The EV71 virus was provided from the Division of Vaccine Research of the Korea Center Disease Control and Prevention (KCDC, Cheongwon, Korea), and CVB3 and CVA16 were obtained by Chungcheongnam-do Health and Environment Research Institute in Korea. The viruses were propagated in African green monkey kidney (Vero) cells (ATCC, CCR-81) at 37°C. HRV1B was purchased from ATCC (Manassas, VA, USA), and propagated at 32°C in the human cervical cancer (HeLa) cell line (ATCC, CCR-2). Vero cells and Hela cells were propagated in minimal essential medium (MEM) adding with fetal bovine serum (10% FBS) and antibiotic-antimycotic solution (0.01%). The MEM (Cat NO. 11095080), FBS (Cat NO. 16000044), trypsin-EDTA (Cat NO. 15400054) and antibiotic-antimycotic solutions (Cat NO. 15240096) were purchased from Gibco BRL (Invitrogen Life Technologies, Karlsruhe, Germany). Sulforhodamine B (SRB, Cat NO. S1402) and Q3G were provided by Sigma-Aldrich (St. Louis, MO, USA). Q3G were of HPLC analytical grade with purity of 98%. Tissue culture plates (Cat NO. 353072) were provided from Falcon (BD Biosciences, San Jose, CA, USA). Rupintrivir (Santa Cruz Biotechnology, Dallas, TX, USA, Cat NO. AG7088) was purchased and dissolve

Antiviral activity was analyzed by the SRB method, as previously reported (8). The day before infection, after seeding of cells in a 96-well plate with 3 × 10⁴ cells per well, the cells cultured for 1 day. The next day, add 10 µL of Q3G to 90 µL of a diluted virus suspension containing the virus stock of Tissue Culture Infectious Dose (TCID₅₀) with 1% FBS and investigate for cytopathic effect (CPE) after 2 days post infection. The antiviral activity of Q3G was determined with four concentrations ranging from 0.4 μg/mL to 50 μg/mL. The cells were propagated at 37°C for 2 days under 5% CO₂. After 2 days, the cell supernatant was discarded, washed with PBS, and the culture was terminated. Then, cold acetone was added and incubated at -20°C for 30 minutes to fix the cells. After removing the acetone, the plate was dried for 30 min in a drying oven, after which, 0.4% SRB (w/v) solutions in 1% acetic acid were given to the plates and cultured for 1 h at room temperature. After 1 h, the SRB solution was sucked and the plates were washed with 200 µL of 1% acetic acid. After drying of overnight, the bounded SRB was solubilized by a 10 mM Tris-based solution. The absorbance of the plates was measured at 562 nm using a SpectraMax i3 microplate reader (Molecular Devices, Palo Alto, CA, USA). The results were converted into % of the controls, and the % protection achieved by the experimental compound in the virus-infected cells. The formula was [(OD_t)_virus − (OD_c)_virus] ÷ [(OD_c)_mock − (OD_c)_virus] × 100%. The (OD_t)_virus is the optical density (OD) obtained with a given concentration of the experimental compound in virus-infected cells. The (OD_c)_virus is the OD of the non-experimental compound-treated in virus-infected cells. The (OD_c)_mock is the OD in normal cells.

The Total RNAs were extracted by an RNA Extraction Mini Kit (Qiagen, Hilden, Germany). Taqman real-time PCR and reverse transcription PCR were conducted using AgPath-ID™ One-Step RT-PCR Reagents (Applied Biosystems, Waltham, MA, USA) on a Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA). EV71, CVB3 and CVA16 5′ NCR genes and HRV1B 5′ NCR genes were detected using qRT-PCR. The following EV71, CVB3 and CVA16 primers and probe were used: 5′ NCR forward primer 5′- AATCCTGTCACCTCTGACTAAGG-3′, 5′ NCR reverse primer 5′-CATTYTGGACAAAKCGTCTACG-3′, and 5′ NCR probe primer 5′-TGCAGTCCTCGCTCAC-3′. The following HRV1B primers and probe were used: forward primer 5′- AATCCTGTCACCTCTGACTAAGG-3′, reverse primer 5′-CATTYTGGACAAAKCGTCTACG-3′, and probe primer 5′-TGCAGTCCTCGCTCAC-3′. The following GAPDH primers and probe were used: forward primer 5′-GGTCTCCTCTGACTTCAACA-3′, reverse primer 5′-AGCCAAATTCGTTGTCATAC-3′, and probe primer 5′-CCCTCAACGACCACTTTGTCAAG-3′. The cycling conditions were as follows: heating at 45°C for 10 min for reverse transcription, reverse transcription inactivation and initial denaturation condition at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and at 60°C for 45 s. The results were obtained using the real-time system AB 7900HT software (Life Technologies, Carlsbad, CA, USA), and all values were normalized to GAPDH levels.

Vero cells and Hela cells infected with EV71, CVB3, CVA16, and HRV1B at the Tissue Culture Infectious Dose (TCID₅₀) were harvested at the indicated time points including 4, 6, 8 and 10 h post-infection. After adding of Q3G at virus co-infection, total RNA was extracted at the indicated time of post-infection, and the level of 5` non-coding region (5` NCR) gene of viruses was analyzed using real time-PCR.

TOA analysis was performed to determine the mechanism of action of Q3G. Vero and Hela cells were treated with Q3G of 10 mg/mL at –1 h (1 h before), 0 h (during), or after infection (1, 2, 4, 6, 8 and 10 hours) of virus infection. At 12 h post-infection, virus-specific 5` NCR gene expression was analyzed using real-time PCR.

We compared multiple groups using a one-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered significant at a 95% confidence interval.

Based on the results of previous studies that Q3G exhibits wide range of antiviral activity against RNA viruses such as zika virus (9) and influenza virus (10), we sought to determine whether Q3G also has antiviral activity against other RNA viruses. Therefore, we tested the antiviral activity against EV71, CVB3, CVA16 and HRV1B belonging to the Picornaviridae family using Q3G. As a result of evaluating virus-induced CPE in cells through SRB analysis, infection with EV71 induced about 80% of apoptosis in Vero cells. However, 50 μg/mL of Q3G inhibited EV71-induced CPE by 100% (Fig. 1A). The IC₅₀ value of Q3G against EV71 was found to be 27.26 μg/mL (Table 1). In Vero cells, CVA16 infection induced about 80% CPE and CVB3 induced about 70% CPE, whereas Q3G at a concentration of 2 μg/mL inhibited CVA16-induced CPE by about 80%, and CVB3 induced CPE about 60% inhibition. Q3G at a concentration of 0.4 μg/mL inhibited CVA16-induced CPE by about 50%, but showed no antiviral activity in CVB3 (Figs. 1B, 1C). In addition, for CVA16 and CVB3, IC₅₀ values were confirmed at the concentrations of 0.74 μg/mL and 6.56 μg/mL, respectively (Table 1). Next, HRV1B induced about 80% of cytotoxicity in Hela cells, but Q3G adding of 0.4 μg/mL inhibited HRV1B-induced CPE by about 50%, thereby exhibiting high antiviral activity (Fig. 1D). The IC50 value of Q3G against HRV1B was found to be 8.01 μg/mL concentration (Table 1). Rupintrivir used as a positive control showed high antiviral activity against all viruses except CVB3 up to the lowest concentration used in the experiment, 16 ng/ml. CVB3 showed about 60% inhibitory activity at a concentration of 80 ng/ml (Fig. 1). The cytotoxicity of Q3G was not observed even at the highest concentration used in the experiment, the highest concentration of 50 μg/mL, for the cytotoxicity of Q3G to Vero cells and Hela cells even at the highest concentration used in the experiment (Figs. 1E, 1F).

We performed time course and time-of-addition experiments to analyze the Q3G action, antiviral mode of against NPEVs. For the time course experiment, real-time PCR analysis was conducted at 4, 6, 8, and 10 h after the EV71, CVA16, CVB3-infected Vero cells and HRV1B-infected Hela cells which were treated with 10 µg/ml Q3G. The 5' NCR gene of EV71 and CVA16 was detected at 6 hours after infection, and the 5ours after infection. However, Q3G strongly suppressed viral RNA expression up to 10 hours after CVA16, CVB3 and HRV1B' NCR gene of CVB3 was detected at 8 hours after infection. The 5`NCR gene of HRV1B was detected at 4 h infection, while EV71 gene expression until 8 hours after infection (Fig. 2). To investigate which step was affected by Q3G, we performed a time-of-addition experiment in which 10 µg/ml Q3G was added to the culture media at each indicated time of post-infection. And the expression of 5` NCR gene in EV71, CVA16, CVB3 and HRV1B were analyzed at 14 hours after infection. When EV71, CVA16, CVB3 and HRV1B were infected to Vero cells or Hela cells which were treated Q3G before 1 hour, but the virus infection was not inhibited. As a result, it was considered that Q3G did not shows inhibition in the entry stage of EV71, CVA16, CVB3 and HRV1B into the cell. However, when treating Q3G in 1, 2, and 4 hours after infection of EV71, CVA16, CVB3 and HRV1B result shows that viral RNA expression was suppressed in cells (Fig. 3). These results suggest that Q3G suppresses the immediate early stage of the cycle of viral replication.