Assuntos
Vírus da Encefalite Equina do Leste/fisiologia , Encefalomielite Equina do Oeste/diagnóstico , Encefalomielite Equina do Oeste/virologia , Adolescente , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina do Oeste/imunologia , Evolução Fatal , Humanos , Masculino , Filogenia , RNA Viral/genética , Uruguai , Proteínas não Estruturais Virais/genéticaRESUMO
In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.
Assuntos
Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Encefalomielite Equina/diagnóstico , Encefalomielite Equina do Oeste/diagnóstico , Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Animais , Primers do DNA/genética , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Oeste/genética , Encefalomielite Equina/virologia , Encefalomielite Equina do Oeste/virologia , Doenças dos Cavalos/virologia , Cavalos , Humanos , Programas de Rastreamento/métodos , Sondas de Oligonucleotídeos/genética , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
During the monitoring of arbovirus seroprevalence in wild birds collected in California, we inadvertently made two isolates of western equine encephalomyelitis virus (WEEV) from California quail sera being tested by plaque reduction neutralization assay for antibodies against St Louis encephalitis (SLEV) and West Nile (WNV) viruses despite heating the sera at 56 degrees C for 30 minutes. These data prompted us to examine the thermostability of these viruses during heat treatment. The flaviviruses, SLEV and WNV, at titers up to 10(6) plaque-forming units (PFU), were readily inactivated by the standard protocol of heating at 56 degrees C for 30 minutes. In contrast, solutions containing 10(5) and 10(6) PFU of WEEV required 2 hours for complete inactivation. Occasional presence of live virus within sera could lead to false negatives using standard plaque reduction neutralization test protocols.
Assuntos
Vírus da Encefalite de St. Louis/fisiologia , Vírus da Encefalite Equina do Oeste/fisiologia , Temperatura Alta , Manejo de Espécimes/métodos , Vírus do Nilo Ocidental/fisiologia , Animais , Doenças das Aves/diagnóstico , Doenças das Aves/imunologia , Doenças das Aves/virologia , Coturnix , Encefalite de St. Louis/sangue , Encefalite de St. Louis/diagnóstico , Encefalite de St. Louis/veterinária , Encefalomielite Equina do Oeste/sangue , Encefalomielite Equina do Oeste/diagnóstico , Encefalomielite Equina do Oeste/veterinária , Testes Sorológicos , Febre do Nilo Ocidental/sangue , Febre do Nilo Ocidental/diagnóstico , Febre do Nilo Ocidental/veterináriaRESUMO
The E1 and E2 glycoproteins of Western Equine Encephalitis (WEE) are candidate antigens for WEE subunit vaccine development. We have cloned the E1 gene of WEE virus and expressed it in Escherichia coli as inclusion bodies. The inclusion bodies were successfully solubilised, refolded and the immunogenicity of this unglycosylated protein was assessed in mice. Immunization of mice with recombinant E1 protein generated both humoral and cell-mediated immune responses, indicating the recombinant E1 protein is immunogenic. Challenge of E1-immunized mice with live WEE virus demonstrated little or no protection from this E. coli-derived non-glycosylated subunit.
