A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine.

Continuing emergence of variants of concern resulting in reduced SARS-CoV-2 vaccine efficacy necessitates additional prevention strategies. The structure of VLPCOV-01, a lipid nanoparticle-encapsulated, self-amplifying RNA COVID-19 vaccine with a comparable immune response to BNT162b2, was revised by incorporating a modified base, 5-methylcytosine, to reduce reactogenicity, and an updated receptor-binding domain derived from the Brazil (gamma) variant. Interim analyses of a phase 1 dose-escalation booster vaccination study with the resulting construct, VLPCOV-02, in healthy, previously vaccinated Japanese individuals (N = 96) are reported (jRCT2051230005). A dose-related increase in solicited local and systemic adverse events was observed, which were generally rated mild or moderate. The most commonly occurring events were tenderness, pain, fatigue, and myalgia. Serum SARS-CoV-2 immunoglobulin titers increased during the 4 weeks post-immunization. VLPCOV-02 demonstrated a favorable safety profile compared with VLPCOV-01, with reduced adverse events and fewer fever events at an equivalent dose. These findings support further study of VLPCOV-02.

Incorporation of 5 methylcytidine alleviates innate immune response to self-amplifying RNA vaccine.

In order to improve vaccine effectiveness and safety profile of existing synthetic RNA-based vaccines, we have developed a self-amplifying RNA (saRNA)-based vaccine expressing membrane-anchored receptor binding domain (RBD) of SARS-CoV-2 S protein (S-RBD) and have demonstrated that a minimal dose of this saRNA vaccine elicits robust immune responses. Results from a recent clinical trial with 5-methylcytidine (5mC) incorporating saRNA vaccine demonstrated reduced vaccine-induced adverse effects while maintaining robust humoral responses. In this study, we investigate the mechanisms accounting for induction of efficient innate and adaptive immune responses and attenuated adverse effects induced by the 5mC-incorporated saRNA. We show that the 5mC-incorporating saRNA platform leads to prolonged and robust expression of antigen, while induction of type-I interferon (IFN-I), a key driver of reactogenicity, is attenuated in peripheral blood mononuclear cells (PBMCs), but not in macrophages and dendritic cells. Interestingly, we find that the major cellular source of IFN-I production in PBMCs is plasmacytoid dendritic cells (pDCs), which is attenuated upon 5mC incorporation in saRNA. In addition, we demonstrate that monocytes also play an important role in amplifying proinflammatory responses. Furthermore, we show that the detection of saRNA is mediated by a host cytosolic RNA sensor, RIG-I. Importantly, 5mC-incorporating saRNA vaccine candidate produced robust IgG responses against S-RBD upon injection in mice, thus providing strong support for the potential clinical use of 5mC-incorporating saRNA vaccines.

Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. A phase 1 study of VLPCOV-01 is conducted (jRCT2051210164). Participants who completed two doses of the BNT162b2 mRNA vaccine previously are randomized to receive one intramuscular vaccination of 0.3, 1.0, or 3.0 µg VLPCOV-01, 30 µg BNT162b2, or placebo. No serious adverse events have been reported. VLPCOV-01 induces robust immunoglobulin G (IgG) titers against the RBD protein that are maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,037 (95% confidence interval [CI] 1,272-19,940) at 0.3 µg to 12,873 (95% CI 937-17,686) at 3 µg compared with 3,166 (95% CI 1,619-6,191) with 30 µg BNT162b2. Neutralizing antibody titers against all variants of SARS-CoV-2 tested are induced. VLPCOV-01 is immunogenic following low-dose administration. These findings support the potential for saRNA as a vaccine platform.

saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern.

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates.

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 x 10(6)pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

BACKGROUND: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. CONCLUSIONS/SIGNIFICANCE: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

Development and preclinical evaluation of an alphavirus replicon vaccine for influenza.

We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

Restricted and selective tropism of a Venezuelan equine encephalitis virus-derived replicon vector for human dendritic cells.

Dendritic cells (DCs) consist of heterogeneous phenotypic populations and have diverse immunostimulatory functions dependent on both lineage and functional phenotype, but as exceptionally potent antigen-presenting cells, they are targets for generating effective antigen-specific immune responses. A promising replicon particle vector derived from Venezuelan equine encephalitis virus (VEE) has been reported to transduce murine footpad DCs. However, the receptive DC subset, the degree of restriction for this tropism, and the extent of conservation between rodents and humans have not been well characterized. Using fresh peripheral blood DCs, mononuclear cells, monocyte-derived macrophages, and monocyte-derived DCs, our results demonstrate conservation of VEE replicon particle (VRP) tropism for DCs between humans and rodents. We observed that a subset of immature myeloid DCs is the target population, and that VRP-transduced immature DCs retain intact functional capacity, for example, the ability to resist the cytopathic effects of VRP transduction and the capacity to acquire the mature phenotype. These studies support the demonstration of selective VRP tropism for human DCs and provide further insight into the biology of the VRP vector, its parent virus, and human DCs.

VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model.

The ability to overcome intrinsic tolerance to a strict "self" tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent "self" TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.

Preconceptual administration of an alphavirus replicon UL83 (pp65 homolog) vaccine induces humoral and cellular immunity and improves pregnancy outcome in the guinea pig model of congenital cytomegalovirus infection.

Development of a vaccine against congenital cytomegalovirus (CMV) infection is a major public health priority. We report the use of a propagation-defective, single-cycle, RNA replicon vector system, derived from an attenuated strain of the alphavirus Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRPs) expressing GP83, the guinea pig CMV (GPCMV) homolog of the human CMV pp65 phosphoprotein. Vaccination with VRP-GP83 induced antibodies and CD4(+) and CD8(+) T cell responses in GPCMV-seronegative female guinea pigs. Guinea pigs immunized with VRP-GP83 vaccine or with a VRP vaccine expressing influenza hemagglutinin (VRP-HA) were bred for pregnancy and subsequent GPCMV challenge during the early third trimester. Dams vaccinated with VRP-GP83 had improved pregnancy outcomes, compared with dams vaccinated with the VRP-HA control. For VRP-GP83-vaccinated dams, there were 28 live pups and 4 dead pups (13% mortality) among 10 evaluable litters, compared with 9 live pups and 12 dead pups (57% mortality) among 8 evaluable litters in the VRP-HA-vaccinated group (P<.001, Fisher's exact test). Improved pregnancy outcome was accompanied by reductions in maternal blood viral load, measured by real-time polymerase chain reaction. These results indicate that cell-mediated immune responses directed against a CMV matrix protein can protect against congenital CMV infection and disease.

Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice.

The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.

Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models.

The central objective of this research was to test molecularly defined, live attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidates that were produced through precise genetic manipulation of rationally selected viral nucleotide sequences. Molecular clones of vaccine candidates were constructed by inserting either three independently attenuating mutations or a PE2 cleavage-signal mutation with a second-site resuscitating mutation into full-length cDNA clones. Vaccine candidate viruses were recovered through DNA transcription and RNA transfection of cultured cells, and assessed in rodent and non-human primate models. Based on results from this assessment, one of the PE2 cleavage-signal mutants, V3526, was determined to be the best vaccine candidate for further evaluation for human use.

Venezuelan equine encephalitis replicon immunization overcomes intrinsic tolerance and elicits effective anti-tumor immunity to the 'self' tumor-associated antigen, neu in a rat mammary tumor model.

Many tumor-associated antigens (TAAs) represent 'self' antigens and as such, are subject to the constraints of immunologic tolerance. There are significant barriers to eliciting anti-tumor immune responses of sufficient magnitude. We have taken advantage of a Venezuelan equine encephalitis-derived alphavirus replicon vector system with documented in vivo tropism for immune system dendritic cells. We have overcome the intrinsic tolerance to the 'self' TAA rat neu and elicited an effective anti-tumor immune response using this alphavirus replicon vector system and a designed target antigen in a rigorous rat mammary tumor model. We have demonstrated the capacity to generate 50% protection in tumor challenge experiments (p = 0.004) and we have confirmed the establishment of immunologic memory by both second tumor challenge and Winn Assay (p = 0.009). Minor antibody responses were identified and supported the establishment of T helper type 1 (Th1) anti-tumor immune responses by isotype. Animals surviving in excess of 300 days with established effective anti-tumor immunity showed no signs of autoimmune phenomena. Together these experiments support the establishment of T lymphocyte dependent, Th1-biased anti-tumor immune responses to a non-mutated 'self' TAA in an aggressive tumor model. Importantly, this tumor model is subject to the constraints of immunologic tolerance present in animals with normal developmental, temporal, and anatomical expression of a non-mutated TAA. These data support the continued development and potential clinical application of this alphaviral replicon vector system and the use of appropriately designed target antigen sequences for anti-tumor immunotherapy.

Recombinant chimeric western and eastern equine encephalitis viruses as potential vaccine candidates.

Chimeric cDNA clones, pMWE1000 and pMWE2000, differing by five nucleotides at their 5' termini, were constructed of the 5' two-thirds of the western equine encephalitis (WEE) virus genome (encoding nonstructural proteins) and the 3' one-third of the eastern equine encephalitis (EEE) virus genome (encoding structural proteins). The WEE virus sequences were derived from full-length cDNA clones, pWE1000 and pWE2000, which were isogenic except for five nucleotide differences at their 5' termini and were responsible for significant differences in mouse virulence. Each cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length RNA. Transfection of BHK-21 cells with the chimeric RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 10(5) PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900-7909).

An outbreak of West Nile virus in a New York City captive wildlife population.

An outbreak of West Nile virus (WNV) in and around New York City during the late summer of 1999 was the cause of extensive mortality among free-ranging birds. Within the Bronx Zoo/Wildlife Conservation Park, viral activity was also observed and produced some morbidity and mortality among specimens in the zoo's bird collection and probably caused morbidity in at least one specimen from the zoo's mammal collection. To determine the extent of the outbreak and attempt to ascertain the temporal appearance of virus within the park, a serologic survey of birds and mammals was performed. The survey showed that 34% of tested birds (125 of 368; 124 species) were positive for antibody to WNV. The virus caused a disease to infection ratio of 22% (27 of 125) among birds with a 70% (19 of 27) case fatality rate. In contrast, only 8% of the mammals (9 of 117; 35 species) possessed antibody to WNV and there was no virus-associated mortality. Testing of banked and fresh sera obtained from both birds and mammals revealed that there was no evidence of WNV circulation before the 1999 outbreak and that birds introduced into the park were not the source of the New York outbreak. West Nile virus RNA was detected in tissues from one bird that died in February 2000, long after the end of the mosquito transmission season. The potential importance of zoologic parks as possible sentinels for emerging diseases is discussed.

Immune protection against staphylococcal enterotoxin-induced toxic shock by vaccination with a Venezuelan equine encephalitis virus replicon.

A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-replicating RNA, termed "replicon," containing the VEE nonstructural genes and cis-acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice inoculated subcutaneously with mSEB-VRPs were protected (15 of 20 mice) from a challenge with 5 median lethal dose units of wild-type (wt) SEB. T cells from mice vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall response to the inactivated mSEB polypeptide. The profile of cytokines measured after challenge with wt SEB suggested that the mode of protection was predominantly Th1 dependent. Our results suggest that the VEE replicon is a practical and convenient model system for evaluating efficacy of vaccines for the control of bacterial diseases.

Geographic distribution of Venezuelan equine encephalitis virus subtype IE genotypes in Central America and Mexico

Phylogenetic analysis of 20 strains of Venezuelan equine encephalitis (VEE) virus subtype IE isolated from 1961 to 1996 in Mexico and throughout Central America showed that VEE virus subtype IE was monophyletic with respect to other VEE virus subtypes. Nonetheless, there were at least three distinct geographically separated VEE virus IE genotypes: northwestern Panama, Pacific coast (Mexico/Guatemala), and Gulf/Caribbean coast (Mexico/Belize). Strains from the Caribbean coast of Guatemala, Honduras, and Nicaragua may cluster with the Gulf/Caribbean genotype, but additional isolates from the region between Guatemala and Panama will be required to firmly establish their phylogenetic position. Viruses associated with two separate equine epizootics in Mexico in the 1990s were phlogenticaly related to nonepizootic viruses from neighbouring Guatemala and may represent the emergence or re-emergence of equine-virulent VEE virus subtype IE in Middle America.(AU)