*2.1. Compounds*

In total, 12 oleanane-type triterpenoid compounds (Figure S1 and Figure 1A), including oleanolic acid and its 11 derivatives, and their antiviral activities against H5N1 IAV infection in A549 cells were studied. Oleanolic acid was purchased from Chengdu Gelipu Biotechnology Co., Ltd. (Chengdu, China). Seven derivatives named OA-1, OA-2, OA-3, OA-4, OA-5, OA-6 and OA-7 were synthesized as described previously [27]. Four new derivatives named OA-8, OA-9, OA-10 and OA-11, which were confirmed by data from nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis (shown in the supplementary material), were synthesized by a method similar to that described previously [27]. Briefly, the known saponin 3-(β-d-glucopyranosyl)-oleanolic acid benzyl ester or 3-(α-d-mannopyranosyl)-oleanolic acid benzyl ester was subjected to PivCl in the presence of pyridine to selectively protect the 3,6-OHs of the β-glucopyranosyl or α-mannopyranosyl residue, and subsequent glycosylation of the 2, 4-OHs in sugars with 2,3,4-tri-*O*-acetyl-L-rhamnopyranosyl trichloroacetimidate using TMSOTf as the promoter, followed by deprotection of the acyl groups with NaOH to produce two tridesmosidic oleanolic acid benzyl ester saponins, respectively. Hydrogenolysis of the above oleanolic acid benzyl ester saponins over palladium/carbon in THF-MeOH led to OA-9 and 3-O-β-chacotriosyl oleanolic acid, was carried out to furnish OA-10 or OA-11 by four consecutive acetylation, acyl chlorination, amide condensation reaction and alkaline hydrolysis, respectively. The purities of all oleanane-type triterpenoids were ≥98% by HPLC analysis. Ribavirin hydrochloride was purchased from Guangdong Starlake Bioscience Co. Ltd. (Zhaoqing, China) with purities ≥ 99%. Peramivir in sterile 0.9% NaCl solution (0.3 g/100 mL) was purchased from Guangzhou Nucien Pharmaceutical Co., Ltd. (Guanzhou, China). Arbidol hydrochloride was purchased from Dalian Melun Biological Technology Co., Ltd. (Dalian, China) with purities ≥98%. Oleanane-type triterpenoids were dissolved in dimethyl sulfoxide (DMSO) and diluted with PBS to <0.4% DMSO for in vitro experiments. Ribavirin hydrochloride was dissolved in PBS for in vitro experiments.

**Figure 1.** OA-10 inhibited H5N1 IAV replication in A549 cells with minimal cytotoxicity. (**A**) Chemical structure of OA-10. (**B**) Antiviral activity of OA-10 against H5N1 IAV infection. Confluent A549 cells grown in 96-well plates were infected with H5N1 IAV (0.1 MOI) for 1 h at 37 ◦C and then cultured in fresh medium containing various concentrations of OA-10 or 15 μM peramivir (Per). At 24 hpi, the cells were fixed with paraformaldehyde and the viral NP expression was detected by indirect immunofluorescence assay (IFA). Representative IFA images from three independent experiments are shown. Scale bar: 250 μm. (**C**) Cytotoxicity of OA-10 in A549 cells. The results are expressed as percentages (%) of mock treated cells.

#### *2.2. Cell Lines and Influenza Virus*

A549 (human lung carcinoma) cells and MDCK (Madin-Darby canine kidney) cells used for IAV infection were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, UT, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL of penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2. All cells were obtained from the Center of Cellular Resource, Chinese Academy of Sciences (Shanghai, China).

Avian influenza A virus strain A/Duck/Guangdong/99(H5N1 IAV) and A/Chicken/Guangdong/16/ 1996 (H9N2) were kindly provided by the Veterinary Technology Center of South China Agricultural University (Guangzhou, China). The H1N1 IAV strain A/Puerto Rico/8/34 (PR8) and H3N2 IAV strain A/Guangdong/Dongguan/1100/2006 viruses were obtained from the Chinese Center for Disease Control and Prevention (Beijing, China). Virus stocks were passaged in 10-day old embryonated chicken eggs for 48 h. The allantoid fluid was harvested and aliquots were stored at −80 ◦C until required. Experiments involving H5N1 virus were conducted in a physical containment level three (PC3) laboratory.

#### *2.3. Cytotoxicity Assay*

The cytotoxicity of tested compounds was evaluated using a MTT assay as described previously by Luo [28]. Briefly, cells were grown in 96-well plates for 24 h. The medium was replaced with fresh one containing serially diluted compounds and the cells were further incubated for 24 h, 48 h or 72 h. The culture medium was removed and replaced with 100 μL 3-(4,5-dimethylthiozol-2-yl)-3,5-dipheryl tetrazolium bromide (MTT; Sigma-Aldrich, MA, USA) solution (1 mg/mL in PBS) and incubated at 37 ◦C for 4 h. After removal of the supernatant, 150 μL of DMSO was added to all of the wells to dissolve the formazan crystals for 10 min at 37 ◦C. Cell viability was then measured as the absorbance

at 490 nm with a microplate reader (Thermo fisher scientific, MA, USA) and expressed as a percentage of the control level. The mean optical density (OD) values from six replicated wells per treatment were used as the cell viability index. The 50% cytotoxic concentration (CC50) was calculated by GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA).
