Endemic eastern equine encephalitis in the Amazon region of Peru

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.     

Genetic Diversity of Venezuelan Alphaviruses and Circulation of a Venezuelan Equine Encephalitis Virus Subtype IAB Strain During an Interepizootic Period

Several species of alphaviruses have been previously described in the Americas, some of which are associated with encephalitis and others are associated with arthralgia. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are endemic to Venezuela, with the former being responsible for major outbreaks of severe and often fatal disease in animals and humans. The aim of this study was to analyze the genetic diversity of Venezuelan alphaviruses isolated during two decades (1973–1999) of surveillance in northern Venezuela. Phylogenetic analysis indicated the circulation of a VEEV subtype IAB strain 8 years after the last reported outbreak. Thirteen strains within two subclades of South American lineage III of EEEV were also found in Venezuela. Considerable genetic variability was observed among Venezuelan Una virus strains, which were widely distributed among the clades. The first Venezuelan Mayaro sequence was also characterized.     

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.     

A multi-year study of mosquito feeding patterns on avian hosts in a southeastern focus of eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV) is a mosquito-borne pathogen that cycles in birds but also causes severe disease in humans and horses. We examined patterns of avian host use by vectors of EEEV in Alabama from 2001 to 2009 using blood-meal analysis of field-collected mosquitoes and avian abundance surveys. The northern cardinal (Cardinalis cardinalis) was the only preferred host (fed on significantly more than expected based on abundance) of Culiseta melanura, the enzootic vector of EEEV. Preferred hosts of Culex erraticus, a putative bridge vector of EEEV, were American robin (Turdus migratorius), Carolina chickadee (Poecile carolinensis), barred owl (Strix varia), and northern mockingbird (Mimus polyglottis). Our results provide insight into the relationships between vectors of EEEV and their avian hosts in the Southeast and suggest that the northern cardinal may be important in the ecology of EEEV in this region.     

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.     

Western equine encephalitis virus is a recombinant virus.

The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.     

Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America.

Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. Here we describe serologic and phylogenetic analyses of representatives of these varieties, spanning the entire temporal and geographic range available. Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range. Plaque reduction neutralization tests with representatives from the 4 major lineages indicated that each represents a distinct antigenic subtype. A taxonomic revision of the EEE complex is proposed.     

Serosurveillance of eastern equine encephalitis virus in amphibians and reptiles from Alabama, USA

Eastern equine encephalitis virus (EEEV) is among the most medically important arboviruses in North America, and studies suggest a role for amphibians and reptiles in its transmission cycle. Serum samples collected from 351 amphibians and reptiles (27 species) from Alabama, USA, were tested for the presence of antibodies against EEEV. Frogs, turtles, and lizards showed little or no seropositivity, and snakes had high seropositivity rates. Most seropositive species were preferred or abundant hosts of Culex spp. mosquitoes at Tuskegee National Forest, that target ectothermic hosts. The cottonmouth, the most abundant ectotherm sampled, displayed a high prevalence of seropositivity, indicating its possible role as an amplification and/or over-wintering reservoir for EEEV.     

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.     

Isolation of eastern equine encephalitis virus and West Nile virus from crows during increased arbovirus surveillance in Connecticut, 2000

The emergence of the West Nile virus (WNV) in the northeastern United States has drawn emphasis to the need for expanded arbovirus surveillance in Connecticut. Although the state of Connecticut began a comprehensive mosquito-screening program in 1997, only since 1999 have there been efforts to determine the prevalence of arboviruses in bird populations in this state. Herein, we report on our results of an arbovirus survey of 1,704 bird brains. Included in this report are the first known isolations of eastern equine encephalitis virus (EEEV) from crows and data on the geographic and temporal distribution of 1,092 WNV isolations from crow species. Moreover, these nine isolations of EEEV identify regions of Connecticut where the virus is rarely found. With the exception of WNV and EEEV, no other arboviruses were isolated or detected. Taken together, these data illustrate the distribution of avian borne EEEV and WNV in 2000 and support the need for ongoing avian arbovirus surveillance in Connecticut.     

PERK Is Critical for Alphavirus Nonstructural Protein Translation

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4–5 log₁₀. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.     

Detection of Venezuelan equine encephalitis virus in rural communities of Southern Florida by exposure of sentinel hamsters.

Venezuelan equine encephalitis (VEE) virus strains were recovered from sentinel hamsters exposed in close proximity to homes in rural South Florida. Sentinel hamster surveillance methods over extended periods offer one effective way of uncovering VEE virus activity in relation to human habitation.     

Cross-Strain Neutralizing and Protective Monoclonal Antibodies against EEEV or WEEV

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.     

Transmission potential of two chimeric Chikungunya vaccine candidates in the urban mosquito vectors, Aedes aegypti and Ae. albopictus

Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus that has caused major epidemics in Africa and Asia. We developed chimeric vaccine candidates using the non-structural protein genes of either Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain TC-83 or a naturally attenuated strain of eastern equine encephalitis virus (EEEV) and the structural genes of CHIKV. Because the transmission of genetically modified live vaccine strains is undesirable because of the potentially unpredictable evolution of these viruses as well as the potential for reversion, we evaluated the ability of these vaccines to infect the urban CHIKV vectors, Aedes aegypti and Ae. albopictus. Both vaccine candidates exhibited significantly lower infection and dissemination rates compared with the parent alphaviruses. Intrathoracic inoculations indicated that reduced infectivity was mediated by midgut infection barriers in both species. These results indicate a low potential for transmission of these vaccine strains in the event that a vaccinee became viremic.     

Tracking eastern equine encephalitis virus perpetuation in the northeastern United States by phylogenetic analysis

Epidemics and epizootics of eastern equine encephalitis virus (EEEV) occur sporadically in temperate regions where transmission is seasonal from late summer to early fall. These outbreaks may derive from virus that overwinters locally or perhaps results from reintroduction of virus from other sites. To evaluate these possibilities, we compared the phylogenetic relationships of EEEV isolates obtained from mosquitoes collected during statewide arbovirus surveillance in Connecticut, in addition to isolates from concurrent outbreaks in southern New Hampshire and upstate New York. In Connecticut, viral isolates grouped into temporally discrete clades by year of isolation or over 2 years of sampling. Two or more clades arose in 2000, 2001, 2003, 2004, and 2006, possibly the result of separate introduction events into the state, whereas viruses from upstate New York and New Hampshire segregated into single clades that persisted for 2 or more years. New Hampshire viruses shared recent common ancestry to those isolated in Connecticut suggesting viral dispersal among these regions. These results provide additional evidence for independent episodes of EEEV overwintering in northern foci.     

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.     

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.     

实时RT-PCR法检测感染小鼠不同组织标本中的委内瑞拉马脑炎病毒

目的为委内瑞拉马脑炎病毒所致疾病的早期诊断和流行病学研究提供技术支持。方法建立了委内瑞拉马脑炎病毒特异、敏感和快速的实时RT-PCR法,并对委内瑞拉马脑炎病毒感染昆明小鼠的不同组织标本中的病毒载量进行了定量检测。结果本研究建立的实时RT-PCR法敏感(10TCID50/ml)、特异(对其它甲病毒成员,如东部马脑炎病毒和西部马脑炎病毒检测为阴性)。该方法可从委内瑞拉马脑炎病毒感染小鼠后第1d的血液、脾脏、脑组织中检测到不同含量的病毒核酸,而在感染后第3d仅从脾脏和脑组织中检测到病毒核酸。结论实时RT-PCR法不仅是一种敏感、准确的检测方法,可用来了解病毒感染量与疾病严重程度的关系和评价病毒病疫苗的免疫效果,而且也为VEEV疾病的临床诊断和流行病学监测提供工具。     

Field Investigations of Winter Transmission of Eastern Equine Encephalitis Virus in Florida

Studies investigating winter transmission of Eastern equine encephalitis virus (EEEV) were conducted in Hillsborough County, Florida. The virus was detected in Culiseta melanura and Anopheles quadrimaculatus in February 2012 and 2013, respectively. During the winter months, herons were the most important avian hosts for all mosquito species encountered. In collections carried out in the summer of 2011, blood meals taken from herons were still common, but less frequently encountered than in winter, with an increased frequency of mammalian- and reptile-derived meals observed in the summer. Four wading bird species (Black-crowned Night Heron [Nycticorax nycticorax], Yellow-crowned Night Heron [Nyctanassa violacea], Anhinga [Anhinga anhinga], and Great Blue Heron [Ardea herodias]) were most frequently fed upon by Cs. melanura and Culex erraticus, suggesting that these species may participate in maintaining EEEV during the winter in Florida.