Analysis of the antiviral activity of γ-PGA against mouse and human noroviruses. (a) RAW264.7 cells infected with the indicated MOI of MNV-1 were treated with 100 nM 2,000-kDa γ-PGA. Viral genome copy number and infectious virus titer in the culture media were determined by RT-qPCR at 40 h post-infection. (b) The HG23 cell line harboring a Norwalk virus sub-genomic replicon was transfected with plasmids expressing TLR4, MD2, and CD14 individually. After incubation for 6 h, transfected cells were treated with γ-PGA (100 nM) for 2 days, prior to RT-qPCR quantification of replicon RNA titer two days later. Neo, neomycin phosphotransferase-coding gene conferring resistance to G418. (c) RAW264.7 cells were pre-treated for 6 h before MNV-1 infection or treated after the viral infection with increasing concentrations of 2,000-kDa γ-PGA. Viral genome titer was determined at 30 h post-infection. (d,e) Inhibition of MNV-1 entry by γ-PGA. Q-dot (QD)-conjugated MNV-1, which was pre-incubated with 2,000-kDa γ-PGA (100 nM) for 30 min, was applied to RAW264.7 cells. Two hours after adsorption, viral entry was quantified by fluorescence microscopy (d), and the fluorescence intensity signals were quantified by image analysis using ImageJ software (e). BF, bright field image. Scale bar, 50 μm. Shown as an inset in (d) is the transmission electron micrograph of purified MNV particles negatively stained with uranyl acetate (Scale bar, 100 nM). (f) Expression of TLR1-9 in RAW264.7 cells infected with MNV-1 (MOI of 0.005). Shown are the GAPDH-normalized TLR expression levels relative to those in mock-treated cells, analyzed by RT-qPCR at 24 h post-infection. Results are from two independent experiments, each analyzed by triplicate assay. (g) Immunoblot assay representing the activation of IRF-3 by γ-PGA (100 nM) and MNV-1 infection (MOI of 0.005) without affecting TLR2, TLR3, and TLR4 expression levels. pIRF-3, phosphorylated IRF-3. In all panels, data are mean ± s.d. Statistical significance of differences between groups was determined via two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; n.s., not significant.