Severe encephalitis in cynomolgus macaques exposed to aerosolized Eastern equine encephalitis virus

Cynomolgus macaques exposed to an aerosol containing a virulent strain of eastern equine encephalitis (EEE) virus developed neurological signs indicating encephalitis that corresponded with the onset of fever and an elevated heart rate. Viremia was either transient or undetectable even in animals that succumbed to the illness. The onset of illness was dose dependent, but once a febrile response was observed, macaques were moribund within 36 h. Simultaneously, a prominent leukocytosis was seen; 1 day before being moribund, macaques had a white blood cell count >20,000 cells/ microL. The leukocytes were predominantly granulocytes. Increases in serum levels of blood urea nitrogen, sodium, and alkaline phosphatase were also seen. The rapid onset and severity of neurological signs mirror what has been reported for human cases of disease caused by EEE.     

Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America.

One of the most important questions in arbovirology concerns the origin of epidemic Venezuelan equine encephalitis (VEE) viruses; these viruses caused periodic, extensive epidemics/epizootics in the Americas from 1938-1973 (reaching the United States in 1971) but had recently been presumed extinct. We have documented the 1992 emergence of a new epidemic/epizootic VEE virus in Venezuela. Phylogenetic analysis of strains isolated during two outbreaks indicated that the new epidemic/epizootic virus(es) evolved recently from an enzootic VEE virus in northern South America. These results suggest continued emergence of epizootic VEE viruses; surveillance of enzootic viruses and routine vaccination of equines should therefore be resumed.     

Association of Tonate virus (subtype IIIB of the Venezuelan equine encephalitis complex) with encephalitis in a human.

Tonate virus, subtype IIIB of the Venezuelan equine encephalitis (VEE) complex, was first isolated in 1973 in French Guiana, South America. However, very little is known about its pathogenicity; it was considered to be responsible for only mild dengue-like syndromes. In 1998, a 2-month-old boy living along the Oyapock river in French Guiana was hospitalized for fever and generalized status myoclonus, and despite treatment the patient died 72 h after admission. Testing showed the presence of IgM specific for viruses of the VEE complex. A sensitive seminested polymerase chain reaction derived from a previous study was developed to detect viruses from the VEE complex, since no virus could be recovered from clinical specimens cultured on mosquito cells or from intracerebral inoculation into newborn mice. The genome of a virus from the VEE complex was detected in postmortem brain biopsies, and Tonate virus was identified by direct sequencing. This is the first reported case of human encephalitis due to Tonate virus.     

Vector competence of five common mosquito species in the People's Republic of China for Western equine encephalitis virus

Two strains of the Western equine encephalitis virus (WEEV) were first detected and isolated in China in 2001. The maintenance and transmission cycles of WEEV in China are currently not well understood, and the mosquito vectors involved in these cycles are unknown. To understand the ability of the local mosquitoes in China to transmit WEEV, the vector competence of five mosquito species, namely, Culex pipiens pallens Coquillett, Cx. p. quinquefasciatus Say, Aedes (Stegomyia) albopictus Skuse, Ae. (Stegomyia) aegypti Linnaeus, and C. tritaeniorhynchus Giles, for WEEV were evaluated. Infection rates for Cx. p. pallens, Cx. p. quinquefasciatus, Cx. tritaeniorhynchus, Ae. Albopictus, and Ae. aegypti were 46%, 60%, 80%, 37%, and 25%, respectively. Dissemination rates for the same species were 60%, 61%, 75%, 55%, and 50%, respectively. Transmission rates were 41%, 53%, 57%, and 45% for Cx. p. pallens, Cx. p. quinquefasciatus, Ae. Albopictus, and Ae. Aegypti, respectively. Infection rates were significantly different between species, but the difference between dissemination and transmission rates were nonsignificant. These results suggest that several local mosquito species in China are competent laboratory vectors for WEEV.     

Aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques

Cynomolgus macaques were exposed by aerosol to a virulent strain of western equine encephalitis virus (WEEV). Between 4 and 6 days after exposure, macaques had a significantly elevated temperature that lasted for 3-4 days. Clinical signs of encephalitis began as the body temperature decreased, and then they rapidly increased in severity. Cynomolgus macaques with clinical signs of encephalitis had elevated white cell counts in the blood caused mostly by increased numbers of segmented neutrophils and monocytes. Elevated serum glucose levels also correlated with the severity of the clinical signs of encephalitis. Three cynomolgus macaques died; immunohistochemical evidence of viral antigen was present in the brain and central nervous system (CNS). Microscopic analysis also revealed a marked lymphocytic infiltrate in the CNS. Cynomolgus macaques will serve as a useful model of aerosol exposure to WEEV for the evaluation of potential vaccine candidates.     

Necrotizing myocarditis in mice infected with Western equine encephalitis virus: Clinical, electrocardiographic, and histopathologic correlations

Western equine encephalitis (WEE) virus was found in myocardial tissue of adult mice during the first five days after inoculation of the virus, with a peak titer (5.0 log plaque-forming units/g) at 24 hr. Light microscopy revealed a multifocal necrotizing myocarditis with a prominent inflammatory response and hyaline and granular degeneration of myofibers. Electron microscopy showed cytoplasmic viral nucleoids and budding and free mature WEE viral particles. Serial electrocardiograms showed the development of disturbances of rate and rhythm, defects in conduction, marked elevation in the ST segment, and low voltage. Myocarditis has not been previously recognized as a complication of alphavirus infection in humans. and we found no evidence for myocardial damage in 11 persons with acute WEE virus infections studied electrocardiographically in 1975. Demonstration of myocarditis in the WEE virus-infected mouse, however, suggests the need to monitor human patients for possible cardiac involvement during future epidemics of WEE virus infection.     

Competency of reptiles and amphibians for eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV) is endemic throughout most of the eastern United States. Although it is transmitted year round in Florida, transmission elsewhere is seasonal. The mechanism that enables EEEV to overwinter in seasonal foci remains obscure. In previous field studies, early season EEEV activity was detected in mosquito species that feed primarily upon ectothermic hosts, suggesting that reptiles and amphibians might represent overwintering reservoir hosts for EEEV. To determine if this might be possible, two commonly fed upon amphibian and reptile species were evaluated as hosts for the North American subtype I strain of EEEV. Neither amphibian species was a competent host. However, circulating viremias were detected in both reptile species examined. Hibernating infected garter snakes remained viremic after exiting hibernation. These data suggest that snakes may represent an overwintering host for North American EEEV.     

Phylogenetic analysis of eastern equine encephalitis virus isolates from Florida

Florida has the highest degree of endemicity for eastern equine encephalitis virus (EEEV) of any state in the United States and is the only state with year-round transmission of EEEV. To further understand the viral population dynamics in Florida, the genome sequence of six EEEV isolates from central Florida were determined. These data were used to identify the most polymorphic regions of the EEEV genome from viruses isolated in Florida. The sequence of these polymorphic regions was then determined for 18 additional Florida isolates collected in four geographically distinct regions over a 20-year period. Phylogenetic analyses of these data suggested a rough temporal association of the Florida isolates, but no clustering by region or by source of the isolate. Some clustering of northeastern isolates with Florida isolates was seen, providing support for the hypothesis that Florida serves as a reservoir for the periodic introduction of EEEV into the northeastern United States.     

Genetic evidence for the origins of Venezuelan equine encephalitis virus subtype IAB outbreaks.

Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.     

Pseudotyped viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus.

Virus envelope proteins are the primary targets of neutralizing antibody responses. The epitopes recognized differ sufficiently between virus subtypes and species to distinguish viruses and provide an important basis for disease diagnosis. Venezuelan equine encephalitis virus (VEEV) causes acute febrile illness in humans and has high mortality in equines. The most specific detection methods for serum antibodies use live virus in neutralization assays or in blocking enzyme linked immunosorbent assays. However, work with Venezuelan equine encephalitis virus requires biosafety level 3 containment and select agent security in the United States. We report two new assays for detection of Venezuelan equine encephalitis virus neutralizing antibody responses, based on virus pseudotypes. The first provides detection by marker gene expression after 20 hours and is particularly suited for high-throughput screening; the second uses a new, rapid virus entry assay to give readouts within 1 hour. Both assays are safe, sensitive, and in general recapitulate neutralizing antibody titers obtained by conventional plaque reduction assays. Each is suitable as a rapid primary screen for detection of neutralizing antibodies against Venezuelan equine encephalitis virus.     

委内瑞拉马脑炎疫苗研究进展

委内瑞拉马脑炎是一种由蚊虫传播的人兽共患病毒性传染病. 我国虽然尚未发现委内瑞拉马脑炎病例,但随着国际交流的日益频繁,该病传入我国的风险大大增加. 该病目前尚无特效药及疗法,预防接种安全高效的疫苗对减少疾病暴发起着非常关键的作用. 本文对委内瑞拉马脑炎疫苗的最新研究进展进行综述,旨在为该病的新型疫苗研究及防控策略的制定提供参考.     

The systemic pathology of Venezuelan equine encephalitis virus infection in humans

The histopathology of fatal Venezuelan equine encephalitis (VEE) in humans has not been well documented. To evaluate the spectrum of disease in man, the histologic slides of the 21 autopsied patients who died with documented VEE infection during the 1962-63 VEE epidemic in Zulia, Venezuela were reviewed. The main histopathologic lesion observed in multiple organs and tissues, especially the brain, gastrointestinal tract, and lungs, was moderate to marked diffuse congestion and edema with hemorrhage. In the central nervous system (CNS), mild or focal mixed inflammatory cell infiltrates were present in the leptomeninges and perivascular spaces (65%). Meningoencephalitis associated with intense necrotizing vasculitis was observed in 2 patients (10%), and cerebritis was observed in 5 cases (25%). There was a striking depletion of lymphocytes with vascular thrombosis and necrosis of follicles in lymph nodes (77%), spleen (69%), and the gastrointestinal tract (90%). Widespread hepatocellular degeneration and individual cell necrosis was observed in 61% of the cases. Most patients (90%) had interstitial pneumonia, frequently complicated by acute bronchopneumonia (33%). Overall, the lesions observed in the CNS and reticuloendothelial tissues are comparable to what is observed in experimental animals; however, extensive hepatocellular degeneration and interstitial pneumonia are not prominent pathologic features of VEE in animals. The findings are consistent with the hypothesis that lymphoid and reticuloendothelial tissues are the targets in VEE virus infection in humans, and that many of the histopathologic changes are attributable to primary lymphoid and endothelial cell injury.     

Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.

Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.     

Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.

A candidate live-attenuated virus vaccine for protection against Venezuelan equine encephalitis (VEE) (designated V3526) was tested in mice to measure the magnitude, duration, and kinetics of virus replication in the blood and the central nervous system and its phenotypic stability after multiple passages in mice and cell culture. All results were compared to parallel experiments with parental virus and the existing VEE virus vaccine, TC-83. Maximum virus titers in the brains of V3526-inoculated mice were between 10- and 100-fold less than those observed in brains of mice inoculated intracranially (i.c.) with either the parental virus or TC-83. Neither V3526 nor TC-83 was lethal in BALB/c mice inoculated i.c.. However, mice inoculated with TC-83 developed acute symptoms lasting at least 14 days. In contrast, i.c. inoculation of TC-83 was uniformly lethal for C3H/HeN mice. V3526 was avirulent in both BALB/c and C3H/HeN mice after i.c. inoculation. The virulence characteristics of V3526 remained unchanged after five serial i.c. passages in mouse brains or after five cell culture passages. Finally, pathologic changes induced after i.c. inoculation of V3526 were consistently less severe and of shorter duration than those observed in TC-83-inoculated mice. Based on these results, V3526 is stable and appears to be significantly less neurovirulent in mice than TC-83.     

Genetic diversity and relationships among Venezuelan equine encephalitis virus field isolates from Colombia and Venezuela.

During field studies of enzootic Venezuelan equine encephalitis (VEE) viruses associated with epizootic emergence, a large number of virus isolates were made in sylvatic foci of Venezuela and Colombia. To rapidly characterize these isolates, antigenic subtypes were determined by means of immunofluorescence and by single-strand conformational polymorphism (SSCP) analysis by use of an 856-bp fragment from the P62 gene, which we used to distinguish genetic variants. Representative isolates were sequenced to assess the sensitivity of SSCP to detect genetic differences. The SSCP analysis distinguished isolates differing by as little as 1 nucleotide; overall, differences of > or = 1 nucleotide were recognized 89% of the time, and the sensitivity to distinguish strains that differed by only 1 or 4 nucleotides was 17 and 57%, respectively. Phylogenetic analyses of representative sequences showed that all recent isolates from the Catatumbo region of western Venezuela and the middle Magdalena Valley of Colombia were closely related to epizootic subtype IAB and IC strains; strains from Yaracuy and Miranda States were more distantly related. Cocirculation of the same virus genotype in both Colombian and Venezuelan foci indicated that these viruses are readily transported between enzootic regions separated by > 300 km. The SSCP analysis appears to be a simple, fast, and relatively efficient method of screening VEE virus isolates to identify meaningful genetic variants.