Cell and virus culture
Madin Darby canine kidney (MDCK, CCL-34), Vero-E6 (CRL-1586), 293T (CRL-3216), and Calu-3 (HTB-55) cells obtained from ATCC (Manassas, VA, USA) were cultured in Dulbecco minimal essential medium (DMEM) or MEM supplemented with 10% fetal bovine serum (FBS), 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. The virus strains used in this study included A/Hong Kong/415742M/2009(H1N1), A/Hong Kong/4801/2014(H3N2), A/Netherlands/219/2003(H7N7), A/Anhui/1/2013(H7N9), influenza B/TW/70555/05(FluB), SARS-CoV, and SARS-CoV-2(16, 37).
Plaque reduction assay
Peptides were synthesized by ChinaPeptide. Antiviral activity of peptides was measured using a plaque reduction assay. Briefly, peptides or bovine serum albumin (BSA, 0.2–50.0 μg ml−1) were premixed with 50 PFU of virus in PBS at room temperature. After 45-60 min of incubation at room temperature, peptide-virus mixture was transferred to MDCK or Vero-E6 cells, correspondingly. At 1 h post infection, infectious media were removed and 1% low melting agar was added to cells. Cells were fixed using 4% formalin at 2-3 day post infection. Crystal violet (0.1%) was added for staining, and the number of plaques was counted.
Viral RNA extraction and RT-qPCR
Viral RNA was extracted by Viral RNA Mini Kit (QIAGEN, Cat# 52906, USA) according to the manufacturer’s instructions. Extracted RNA was reverse transcribed to cDNA using PrimeScript II 1st Strand cDNA synthesis Kit (Takara, Cat# 6210A) with GeneAmp® PCR system 9700 (Applied Biosystems, USA). The cDNA was then amplified using specific primers (Supplementary Table 1) for detecting A(H1N1) virus using LightCycle® 480 SYBR Green I Master (Roach, USA). For quantitation, 10-fold serial dilutions of standard plasmid equivalent to 101 to 106 copies per reaction were prepared to generate the calibration curve. Real-time qPCR experiments were performed using LightCycler® 96 system (Roche, USA).
Antiviral multicycle growth assay
Influenza viruses were pretreated by peptide and then infected MDCK cells (0.005 MOI). After 1h infection, infectious media were removed and fresh media with supplemental peptides were added to infected cells for virus culture. At 18 h post infection, the supernatants of infected cells were collected for RT-qPCR assay to determine the viral titers in cell supernatants.
Cytotoxicity assay
Cytotoxicity of peptides was determined by the detection of 50% cytotoxic concentration (CC50) using a tetrazolium-based colorimetric MTT assay. Briefly, MDCK cells were seeded in 96-well cell culture plate at an initial density of 2 × 104 cells per well in DMEM supplemented with 10% FBS and incubated for overnight. Cell culture media were removed and then DMEM supplemented with various concentrations of peptides and 1% FBS were added to each well. After 24 h incubation at 37 °C, MTT solution (5 mg ml−1, 10 μl per well) was added to each well for incubation at 37 °C for 4 h. Then, 100 μl of 10% SDS in 0.01M HCl was added to each well. After further incubation at room temperature with shaking overnight, the plates were read at OD570 using VictorTM X3 Multilabel Reader (PerkinElmer, USA). Cell culture wells without peptides were used as the experiment control and medium only served as a blank control.
Hemolysis and hemolysis inhibition assay
Serially diluted peptide P16 in PBS were incubated with turkey red blood cells for 1 h at 37°C. PBS was used as a 0% lysis control and 0.1% Triton X-100 as 100% lysis control. Plates were centrifuged at 350 g for 3 min to pellet non-lysed red blood cells. Supernatants used to measure hemoglobin release were detected by absorbance at 450 nm. For hemolysis inhibition assay, P16 (200 μg ml-1), P9R (200 μg ml-1) or arbidol (100 μg ml-1) were mixed with or without same volume of H1N1 virus (HA titer >128) for 1 h, and then 60 μl of 2% turkey red blood cells was added for 15 min. PBS and Triton X-100 (0.1%) were included as the negative and positive control of hemolysis. The precipitated erythrocytes were incubated with sodium citrate solution (pH of 4.9) for 25 min. The hemoglobin release in supernatants was detected at 450 nm.
Transmission electron microscopy assay
To determine the effect of P16 on viral particles, A(H1N1) virus was pretreated by 200 μg ml-1 of P16, PBS or Triton X-100 (0.15%) for 1h. The virus was fixed by formalin for overnight and then applied to continuous carbon grids. The grids were transferred into 4% uranyl acetate and incubated for 1 min. After removing the solution, the grids were air-dried at room temperature. For each sample, two-three biological samples were done for taking TEM images by FEI Tecnal G2-20 TEM.
H5N1 pseudovirus assay
H5N1 pseudotype virus(38) bearing H5N1 HA and NA was pretreated with PBS or P16 in PBS and then incubated at RT for 1h. MDCK cells were infected with the treated pseudotype virus for 1h. MDCK cells without pseudotype virus infection were served as the baseline control of luciferase protein. After 18h cell culture, cell lysates were collected and the luciferase protein was measured by Luciferase assay system (Promega) in a Victor X3 Multilabel reader (PerkinElmer). The luminescence reading was normalized to 1mg protein.
Virus induced cell fusion assay
MDCK cells were transfected with pGFP. Eight hour later, cells were infected with 1MOI of FluB virus. At 18h post infection, cells were treated by PBS or P16 for 1h and then cells were treated by pH5.0 or pH 7.4 for 10 min. After removing the pH buffer, cells were cultured at 37 °C for 4 h with full media. Fusion pictures were taken at 4h after pH treatment.
HA mediated cell fusion assay
The 293T cells were co-transfected with pGFP and pH7N7-HA. At 24h post transfection, cells were treated by PBS or P16 for 1h and then treated by pH5.0 or pH 7.4 for 10 min. After removing the pH buffer, cells were cultured at 37 °C for 4 h with full media. Fusion pictures were taken at 4 h after pH treatment.
Spike-ACE2 mediated cell fusion assay
The pSpike of SARS-CoV-2, pACE2-human, or pGFP were transfected to 293T cells for protein expression. After 24 hours, to trigger the spike-ACE2 mediated cell fusion, 293T-Spike-GFP cell were co-cultured with 293T-ACE2 with or without the supplement of peptide or drug. The 293T-GFP cells were co-cultured with 293T-ACE2 cells as the no-fusion negative control. After 8 h of co-culture, five fields were randomly selected in each well to take the cell fusion pictures by fluorescence microscopes.
Peptide binding assay
Peptides (1.0-3.0 μg per well) dissolved in H2O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4°C overnight. For virus or spike protein binding to peptides, viruses or spike protein were diluted in PBS and then were added to ELISA plate for binding to the coated peptides at room temperature for 1h. After washing the unbound viruses or spike protein, the bound viruses were lysed by RLT buffer of RNeasy Mini Kit (Qiagen, Cat# 74106) for viral RNA extraction. Viral RNA copies of binding viruses were measured by RT-qPCR. The bound spike protein detected by anti-His-HRP (Invitrogen, Cat# R93125, 1: 2,000) by reading OD450.
Endosomal acidification assay
Endosomal acidification was detected with a pH-sensitive dye (pHrodo Red dextran, Invitrogen, Cat#P10361) according to the manufacturer’s instructions as previously described but with slight modification(3). First, MDCK cells were treated with BSA (25.0 μg ml−1), P16 (25.0 μg ml−1) and bafilomycin A1 (50 nM) at 4 °C for 15 min. Second, MDCK cells were added with 100 μg ml−1 of pH-sensitive dye and DAPI and then incubated at 4 °C for 15 min. Before taking images, cells were further incubated at 37 °C for 15 min and then cells were washed twice with PBS. Finally, PBS was added to cells and images were taken immediately with confocal microscope (Carl Zeiss LSM 700, Germany).
Rearing of virus resistant mutant
A(H1N1) virus was pretreated by P16 and then infected MDCK cells. After 1 h infection, MEM media with the supplement P16 were added to cells for virus culture. At 24 h post infection, viruses were collected for next passages. After 20 passages, passaged viruses (P20 and P15) and virus without passage (P0) were pretreated by P16 or PBS with the indicated concentration of P16 for MDCK infection. At 18 h post infection, viral loads in cell supernatants were measured by RT-qPCR to identify the sensitivity of passaged viruses to P16.
Hemagglutination inhibition assay
HA titers of H1N1 and FluB viruses were tested by TRBC. Viruses (8HA titer) were premixed with peptides or PBS for 1h and then equal volume of TRBC was added to virus for incubation at room temperature for 30 min. The precipitates of TRBC were record for calculating the HAI activity. TRBC with untreated virus and neutralization antibody from serum were served as negative and positive control of hemagglutination inhibition.
Human Ex Vivo Lung Tissues
Human lung tissues for ex vivo studies were obtained from patients undergoing surgical operations at Queen Mary Hospital, Hong Kong, as we previously described (39). All donors gave written consent as approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364). The freshly obtained lung tissues were processed into small pieces and then were infected with SARS-CoV-2 (2 × 104 PFU) in 500 µl advanced Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 medium (Gibco, Thermo Fisher Scientific) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and chloroquine (5 µg/ml). After 18 hours, the inoculum was removed and the specimens were washed one time with PBS. The infected human lung tissues were then cultured in 0.5 ml of advanced DMEM/F12 medium with chloroquine (5 µg/ml). Supernatants were collected at 60 hours post inoculation for plaque assays.
Antiviral analysis in vivo
BALB/c female mice (10-12 weeks for H1N1 virus and 10-12 months for SARS-CoV) and female hamsters (4-6 weeks for SARS-CoV-2) were kept in biosafety level 2 laboratory (housing temperature between 22~25 °C with dark/light cycle) and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating procedures of the approved biosafety level 2 animal facilities and were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong(40). To evaluate the therapeutic effect, mice were intranasally inoculated with 3 LD50 of A(H1N1) virus or hamsters were intranasally inoculated with 5000 PFU of SARS-CoV-2 and then intranasally inoculated with PBS, P16, zanamivir, or chloroquine at 6-8 h after the viral inoculation. Two more doses were given to the challenged mice on the following day. Survival and general conditions were monitored for 14 days or until death. Viral loads in lungs were measured at day 2 post infection. To evaluate the prophylactic effect, mice were intranasally inoculated with chloroquine at 1-day or 1-min before viral inoculation with 200 PFU of A(H1N1) virus or 5000 PFU of SARS-CoV to mouse lungs. To evaluate the antiviral activity of chloroquine atomization, mice were given the atomized chloroquine and then A(H1N1) or SARS-CoV were inoculated to mouse lungs. The viral loads in lungs were measured at day 1 post infection.