2’-Fucosyllactose Inhibits Coxsackievirus Class A Type 9 Infection by Blocking Virus Attachment and Internalisation

Figures

• 

  Figure 1. Hits of anti-CV-A9 drug screening from 16 breast milk ingredients. A total of 5 hits (MFGM, WPC, 2’-FL, LF, and OPN) were found with significant inhibition against CV-A9 indicated by the blue bars; OPN and WPC with an inhibition rate of <90% were indicated in light blue. Ineffective drugs are represented by red bars and control by purple. The Y-axis represent the relative viral load. The relative viral mRNA expression and inhibition rate were calculated based on the Ct value, denoted by B and I, respectively. The formula is as follows: A = expression level of CV-A9 mRNA/GAPDH mRNA = 2(CtGAPDH − CtCV-A9); Bingredient (%) = (Aingredient/Acontrol) × 100%; Iingredient (%) = 1 − (Bingredient − Bcontrol) × 100%. Data represent the mean of the three replicate results. Error bars represent ±1 SD, and the experiments were repeated at least twice.

• 

  Figure 2. The anti-CV-A9 activity of 2’-Fucosyllactose (2’-FL). (A) 2’-FL’s inhibition rate against CV-A9 on RD and Huh7. Cells were infected with CV-A9 at MOI = 0.001 and treated with 2’-FL at a final concentration of 20, 10, and 5 mg/mL for 48 h. The intracellular viral load was quantified using qRT-PCR and normalised by GAPDH level. (B) The anti-CV-A9 EC50 and CC50 of 2’-FL on RD and Huh7. Cells were infected with CV-A9 at MOI = 0.001 and treated with 2’-FL at various concentrations (from 40 mg/mL to 0.078 mg/mL) to determine viral inhibition and cell viability. Concentration ≥5 mg/mL inhibited CV-A9 infection. The left and right Y-axis represent the mean percentage of virus yield and cytotoxicity inhibition, respectively. (C) 2’-FL blocks the infection without destroying the virus structure. CV-A9 was pre-incubated with DMEM or 2’-FL (10 mg/mL) for 2 h before plaque assay. Both treatment groups exhibited the same viral titres. MOI: multiplicity of infection; qRT-PCR: quantitative real-time polymerase chain reaction; EC50: concentration for 50% of maximal effect; CC50: cytotoxicity concentration 50%; mRNA: messenger RNA. The experiments were repeated at least twice. Data represent the…

• 

  Figure 3. 2’-FL acts on cells to block infection. (A) Cells were incubated with 2′-FL at a final concentration of 20 mg/ml and 10 mg/ml for 4 h at 37 ℃, followed by CV-A9 infection (MOI = 0.001) in the presence of 2’-FL at the same concentration. At 48 h.p.i., the intracellular viral load was quantified using qRT-PCR and normalised by GAPDH level. (B) CV-A9 were incubated with 2’-FL at a final concentration of 20 mg/ml and 10 mg/ml for 4 h at 4 ℃ and added into RD cells at MOI = 0.001. At 48 h.p.i., the intracellular viral load was quantified using qRT-PCR and normalised by GAPDH level. (C) 2’-FL (20 mg/ml) was used to pre-treat RD cells for 24 h. Then the supernatant was removed and replaced with CV-A9 virus solution (MOI = 0.001) for 24 h. Cells without drug treatment but only with virus treatment were the control group. The intracellular viral load was quantified using qRT-PCR and normalised by GAPDH level. **** p < 0.0001; *** p < 0.001; ns: no significant difference; Student’s t-test compared with control. Data represent the mean of the three replicate results. Error bars represent ±1 SD, and the experiments were repeated at least twice.

• 

  Figure 4. 2’-FL inhibits CV-A9 infection by blocking viral cell entry. (A) Time-of-addition experiment of 2’-FL (10 mg/mL). The detailed steps for the time-of-addition experiment were described in the method section, the viral and GAPDH mRNA levels in the infected cell were quantified using qRT-PCR at 24 h.p.i. The viral load in the supernatant was measured by plaque assay. Time course curve (B) and single-step growth curve (C) of CV-A9 production (top) in RD cells with or without a single dose of 2’-FL (10 mg/mL) or LF (0.156 mg/mL) treatment after 2 h.p.i. Supernatant and cells were harvested at 4, 6, 10, 12, 24, 36, and 48 h.p.i. for qRT-PCR. Plaque assay corresponding to the CV-A9 single-step growth curve (bottom). (D,E) Illustrates the time periods when 2’-FL was present in the cell culture. The arrows indicate the time period 2’-FL (10 mg/mL) exists in the cell culture (left). RD cells were infected with CV-A9 at MOI = 5, with cells following the corresponding drug treatment. At 12 h.p.i., the viral yield in cells was quantified using qRT-PCR and normalised by GAPDH level (right). Determining the effectiveness of 2’-FL on (F) viral attachment and (G) viral internalisation. Virus…

• 

  Figure 5. Docking analysis visualisation of 2’-FL binding with (A) ITGB6 (PDB: 4UM8), (B) FCGRT (PDB: 1EXU), and (C) β2M (PDB: 1A1M). The yellow dots show H-bonds. The ITGB6-2’-FL complex structure highlighting the ITGB6 (blue), 2’-FL (magenta) and residue I216 (green); the FCGRT-2’-FL complex structure highlighting the FCGRT (blue), 2’-FL (magenta) and residue W51 (green); The β2M-2’-FL complex structure highlighting the β2M (blue), 2’-FL (magenta) and residue Q155, D4 and R62 (green).

• 

  Figure 6. Schematic overview on the antiviral role of 2’-FL. Similar to cell surface glycan (such as HBGA), 2’-FL can act as a soluble decoy receptor to bind to the virus to block infection; 2’-FL can also block viral attachment by binding to cell surface receptors.