RESUMO
West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log(10) greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), did not occur until day 7. At these later time points (7 and 9 days PI), nocodazole-treated WNV-infected animals attained viral titers in these tissues similar to titers in the untreated WNV-infected control animals. These results demonstrate that a single dose of nocodazole delays, but does not block, WNV infection of the brain.
Assuntos
Encéfalo/virologia , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia , Internalização do Vírus/efeitos dos fármacos , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/fisiologia , Animais , Encéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , RNA Viral/genética , Fatores de Tempo , Carga Viral , Febre do Nilo Ocidental/patologia , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/isolamento & purificaçãoRESUMO
We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Genes de Imunoglobulinas , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/imunologiaRESUMO
This report describes the clinical, laboratory, and epidemiological findings on 27 cases of Mayaro virus (MV) disease, an emerging mosquito-borne viral illness that is endemic in rural areas of tropical South America. MV disease is a nonfatal, dengue-like illness characterized by fever, chills, headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash of 3-5 days' duration. Severe joint pain is a prominent feature of this illness; the arthralgia sometimes persists for months and can be quite incapacitating. Cases of two visitors from the United States, who developed MV disease during visits to eastern Peru, are reported. MV disease and dengue are difficult to differentiate clinically.
Assuntos
Infecções por Alphavirus/diagnóstico , Alphavirus/isolamento & purificação , Adulto , Distribuição por Idade , Alphavirus/classificação , Alphavirus/genética , Alphavirus/imunologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/virologia , Animais , Anticorpos Antivirais/sangue , Culicidae , DNA Viral/análise , Feminino , Humanos , Insetos Vetores , Pessoa de Meia-Idade , Peru/epidemiologia , Estações do Ano , Análise de Sequência de DNA , ZoonosesRESUMO
We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.
Assuntos
Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/veterinária , Doenças dos Cavalos/virologia , Interferon Tipo I/farmacologia , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Resistência Microbiana a Medicamentos/genética , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Glicoproteínas/genética , Cavalos , Humanos , Camundongos , Virulência/genética , Zoonoses/virologiaRESUMO
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/química , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Epitopos de Linfócito B/química , Testes de Inibição da Hemaglutinação , Humanos , Jamaica , Masculino , Fusão de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Testes de Neutralização , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos , Conformação Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/químicaRESUMO
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (Mabs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-bourne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independently regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus haemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDA tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-bourne encephalitis virus, determination of domain C was more problematic: however, at least four epitopes and biochemical characteristics consistent with C-domain epitopes(AU)
Assuntos
21003 , Humanos , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Proteínas do Envelope Viral/imunologia , Testes de Inibição da Hemaglutinação , Jamaica , Fusão de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Testes de Neutralização , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos , Conformação Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/química , Anticorpos Monoclonais/imunologia , Antígenos Virais/química , Sítios de Ligação , Ligação Competitiva , Linhagem CelularRESUMO
A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.
Assuntos
Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/epidemiologia , Adolescente , Adulto , Idoso , Instituições de Assistência Ambulatorial , Anticorpos Antivirais/análise , Células Cultivadas , Criança , Pré-Escolar , Vírus da Encefalite Equina Venezuelana/classificação , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/sangue , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lactente , Malária/diagnóstico , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Peru/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Vigilância da População , RNA Viral/análise , RNA Viral/genética , Estudos Soroepidemiológicos , SorotipagemRESUMO
Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.
Assuntos
Antígenos Virais/genética , Vírus da Encefalite Equina Venezuelana/imunologia , RNA Mensageiro/química , RNA Viral/química , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/química , Capsídeo/genética , DNA Complementar/química , Vírus da Encefalite Equina Venezuelana/classificação , Vírus da Encefalite Equina Venezuelana/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
We used previously characterized murine monoclonal antibodies to develop a panel useful in subtyping Venezuelan equine encephalitis (VEE) viruses by an indirect fluorescent antibody assay. This panel worked well with either prototype VEE viruses or a series of more recent VEE virus isolates. The panel is particularly useful for rapidly differentiating VEE viruses with epidemic-epizootic potential from other endemic varieties of this virus. Using this panel, we identified an antigenic variant of prototype VEE subtype 1E virus currently present in Mexico. This antigenic change in the E2 glycoprotein was confirmed by enzyme-linked immunosorbent assay. Because VEE virus virulence has been associated in part with the E2 glycoprotein, this observed antigenic change in the 1E virus E2 glycoprotein may explain the apparent equine virulence of this unusual VEE 1E virus.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Animais , Vírus da Encefalite Equina Venezuelana/imunologia , Cavalos , HumanosRESUMO
An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.