RESUMO
Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Arbovírus/isolamento & purificação , Quirópteros/virologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Alphavirus/classificação , Alphavirus/genética , Infecções por Alphavirus/virologia , Animais , Infecções por Arbovirus/veterinária , Infecções por Arbovirus/virologia , Arbovírus/classificação , Arbovírus/genética , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Filogenia , UruguaiRESUMO
Madariaga virus (MADV), previously known as South American eastern equine encephalitis virus (SA EEEV; family Togaviridae, genus Alphavirus), is a mosquito-borne virus associated mainly with equine disease. In 2010, the first human outbreak by MADV was reported in Central America, but the mosquito vectors and vertebrate hosts involved in the outbreak were not identified. In Argentina, the first epizootic of MADV was in 1930, and since then, several epizootics by MADV have been reported. However, the potential vectors and hosts involved in the transmission cycle remain unknown. In the present study, MADV was detected in Culex (Culex) spp. mosquitoes and the phylogenetic analysis showed that the MADV fragment amplified grouped with the lineage/subtype III of the SA EEEV complex. Our results provide information about the natural infection with MADV in mosquitoes collected in a wild environment of Argentina and its genetic relatedness.
Assuntos
Alphavirus/isolamento & purificação , Culex/virologia , Surtos de Doenças , Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina/virologia , Alphavirus/genética , Animais , Argentina/epidemiologia , Vírus da Encefalite Equina do Leste/genética , Encefalomielite Equina/epidemiologia , Humanos , FilogeniaRESUMO
Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America.
Assuntos
Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/transmissão , Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina do Leste/epidemiologia , Encefalomielite Equina do Leste/transmissão , Animais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Doenças Transmissíveis Importadas/virologia , Culex/virologia , Surtos de Doenças , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Encefalomielite Equina do Leste/virologia , Feminino , Haiti/epidemiologia , Humanos , Masculino , Filogenia , RNA Viral/sangue , Instituições AcadêmicasRESUMO
In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 µg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 µg/mL for VEE and 20-250 µg/mL for EEE and WEE VLPs.
Assuntos
Eletroforese Capilar/métodos , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Fluorescência , Corantes Fluorescentes/química , Humanos , Sensibilidade e Especificidade , Vacinas de Partículas Semelhantes a Vírus/químicaRESUMO
Madariaga virus (MADV), the new species designation for the South American isolates of eastern equine encephalitis virus (EEEV), is genetically divergent and substantially different in ecology and pathogenesis from North American EEEV strains. We isolated and characterized a MADV isolate obtained from a horse in Brazil. Our results support previous phylogenetic studies showing there are three genetically distinct MADV lineages. The MADV isolate from Paraíba State belongs to the South American lineage III and is closely related to Peruvian, Colombian and Venezuelan isolates.
Assuntos
Vírus da Encefalite Equina do Leste , Encefalomielite Equina/veterinária , Doenças dos Cavalos/virologia , Aedes/citologia , Aedes/virologia , Animais , Encéfalo/virologia , Brasil , Células Cultivadas , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina/virologia , Cavalos , Camundongos , FilogeniaRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/genética , RNA Viral/genética , Alphavirus/genética , Animais , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Cavalos/virologia , Humanos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , América do SulRESUMO
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.
Assuntos
Cricetinae/virologia , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina Venezuelana/genética , RNA Viral/genética , Alphavirus/genética , Alphavirus/isolamento & purificação , Animais , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Variação Genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VenezuelaRESUMO
Background: Eastern equine encephalitis (EEE) is a highly lethal zoonotic disease caused by Eastern equine encephalitisvirus (EEEv), an RNA virus of the genus Alphavirus, family Togaviridae. The transmission of these alphaviruses is throughmosquitoes, mainly species of Culex, Aedes, Anopheles and Culiseta. Horses and humans are considered accidental hostsand the main reservoirs are birds and some wild rodents. The disease has been identified in Brazil by serological studies,but investigations with clinico-pathological descriptions are scarce. The present study aimed to describe the epidemiologyand clinical-pathological findings of four cases of EEE in horses from Midwestern Brazil.Materials, Methods & Results: Four confirmed cases of EEE in horses from the necropsy and histopathology files of theLaboratory of Veterinary Pathology (LPV) of the University of Brasília (UnB) were reviewed. Cases occurred betweenJanuary 2005 and April 2012. Samples of brain and spinal cord samples were fixed in formalin 10%, processed routinelyfor histopathology, and stained by hematoxylin and eosin (HE). Immuno-histochemistry (IHC) with the peroxidase streptoavidin-biotin method was done to confirm the diagnosis of EEEv infection in all cases. The slides were incubated withthe anti-EEEv monoclonal primary antibody (overnight, 1:100 dilution). The disease affected both young and adult horses.One case occurred in the summer and the other three in the fall. Clinical signs more frequently observed included circling,blindness, paresis, paralysis, somnolence, ataxia, head pressing, and recumbence...(AU)
Assuntos
Animais , Cavalos/virologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina/epidemiologia , Encefalomielite Equina/patologia , Encefalomielite Equina/veterináriaRESUMO
Background: Eastern equine encephalitis (EEE) is a highly lethal zoonotic disease caused by Eastern equine encephalitisvirus (EEEv), an RNA virus of the genus Alphavirus, family Togaviridae. The transmission of these alphaviruses is throughmosquitoes, mainly species of Culex, Aedes, Anopheles and Culiseta. Horses and humans are considered accidental hostsand the main reservoirs are birds and some wild rodents. The disease has been identified in Brazil by serological studies,but investigations with clinico-pathological descriptions are scarce. The present study aimed to describe the epidemiologyand clinical-pathological findings of four cases of EEE in horses from Midwestern Brazil.Materials, Methods & Results: Four confirmed cases of EEE in horses from the necropsy and histopathology files of theLaboratory of Veterinary Pathology (LPV) of the University of Brasília (UnB) were reviewed. Cases occurred betweenJanuary 2005 and April 2012. Samples of brain and spinal cord samples were fixed in formalin 10%, processed routinelyfor histopathology, and stained by hematoxylin and eosin (HE). Immuno-histochemistry (IHC) with the peroxidase streptoavidin-biotin method was done to confirm the diagnosis of EEEv infection in all cases. The slides were incubated withthe anti-EEEv monoclonal primary antibody (overnight, 1:100 dilution). The disease affected both young and adult horses.One case occurred in the summer and the other three in the fall. Clinical signs more frequently observed included circling,blindness, paresis, paralysis, somnolence, ataxia, head pressing, and recumbence...
Assuntos
Animais , Cavalos/virologia , Encefalomielite Equina/epidemiologia , Encefalomielite Equina/patologia , Encefalomielite Equina/veterinária , Vírus da Encefalite Equina do Leste/isolamento & purificaçãoRESUMO
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.).
Assuntos
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
Nove casos de encefalomielite equina foram estudados na Ilha de Marajó, estado do Pará, Brasil. Os equinos apresentavam dificuldade em se manter em estação, andavam em círculo, tinham acentuada depressão, pálpebras cerradas, paralisia da língua, tremores musculares, bruxismo, anorexia e desidratação. Alguns apresentavam diminuição dos reflexos auricular, palpebral, de ameaça, diminuição do tônus da língua e taquicardia. Posição de auto-auscultação foi observada com frequência. Os animais muitas vezes eram encontrados apoiados em troncos e cercas para se manterem em estação. À necropsia verificou-se hemorragia das leptomeninges e da medula, alguns apresentaram ainda aderência das leptomeninges. À histopatologia verificou-se encefalite difusa que afetava principalmente a substância cinzenta, com meningite e coroidite. Foi observada perivasculite mononuclear. Em dois equinos identificou-se o vírus da encefalomielite equina Leste pela reação de Semi-Nested transcrição reversa de polimerase em cadeia (Semi-Nested RT-PCR).(AU)
Nine cases of equine encephalomyelitis on Marajó Island, state of Pará, Brazil, were studied. The affected horses had difficulty to stand, walked in circles, with marked depression, closed eyelids, tongue paralysis, muscle tremors, bruxism, anorexia and dehydration. Some had their ear and eyelid reflexes diminished, decreased tongue tone and tachycardia; laid down frequently they kept their head on the chest. Often they were seen resting their head on tree trunks or fences. At necropsy, hemorrhages of the meninges and spinal cord, and in some animals also adhesion of the meninges were found. Histologically there was diffuse encephalitis affecting mainly the gray matter, with meningitis and choroiditis. Presence of perivascular cuffs consisting of mononuclear inflammatory cells was observed. From two horses the Eastern equine encephalitis virus was identified by semi-nested reverse transcription polymerase chain reaction (semi-nested RT-PCR).(AU)
Assuntos
Animais , Cavalos/virologia , Encefalomielite Equina do Leste/diagnóstico , Vírus da Encefalite Equina do Leste/isolamento & purificação , Autopsia/veterinária , Sinais e SintomasRESUMO
Nove casos de encefalomielite equina foram estudados na Ilha de Marajó, estado do Pará, Brasil. Os equinos apresentavam dificuldade em se manter em estação, andavam em círculo, tinham acentuada depressão, pálpebras cerradas, paralisia da língua, tremores musculares, bruxismo, anorexia e desidratação. Alguns apresentavam diminuição dos reflexos auricular, palpebral, de ameaça, diminuição do tônus da língua e taquicardia. Posição de auto-auscultação foi observada com frequência. Os animais muitas vezes eram encontrados apoiados em troncos e cercas para se manterem em estação. À necropsia verificou-se hemorragia das leptomeninges e da medula, alguns apresentaram ainda aderência das leptomeninges. À histopatologia verificou-se encefalite difusa que afetava principalmente a substância cinzenta, com meningite e coroidite. Foi observada perivasculite mononuclear. Em dois equinos identificou-se o vírus da encefalomielite equina Leste pela reação de Semi-Nested transcrição reversa de polimerase em cadeia (Semi-Nested RT-PCR).
Nine cases of equine encephalomyelitis on Marajó Island, state of Pará, Brazil, were studied. The affected horses had difficulty to stand, walked in circles, with marked depression, closed eyelids, tongue paralysis, muscle tremors, bruxism, anorexia and dehydration. Some had their ear and eyelid reflexes diminished, decreased tongue tone and tachycardia; laid down frequently they kept their head on the chest. Often they were seen resting their head on tree trunks or fences. At necropsy, hemorrhages of the meninges and spinal cord, and in some animals also adhesion of the meninges were found. Histologically there was diffuse encephalitis affecting mainly the gray matter, with meningitis and choroiditis. Presence of perivascular cuffs consisting of mononuclear inflammatory cells was observed. From two horses the Eastern equine encephalitis virus was identified by semi-nested reverse transcription polymerase chain reaction (semi-nested RT-PCR).
Assuntos
Animais , Cavalos/virologia , Encefalomielite Equina do Leste/diagnóstico , Vírus da Encefalite Equina do Leste/isolamento & purificação , Autopsia/veterinária , Sinais e SintomasRESUMO
Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is an arbovirus that causes severe disease in humans in North America and in equids throughout the Americas. The enzootic transmission cycle of EEEV in North America involves passerine birds and the ornithophilic mosquito vector, Culiseta melanura, in freshwater swamp habitats. However, the ecology of EEEV in South America is not well understood. Culex (Melanoconion) spp. mosquitoes are considered the principal vectors in Central and South America; however, a primary vertebrate host for EEEV in South America has not yet been identified. Therefore, to further assess the reservoir host potential of wild rodents and wild birds, we compared the infection dynamics of North American and South American EEEV in cotton rats (Sigmodon hispidus) and house sparrows (Passer domesticus). Our findings suggested that each species has the potential to serve as amplification hosts for North and South America EEEVs.
Assuntos
Vetores de Doenças , Vírus da Encefalite Equina do Leste , Encefalomielite Equina do Leste/veterinária , Doenças dos Cavalos/transmissão , Sigmodontinae/virologia , Pardais/virologia , Animais , Anticorpos Antivirais/sangue , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina do Leste/transmissão , Encefalomielite Equina do Leste/virologia , Doenças dos Cavalos/virologia , Cavalos , América do Norte , América do Sul , Especificidade da EspécieRESUMO
Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10(4)pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n=6) manifested a viremia averaging 3.2 days (+/-0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n=6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (+/-0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.
Assuntos
Aotidae/imunologia , Aotidae/virologia , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Leste/patogenicidade , Encefalomielite Equina do Leste/imunologia , Encefalomielite Equina do Leste/virologia , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Modelos Animais de Doenças , Vírus da Encefalite Equina do Leste/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Cavalos , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Imunoglobulina M/análise , Imunoglobulina M/biossíntese , Injeções Subcutâneas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio de Placa Viral , Viremia/virologiaRESUMO
In support of efforts to develop rapid diagnostic assays for use in the field, reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect arboviruses circulating in the Amazon Basin region of Peru. Previous knowledge of arthropod/pathogen relationships allowed a focused evaluation to be conducted in November 2000 that assessed the feasibility and reliability of a mobile, rapid, field-expedient RT-PCR diagnostic system aimed at detecting eastern equine encephalitis virus (EEEV) in Culex (Melanoconion) pedroi mosquitoes. Modifications were made to a commercially available mobile molecular laboratory kit and assay procedures were tailored for use under harsh environmental conditions with field-collected and field-processed mosquitoes. From CO2 baited mosquito light traps, 3,227 Cx. (Mel.) pedroi mosquitoes were collected and sorted into 117 pools. The pools were processed and assayed in the field by RT-PCR and five of those pools were found positive for EEEV. Laboratory sequence analysis confirmed the presence of two distinct subtypes of EEEV.
Assuntos
Culex/virologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Insetos Vetores/virologia , Ochlerotatus/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , DNA Complementar/química , DNA Complementar/metabolismo , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Peru , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.
Assuntos
Culicidae/virologia , Vírus da Encefalite Equina do Leste/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Etilaminas/farmacologia , Flavivirus/efeitos dos fármacos , Orthobunyavirus/isolamento & purificação , Animais , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Feminino , Flavivirus/isolamento & purificação , Orthobunyavirus/efeitos dos fármacos , América do SulRESUMO
Objetivo: No período de 1996 a 1999, um agente viral causador de encefalomielite afetou as populaçöes de eqüinos em diferentes regiöes do Estado do Paraná, Brasil. Objetivou-se realizar pesquisa sorológica na tentativa de isolar o vírus causador da doença. Métodos: Em quatro municípios do Estado do Paraná, Brasil, foram coletados culicídeos com armadilha Shannon e isca humana, identificados e processados para isolamento de vírus. Em dois municípios estudados foram colhidas amostras de sangue de eqüinos para isolamento de vírus e para pesquisa sorológica. Os soros foram analisados pelo teste de inibiçäo da hemaglutinaçäo frente a diferentes antígenos de Alphavirus e Flavivirus. Aqueles que revelaram reaçöes positivas-cruzadas foram analisados pelo teste de neutralizaçäo. Resultados: Foram coletados culicídeos dos gêneros: Culex, Aedes, Mansonia, Coquillettidia, Psorophora, Sabethes, Wyeomyia e Limatus. Embora näo sendo isolado o agente viral, foram detectados anticorpos hemaglutinantes para o vírus Encefalomielite eqüina do Leste, Mucambo, Pixuna, Maguari e St. Luis. Em doze amostras de soros foram detectados anticorpos neutralizantes para o vírus Encefalomielite eqüina do Leste. Conclusöes: Foram coletadas espécies de culicídeos, considerados na bibliografia como vetores de vírus causadores de encefalomielite buniavírus e outras arboviroses de importância epidemiológica. Pela presença de sintomas de encefalomielite e de anticorpos para o vírus Encefalomielite eqüina do Leste nos soros de cavalos, supöe-se ser esse o vírus causador da doença nos eqüinos das regiöes estudadas
Assuntos
Animais , Alphavirus/isolamento & purificação , Encefalomielite Equina/sangue , Insetos Vetores , Culicidae/virologia , Doenças dos Cavalos , Cavalos/virologia , Testes Sorológicos , Vírus da Encefalite Equina do Leste/isolamento & purificaçãoRESUMO
This is a review of the arboviruses in Argentina belonging to families Flaviridae, Bunyaviridae and Rhabdoviridae. Of the many viruses belonging to these families, the flavivirus St. Louis encephalitis (SLE), has been most intensively studied. SLE virus strains have been recovered from three sources: 2 strains from humans with an undifferentiated, febrile disease; 6 from mosquitoes; and 2 from rodents. The viruses recovered from rodents are attenuated and those from mosquitoes are virulent based on a neuroinvasiveness test in mice; the degree of virulence of the mosquito strains remain to be analyzed. Serological surveys indicate a wide distribution and endemicity of SLE virus in the temperate and subtropical areas (central and northern Argentina), but no data are available from the andean region or from the South. The virulent SLE virus strains appear to be transmitted between Culex (Cx.)spp. from which they were isolated, and wild birds, based on antobody prevalence. A urban cycle may involve Cx. quinquefasciatus (source of a viral isolate and a competent experimental vector) and abundant birds (house sparrows, doves, and/or chickens), chickens are experimentally competent host species. Despite similarities in the ecology of SLE between Argentina and North America, urban outbreaks of SLE have not been recognized. Possible explanations for this discrepancy include virus strain differences in virulenc, ecologic factors determining the rate of virus transmission, and the lack of disease recognition and specific laboratory diagnosis of human meningoencephalitis. The transmission cycle of attenuated SLE virus strains isolated from rodentshas not been studied. Ilheus virus has isolated only once from a human being. The available serological data are difficult to interpret due to cross-reactivity with other flaviviruses, and the ecologyand medical importance of this agent remain uncertain. Dengue has not been recognized in Argentina since 1916, although is vector, Aedes aegypti, was not erradicated until 1963. Dengue was previously present in coastal localities of Chaco. Corrientes and Misiones Provinces. Within the last few years, Argentina was reinfested by Ae. aegypti. Although no human cases have vet been reported, outbreaks of dengue in bordering countries (Brazil, Paraguay, Bolivia) since 1986, cleary signal that the country in once againat risk of importantion ans spread of the virus
Assuntos
Vírus da Encefalite Equina do Leste/isolamento & purificação , Encefalomielite Equina , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/epidemiologia , Infecções por Togaviridae/diagnóstico , Infecções por Togaviridae/epidemiologia , Vírus da Encefalite Equina do Oeste/isolamento & purificaçãoRESUMO
We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates. In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA- and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA- and SA-serotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NA- and SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.
Assuntos
Vírus da Encefalite Equina do Leste/classificação , Animais , Aves/virologia , Culicidae/virologia , Cães/virologia , Eletroforese em Gel de Poliacrilamida , Vírus da Encefalite Equina do Leste/isolamento & purificação , Glicoproteínas/análise , Glicoproteínas/isolamento & purificação , Cavalos/virologia , Humanos , Mapeamento de Peptídeos , Sorotipagem , América do Sul , Estados Unidos , Proteínas Virais/análise , Proteínas Virais/isolamento & purificação , Vírion/químicaRESUMO
Se muestran los resultados obtenidos de la utilización de un ElISA de doble anticuerpo y el método de inmunofluorescencia indirecta para la identificación de aislamientos del virus de la encefalomielitis equina del este realizados en los sitemas celulares Vero XL-1. Los resultados obtenidos mediante ambas técnicas fueron del 100 por ciento de coincidencia con la identificación por neutralización. Utilizando las primeras se pudo detectar el virus entre 6 y 8 horas posinoculación. Los resultados obtenidos utilizandon las células XL-2 fueron mejores que con las células Vero