The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model

Abstract

Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2'-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.

Fig 1

Antiviral activity of 2CMC against the Norwalk virus replicon. HG23 cells were exposed to serial concentrations of 2CMC for a period of 72 h, after which cell monolayers were collected for quantification of RNA loads by qRT-PCR. Intracellular RNA loads are represented by the variation in Norwalk virus replicon and β-actin C_T values (A) and by relative genome quantification of the Norwalk virus replicon versus β-actin (B). Dotted lines represent 50 and 90% reductions of the relative Norwalk virus replicon RNA levels. Results are mean values ± SEM of five independent experiments.

Fig 2

Effect of 2CMC on Norwalk virus replicon clearance from HG23 cells. (A) In the clearance phase, cells were treated for six consecutive passages with 2CMC (at concentrations of 6.25 to 25 μg/ml) in the absence of G418 selective pressure and the ratio of viral RNA to beta-actin RNA was determined (NT, not tested). (B) In the rebound phase (for cells treated with 25 μg/ml 2CMC), 2CMC was omitted from the culture medium but cells were again cultured under the selective pressure of G418 (1.25 mg/ml). The graph shows the number of cells/25-cm² T flask for three consecutive rebound passages with cells from each clearance passage, namely, passages 2, 4, and 6 (ND, not detected). The dotted line represents the number of cells that was set as the starting point to start a new culture. Results are representative of two independent experiments.

Fig 3

Survival curves of 2CMC-treated (n = 15) and untreated (n = 12) MNV-infected AG129 mice. Treatment with 2CMC (100 mg/kg/day) was initiated 1 h before infection. The compound was administered twice daily by the subcutaneous route for 7 consecutive days. The graph depicts Kaplan-Meier survival curves as percent survival versus time postinfection.

Fig 4

Weight variation of MNV-infected AG129 mice treated with 2CMC (n = 15) or left untreated (n = 12). The graph shows percent body weight variation (compared to day 0 p.i.) versus time postinfection and mean values ± SEM. Values obtained with 2CMC-treated and untreated groups on the same days postinfection were compared.

Fig 5

Fecal consistency of MNV-infected AG129 mice treated with 2CMC or left untreated. Scores for consistency of stool (0, normal feces; 1, mixed stool samples containing both solid and pasty feces; 2, pasty feces; 3, semiliquid feces; 4, liquid feces) versus time postinfection. Data are expressed as mean values ± SEM. In each group, 6 to 15 individual stool samples were collected each day, with the exceptions of the untreated group at 4 days p.i. and the 2CMC-treated group at 11 days p.i., when 3 stool samples were collected. Values obtained with the 2CMC-treated and untreated groups on the same days postinfection were compared.

Fig 6

Viral RNA loads in stool samples of 2CMC-treated or untreated MNV-infected AG129 mice. Data are presented as log₁₀ RNA copy numbers per gram of stool versus time postinfection for untreated or 2CMC-treated animals. In each group, RNA was quantified in 6 to 15 individual stool samples per day, with the exceptions of the untreated group at 4 days p.i. and the 2CMC-treated group at 11 days p.i., when 3 stool samples were collected. The dotted line represents the LOD. Values obtained with the 2CMC-treated and untreated groups on the same days postinfection were compared.

Fig 7

(A) Viral RNA loads in organs of 2CMC-treated or untreated MNV-infected AG129 mice. Data are presented as log₁₀ RNA copy numbers per gram of organ at 3 days p.i. for the small intestines, large intestines, mesenteric lymph nodes, spleens, and lungs of untreated or 2CMC-treated animals. The dotted line represents the LOD. An asterisk indicates that a P value of <0.05 was determined with the Mann-Whitney test, and medians obtained with 2CMC-treated and untreated groups at 3 days p.i. were compared. (B to E) Histopathology of the small intestines of 2CMC-treated and untreated MNV-infected AG129 mice. Images show H&E staining of the small intestines of representative untreated AG129 mice (B and C) and 2CMC-treated mice (D and E) at ×50 (B and D) and ×200 (C and E) magnifications.