Cells, Viruses and natural compounds
Madin-Darby canine kidney (MDCK), human non small cell lung cancer (A549) and human embryonic kidney (293T) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1,000 units/mL penicillin and 100µg/ml of streptomycin (Invitrogen, USA). All cells were grown at 37 ℃ in 5% CO2 incubator.
A recombinant influenza A reporter virus (PR8-PB2-Gluc) that had been engineered to express Gaussia luciferase of which inserted into the PB2 segment, and the wild-type influenza A/Puerto Rico/8/34 (A/H1N1/PR8) were rescued and generated as previously described16-19. Oseltamivir-resistant influenza A/H1N1/pdm(09) virus containing NA/H274Y mutant site was provided by Beijing CDC, China, influenza A/Brisbane/10/2007(H3N2) was provided by Chinese Academy of Medical Sciences, and influenza B-Yamagata-like and B-Victoria-like strains were provided by Shandong CDC (Jinan, China). Infections were performed in Opti-MEM containing 1.5 µg/mL N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich, St. Louis, MO, USA).
The compound library containing 891 natural products dissolved in 100 µL DMSO at a stored concentration of 10 mM, was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). Samples were arranged in 96-well plates and diluted with Opti-MEM containing 1.5 µg/mL TPCK-trypsin to 20 µM screened concentration.
Cell-Based High-throughput antiviral screening
The replication-competent reporter IAV carrying Gaussia luciferase gene (PR8-PB2-Gluc) has been reported as a sensitive and convenient tool to characterize the virus titer [23]. A cell-based phenotypic high-throughput screening (HTS) that allows for multicycle viral replication to identify potential inhibitors was carried out as previously described [24,25]. Briefly, the adherent growth of monolayer MDCK cells growing in white, flat-bottom, 96-well culture plates (PerkinElmer, Waltham, MA) were infected with PR8-PB2-Gluc virus at 0.01 multiplicities of infection (MOI) in presence of test samples of 20 µM. After incubation 36 hours (hrs), infections were determined through the Guassia luciferase activity which was measured using Pierce Guassia luciferase glow assay kit (Thermo scientific, Rockford, IL, USA) following the manufacturer’s instructions. Mock infected cells were used as blank control. DMSO and baloxavir acid (BA, diluted to 20 nM concentration) were set as negative and positive control, respectively. The compounds with inhibition rate above 80% were selected for detection of cytotoxicity.
Cytotoxicity assay
The cytotoxicity analysis was performed according to the manufacturer's instructions of CCK-8 kit (MCE). As described previously, MDCK cells at a density of 10000 cells/well grown in 96-well assay plates were treated with specified screening concentration and incubated for 36 hrs. Cell viability was assessed by measuring the absorbance at a wavelength of 450 nm of a microplate reader.
Dose-response assay
The dose-effect relationship was verified for the selected active natural product. Serial diluted samples were added into infected cells and mock cells, separately to obtain IC50 (50% inhibiting concentration) and CC50 (50% cytotoxic concentration) by fitting dose-response curves with a four-parameter logistic regression to the data in GraphPad Prism software (version 5.02, La Jolla, CA, United States), and the selectivity index (SI) was further analyzed.
Viral Titer Reduction Assay
Viral yield reduction assay was performed as previously described22. Briefly, MDCK cells growing in 24-well plates were infected with A/H1N1/PR8, oseltamivirresistant influenza A/H1N1/pdm(09), A/H3N2/Brisbane, B-Yamagata-like, and B-Victoria-like strains at MOI of 0.01 with or without various concentrations of ALLO, respectively. The culture supernatants were harvested at 36 hrs post-infection (p.i.), and virus titers (TCID50/mL) were determined using cytopathic effect (CPE) experiment. DMSO (0.2%) and uninfected MDCK cells were used as the negative control and the blank control, BA at different concentrations was designed as an antiviral positive drug control group.
Immunofluorescence (IF) analysis
The procedures used for NP immunofluorescence staining were described previously [26]. In a brief, MDCK cells were grown on coverslips and incubated in DMEM containing 2% fetal bovine serum followed by the infection with A/H1N1/PR8 (MOI = 5). After added compound 8 hrs, cells were fixed with 4% PFA for 30 min at room temperature and then were permeabilized with 0.25% TritonX-100 (Sigma-Aldrich, USA). Influenza nucleoprotein (NP) in the cells were detected using anti-NP antibodies (Gene Tex, USA) followed by CoraLite488-conjugated affinipure goat anti-rabbit IgG (H+L) (Proteintech, USA). Nuclear staining with DAPI (40,6-diamidino-2-phenylindole, Solarbio, Beijing) was also performed.
Time of Addition Assay
A classic experiment to further investigate which stage of viral life cycle ALLO disturbed was performed as previously reported [27]. MDCK cells seeded in 24-well plates at a density of 1×105 cells/well were infected with 0.1 MOI PR8-wt virus, and interfered with 20 µM compounds at indicated time (-2, 0, 2, 4, 6, 8) to investigate the period of the viral life cycle targeted by ALLO. At 12 hrs pi, the expression of newly produced virion was analyzed by viral titer test.
Mini-replicon System
The polymerase activity assay was determined by using viral ribonucleoprotein complex (vRNP) minigenome system in A549 cells as previously described [26, 28,29]. In a brief, A549 cells grown in 6-well plates were transfected with viral polymerase plasmids (PB2/pCAGGS, PB1/pCAGGS, PA/pCAGGS, and NP/pCAGGS) and pPolI-Fluc (a firefly luciferase reporter plasmid) together with hRluc-TK (Renilla luciferase plasmid) using Lipofectamine 2000 reagent. At 5 hrs co-transfection, the cells were washed with PBS, re-suspended with fresh DMEM (phenol red free) and seeded into white, 96-well plates at a density of 10,000 cells per well, with various concentrations of ALLO or 0.5% DMSO. After incubation at 37 ℃ for 24 hrs, luciferase activity was measured by dual-luciferase reporter system (Promega, Madison, WI USA) according to manufacturer’s instruction.
Statistical Analysis
In order to quantify the robustness of the screen, Z’ factor was calculated from the normalized signals from positive and negative control wells on each plate as follows: Z’ = 1 - 3 × (SD of positive control + SD of negative control) / (mean of negative control - mean of positive control). SD represents the standard deviation. Z’ value between 0.5 and 1.0 is regarded as appropriate for an HTS assay [30].
The antiviral inhibition rate was calculated according measured luciferase expression with the following equation: inhibition rate = (signal of negative control - signal of tested compound) / (signal of negative control - signal of positive control) × 100%.