RESUMO
The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.
Assuntos
Doenças dos Cavalos/diagnóstico , Vírus da Influenza A Subtipo H3N8 , Vírus da Influenza A Subtipo H7N7 , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taq Polimerase , Animais , Embrião de Galinha , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A Subtipo H3N8/classificação , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A Subtipo H7N7/classificação , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/isolamento & purificação , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/virologia , Sensibilidade e Especificidade , Proteínas da Matriz Viral/genéticaRESUMO
Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 specific inhibitor Ac-DEVD-CHO, a general caspase inhibitor Boc-D-FMK provided much stronger inhibition of EIV-induced cytopathic effect and apoptosis. Our results suggest that EIV may activate more than one caspase. Caspase activation and cleavage of its cellular targets may play a critical role in EIV-mediated cytotoxicity.