*2.5. In Vitro Virus Growth Inhibition Assay*

A549 monolayers were infected with the virus for 1 h. Supernatants were removed, and cells were then incubated with DMEM containing serial concentrations of test compound. Cells and supernatants were collected at indicated time points post-infection and in total subjected to three freeze-thaw cycles at −80 ◦C and 4 ◦C to ensure maximal release of cellular virions [29]. Final viral titers in the supernatants were determined by an end point dilution assay using MDCK cells and expressed as log10TCID50 /0.1 mL.

#### *2.6. Real-Time Reverse-Transcription PCR (RT-PCR)*

Total RNA was extracted from cells or mixtures of cells and supernatants using the total RNA rapid extraction kit (Fastagen, Shanghai, China) following the manufacturer's instructions. RNA was reverse-transcribed into first-strand cDNA using a reverse transcription kit (TaKaRa, Japan). PCR amplification was performed using 1 μL of reverse-transcribed product with primers designed for IAV-NP and GAPDH (glyceraldehyde-3-phosphate dehydrogenase, used as the endogenous control). The primers used for PCR amplification are listed in Table 1 [30]. Real-time PCR was performed using 2×RealStar Green Power Mixture (containing SYBR Green I Dye) (Genstar, Beijing, China) on a CFX96 Real-time PCR system (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was calculated by 2−ΔΔCT method using DMSO-treated infected cells as reference samples for determining IAV-NP [31,32]. To assess the effect of OA-10 on transcriptional activation of NP in IAV infected cells, the relative fold change of each NP gene expression was calculated and compared between OA-10-treated virus-infected and virus-infected cells.


**Table 1.** Real-time PCR primer sequences.

<sup>a</sup> F: forward primer; R: reverse primer

#### *2.7. Time Course Inhibition Assay*

To estimate the influence of OA-10 on the IAV replication cycle, A549 cells were grown in 24-well plates to confluence and then infected with H5N1 IAV (0.1 MOI) for 1 h at 37 ◦C. OA-10 or ribavirin or peramivir was added before, during or after IAV infection. For pretreatment, cells were incubated with the indicated compound for 2 h at 37 ◦C, followed by three washes with PBS and then infected with H5N1 IAV for 1 h. For co-treatment, cells were simultaneously incubated with H5N1 IAV and the compound. After 1 h, the virus–drug mixture was removed, and the cells were washed three times with PBS before fresh medium was added. For post-treatment, cells were first infected with H5N1 IAV for 1 h followed by three washes with PBS and then incubated in the fresh medium containing the compound. At 24 hpi, progeny viruses in the supernatants were determined by an end point dilution assay and the extent of virus infection in the cells was assessed by IFA for NP protein, respectively.

To determine the specific stage(s) of the viral life cycle affected by OA-10, a time-of-addition assay was performed as described by Luo et al. [28]. Briefly, confluent monolayers of A549 cells grown in 24-well plates were infected with H5N1 IAV (1.0 MOI) for 1 h at 37 ◦C. Cells were washed three times with PBS to remove unbound viruses and incubated in fresh medium. In total, 80 μM of OA-10 or ribavirin or peramivir was then added for 0 to 2, 2 to 4, 4 to 6, 6 to 8 h or 0 to 8 hpi. After each incubation period, the cells were washed three times with PBS and incubated with fresh medium at 37 ◦C. At 8 hpi, the cells were subjected to viral NP protein analysis using IFA and viral mRNA analysis using RT-PCR, respectively.

## *2.8. Hemagglutination Inhibition Assay (HAI)*

The inhibitory activity of OA-10 on HA-mediated hemagglutination of chicken red blood cells (RBCs) was assessed by an HAI assay. Briefly, 25 μL of A/Duck/Guangdong/99(H5N1) (4 hemagglutination units) was incubated with 25 μL H5 standard antiserum (H5 antiserum were provided by the Veterinary Technology Center of South China Agricultural University, Guangzhou, China) or OA-10 at indicated concentrations for 30 min at RT. Then, 50 μL chicken RBCs (0.5%) in saline solution were added to each well and incubated at 37 ◦C for 30 min. The plates were taken images at 45 degree inclination for recording hemagglutination and the fluidity of deposited RBCs.

#### *2.9. Hemolysis Inhibition Assay (HIA)*

The inhibitory effects of compounds on virus-induced hemolysis at low pH were determined by a procedure described previously by Basu et al. [33]. Briefly, chicken RBCs were washed twice with PBS and resuspended to 2% (vol/vol) in PBS and stored at 4 ◦C until use; 100 μL of compound diluted in PBS was then mixed with an equal volume of H5N1 IAV (108 PFU/mL) in a 96-well plate. After incubating the virus-compound mixture at room temperature for 30 min, 200 μL of 2% chicken RBCs (pre-warmed at 37 ◦C) was added. The mixture was incubated at 37 ◦C for another 30 min. To trigger hemolysis, 100 μL of sodium acetate-acetic acid buffer solution (0.5 M, pH 5.2) was added and mixed with the RBC suspension. The mixture was incubated at 37 ◦C for another 30 min for HA acidification and hemolysis. To separate unlysed RBC from the lysed ones, plates were centrifuged at the end of

incubation at 1200 rpm for 6 min; 300 μL of the supernatant was transferred to another flat-bottom 96-well plate. The OD540 was read on a microtiter plate reader. IC50 is defined as the compound concentration that generates 50% protection on chicken RBCs lysed by virus.
