Venezuelan Equine Encephalitis Virus Subtype Research Articles

Host associations of Culex panocossa (Diptera: Culicidae) in southern Florida and its implications for arbovirus transmission.

Culex panocossa, Dyar and Knab, an important enzootic vector of Venezuelan equine encephalitis virus subtype ID in Central and South America, was found to have invaded and become established in southern Florida in 2016. No information is currently available regarding the ecology of this invasive mosquito in the United States. Here, we use PCR-based blood meal analysis to investigate vertebrate host associations of Cx. panocossa from Florida to provide information necessary for determining the potential importance of this mosquito for arbovirus transmission in the United States. Culex panocossa fed mainly upon birds (49.5%) but took a substantial fraction of blood meals from mammals (33.3%) and reptiles (17.1%). By feeding upon amplifying hosts of Everglades virus (hispid cotton rat) and eastern equine encephalitis virus (wading birds) and humans, Cx. panocossa could act as a bridge vector for these pathogenic Alphaviruses in Florida, potentially resulting in increased human disease.

Defining the Cynomolgus Macaque (Macaca fascicularis) Animal Model for Aerosolized Venezuelan Equine Encephalitis: Importance of Challenge Dose and Viral Subtype.

Venezuelan equine encephalitis virus (VEEV) outbreaks occur sporadically. Additionally, VEEV has a history of development as a biothreat agent. Yet, no FDA-approved vaccine or therapeutic exists for VEEV disease. The sporadic outbreaks present a challenge for testing medical countermeasures (MCMs) in humans; therefore, well-defined animal models are needed for FDA Animal Rule licensure. The cynomolgus macaque (CM) model has been studied extensively at high challenge doses of the VEEV Trinidad donkey strain (>1.0 × 108 plaque-forming units [PFU]), doses that are too high to be a representative human dose. Based on viremia of two subtypes of VEEV, IC, and IAB, we found the CM infectious dose fifty (ID50) to be low, 12 PFU, and 6.7 PFU, respectively. Additionally, we characterized the pattern of three clinical parameters (viremia, temperature, and lymphopenia) across a range of doses to identify a challenge dose producing consistent signs of infection. Based on these studies, we propose a shift to using a lower challenge dose of 1.0 × 103 PFU in the aerosol CM model of VEEV disease. At this dose, NHPs had the highest viremia, demonstrated a fever response, and had a measurable reduction in complete lymphocyte counts-biomarkers that can demonstrate MCM efficacy.

Everglades virus: an underrecognized disease-causing subtype of Venezuelan equine encephalitis virus endemic to Florida, USA.

Everglades virus (EVEV) is subtype II of the Venezuelan equine encephalitis virus (VEEV) complex (Togaviridae: Alphavirus), endemic to Florida, USA. EVEV belongs to a clade that includes both enzootic and epizootic/epidemic VEEV subtypes. Like other enzootic VEEV subtypes, muroid rodents are important vertebrate hosts for EVEV and certain mosquitoes are important vectors. The hispid cotton rat Sigmodon hispidus and cotton mouse Peromyscus gossypinus are important EVEV hosts, based on natural infection (virus isolation and high seropositivity), host competence (experimental infections), and frequency of contact with the vector. The mosquito Culex (Melanoconion) cecedei is the only confirmed vector of EVEV based upon high natural infection rates, efficient vector competence, and frequent feeding upon muroid rodents. Human disease attributed to EVEV is considered rare. However, cases of meningitis and encephalitis are recorded from multiple sites, separated by 250 km or more. Phylogenetic analyses indicate that EVEV is evolving, possibly due to changes in the mammal community. Mutations in the EVEV genome are of concern, given that epidemic strains of VEEV (subtypes IAB and IC) are derived from enzootic subtype ID, the closest genetic relative of EVEV. Should epizootic mutations arise in EVEV, the abundance of Aedes taeniorhynchus and other epizootic VEEV vectors in southern Florida provides a conducive environment for widespread transmission. Other factors that will likely influence the distribution and frequency of EVEV transmission include the establishment of Culex panocossa in Florida, Everglades restoration, mammal community decline due to the Burmese python, land use alteration by humans, and climate change.

Detection of Venezuelan Equine Encephalitis Virus from Brain Samples of Equines with Encephalitis

Alphavirus species are globally distributed zoonoses primarily transmitted by arthropods. The Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are endemic in Costa Rica. This study aims to detect these viruses in brain samples from equines displaying nervous signs. For this purpose, four published Universal RT-PCR methods were compared. The most sensitive and specific RT-PCR method was used to test a total of 70 brain samples, including 40 from bovines and 30 from equines, all exhibiting nervous signs. In the positive cases, eight different brain regions were extracted and tested using this RT-PCR. Positive cases were confirmed through sequencing. Torii RT-PCR demonstrated the highest sensitivity and specificity for diagnosing VEEV and EEEV/Sind among the four Universal RT-PCR assays. Not all assessed brain regions showed DNA amplification. None of the bovine brains was positive, and out of the 30 equine brain samples, only four tested positive, and sequencing confirmed two of these samples as VEEV subtype IE. Torii RT-PCR successfully detected VEEV in pools of the hippocampus, spinal cord, and basal nuclei, making these brain regions suitable for diagnosing this virus. None of the samples were positive for EEEV or WEEV.

Serological and Molecular Evidence of the Circulation of the Venezuelan Equine Encephalitis Virus Subtype IIIA in Humans, Wild Vertebrates and Mosquitos in the Brazilian Amazon.

Understanding the interaction between viruses and ecosystems in areas with or without anthropic interference can contribute to the organization of public health services, as well as prevention and disease control. An arbovirus survey was conducted at Caxiuanã National Forest, Pará, Brazil, where 632 local residents, 338 vertebrates and 15,774 pools of hematophagous arthropods were investigated. Neutralization antibodies of the Venezuelan Equine Encephalitis virus, subtype IIIA, Mucambo virus (MUCV) were detected in 57.3% and 61.5% of humans and wild vertebrates, respectively; in addition, genomic fragments of MUCV were detected in pool of Uranotaenia (Ura.) geometrica. The obtained data suggest an enzootic circulation of MUCV in the area. Understanding the circulation of endemic and neglected arboviruses, such as MUCV, represents an important health problem for the local residents and for the people living in the nearby urban centers.

Phylogenetic and Mutation Analysis of the Venezuelan Equine Encephalitis Virus Sequence Isolated in Costa Rica from a Mare with Encephalitis.

Venezuelan Equine Encephalitis virus (VEEV) is an arboviral pathogen in tropical America that causes lethal encephalitis in horses and humans. VEEV is classified into six subtypes (I to VI). Subtype I viruses are divided into epizootic (IAB and IC) and endemic strains (ID and IE) that can produce outbreaks or sporadic diseases, respectively. The objective of this study was to reconstruct the phylogeny and the molecular clock of sequences of VEEV subtype I complex and identify mutations within sequences belonging to epizootic or enzootic subtypes focusing on a sequence isolated from a mare in Costa Rica. Bayesian phylogeny of the VEEV subtype I complex tree with 110 VEEV complete genomes was analyzed. Evidence of positive selection was evaluated with Datamonkey server algorithms. The putative effects of mutations on the 3D protein structure in the Costa Rica sequence were evaluated. The phylogenetic analysis showed that Subtype IE-VEEV diverged earlier than other subtypes, Costa Rican VEEV-IE ancestors came from Nicaragua in 1963 and Guatemala in 1907. Among the observed non-synonymous mutations, only 17 amino acids changed lateral chain groups. Fourteen mutations located in the NSP3, E1, and E2 genes are unique in this sequence, highlighting the importance of E1-E2 genes in VEEV evolution.

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-transmitted neurotropic alphavirus that causes encephalitis and death in humans1. VEEV is a biodefense concern because of its potential for aerosol spread and lack of sufficient countermeasures. The host factors required for VEEV entry and infection remain poorly characterized. Using a genome-wide CRISPR/Cas9-based screen, we identify Ldlrad3, a highly conserved member of the scavenger receptor superfamily, as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation. Ldlrad3 binds directly to VEEV particles and enhances virus attachment and internalization into cells. Genetic studies indicate that domain 1 (D1) of Ldlrad3 is necessary and sufficient to support VEEV infection, and both anti-Ldlrad3 antibodies and a Ldlrad3-D1-Fc fusion protein block VEEV infection in cell culture. Remarkably, VEEV pathogenesis is abrogated in mice with deletions in Ldlrad3, and administration of Ldlrad3-D1-Fc abolishes disease caused by multiple VEEV subtypes including highly virulent strains. The development of a decoy receptor fusion protein creates a strategy for preventing severe VEEV infection and disease in humans.

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.

Patterns of Abundance, Host Use, and Everglades Virus Infection in Culex (Melanoconion) cedecei Mosquitoes, Florida, USA.

Everglades virus (EVEV), subtype II within the Venezuelan equine encephalitis (VEE) virus complex, is a mosquitoborne zoonotic pathogen endemic to south Florida, USA. EVEV infection in humans is considered rare, probably because of the sylvatic nature of the vector, the Culex (Melanoconion) cedecei mosquito. The introduction of Cx. panocossa, a tropical vector mosquito of VEE virus subtypes that inhabits urban areas, may increase human EVEV exposure. Field studies investigating spatial and temporal patterns of abundance, host use, and EVEV infection of Cx. cedecei mosquitoes in Everglades National Park found that vector abundance was dynamic across season and region. Rodents, particularly Sigmodon hispidus rats, were primary vertebrate hosts, constituting 77%–100% of Cx. cedecei blood meals. Humans were fed upon at several locations. We detected EVEV infection in Cx. cedecei mosquitoes in lower and upper regions of Everglades National Park only during the wet season, despite an abundance of Cx. cedecei mosquitoes at other sampling times.

A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

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.

IRES-based Venezuelan equine encephalitis vaccine candidate elicits protective immunity in mice

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes periodic outbreaks that impact equine and human populations in the Americas. One of the VEEV subtypes located in Mexico and Central America (IE) has recently been recognized as an important cause of equine disease and death, and human exposure also appears to be widespread. Here, we describe the use of an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus to stably attenuate VEEV, creating a vaccine candidate independent of unstable point mutations. Mice infected with this virus produced antibodies and were protected against lethal VEEV challenge. This IRES-based vaccine was unable to establish productive infection in mosquito cell cultures or in intrathoracically injected Aedes taeniorhynchus, demonstrating that it cannot be transmitted from a vaccinee. These attenuation, efficacy and safety results justify further development for humans or equids of this new VEEV vaccine candidate.

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

Antibody to the E3 Glycoprotein Protects Mice against Lethal Venezuelan Equine Encephalitis Virus Infection

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.

Immunogenicity and protective efficacy of a DNA vaccine against Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge.

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

BackgroundVenezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.MethodsWe used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.FindingsUsing 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.ConclusionsThe VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

Vector competence of Culex (Melanoconion) taeniopus for equine-virulent subtype IE strains of Venezuelan equine encephalitis virus.

The mosquito Culex (Melanoconion) taeniopus is a proven vector of enzootic Venezuelan equine encephalitis virus (VEEV) subtype IE in Central America. It has been shown to be highly susceptible to infection by this subtype, and conversely to be highly refractory to infection by other VEEV subtypes. During the 1990s in southern coastal Mexico, two VEE epizootics in horses were attributed to subtype IE VEEV. These outbreaks were associated with VEEV strains with an altered infection phenotype for the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. To determine the infectivity for the enzootic vector, Culex taeniopus, mosquitoes from a recently established colony were orally exposed to VEEV strains from the outbreak. The equine-virulent strains exhibited high infectivity and transmission potential comparable to a traditional enzootic subtype IE VEEV strain. Thus, subtype IE VEEV strains in Chiapas are able to efficiently infect enzootic and epizootic vectors and cause morbidity and mortality in horses.

Experimental Infections of Oryzomys couesi with Sympatric Arboviruses from Mexico

Coues rice rat (Oryzomys couesi), a species abundant throughout Central America, was evaluated experimentally for the ability to serve as an amplifying host for three arboviruses: Patois (Bunyaviridae, Orthobunyavirus), Nepuyo (Orthobunyavirus), and Venezuelan equine encephalitis virus subtype ID (Togaviridae, Alphavirus). These three viruses have similar ecologies and are known to co-circulate in nature. Animals from all three cohorts survived infection and developed viremia with no apparent signs of illness and long-lasting antibodies. Thus, O. couesi may play a role in the general maintenance of these viruses in nature.

Comparison of the immunological responses and efficacy of gamma-irradiated V3526 vaccine formulations against subcutaneous and aerosol challenge with Venezuelan equine encephalitis virus subtype IAB

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel™ further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.

Genetic Characterization of Venezuelan Equine Encephalitis Virus from Bolivia, Ecuador and Peru: Identification of a New Subtype ID Lineage

Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. A passive surveillance study was conducted in Peru, Bolivia and Ecuador to determine the arboviral etiology of febrile illness. Patients with suspected viral-associated, acute, undifferentiated febrile illness of <7 days duration were enrolled in the study and blood samples were obtained from each patient and assayed by virus isolation. Demographic and clinical information from each patient was also obtained at the time of voluntary enrollment. In 2005–2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. Since 1993, VEEV has been continuously isolated from patients in Loreto, Peru, and more recently (2005), in Madre de Dios, Peru. We performed phylogenetic analyses with VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that VEEV subtype ID Panama/Peru genotype is the predominant one circulating in Peru. We also demonstrated that VEEV subtype ID strains circulating in Ecuador belong to the Colombia/Venezuela genotype and VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new ID genotype. In summary, we identified a new major lineage of enzootic VEEV subtype ID, information that could aid in the understanding of the emergence and evolution of VEEV in South America.