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Madariaga virus (MADV) and Venezuelan equine encephalitis virus (VEEV) are emerging arboviruses affecting rural and remote areas of Latin America. However, there are limited clinical and epidemiological reports available, and outbreaks are occurring at an increasing frequency. We addressed this gap by analyzing all the available clinical and epidemiological data of MADV and VEEV infections recorded since 1961 in Panama. A total of 168 of human alphavirus encephalitis cases were detected in Panama from 1961 to 2023. Here we describe the clinical signs and symptoms and epidemiological characteristics of these cases, and also explored signs and symptoms as potential predictors of encephalitic alphavirus infection when compared to those of other arbovirus infections occurring in the region. Our results highlight the challenges clinical diagnosis of alphavirus disease in endemic regions with overlapping circulation of multiple arboviruses.
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Anthropogenic activities are altering ecosystem stability and climate worldwide, which is disturbing and shifting arbovirus vector distributions. Although the overall geographic range of some epidemiologically important species is recognized, the spatiotemporal variation for other species in the context of climate change remains poorly understood. Here we predict the current potential distribution of 9 species of Culex (Melanoconion) based on an ecological niche modeling (ENM) approach and assess spatiotemporal variation in future climate change in the Neotropics. The most important environmental predictors were the mean temperature of the warmest season (27 °C), precipitation during the driest month (50 mm), and precipitation during the warmest season (>200 mm). The best current model for each species was transferred to the future general circulation model IPSL-CM6A-LR, using 2 shared socioeconomic pathway scenarios (ssp1-2.6, ssp5-8.5). Under both scenarios of climatic change, an expansion of suitable areas can be observed followed by a strong reduction for the medium-long future under the worst scenario. The multivariate environmental similarity surface analysis indicated future novel climates outside the current range. However, none of the species would occur in those areas. Even if many challenges remain in improving methods for forecasting species responses to global climate change and arbovirus transmission, ENM has strong potential to be applied to the geographic characterization of these systems. Our study can be used for the monitoring of Culex (Melanoconion) species populations and their associated arboviruses, contributing to develop region-specific public health surveillance programs.
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Arbovírus , Culex , Culicidae , Animais , Ecossistema , Saúde Pública , Mosquitos Vetores , Arbovírus/fisiologia , Mudança ClimáticaRESUMO
The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.
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Anticorpos Antivirais , Vírus da Encefalite Equina Venezuelana , Animais , Cavalos , Humanos , Vírus da Encefalite Equina Venezuelana/genética , Anticorpos Neutralizantes/farmacologia , Receptores Fc , Imunoglobulina GRESUMO
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.
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Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Vacinas Virais , Animais , Cavalos , Humanos , Macaca fascicularis , Viremia/tratamento farmacológico , Modelos Animais de DoençasRESUMO
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.
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Alphavirus , Culicidae , Dengue , Vírus da Encefalite Equina do Leste , Encefalomielite Equina do Leste , Encefalomielite Equina Venezuelana , Humanos , Animais , Cavalos/genética , Vírus da Encefalite Equina do Leste/genética , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/epidemiologia , Culicidae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Filogenia , Estudos Prospectivos , Vigilância em Saúde Pública , Estudos Retrospectivos , Alphavirus/genética , RNARESUMO
Venezuelan equine encephalitis virus (VEEV) is a disease typically confined to South and Central America, whereby human disease is characterised by a transient systemic infection and occasionally severe encephalitis, which is associated with lethality. Using an established mouse model of VEEV infection, the encephalitic aspects of the disease were analysed to identify biomarkers associated with inflammation. Sequential sampling of lethally challenged mice (infected subcutaneously) confirmed a rapid onset systemic infection with subsequent spread to the brain within 24 h of the challenge. Changes in inflammatory biomarkers (TNF-α, CCL-2, and CCL-5) and CD45+ cell counts were found to correlate strongly to pathology (R>0.9) and present previously unproven biomarkers for disease severity in the model, more so than viral titre. The greatest level of pathology was observed within the olfactory bulb and midbrain/thalamus. The virus was distributed throughout the brain/encephalon, often in areas not associated with pathology. The principal component analysis identified five principal factors across two independent experiments, with the first two describing almost half of the data: (1) confirmation of a systemic Th1-biased inflammatory response to VEEV infection, and (2) a clear correlation between specific inflammation of the brain and clinical signs of disease. Targeting strongly associated biomarkers of deleterious inflammation may ameliorate or even eliminate the encephalitic syndrome of this disease.
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Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Humanos , Cavalos , Camundongos , Animais , Fator de Necrose Tumoral alfa , Vírus da Encefalite Equina Venezuelana/fisiologia , Encéfalo , Inflamação/patologia , Quimiocinas , LeucócitosRESUMO
Self-replicating RNA (repRNA) derived from Venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, inflammatory cytokines served as genes of interest and were cloned in their wild type and several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region could significantly reduce the recognition by innate immunity as evidenced by the decreased production of type I interferon, and enhance transgene expression in host cells. Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEE-derived repRNA-based gene therapies.
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Vírus da Encefalite Equina Venezuelana , Animais , Vírus da Encefalite Equina Venezuelana/genética , Linhagem Celular , Fases de Leitura Aberta , RNA/metabolismo , Replicon/genética , Mutação , Expressão Gênica , Mamíferos/genéticaRESUMO
INTRODUCTION: Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) viruses are zoonotic pathogens affecting humans, particularly equines. These neuroarboviruses compromise the central nervous system and can be fatal in different hosts. Both have significantly influenced Colombia; however, few studies analyse its behaviour, and none develop maps using geographic information systems to characterise it. OBJECTIVE: To describe the temporal-spatial distribution of those viruses in Colombia between 2008 and 2019. METHODS: Retrospective cross-sectional descriptive study, based on weekly reports by municipalities of the ICA, of the surveillance of both arboviruses in equines, in Colombia, from 2008 to 2019. The data were converted into databases in Microsoft Access 365®, and multiple epidemiological maps were generated with the Kosmo RC1®3.0 software coupled to shape files of all municipalities in the country. RESULTS: In the study period, 96 cases of EEE and 70 of VEE were reported, with 58% of EEE cases occurring in 2016 and 20% of EEV cases in 2013. The most affected municipalities for EEE corresponded to the department of Casanare: Yopal (20), Aguazul (16), and Tauramena (10). In total, 40 municipalities in the country reported ≥1 case of EEE. CONCLUSIONS: The maps allow a quick appreciation of groups of neighbouring municipalities in different departments (1° political division) and regions of the country affected by those viruses, which helps consider the expansion of the disease associated with mobility and transport of equines between other municipalities, also including international borders, such as is the case with Venezuela. In that country, especially for EEV, municipalities in the department of Cesar are bordering and at risk for that arboviral infection. there is a high risk of equine encephalitis outbreaks, especially for VEE. This poses a risk also, for municipalities in the department of Cesar, bordering with Venezuela.
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Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Cavalos , Animais , Colômbia/epidemiologia , Estudos Transversais , Encefalomielite Equina Venezuelana/epidemiologia , Sistemas de Informação Geográfica , Cavalos/virologia , Estudos RetrospectivosRESUMO
Arthropod-borne viruses (arboviruses) are a diverse group of ribonucleic acid (RNA) viruses, with the exception of African swine fever virus, that are transmitted by hematophagous arthropods to a vertebrate host. They are the important cause of many diseases due to their ability to spread in different environments and their diversity of vectors. Currently, there is no information on the geographical distribution of the diseases because the routes of transmission and the mammals (wild or domestic) that act as potential hosts are poorly documented or unknown. We conducted a systematic review from 1967 to 2021 to identify the diversity of arboviruses, the areas, and taxonomic groups that have been monitored, the prevalence of positive records, and the associated risk factors. We identified forty-three arboviruses in nine mammalian orders distributed in eleven countries. In Brazil, the order primates harbor the highest number of arbovirus records. The three most recorded arboviruses were Venezuelan equine encephalitis, Saint Louis encephalitis and West Nile virus. Serum is the most used sample to obtain arbovirus records. Deforestation is identified as the main risk factor for arbovirus transmission between different species and environments (an odds ratio of 1.46 with a 95% confidence interval: 1.34-1.59). The results show an increase in the sampling effort over the years in the neotropical region. Despite the importance of arboviruses for public health, little is known about the interaction of arboviruses, their hosts, and vectors, as some countries and mammalian orders have not yet been monitored. Long-term and constant monitoring allows focusing research on the analysis of the interrelationships and characteristics of each component animal, human, and their environment to understand the dynamics of the diseases and guide epidemiological surveillance and vector control programs. The biodiversity of the Neotropics should be considered to support epidemiological monitoring strategies.
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Vírus da Febre Suína Africana , Arbovírus , Animais , Suínos , Cavalos , Humanos , Mamíferos , Saúde Pública , Monitoramento EpidemiológicoRESUMO
The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.
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Domínio Catalítico , Cisteína Proteases , Vírus da Encefalite Equina Venezuelana , Animais , Humanos , Cristalografia por Raios X , Cisteína Proteases/química , Cisteína Proteases/genética , Vírus da Encefalite Equina Venezuelana/enzimologia , Cavalos , Simulação de Dinâmica MolecularRESUMO
Venezuelan equine encephalitis virus (VEEV) is an alphavirus transmitted by mosquitos that can cause a febrile illness and induce severe neurological complications in humans and equine populations. Currently there are no FDA approved vaccines or antiviral treatments to combat VEEV. Proteomic techniques were utilized to create an interactome of the E1 fusion glycoprotein of VEEV. VEEV E1 interacted with a number of cellular chaperone proteins including protein disulfide isomerase family A member 6 (PDIA6). PDI inhibition through LOC14 and/or nitazoxanide treatment effectively decreased production of VEEV and other alphaviruses in vitro, including eastern equine encephalitis virus, Sindbis virus, and chikungunya virus. Decreased oxidoreductive capabilities of PDIs through LOC14 or nitazoxanide treatment impacted both early and late events in viral replication, including the production of non-infectious virions and decreased VEEV E1 disulfide bond formation. Results from this study identified PDIs as critical regulators of alphavirus replication and potential therapeutic targets.
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Alphavirus , Vírus Chikungunya , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Humanos , Animais , Cavalos , Proteômica , Linhagem Celular , Replicação Viral , Encefalomielite Equina Venezuelana/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/farmacologia , Isomerases de Dissulfetos de Proteínas/uso terapêuticoRESUMO
The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.
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Jacarés e Crocodilos , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Animais , Cavalos , Vírus da Encefalite Equina Venezuelana/fisiologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/prevenção & controle , Bioprospecção , Replicação Viral , PeptídeosRESUMO
The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.
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Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Humanos , Animais , Cavalos , Vírus da Encefalite Equina Venezuelana/fisiologia , Barreira Hematoencefálica , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/prevenção & controle , Células Endoteliais , Sistemas MicrofisiológicosRESUMO
Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.
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Vírus da Encefalite Equina Venezuelana , RNA Interferente Pequeno , Animais , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/fisiologia , Cavalos , Humanos , RNA Helicases , RNA Interferente Pequeno/farmacologia , Replicação ViralRESUMO
The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.
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Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Vacinas Virais , Aerossóis , Animais , Anticorpos Antivirais , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/prevenção & controle , Cavalos , Macaca fascicularis , RepliconRESUMO
Background: Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas. There are no approved vaccines or antivirals. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, albeit less so for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that altered the error incorporation rate of the RNA-dependent RNA polymerase (RdRp). Methods: The research presented here examines the impact of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains. Results: Our results show that the V3526 RdRp mutants exhibited reduced tissue tropism in the spleen and kidney compared to wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing showed that the RdRp mutations could revert to wild-type V3526. Conclusions: The observed genotypic reversion is likely of limited concern because wild-type V3526 is still an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.
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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.
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Vírus da Encefalite Equina Venezuelana/genética , Genoma Viral , RNA Viral , Inativação de Vírus , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar/biossíntese , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/fisiologia , RNA Viral/química , RNA Viral/fisiologia , Ribonucleases/metabolismo , Células VeroRESUMO
We report a case of vertical transmission of Tonate virus in a pregnant woman from French Guiana. The fetus showed severe necrotic and hemorrhagic lesions of the brain and spinal cord. Clinicians should be made aware of possible adverse fetal outcomes in pregnant women infected with Tonate virus.
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Alphavirus , Encéfalo , Feminino , Guiana Francesa/epidemiologia , Humanos , Transmissão Vertical de Doenças Infecciosas , GravidezRESUMO
Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.
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Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/virologia , Proteínas do Envelope Viral/imunologia , Replicação Viral/efeitos dos fármacos , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas , Encefalomielite Equina Venezuelana/terapia , Glicoproteínas/imunologia , Imunoterapia , Camundongos , Ligação Proteica , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia , Vírion/metabolismoRESUMO
There is an urgent need for novel strategies for the treatment of emerging arthropod-borne viral infections, including those caused by dengue virus (DENV) and Venezuelan equine encephalitis virus (VEEV). We prepared and screened focused libraries of 4-anilinoquinolines and 4-anilinoquinazolines for antiviral activity and identified three potent compounds. N-(2,5-dimethoxyphenyl)-6-(trifluoromethyl)quinolin-4-amine (10) inhibited DENV infection with an EC50 = 0.25 µM, N-(3,4-dichlorophenyl)-6-(trifluoromethyl)quinolin-4-amine (27) inhibited VEEV with an EC50 = 0.50 µM, while N-(3-ethynyl-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine (54) inhibited VEEV with an EC50 = 0.60 µM. These series of compounds demonstrated nearly no toxicity with CC50 values greater than 10 µM in all cases. These promising results provide a future prospective to develop a clinical compound against these emerging viral threats.