Please cite this paper as: He et al. (2013) Amaryllidaceae alkaloids inhibit nuclear‐to‐cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1. Influenza and Other Respiratory Viruses 7(6), 922–931. 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.

While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB2 residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that VK627 and rTsE627K infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rVK627E and TsE627. Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by VK627 and rTsE627K infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung. These data demonstrated that VK627 and rTsE627K infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV.