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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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During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy.
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Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58⯰C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.
Assuntos
Desinfetantes/farmacologia , Desinfecção , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos da radiação , Temperatura Alta , Animais , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos da radiação , Desinfecção/métodos , Células VeroRESUMO
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.
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Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/epidemiologia , Evolução Molecular , Doenças dos Cavalos/virologia , América , Sequência de Aminoácidos , Animais , Culex/virologia , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Doenças dos Cavalos/epidemiologia , Cavalos/virologia , Humanos , Insetos Vetores/virologia , FilogeniaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broad panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Finally, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.
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Vírus da Encefalite Equina Venezuelana/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Animais , Cricetinae/virologia , Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/epidemiologia , Variação Genética , Genoma Viral , Genótipo , Interações Hospedeiro-Patógeno/genética , México/epidemiologia , Camundongos Endogâmicos/virologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , FenótipoRESUMO
Since chikungunya virus (CHIKV) was introduced into the Americas in 2013, its geographic distribution has rapidly expanded. Of 119 serum samples collected in 2014 from febrile patients in southern Mexico, 79% were positive for CHIKV or IgM against CHIKV. Sequencing results confirmed CHIKV strains closely related to Caribbean isolates.
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Febre de Chikungunya/epidemiologia , Vírus Chikungunya/patogenicidade , Surtos de Doenças/estatística & dados numéricos , Febre de Causa Desconhecida/etiologia , Animais , Anticorpos Antivirais/sangue , Febre de Chikungunya/patologia , Culicidae/virologia , Febre de Causa Desconhecida/epidemiologia , Humanos , Insetos Vetores/patogenicidade , Insetos Vetores/virologia , México/epidemiologia , Dados de Sequência MolecularRESUMO
In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.
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Infecções por Alphavirus/epidemiologia , Alphavirus/genética , Evolução Biológica , Biota/imunologia , Surtos de Doenças , Alphavirus/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Filogenia , Venezuela/epidemiologiaRESUMO
Mayaro virus (MAYV) is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease.
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Alphavirus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células Cultivadas , Camundongos , América do Sul , Vacinas Atenuadas/imunologia , Replicação ViralRESUMO
Pools of mosquitoes were tested for insect-specific viruses using cytopathic effect (CPE) assays on Aedes albopictus (C6/36) cells. Illumina sequencing of RNA from pool TR7094, which produced extensive CPE 2 days post-infection, yielded the complete genome sequences of a previously unknown Bunyavirus, designated Cumuto virus (CUMV), and a second virus designated Wallerfield virus (WALV). WALV shared highest amino acid identity (60.1 %) with Dezidougou virus from Côte d'Ivoire, a positive-sense, single-strand RNA, insect-specific virus belonging to the newly proposed genus Negevirus associated with mosquitoes and phlebotomine sandflies. The S, M and L segments of CUMV were most closely related to those of Gouleako virus, also from Côte d'Ivoire (amino acid identities of 36 %, 38% and 54 % respectively). Neither virus produced CPE on vertebrate cells, or illness in newborn mice. Isolation and characterization of these viruses increase our knowledge of the geographical distribution, diversity and host range of mosquito-specific bunyaviruses and negeviruses.
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Bunyaviridae/classificação , Bunyaviridae/isolamento & purificação , Culex/virologia , Animais , Bunyaviridae/genética , Linhagem Celular , Efeito Citopatogênico Viral , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de DNA , Trinidad e TobagoRESUMO
BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).
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Surtos de Doenças , Vírus da Encefalite Equina do Leste , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina do Leste , Encefalomielite Equina Venezuelana , Adolescente , Animais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina do Leste/epidemiologia , Encefalomielite Equina do Leste/veterinária , Encefalomielite Equina Venezuelana/epidemiologia , Encefalomielite Equina Venezuelana/veterinária , Evolução Fatal , Feminino , Doenças dos Cavalos/epidemiologia , Cavalos , Humanos , Lactente , Masculino , Panamá/epidemiologia , Filogenia , RNA Viral/sangueRESUMO
The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.
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Proteínas do Capsídeo/biossíntese , Vírus da Encefalite Equina Venezuelana/imunologia , Expressão Gênica , Biossíntese de Proteínas , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologia , Aedes , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Instabilidade Genômica , Humanos , Camundongos , Análise de Sobrevida , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
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.
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Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/imunologia , Vírus da Encefalomiocardite/genética , Camundongos , Biossíntese de Proteínas , Análise de Sobrevida , Vacinação/efeitos adversos , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/genéticaRESUMO
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.
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Infecções por Arbovirus/virologia , Arbovírus/patogenicidade , Sigmodontinae , Animais , Anticorpos Antivirais/sangue , Infecções por Arbovirus/imunologia , Testes de Inibição da Hemaglutinação , MéxicoRESUMO
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.
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Doenças Transmissíveis Emergentes/veterinária , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/veterinária , Doenças dos Cavalos/transmissão , Animais , Animais Selvagens/virologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , Surtos de Doenças/veterinária , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Ecossistema , Encefalomielite Equina Venezuelana/epidemiologia , Encefalomielite Equina Venezuelana/transmissão , Encefalomielite Equina Venezuelana/virologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Cavalos , México/epidemiologia , Roedores/virologia , Viremia/imunologia , Viremia/veterináriaRESUMO
Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.
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Vírus Chikungunya/química , Vírus da Encefalite Equina Venezuelana/química , Proteínas não Estruturais Virais/química , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Vírus Chikungunya/genética , Sequência Conservada , Vírus da Encefalite Equina Venezuelana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genéticaRESUMO
The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.