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.

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.).

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.

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.

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.

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.

[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.

Venezuelan equine encephalomyelitis and African horse sickness. Current status and review.

The arthropod-borne virus (arbovirus) diseases of livestock have worldwide impact. The prevention of an introduction of an exotic disease and the control of one subsequent to an introduction will require the attention, cooperation, and support of the livestock industry, regulatory agencies, and researchers. The most effective protection of our livestock industries is to prevent the introduction of an exotic disease agent. This implies complete restriction of animal imports and exports. However, "zero risk" is an unacceptable option in today's world of internationally integrated and interdependent agriculture. Scientifically sound and factually based regulatory decisions must be combined with continued vigilance and preparedness, as well as appropriately directed surveillance and research, to protect the world agricultural marketplace. Two exotic arbovirus diseases that are of current concern to the livestock industries and regulatory officials of the North American and Caribbean Basin countries are VEE and AHS. Devastating epizootics of VEE in equines have occurred frequently in the Western Hemisphere, but no recent epizootic activity has been documented. Naturally occurring foci of sylvatic, equine nonpathogenic VEE virus subtypes, however, do exist in the tropical countries of the hemisphere. The relationship of these sylvatic virus foci to the origin of equine virulent epizootic VEE virus subtypes is unknown. AHS epizootics had been confined to Africa, the Middle East, and the Indian subcontinent until recent outbreaks in 1966 and 1987-1990 in Spain. With the recurrence of AHS in Spain for the past four years, concern about the possible introduction into potential vector species and equines in the Western Hemisphere has increased. This review addresses the current VEE and AHS virus activity and the potential for outbreaks in the Western Hemisphere.

Venezuelan equine encephalitis virus: horse virulence of P-676 and MF-8 small and minute plaques.

The P-676 and MF-8 epizootic strains of Venezuelan equine encephalitis (VEE) virus were found to contain a minute plaque (MP), different from the predominant small plaque (SP) present in these virus strains. The MP and SP were stable after passages in Vero cells, mice, or horses. Equines were inoculated with the SP or MP of the P-676 and MF-8 strains. Inoculation of either P-676 SP or MP into horses induced high fever and viremia but no signs of encephalitis or death. Four horses infected with MF-8 SP became very ill, with high fever and viremia; three of the inoculated animals died. Four horses were infected with MF-8 MP; only showed viremia but appeared asymptomatic and afebrile. Neutralization tests with immune sera from the infected equines showed that the P-676 SP and MP appear distinct, while a clear difference cannot be observed with MF-8 SP and MP.

Further observations on infections of guinea pigs with Venezuelan encephalitis virus strains.

Guinea pigs from a Guatemalan colony died after subcutaneous inoculating of moderately small doses of equine-benign strains of Venezuelan encephalitis (VE) virus of hemagglutination-inhibition subtype I-E from enzootic habitats in Mexico and Guatemala. Thus these guinea pigs were unlike English short hair and inbred 13 guinea pigs, which usually survive infections with equine-benign VE strains of subtype I-E. We therefore caution others that not all strains of guinea pigs can be used to evaluate the potential equine virulence of VE viruses.

Transplacental transmission of Venezuelan equine encephalitis virus in horses.

Transplacental passage of Venezuelan equine encephalitis virus, epizootic strain P-676, occurred in four of nine fetuses studied. The mares were infected near term. Virus was recovered in high titer from fetal blood and organs, while no virus was detected in maternal blood but neutralizing antibodies were present. No evidence of in utero infection was found in two fetuses from mares infected with MF-8, another epizootic strain of Venezuelan equine encephalitis virus.

Venezuelan encephalitis virus infection in neotropical bats. III. Experimental studies on virus excretion and non-arthropod transmission.

A total of 80 Neotropical bats of five species was inoculated with one of four strains of Venezuelan encephalitis (VE) virus. Virus was detected in the oropharynges of 56% of bats, and most regularly in Artibeus jamaicensis (75%). Titers of virus in oropharyngeal secretions were occasionally very high (8.5 log10 SMicLD50/ml in one A. jamaicensis). Only 2 of 123 urine samples from 50 bats and 2 of 86 fecal samples from 46 bats yielded VE virus. No contact or aerosol virus transmission from bat to bat was detected. VE virus passed transplacentally from two infected mothers to their fetuses, which were aborted. Virus did not pass from one infected mother to her nursing young.

Experimental infection of birds with epidemic Venezuelan encephalitis virus.

Sixty-three birds representing 13 species were inoculated with a strain of epidemic Venezuelan encephalitis (VE) virus from the 1971 Texas outbreak. More than 95% of the birds became infected. Mortality which could be attributed to infection with VE virus was very low. Viremia persisted 2-6 days. Peak viremia levels ranged from 10(3.2) to 10(8.2) suckling mouse intracranial 50% lethal doses per milliliter (SMICLD50/ml). Blood virus levels were highest in juvenile Louisiana Herones, adult Robins and adult Mockingbirds and were lowest in juvenile Common Egrets. Most bird species had blood virus levels about 10(5) SMICLD50/ml (high vector infection potential) for 2-3 days. Neutralizing antibody response was more uniform and frequent in herons (95%) than in passerines (56%). The role of birds in the epidemiology of Venezuelan is discussed.

Responses of guinea pigs to infections with strains of Venezuelan encephalitis virus, and correlations with equine virulence.

Five equine-virulent strains of Venezuelan encephalitis (VE) virus consistently killed guinea pigs (strain English short hair) inoculated subcutaneously, whereas ten equine benign strains did not. Two enzootic eastern Panamanian and Colombian strains of hemagglutination-inhibition antigenic subtype I-D and of undetermined virulence for equine animals also killed guinea pigs. This subtype of VE virus may represent an excpetion to a positive correlation between equine virulence and guinea pig lethality since another strain of ts subtype has been reported to be benign in horses inoculated subcutaneously. Within the strains tested, viruses of subtypes I-ABC and I-D were lethal for guinea pigs, and viruses of other subtypes were benign. There was no correlation between guinea pig and human virulence of VE strains.

Venezuelan encephalitis virus infection in neotropical bats. I. Natural infection in a Guatemalan enzootic focus.

A serological survey of 939 Neotropical bats of 22 species from an enzootic focus of Venezuelan encephalitis (VE) virus on the Pacific lowlands of Guatemala during 1971--1975 revealed VE virus specific antibodies in seven species, three belonging to the genus Artibeus. VE virus was isolated from the blood of one Uroderma bilobatum. Antibody frequency was considerably lower in bats than in terrestrial mammals, and tended to vary within any given species from locality to locality and from year to year. At the village of La Avellana where VE virus was most active, antibody rates in the two best-sampled Artibeus species were 0.14 in 1971, 0.11 in 1972, 0.03 in 1973, and 0.11 in 1975. The positive rate of 0.10 over all years at La Avellana for the genus Artibeus suggests that these and possibly other bats regularly are infected by VE virus, and may possibly serve as alternate hosts to maintain virus circulation if most terrestrial animals become immune.

Venezuelan encephalitis virus infection in neotropical bats. II. Experimental infections.

Eighty-nine Neotropical bats of five species were inoculated subcutaneously with epizootic or enzootic strains of Venezuelan encephalitis (VE) virus. Viremia was detected in 92.5% of all bats, but no illness attributable to virus infection was observed. Detectable viremias averaged slightly over 4 days in Artibeus jamaicensis and A. lituratus, and 2.8 days in Phyllostomus discolor, and maximal viremia titers in these three species averaged 6.9, 6.6, and 4.6 log10 SMicLD50 per ml of blood, respectively. In general Artibeus developed and maintained detectable levels of both hemagglutination-inhibition (HI) and neutralizing antibody for as long as tested (up to 506 days), although HI antibody to enzootic VE virus strains disappeared in some A. lituratus. The detectable antibody response of P. discolor was slower and of lower magnitude and shorter duration than that of Artibeus, although individual P. discolor which had lost detectable HI and N antibody resisted challenge. Vertical passage of antibody was observed in three A. lituratus offspring. Artibeus jamaicensis were found to be only slightly less susceptible to VE virus infection than a U.S. subspecies of the cotton rat (Sigmodon hispidus). No virus was recovered by mouse inoculation of organ pools of bats killed as early as 2 days and as late as 299 days after the last day of detectable viremia.

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.

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.