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Journal of Virology, July 2005, p. 9128-9133, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.9128-9133.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Envelope Glycoprotein Mutations Mediate Equine Amplification and Virulence of Epizootic Venezuelan Equine Encephalitis Virus

Ivorlyne P. Greene,^1,2 Slobodan Paessler,^1,2 Laura Austgen,³ Michael Anishchenko,^1,2 Aaron C. Brault,^1,2, Richard A. Bowen,³ and Scott C. Weaver^1,2*

Center for Biodefense and Emerging Infectious Diseases,¹ Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609,² Animal Reproduction and Biotechnology Lab, Colorado State University, Ft. Collins, Colorado 80522³

Received 14 February 2004/ Accepted 26 March 2005

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Epidemics of Venezuelan equine encephalitis (VEE) result from high-titer equine viremia of IAB and IC subtype viruses that mediate increased mosquito transmission and spillover to humans. Previous genetic studies suggest that mutations in the E2 envelope glycoprotein allow relatively viremia-incompetent, enzootic subtype ID strains to adapt for equine replication, leading to VEE emergence. To test this hypothesis directly, chimeric VEEV strains containing the genetic backbone of enzootic subtype ID strains and the partial envelope glycoprotein genes of epizootic subtype IC and IAB strains, as well as reciprocal chimeras, were used for experimental infections of horses. Insertion of envelope genes from two different, closely related enzootic subtype ID strains into the epizootic backbones resulted in attenuation, demonstrating that the epizootic envelope genes are necessary for the equine-virulent and viremia-competent phenotypes. The partial epizootic envelope genes introduced into an enzootic ID backbone were sufficient to generate the virulent, viremia-competent equine phenotype. These results indicate that a small number of envelope gene mutations can generate an equine amplification-competent, epizootic VEEV from an enzootic progenitor and underscore the limitations of small animal models for evaluating and predicting the epizootic phenotype.

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Venezuelan equine encephalitis (VEE) is a reemerging viral disease of humans, equines, and other domestic animals that has occurred sporadically in the Americas since the 1920s, with up to hundreds of thousands of cases during major outbreaks (12, 26, 34). The transmission cycle of VEE virus (VEEV) during epidemics involves equines serving as amplification hosts and floodwater mosquitoes as vectors. All VEEV strains isolated during major outbreaks belong to serotypes IAB and IC and produce encephalitis preceded by high-titer viremia in horses, donkeys, and mules. Equine virulence and viremia induction are the most important hallmarks of the "epizootic" phenotype; equine viremia mediates high levels of virus circulation when large populations of susceptible mosquitoes are present for transmission (31). Humans who live in close association with infected equines in agricultural settings become infected via tangential spillover, resulting in epidemic disease. In contrast, other serotypes of VEEV and related alphaviruses in the VEE complex, designated enzootic strains, circulate without apparent disease in sylvatic or swamp habitats. The enzootic strains are relatively avirulent for equines and generate little or no equine viremia, rendering them incapable of amplification to produce epidemics (31).

A member of the Alphavirus genus in the Togaviridae family, VEEV is an enveloped virus with a nonsegmented, positive-sense RNA genome of approximately 11.4 kb containing a 5'-methylguanylate cap and a 3'-polyadenylate tail (14). Upon arrival in the cytoplasm following receptor-mediated endocytosis, the viral genome is translated by cellular components into four nonstructural proteins (nsP1 to -4) that participate in viral replication. The 3' one-third of the genome is expressed from a 26S subgenomic message that encodes three major structural proteins, the capsid, E2 and E1 envelope glycoproteins, that are involved in packaging of the viral genome and in the production of infectious viral particles via budding from the plasma membrane (23).

Serotype IAB and IC epizootic VEEV strains are believed to arise from enzootic subtype ID VEEV progenitors via mutations in the E2 protein and possibly other genes and genome regions (4). Previously, chimeric VEEV strains containing the genetic backbone of enzootic strains and either the full complement of structural genes (17) or the partial envelope glycoprotein genes from epizootic strains (10), as well as reciprocal constructs, were used to map the equine virulence and epizootic phenotypes. The guinea pig, the small animal model studied to date that most closely resembles equines in its differential response to enzootic versus epizootic VEEV strains (20, 21), was used to assess the epizootic phenotype in these studies. An intermediate guinea pig viremia and virulence phenotype exhibited by all of the chimeric VEEV strains suggested that both envelope and nonenvelope genes and genome regions are determinants of the epizootic phenotype (10). However, unlike equines that almost invariably survive enzootic VEEV infection without detectable disease and with little or no viremia, guinea pigs become viremic and suffer fatal disease after infection by some enzootic strains, including those of the serotype ID lineage believed to have generated all IAB and IC epizootic strains (4). This limitation of the guinea pig model indicates the need to confirm the results of reverse genetic studies with experimental equine infections.

We therefore infected horses with wild-type enzootic and epizootic VEEV strains derived from infectious cDNA clones, as well as with chimeric strains that differ only in seven amino acid residues within the envelope glycoproteins. Our results demonstrated that these envelope gene regions are both necessary for and sufficient to generate the epizootic, equine amplification-competent phenotype from an enzootic VEEV strain.

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Cell cultures. Vero and BHK cells were obtained from the American Type Culture Collection (Manassas, Va.). They were propagated in Eagle's minimal essential medium supplemented with 5% fetal bovine serum and gentamicin (10 µ/ml).

Plasmids. Recombinant plasmids encoding cDNA copies of the RNA genomes of relevant epizootic and enzootic VEEV strains were described previously (1, 7, 10, 13). The epizootic subtype IC strain 3908 (EpA) was isolated from a febrile human during the major 1995 Venezuelan epidemic (36). It was passaged once in C6/36 mosquito cells prior to RNA extraction and cDNA clone (pM1-3908) production (7). The Trinidad donkey (TrD) subtype IAB strain was isolated in 1943 and passaged once in a guinea pig brain, six times in Vero cells, and once in BHK cells before RNA extraction and cloning as described previously (13). This parent virus is highly virulent for horses (R.A.B., unpublished), and virus derived from the clone is identical to the parent in a variety of in vitro assays and in mouse infections (S.C.W., unpublished). Enzootic subtype ID strain ZPC 738 (EnA) was isolated from a sentinel hamster in 1997 in Venezuela (28) and was passaged once in Vero cells prior to RNA extraction and cDNA clone (pM1-738) construction (1). Enzootic strain 66637 (EnB) was isolated from a sentinel hamster in Venezuela in 1981 (24) and was passaged once in baby mice and once in Vero cells. Both of the enzootic ID strains were demonstrated previously to be avirulent for horses (29).

Chimeras (Fig. 1) were generated by swapping cDNA fragments to produce plasmids containing an SP6 promoter, 5'-untranslated region (UTR), nsP1 to -4, subgenomic 26S promoter, and 3'-UTR sequences of the backbone virus; the C-terminal 111 amino acids in the capsid, the complete PE2 and 6k genes, and the N-terminal 108 amino acids of the E1 protein gene were derived from the second virus strain to generate chimeras. The 3908/ZPC738-E2 (Ep/EnA-E2) and ZPC738/3908-E2 (En/EpA-E2) chimeric clones were described previously (10), and the 3908/66637-E2 (Ep/EnB-E2) and TrD/66637-E2 (TrD/EnB-E2) clones were generated using the same methods (7). The amino acid differences between EnA and EpA in the swapped fragments included only one residue in the E3 protein, five in E2, and a single difference in E1 (Table 1) (10). EnB, which is slightly more distantly related to epizootic strains including EpA than is enzootic strain EnA (28), had three additional amino acid differences in the E3, E2, and 6k genes of the swapped fragment, respectively, compared to both of the other VEEV strains (Table 1). The IAB strain TrD is similar in amino sequence to EpA but has several additional, unique amino acid differences in the fragment exchanged, compared to all others used in this study (Table 1).

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FIG. 1. Genome maps of parental infectious clones and reciprocal envelope chimeras showing the DNA fragments swapped. The exchanged fragments between AflII and SnabI restriction sites at genomic positions 8031 and 10298 contained the last 111 codons in the capsid protein gene, the complete PE2 and 6k protein genes, and the first 108 codons of the E1 protein gene of the epizootic (Ep) parent in the backbone of the enzootic (En) parent, and vice versa. Shaded regions correspond to genome regions obtained from epizootic viruses.

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TABLE 1. Genetic differences among VEEV strains in the fragment swapped to generate chimeric viruses

In vitro transcriptions and transformations. Infectious clone plasmid DNA was linearized with NotI or MluI restriction enzymes (New England Biolabs, Beverly, MA), and RNA was transcribed in the presence of m⁷G(5')ppp(5')A cap analogue (New England Biolabs) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK-21 cells. Viruses were harvested from the cell culture medium 24 h after electroporation and stored at –80°C. The genetic and phenotypic accuracy of the cDNA clones and viruses was confirmed previously for the parental clones and the chimeric Ep/EnA-E2 and En/EpA-E2 clones by sequencing, infectious center assays, immunofluorescence and plaque size assays, in vitro and in vivo replication kinetics, and hemagglutination assays (1, 10). The same characterization was applied to the Ep/EnB-E2 and TrD/EnB-E2 clones and indicated wild-type, enzootic phenotypes (data not shown).

Antibody assays. All horses were screened for alphavirus antibodies prior to experimental infection using 80% endpoint plaque reduction neutralization tests (PRNT) with the following viruses that represent those typically administered to North American horses in multivalent inactivated vaccines: VEEV strain TC-83, eastern equine encephalitis virus strain 82V2137, and western equine encephalitis virus Fleming strain. Following infection, seroconversion was assessed using PRNT with the homologous enzootic ID and epizootic IC strains or with the strain contributing the PE2 gene for chimeric infections.

Virus titrations. Virus titers were determined by plaque assay using Vero cell monolayers in six-well plates as described previously (2).

Horses. Mixed-breed horses 1 to 12 years of age, obtained from a local horse seller, were placed into the animal biosafety level 3 (ABSL-3) large animal facility at Colorado State University and acclimated 3 to 7 days prior to infection. For the duration of the study, they were fed a pelleted ration plus a small amount of mixed grain twice daily and provided water ad libitum.

Infection of horses. Four horses each were infected with the wild-type VEEV strains EpA and EnA rescued from the infectious cDNA clones. These VEEV strains were previously shown to be indistinguishable from their parent strains in cell culture replication and mouse virulence (10). Three horses were infected with strain En/EpA-E2 containing the epizootic PE2 gene in the enzootic backbone, and two horses each were infected with the reciprocal chimeras Ep/EnA-E2 and Ep/EnB-E2. One horse was infected with the TrD/EnB-E2 chimera. All horses were inoculated subcutaneously in the left shoulder with 2,000 PFU in a volume of 1.0 ml of phosphate-buffered saline. Horses were bled daily from the jugular vein, and rectal temperatures were recorded twice daily. Horses exhibiting severe encephalitis signs and/or anorexia for 48 h were euthanized by an intravenous overdose of pentobarbital. All surviving horses were bled and sacrificed 14 to 18 days postinfection.

Statistical analysis. One-way analysis of variance with the Tukey-Kramer multiple-comparisons test and the Kruskal-Wallis test with Dunn's multiple-comparisons test were performed for comparisons of peak viremia magnitude and febrile response among virus strains using GraphPad InStat version 3.05 for Windows 95/NT (GraphPad Software, San Diego, CA.; www.graphpad.com).

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Clinical disease. Most horses exhibited a febrile response within 36 h of VEEV infection (Fig. 2). As expected, the epizootic strain EpA produced the highest febrile response at most time points. The Ep/EnA-E2 chimera produced an intermediate febrile response, especially during the first 3 days of infection, while the Ep/EnB-E2 and TrD/EnB-E2 chimeras generated body temperatures similar to those of the enzootic strain (Fig. 2).

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FIG. 2. Febrile responses of horses infected with epizootic strain EpA (four horses), enzootic strain EnA (four horses), and chimeric VEEV strains En/EpA-E2 (three horses), Ep/EnA-E2 (two horses), and TrD/EnB-E2 (one horse). Vertical bars represent standard errors of the means. Horizontal, dashed lines show the normal rectal temperature range for horses.

All four horses infected with strain 3908 (EpA) developed severe disease characterized by lethargy and anorexia. Three of these horses also developed signs of clinical encephalitis beginning on days 6 to 8 postinfection, including ataxia, depression, and pressing the head against the side of the stall. The three horses infected with chimeric strain En/EpA-E2 also developed clinical encephalitis that was similar to that produced by the epizootic strain. In contrast, neither the enzootic strain EnA nor the Ep/EnA-E2, Ep/EnB-E2, and TrD/EnB-E2 chimeras produced clinical encephalitis in any of the horses.

Viremia. As in previous studies using the parent virus (29), enzootic subtype ID strain EnA rescued from the cDNA clone generated no detectable viremia (<0.6 PFU/ml serum) in the four infected horses (Fig. 3). In contrast, the epizootic subtype IC strain EpA generated a peak mean viremia titer of 3.5 log₁₀ PFU/ml, 2 days after infection, with a viremia duration of 4 days in three horses and 2 days in the other horse. The Ep/EnA-E2, Ep/EnB-E2, and TrD/EnB-E2 chimeras were phenotypically indistinguishable from the enzootic ID strain, with undetectable viremia, while the reciprocal En/EpA-E2 chimera produced a mean peak viremia (3.8 log₁₀ PFU/ml) similar to EpA. Viremia generated by the En/EpA-E2 chimera lasted only 3 days in all three horses, in contrast to 4 days of viremia in 3/4 of the horses infected with strain EpA. The timing of viremia generally correlated with the febrile responses.

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FIG. 3. Mean viremia titers of horses infected with epizootic EpA (four horses) and chimeric VEEV strain En/EpA-E2 (three horses). Strains EnA (four horses), Ep/EnA-E2 (two horses), Ep/EnB-E2 (two horses), and TrD/EnB-E2 (one horse) produced no viremia detectable by plaque assay on Vero cells. Vertical bars represent standard errors of the means. The y-axis scale begins at the detection limit of the plaque assay (0.6 log10 PFU/ml).

Seroconversion. All horses that survived for 14 to 18 days after infection seroconverted, with PRNT titers ranging from 1:20 to 1:80.

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Mechanisms of VEE emergence. Several mechanisms have been hypothesized for the emergence of epizootic VEE (12). (i) The first mechanism is emergence from epizootic subpopulations within enzootic VEEV populations, a hypothesis not supported by examination of low-passage enzootic isolates using biochemical separation methods capable of detecting minority epizootic populations (22). (ii) The second mechanism is initiation of outbreaks by the administration of inadequately inactivated VEEV vaccines, a hypothesis supported by the extreme genetic conservation of subtype IAB strains from which inactivated vaccines were made from the late 1930s until the early 1970s, when these vaccines were discontinued in favor of the live-attenuated TC-83 strain (15, 35). However, this hypothesis cannot explain three epizootics caused by subtype IC strains, which have not been used for vaccine production. (iii) The third mechanism is initiation of outbreaks from cryptic transmission cycles of epizootic subtype IAB and IC strains that have never been detected in nature. Despite intensive surveillance in regions of Venezuela and Colombia where epizootics appear to begin, such cycles have not been reported. (iv) Finally, the fourth mechanism involves periodic evolution of epizootic subtype IAB and IC strains from enzootic VEEV progenitors. This hypothesis has been supported by extensive phylogenetic studies (4, 18, 33) and by the epidemiologic and geographic link between closely related enzootic ID and epizootic IC strains from the 1992-to-1993 Venezuelan epizootic (19, 28). Another hypothesis consistent with genetic studies of VEEV strains isolated during the 1995 epidemic is that epidemics could result from the accidental release from laboratories via human infections with epizootic strains (5).

Our data on the epizootic phenotypes of chimeric enzootic/epizootic VEEV strains provide additional support for the hypothesis that epizootic strains evolve periodically from enzootic progenitors via small numbers of mutations in the envelope glycoproteins. By swapping a genetic fragment encoding only seven amino acid differences in the E3 (1), E2 (5), and E1 (1) envelope glycoprotein genes, we generated the epizootic, equine amplification-competent phenotype from the genetic backbone of a Venezuelan subtype ID strain that is closely related to the phylogenetically predicted ancestor of strain EpA and all other epizootic VEEV strains. Although mutations outside this envelope glycoprotein gene region may contribute slightly to equine disease and viremia, such as the viremia duration to day 4 that was exhibited by most horses infected with the epizootic IC strain but not by those infected with the chimera, the envelope mutations included in our chimeric strains appear to be the major determinants of the epizootic phenotype, especially the viremia titers that are responsible for epidemic transmission via mosquito vectors. Envelope glycoprotein mutations have also been shown to mediate changes in the vector host range of VEEV (6, 7), and so determining whether the same mutations affect both mosquito and equine phenotypes will be of epidemiological interest.

Identification of which of these seven amino acid substitutions is critical to the epizootic phenotype awaits further studies using site-directed mutagenesis of the ID genome, which are under way in our laboratory. This information will be critical to not only to understanding the molecular genetic basis of the equine amplification and virulence phenotypes but also to predicting the frequency at which the epizootic mutants are generated in nature via the error-prone replication of RNA viruses (11), including alphaviruses (32). An estimation of this frequency will help to determine if VEE outbreaks are limited more by genetic constraints (an infrequent occurrence of epizootic mutants) or by ecological and epidemiological conditions conducive to efficient transmission among equines by mosquitoes.

Equine pathogenesis of VEEV. In general, VEEV infection of equines can result in (i) inapparent infection, followed by seroconversion; (ii) systemic disease characterized by tachycardia, fever, depression, and anorexia; or (iii) encephalitic disease that follows systemic disease and is often fatal (12, 16). The epizootic strains induce high-level viremia, and VEEV can be isolated from the blood as early as 1 to 2 days after experimental infection, indicating rapid initiation of productive replication. High-level viremia, fever, and lymphopenia correlate with the development of encephalitis (25; R.A.B., unpublished). Horses infected with both epizootic and enzootic strains develop neutralizing antibodies soon after the clearance of virus from the blood, and antibodies usually persist for at least months and probably years in survivors (25, 27). Preexisting VEEV neutralizing antibodies in horses provide protection from fatal VEE and suppress viremia, as shown in subsequent experimental challenge using equine-virulent strains (25).

In the past, only subtype IAB/C strains of VEEV have been implicated in major epizootics (30). These strains can induce high-level viremia and are usually neurovirulent in equines. The pathogenesis of epizootic and enzootic strains in equines is inadequately understood, and we attempted in our experiments to locate the genetic determinants responsible for virulence. Our results underscore the importance of the envelope glycoproteins in the generation of viremia and encephalitic disease. At the moment, we can only speculate about the mechanisms responsible for these phenotypes. However, in previous studies, we found no evidence for reduced replication of the enzootic strain or of chimeric viruses in vitro or in a rodent model (10). In addition, as previously reported the parental viral strains used in this study possess similar levels of resistance/sensitivity to type I murine interferon in vitro, indicating either the unimportance of this antiviral mechanism or the unsuitability of the murine model. Other hypotheses for the difference in enzootic versus epizootic strain pathogenesis, such as more efficient enzootic VEEV clearance from the blood of horses to reduce viremia or the differences in tissue tropism in equines, should be tested. Interestingly, positive-charge amino acid replacements (Arg, Lys) were implicated phylogenetically (4) and were contained in the epizootic fragments shown in this study (Table 1) to transform enzootic VEEV to the equine amplification-competent phenotype. These replacements are similar to E2 mutations known to attenuate alphaviruses, including VEEV, following selection for glycosaminoglycan binding during cell culture passage (3, 4, 8, 9). Understanding their effect on replication and pathogenesis in equines, which respond in a uniquely differential manner to epizootic versus enzootic VEEV strains, will be critical to elucidating the molecular mechanism of VEE emergence.

 
I.P.G. was supported by an NIH supplemental fellowship to grant AI10984 and CDC Vector-Borne Infectious Disease Training Program 417760. S.P. was supported by NIH Emerging and Tropical Diseases T32 training grant AI107536 and by NIH K08 award AI05949. A.C.B. was supported by the James McLaughlin Infection and Immunity Fellowship Fund and NIH Emerging and Tropical Infectious Diseases T32 training grant AI-107526. This research was supported by NIH grants AI39800 and AI38807.

 
* Corresponding author. Mailing address: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. Phone: (409) 747-0758. Fax: (409) 747-2415. E-mail: sweaver{at}utmb.edu.

Present address: Center for Vector-borne Diseases and Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Journal of Virology, July 2005, p. 9128-9133, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.9128-9133.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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