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.     

IRES-Containing VEEV Vaccine Protects Cynomolgus Macaques from IE Venezuelan Equine Encephalitis Virus Aerosol Challenge

Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas that is responsible for severe, sometimes fatal, disease in humans and horses. We previously described an IRES-based VEE vaccine candidate based up the IE serotype that offers complete protection against a lethal subtype IE VEEV challenge in mice. Here we demonstrate the IRES-based vaccine’s ability to protect against febrile disease in cynomolgus macaques. Vaccination was well tolerated and elicited robust neutralizing antibody titers noticed as early as day 14. Moreover, complete protection from disease characterized by absence of viremia and characteristic fever following aerosolized IE VEEV challenge was observed in all vaccinees compared to control animals, which developed clinical disease. Together, these results highlight the safety and efficacy of IRES-based VEEV vaccine to protect against an endemic, pathogenic VEEV IE serotype.     

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.     

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.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge.

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.     

Possible Evidence for Interference with Venezuelan Equine Encephalitis Virus Vaccination of Equines by Pre-Existing Antibody to Eastern or Western Equine Encephalitis Virus, or Both

During 1971, an epizootic of Venezuelan equine encephalitis (VEE) reached the United States. Laboratory tests were performed on a large number of sick, healthy, unvaccinated, and vaccinated horses. Neutralization (N) tests in cell cultures revealed that 153 of 193 (79.3%) equines outside the state of Texas and 175 of 204 (85.8%) within Texas (82.6% overall) had detectable N antibody to VEE virus a week or more after vaccination. Twenty-six of 40 (65%) non-Texas equines and 18 of 29 (62%) Texas equines which had no detectable antibody against VEE virus a week or more after vaccination had N antibody against Eastern equine encephalitis (EEE) or Western equine encephalitis (WEE) virus or both, whereas only 50 of 153 (32.7%) non-Texas equines and 82 of 175 (46.9%) Texas equines with demonstrable N antibody against VEE also had N antibody against EEE and/or WEE virus. In vaccinated equines, significant negative correlations were found between the occurrence of antibody to VEE and antibody to EEE and/or WEE virus. These findings support the hypothesis that pre-existing antibody to EEE and/or WEE virus may modify or interfere with infection by VEE virus. The epizoologic significance of this possibility is discussed briefly.     

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.     

Gamma-Irradiated Venezuelan Equine Encephalitis Vaccines

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.     

Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses

Although alphaviruses have been extensively studied as model systems for the structural organization of enveloped viruses, no structures exist for the phylogenetically distinct eastern equine encephalomyelitis (EEE)-Venezuelan equine encephalomyelitis (VEE) lineage of New World alphaviruses. Here we report the 25-A structure of VEE virus, obtained from electron cryomicroscopy and image reconstruction. The envelope spike glycoproteins of VEE virus have a T=4 icosahedral arrangement, similar to that observed in Old World Sindbis, Semliki Forest, and Ross River alphaviruses. However, VEE virus has pronounced differences in its nucleocapsid structure relative to nucleocapsid structures repeatedly observed in Old World alphaviruses.     

Serologic evidence of Venezuelan equine encephalitis in some wild and domestic populations of southern Texas.

More than 2,500 sera from approximately 30 wild and domestic species in southern Texas were tested for neutralizing antibodies to Venezuelan equine encephalitis (VEE). Virus isolations were also attempted from blood and tissue samples of many of the wild specimens. VEE neutralizing substances were present in a variety of species collected prior to the 1971 epizootic, suggesting that VEE was present and perhaps enzootic in this area before the recent epizootic. Serologic results of this study suggest that deer (Odocoileus virginianus) and feral swine (Sus scrofa) may serve as good indicators or sentinels of VEE transmission. The reservoir of VEE was not established, but results of the study suggest that a number of species or a combination of animal host populations including deer, feral swine, and peccaries (Pecari angulatus) may be involved in the eizootiologyof VEE in southern Texas.     

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses

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.     

Tyrosine hydroxylase activity in Venezuelan equine encephalomyelitis virus infection

In rats inoculated with the Venezuelan equine encephalomyelitis (VEE) virus, a significant decrease was found in the tyrosine hydroxylase activity of neostriatum, midbrain, and hypothalamus during the acute phase of the infection. In animals that survived the acute infection, we observed no changes in the enzymatic activity in the same regions studied. Our findings suggest a vulnerability of the dopaminergic and noradrenergic pathways to the infection produced by VEE virus.     

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Melatonin Decreases Nitric oxide Production, Inducible Nitric oxide Synthase Expression and Lipid Peroxidation Induced by Venezuelan Encephalitis Equine Virus in Neuroblastoma Cell Cultures

Increased expression of inducible nitric oxide synthase has been shown in murine Venezuelan equine encephalitis (VEE) virus infection. In this experimental model, melatonin (MTL) treatment has shown to be beneficial. The aim of this study was to determine the effect of VEE virus on the nitric oxide (NO) production and lipid peroxidation in neuroblastoma cell cultures, and to investigate the role of MTL during cell-virus interaction. Neuroblastoma cells were co-cultured with VEE virus and treated with MTL at doses ranging from 0 to 1.8 mM, for 6, 12, 24 and 48 h. NO and lipid peroxidation were measured in culture supernatants and in the cellular content by nitrite concentration and thiobarbituric acid assay, respectively. Expression of inducible nitric oxide synthase (iNOS) was determined by indirect immunofluorescence. Increased production of NO and lipid peroxidation products were found in supernatants and cellular contents of VEE virus treated cultures. Both NO and lipid peroxidation were decreased by MTL treatment in a time dependent manner. Increased iNOS expression was observed in VEE virus infected cultures that was reduced by MTL treatment. These results could be related to the beneficial role of MTL in the VEE experimental disease and address the possible therapeutic potential of the hormone in human VEE virus infection.     

Analysis of the survival of Venezuelan equine encephalomyelitis virus and possible viral simulants in liquid suspensions

Aims: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. Methods and Results: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4°C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30°C were very similar (between 0·12 and 0·14 log₁₀ per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21°C, VEE and SV were inactivated at twice the rate measured in dd water. Conclusions: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. Significance and Impact of the Study: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE.     

High Intensity Light Increases Olfactory Bulb Melatonin in Venezuelan Equine Encephalitis Virus Infection

In mice infected with the Venezuelan equine encephalomyelitis (VEE) virus and exposed to high intensity light (2500 lux) with a 12 h light: 12 h dark photoperiod, a significant increase in the levels of melatonin in the olfactory bulb was observed. The significance of these findings deserves further studies to understand the mechanisms involved in this effect since the olfactory bulbs have been proposed as first portal for VEE virus entry into the CNS. The increase in melatonin content could represent one of the mechanisms of defense against the viral attack.     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.