Susceptibility of Peruvian mosquitoes to eastern equine encephalitis virus

Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of 4105 plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedesfulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophoraferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of approximately 10(8) PFU/ml, Cx. pedroi, Ae.II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.     

Spread of Venezuelan equine encephalitis virus in mice olfactory tract

Summary Spread of Venezuelan equine encephalitis (VEE) virus and damage of the central nervous system (CNS) in mice infected by respiratory route was studied. Virus concentration in organs and blood, dose-effect relationships, and ultrastructural lesions in various tissues were examined in immune and normal mice. We showed, via three independent methods — characteristic curve investigations, tissue virus concentration dynamics, and ultrastructural methods — the spread of VEE virus through the olfactory tract into the brain of immune mice. From these experiments it was concluded that in case of respiratory challenge VEE virus can enter the CNS of normal mice by both vascular and olfactory pathways, while in immune mice the main route is olfactory.     

Type I interferon induction is correlated with attenuation of a South American eastern equine encephalitis virus strain in mice

North American eastern equine encephalitis virus (NA-EEEV) strains cause high mortality in humans, whereas South American strains (SA-EEEV) are typically avirulent. To clarify mechanisms of SA-EEEV attenuation, we compared mouse-attenuated BeAr436087 SA-EEEV, considered an EEEV vaccine candidate, with mouse-virulent NA-EEEV strain, FL93-939. Although attenuated, BeAr436087 initially replicated more efficiently than FL93-939 in lymphoid and other tissues, inducing systemic IFN-α/β release, whereas FL93-939 induced little. BeAr436087 was more virulent than FL93-939 in IFN-α/β-deficient mice, confirming that type I IFN responses determined attenuation, but the viruses were similarly sensitive to IFN-α/β priming in vitro. Infection with BeAr436087 protected against FL93-939 disease/death, even when given 8 h afterward, suggesting that the environment produced by BeAr436087 infection attenuated FL93-939. We conclude that avoidance of IFN-α/β induction is a major virulence factor for FL93-939. Furthermore, BeAr436087 could be used for vaccination and therapeutic treatment in the event of exposure to NA-EEEV during a bioterrorism attack.     

Identification of an antigenic subtype of eastern equine encephalitis virus isolated from a human.

Eastern equine encephalitis (EEE) virus was isolated from the cerebrospinal fluid of a 6-year-old male who had clinically diagnosed aseptic meningitis and subsequently died. Several standard serologic tests that use polyclonal antibody and indirect immunofluorescence and hemagglutination inhibition tests that use monoclonal antibody provided evidence that the isolate was an antigenic subtype of prototype North American EEE virus. We believe that this is the first evidence of an antigenic subtype of EEE virus.     

Human eastern equine encephalitis: immunohistochemistry and ultrastructure.

The brain of a 7-year-old boy who died of eastern equine encephalitis (EEE) was examined by immunohistochemical and ultrastructural techniques to detect the presence and distribution of viral antigen. A mouse polyclonal antibody was most effective for demonstrating the presence of antigen previously unreported in this disease in humans. Antigen was localized to the perikaryon and dendrites of neurons; little was detected in glial cells. Cell death by apoptosis was conspicuous, but it was primarily identified in glial and inflammatory cells. Neuronal death was most commonly marked by cytoplasmic swelling or eosinophilia and nuclear pyknosis. A disassociation between the degree of inflammation and the presence of antigen was noted, especially in cerebral cortex and spinal cord, presumably where infected cells already had been cleared. Ultrastructurally, rare mature viral particles were seen in extracellular spaces.     

Habitat associations of eastern equine encephalitis transmission in Walton County Florida

Eastern Equine Encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) a highly pathogenic mosquito-borne virus is endemic to eastern North America. The ecology of EEEV in Florida differs from that in other parts of the United States; EEEV in the northeastern United States is historically associated with freshwater wetlands. No formal test of habitat associations of EEEV in Florida has been reported. Geographical Information Sciences (GIS) was used in conjunction with sentinel chicken EEEV seroconversion rate data as a means to examine landscape features associated with EEEV transmission in Walton County, FL. Sentinel sites were categorized as enzootic, periodically enzootic, and negative based on the number of chicken seroconversions to EEEV from 2005 to 2009. EEEV transmission was then categorized by land cover usage using Arc GIS 9.3. The land classification data were analyzed using the Kruskal-Wallis test for each land use class to determine which habitats may be associated with virus transmission as measured by sentinel chicken seroconversion rates. The habitat class found to be most significantly associated with EEEV transmission was tree plantations. The ecological factor most commonly associated with reduced levels of EEEV transmission was vegetated nonforest wetlands. Culiseta melanura (Coquillett), the species generally considered to be the major enzootic EEEV vector, was relatively evenly distributed across all habitat classes, while Aedes vexans (Meigen) and Anopheles crucians Weidemann were most commonly associated with tree plantation habitats.     

Genomic sequencing of Highlands J virus: A comparison to western and eastern equine encephalitis viruses

Highlands J virus (HJV) is a member of the genus Alphavirus, family Togaviridae. HJV is the sole representative of the western equine encephalitis (WEE) serocomplex found in the eastern United States, and circulates in nature in an apparently identical transmission cycle as eastern equine encephalitis virus (EEEV). North American representatives of the WEE serocomplex [HJV, WEE virus (WEEV), and Fort Morgan virus (FMV)] are believed to be derived from a recombination event involving EEEV and a Sindbis (SIN)-like virus, such that the nonstructural polyprotein, the capsid, and the terminal end of the 3′ UTR are derived from EEEV, while the surface glycoproteins (E1 and E2) and small peptides (E3 and 6K) encoded in the subgenomic RNA are derived from the SIN-like virus. In this report, the complete nucleotide sequence of HJV is described, along with a comparative analysis of the HJV nonstructural polyprotein to WEEV and EEEV.     

Estimation of dispersal distances of Culex erraticus in a focus of eastern equine encephalitis virus in the southeastern United States

Patterns of mosquito dispersal are important for predicting the risk of transmission of mosquito-borne pathogens to vertebrate hosts. We studied dispersal behavior of Culex erraticus (Dyar & Knab), a potentially significant vector of eastern equine encephalitis virus (EEEV) that is often associated with foci of this pathogen in the southeastern United States. Using data on the relative density of resting adult female Cx. erraticus around known emergence sites in Tuskegee National Forest, Alabama, we developed a model for the exponential decay of the relative density of adult mosquitoes with distance from larval habitats through parameterization of dispersal kernels. The mean and 99th percentile of dispersal distance for Cx. erraticus estimated from this model were 0.97 and 3.21 km per gonotrophic cycle, respectively. Parameterized dispersal kernels and estimates of the upper percentiles of dispersal distance of this species can potentially be used to predict EEEV infection risk in areas surrounding the Tuskegee National Forest focus in the event of an EEEV outbreak. The model that we develop for estimating the dispersal distance of Cx. erraticus from collections of adult mosquitoes could be applicable to other mosquito species that emerge from discrete larval sites.     

Lymphoreticular and myeloid pathogenesis of Venezuelan equine encephalitis in hamsters.

Ultrastructural, histopathologic, and virologic studies of adult hamsters infected with virulent Venezuelan equine encelphalomyelitis (VEE) virus (Subtype I-B) demonstrated precise chronologic and topographic progression of lesions and viral replication in extraneural sites. Thymus contained the earliest lesions and the highest initial and subsequent viral titers. No particular cytotropism was observed as highly efficient viral replication and severe cytonecrosis proceded. Early cortical necrosis of splenic periarteriolar lymphocytic sheath was followed by lymphoblastoid repopulation of the peripheral zone. Massive bone marrow necrosis was accompained by ultrastructural evidence of VEE viral particle production in reticulum cells, rubricytes, myeloid cells, lymphoblastoid cells, and megakaryocytes. Speed, efficiency, destructiveness, and relative sensitivity of virtually all lymphoreticular and hematopoetic cells were hallmarks of virulent VEE infection in the hamster.     

Experimental transmission of eastern equine encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae)

We evaluated the potential for Ochlerotatus j. japonicus (Theobald), a newly recognized invasive mosquito species in the United States, to transmit eastern equine encephalitis (EEE) virus. Aedes albopictus (Skuse) and Culex pipiens (L.) were similarly tested for comparison. Ochlerotatus j. japonicus and Ae. albopictus became infected and transmitted EEE virus by bite after feeding on young chickens 1 d after they had been inoculated with EEE virus (viremias ranging from 10(7.0-8.7) plaque-forming units [PFU]/ml of blood). No Cx. pipiens (n = 20) had detectable levels of virus 14 d after feeding on an EEE-virus infected chicken with a viremia of 10(8.1) PFU per ml of blood. Depending on the viral titer in the donor chicken, infection rates ranged from 55-100% for Oc. j. japonicus and 93-100% for Ae. albopictus. In these two species, dissemination rates were identical to or nearly identical to infection rates. Depending on the viral titer in the blood meal, estimated transmission rates ranged from 15 to 25% for Oc. j. japonicus and 59-63% for Ae. albopictus. Studies of replication of EEE virus in Oc. j. japonicus showed that there was an "eclipse phase" in the first 4 d after an infectious blood meal, that viral titers peak by day 7 at around 10(5.7) per mosquito, and that virus escaped the mid-gut as soon as 3 d after the infectious blood meal. These data, combined with the opportunistic feeding behavior of Oc. j. japonicus in Asia and the reported expansion of its range in the eastern United States, indicate that it could function as a bridge vector for EEE virus between the enzootic Culiseta melanura (Coquillett)-avian cycle and susceptible mammalian hosts.     

Early expression of IFN-alpha/beta and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice.

To investigate the roles of type I interferon (IFN-alpha/beta) and other mediators of innate immune responses (e.g., inducible nitric oxide synthase [iNOS]) in early dissemination of Venezuelan equine encephalitis virus (VEE) infection, we used mice with targeted deletions in either their IFN-alpha/beta-receptor (IFNAR-1-/-) or interferon regulatory factor 2 (IRF-2-/-) genes. Following footpad infection, both IFNAR-1-/- and IRF-2-/- mice were more susceptible than control mice to VEE. The IFNAR-1-/- mice also exhibit accelerated VEE dissemination to serum, spleen, and brain, and compared with control mice, they evidenced faster kinetics in the upregulation of proinflammatory genes. In contrast, in IRF-2-/- mice, iNOS gene induction was completely absent following peripheral virulent VEE infection. In evaluating the role of cells involved in iNOS production, primary microglial cell cultures were found to be highly permissive to VEE infection. Moreover, VEE infection increased levels of nitric oxide (NO) in resting microglial cultures but decreased NO production in IFN-gamma-stimulated microglia. Thus, these findings suggest that reactive nitrogen species play an important contributory role in VEE dissemination and survival of the host. Our results further suggest the necessity for a carefully balanced host response that follows a middle course between immunopathology and insufficient inflammatory response to VEE infection.     

Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge

An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.     

Human eastern equine encephalitis. Electron microscopic study of a brain biopsy

An electron microscopic study was done on brain biopsy tissue from an eight-month-old female with acute eastern equine encephalitis diagnosed by indirect immunofluorescence. Rare clusters of round virions were found almost exclusively in the extracellular space. All virions observed had spiked envelopes, and their sizes averaged approximately 55 nm. Also found were rare enveloped virions along with degenerate organelles in a membrane-bound structure in the cytoplasm of macrophages. Intracytoplasmic development of virions was not found. Tubuloreticular complexes were seen in the endothelial cells and macrophages. This is the first report of an electron microscopic study on biopsy material from a case of human eastern equine encephalitis. It will extend the usefulness of brain biopsy in the diagnosis of acute encephalitis.     

Effect of cordycepin on the replication of western equine encephalitis virus

Summary Cordycepin (3-deoxyadenosine) inhibited viral RNA synthesis in the replication of western equine encephalitis virus, thereby causing a reduction of virus production. The rate of inhibition of viral RNA synthesis was dependent on drug concentration and the period of treatment with the drug. These results suggest that the virus RNA synthesizing system is sensitive to the drug.With 3 Figures