Complete genomic sequences of Venezuelan equine encephalitis virus subtype IIID isolates from mosquitoes.

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

Comparison of Aerosol- and Percutaneous-acquired Venezuelan Equine Encephalitis in Humans and Nonhuman Primates for Suitability in Predicting Clinical Efficacy under the Animal Rule.

Licensure of medical countermeasure vaccines to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires the use of the Animal Rule to assess vaccine efficacy, because human studies are not feasible or ethical. We therefore performed a retrospective study of VEE cases that occurred in at-risk laboratory workers and support personnel during the United States Biowarfare Program (1943-1969) to better define percutaneous- and aerosol-acquired VEE in humans and to compare these results with those described for the NHP model (in which high-dose aerosol VEEV challenge led to more severe encephalitis than parenteral challenge). Record review and analysis of 17 aerosol- and 23 percutaneous-acquired human cases of VEE included incubation period, symptoms, physical examination findings, and markers of infection. Human VEE disease by both exposure routes presented as acute febrile illness, typically with fever, chills, headache, back pain, malaise, myalgia, anorexia, and nausea. Aerosol exposure more commonly led to upper respiratory tract-associated findings of sore throat (59% compared with 26%), pharyngeal erythema (76% compared with 52%), neck pain (29% compared with 4%), and cervical lymphadenopathy (29% compared with 4%). Other disease manifestations, including encephalitis, were similar between the 2 exposure groups. The increase in upper respiratory tract findings in aerosol-acquired VEE in humans has not previously been reported but is supported by the mouse model, which showed nasal mucosal necrosis, necrotizing rhinitis, and an increase in upper respiratory tract viral burden associated with aerosol VEEV challenge. Fever, viremia, and lymphopenia were common markers of VEE disease in both humans and NHP, regardless of the exposure route. Taken collectively, our findings provide support for use of the nonlethal NHP model for advanced development of medical countermeasures against aerosol- or percutaneous-acquired VEE.

Isolation of Complete Equine Encephalitis Virus Genome from Human Swab Specimen, Peru.

While studying respiratory infections in Peru, we identified Venezuelan equine encephalitis virus (VEEV) in a nasopharyngeal swab, indicating that this alphavirus can be present in human respiratory secretions. Because VEEV may be infectious when aerosolized, our finding is relevant for the management of VEEV-infected patients and for VEEV transmission studies.

Genetic and evolutionary characterization of Venezuelan equine encephalitis virus isolates from Argentina.

Venezuelan equine encephalitis viruses (VEEV) are emerging pathogens of medical and veterinary importance circulating in America. Argentina is a country free from epizootic VEEV activity, with circulation of enzootic strains belonging to Rio Negro virus (RNV; VEEV subtype VI) and Pixuna virus (PIXV, VEEV subtype IV). In this work, we aim to report the sequencing and phylogenetic analyses of all Argentinean VEE viruses, including 7 strains previously isolated from mosquitoes in 1980, 5 sequences obtained from rodents in 1991 and 11 sequences amplified from mosquitoes between 2003 and 2005. Two genomic regions, corresponding to the non-structural protein 4 (nsP4) and the protein E3/E2 (PE2) genes were analyzed, but only 8 samples could be amplified in the last one (longer and more variable fragment of 702 bp). For both genomic fragments, phylogenetic trees showed the absence of lineages within RNV group, and a close genetic relationship between Argentinean strains and the prototype strain BeAr35645 for PIXV clade. The analysis of nsP4 gene opens the possibility to propose a possible geographic clustering of strains within PIXV group (Argentina and Brazil). Coalescent analysis performed on RNV sequences suggested a common ancestor of 58.3 years (with a 95% highest posterior density [HPD] interval of 16.4-345.7) prior to 1991 and inferred a substitution rate of 9.8×10(-5)substitutions/site/year, slightly lower than other enzootic VEE viruses. These results provide, for the first time, information about genetic features and variability of all VEEVs detected in Argentina, creating a database that will be useful for future detections in our country. This is particularly important for RNV, which has indigenous circulation.

Pathogenesis of Venezuelan equine encephalitis.

Equine encephalids have high mortality rates and represent a significant zoonotic public health threat. Of these the most pathogenic viruses to equids are the alphaviruses in the family Togaviridae. The focus of this review Venezualen equine encephalitis virus (VEEV) has caused the most widespread and recent epidemic outbreaks of disease. Circulation in naturally occuring rodent-mosquito cycles, results in viral spread to both human and equine populations. However, equines develop a high titer viremia and can transmit the virus back to mosquito populations. As such, the early recognition and control of viral infection in equine populations is strongly associated with prevention of epidemic spread of the virus and limiting of disease incidence in human populations. This review will address identification and pathogenesis of VEEV in equids vaccination and treatment options, and current research for drug and vaccine development.

Vector-borne transmission imposes a severe bottleneck on an RNA virus population.

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study.

Candidate vectors and rodent hosts of Venezuelan equine encephalitis virus, Chiapas, 2006-2007.

Enzootic Venezuelan equine encephalitis virus (VEEV) has been known to occur in Mexico since the 1960s. The first natural equine epizootic was recognized in Chiapas in 1993 and since then, numerous studies have characterized the etiologic strains, including reverse genetic studies that incriminated a specific mutation that enhanced infection of epizootic mosquito vectors. The aim of this study was to determine the mosquito and rodent species involved in enzootic maintenance of subtype IE VEEV in coastal Chiapas. A longitudinal study was conducted over a year to discern which species and habitats could be associated with VEEV circulation. Antibody was rarely detected in mammals and virus was not isolated from mosquitoes. Additionally, Culex (Melanoconion) taeniopus populations were found to be spatially related to high levels of human and bovine seroprevalence. These mosquito populations were concentrated in areas that appear to represent foci of stable, enzootic VEEV circulation.

The crab hole mosquito blues.

Venezuelan equine encephalomyelitis (VEE) epizoodemics were reported at 6-10-year intervals in northern South America beginning in the 1920s. In 1937, epizootic VEE virus was isolated from infected horse brain and shown as distinct from the North American equine encephalomyelitis viruses. Subsequently, epizootic and sylvatic strains were isolated in distinct ecosystems; isolates were characterized serologically as epizootic subtype I, variants A/B and C; or sylvatic (enzootic) subtype I, variants D, E, and F, and subtypes II, III, and IV. In 1969, variant I-A/B virus was transported from a major outbreak in northern South America to the borders of El Salvador, Guatemala, and Honduras. This musical poem describes the history and ecology of VEE viruses and the epidemiology of an unprecedented 1969 movement of VEE viruses from South America to equids and humans in Central America from Costa Rica to Guatemala and Belize and in Mexico and the United States that continued until 1972.

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.

Venezuelan equine encephalitis virus in Iquitos, Peru: urban transmission of a sylvatic strain.

Enzootic strains of Venezuelan equine encephalitis virus (VEEV) have been isolated from febrile patients in the Peruvian Amazon Basin at low but consistent levels since the early 1990s. Through a clinic-based febrile surveillance program, we detected an outbreak of VEEV infections in Iquitos, Peru, in the first half of 2006. The majority of these patients resided within urban areas of Iquitos, with no report of recent travel outside the city. To characterize the risk factors for VEEV infection within the city, an antibody prevalence study was carried out in a geographically stratified sample of urban areas of Iquitos. Additionally, entomological surveys were conducted to determine if previously incriminated vectors of enzootic VEEV were present within the city. We found that greater than 23% of Iquitos residents carried neutralizing antibodies against VEEV, with significant associations between increased antibody prevalence and age, occupation, mosquito net use, and overnight travel. Furthermore, potential vector mosquitoes were widely distributed across the city. Our results suggest that while VEEV infection is more common in rural areas, transmission also occurs within urban areas of Iquitos, and that further studies are warranted to identify the precise vectors and reservoirs involved in urban VEEV transmission.

Susceptibility of Ae. aegypti (Diptera: Culicidae) to infection with epidemic (subtype IC) and enzootic (subtypes ID, IIIC, IIID) Venezuelan equine encephalitis complex alphaviruses.

To test the hypothesis that enzootic and epidemic Venezuelan equine encephalitis (VEE) complex alphaviruses can infect and be transmitted by Ae. aegypti, we conducted a series of experimental infection studies. One set of experiments tested the susceptibility of geographic strains of Ae. aegypti from Peru and Texas (U.S.A.) for epidemic (subtype IC) and enzootic (subtype ID) strains from Colombia/Venezuela, whereas the second set of experiments tested the susceptibility of Ae. aegypti from Iquitos, Peru, to enzootic VEE complex strains (subtypes ID, IIIC, and IIID) isolated in the same region, at different infectious doses. Experimental infections using artificial bloodmeals suggested that Ae. aegypti mosquitoes, particularly the strain from Iquitos, Peru, is moderately to highly susceptible to all of these VEE complex alphaviruses. The occurrence of enzootic VEE complex viruses circulating endemically in Iquitos suggests the possibility of a dengue-like transmission cycle among humans in tropical cities.

[Mosquitos (Diptera: Culicidae) in the small village where a human case of Venezuelan equine encephalitis was recorded]. / Mosquitos (Diptera: Culicidae) en el caserío de Chingalé, Santander, donde se registró un caso humano de encefalitis equina venezolana.

INTRODUCTION: The enzootic focus of subtype ID of Venezuelan equine encephalitis (VEE) virus in the Central Magdalena region (central Colombia) occasionally produces human cases. The report of a VEE infection in a three-year-old girl in the small Chingalé, municipalitype of Puerto Wilches, Santander, motivated this study. OBJECTIVE: The village of Chingalé was evaluated as the probable site of infection. MATERIALS AND METHODS: In June 2005, mosquitoes were collected with CDC light traps in and outside of dwellings in the village. Trinidad traps were placed in nearby vegetation, and hamsters were used as sentinel animals near homes. RESULTS: One hundred and seven samples, consisting of 14,423 mosquitoes of 35 species were collected. The relative abundance of incriminated vectors of subtype ID of VEE, Culex (Melanoconion) pedroi and Cx. (Mel.) ocossa, was generally low (<4%), but both species were more frequent outside of dwellings than indoors. Cx. (Mel.) ocossa was collected in CDC traps and was more frequent indoors,whereas Cx. (Mel.) pedroi was found in the Trinidad traps. In addition, Psorophora confinnis was present, recognized as a potential vector of the epidemo/epizootic subtype. Mansonia indubitans, another recognized vector, was present at high frequency within dwellings. The exposed hamsters did not become infected. CONCLUSION: The child may have been infected in or near her home, although the epidemiologic cycle of the virus was not demonstrated within the village of Chingalé. Possibly, infected Culex mosquitoes of the subgenus Melanoconion carried the virus into the village from a neighboring habitat.

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.

Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: infection initiated by a small number of susceptible epithelial cells and a population bottleneck.

We evaluated infection of Aedes taeniorhynchus mosquitoes, vectors of Venezuelan equine encephalitis virus (VEEV), using radiolabeled virus and replicon particles expressing green (GFP) or cherry fluorescent protein (CFP). More epidemic VEEV bound to and infected mosquito midguts compared to an enzootic strain, and a small number of midgut cells was preferentially infected. Chimeric replicons infected midgut cells at rates comparable to those of the structural gene donor. The numbers of midgut cells infected averaged 28, and many infections were initiated in only 1-5 cells. Infection by a mixture of GFP- and CFP-expressing replicons indicated that only about 100 midgut cells were susceptible. Intrathoracic injections yielded similar patterns of replication with both VEEV strains, suggesting that midgut infection is the primary limitation to transmission. These results indicate that the structural proteins determine initial infection of a small number of midgut cells, and that VEEV undergoes population bottlenecks during vector infection.

Infection and dissemination of Venezuelan equine encephalitis virus in the epidemic mosquito vector, Aedes taeniorhynchus.

The mosquito Aedes taeniorhynchus is an important epidemic vector of Venezuelan equine encephalitis virus (VEEV), but detailed studies of its infection are lacking. We compared infection by an epidemic VEEV strain to that by an enzootic strain using virus titrations, immunohistochemistry, and a virus expressing the green fluorescent protein. Ae. taeniorhynchus was more susceptible to the epidemic strain, which initially infected the posterior midgut and occasionally the anterior midgut and cardia. Once dissemination beyond the midgut occurred, virus was present in nearly all tissues. Transmission of the epidemic strain to mice was first detected 4 days after infection. In contrast, the enzootic strain did not efficiently infect midgut cells but replicated in muscles and nervous tissue on dissemination. Because VEEV emergence can depend on adaptation to epidemic vectors, these results show that epidemic/enzootic strain comparisons not only comprise a useful model system to study alphavirus transmission by mosquitoes, but also have important public health implications.

Challenges in biodefense research and the role of US Army veterinary pathologists.

For years the nation's development of medical countermeasures to biowarfare agents has primarily existed as the domain of the United States military, but it has taken on increased urgency in the last few years. The realization that the civilian population is also at risk from biological agents has resulted in the institution of new biodefense programs at a variety of nonmilitary organizations. USAMRIID, a long-time leader in the nation's biodefense effort, will soon be joined by other US government agencies as part of a planned National Interagency Biodefense Campus at Fort Detrick Maryland. US Army veterinary pathologists at USAMRIID have played an important role in the nation's biodefense effort, along with our veterinary colleagues representing other specialties, our military colleagues in other Army Medical Department corps, and our civilian colleagues. Together, we will continue to strive to develop the diagnostics, vaccines, therapeutic agents, and operational practices that are required to meet the great demands posed by the threat of biowarfare agents.

Venezuelan equine encephalitis virus transmission and effect on pathogenesis.

Quantifying the dose of an arbovirus transmitted by mosquitoes is essential for designing pathogenesis studies simulating natural infection of vertebrates. Titration of saliva collected in vitro from infected mosquitoes may not accurately estimate titers transmitted during blood feeding, and infection by needle injection may affect vertebrate pathogenesis. We compared the amount of Venezuelan equine encephalitis virus collected from the saliva of Aedes taeniorhynchus to the amount injected into a mouse during blood feeding. Less virus was transmitted by mosquitoes in vivo (geometric mean 11 PFU) than was found for comparable times of salivation in vitro (mean saliva titer 74 PFU). We also observed slightly lower early and late viremia titers in mice that were needle injected with 8 PFU, which represents the low end of the in vivo transmission range. No differences in survival were detected, regardless of the dose or infection route.

Neurologists and the threat of bioterrorism.

Neurologists are most likely to become involved in primarily diagnosing those bioterrorist attacks utilising botulinum toxin. Oral ingestion, or possibly inhalation, are likely routes of delivery. The characteristic descending paralysis starts in the extraocular and bulbar muscles, with associated autonomic features. Repetitive nerve stimulation usually shows an incremental muscle response. Treatment is supportive. The differential diagnosis is from naturally occurring paralysing illnesses such as Guillain-Barré syndrome, myasthenic crisis or diphtheria, from paralysing seafood neurotoxins (tetrodotoxin, saxitoxin), snake envenomation, and from chemical warfare poisoning by organophosphates. Primary neurological infections are less feasible for use as bioweapons. There are theoretical possibilities of Venezuelan equine encephalitis transmission by inhalation and secondary zoonotic transmission cycles sustained by horses and mosquitoes. Severe haemorrhagic meningitis regularly occurs in anthrax, usually in the aftermath of severe systemic disease likely to have been transmitted by spore inhalation. Panic and psychologically determined 'me-too' symptomatology are likely to pose the biggest diagnostic and management burden on neurologists handling bioterrorist attack on an institution or a random civilian population. Indeed civilian panic and disablement of institutional operations are likely to be prominent intentions of any bioterrorist attack.

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for a subtype IIIC virus in the Venezuelan equine encephalomyelitis complex isolated from mosquitoes captured in Peru.

We evaluated mosquitoes collected in the Amazon Basin, near Iquitos, Peru, for their susceptibility to a subtype IIIC strain of the Venezuelan equine encephalomyelitis complex. This virus had been previously isolated from a pool of mixed Culex vomerifer and Cx. gnomatos captured near Iquitos, Peru, in 1997. After feeding on hamsters with viremias of about 10(8) plaque-forming units of virus per ml, Cx. gnomatos was the most efficient vector. Other species, such as Ochlerotatus fulvus and Psorophora cingulata, although highly susceptible to infection, were not efficient laboratory vectors of this virus due to a significant salivary gland barrier. The Cx. (Culex) species, consisting mostly of Cx. (Cux.) coronator, were nearly refractory to subtype IIIC virus and exhibited both midgut infection as well as salivary gland barriers. Additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of subtype IIIC virus are needed to determine the role that these species play in the maintenance and spread of this virus in the Amazon Basin region.