Interfering With Lipid Raft Association: A Mechanism to Control Influenza Virus Infection By Sambucus Nigra

Abstract

Sambucus nigra (elder) are broadly used species to treat microbial infections. The potential antiviral activity and mechanism action of elder fruit (EF) in human epithelium cell (A549) cultures infected with H9N2 influenza virus were determined. The effect of various concentrations of EF on influenza virus replication was examined by using virus titration, quantitative real time RT-PCR, fusion and lipid raft assays following two treatment procedures: A) pre-treated H9N2 virus with each concentration of EF extract and transfection of A549 cell cultures, and B) each concentrations of EF was added to H9N2 virus infected-cell cultures following virus adsorption. In both treatments with lower doses of EF increased viral titer as well as synthesized viral nucleoprotein as indicating the herb had no inhibitory effects on virus replication. In (B) trial with higher doses, 40 and 80 μg/mL of EF, a significant decrease in virus titer and viral protein synthesis were shown in EF treated cells indicating the herb affect either entry of viruses or inhibition virus particle release. The results suggest that EF treatment of the influenza virus infected-human epithelial cells may involve in lipid raft association which function as platform for formation of viral membrane fusion and budding. Differencesin treatment time and dose of EF extract in infected cells with influenza virus have a marked effect on the efficacy of the herb.

Keywords: Antiviral activity; Hemagglutinin; Influenza virus; Lipid raft; Sambucus nigra.

Figure 1

Microscopic analyses of H9N2 influenza virus infected A549 cells after 48 h incubation with different concentrations of Sambucus nigra (EF) extract (100X magnification).the influenza-virus infected A549 cells became rounded and eventually detached in (A) treatment with 40 and 80μg/mL of EF in compared with the uninfected cells (mock), while the CPE was not observed in (B) trial

Figure 2

Inhibition of H9N2 influenza virus replication in the presence of different concentrations of Sambucus nigra (EF) extract was determined. The titer of virus in A549 supernatants was assayed by TCID₅₀ in trials (A) and (B) with 40 and 80 μg/mL of EF compared to the virus-infected and EF-untreated cells (mock

Figure 3

Triton X-100 solubilization analysis of A549cells infected with H9N2influenza virus in pre- and post treatments of Sambucus nigra (EF). Equivalent aliquots from soluble material (S) and insoluble (I) fractions of 80 mg/mL of EF in trials (A) and (B) were subjected to SDS-PAGE. Then viral HA and NP were detected by Western blot using specific antibodies. HA incorporated into lipid raft during virus attachment to the cell membrane. In post treatment with the higher dose of EF, the solubility of HA was affected due to the disruption of lipid raft, whereas the solubility of the protein remained unaffected in control (C). The NP floated to the top fraction in (A) treatment