RESUMO
Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-ß promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1ß and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.
Assuntos
Proteínas Aviárias/genética , Patos , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Doenças das Aves Domésticas/imunologia , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Fibroblastos/fisiologia , Fibroblastos/virologia , Imunidade Inata , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/genética , Influenza Aviária/virologia , Dados de Sequência Molecular , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Alinhamento de Sequência/veterinária , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: Few drugs are currently licensed to treat influenza A infection, and new therapies are needed, especially for highly pathogenic strains. Traditional medicinal plants, such as Lycoris radiata, are a potential source of new antiviral agents. OBJECTIVE: To test 15 Amaryllidaceae alkaloids isolated from the bulbs of L. radiata in vitro for antiviral activities against influenza virus type A, A/Chicken/GuangDong/178/2004 (H5N1, 178). METHODS: Antiviral activities of the compounds were tested in time-of-addition assays, hemagglutination inhibition (HI) assays, neuraminidase (NA) activity assays, and viral entry inhibition assays using H5N1-HIV pseudoviruses. Effects of the compounds on localization and activity of the viral ribonucleoprotein (RNP) were determined by immunofluorescence and an RNP minigenome assay, respectively. RESULTS: Among the alkaloids, lycorine (AA1), hippeastrine (AA2), hemanthamine (AA3) and 11-hydroxy vittatine (AA4) exhibited antiviral activities, with EC90 values of 0·52, 82·07, 4·15, and 13·45 µm, respectively. These compounds did not affect the function of the outer membrane proteins or the viral entry process and viral RNP activity. As AA1 and AA3 exhibited stronger antiviral activities, they were further analyzed. Intracellular nucleoprotein (NP) localization showed that AA1 and AA3 inhibited the RNP complex in the nucleus at an early stage of a single-round and multi-round of replication. CONCLUSION: Four Amaryllidaceae alkaloids were first determined that could exert anti-influenza activities after virus entry into cells. Furthermore, AA1 and AA3 could inhibit nuclear-to-cytoplasmic export of the RNP complex of virus replication. Thus, these compounds may be developed further as anti-influenza drug candidates.