ASSOCIATION OF VENEZUELAN EQUINE ENCEPHALITIS VIRUS SUBTYPE IE WITH TWO EQUINE EPIZOOTICS IN MEXICO

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1 Am. J. Trop. Med. Hyg., 59(1), 1998, pp Copyright 1998 by The American Society of Tropical Medicine and Hygiene ASSOCIATION OF VENEZUELAN EQUINE ENCEPHALITIS VIRUS SUBTYPE IE WITH TWO EQUINE EPIZOOTICS IN MEXICO M. STEVEN OBERSTE, MOISES FRAIRE, ROBERTO NAVARRO, CRISTOBAL ZEPEDA, MARIA L. ZARATE, GEORGE V. LUDWIG, JOHN F. KONDIG, SCOTT C. WEAVER, JONATHAN F. SMITH, AND REBECA RICO-HESSE Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland; U.S.-Mexico Exotic Animal Disease Commission, Palo Alto, Mexico DF, Mexico and Tuxtla Gutierrez, Chiapas, Mexico; Instituto de Salud y Enfermedades Tropicales, Mexico DF, Mexico; Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas; Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas Abstract. Two outbreaks of encephalitis consistent with an etiology of Venezuelan equine encephalitis (VEE) virus occurred in equines on the Pacific coast of southern Mexico in 1993 (Chiapas State) and in 1996 (Oaxaca State). In Chiapas, there were 125 cases, of which 63 were fatal and in Oaxaca, there were 32 cases and 12 fatalities. Virus was isolated from two horses from each outbreak, including three brain isolates and one from blood. Virus isolates ( , ISET-Chi93, Oax131, and Oax142) were shown by indirect immunofluorescence, hemagglutination inhibition, monoclonal antibody ELISA, and nucleotide sequencing to be VEE virus, subtype IE, a type previously thought to be equine-avirulent. Genetic characterization and phylogenetic analysis indicated that the outbreak viruses were identical or nearly identical to one another and that they were closely related to equine-avirulent IE strains from Guatemala and the Gulf coast of Mexico. In a plaque-reduction neutralization test, sera collected from healthy horses in Chiapas and Oaxaca reacted significantly better with isolate than with Guatemala IE isolate 68U201, suggesting that subtle genetic changes may have resulted in alteration of neutralization domains. It is not clear whether these differences may also influence equine virulence. However, renewed VEE virus subtype IE activity in Mexico, and its apparent conversion to equine virulence, underscores the need for increased surveillance, additional laboratory and epidemiologic studies in VEE-endemic regions, and possibly new vaccines. Venezuelan equine encephalitis (VEE) virus has caused numerous equine epizootics and human outbreaks during the last 60 years in tropical and subtropical regions of the Americas. 1 Serologic variants IAB and IC cause severe neurologic disease in equines, with a case-fatality rate of 30 80%. Other subtypes and variants within the VEE virus complex (ID, IE, IF, and II-VI) have not been associated with epizootics or equine virulence, but most of these enzootic strains appear to be virulent for humans. Major equine epizootics had been restricted to South America until 1969, when a subtype IAB virus thought to be related to strains from an Ecuador outbreak infected equines in Guatemala and spread both north and south, eventually involving most of Central America, as well as Mexico and southern Texas. The epizootic was brought under control in 1972 by mass equine vaccination, mosquito abatement, and other public health measures. Between epizootics, the causative IAB and IC viruses could not be isolated, which indicated that either the epizootic strains were maintained in unidentified reservoirs or cryptic enzootic cycles or that they repeatedly arose from enzootic strains by accumulation of appropriate mutations. Recent studies have provided support for both possibilities. Nucleotide sequence analyses have demonstrated close genetic relationships between IC and ID strains, suggesting that IC viruses may emerge from enzootic ID progenitors. 2,3 However, partial sequence analysis of isolates obtained during the 1995 IC epizootic in Venezuela and Colombia demonstrated that the causative virus was essentially identical to the virus that caused an epizootic in the same area in 1963, and to a virus that was isolated in 1983, in the absence of a major epizootic. 4 This observation suggests that this epizootic virus has been maintained, essentially unchanged, in some unidentified enzootic cycle or latent infection. The IAB virus strains have not been isolated in the wild since With the exception of IAB viruses isolated during the epizootic period, the only VEE serotype found in Mexico and Central America (north of Panama) is subtype IE. 1 The prototype VEE virus subtype IE strain MenaII was isolated from an infected human in northwest Panama in 1962, and other IE viruses have been isolated in the same area since that time. 5,6 The VEE virus subtype IE was isolated from mosquitoes and sentinel hamsters in Veracruz State, Mexico during the period , 7 9 in Nicaragua in 1968, 6 and in Belize, Guatemala, and Honduras during the period Subtype IE viruses have generally been regarded as equine avirulent, based on experimental infection of horses with IE strains isolated in Mexico and Nicaragua in the 1960s and on observation of naturally infected horses and burros in Mexico. 17 However, an outbreak of equine encephalitis, consistent with VEE virus etiology, occurred in Tamaulipas State on Mexico s northeast coast in 1966 prior to the introduction of classic epizootic VEE IAB strains to the region; 18,19 no virus isolates are available from that outbreak for serologic and/or genetic characterization. From June to July 1993, an outbreak of equine encephalitis involving 125 horses (including 63 deaths) occurred in Chiapas, the southernmost state of Mexico. A second equine epizootic occurred in adjacent Oaxaca State from June to July, 1996 and involved 32 horses (including 12 deaths). In both cases, initial epidemiologic and serologic studies pointed to VEE virus as the etiologic agent, and VEE virus was isolated from two of the affected animals in each outbreak. In this report, we present studies that have shown that virus isolates obtained from dead or sick horses during this epizootic are IE strains and are similar to known enzootic strains that have circulated in nearby areas of Mexico and Guatemala for at least 30 years. The possible origins and public health implications of the emergence of these virus isolates are discussed.

2 VEE VIRUS SUBTYPE IE EQUINE EPIZOOTICS IN MEXICO 101 MATERIALS AND METHODS Specimen collection. Brain and/or blood samples were collected by veterinarians from three dead horses ( #7, collected in Mapastepec, Chiapas on June 29, 1993; #131, collected in Chahuites, Oaxaca on June 28, 1996; #142, collected in Tapanatepec, Oaxaca on July 20, 1996) and from one sick horse (ISET-Chi93, collected in Chiapas in July 1993), respectively. Virus ISET- Tab91 was isolated from a pool of Culex spp. mosquitoes in April 1991 during a human disease outbreak of unknown etiology in the State of Tabasco. Additional sera were collected from 59 healthy horses in four states in southern Mexico during 1995 and 1996: Oaxaca, Chiapas, Tabasco, and Yucatan. Virus isolation, stock viruses, and antigenic tests. Specimens were tested for the presence of virus by intracerebral inoculation of newborn mice. Virus obtained from mouse brain suspension was amplified by one passage in either Vero or baby hamster kidney cell culture prior to antigenic and genetic characterization. Samples of these infected cells were used in an indirect immunofluorescent antibody test (using anti-vee, anti Western equine encephalitis [WEE], and anti Eastern equine encephalitis [EEE] hyperimmune mouse ascitic fluids and goat anti-mouse conjugate) to identify the viruses as VEE. The five Mexican virus isolates (from 1991 to 1996) were characterized antigenically, first in a hemagglutination inhibition (HI) assay using rabbit polyclonal antisera specific for the E2 glycoprotein of various VEE virus subtypes, 20 and then in an ELISA using three monoclonal antibodies (VEE 3B4C-4, 1A1B-9, and 1A2B- 10) whose specificities have been well-characterized Sera from healthy horses were tested for VEE virus reactivity and subtype specificity by a plaque-reduction neutralization test (PRNT) against VEE IAB (Trinidad donkey strain) and VEE IE ( and 68U201 strains) viruses. For the PRNT, 0.1 ml of each serum sample was mixed with equal volumes of VEE virus diluted to contain approximately 200 plaque-forming units. After overnight incubation at 4 C, 0.1 ml of the mixture was added in duplicate to monolayers of Vero-76 cells (CRL 1587; American Type Culture Collection, Rockville, MD) in six-well plates. Inoculated cultures were incubated at 37 C for 1 hr, then overlaid with basal medium with Earle s salts (Gibco/BRL, Gaithersburg, MD) containing nonessential amino acids (Gibco/BRL), 0.6% (w/ v) agarose, 10% fetal bovine serum, 10 mm HEPES buffer, penicillin (100 U/ml), streptomycin (100 g/ml), gentamicin (50 g/ml), and amphotericin B (0.25 g/ml). After incubation for 24 hr at 37 C, the cultures were overlaid with the same medium described above containing 0.017% (w/v) neutral red. Plaques were counted after an additional 24 hr incubation at 37 C. Reduction of plaque counts by 80% or more when compared with negative controls was considered positive for VEE virus neutralizing (N) antibody. For the purpose of comparison, a four-fold or greater difference in neutralization titer against any virus was considered significant. Sequencing and sequence analysis. Isolation of RNA and reverse transcription polymerase chain reaction (RT- PCR) were carried out as described previously. 24 Viral RNA was extracted from infected cell culture supernatant using Trizol-LS (Life Technologies, Inc., Gaithersburg, MD) and cdna was synthesized using an oligonucleotide primer annealing in the E2 glycoprotein gene (VEE-116; TACA- CCCAYTTRTCRTTCTG, nucleotides ). A fragment encompassing most of the PE2 glycoprotein gene (nucleotides ) was amplified by PCR using primers VEE-115 (GAGAAYWGCGAGCARTGGTC, nucleotides ) and VEE-116. The sequenced segment includes the coding region for all 58 amino acids of E3 and for amino acids 1 to 231 of E2. 25 The cdna was primed with an oligonucleotide complementary to the 3 end of the genome (GAAATATTAAAAAC) and the structural gene region was amplified as a set of overlapping PCR products using primers derived from the sequence of IE-68U The PCR products were purified using the Wizard PCR Preps kit (Promega, Inc., Madison, WI) and sequenced directly on a model 373A automated sequencer (Applied Biosystems Division, Perkin Elmer, Inc., Foster City, CA). The sequences were compared with one another and with the sequences of other VEE virus strains using the programs of the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc., Madison, WI). Phylogenetic relationships were inferred using the PAUP 26 and PHYLIP 27 programs. Parsimony analysis was performed by the heuristic algorithm and unordered characters, implemented in PAUP, and sequences were added at random with 100 replications. A distance matrix was generated for neighbor-joining analysis by the one-parameter formula of Jukes and Cantor. 28 Confidence values were placed on groupings within trees by bootstrap analysis with 200 resamplings. The sequences reported in this paper have been deposited in the GenBank database with accession numbers U96403-U RESULTS Chiapas outbreak. During June and July 1993, 125 cases of equine encephalitis were reported among horses in eight municipalities of Chiapas State, Mexico (Figure 1). Of the affected animals, 63 died with a clinical diagnosis of encephalitis consistent with VEE (50% case-fatality rate); the total equine population (susceptible horses) on the affected ranches was 417. Clinical signs ranged from anorexia, fever, frothing at the mouth, edema of the legs and neck, ataxia, walking in circles, blindness, and diverse lesions caused by colliding with walls, fences, etc. We received one brain isolate ( #7) from a three-year-old male horse that died on June 29 in Mapastapec municipality; isolate ISET-Chi93 was from the blood of a sick horse collected in Chiapas, with no other information given. Movement of animals into and out of the affected area was controlled by placing check points along highway 200, the major road through the area. Once VEE was confirmed as the cause of the outbreak, a campaign was initiated on July 16, 1993 to vaccinate equines in the area with a live-attenuated vaccine derived from TC- 83. In a four-week period, approximately 38,000 horses and burros were vaccinated in the outbreak area and surrounding regions. The vaccination program was limited to the coastal areas of Chiapas and Oaxaca states, using vaccine manufactured in Mexico. Ultra-low dose malathion application was done in the 61 ranches with equine cases in the eight-municipality area to control the mosquito population. The last

3 102 OBERSTE AND OTHERS FIGURE 1. Map of Mexico indicating sites where virus isolates and horse sera were obtained. Closed circles serum collection sites; open circles sites where Venezuelan equine encephalitis virus was isolated from four affected horses; 1 Mapastepec; 2 Chahuites; 3 Tapanatepec. The fourth isolate was from an unknown location in Chiapas. The star indicates Mexico City (the capital). known equine case occurred on July 25, 1993, and the equine transport quarantine, which began on July 10, was lifted on August 27. No human cases of encephalitis were reported during the outbreak period, although the Ministry of Health had increased surveillance. Oaxaca outbreak. From June 14 to July 23, 1996, equine encephalitis was reported in horses in three municipalities of Oaxaca state: Chahuites, Tapanatepec, and Matias Romero, all located along the Pacific Coast, in the Isthmus of Tehuantepec (Figure 1). A total of 32 cases was reported, including 12 deaths (37% case-fatality rate); there were no cases in burros or mules. Of 380 serum samples, 154 were positive for VEE virus antibody by the HI test; six brain samples yielded two virus isolates (described here), while 23 blood samples also yielded two virus isolates (not included here). Sample number 131 (brain) was from an eight-old male foal that died on June 25 in Chahuites municipality; sample 142 (brain) was from a 4 5-year-old mare that died on July 5 in Tapanatepec municipality. A quarantine was placed on this region of southern Oaxaca and a massive equine vaccination program was carried out at 228 sites, from June 24 to July 15; a total of 15,618 equines were vaccinated in 32 municipalities. Approximately half of the vaccination area had already been covered in 1993 in response to the Chiapas epidemic; Chahuites, the municipality with the highest number of cases (23), had received broad vaccination coverage in There were no reported cases of human disease associated with the equine encephalitis outbreak in Oaxaca. Virus isolation and identification. Virus was isolated from samples (brain) and ISET-Chi93 (blood) from Chiapas and samples 131 (brain) and 142 (brain) from Oaxaca by intracerebral inoculation of newborn mice. The four virus isolates were all presumptively identified as VEE by an indirect immunofluorescent antibody test using antisera specific for VEE, WEE, or EEE viruses. The antigenic subtype of the virus isolates was determined by HI using E2 glycoprotein polyclonal sera and by ELISA using a panel of VEE virus-specific monoclonal antibodies. Serologic typing identified all equine isolates as VEE virus IE, a variety not previously known to cause encephalitis in horses. The HI test classified these viruses as either IE or IF serotypes (only a two-fold difference in titer), but clearly distinguished them from IAB, IC, or ID serotype viruses (four-fold or more difference). The more sensitive ELISA with monoclonal antibodies was able to distinguish the Mexican viruses from all other serotype I prototype strains, including the TC-83 vaccine strain. All five viruses reacted as the Mena II strain, the IE serogroup prototype virus, by end-point dilution of the three monoclonal antibodies (reciprocal endpoint titers were 100, 10,000, and 10,000, respectively), thus identifying them as IE viruses. The typing of one of the isolates, , was verified by RT-PCR amplification and sequencing of 518 nucleotides at the 3 end of nonstructural gene 4 and comparison with the homologous region of VEE virus strains of known subtype/variety. Genetic characterization. Since VEE virus subtype IE was not previously known to cause equine disease, the entire structural gene sequence (26S region) of was sequenced to determine whether changes in the structural proteins might be responsible for the observed equine virulence. The nucleotide sequences encoding the E2 glycoprotein precursor, PE2, of the second Chiapas isolate, the two Oaxaca isolates, and the 1991 Tabasco isolate were also determined (868 nucleotides). Overall, the 26S nucleotide sequence of (3,920 nucleotides) was 1.5% divergent from the homologous sequence of IE-68U201, a 1968 isolate from southeastern Guatemala, and 7.0% divergent from IE- MenaII, a 1962 isolate from northwestern Panama (Table 1). The predicted complete structural polyprotein sequences of differed from 68U201 at three residues (99.8% identity), all in E2 (Ser 218 Asn, Leu 389 Phe, Trp 390 Cys), and from MenaII at 21 residues (98.3% identity) in capsid, E2 and E1 (Table 1 and Figure 2). To determine the

4 VEE VIRUS SUBTYPE IE EQUINE EPIZOOTICS IN MEXICO 103 TABLE 1 Nucleotide and predicted amino identities comparing Chiapas and Oaxaca outbreak isolates to other Venezuelan equine encephalitis virus IE strains* * Comparisons based on 868 nucleotides at the 5 end of the PE2 gene and its predicted 289 amino acid translation product. Percent nucleotide identity is shown above the diagonal; percent predicted amino acid identity is shown below the diagonal. relatedness of the outbreak isolates to other IE strains, we sequenced 868 nucleotides at the 5 end of the PE2 gene of ISET-Chi93, Oax131, Oax142, ISET-Tab91, and 63U16. Within the predicted partial PE2 glycoprotein sequence (289 amino acids), the Chiapas and Oaxaca isolates were all identical to one another, 99.6% identical to 68U201, and 97.6% identical to MenaII (Table 1 and Figure 2). Isolates ISET- Tab91 and 63UI6 were 99.6% identical to one another in amino acid sequence and 99.3% identical to the other Mexican isolates. The complete predicted PE2 sequence of was different from both 68U201 and MenaII at only one residue, E2-218 (serine replaced by asparagine); that is, at every other site the sequence was identical to that of either 68U201 or MenaII. All four outbreak viruses contain asparagine at this position, whereas all four other IE strains contain serine (Figure 2). Equine-virulent prototype strains IAB-TRD and IC-P676 contain serine at this position, as do 68U201, MenaII, ID-3880, and II-Fe3-7c, suggesting that Asn 218 alone does not control equine virulence. It remains possible, however, that Asn 218 plays a role in equine virulence, in concert with other changes specific to the Mexican IE strains. Phylogenetic analysis using the 868 nucleotide portion of the PE2 gene of , ISET-Chi93, Oax131, Oax142, and ISET-Tab91 compared with the homologous region of IE-63U16, IE-68U201, IE-MenaII, IAB-TRD, IC-P676, ID- 3880, and II-Fe3-7c showed that the Chiapas and Oaxaca isolates were most closely related to 68U201 (Figure 3), which was isolated in Guatemala 25 years previously, in an area approximately 300 km southeast of the Chiapas outbreak area. The Veracruz and Tabasco isolates were most closely related to one another and to the Chiapas/Oaxaca/ Guatemala group, and more distant from MenaII. As a group, the IE isolates were distinct from the serologically distantly related IAB, IC, ID, and II strains, as described previously. 2,25,29 Serologic surveillance. Prior to a vaccination campaign, sera were collected from healthy horses in the states of Chiapas, Oaxaca, Yucatan, and Tabasco in 1995 and 1996 and tested for the presence of VEE virus N antibodies (Figure 4). The Oaxaca horses were contacts of sick animals bled during the outbreak. Forty-nine of 59 animals possessed N antibody to VEE virus. Forty-three of the 49 VEE-positive animals had at least a four-fold greater titer against the 1993 Chiapas IE isolate ( strain) than against IAB virus (Trinidad donkey strain) (Figure 4B). None of the 49 antibody-positive animals showed a greater titer to Trinidad donkey than to Interestingly, only 10 of 49 VEEpositive animals had at least a four-fold greater titer against IE-68U201 than against Trinidad donkey (Figure 4A) and two animals had at least a four-fold greater titer against Trinidad donkey than against 68U201. When N titers against the two IE strains were compared, 34 of 49 VEE-positive horses had at least a four-fold greater titer against than 68U201, while none of the horses responded better against 68U201 than against (Figure 4C). The N titers from all horses sampled in Chiapas and Oaxaca were at least four-fold greater against than against 68U201. While some horses from Tabasco possessed serum N antibodies that reacted significantly better against than 68U201, other horses did not. None of the horses sampled from Yucatan possessed N antibody with higher titers to than to 68U201 (Figure 4C). DISCUSSION To our knowledge, the outbreaks described here represent the first cases of epizootic equine encephalitis from which an enzootic strain of VEE virus has been isolated. The fact that three of these isolates were obtained from the brains of horses that had died of encephalitis, and the other was obtained from the serum of a sick horse with an active viremia, suggests that this VEE virus IE strain possessed at least some of the phenotypic characteristics normally associated with IAB and IC epizootic strains. The high sequence similarities observed between the VEE virus IE strains isolated from the Chiapas and Oaxaca epizootics and the IE strains that have been and continue to be isolated in neighboring regions of Mexico and Guatemala suggest that either the equine virulence of these strains was undetected in previous experimental infection studies or that subtle genetic changes are sufficient to significantly increase equine virulence. These results point to the urgent need for equine pathogenicity studies with VEE viruses isolated in Mexico because equinevirulent viruses may potentially be introduced into countries such as the United States, which do not perform routine VEE vaccination. The genetic loci or mutations controlling alphavirus virulence, equine viremia levels, and efficient replication in epizootic mosquitoes are poorly understood, although there is some indication that at least some of these traits may be controlled by nonstructural genes. 25,30 Recent studies in which site-directed mutagenesis procedures were used to alter the nucleotide sequence of VEE virus IAB structural genes have shown that even single nucleotide substitutions can result in dramatic changes in virulence, viremia levels, and tissue tropism. 31,32 Rigorous identification of the loci controlling the virulence and epizootic potential of the VEE virus IE strains would likely require similar studies with fulllength IE molecular clones and relevant animal models. However, it would appear clear from the present data that VEE viruses that can be genetically and serologically typed as IE have epizootic potential, at least under some conditions.

5 104 OBERSTE AND OTHERS FIGURE 2. Alignment of partial predicted PE2 glycoprotein sequences of Mexico and Guatemala IE strains of Venezuelan equine encephalitis virus. Residues that are identical in all strains are boxed. Western equine encephalitis virus is believed to have arisen by recombination between EEE virus and a Sindbis virus like progenitor, in which the nonstructural genes and some of the nucleocapsid gene were derived from EEE virus and the remainder of the structural protein genes were derived from the Sindbis virus like progenitor. 33 To determine whether the unexpected properties of the Mexico epizootic IE strains could have resulted from a similar recombination event, we have sequenced part of nonstructural gene 3 (ns3) of The 3 half of ns3 is highly variable among different serologic variants and IE strains have distinctive sequence motifs that clearly differentiate them from IAB, IC, or ID viruses. 25 The sequence of ns3 is similar to that of 68U201 and MenaII, which contain the IE-specific motifs, suggesting that did not arise by recombination of IAB or IC viruses with a IE virus. The VEE virus IE strains are genetically and antigenically quite distinct from the IAB strains. 2,6,25,29 Cross-neutralization

6 VEE VIRUS SUBTYPE IE EQUINE EPIZOOTICS IN MEXICO 105 FIGURE 3. Phylogenetic tree showing the relationship of Chiapas, Oaxaca, and Tabasco isolates of Venezuelan equine encephalitis virus to isolates IE-63UI6, IE-68U201, IE-MenaII, IAB-TRD, IC-P676, IC-1289, ID-3880, and II-Fe3-7c based on alignment of their partial PE2 nucleotide sequences (868 nucleotides). Branch lengths are shown above the branches and bootstrap values (percent of 200 replicates) are shown in parentheses below the branches. levels between these strains are low, and individuals vaccinated with the TC-83 vaccine, which was derived from the IAB Trinidad donkey strain, develop low or insignificant titers to prototype IE strains. 34,35 There have been no longterm antibody cross-neutralization studies conducted in horses, but the cross-protecting neutralizing antibody response induced by vaccination of humans or mice with TC-83 is limited and decreases over time. 34,35 Furthermore, at least two individuals who were vaccinated with TC-83, and who responded successfully with adequate homologous neutralization titers, nonetheless developed apparently unmodified VEE as a result of laboratory infections with the IE-68U201 strain, and subsequently responded with significantly elevated neutralization titers to IE-68U201 (Jahrling PB, unpublished data). Therefore, in consideration of the fact that historically the most efficient means of preventing or controlling VEE epizootics has been equine vaccination with TC- 83, or vaccines derived from TC-83, the emergence of a VEE virus IE strain with epizootic potential may constitute a significant veterinary or public health concern. In Mexico, government-sponsored, routine, large-scale vaccination of equines was stopped after 1991 because the country was considered to be free of epizootic VEE virus strains. Equine vaccination with a TC-83-like vaccine was used in response to the Chiapas and Oaxaca outbreaks, in conjunction with vector control and restrictions on equine movements; however, the relative effectiveness of these measures could not be determined. There is currently no IE vaccine available, but efforts are underway to generate an IE-specific product 25 using essentially the same site-directed mutagenesis procedures that were used to generate new, live-attenuated, IAB serotype vaccine candidates. 31 Serologic results from horses residing in the same areas where the original Chiapas VEE virus isolates and subsequent Oaxaca isolates were obtained indicate that VEE IE virus was circulating along the Pacific coast of southern Mexico during the time between the two outbreaks. In addition, the results show that IE viruses were also circulating among horses in states along the Gulf coast of Mexico. There is also serologic evidence to suggest that the IE strains on the Pacific coast of Mexico were more closely related to a Chiapas isolate than to an older IE isolate from Guatemala (68U201). This relationship is consistent among all horses sampled along the Pacific coast, but not consistent among all horses sampled along the Gulf coast, suggesting that multiple IE virus lineages may be cocirculating in southern Mexico. These data suggest that VEE viruses circulating on the Pacific coast of Mexico may differ from previously known

7 106 OBERSTE AND OTHERS FIGURE 4. Serologic responses of horses in plaque-reduction neutralization tests (PRNTs) against Venezuelan equine encephalitis virus subtypes IAB and IE (two isolates). Titers are expressed as the reciprocal of the greatest serum dilution exhibiting 80% plaque reduction of the virus being tested in comparison with negative control sera. Horse sera with identical neutralization patterns appear as a single point on the plot. IE strains in at least some of the critical neutralizing domains. Whether these differences are also involved in increased equine virulence remains unknown. In contrast to previous VEE epizootics caused by subtype IAB and IC viruses, 1,4,23 there was apparently no human disease associated with the outbreaks in Chiapas and Oaxaca. Since other IE strains are capable of infecting humans and causing disease, there is no reason to believe that the Chiapas and Oaxaca strains would be avirulent for humans. More likely explanations for the apparent lack of human disease during the equine outbreaks are that human disease was mild and therefore unreported or that appropriate anthropophilic mosquito vector species were absent during the outbreak, such that virus was not efficiently transmitted to humans. Ecologic studies in the affected areas of Chiapas and Oaxaca are needed to determine which mosquito species are important VEE virus IE vectors on the Pacific coast of Mexico and to determnine the reservoir(s) of VEE IE virus in the region. Renewed VEE activity in Mexico, together with recent outbreaks in Venezuela, Colombia, and Peru 4,23,36,37 underscores the re-emergence of VEE as a threat to human and veterinary health in tropical regions of the Americas. Increased surveillance is needed throughout the VEE-endemic region, as are additional studies to determine the extent of antigenic and genetic diversity among strains capable of initiating epizootics, and whether existing vaccines are adequate to protect against such strains. Acknowledgments: We thank Cathy Lind for technical assistance and David Alstad, Peter Fernandez, Rafael N. Garcia, James Pearson, Elisa Rubi, and Diodoro Batalla for help in obtaining information and isolates from these outbreaks. Financial support: This research was supported in part by the U.S. National Institutes of Health grant AI Authors addresses: M. Steven Oberste, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop G-17, 1600 Clifton Road, NE, Atlanta, GA Moises Fraire and Cristobal Zepeda, U.S.- Mexico Exotic Animal Disease Commission, Km Carr. Mexico-Toluca, Palo Alto, Del. Cuajimalpa, Mexico, DF, Mexico Roberto Navarro, U.S.-Mexico Exotic Animal Disease Commission, Carr. Tuxtla-Chicoasen, Fracc. Los Laguitos, Tuxtla Gutierrez, Chiapas, Mexico. Maria L. Zarate, Instituto de Salud y Enfermedades Tropicales, Cerrada de Linares #1 Bis, Col. Roma, Mexico, DF, Mexico George V. Ludwig, John F. Kondig, and Jonathan F. Smith, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD Scott C. Weaver, Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, TX Rebeca Rico-Hesse, Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX Reprint requests: M. Steven Oberste, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop G-17, 1600 Clifton Road NE, Atlanta, GA REFERENCES 1. Walton TE, Grayson MA, Venezuelan equine encephalopmyelitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Volume IV. Boca Raton, FL: CRC Press, Weaver SC, Bellew LA, Rico-Hesse R, Phylogenetic analysis of alphaviruses in the Venezuelan equine encephalitis complex and identification of the source of epizootic viruses. Virology 191: Kinney RM, Tsuchiya KR, Sneider JM, Trent DW, Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus. Virology 191: Weaver SC, Salas R, Rico-Hesse R, Ludwig GV, Oberste MS, Boshell J, Tesh RB, Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. Lancet 348:

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