Interferons and ribavirin effectively inhibit Norwalk virus replication in replicon-bearing cells

Abstract

The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-alpha) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-gamma and ribavirin for their effects on NV replication in the cells. Like IFN-alpha, IFN-gamma inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED(50)) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED(50) calculated as approximately 40 microM. The combination of IFN-alpha and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 microM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis.

FIG. 1.

Effects of IFN-α and IFN-γ on NV replication in HG23 cells. (A) Effects of IFN-α and IFN-γ on the expression of NV genome. One-day-old semiconfluent NV replicon-bearing HG23 cells were incubated with various concentrations (units/ml) of IFN-α and IFN-γ for 72 h, and then total RNA was prepared for real-time qRT-PCR to detect the NV genome. The reduction of NV genome by IFNs was calculated by the comparison to that with mock (medium) treatment. Error bars represent standard deviations from at least three independent experiments. RNA levels for cells subjected to a treatment that were significantly reduced (P < 0.05) compared to the RNA level of mock-treated cells are indicated by asterisks. (B) Effect of IFN-γ on the expression of NV ProPol or neomycin phosphotransferase in HG23 cells. One-day-old semiconfluent HG23 cells were incubated with 0 (mock) or 100 units/ml of IFN-γ for 72 h. The cells were then fixed with 100% methanol and stained with antibodies to NV ProPol (top panels) or neomycin phosphotransferase II (bottom panels) and FITC-conjugated secondary antibody. The negative control includes parental Huh-7 cells treated with 100 units/ml of IFN-γ with the same staining.

FIG. 2.

Effect of ribavirin on NV replication in HG23 cells. (A) Effect of ribavirin on the expression of the NV genome. One-day-old semiconfluent HG23 cells were incubated with various concentrations of ribavirin ranging from 0 (mock) to 100 μM for 48 h, 72 h, and 96 h, and then total RNA was prepared for real-time qRT-PCR to detect the NV genome. The reduction of NV genome by IFNs was calculated by comparison to that with mock treatment. RNA levels for cells subjected to a treatment that were significantly reduced (P < 0.05) compared to the RNA level of mock-treated cells are indicated by asterisks. Error bars represent standard deviations from at least three independent experiments. (B) Effect of ribavirin on the expression of neomycin phosphotransferase II in HG23 cells. One-day-old semiconfluent HG23 cells were incubated with various concentrations (0 [mock], 10, 50, 100, or 200 μM) of ribavirin for 72 h. The cells were then fixed with 100% methanol and stained with antibody to neomycin phosphotransferase II and FITC-conjugated secondary antibody. The negative control includes parental Huh-7 cells with the same staining.

FIG. 3.

Effect of the combination of IFN-α and ribavirin on NV replication in HG23 cells. (A) Effect of ribavirin alone or IFN-α and ribavirin on the expression of the NV genome. One-day-old semiconfluent HG23 cells were incubated with various concentrations of ribavirin ranging from 0 (mock) to 100 μM with (+) or without (−) 2 units/ml of IFN-α. After 72 h of incubation, total RNA was prepared for real-time qRT-PCR to detect the NV genome. The reduction of NV genome by the different treatments was calculated by comparison to the NV RNA level of the control (mock treated). Error bars represent standard deviations from at least three independent experiments. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blot. (B) Effect of ribavirin alone or IFN-α and ribavirin on the expression of NPT II detected by Western blot analysis. One-day-old semiconfluent HG23 cells were incubated with various concentrations of ribavirin ranging from 0 (mock) to 100 μM with or without 2 units/ml of IFN-α. After 72 h of incubation, cell lysate was prepared for Western blot analysis using antibody to neomycin phosphotransferase II. The bar graph shows relative values (the value of mock-treated cells was set at 100%) of expression of NPT II (NPT II-tVP1) in cells treated with ribavirin alone or with ribavirin and IFN-α. The intensity of each band in the blots was measured by scanning and represented the ratio of β-actin to NPT II-tVP1. RNA levels or NPT II levels for cells treated with both IFN-α and ribavirin that were significantly different (P < 0.05) from the value for cells treated with ribavirin only are indicated by asterisks.

FIG. 4.

Effect of ribavirin on MNV-1 in RAW267.4 cells. (A) Confluent RAW267.4 cells in six-well plates were treated with various concentrations of ribavirin (0 to 200 μM) for 6 h before MNV-1 was added to the same medium at an MOI of 5. The virus-infected cells were incubated for an additional for 24 h, and then virus replication was measured using the TCID₅₀ assay, after the plates had been frozen and thawed three times. (B) Cells were mock treated or treated with ribavirin (100 μM) for 6 h, and then MNV-1 (MOI of 5) was added to the same medium for 12 h, 24 h, or 48 h. Virus titers were measured by the TCID₅₀ assay. MNV-1 titers for cells subjected to a treatment that were significantly reduced (P < 0.05) compared to the MNV-1 titer for mock-treated cells are indicated by asterisks. Error bars represent standard deviations from at least three independent experiments for panels A and B.

FIG. 5.

Effect of supplementation of guanosine on NV replication in ribavirin-treated HG23 cells and effect of MPA on NV replication. (A) Effect of ribavirin alone or with guanosine (100 μM) on the NV genome in HG23 cells. One-day-old semiconfluent HG23 cells were incubated with various concentrations of ribavirin ranging from 0 (mock) to 100 μM with (+) or without (−) 100 μM guanosine. After 72 h of incubation, total RNA was prepared for real-time qRT-PCR to detect the NV genome. The reduction of NV genome by the treatments was calculated by comparison to the value for the mock-treated control. RNA levels for cells treated by guanosine and ribavirin that were significantly different (P < 0.05) from the RNA level of cells treated with ribavirin alone are indicated by asterisks. (B) Effect of MPA on NV replication. One-day-old semiconfluent HG23 cells were incubated with various concentrations of MPA ranging from 0 (mock) to 2 μM for 72 h. After the incubation, total RNA was prepared for real-time qRT-PCR to detect the NV genome. The reduction of NV genome in cells subjected to different treatments was calculated through comparison to mock-treated cells. RNA levels for cells subjected to a treatment that were significantly reduced (P < 0.05) compared to the RNA level of mock-treated cells are indicated by asterisks. Error bars represent standard deviations from at least three independent experiments for panels A and B.