Commensal microbes and interferon-λ determine persistence of enteric murine norovirus infection

Abstract

The capacity of human norovirus (NoV), which causes >90% of global epidemic nonbacterial gastroenteritis, to infect a subset of people persistently may contribute to its spread. How such enteric viruses establish persistent infections is not well understood. We found that antibiotics prevented persistent murine norovirus (MNoV) infection, an effect that was reversed by replenishment of the bacterial microbiota. Antibiotics did not prevent tissue infection or affect systemic viral replication but acted specifically in the intestine. The receptor for the antiviral cytokine interferon-λ, Ifnlr1, as well as the transcription factors Stat1 and Irf3, were required for antibiotics to prevent viral persistence. Thus, the bacterial microbiome fosters enteric viral persistence in a manner counteracted by specific components of the innate immune system.

Fig. 1. Pretreatment with an antibiotic cocktail prevents establishment of persistent intestinal infection by murine norovirus strain CR6

Mice were treated with Abx (vancomycin, neomycin, ampicillin, and met:ronidazole) for two weeks prior to oral infection with 10⁶ pfu of CR6. (A and B) Time course of MNoV genome copies shed into fecal pellets with ti.me points at 4 hrs and 1, 3, 7, and 14 days with individual data points at day 14 in (B). Results analyzed by two-way analysis of variance (ANOVA) with Sidak's multiple-comparisons test. ANOVA P < 0.0001. N = 7 to 33 mice per cohort per ti.me point combined from 3 independent experiments. (C and D) MNoV genome copies detected in ileum, colon or MLN at day 3 (C) or 14 (D) post-CR6 infection. Analyzed by Mann-Whitney test. N= 10 to 16 mice per cohort combined from three or four independent experiments. **P < 0.01, ***P< 0.001.

Fig. 2. Bacterial depletion prevents CR6 infection, and reconstitution of the intestinal microbiota rescues infection

(A) 16S rDNA copies per fecal pellet as detected by quantitative real-time polymerase chain reaction of the V4 hypervariable region of the l 6S rRNA gene. Fecal pellets were collected after 2 weeks of the indicated antibiotic treatment. Neo, neomycin; Metro, metronidazole; Vane, vancomycin; Amp, ampicillin. ANOVA P < 0.0001; N= 8 to 22 mice per cohort combined from two to five independent experiments. (Band C) Mice received no Abx, continuous Abx treatment (+Abx), or discontinuance of Abx with no fecal transplantation (Stop, No Ff), transplantation from control untreated mice (Stop, +Ff), or transplantation from control Abx-treated mice (Stop, +Abx Ff). MNoV genome copies were detected at day 14 in fecal pellets (B) or indicated tissues (C). ANOVA P < 0.0001; N = 4 to 9 mice per cohort combined from three independent experiments. All data were analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons test. **P < 0.01, ***P < 0.001.

Fig. 3. The intestinal microbiota is dispensable for extraintestinal infection of MNoV and initial viral trafficking

(A) MNoV genome copies in indicated tissues at day 14 after oral CR6 infection of Ifnar1^−/− mice. Analyzed by Mann-Whitney test; N = 6 to 14 mice per cohort combined from three independent experiments. (B and C) MNoV genome copies detected in tissues at day 3 (B) or day 14 (C) after oral CR6, CW3, or CR6^VP1-CW3 infection. Results from each tissue type were analyzed by one-way ANOVA with Tukey’s multiple-comparisons test. ANOVA P < 0.0001 except day 14 spleen, where ANOVA P < 0.01; N = 6 to 11 mice per cohort combined from three independent experiments. (D) MNoV genome copies detected in the indicated tissues (PP, Peyer’s patches; Ileum, ileal tissue with no PP) at day 3 after oral CR6 infection. Analyzed by Mann-Whitney test; N = 6 or 7 mice per cohort combined from three independent experiments. *P < 0.05, ***P < 0.001; ns, not significant.

Fig. 4. The IFN-λ pathway regulates antibiotic-dependent clearance of CR6

(A) MNoV was detected in fecal pellets of control B6 mice at day 7 after infection of 10⁵, 10⁶, or 10⁷ pfu of CR6. Percentages of infected mice are shown. Numbers of mice are shown in table S1. Analyzed by contingency table analysis with Fisher’s exact test. (B) Mice deficient for the indicated genes or control mice (B6) were pretreated with Abx, then infected with 10⁶ pfu of CR6. Infectivity was assessed according to detection of CR6 in fecal pellets at day 7 after infection; percentages of mice that became infected are shown. Numbers of mice are shown in table S1. Analyzed by contingency table analysis with Fisher’s exact test. Each genotype was assessed in at least three independent experiments. (C) MNoV genome copies detected in ileum at day 14 after CR6 infection. Analyzed by one-way ANOVA with Tukey’s multiple-comparisons test. ANOVA P < 0.0001; N = 6 to 15 mice per cohort combined from two to seven independent experiments. (D) MNoV was detected in fecal pellets at day 7 after infection with 10⁵ or 10⁶ pfu of CR6. Percentages of infected mice are shown. Numbers of mice are shown in table S1. Analyzed by contingency table analysis with Fisher’s exact test. Each genotype was assessed in at least four independent experiments. Comparisons of +Abx, 10⁵ pfu of CR6 infections to B6, +Abx, 10⁵ pfu of CR6 infections shown in (A) by Fisher’s exact test are all P < 0.01. *P < 0.05, **P < 0.01, ***P < 0.001.