistency observed in SA EEEV evolutionary rates this limited host and virus mobility leads to geographically suggests long-term adaptation to its ecology and stability in its defined transmission foci with independent evolution. environment. Nonsynonymous (dN)-to-synonymous (dS) mu- As the closest relative to EEEV, VEEV circulates sympat- tation ratios (data not shown) in SA EEEV lineages II and III rically with SA EEEV and provides a prototypical example of suggested similar degrees of purifying selection. This may in- the evolutionary pattern generated by an arbovirus that relies dicate that EEEV has reached a high level of fitness for cir- primarily on terrestrial mammalian vertebrate hosts for its culation in South and Central America, thus stabilizing its enzootic maintenance. A comparison between SA EEEV and evolutionary rates. Although still dominated by purifying se- VEEV subtypes ID and IE revealed similar patterns of genetic lection, higher dN/dS ratios were observed for NA EEEV, with divergence characterized by the evolution of multiple subtypes that of the pre-1977 group exceeding that of the post-1977 and lineages and highly conserved geographic groupings that group. This pattern is consistent with progressive adaption of lack temporal clustering. Comparable to those observed with EEEV to its transmission cycle in North America, possibly VEEV subtypes ID/IE, the geographic scales defining SA EEEV reflecting its relatively recent introduction or anthropogenic clusters are highly focal, on the order of a few hundred miles changes in it habitat. However, a decline in the dN/dS ratios or less. This pattern suggests a mode of transmission that limits was also associated with increasing evolutionary rates, suggest- dispersal of EEEV in SA and is consistent with the use of ing that positive selection is an unlikely driving force behind mammalian vertebrate hosts as reservoirs and amplifiers. In this rate change. contrast, NA EEEV demonstrates a similar degree of genetic An alternative explanation for the apparent increase in the conservation over its entire geographic range, up to thousands EEEV evolutionary rate in North America is genetic drift. of miles, which is consistent with wide dispersal of the virus by Recent studies have focused on NA EEEV transmission in the avian hosts. northeastern United States and provide evidence for episodic Although VEEV and SA EEEV overlap in their range of overwintering, regionally independent evolution, and epizootic transmission and share similar evolutionary profiles, their de- clustering (5, 69). While the precise mechanisms are unclear, gree of ecological similarity is unknown. Members of the Culex viral overwintering in temperate regions could impose focal (Melanoconion) subgenus have been implicated as the primary bottlenecks, and surviving populations may be more subject vectors of both enzootic VEEV (15, 42, 51, 52, 59, 67) and SA Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. to rapid genetic drift and seasonal competition with southern EEEV (28, 33, 53, 58) in Central and South America. While strains reintroduced from areas of continuous transmission. these mosquitoes are known to feed on a variety of vertebrates, In addition, recent work suggests that, in some areas, NA a primary vertebrate host(s) for SA EEEV has not yet been EEEV transmission may deviate from the typical avian-mos- identified. Field isolations, seroprevalence among wild birds, quito enzootic cycle to involve ectothermic hosts, such as rep- rodents, marsupials, and reptiles, and experimental data (N. C. tiles and amphibians, and herpetophilic mosquito vectors (14, Arrigo, unpublished data) indicate that both mammalian and 16). Changes in vector and host ecology in these southeastern avian species are susceptible to infection (12, 17, 31, 45, 56, 57, foci could impact the spatial and temporal transmission pat- 58); however, their involvement in maintaining enzootic trans- terns by affecting virus dispersal and reducing virus popula- mission of SA EEEV is unclear. Additional ecological and tions, thereby providing additional opportunities for founder experimental data are needed to implicate a particular type of effects and genetic drift. Because these dynamics could con- vertebrate host responsible for the maintenance of SA EEEV. tribute to variability in EEEV evolutionary rates, it may be Systematics of EEEV. In the early 1980s, the classifications important to monitor the evolutionary progression of NA of numerous arboviruses, including EEEV, were proposed EEEV when considering predictive factors of epizootic/epi- based solely on their antigenic properties (10). Different vi- demic emergence and adaptation to new environments. ruses were delineated by a fourfold or greater difference in Implications for understanding EEEV ecology. The dichot- antibody cross-reactivity in both directions, i.e., the heterolo- omy between NA and SA EEEV was further underscored by gous versus homologous antibody titers of sera from 2 viruses. their distinct genetic and phylogenetic patterns. The highly con- A fourfold or greater difference in only one direction desig- served, monophyletic, and temporally dominated relationships nated a subtype, while antigenic varieties were distinguishable among strains of NA EEEV starkly contrast with the highly only with special serological tests (e.g., kinetic hemagglutina- divergent, polyphyletic, cocirculating, and geographically asso- tion inhibition). According to this definition, all EEEV strains ciated relationships among SA EEEV strains. The mainte- were originally classified as a single virus consisting of two anti- nance of NA EEEV by highly mobile avian hosts, with their genic varieties, NA and SA (11). Later, cross-neutralization ability to widely disperse the virus, is hypothesized to deter- testing with representatives from each phylogenetically identi- VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1023 fied EEEV lineage divided EEEV into 4 antigenic subtypes, de- virus species, given their distinct geographic, epidemiologic, spite some relationships with greater than fourfold differences in ecologic, pathogenic, genetic, phylogenetic, and evolutionary cross-reactivity in both directions (7). characteristics. This revision, based on polythetic criteria, would The International Committee on Taxonomy of Viruses (ICTV) provide a more medically and scientifically accurate represen- has more recently revised the definition of a virus species to be tation of the viruses comprising the EEE complex. Reclassifi- a “polythetic class of viruses that constitute a replicating lin- cation of individual SA EEEV subtypes is not warranted based eage and occupy a particular ecological niche” (24, 54). This solely on genetic differences, as the lack of information on definition incorporates the notion of multiple characteristics potential ecologic differences within South America precludes defining a virus species, including but not limited to genetic the evaluation of polythetic criteria. Because NA EEEV strains and phylogenetic relationships, geographic distribution, differ- are considered the prototypes, we propose a revision of all SA ences in ecology and transmission cycles, pathogenicity, mor- EEEV strains to a new species called Madariaga virus phology, replication patterns, and antigenicity. Genetic diver- (MADV), based on the location of the earliest strain isolated sity resulting in distinct phylogenetic lineages can often reflect in 1930 from General Madariaga Partido, Buenos Aires Prov- differences in ecological niche and evolutionary history; there- ince, Argentina (39, 41). fore, they often dominate the current classification of novel virus species. For example, the newly discovered Lujo virus ACKNOWLEDGMENTS (family Arenaviridae) (8) and Bundibugyo ebolavirus (family We thank Robert Tesh and Hilda Guzman of the World Reference Filoviridae) (49) were designated novel species primarily based Center for Emerging Viruses and Arboviruses (UTMB) for providing on their nucleotide sequence divergence of at least 21.5% and many of the EEEV isolates used in this study and Sara M. Volk for 32%, respectively, which also corresponded to unique geo- valuable theoretical and technical expertise in the evolutionary graphic isolation and pathogenic properties. analysis. N.C.A. was supported by the TO1/CCT622892 Fellowship Training The ability to analyze genetic relationships has also led to Grant in Vector-Borne Infectious Diseases from the Centers for Dis- the reconsideration of established Alphavirus taxonomy, result- ease Control and Prevention and by Biodefense Training Program ing in recommendations that have subsequently been accepted NIH T32 training grant AI-060549. A.P.A. was supported by the James by the ICTV. Tonate virus was designated a species unique W. McLaughlin Fellowship Fund. This work was supported by the from Mucambo virus within subtype III of the VEE complex John S. Dunn Research Foundation and NIH grant U54 AI-057156 from the National Institute of Allergy and Infectious Diseases to based on 16% nucleotide and 7% amino acid sequence diver- S.C.W. through the Western Regional Center of Excellence for Bio- gence as well as antigenic differences and the use of different defense and Emerging Infectious Diseases Research. reservoir hosts (37). The distinction of the Mayaro virus and Una virus species was also supported by recent molecular ep- REFERENCES idemiological studies, despite their previous conspecific desig- 1. Aguilar, P. V., A. P. Adams, E. Wang, W. Kang, A. Carrara, M. Anishchenko, I. Frolov, and S. C. Weaver. 2008. Structural and nonstructural protein nations based on antigenic relationships (36). These viruses genome regions of eastern equine encephalitis virus are determinants of exhibit 55% nucleotide sequence divergence, and their phylo- interferon sensitivity and murine virulence. J. Virol. 82:4920–4930. genetic patterns also suggest differences in the use of reservoir 2. Aguilar, P. V., I. P. Greene, L. L. Coffey, G. Medina, A. C. Moncayo, M. Anishchenko, G. V. Ludwig, M. J. Turell, M. L. O’Guinn, J. Lee, R. B. Tesh, hosts and the occupation of distinct ecological niches. With up D. M. Watts, K. L. Russell, C. Hice, S. Yanoviak, A. C. Morrison, T. A. Klein, to 24% nucleotide and 11% amino acid sequence divergence D. J. Dohm, H. Guzman, A. P. Travassos da Rosa, C. Guevara, T. Kochel, J. Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. between lineages of NA and SA EEEV, the genetic and phy- Olson, C. Cabezas, and S. C. Weaver. 2004. Endemic Venezuelan equine encephalitis in northern Peru. Emerg. Infect. Dis. 10:880–888. logenetic diversities observed in our study were consistent with 3. Aguilar, P. V., S. Paessler, A. S. Carrara, S. Baron, J. Poast, E. Wang, A. C. the examples described above and with the 21% nucleotide Moncayo, M. Anishchenko, D. Watts, R. B. Tesh, and S. C. Weaver. 2005. Variation in interferon sensitivity and induction among strains of eastern and 8% amino acid sequence divergence generally observed equine encephalitis virus. J. Virol. 79:11300–11310. among different Alphavirus species of the same antigenic com- 4. Aguilar, P. V., R. M. Robich, M. J. Turell, M. L. O’Guinn, T. A. Klein, A. plex (37). Huaman, C. Guevara, Z. Rios, R. B. Tesh, D. M. Watts, J. Olson, and S. C. Weaver. 2007. Endemic eastern equine encephalitis in the Amazon region of The current ICTV species definition encompasses several Peru. Am. J. Trop. Med. Hyg. 76:293–298. characteristics that are applicable to public health programs 5. Armstrong, P. M., T. G. Andreadis, J. F. Anderson, J. W. Stull, and C. N. aimed at prevention and intervention. Perceptions of EEEV Mores. 2008. Tracking eastern equine encephalitis virus perpetuation in the northeastern United States by phylogenetic analysis. Am. J. Trop. Med. Hyg. often focus on NA strain characteristics, namely, the avian- 79:291–296. mosquito transmission cycle, geographic range, highly patho- 6. Auguste, A. J., S. M. Volk, N. C. Arrigo, R. Martinez, V. Ramkissoon, A. P. Adams, N. N. Thompson, A. A. Adesiyun, D. D. Chadee, J. E. Foster, A. P. A. genic nature resulting in severe human and equine encephali- Travassos da Rosa, R. B. Tesh, S. C. Weaver, and C. V. F. Carrington. 2009. tis, and highly conserved genetic nature. However, the distinct Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine characteristics of SA EEEV are not reflected by this depiction. encephalitis complex subtype IIIA) in Trinidad. Virology 392:123–130. 7. Brault, A. C., A. M. Powers, C. L. Chavez, R. N. Lopez, M. F. Cachon, L. F. Importantly, unlike NA EEEV, SA EEEV has little to no Gutierrez, W. Kang, R. B. Tesh, R. E. Shope, and S. C. Weaver. 1999. association with human disease, despite evidence of human Genetic and antigenic diversity among eastern equine encephalitis viruses exposure in areas of endemic and epizootic activity (4, 17, 18, from North, Central, and South America. Am. J. Trop. Med. Hyg. 61:579– 586. 40). Differential replication in lymphoid tissues of mice and 8. Briese, T., J. T. Paweska, L. K. McMullan, S. K. Hutchison, C. Street, G. differences in interferon induction and sensitivity (3, 26) may Palacios, M. L. Khristova, J. Weyer, R. Swanepoel, M. Egholm, S. T. Nichol, and W. I. Lipkin. 2009. Genetic detection and characterization of Lujo virus, contribute to the observed attenuation of SA EEEV, further a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS distinguishing it pathogenically from NA EEEV. Pathog. 5:e1000455. Considering the goal of classification as a means to facilitate 9. Calisher, C. H., and N. Karabatsos. 1988. Arbovirus serogroups: definition and geographic distribution, p. 19–57. In T. P. Monath (ed.), The arbovi- the understanding of a virus taxon from multiple perspectives, ruses: epidemiology and ecology, vol. I. CRC Press, Boca Raton, FL. we recommend designating NA and SA EEEV as separate 10. Calisher, C. H., R. E. Shope, W. Brandt, J. Casals, N. Karabatsos, F. A. 1024 ARRIGO ET AL. J. VIROL. Murphy, R. B. Tesh, and M. E. Wiebe. 1980. Proposed antigenic classifica- 37. Powers, A. M., A. C. Brault, Y. Shirako, E. G. Strauss, W. Kang, J. H. tion of registered arboviruses I. Togaviridae, Alphavirus. Intervirology 14: Strauss, and S. C. Weaver. 2001. Evolutionary relationships and systematics 229–232. of the alphaviruses. J. Virol. 75:10118–10131. 11. Casals, J. 1964. Antigenic variants of eastern equine encephalitis virus. J. 38. Reisen, W. K., and T. P. Monath. 1988. Western equine encephalomyelitis, Exp. Med. 119:547–565. p. 89–137. In T. P. Monath (ed.), The arboviruses: epidemiology and ecology, 12. Causey, O. R., R. E. Shope, P. Sutmoller, and H. Laemmert. 1962. Epizootic vol. V. CRC Press, Boca Raton, FL. eastern equine encephalitis in the Bratanca region of Para, Brazil. Rev. 39. Sabattini, M. S., G. Aviles, and T. P. Monath. 1998. Historical, epidemio- Servicio Especial Saude Publica 12:39–45. logical, and ecological aspects of arboviruses in Argentina: Togaviridae, 13. Cilnis, M. J., W. Kang, and S. C. Weaver. 1996. Genetic conservation of Alphavirus, p. 140. In A. P. A. Travassos da Rosa, P. F. C. Vasconcelos, and Highlands J. viruses. Virology 218:343–351. J. F. S. Travassos da Rosa (ed.), An overview of arbovirology in Brazil and 14. Cupp, E. W., K. Klingler, H. K. Hassan, L. M. Viguers, and T. R. Unnasch. neighbouring countries. Instituto Evandro Chagas, Belem, Brazil. 2003. Transmission of eastern equine encephalomyelitis virus in central 40. Sabattini, M. S., J. F. Daffner, T. P. Monath, T. I. Bianchi, C. B. Cropp, C. J. Alabama. Am. J. Trop. Med. Hyg. 68:495–500. Mitchell, and G. Aviles. 1991. Localized eastern equine encephalitis in San- 15. Cupp, E. W., W. F. Scherer, and J. V. Ordonez. 1979. Transmission of tiago del Estero Province, Argentina, without human infection. Medicina Venezuelan encephalitis virus by naturally infected Culex (Melanoconion) 51:3–8. opisthopus. Am. J. Trop. Med. Hyg. 28:1060–1063. 41. Sabattini, M. S., T. P. Monath, C. J. Mitchell, J. F. Daffner, G. S. Bowen, R. 16. Cupp, E. W., D. Zhang, X. Yue, M. S. Cupp, C. Guyer, T. R. Sprenger, and Pauli, and M. S. Contigiani. 1985. Arbovirus investigations in Argentina, T. R. Unnasch. 2004. Identification of reptilian and amphibian blood meals 1977–1980. I. Historical aspects and description of study sites. Am. J. Trop. from mosquitoes in an eastern equine encephalomyelitis virus focus in cen- Med. Hyg. 34:937–944. tral Alabama. Am. J. Trop. Med. Hyg. 71:272–276. 42. Scherer, W. F., S. C. Weaver, C. A. Taylor, E. W. Cupp, R. W. Dickerman, 17. de Souza Lopes, O., and L. de Abreu Sacchetta. 1974. Epidemiological and H. H. Rubino. 1987. Vector competence of Culex (Melanoconion) studies on Eastern equine encephalitis virus in Sao Paulo, Brazil. Rev. Inst. taeniopus for allopatric and epizootic Venezuelan equine encephalomyelitis Med. Trop. Sao Paulo 16:253–258. viruses. Am. J. Trop. Med. Hyg. 36:194–197. 18. Dietz, W. H., Jr., P. Galindo, and K. M. Johnson. 1980. Eastern equine 43. Scott, T. W., and S. C. Weaver. 1989. Eastern equine encephalomyelitis virus: encephalomyelitis in Panama: the epidemiology of the 1973 epizootic. Am. J. epidemiology and evolution of mosquito transmission. Adv. Virus Res. 37: Trop. Med. Hyg. 29:133–140. 277–328. 19. Drummond, A. J., S. Y. Ho, M. J. Phillips, and A. Rambaut. 2006. Relaxed 44. Shapiro, B., A. Rambaut, and A. J. Drummond. 2006. Choosing appropriate phylogenetics and dating with confidence. PLoS Biol. 4:e88. substitution models for the phylogenetic analysis of protein-coding se- 20. Drummond, A. J., and A. Rambaut. 2007. BEAST: Bayesian evolutionary quences. Mol. Biol. Evol. 23:7–9. analysis by sampling trees. BMC Evol. Biol. 7:214. 45. Shope, R. E., A. H. D. Andrade, G. Bensabath, O. R. Causey, and P. S. 21. Drummond, A. J., A. Rambaut, B. Shapiro, and O. G. Pybus. 2005. Bayesian Humphrey. 1966. The epidemiology of EEE WEE, SLE and Turlock viruses, coalescent inference of past population dynamics from molecular sequences. with special reference to birds, in a tropical rain forest near Belem, Brazil. Mol. Biol. Evol. 22:1185–1192. Am. J. Epidemiol. 84:467–477. 22. Elvinger, F., A. D. Liggett, K. N. Tang, L. R. Harrison, J. R. Cole, Jr., C. A. 46. Strauss, E. G., and J. H. Strauss. 1986. Structure and replication of the Baldwin, and W. B. Nessmith. 1994. Eastern equine encephalomyelitis virus alphavirus genome, p. 35–90. In S. Schlesinger and M. Schlesinger (ed.), The infection in swine. J. Am. Vet. Med. Assoc. 205:1014–1016. togaviruses and flaviviruses. Plenum Press, New York, NY. 23. Farrar, M. D., D. L. Miller, C. A. Baldwin, S. L. Stiver, and C. L. Hall. 2005. 47. Swofford, D. L. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Eastern equine encephalitis in dogs. J. Vet. Diagn. Invest. 17:614–617. other methods), version 4. Sinauer Associates, Sunderland, MA. 24. Fauquet, C. M., M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball 48. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: (ed.). 2005. Virus taxonomy: eighth report of the International Committee improving the sensitivity of progressive multiple sequence alignment through on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA. sequence weighting, position-specific gap penalties and weight matrix choice. 25. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using Nucleic Acids Res. 22:4673–4680. the bootstrap. Evolution 39:783–791. 49. Towner, J. S., T. K. Sealy, M. L. Khristova, C. G. Albarino, S. Conlan, S. A. 26. Gardner, C. L., J. Yin, C. W. Burke, W. B. Klimstra, and K. D. Ryman. 2009. Reeder, P. L. Quan, W. I. Lipkin, R. Downing, J. W. Tappero, S. Okware, J. Type I interferon induction is correlated with attenuation of a South Amer- Lutwama, B. Bakamutumaho, J. Kayiwa, J. A. Comer, P. E. Rollin, T. G. ican eastern equine encephalitis virus strain in mice. Virology 390:338–347. Ksiazek, and S. T. Nichol. 2008. Newly discovered Ebola virus associated 27. Hayes, C. G., and R. C. Wallis. 1977. Ecology of Western equine encepha- with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 4:e1000212. lomyelitis in the eastern United States. Adv. Virus Res. 21:37–83. 50. Tully, T. N., Jr., S. M. Shane, R. P. Poston, J. J. England, C. C. Vice, D. Y. 28. Kondig, J. P., M. J. Turell, J. S. Lee, M. L. O’Guinn, and L. P. Wasieloski, Cho, and B. Panigrahy. 1992. Eastern equine encephalitis in a flock of emus Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45. Jr. 2007. Genetic analysis of South American eastern equine encephalomy- (Dromaius novaehollandiae). Avian Dis. 36:808–812. elitis viruses isolated from mosquitoes collected in the Amazon Basin region 51. Turell, M. J., J. Barth, and R. E. Coleman. 1999. Potential for Central American of Peru. Am. J. Trop. Med. Hyg. 76:408–416. mosquitoes to transmit epizootic and enzootic strains of Venezuelan equine 29. Mendez, W., J. Liria, J. C. Navarro, C. Z. Garcia, J. E. Freier, R. Salas, S. C. encephalitis virus. J. Am. Mosq. Control Assoc. 15:295–298. Weaver, and R. Barrera. 2001. Spatial dispersion of adult mosquitoes 52. Turell, M. J., J. W. Jones, M. R. Sardelis, D. J. Dohm, R. E. Coleman, D. M. (Diptera: Culicidae) in a sylvatic focus of Venezuelan equine encephalitis Watts, R. Fernandez, C. Calampa, and T. A. Klein. 2000. Vector competence virus. J. Med. Entomol. 38:813–821. of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic 30. Mitchell, C. J., C. D. Morris, G. C. Smith, N. Karabatsos, D. Vanlanding- strains of Venezuelan equine encephalomyelitis virus. J. Med. Entomol. ham, and E. Cody. 1996. Arboviruses associated with mosquitoes from nine 37:835–839. Florida counties during 1993. J. Am. Mosq. Control Assoc. 12:255–262. 53. Turell, M. J., M. L. O’Guinn, D. Dohm, M. Zyzak, D. Watts, R. Fernandez, 31. Monath, T. P., M. S. Sabattini, R. Pauli, J. F. Daffner, C. J. Mitchell, G. S. C. Calampa, T. A. Klein, and J. W. Jones. 2008. Susceptibility of Peruvian Bowen, and C. B. Cropp. 1985. Arbovirus investigations in Argentina, 1977– mosquitoes to eastern equine encephalitis virus. J. Med. Entomol. 45:720– 1980. IV. Serologic surveys and sentinel equine program. Am. J. Trop. Med. 725. Hyg. 34:966–975. 54. van Regenmortel, M. H. V. 2000. Introduction to the species concept in virus 32. Morris, C. D. 1988. Eastern equine encephalomyelitis, p. 1–36. In T. P. taxonomy, p. 3–16. In M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Monath (ed.), The arboviruses: epidemiology and ecology, vol. III. CRC Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, Press, Boca Raton, FL. D. J. McGeogh, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy. 33. O’Guinn, M. L., J. S. Lee, J. P. Kondig, R. Fernandez, and F. Carbajal. 2004. Classification and nomenclature of viruses, seventh report of the Interna- Field detection of eastern equine encephalitis virus in the Amazon Basin tional Committee on Taxonomy of Viruses. Academic Press, San Diego, CA. region of Peru using reverse transcription-polymerase chain reaction 55. van Regenmortel, M. H. V., C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, adapted for field identification of arthropod-borne pathogens. Am. J. Trop. M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeogh, C. R. Med. Hyg. 70:164–171. Pringle, and R. B. Wickner (ed.). 2000. Virus taxonomy. Classification and 34. Platteborze, P. L., J. P. Kondig, R. J. Schoepp, and L. P. Wasieloski. 2005. nomenclature of viruses, seventh report of the International Committee on Comparative sequence analysis of the eastern equine encephalitis virus Taxonomy of Viruses. Academic Press, San Diego, CA. pathogenic strains FL91-4679 and GA97 to other North American strains. 56. Walder, R., and O. M. Suarez. 1976. Studies of arboviruses in southwestern DNA Seq. 16:308–320. Venezuela. I. Isolations of Venezuelan and eastern equine encephalitis vi- 35. Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of ruses from sentinel hamsters in the Catatumbo region. Int. J. Epidemiol. DNA substitution. Bioinformatics 14:817–818. 5:375–384. 36. Powers, A. M., P. V. Aguilar, L. J. Chandler, A. C. Brault, T. A. Meakins, D. 57. Walder, R., O. M. Suarez, and C. H. Calisher. 1984. Arbovirus studies in Watts, K. L. Russell, J. Olson, P. F. Vasconcelos, A. T. Da Rosa, S. C. southwestern Venezuela during 1973-1981. II. Isolations and further studies Weaver, and R. B. Tesh. 2006. Genetic relationships among Mayaro and Una of Venezuelan and eastern equine encephalitis, Una, Itaqui, and Moju vi- viruses suggest distinct patterns of transmission. Am. J. Trop. Med. Hyg. ruses. Am. J. Trop. Med. Hyg. 33:483–491. 75:461–469. 58. Walder, R., O. M. Suarez, and C. H. Calisher. 1984. Arbovirus studies in the VOL. 84, 2010 EEEV PATTERNS OF EVOLUTION IN NORTH AND SOUTH AMERICA 1025 Guajira region of Venezuela: activities of eastern equine encephalitis and Holland, and T. W. Scott. 1994. Evolution of alphaviruses in the eastern Venezuelan equine encephalitis viruses during an interepizootic period. equine encephalomyelitis complex. J. Virol. 68:158–169. Am. J. Trop. Med. Hyg. 33:699–707. 65. Weaver, S. C., A. M. Powers, A. C. Brault, and A. D. Barrett. 1999. Molecular 59. Weaver, S. C. 2001. Venezuelan equine encephalitis, p. 539–548. In M. W. epidemiological studies of veterinary arboviral encephalitides. Vet. J. 157: Service (ed.), The encyclopedia of arthropod-transmitted infections. CAB 123–138. International, Wallingford, United Kingdom. 66. Weaver, S. C., R. Rico-Hesse, and T. W. Scott. 1992. Genetic diversity and 60. Weaver, S. C., L. A. Bellew, and R. Rico-Hesse. 1992. Phylogenetic analysis slow rates of evolution in New World alphaviruses. Curr. Top. Microbiol. of alphaviruses in the Venezuelan equine encephalitis complex and identi- Immunol. 176:99–117. fication of the source of epizootic viruses. Virology 191:282–290. 67. Weaver, S. C., W. F. Scherer, C. A. Taylor, D. A. Castello, and E. W. Cupp. 61. Weaver, S. C., A. C. Brault, W. Kang, and J. J. Holland. 1999. Genetic and 1986. Laboratory vector competence of Culex (Melanoconion) cedecei for fitness changes accompanying adaptation of an arbovirus to vertebrate and sympatric and allopatric Venezuelan equine encephalomyelitis viruses. invertebrate cells. J. Virol. 73:4316–4326. Am. J. Trop. Med. Hyg. 35:619–623. 62. Weaver, S. C., C. Ferro, R. Barrera, J. Boshell, and J. C. Navarro. 2004. 68. Weaver, S. C., T. W. Scott, and R. Rico-Hesse. 1991. Molecular evolution of Venezuelan equine encephalitis. Annu. Rev. Entomol. 49:141–174. eastern equine encephalomyelitis virus in North America. Virology 182:774– 63. Weaver, S. C., A. Hagenbaugh, L. Bellew, and C. H. Calisher. 1992. Genetic 784. characterization of an antigenic subtype of eastern equine encephalomyelitis 69. Young, D. S., L. D. Kramer, J. G. Maffei, R. J. Dusek, P. B. Backenson, C. N. virus. Arch. Virol. 127:305–314. Mores, K. A. Bernard, and G. D. Ebel. 2008. Molecular epidemiology of 64. Weaver, S. C., A. Hagenbaugh, L. A. Bellew, L. Gousset, V. Mallampalli, J. J. eastern equine encephalitis virus, New York. Emerg. Infect. Dis. 14:454–460. Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 34.229.63.45.