Eastern, Western, and Venezuelan Equine Encephalitis and West Nile Viruses: Clinical and Public Health Considerations.

The continued recognition and emergence of alphavirus and flavivirus diseases is a growing veterinary and public health concern. As the global environment continues to change, mosquito-borne diseases will continue to evolve and expand. Continued development of readily available vaccines for the prevention of these diseases in humans and animals is essential to controlling epizootics of these diseases. Further research into effective antiviral treatments is also sorely needed. This article describes equine encephalitis viruses with a focus on clinical and public health considerations.

National Seroprevalence and Risk Factors for Eastern Equine Encephalitis and Venezuelan Equine Encephalitis in Costa Rica.

Eastern equine encephalitis and Venezuelan equine encephalitis are endemic neglected tropical diseases in the Americas, causing encephalitis in both horses and humans. In 2013, a cross-sectional study was performed in 243 horses located in the highlands and lowlands throughout Costa Rica. Serum samples were analyzed with an IgG ELISA and confirmed by the plaque-reduction neutralization test (PRNT80). Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) overall seroprevalences by the PRNT80 were 36% (95% confidence interval [CI]: 29.9-42.5; 78/217 horses) and 3% (95% CI: 1.3-5.9; 6/217 horses), respectively. Both the viruses occurred in the lowlands and highlands. Rainfall and altitude were associated with VEEV seropositivity in the univariate analysis, but only altitude <100 meters above sea level was considered a risk factor in the multivariate analysis. No risk factors could be identified for the EEEV in the multivariate analysis. This is the first study that estimates the seroprevalence of the EEEV and VEEV in Costa Rican horses. The VEEV is widely distributed, whereas the EEEV occurs at a much lower frequency and only in specific areas. Clinical cases and occasional outbreaks of both viruses are to be expected.

[Do some conditions contribute to the reemergence of the Venezuelan equine encephalitis virus in the Colombian Alta Guajira?]. / ¿Existen condiciones que favorecen la reaparición del virus de la encefalitis equina venezolana en la Alta Guajira colombiana?

INTRODUCTION: In the last 18 years, epizootics of Venezuelan equine encephalitis have not occurred in places with historic epidemic register (1925-1995) in the Guajira Peninsula, Colombia. OBJECTIVE: To assess if the Guajira Peninsula, Colombia, still maintains the epidemiological conditions for Venezuelan equine encephalitis virus reemergence. MATERIALS AND METHODS: Research was carried out in places affected by the 1995 epidemic. We evaluated: 1) abundance and seasonal variation of vector mosquito populations; 2) availability of mammals that are potential amplifiers of the virus, and 3) knowledge among the community about the disease and its vectors. RESULTS: Most of the 16 mosquito species were found during the rainy season. Aedes taeniorhynchus and Psorophora confinnis showed direct relation with rainfall and temperature. In contrast, the dominant species, Deinocerites atlanticus , was always present in the collections, regardless of climatic conditions. No IgG antibodies were found in humans younger than 17 years old, goats or bovine sera. One third of those interviewed remembered the last epidemic and had basic understanding of the disease. Only 20% of the families were owners of equines, and 8% was informed of the importance of equine vaccination. CONCLUSIONS: Some epidemiological conditions that eventually could help epizootic Venezuelan equine encephalitis virus reemergence are maintained. However, an abrupt decrease in the number of susceptible equines was found in the area. Apparently, this new condition has not allowed the virus reemergence and is the biggest observed change.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

¿Existen condiciones que favorecen la reaparición del virus de la encefalitis equina venezolana en la Alta Guajira colombiana? / Do some conditions contribute to the reemergence of the Venezuelan equine encephalitis virus in the Colombian Alta Guajira?

Introducción. En los últimos 18 años, no se han vuelto a registrar epizootias de encefalitis equina venezolana en áreas con antecedentes históricos de epidemia (1925-1995) en la península de La Guajira. Objetivo. Establecer si en la Alta Guajira colombiana se mantienen las condiciones que favorecen la reaparición de las cepas epizoóticas del virus de la encefalitis equina venezolana. Materiales y métodos. La investigación se realizó en las localidades afectadas por la epidemia de 1995, y se evaluaron los siguientes aspectos: 1) abundancia y variación estacional de las poblaciones de los mosquitos vectores; 2) disponibilidad de mamíferos sensibles amplificadores del virus, y 3) conocimiento de los habitantes sobre la enfermedad y sus vectores. Resultados. La mayoría de las especies de mosquitos, incluidas las de los vectores, se encontraron durante la estación lluviosa. Aedes taeniorhynchus y Psorophora confinnis presentaron relación con la precipitación y la temperatura. Deinocerites atlanticus estuvo siempre presente, independientemente de las condiciones climáticas. No se encontraron anticuerpos de tipo IgG en los sueros de humanos menores de 17 años, tampoco en los de los animales caprinos y bovinos analizados. En la encuesta sobre conocimientos, se detectó que la tercera parte de los encuestados recordaba la última epidemia y tenía conocimientos básicos sobre la enfermedad. Solamente 20 % de las familias poseía équidos y el 8 % estaba informado sobre la necesidad de vacunarlos. Conclusiones. Se mantienen algunas condiciones epidemiológicas que eventualmente podrían favorecer la reaparición del virus, pero el número de equinos en riesgo es escaso, lo cual constituye el cambio más notable y lo que, aparentemente, ha impedido la reaparición del virus.

Introduction: In the last 18 years, epizootics of Venezuelan equine encephalitis have not occurred in places with historic epidemic register (1925-1995) in the Guajira Peninsula, Colombia. Objective: To assess if the Guajira Peninsula, Colombia, still maintains the epidemiological conditions for Venezuelan equine encephalitis virus reemergence. Materials and methods: Research was carried out in places affected by the 1995 epidemic. We evaluated: 1) abundance and seasonal variation of vector mosquito populations; 2) availability of mammals that are potential amplifiers of the virus, and 3) knowledge among the community about the disease and its vectors. Results: Most of the 16 mosquito species were found during the rainy season. Aedes taeniorhynchus and Psorophora confinnis showed direct relation with rainfall and temperature. In contrast, the dominant species, Deinocerites atlanticus , was always present in the collections, regardless of climatic conditions. No IgG antibodies were found in humans younger than 17 years old, goats or bovine sera. One third of those interviewed remembered the last epidemic and had basic understanding of the disease. Only 20% of the families were owners of equines, and 8% was informed of the importance of equine vaccination. Conclusions: Some epidemiological conditions that eventually could help epizootic Venezuelan equine encephalitis virus reemergence are maintained. However, an abrupt decrease in the number of susceptible equines was found in the area. Apparently, this new condition has not allowed the virus reemergence and is the biggest observed change.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Experimental infection of potential reservoir hosts with Venezuelan equine encephalitis virus, Mexico.

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.

Venezuelan equine encephalitis virus infection of cotton rats.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogen of equids and humans, but infection of its rodent reservoir hosts has received little study. To determine whether responses to infection vary among geographic populations, we inoculated 3 populations of cotton rats with 2 enzootic VEEV strains (Co97-0054 [enzootic ID subtype] and 68U201 [enzootic IE subtype]). The 3 populations were offspring from wild-caught cotton rats collected in a VEE-enzootic area of south Florida, USA; wild-caught cotton rats from a non-VEE-enzootic area of Texas, USA; and commercially available (Harlan) colony-reared cotton rats from a non-VEE-enzootic region. Although each population had similar early viremia titers, no detectable disease developed in the VEE-sympatric Florida animals, but severe disease and death affected the Texas and Harlan animals. Our findings suggest that the geographic origins of cotton rats are important determinants of the outcome of VEE infection and reservoir potential of these rodents.

Recombinant alphaviruses are safe and useful serological diagnostic tools.

Serological assays for diagnosis of Venezuelan equine encephalitis virus (VEEV) currently require bio-safety level 3 facilities and select agent certification to produce antigens, reference sera, or viral stocks. Rapid identification of VEEV infection is required to respond to human and equine outbreaks of encephalitis caused by that virus and can be useful for epidemiologic surveillance. Alphavirus (Sindbis)-based recombinant viruses that express VEEV structural proteins are attenuated in animal models, thus representing an alternative to the handling of virulent infectious virus. Virus and viral antigens from recombinant Sindbis/VEE constructs engineered to express structural proteins from multiple VEEV subtypes were evaluated as diagnostic reagents in VEEV-specific serological assays, e.g., plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) assay, and complement fixation (CF) test. Chimeric viruses were produced efficiently in cell culture and were as effective as the parental virus for identifying infection of humans, horses, and rodents in these serological assays.

Testosterone correlates with Venezuelan equine encephalitis virus infection in macaques.

Here we briefly report testosterone and cytokine responses to Venezuelan equine encephalitis virus (VEEV) in macaques which were used as part of a larger study conducted by the Department of Defense to better characterize pathological responses to aerosolized VEEV in non-human primates. Serial samples were collected and analyzed for testosterone and cytokines prior to and during infection in 8 captive male macaques. Infected animals exhibited a febrile response with few significant changes in cytokine levels. Baseline testosterone levels were positively associated with viremia following exposure and were significantly higher than levels obtained during infection. Such findings suggest that disease-induced androgen suppression is a reasonable area for future study. Decreased androgen levels during physiological perturbations may function, in part, to prevent immunosuppression by high testosterone levels and to prevent the use of energetic resources for metabolically-expensive anabolic functions.

[Venezuelan equine encephalitis: state-of-the-art].

The paper provides the currently available data on the global prevalence of Venezuelan equine encephalitis (VEE), its epidemiology, clinical picture, and specific prevention in human beings. It also discussed the problem of potential use of the causative agent of VEE as a subject of bioterrorism.

Venezuelan equine encephalitis virus, southern Mexico.

Equine epizootics of Venezuelan equine encephalitis (VEE) occurred in the southern Mexican states of Chiapas in 1993 and Oaxaca in 1996. To assess the impact of continuing circulation of VEE virus (VEEV) on human and animal populations, serologic and viral isolation studies were conducted in 2000 to 2001 in Chiapas State. Human serosurveys and risk analyses indicated that long-term endemic transmission of VEEV occurred among villages with seroprevalence levels of 18% to 75% and that medical personnel had a high risk for VEEV exposure. Seroprevalence in wild animals suggested cotton rats as possible reservoir hosts in the region. Virus isolations from sentinel animals and genetic characterizations of these strains indicated continuing circulation of a subtype IE genotype, which was isolated from equines during the recent VEE outbreaks. These data indicate long-term enzootic and endemic VEEV circulation in the region and continued risk for disease in equines and humans.

Vector competence of rural and urban strains of Aedes (Stegomyia) albopictus (Diptera: Culicidae) from São Paulo State, Brazil for IC, ID, and IF subtypes of Venezuelan equine encephalitis virus.

Aedes albopictus (Skuse) is an Asiatic mosquito species that has spread and colonized all continents except Antarctica. It has major public health importance because it is a potential vector of several pathogens. The objectives of our study were to analyze the vector competence of urban and rural strains of Ae. albopictus from São Paulo State (Brazil) for Venezuelan equine encephalitis virus (VEE) subtypes IC, ID, and IF, and to evaluate the effect of infection with subtype IC of VEE on mosquito longevity. Both mosquito strains were susceptible to subtypes IC and ID, but the infection rate for subtype IF was low. Infection and transmission rates of Ae. albopictus for subtype IC were similar to those reported for Ochlerotatus taeniorhynchus (Wiedemann). The high infection, dissemination, and transmission rates for subtype ID reported for Oc. fulvus (Wiedemann) and Culex (Melanoconion) spp. are comparable with those found in this study. We found significant differences in the susceptibility to subtype IC between rural and urban populations of São Paulo. Significant survival rate differences were observed between uninfected and infected mosquitoes, but there were no differences in survival between rural and urban mosquito strains.

Pathogenicity of a Venezuelan equine encephalomyelitis serotype IE virus isolate for ponies.

The enzootic or endemic strains of Venezuelan equine encephalomyelitis (VEE) virus (ID, IE, IF, and II-VI) are considered avirulent. In 1993 and 1996, outbreaks of encephalitis occurred in the horse populations in the Chiapas and Oaxaca provinces of Mexico, respectively. In both instances, enzootic VEE virus subserotype IE was isolated from brain tissues of dead horses. The present study investigated the pathogenicity of the Chiapas viral isolate (NVSL VEE IE 93-42124) in ponies. Three ponies were inoculated intradermally with 4, 5, and 6 logs, respectively, of the NVSL VEE IE 93-42124 viral isolate. All ponies showed fluctuations in body temperature, encephalitis, and other signs of infection with VEE virus. Virus was isolated only from the blood of ponies from day 1 to day 3 postinfection. Microscopic examination of hematoxylin and eosin-stained tissue sections showed mild to moderate nonsuppurative encephalitis, perivascular cuffing by mononuclear cells, gliosis, and meningoencephalitis. Antibody (IgM) to VEE virus IE was unable to differentiate between various subserotypes of VEE I viruses (serotypes IAB, IC, ID, and IF). Virus neutralizing antibody titers to heterologous VEE I viruses were 10-100-fold less than those for NVSL VEE IE 93-42124 virus and Mena II, a human isolate of VEE IE virus. The study confirmed that NVSL VEE IE 93-42124 virus, which was isolated from a brain of a horse during an outbreak of VEE in Chiapas, Mexico, was pathogenic for ponies.

Limited potential for mosquito transmission of genetically engineered, live-attenuated western equine encephalitis virus vaccine candidates.

Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.

Equine amplification and virulence of subtype IE Venezuelan equine encephalitis viruses isolated during the 1993 and 1996 Mexican epizootics.

To assess the role of horses as amplification hosts during the 1993 and 1996 Mexican Venezuelan equine encephalitis (VEE) epizootics, we subcutaneously infected 10 horses by using four different equine isolates. Most horses showed little or no disease and low or nonexistent viremia. Neurologic disease developed in only 1 horse, and brain histopathologic examination showed meningeal lymphocytic infiltration, perivascular cuffing, and focal encephalitis. Three animals showed mild meningoencephalitis without clinical disease. Viral RNA was detected in the brain of several animals 12-14 days after infection. These data suggest that the duration and scope of the recent Mexican epizootics were limited by lack of equine amplification characteristic of previous, more extensive VEE outbreaks. The Mexican epizootics may have resulted from the circulation of a more equine-neurotropic, subtype IE virus strain or from increased transmission to horses due to amplification by other vertebrate hosts or transmission by more competent mosquito vectors.

Positively charged amino acid substitutions in the e2 envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus.

Epidemic-epizootic Venezuelan equine encephalitis (VEE) viruses (VEEV) have emerged repeatedly via convergent evolution from enzootic predecessors. However, previous sequence analyses have failed to identify common sets of nucleotide or amino acid substitutions associated with all emergence events. During 1993 and 1996, VEEV subtype IE epizootics occurred on the Pacific Coast of the states of Chiapas and Oaxaca in southern Mexico. Like other epizootic VEEV strains, when inoculated into guinea pigs and mice, the Mexican isolates were no more virulent than closely related enzootic strains, complicating genetic studies of VEE emergence. Complete genomic sequences of 4 of the Mexican strains were determined and compared to those of closely related enzootic subtype IE isolates from Guatemala. The epizootic viruses were less than 2% different at the nucleotide sequence level, and phylogenetic relationships confirmed that the equine-virulent Mexican strains probably evolved from enzootic progenitors on the Pacific Coast of Mexico or Guatemala. Of 35 amino acids that varied among the Guatemalan and Mexican isolates, only 8 were predicted phylogenetically to have accompanied the phenotypic change. One mutation at position 117 of the E2 envelope glycoprotein, involving replacement of Glu by Lys, resulted in a small-plaque phenotype characteristic of epizootic VEEV strains. Analysis of additional E2 sequences from representative enzootic and epizootic VEEV isolates implicated similar surface charge changes in the emergence of previous South American epizootic phenotypes, indicating that E2 mutations are probably important determinants of the equine-virulent phenotype and of VEE emergence. Maximum-likelihood analysis indicated that one change at E2 position 213 has been influenced by positive selection and convergent evolution of the epizootic phenotype.

Venezuelan equine encephalomyelitis.

The emergence of epidemic VEE viruses has been reported ever since the virus was first described; this phenomenon is likely to continue to occur because of the high mutation rate of these RNA viruses. A vaccine that was first developed by the US Military for human use has proved helpful in curtailing the spread of VEE virus during epizootics of the disease in equids but not during human epidemics. It has not, however, eliminated the source of these highly pathogenic and transmissible viruses. Occurrences of VEE in equids in Mexico in recent years suggest that the present vaccine is not effective in interrupting transmission of new epizootic viruses arising from what were previously known as avirulent enzootic cycles. Future vaccines against VEE should be based on immunogens derived from enzootic viruses to interrupt VEE virus transmission at the source itself rather than waiting for virulent phenotypes of VEE virus to emerge.

Vector competence of three Venezuelan mosquitoes (Diptera: Culicidae) for an epizootic IC strain of Venezuelan equine encephalitis virus.

Experimental studies were undertaken to evaluate the vector competence of selected mosquito species [Aedes taeniorhynchus (Wiedemann), Culex declarator Dyar and Knab, and Mansonia titillans (Walker)] from northwestern Venezuela for the epizootic (IC) strain of Venezuelan equine encephalitis (VEE) virus that was responsible for the 1995 outbreak of VEE in this area. Ae. taeniorhynchus was highly susceptible to infection (94% of 35), and 89% had a disseminated infection. Virus-exposed Ae. taeniorhynchus that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE virus. In contrast, only 1 of 28 (4%) Cx. declarator was infected, and that individual did not develop a disseminated infection. Ma. titillans was moderately susceptible (3 of 8 infected, 38%), and 2 (25%) of these had a disseminated infection. These data indicate that Ae. taeniorhynchus was an important epizootic vector during the 1995 VEE outbreak in Columbia and Venezuela.

Limited potential for mosquito transmission of genetically engineered, live-attenuated Venezuelan equine encephalitis virus vaccine candidates.

In an attempt to improve the current live-attenuated vaccine (TC-83) for Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts.