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1.  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.
PMCID: PMC2815040  PMID: 19903494
Venezuelan equine encephalitis virus (VEEV); Formalin inactivated vaccines; Gamma irradiated vaccines; Neurovirulence; Alphavirus
2.  The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity 
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.
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.
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.
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.
PMCID: PMC2903468  PMID: 20644615
3.  Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo 
PLoS ONE  2012;7(5):e37242.
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.
In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.
The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.
PMCID: PMC3364240  PMID: 22666347
4.  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.
PMCID: PMC4072787  PMID: 24967809
5.  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.
PMCID: PMC187387  PMID: 12915543
6.  Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains 
Virology Journal  2009;6:206.
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.
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.
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
PMCID: PMC2783036  PMID: 19925641
7.  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.
PMCID: PMC3861189  PMID: 24349588
8.  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.
PMCID: PMC3122536  PMID: 21450977
9.  Genetic Characterization of Venezuelan Equine Encephalitis Virus from Bolivia, Ecuador and Peru: Identification of a New Subtype ID Lineage 
Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. A passive surveillance study was conducted in Peru, Bolivia and Ecuador to determine the arboviral etiology of febrile illness. Patients with suspected viral-associated, acute, undifferentiated febrile illness of <7 days duration were enrolled in the study and blood samples were obtained from each patient and assayed by virus isolation. Demographic and clinical information from each patient was also obtained at the time of voluntary enrollment. In 2005–2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. Since 1993, VEEV has been continuously isolated from patients in Loreto, Peru, and more recently (2005), in Madre de Dios, Peru. We performed phylogenetic analyses with VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that VEEV subtype ID Panama/Peru genotype is the predominant one circulating in Peru. We also demonstrated that VEEV subtype ID strains circulating in Ecuador belong to the Colombia/Venezuela genotype and VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new ID genotype. In summary, we identified a new major lineage of enzootic VEEV subtype ID, information that could aid in the understanding of the emergence and evolution of VEEV in South America.
Author Summary
Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. In 2005–2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. We characterize recent VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that most VEEV from Peru grouped within a particular genetic lineage known to circulate in Panama and Peru whereas the VEEV circulating in Ecuador belong to a genetic lineage that circulates in Colombia and Venezuela. Importantly, the VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new genetic lineage. This finding could aid in the understanding of the emergence and evolution of VEEV in South America and underscores the need for continuous monitoring for VEEV activity.
PMCID: PMC2734058  PMID: 19753102
10.  High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing 
PLoS Pathogens  2010;6(10):e1001146.
We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.
Author Summary
Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that was first identified in Venezuela in 1938. VEEV normally circulates in rodent populations, but during outbreaks it can jump to horses and humans where it can cause debilitating and potentially fatal disease. There are currently no vaccines or antiviral agents against VEEV licensed for use in humans. In this study, we describe a technique that we have developed that allows for the rapid identification of viral mutants that can be useful for studying the basic biology of viral replication. These mutants can also be used to generate vaccines that protect against infection with wild-type virus. We demonstrate the utility of this technique by identifying over 200 mutations spread throughout VEEV genome that make the virus unable to replicate efficiently at higher temperatures (37°C or 40°C.) Furthermore, we show that two of the mutant viruses work as vaccines, and protect mice against lethal infection with VEEV. This technique can be applied to studying other viruses, and may allow for the rapid identification of numerous vaccine candidates.
PMCID: PMC2954836  PMID: 20976195
11.  IRES-driven Expression of the Capsid Protein of the Venezuelan Equine Encephalitis Virus TC-83 Vaccine Strain Increases Its Attenuation and Safety 
The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.
Author Summary
Venezuelan equine encephalitis virus (VEEV) is transmitted by mosquitoes and widely distributed in Central and South America, causing regular outbreaks in horses and humans. Often misdiagnosed as dengue, VEEV infection in humans can lead to lifelong neurological sequelae and is fatal in up to >80% of equine cases, representing a significant socio-economic burden and constant public health threats for developing countries of Latin America. The only available vaccine, the live-attenuated TC-83 strain, is restricted to veterinary use due to its high reactogenicity in humans and risk for reversion to virulence, which could initiate an epidemic. By using an attenuation approach that allows the modulation of the virus capsid protein expression, we generated a new version of TC-83 that is more attenuated but still induces a protective immune response in mice. Additionally, this new vaccine cannot infect mosquitoes, which prevents the risk of spreading in nature. The attenuation approach we describe can be applied to a lot of other alphaviruses to develop vaccines against diseases regularly emerging and threatening developing countries.
PMCID: PMC3649961  PMID: 23675542
12.  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.
PMCID: PMC3767167  PMID: 23351391
Venezuelan equine encephalitis virus; vaccine; alphavirus; internal ribosome entry site
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.
PMCID: PMC1395430  PMID: 16501087
14.  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.
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.
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.
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.
PMCID: PMC2543005  PMID: 18764933
15.  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.
PMCID: PMC3097527  PMID: 21489591
Venezuelan equine encephalitis virus; Alphavirus; human monoclonal antibody; envelope glycoprotein; antibody prophylaxis; antibody therapy
16.  Genetic and Anatomic Determinants of Enzootic Venezuelan Equine Encephalitis Virus Infection of Culex (Melanoconion) taeniopus 
Venezuelan equine encephalitis (VEE) is a re-emerging, mosquito-borne viral disease with the potential to cause fatal encephalitis in both humans and equids. Recently, detection of endemic VEE caused by enzootic strains has escalated in Mexico, Peru, Bolivia, Colombia and Ecuador, emphasizing the importance of understanding the enzootic transmission cycle of the etiologic agent, VEE virus (VEEV). The majority of work examining the viral determinants of vector infection has been performed in the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. Based on the fundamental differences between the epizootic and enzootic cycles, we hypothesized that the virus-vector interaction of the enzootic cycle is fundamentally different from that of the epizootic model. We therefore examined the determinants for VEEV IE infection in the enzootic vector, Culex (Melanoconion) taeniopus, and determined the number and susceptibility of midgut epithelial cells initially infected and their distribution compared to the epizootic virus-vector interaction. Using chimeric viruses, we demonstrated that the determinants of infection for the enzootic vector are different than those observed for the epizootic vector. Similarly, we showed that, unlike A. taeniorhynchus infection with subtype IC VEEV, C. taeniopus does not have a limited subpopulation of midgut cells susceptible to subtype IE VEEV. These findings support the hypothesis that the enzootic VEEV relationship with C. taeniopus differs from the epizootic virus-vector interaction in that the determinants appear to be found in both the nonstructural and structural regions, and initial midgut infection is not limited to a small population of susceptible cells.
Author Summary
Venezuelan equine encephalitis virus (VEEV) is transmitted to humans and horses by mosquitoes in Mexico, Central and South America. These infections can lead to fatal encephalitis in humans as well as horses, donkeys and mules, and there are no licensed vaccines or treatments available for humans. VEEV circulates in two distinct transmission cycles (epizootic and enzootic), which are differentiated by the ecological niche that each virus inhabits. Epizootic strains, those that cause major outbreaks in humans and equids, have been studied extensively and have been used primarily to develop and test several vaccine candidates. In this study, we demonstrate some important differences in the roles of different viral genes between enzootic/endemic versus epizootic VEEV strains that affect mosquito infection as well as differences in the way that enzootic VEEV more efficiently infects the mosquito initially. Our findings have important implications for designing vaccines and for understanding the evolution of VEEV-mosquito interactions.
PMCID: PMC3317907  PMID: 22509419
17.  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.
PMCID: PMC2293000  PMID: 18256144
18.  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.
PMCID: PMC2593782  PMID: 19079600
19.  Efficient Functional Pseudotyping of Oncoretroviral and Lentiviral Vectors by Venezuelan Equine Encephalitis Virus Envelope Proteins 
Journal of Virology  2005;79(2):756-763.
Murine oncoretroviruses and lentiviruses pseudotyped with envelope proteins of alphaviruses have shown great potential in providing broad-host-range, stable vectors for gene therapy. Unlike vesicular stomatitis virus G protein-pseudotyped vectors, they are not neutralized by complement and do not appear to cause significant tissue damage. Here we report the production of murine oncoretroviral and lentiviral vectors pseudotyped with the envelope proteins of Venezuelan equine encephalitis virus (VEEV). When optimized, these pseudotypes achieve titers of 106 CFU/ml, which is 5- to 10-fold higher than for previous vectors pseudotyped with envelope proteins from other alphaviruses. They can also be concentrated or stored frozen without significant loss of infectivity. Consistent with the tropism of the envelope donor, they transduce a broad array of human cell types, including lung epithelial cells, neuronal cells, lymphocytes, and fibroblasts. Infection is blocked by agents that inhibit endosomal acidification and by neutralizing antibodies against VEEV. These observations indicate that the pseudotypes present native epitopes on their surface and enter through a VEEV envelope-dependent, pH-sensitive mechanism. The fact that the pseudotypes are unaffected by sera reactive to other alphaviruses indicates that they may be useful when successive gene therapies are required in the presence of an active immune response. In this case, having an array of alphavirus-based vectors with similar cell tropisms would be highly advantageous. These vectors may also be useful in diagnostic assays in which infectious VEEV is undesirable but immune reactivity to native epitopes is required.
PMCID: PMC538582  PMID: 15613303
20.  The Role of IKKβ in Venezuelan Equine Encephalitis Virus Infection 
PLoS ONE  2014;9(2):e86745.
Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKβ component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKβ component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKβ in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKβ function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKβ function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKβ for VEEV replication, we over-expressed IKKβ in cells and observed an increase in viral titers. In contrast, studies carried out using IKKβ−/− cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKβ may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKβ protein may be critically involved in VEEV replication.
PMCID: PMC3929299  PMID: 24586253
21.  Serological Evidence of Flaviviruses and Alphaviruses in Livestock and Wildlife in Trinidad 
Vector Borne and Zoonotic Diseases  2012;12(11):969-978.
Seroprevalence rates of selected arboviruses in animal populations in Trinidad were determined using serum samples collected between 2006 and 2009 from horses (n=506), cattle (n=163), sheep (n=198), goats (n=82), pigs (n=184), birds (n=140), rodents (n=116), and other vertebrates (n=23). The sera were screened for antibodies to West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Ilheus virus (ILHV), Bussuquara virus (BSQV), Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), using hemagglutination inhibition assay (HIA) and epitope-blocking enzyme-linked immunosorbent assays (ELISA). Antibodies to SLEV were detected in a total of 49 (9.7%) horses, 8 (4.9%) cattle, 1 (1.2%) goat, 2 (1.4%) wild birds, and 3 (2.2%) wild rodents by both methods. In contrast, antibodies to EEEV, VEEV, and WNV were detected only in horses, at rates of 4.3%, 0.8%, and 17.2%, respectively, by ELISA, and IgM capture ELISA was WNV-positive in 3 (0.6%) of these sera. Among locally bred unvaccinated horses that had never left Trinidad, seroprevalence rates against WNV were 12.1% and 17.2% by ELISA and HIA, respectively. The presence of WNV- and SLEV-specific antibodies in a representative sample of horse sera that were both ELISA- and HIA-seropositive was confirmed by plaque reduction neutralization testing (PRNT). Antibodies to ILHV and BSQV were not detected in any of the serum samples tested (i.e., sera from horses, other livestock, and wild birds in the case of ILHV, and wild mammals in the case of BSQV). The data indicate the presence of WNV in Trinidad, and continuing low-level circulation of SLEV, EEEV, and VEEV.
PMCID: PMC3491626  PMID: 22989182
Alphavirus; Arbovirus; Eastern equine encephalitis virus; Flavivirus; Seroprevalence; St. Louis encephalitis virus; Trinidad; Venezuelan equine encephalitis virus; West Nile virus
22.  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.
PMCID: PMC3319612  PMID: 22496857
23.  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.
PMCID: PMC2447162  PMID: 18468653
Venezuelan equine encephalitis virus; Sindbis virus; pathogenic alphaviruses; antiviral agents; antisense therapy; morpholino oligomers
24.  Venezuelan Equine Encephalitis Virus Activity in the Gulf Coast Region of Mexico, 2003–2010 
Venezuelan equine encephalitis virus (VEEV) has been the causative agent for sporadic epidemics and equine epizootics throughout the Americas since the 1930s. In 1969, an outbreak of Venezuelan equine encephalitis (VEE) spread rapidly from Guatemala and through the Gulf Coast region of Mexico, reaching Texas in 1971. Since this outbreak, there have been very few studies to determine the northward extent of endemic VEEV in this region. This study reports the findings of serologic surveillance in the Gulf Coast region of Mexico from 2003–2010. Phylogenetic analysis was also performed on viral isolates from this region to determine whether there have been substantial genetic changes in VEEV since the 1960s. Based on the findings of this study, the Gulf Coast lineage of subtype IE VEEV continues to actively circulate in this region of Mexico and appears to be responsible for infection of humans and animals throughout this region, including the northern State of Tamaulipas, which borders Texas.
Author Summary
Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. In 1969, an outbreak of Venezuelan equine encephalitis (VEE) spread rapidly from Guatemala and through the Gulf Coast region of Mexico, reaching Texas in 1971. Since this outbreak, there has been very little done to understand the ecology of VEEV in this region. Here, we present that the results of recent field studies that focus on confirming the continued existence of enzootic VEEV in the Gulf Coast region of Mexico. We performed serological analyses of sera collected between 2003 and 2010 from humans, cattle, horses, and dogs in various regions along the Gulf Coast of Mexico, and these data were complemented by wildcaught rodent serosurveys. Additionally, phylogenetic analyses were performed on VEEV isolates from this region to determine whether there have been substantial genetic changes in these viruses since the 1960s.
PMCID: PMC3486887  PMID: 23133685
25.  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.
PMCID: PMC2753124  PMID: 19656875

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