Venezuelan Equine Encephalitis Complex Research Articles

Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

Venezuelan Equine Encephalitis Complex Alphavirus in Bats, French Guiana.

Although essential for control strategies, knowledge about transmission cycles is limited for Venezuelan equine encephalitis complex alphaviruses (VEEVs). After testing 1,398 bats from French Guiana for alphaviruses, we identified and isolated a new strain of the encephalitogenic VEEV species Tonate virus (TONV). Bats may contribute to TONV spread in Latin America.

Serologic Evidence of Zoonotic Alphaviruses in Humans from an Indigenous Community in the Peruvian Amazon.

Alphaviruses (Togaviridae, Alphavirus) are arthropod-borne single-stranded RNA pathogens that cause febrile and neurologic disease in much of Latin America. However, many features of Alphavirus epidemiology remain unknown. In 2011, we undertook a cross-sectional study in Nueva Esperanza, an indigenous community in the Peruvian Amazon. Here, we present the first serologic evidence of Mayaro (MAYV), Venezuelan equine encephalitis (VEE) complex alphavirus, Una (UNAV), and Madariaga (MADV) viruses reported in humans (24%, 16%, 13%, and 1.5%, respectively) from an Amazonian indigenous community in Peru. Hunting activity and cohabiting with hunters were the main risk factors for Mayaro seroconversion, but only hunting was associated with UNAV seropositivity. Our results suggest that alphavirus infection in this region is common, but we highlight the high UNAV seroprevalence found and corroborate the low MADV prevalence reported in this region. Furthermore, MAYV-neutralizing antibodies were also detected in stored samples from wild animals (18%) hunted by Nueva Esperanza inhabitants and another mestizo community located close to Iquitos. Further serological surveys of VEE complex alphaviruses, UNAV, and MADV in wild animals and assessing the ability of the MAYV seropositive species to transmit the virus will be relevant.

Evolution and spread of Venezuelan equine encephalitis complex alphavirus in the Americas.

Venezuelan equine encephalitis (VEE) complex alphaviruses are important re-emerging arboviruses that cause life-threatening disease in equids during epizootics as well as spillover human infections. We conducted a comprehensive analysis of VEE complex alphaviruses by sequencing the genomes of 94 strains and performing phylogenetic analyses of 130 isolates using complete open reading frames for the nonstructural and structural polyproteins. Our analyses confirmed purifying selection as a major mechanism influencing the evolution of these viruses as well as a confounding factor in molecular clock dating of ancestors. Times to most recent common ancestors (tMRCAs) could be robustly estimated only for the more recently diverged subtypes; the tMRCA of the ID/IAB/IC/II and IE clades of VEE virus (VEEV) were estimated at ca. 149–973 years ago. Evolution of the IE subtype has been characterized by a significant evolutionary shift from the rest of the VEEV complex, with an increase in structural protein substitutions that are unique to this group, possibly reflecting adaptation to its unique enzootic mosquito vector Culex (Melanoconion) taeniopus. Our inferred tree topologies suggest that VEEV is maintained primarily in situ, with only occasional spread to neighboring countries, probably reflecting the limited mobility of rodent hosts and mosquito vectors.

Specific detection of all members of the Venezuelan Equine Encephalitis complex: Development of a RT-Nested PCR

Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rapid and specific detection of these viruses, and may be useful for diagnosis and surveillance.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.

Cocirculation of Rio Negro Virus (RNV) and Pixuna Virus (PIXV) in Tucumán province, Argentina

Venezuelan equine encephalitis complex includes viruses considered emerging pathogens for humans and animals in the Americas. Two members of this complex have been detected previously in Argentina: Rio Negro Virus (RNV), detected in mosquitoes from Chaco province and rodents from Formosa province, and Pixuna Virus (PIXV), detected in mosquitoes from Chaco province. To carry out surveillance studies in other parts of the country, detection of a 195-bp fragment of alphaviruses by RT-nested PCR was performed in mosquito samples from San Miguel de Tucumán city. Four pools resulted positive and three were sequenced. Two amplicons grouped with RNV and one with PIXV. This is the first report of viral activity of members of the Venezuelan equine encephalitis complex in north-eastern Argentina.

Enzootic Activity of Pixuna and Rio Negro Viruses (Venezuelan Equine Encephalitis complex) in a Neotropical Region of Argentina

Venezuelan Equine Encephalitis complex viruses cause epidemics and epizootics periodically in some regions of the Americas. In Argentina, only enzootic Rio Negro virus (AG80-663) (RNV) has been isolated. To survey and identify activity of viruses that belong to Venezuelan Equine Encephalitis complex in a template region of the country, a generic Alphavirus RT-Nested PCR was performed in 99 mosquito pools collected in Chaco province. Five pools were positive, and amplicons were sequenced: four of them clustered with RNV(AG80-663) and one with Pixuna virus. This is the first report of the circulation of Pixuna virus in Argentina, and it confirms enzootic and endemic activity of RNV(AG80-663) in neotropical regions of this country.

Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 x 10(-4) substitutions/site/yr.

Identification of <I>Culex</I> (<I>Melanoconion</I>) Species of the United States Using Female Cibarial Armature (Diptera: Culicidae)

Species within the subgenus Culex (Melanoconion) Theobald are the primary enzootic vectors of viruses in the Venezuelan equine encephalitis complex including Everglades virus, and probable enzootic vectors of eastern equine encephalitis and West Nile viruses. Adult females of this subgenus are often difficult or impossible to identify to species based on external morphological characters. The use of female cibarial armature allows for the identification of field-collected adult female specimens of Culex (Melanoconion). The cibarial armatures are described and illustrated for all species from the United States and a key to species using this character is presented.

Susceptibility of <I>Ae. aegypti</I> (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.

Serologic Evidence of Widespread Everglades Virus Activity in Dogs, Florida

Everglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis complex, circulates among rodents and vector mosquitoes in Florida and occasionally infects humans. It causes febrile disease, sometimes accompanied by neurologic manifestations. Although previous surveys showed high seroprevalence in humans, EVEV infections may be underdiagnosed because the disease is not severe enough to warrant a clinic visit or the undifferentiated presentations complicate diagnosis. Documented EVEV activity, as recent as 1993, was limited to south Florida. Using dogs as sentinels, a serosurvey was conducted to evaluate whether EVEV circulated recently in Florida and whether EVEV's spatial distribution parallels that of the mosquito vector, Culex cedecei. Four percent of dog sera contained neutralizing EVEV antibodies, and many seropositive animals lived farther north than both recorded EVEV activity and the principal vector. These results indicate that EVEV is widespread in Florida and may be an important, unrecognized cause of human illness.

Transmission of a Venezuelan Equine Encephalitis Complex Alphavirus byCulex(Melanoconion)gnomatos(Diptera: Culicidae) in Northeastern Peru

Venezuelan equine encephalitis (VEE) complex alphaviruses are serious health threats in the Americas and regularly infect humans living in or near Amazonian rain forests. As part of a larger surveillance program, we placed six hamster-baited mosquito traps in a disturbed white sand forest of northeastern Peru for 3 d. Virus isolations from hamster serum and trapped mosquito pools demonstrated that a VEE subtype IIIC alphavirus was transmitted to a hamster by the mosquito Culex (Melanoconion) gnomatos Sallum, Hutchings & Ferreira. This species, like the other seven proven VEE complex alphavirus vectors, is a member of the Spissipes section of this subgenus. The composition of mosquitoes collected at the site over the sampling period was typical for the region.

Seroprevalence of antibodies to Venezuelan equine encephalitis complex (subtypes IAB and VI) in humans from General Belgrano island, Formosa, Argentina.

This work presents the results of the detection of antibodies (immunoglobulin G) for subtypes I and VI of VEE viruses complex (Togaviridae family) in people from the General Belgrano island, Formosa province (Argentina). The prevalence of neutralizing (NT) antibodies for subtype VI was from 30% to 70% and the prevalence of antibodies inhibitory of hemagglutination (HI) was of 0% in the first and second inquiry respectively. For the subtype IAB the prevalence of NT antibodies was from 13% to 3.6%, similar to the prevalence total for both subtypes. HI antibodies were not detected in any inquiries for any subtype. It was observed that both subtypes circulate simultaneously, while subtype VI remains constant with some peaks, subtype I was found in low level.

Tonate virus infection in French Guiana: clinical aspects and seroepidemiologic study.

Two recent cases of human infection with Tonate virus, one of which was a fatal case of encephalitis, have renewed interest in these viruses in French Guiana. The clinical aspects of confirmed and probable cases of infection with this virus indicate that it has pathogenic properties in humans similar to those of other viruses of the Venezuelan equine encephalitis complex. To determine the prevalence of antibodies to Tonate virus in the various ethnic groups and areas of French Guiana, 3,516 human sera were tested with a hemagglutination inhibition test. Of these, 11.9% were positive for the virus, but significant differences in seroprevalence were found by age, with an increase with age. After adjustment for age, significant differences were found between places of residence. The prevalence of antibody to Tonate virus was higher in savannah areas, especially in the Bas Maroni (odds ratio [OR] = 22.2, 95% confidence interval [CI] = 15.2-32.4) and Bas Oyapock areas (OR = 13.4; 95% CI = 9.8-18.4). The ethnic differences observed in this study were due mainly to differences in place of residence, except that whites were significantly less frequently infected than other ethnic groups. This study indicates that Tonate virus infection is highly prevalent in French Guiana, especially in savannah areas.

Antibodies to arthropod-borne encephalitis viruses in small mammals from southern Florida.

From 1987 through 1991, blood samples were collected from 10 species of small mammals in Indian River Country, Florida (USA). Sera from 1,347 animals were analyzed for hemagglutination-inhibition (HI) antibody to St. Louis encephalitis (SLE) and eastern equine encephalitis (EEE) viruses. Of these, 75 (5.6%) were positive for HI antibody to SLE virus and 121 (9.0%) were positive for EEE antibody. Sera from five mammalian species were tested for neutralizing (NT) antibody to SLE, EEE, Highlands J (HJ a member of the western equine encephalitis virus complex), or Everglades (EVE, a member of the Venezuelan equine encephalitis complex) viruses. By serum neutralization tests, 26 (46%) of 57 had SLE antibodies, 14 (24%) of 58 had EEE antibodies, two (3.2%) of 63 had HJ antibodies, and 9 (14%) of 63 had EVE antibodies. One Sigmodon hispidus and one Peromyscus gossypinus had NT antibodies both to EEE and HJ viruses. Blood samples from 512 mammals were tested for virus. Isolations of one EVE virus and two unidentified arenaviruses were made from P. gossypinus and one EVE virus isolate was made from a S. hispidus.

Complete Sequence of Venezuelan Equine Encephalitis Virus Subtype IE Reveals Conserved and Hypervariable Domains within the C Terminus of nsP3

The complete nucleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97–100% identity) and more distantly related to VEE strains of other antigenic varieties (83–93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94–95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1–329), which is highly conserved among all alphaviruses, was 93–94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330–563) was only 59–61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts.

Phylogenetic analysis of alphaviruses in the venezuelan equine encephalitis complex and identification of the source of epizootic viruses

We studied the evolution of alphaviruses in the Venezuelan equine encephalitis (VEE) complex using phylogenetic analysis of RNA nucleotide sequences from limited portions of the nsP4, El, and 3′ untranslated genome regions of representative strains. The VEE complex constituted a monophyletic group of viruses (descended from a common ancestor); some serologic VEE varieties such as subtype III formed monophyletic groups while subtype I did not. Subtype II Everglades and variety ID enzootic viruses formed a monophyletic group which also included all epizootic variety IAB and IC VEE isolates. Everglades virus diverged from this ID lineage (colonized North America) ca. 100–150 years ago, followed by divergence of variety IAB and IC epizootic viruses. Variety IAB viruses probably emerged from the variety ID lineage once during the early part of this century, while variety IC viruses evolved at least two times. These results identify the source of epizootic VEE viruses as the variety ID enzootic virus lineage which occurs in northern South America and Panama. Even if variety IAB and IC viruses are extinct, recent, multiple emergences of epizootic viruses from an enzootic lineage suggests that other epizootic VEE viruses may evolve again in the future. The close genetic relationship of subtype II Everglades virus to the variety ID lineage also implies the potential for emergence of equine-virulent VEE viruses in Florida.