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1.  The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity 
Background
Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.
Methods
We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.
Findings
Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.
Conclusions
The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.
Author Summary
Although the murine immune response to Venezuelan equine encephalitis virus (VEEV) is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119.
doi:10.1371/journal.pntd.0000739
PMCID: PMC2903468  PMID: 20644615
2.  A Multisystem Approach for Development and Evaluation of Inactivated Vaccines for Venezuelan Equine Encephalitis Virus (VEEV) 
A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64°C for periods >30 minutes inactivated the virus, but substantially reduced VEEV specific monoclonal antibody binding of the inactivated material. In contrast, V3526 treated either with formalin at concentrations of 0.1% or 0.5% v/v for 4 or 24 hours, or irradiated with 50 kilogray gamma radiation rendered the virus non-infectious while retaining significant levels of monoclonal antibody binding. Loss of infectivity of both the formalin inactivated (fV3526) and gamma irradiated (gV3526) preparations was confirmed via five successive blind passages on BHK-21 cells. Similarly, loss of neurovirulence for fV3526 and gV3526 was demonstrated via intracerebral inoculation of suckling BALB/c mice. Excellent protection against subcutaneous challenge with VEEV IA/B Trinidad donkey strain was demonstrated using a two dose immunization regimen with either fV3526 or gV3526. The combination of in vitro and in vivo assays provides a practical approach to optimize manufacturing process parameters for development of other inactivated viral vaccines.
doi:10.1016/j.jviromet.2009.11.006
PMCID: PMC2815040  PMID: 19903494
Venezuelan equine encephalitis virus (VEEV); Formalin inactivated vaccines; Gamma irradiated vaccines; Neurovirulence; Alphavirus
3.  Treatment of mice with human monoclonal antibody 24 hours after lethal aerosol challenge with virulent Venezuelan equine encephalitis virus prevents disease but not infection 
Virology  2011;414(2):146-152.
We recently described a Venezuelan equine encephalitis virus (VEEV)-specific human monoclonal antibody (MAb), F5 nIgG, that recognizes a new neutralization epitope on the VEEV E2 envelope glycoprotein. In this study, we investigated the ability of F5 nIgG given prophylactically or therapeutically to protect mice from subcutaneous or aerosolized VEEV infection. F5 nIgG had potent ability to protect mice from infection by either route when administered 24 h before exposure; however, mice treated 24 h after aerosol exposure developed central nervous system infections but exhibited no clinical signs of disease. Infectious virus, viral antigen and RNA were detected in brains of both treated and untreated mice 2–6 days after aerosol exposure but were cleared from the brains of treated animals by 14–28 days after infection. This fully human MAb could be useful for prophylaxis or immediate therapy for individuals exposed to VEEV accidentally in the laboratory or during a deliberate release.
doi:10.1016/j.virol.2011.03.016
PMCID: PMC3097527  PMID: 21489591
Venezuelan equine encephalitis virus; Alphavirus; human monoclonal antibody; envelope glycoprotein; antibody prophylaxis; antibody therapy
4.  IRES-Based Venezuelan Equine Encephalitis Vaccine Candidate Elicits Protective Immunity in Mice 
Virology  2013;437(2):81-88.
Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes periodic outbreaks that impact equine and human populations in the Americas. One of the VEEV subtypes located in Mexico and Central America (IE) has recently been recognized as an important cause of equine disease and death, and human exposure also appears to be widespread. Here, we describe the use of an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus to stably attenuate VEEV, creating a vaccine candidate independent of unstable point mutations. Mice infected with this virus produced antibodies and were protected against lethal VEEV challenge. This IRES-based vaccine was unable to establish productive infection in mosquito cell cultures or in intrathoracically injected Aedes taeniorhynchus, demonstrating that it cannot be transmitted from a vaccinee. These attenuation, efficacy and safety results justify further development for humans or equids of this new VEEV vaccine candidate.
doi:10.1016/j.virol.2012.11.013
PMCID: PMC3767167  PMID: 23351391
Venezuelan equine encephalitis virus; vaccine; alphavirus; internal ribosome entry site
5.  Comparative analysis of the alphavirus-based vectors expressing Rift Valley fever virus glycoproteins 
Virology  2007;366(1):212-225.
During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.
doi:10.1016/j.virol.2007.04.014
PMCID: PMC2065871  PMID: 17507072
alphaviruses; recombinant vaccines; chimeric viruses
6.  A DNA Vaccine for Venezuelan Equine Encephalitis Virus Delivered by Intramuscular Electroporation Elicits High Levels of Neutralizing Antibodies in Multiple Animal Models and Provides Protective Immunity to Mice and Nonhuman Primates ▿ 
We evaluated the immunogenicity and protective efficacy of a DNA vaccine expressing codon-optimized envelope glycoprotein genes of Venezuelan equine encephalitis virus (VEEV) when delivered by intramuscular electroporation. Mice vaccinated with the DNA vaccine developed robust VEEV-neutralizing antibody responses that were comparable to those observed after administration of the live-attenuated VEEV vaccine TC-83 and were completely protected from a lethal aerosol VEEV challenge. The DNA vaccine also elicited strong neutralizing antibody responses in rabbits that persisted at high levels for at least 6 months and could be boosted by a single additional electroporation administration of the DNA performed approximately 6 months after the initial vaccinations. Cynomolgus macaques that received the vaccine by intramuscular electroporation developed substantial neutralizing antibody responses and after an aerosol challenge had no detectable serum viremia and had reduced febrile reactions, lymphopenia, and clinical signs of disease compared to those of negative-control macaques. Taken together, our results demonstrate that this DNA vaccine provides a potent means of protecting against VEEV infections and represents an attractive candidate for further development.
doi:10.1128/CVI.00030-11
PMCID: PMC3122536  PMID: 21450977
7.  Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic 
Journal of Virology  2003;77(17):9278-9286.
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.
doi:10.1128/JVI.77.17.9278-9286.2003
PMCID: PMC187387  PMID: 12915543
8.  Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains 
Virology Journal  2009;6:206.
Background
There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.
Results
In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.
Conclusion
A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.
Crown Copyright © 2009
doi:10.1186/1743-422X-6-206
PMCID: PMC2783036  PMID: 19925641
9.  Venezuelan Equine Encephalitis Virus in Iquitos, Peru: Urban Transmission of a Sylvatic Strain 
Enzootic strains of Venezuelan equine encephalitis virus (VEEV) have been isolated from febrile patients in the Peruvian Amazon Basin at low but consistent levels since the early 1990s. Through a clinic-based febrile surveillance program, we detected an outbreak of VEEV infections in Iquitos, Peru, in the first half of 2006. The majority of these patients resided within urban areas of Iquitos, with no report of recent travel outside the city. To characterize the risk factors for VEEV infection within the city, an antibody prevalence study was carried out in a geographically stratified sample of urban areas of Iquitos. Additionally, entomological surveys were conducted to determine if previously incriminated vectors of enzootic VEEV were present within the city. We found that greater than 23% of Iquitos residents carried neutralizing antibodies against VEEV, with significant associations between increased antibody prevalence and age, occupation, mosquito net use, and overnight travel. Furthermore, potential vector mosquitoes were widely distributed across the city. Our results suggest that while VEEV infection is more common in rural areas, transmission also occurs within urban areas of Iquitos, and that further studies are warranted to identify the precise vectors and reservoirs involved in urban VEEV transmission.
Author Summary
Venezuelan equine encephalitis (VEE) is a mosquito-borne viral disease often causing grave illness and large outbreaks of disease in South America. In Iquitos, Peru, a city of 350,000 situated in the Amazon forest, we normally observe 10–14 VEE cases per year associated with people traveling to rural areas where strains VEE virus circulate among forest mosquitoes and rodents. In 2006 we detected a 5-fold increase in human VEE cases, and many of these patients had no travel history outside the city where they lived. In response to this outbreak, we decided to determine if potential carrier mosquitoes were present within the city and if city residents had been previously exposed to the virus. We found that mosquitoes previously shown to transmit the virus in other locations were present—in varying amounts based on location and time of year—throughout Iquitos. A large percentage of the human population (>23%) had antibodies indicating past exposure to the virus. Previous VEE infection was associated with age, occupation, mosquito exposure, and overnight travel. Our data represent evidence of transmission of a forest strain of VEE within a large urban area. Continued monitoring of this situation will shed light on mechanisms of virus emergence.
doi:10.1371/journal.pntd.0000349
PMCID: PMC2593782  PMID: 19079600
10.  Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers 
Virology  2008;376(2):357-370.
The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5′-terminal and AUG translation start site-regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.
doi:10.1016/j.virol.2008.03.032
PMCID: PMC2447162  PMID: 18468653
Venezuelan equine encephalitis virus; Sindbis virus; pathogenic alphaviruses; antiviral agents; antisense therapy; morpholino oligomers
11.  Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2 
PLoS Pathogens  2014;10(6):e1004213.
Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.
Author Summary
Alphaviruses occur worldwide, causing significant diseases such as encephalitis or arthritis in humans and animals. In addition, some alphaviruses, such as VEEV, pose a biothreat due to their high infectivity and lack of available treatments. To discover small molecule inhibitors with lead development potential, we used a cell-based assay to screen 348,140 compounds for inhibition of a VEEV-induced cytopathic effect. The screen revealed a scaffold with high inhibitory VEEV cellular potency and low cytotoxicity liability. While most previously reported anti-alphavirus compounds inhibit host proteins, evidence supported that this scaffold targeted the VEEV nsP2 protein, and that inhibition was associated with viral replication. Interestingly, compound resistance studies with VEEV mapped activity to the N-terminal domain of nsP2, to which no known function has been attributed. Ultimately, this discovery has delivered a small molecule-derived class of potent VEEV inhibitors whose activity is coupled to the nsP2 viral protein, a novel target with a previously unestablished biological role that is now implicated in viral replication.
doi:10.1371/journal.ppat.1004213
PMCID: PMC4072787  PMID: 24967809
12.  Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus 
Journal of Virology  2013;87(9):4952-4964.
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.
doi:10.1128/JVI.03361-12
PMCID: PMC3624300  PMID: 23408633
13.  Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses 
Journal of Virology  2006;80(6):2784-2796.
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
doi:10.1128/JVI.80.6.2784-2796.2006
PMCID: PMC1395430  PMID: 16501087
14.  Venezuelan Equine Encephalitis Virus Disrupts STAT1 Signaling by Distinct Mechanisms Independent of Host Shutoff▿  
Journal of Virology  2009;83(20):10571-10581.
Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-β and IFN-γ. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-β treatment but did inhibit Jak1 and Jak2 activation in response to IFN-γ, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.
doi:10.1128/JVI.01041-09
PMCID: PMC2753124  PMID: 19656875
15.  Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge. 
Journal of Virology  1988;62(12):4697-4702.
Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.
PMCID: PMC253584  PMID: 3184276
16.  Candidate Vectors and Rodent Hosts of Venezuelan Equine Encephalitis Virus, Chiapas, 2006–2007 
Enzootic Venezuelan equine encephalitis virus (VEEV) has been known to occur in Mexico since the 1960s. The first natural equine epizootic was recognized in Chiapas in 1993 and since then, numerous studies have characterized the etiologic strains, including reverse genetic studies that incriminated a specific mutation that enhanced infection of epizootic mosquito vectors. The aim of this study was to determine the mosquito and rodent species involved in enzootic maintenance of subtype IE VEEV in coastal Chiapas. A longitudinal study was conducted over a year to discern which species and habitats could be associated with VEEV circulation. Antibody was rarely detected in mammals and virus was not isolated from mosquitoes. Additionally, Culex (Melanoconion) taeniopus populations were found to be spatially related to high levels of human and bovine seroprevalence. These mosquito populations were concentrated in areas that appear to represent foci of stable, enzootic VEEV circulation.
doi:10.4269/ajtmh.2011.11-0094
PMCID: PMC3225169  PMID: 22144461
17.  Role of adhesion molecules and inflammation in Venezuelan equine encephalitis virus infected mouse brain 
Virology Journal  2011;8:197.
Background
Neuroinvasion of Venezuelan equine encephalitis virus (VEEV) and subsequent initiation of inflammation in the brain plays a crucial role in the outcome of VEEV infection in mice. Adhesion molecules expressed on microvascular endothelial cells in the brain have been implicated in the modulation of the blood brain barrier (BBB) and inflammation in brain but their role in VEEV pathogenesis is not very well understood. In this study, we evaluated the expression of extracellular matrix and adhesion molecules genes in the brain of VEEV infected mice.
Findings
Several cell to cell adhesion molecules and extracellular matrix protein genes such as ICAM-1, VCAM-1, CD44, Cadherins, integrins, MMPs and Timp1 were differentially regulated post-VEEV infection. ICAM-1 knock-out (IKO) mice infected with VEEV had markedly reduced inflammation in the brain and demonstrated a delay in the onset of clinical symptoms of disease. A differential regulation of inflammatory genes was observed in the IKO mice brain compared to their WT counterparts.
Conclusions
These results improve our present understanding of VEEV induced inflammation in mouse brain.
doi:10.1186/1743-422X-8-197
PMCID: PMC3113303  PMID: 21529366
18.  Modulation of GSK-3β Activity in Venezuelan Equine Encephalitis Virus Infection 
PLoS ONE  2012;7(4):e34761.
Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC50 of ∼0.5 µM and a CC50 of >100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection.
doi:10.1371/journal.pone.0034761
PMCID: PMC3319612  PMID: 22496857
19.  Venezuelan Equine Encephalitis Viruses (VEEV) in Argentina: Serological Evidence of Human Infection 
Venezuelan equine encephalitis viruses (VEEV) are responsible for human diseases in the Americas, producing severe or mild illness with symptoms indistinguishable from dengue and other arboviral diseases. For this reason, many cases remain without certain diagnosis. Seroprevalence studies for VEEV subtypes IAB, ID, IF (Mosso das Pedras virus; MDPV), IV (Pixuna virus; PIXV) and VI (Rio Negro virus; RNV) were conducted in persons from Northern provinces of Argentina: Salta, Chaco and Corrientes, using plaque reduction neutralization test (PRNT). RNV was detected in all studied provinces. Chaco presented the highest prevalence of this virus (14.1%). Antibodies against VEEV IAB and -for the first time- against MDPV and PIXV were also detected in Chaco province. In Corrientes, seroprevalence against RNV was 1.3% in the pediatric population, indicating recent infections. In Salta, this was the first investigation of VEEV members, and antibodies against RNV and PIXV were detected. These results provide evidence of circulation of many VEE viruses in Northern Argentina, showing that surveillance of these infectious agents should be intensified.
Author Summary
Venezuelan equine encephalitis viruses (VEEV) are responsible for human diseases in the Americas. They produce severe or mild illnesses with symptoms indistinguishable from dengue and other arboviral diseases; for this reason, many cases remain undiagnosed. We detected neutralizing antibodies (NTAbs) against VEEV IAB, VEEV ID, MDPV (VEEV subtype IF), PIXV (VEEV subtype IV) and RNV (VEEV subtype VI) in human serum samples of Northern provinces of Argentina. Chaco province showed presence of NTAbs against VEEV IAB, MDPV, PIXV and RNV. In Corrientes province, we detected NTAbs against RNV in a pediatric population. NTAbs against PIXV and RNV were also detected in Salta province. These findings demonstrated the circulation of many VEEV strains in Northern Argentina and underscore the need for surveillance of dengue like illness in this region.
doi:10.1371/journal.pntd.0002551
PMCID: PMC3861189  PMID: 24349588
20.  Host responses to live-attenuated Venezuelan equine encephalitis virus (TC-83) 
Human Vaccines & Immunotherapeutics  2012;8(8):1053-1065.
Venezuelan equine encephalitis virus (VEEV) is a positive-strand RNA Alphavirus endemic in Central and South America, and the causative agent of fatal encephalitis in humans. In an effort to better understand the mechanisms of infection, including differences between people who produce a neutralizing antibody response to the vaccine and those who do not, we performed whole genome transcriptional analysis in human PBMCs exposed in vitro to the live-attenuated vaccine strain of VEEV, TC-83. We compared the molecular responses in cells from three groups of individuals: naïve; previously vaccinated individuals who developed a neutralizing antibody response to the vaccine (responders); and those who did not develop a neutralizing antibody response to the vaccine (nonresponders). Overall, the changes in gene expression were more intense for the naïve group after TC-83 challenge and least potent in the nonresponder group. The main canonical pathways revealed the involvement of interferon and interferon-induced pathways, as well as toll-like receptors TLR- and interleukin (IL)-12-related pathways. HLA class II genotype and suppression of transcript expression for TLR2, TLR4 and TLR8 in the nonresponder group may help explain the lack of vaccine response in this study group. Because TL3 and TLR7 transcripts were elevated in all study groups, these factors may be indicators of the infection and not the immunological state of the individuals. Biomarkers were identified that differentiate between the vaccine responder and the vaccine nonresponder groups. The identified biomarkers were contrasted against transcripts that were unique to the naïve population alone upon induction with TC-83. Biomarker analysis allowed for the discernment between the naïve (innate) responses; the responder (recall) responses; and the nonresponder (alternative) changes to gene transcription that were caused by infection with TC-83. The study also points to the existence of HLA haplotypes that may discriminate between vaccine low- and high-responder phenotypes.
doi:10.4161/hv.20300
PMCID: PMC3551876  PMID: 22617845
biomarkers; gene expression; microarray; neutralizing antibody; vaccination; vaccine responder; Venezuelan equine encephalitis virus
21.  Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV) 
BMC Biotechnology  2008;8:66.
Background
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.
Results
In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.
Conclusion
For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.
doi:10.1186/1472-6750-8-66
PMCID: PMC2543005  PMID: 18764933
22.  Experimental Infection of Potential Reservoir Hosts with Venezuelan Equine Encephalitis Virus, Mexico 
Emerging Infectious Diseases  2009;15(4):519-525.
Multiple wild rodent species can serve as amplifying reservoir hosts for virus subtype IE.
In 1993, an outbreak of encephalitis among 125 affected equids in coastal Chiapas, Mexico, resulted in a 50% case-fatality rate. The outbreak was attributed to Venezuelan equine encephalitis virus (VEEV) subtype IE, not previously associated with equine disease and death. To better understand the ecology of this VEEV strain in Chiapas, we experimentally infected 5 species of wild rodents and evaluated their competence as reservoir and amplifying hosts. Rodents from 1 species (Baiomys musculus) showed signs of disease and died by day 8 postinoculation. Rodents from the 4 other species (Liomys salvini, Oligoryzomys fulvescens, Oryzomys couesi, and Sigmodon hispidus) became viremic but survived and developed neutralizing antibodies, indicating that multiple species may contribute to VEEV maintenance. By infecting numerous rodent species and producing adequate viremia, VEEV may increase its chances of long-term persistence in nature and could increase risk for establishment in disease-endemic areas and amplification outside the disease-endemic range.
doi:10.3201/eid1504.081008
PMCID: PMC2671456  PMID: 19331726
Zoonoses; vector-borne infections; bioterrorism agents and preparedness; encephalomyelitis virus; alphaviruses; transmission; Mexico; strains; wild rodents; research
23.  Evaluation in Nonhuman Primates of Vaccines against Ebola Virus 
Emerging Infectious Diseases  2002;8(5):503-507.
Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.
doi:10.3201/eid0805.010284
PMCID: PMC3369765  PMID: 11996686
Keywords: Ebola; macaque; vaccine; Vaccinia virus; replicon
24.  Pegylated Alpha Interferon Is an Effective Treatment for Virulent Venezuelan Equine Encephalitis Virus and Has Profound Effects on the Host Immune Response to Infection 
Journal of Virology  2000;74(11):5006-5015.
Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus endemic in parts of Central and South America. The disease is transmitted by mosquitoes, and the natural reservoir is the small rodent population, with epidemics occurring in horses and occasionally humans. Following infection, VEEV replicates in lymphoid tissues prior to invasion of the central nervous system. Treatment of VEEV-infected BALB/c mice with polyethylene glycol-conjugated alpha interferon (PEG IFN-α) results in a greatly enhanced survival from either a subcutaneous or an aerosol infection. Virus is undetectable within PEG IFN-α-treated individuals by day 30 postinfection (p.i.). Treatment results in a number of changes to the immune response characteristics normally associated with VEEV infection. Increased macrophage activation occurs in PEG IFN-α-treated BALB/c mice infected with VEEV. The rapid activation of splenic CD4, CD8, and B cells by day 2 p.i. normally associated with VEEV infection is absent in PEG IFN-α-treated mice. The high tumor necrosis factor alpha production by macrophages from untreated mice is greatly diminished in PEG IFN-α-treated mice. These results suggest key immunological mechanisms targeted by this lethal alphavirus that can be modulated by prolonged exposure to IFN-α.
PMCID: PMC110852  PMID: 10799574
25.  Venezuelan Equine Encephalitis Virus Capsid Protein Inhibits Nuclear Import in Mammalian but Not in Mosquito Cells▿  
Journal of Virology  2008;82(8):4028-4041.
Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its ∼35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a significant fraction of VEEV capsid associated with nuclear envelope, which suggested that this protein might regulate nucleocytoplasmic trafficking. In this study, we demonstrate that VEEV capsid and its N-terminal sequence efficiently inhibit multiple receptor-mediated nuclear import pathways but have no effect on the passive diffusion of small proteins. The capsid protein of the Old World alphavirus Sindbis virus and the VEEV capsid, with a previously defined frameshift mutation, were found to have no detectable effect on nuclear import. Importantly, the VEEV capsid did not noticeably interfere with nuclear import in mosquito cells, and this might play a critical role in the ability of the virus to develop a persistent, life-long infection in mosquito vectors. These findings demonstrate a new aspect of VEEV-host cell interactions, and the results of this study are likely applicable to other New World alphaviruses, such as eastern and western equine encephalitis viruses.
doi:10.1128/JVI.02330-07
PMCID: PMC2293000  PMID: 18256144

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