Eastern Equine Encephalitis Research Articles

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.

Susceptibility of Peruvian Mosquitoes to Eastern Equine Encephalitis Virus

Susceptibility of Peruvian Mosquitoes to Eastern Equine Encephalitis Virus

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A Five-Amino-Acid Deletion of the Eastern Equine Encephalitis Virus Capsid Protein Attenuates Replication in Mammalian Systems but Not in Mosquito Cells

Eastern equine encephalitis virus (EEEV) is a human and veterinary pathogen that causes sporadic cases of fatal neurological disease. We previously demonstrated that the capsid protein of EEEV is a potent inhibitor of host cell gene expression and that this function maps to the amino terminus of the protein. We now identify amino acids 55 to 75, within the N terminus of the capsid, as critical for the inhibition of host cell gene expression. An analysis of stable EEEV replicons expressing mutant capsid proteins corroborated these mapping data. When deletions of 5 to 20 amino acids within this region of the capsid were introduced into infectious EEEV, the mutants exhibited delayed replication in Vero cells. However, the replication of the 5-amino-acid deletion mutant in C710 mosquito cells was not affected, suggesting that virus replication and assembly were affected in a cell-specific manner. Both 5- and 20-amino-acid deletion mutant viruses exhibited increased sensitivity to interferon (IFN) in cell culture and impaired replication and complete attenuation in mice. In summary, we have identified a region within the capsid protein of EEEV that contributes to the inhibition of host gene expression and to the protection of EEEV from the antiviral effects of IFNs. This region is also critical for EEEV pathogenesis.

Eastern equine encephalitis leading to multi-organ failure and sepsis

Eastern equine encephalitis leading to multi-organ failure and sepsis Anita J. Reddy a,∗, Christopher W. Woods b, Karen E. Welty-Wolf a,b a Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Durham Veterans Administration and Duke University Medical Centers, Durham, NC 27710, United States b Division of Infectious Diseases, Department of Medicine, Durham Veterans Administration and Duke University Medical Centers, Durham, NC 27710, United States

Comparative Analysis of Distribution and Abundance of West Nile and Eastern Equine Encephalomyelitis Virus Vectors in Suffolk County, New York, Using Human Population Density and Land Use/Cover Data

Five years of CDC light trap data from Suffolk County, NY, were analyzed to compare the applicability of human population density (HPD) and land use/cover (LUC) classification systems to describe mosquito abundance and to determine whether certain mosquito species of medical importance tend to be more common in urban (defined by HPD) or residential (defined by LUC) areas. Eleven study sites were categorized as urban or rural using U.S. Census Bureau data and by LUC types using geographic information systems (GISs). Abundance and percent composition of nine mosquito taxa, all known or potential vectors of arboviruses, were analyzed to determine spatial patterns. By HPD definitions, three mosquito species, Aedes canadensis (Theobald), Coquillettidia perturbans (Walker), and Culiseta melanura (Coquillett), differed significantly between habitat types, with higher abundance and percent composition in rural areas. Abundance and percent composition of these three species also increased with freshwater wetland, natural vegetation areas, or a combination when using LUC definitions. Additionally, two species, Ae. canadensis and Cs. melanura, were negatively affected by increased residential area. One species, Aedes vexans (Meigen), had higher percent composition in urban areas. Two medically important taxa, Culex spp. and Aedes triseriatus (Say), were proportionally more prevalent in residential areas by LUC classification, as was Aedes trivittatus (Coquillett). Although HPD classification was readily available and had some predictive value, LUC classification resulted in higher spatial resolution and better ability to develop location specific predictive models.

Comparative Analysis of Distribution and Abundance of West Nile and Eastern Equine Encephalomyelitis Virus Vectors in Suffolk County, New York, Using Human Population Density and Land Use/Cover Data

Comparative Analysis of Distribution and Abundance of West Nile and Eastern Equine Encephalomyelitis Virus Vectors in Suffolk County, New York, Using Human Population Density and Land Use/Cover Data

Structural and Nonstructural Protein Genome Regions of Eastern Equine Encephalitis Virus Are Determinants of Interferon Sensitivity and Murine Virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.

Molecular Epidemiology of Eastern Equine Encephalitis Virus, New York

Perpetuation, overwintering, and extinction of eastern equine encephalitis virus (EEEV) in northern foci are poorly understood. We therefore sought to describe the molecular epidemiology of EEEV in New York State during current and past epizootics. To determine whether EEEV overwinters, is periodically reintroduced, or both, we sequenced the E2 and partial NSP3 coding regions of 42 EEEV isolates from New York State and the Eastern Seaboard of the United States. Our phylogenetic analyses indicated that derived subclades tended to contain southern strains that had been isolated before genetically similar northern strains, suggesting southern to northern migration of EEEV along the Eastern Seaboard. Strong clustering among strains isolated during epizootics in New York from 2003-2005, as well as from 1974-1975, demonstrates that EEEV has overwintered in this focus. This study provides molecular evidence for the introduction of southern EEEV strains to New York, followed by local amplification, perpetuation, and overwintering.

Venezuelan Equine Encephalitis Virus Capsid Protein Inhibits Nuclear Import in Mammalian but Not in Mosquito Cells

Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its approximately 35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a significant fraction of VEEV capsid associated with nuclear envelope, which suggested that this protein might regulate nucleocytoplasmic trafficking. In this study, we demonstrate that VEEV capsid and its N-terminal sequence efficiently inhibit multiple receptor-mediated nuclear import pathways but have no effect on the passive diffusion of small proteins. The capsid protein of the Old World alphavirus Sindbis virus and the VEEV capsid, with a previously defined frameshift mutation, were found to have no detectable effect on nuclear import. Importantly, the VEEV capsid did not noticeably interfere with nuclear import in mosquito cells, and this might play a critical role in the ability of the virus to develop a persistent, life-long infection in mosquito vectors. These findings demonstrate a new aspect of VEEV-host cell interactions, and the results of this study are likely applicable to other New World alphaviruses, such as eastern and western equine encephalitis viruses.

Seroprevalence of Alphaviruses in Chelonians from Southeastern Louisiana

ABSTRACT Eastern equine encephalitis virus, an alphavirus, is the causative agent of eastern equine encephalitis, an arthropod-borne disease endemic to North, Central, and South America. In addition to horses, humans, and birds, the eastern equine encephalitis virus can also infect reptiles. Although experimental studies have demonstrated that reptiles can remain viremic for longer periods of time than mammals and birds, as well as remain viremic through hibernation, the overall role of reptiles in the epidemiology for the virus is unclear. A cross-sectional study was performed to estimate the seroprevalence of eastern equine encephalitis virus in chelonians from southeastern Louisiana. Blood was collected from 117 chelonians in southeastern Louisiana in the summer of 2000 (n=76) and the spring of 2001 (n=41). Hemagglutination inhibition assay and plaque reduction neutralization test were utilized to determine exposure. Six (5%) of the 117 chelonians were seropositive for eastern equine encephalitis virus...

Experimental Infection of Aedes sollicitans and Aedes taeniorhynchus with Two Chimeric Sindbis/Eastern Equine Encephalitis Virus Vaccine Candidates

Two chimeric vaccine candidates for Eastern equine encephalitis virus (EEEV) were developed by inserting the structural protein genes of either a North American (NA) or South American (SA) EEEV into a Sindbis virus (SINV) backbone. To assess the effect of chimerization on mosquito infectivity, experimental infections of two potential North American bridge vectors of EEEV, Aedes sollicitans and Ae. taeniorhynchus, were attempted. Both species were susceptible to oral infection with all viruses after ingestion of high titer blood meals of ca. 7.0 log(10) plaque-forming units/mL. Dissemination rates for SIN/NAEEEV (0 of 56) and SIN/SAEEEV (1 of 54) were low in Ae. taeniorhynchus and no evidence of transmission potential was observed. In contrast, the chimeras disseminated more efficiently in Ae. sollicitans (19 of 68 and 13 of 57, respectively) and were occasionally detected in the saliva of this species. These results indicate that chimerization of the vaccine candidates reduces infectivity. However, its impact on dissemination and potential transmission is mosquito species-specific.

Predicting seasonal abundance of mosquitoes based on off-season meteorological conditions

Modeling mosquito population dynamics has become an important part of understanding the transmission of mosquito-borne arboviruses. Of these models, those including meteorological variables have mainly focused on conditions during or immediately preceding the mosquito breeding season. While these conditions are clearly critical biologically and statistically, it is also biologically plausible that conditions during the off-season may contribute to interannual variation in mosquito population size. To examine the effect of off-season factors, we develop a pair of Poisson regression models for July captures of Aedes sollicitans and Culex salinarius, two East Coast vector species of arboviruses including Eastern equine encephalitis virus and West Nile virus. Model results indicate that average maximum temperature, total heating degree-days, and the total number of days with a minimum temperature below freezing during the winter months was predictive of mosquito populations. In addition, the average maximum relative humidity from the preceding fall and total rainfall and total heating degree-days during the preceding spring were also associated with vector population dynamics. The descriptive and predictive power of these models is discussed.

Analysis of Venezuelan Equine Encephalitis Virus Capsid Protein Function in the Inhibition of Cellular Transcription

The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, approximately 35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C(VEE)33-68 includes two domains with distinct functions: the alpha-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.

AN OUTBREAK OF EASTERN EQUINE ENCEPHALITIS VIRUS IN FREE-RANGING WHITE-TAILED DEER IN MICHIGAN

Eastern equine encephalitis (EEE) virus has been recognized as affecting horses and humans in the eastern United States for 70 yr. Evidence of exposure with EEE virus has been reported in a variety of free-ranging wild birds and mammals but cases of clinical disease are much less commonly reported. In Michigan, reports of outbreaks of EEE virus in equine species extend back more than a half century. We report diagnosis of EEE virus infection of multiple free-ranging white-tailed deer (Odocoileus virginianus) from three Michigan counties during late summer of 2005. Infection was confirmed in seven of 30 deer collected based on reported neurologic signs and results from immunohistochemistry, polymerase chain reaction, and/or virus isolation. One of the deer also was infected with West Nile virus and an eighth deer had microscopic lesions in the cerebrum consistent with those reported for EEE. To our knowledge, this is the first report of multiple cases of EEE in free-ranging white-tailed deer, and highlights several issues of significance to wildlife managers and public health officials.

Host Choice and West Nile Virus Infection Rates in Blood-Fed Mosquitoes, Including Members of theCulex pipiensComplex, from Memphis and Shelby County, Tennessee, 2002–2003

The source of bloodmeals in 2,082 blood-fed mosquitoes collected from February 2002 through December 2003 in Memphis and surrounding areas of Shelby County, Tennessee were determined. Members of the genus Culex and Anopheles quadrimaculatus predominated in the collections. Members of the Cx. pipiens complex and Cx. restuans were found to feed predominately upon avian hosts, though mammalian hosts made up a substantial proportion of the bloodmeals in these species. No significant difference was seen in the host class of bloodmeals in mosquitoes identified as Cx. pipiens pipiens, Cx. p. quinquefasciatus, or hybrids between these two taxa. Anopheles quadrimaculatus and Cx. erraticus fed primarily upon mammalian hosts. Three avian species (the American Robin, the Common Grackle, and the Northern Cardinal) made up the majority of avian-derived bloodmeals, with the American Robin representing the most frequently fed upon avian host. An analysis of these host feeding data using a modification of a transmission model for Eastern Equine encephalitis virus suggested that the American Robin and Common Grackle represented the most important reservoir hosts for West Nile virus. A temporal analysis of the feeding patterns of the dominant Culex species did not support a shift in feeding behavior away from robins to mammals late in the summer. However, a significant degree of temporal variation was noted in the proportion of robin-derived bloodmeals when the data were analyzed by semi-monthly periods throughout the summers of 2002 and 2003. This pattern was consistent with the hypothesis that the mosquitoes were preferentially feeding upon nesting birds.

Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice

We developed chimeric Sindbis (SINV)/eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >10 4 PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines.

Direct broad-range detection of alphaviruses in mosquito extracts

Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.

Eastern Equine Encephalitis in 9 South American Camelids

Background: Eastern equine encephalitis (EEE) virus is a mosquito‐borne togavirus (alphavirus) that causes severe (often fatal) encephalitis in many mammalian species, but it has not been reported previously in South American camelids.Hypothesis: South American camelids can become naturally infected with EEE virus and show encephalitic signs similar to those observed in other affected species.Animals: Nine cases (8 alpacas and 1 llama, aged 3.5 weeks to 12 years) were identified; 4 of 9 were ≤10 weeks old. All cases were from the East Coast of the United States and presented in late summer and fall.Methods: A retrospective study was performed to include confirmed cases of EEE in camelids in North America before 2006.Results: Eight of nine (89%) camelids died or were euthanized in extremis, with the mean time to death of 2 days. Clinical signs were consistent with encephalitis and included fever, lethargy, ataxia, seizures, recumbency, torticollis, opisthotonus, and vestibular signs. No consistent hematologic abnormalities were identified, and cerebrospinal fluid contained an increased protein concentration in the single camelid analyzed. No successful therapy was identified. EEE was confirmed by alphavirus detection by using immunohistochemistry (IHC) and polymerase chain reaction (PCR) in the central nervous system (CNS) and by serology. Findings included polioencephalitis with lymphocytic perivascular cuffing; neutrophil infiltration; gliosis; neuron satellitosis; necrosis; and edema, with intracytoplasmic alphavirus within neurons and glial cells. No virus was detected in extraneural tissues.Conclusions and Clinical Importance: In endemic areas, EEE should be considered a differential diagnosis for young and adult camelids with CNS disease. Brain histopathology with indirect IHC or PCR is diagnostic.

Effect of Salinity on Temephos Toxicity to Larvae of <I>Aedes sollicitans</I> (Diptera: Culicidae)

Aedes sollicitans (Walker) (Diptera: Culicidae) is an important vector of eastern equine encephalitis as well as several other mosquito-borne brain fevers. The larvae are salt-tolerant and develop in salt marshes with highly varying salinity. The effect of salinity on the toxicity of one of the major larvicidal organophosphates, temephos, was evaluated in two groups of larvae raised either in freshwater or water with salinity ranging from 1 to 3.5%. When larvae were raised in freshwater, low salinity (1-3.5%) decreased the toxicity and high salinity (5%) increased the toxicity. In contrast, salinity did not change the toxicity to larvae raised in saltwater. Temephos treatment and salinity seemed to have cross-interaction for the larvae raised in freshwater. High salinity also caused reduction in larval body size, and 5% salinity alone caused mortality for larvae raised in freshwater, suggesting that preadaptation to saltwater in the early instars is essential for survival in later instars at high salinity.