Interferon-λ cures persistent murine norovirus infection in the absence of adaptive immunity

Abstract

Norovirus gastroenteritis is a major public health burden worldwide. Although fecal shedding is important for transmission of enteric viruses, little is known about the immune factors that restrict persistent enteric infection. We report here that although the cytokines interferon-α (IFN-α) and IFN-β prevented the systemic spread of murine norovirus (MNoV), only IFN-λ controlled persistent enteric infection. Infection-dependent induction of IFN-λ was governed by the MNoV capsid protein and correlated with diminished enteric persistence. Treatment of established infection with IFN-λ cured mice in a manner requiring nonhematopoietic cell expression of the IFN-λ receptor, Ifnlr1, and independent of adaptive immunity. These results suggest the therapeutic potential of IFN-λ for curing virus infections in the gastrointestinal tract.

Figure 1. Systemic and intestinal persistence of MNoV are controlled by IFN-αβ and IFN-λ respectively

Mice were orally inoculated with 10⁶ plaque-forming units (PFU) of MNoV strain CR6 and genome copies in the indicated tissue (A) or feces (B and C) were quantitated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Genome copies were compared between control, Stat1^−/− and Ifnar1^−/− mice at day 21 in tissues (A); between control, Stat1^−/−, Ifngr1^−/−, Ifnar1^−/−, Ifnlr1^−/−, and Ifnar1^−/− × Ifngr1^−/− mice at day 14 in feces (B); and between control, Ifnar1^−/−, and Ifnlr1^−/− over time in feces (C). Data shown are pooled from at least two independent experiments with each point representing an individual animal in (A) and (B). Points in (C) represent at least four animals pooled from two to four independent experiments. Dashed line represents limit of detection. Statistical significance determined by one-way (A and B) or two-way (C) analysis of variance (ANOVA). n.s., not significant (P > 0.05); *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001. Error bars in (C) denote SD.

Figure 2. Induction of IFN-λ prevents enteric NoV persistence

(A and B) Control mice were orally inoculated with 10⁶ PFU of either MNoV CW3 or CR6, and total RNA was isolated from MLN, Peyer’s patches, or colon at the indicated times. Relative copy numbers for IFN-β transcripts (A) and IFN-λ transcripts (B) were quantified by qRT-PCR. (C) MNoV genomes, IFN-β transcripts and IFN-λ transcripts in MLNs 48 hours after inoculation were compared between mice infected with CW3^D94E containing either the CW3 or CR6 capsid gene. (D) MNoV genomes in feces from control or Ifnlr1^−/− mice were measured at day 21 after inoculation with 10⁶ PFU of CW3^D94E. Data in (A) to (C) are pooled from three independent experiments for a total of three to four mice per time point. Data points in (D) are individual mice pooled from three independent experiments. Dashed lines represent limit of detection. Statistical significance determined by two-way ANOVA (A and B) or Mann-Whitney test (C and D). n.s., not significant (P > 0.05); *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001. Error bars in (A) and (B) denote SD.

Figure 3. IFN-λ treatment prevents and cures persistent enteric MNoV infection

(A to C) Feces were collected at the indicated day after oral inoculation and MNoV genomes were quantified by qRT-PCR. (A) Mice were injected with 25 µg of IFN-λ or phosphate-buffered saline (PBS) intraperitoneally 1 day after oral inoculation with 10⁶ PFUs of CR6. (B) The IFN-λ-treated mice from (A) were rechallenged with 10⁶ PFUs of CR6 at day 14 after initial infection. (C) Persistent infection with CR6 was established in control, Ifnar1^−/−, or Ifnlr1^−/−mice followed by intraperitoneal injection of 25 µg of IFN-λ on days 21, 23, and 25. Data shown are pooled from two (A) or three (B and C) independent experiments for a total of four to eight mice per time point. Dashed lines represent limit of detection. Statistical significance was determined by two-way ANOVA. n.s., not significant (P > 0.05); *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001. Error bars in (A) and (B) denote SD and in (C) denote SEM.

Figure 4. IFN-λ durably clears enteric MNoV persistence through effects on radiation-insensitive cells in the absence of adaptive immunity

(A) BMDCs were treated with media, 100 IU/mL IFN-β, or 100 ng/mL IFN-λ for 24 hours, then inoculated with CR6 at a multiplicity of infection of five, and viral titers were determined by plaque assay 12 hours later. Data are pooled from three independent experiments performed in triplicate and normalized to untreated. (B and C) Bone marrow chimeras were generated using control and Ifnlr1^−/− donor and recipient mice as indicated and 8 to 10 weeks later were orally inoculated with 10⁶ PFUs of CR6. (B) MNoV shedding in feces was quantified on day 14. (C) A single 25-µg dose of IFN-λ or PBS was administered intraperitoneally 21 days after inoculation and MNoV shedding was quantified on day 23. Data in (B) and (C) are pooled from two independent experiments and shown as individual mice. (D) Control or Rag1^−/− mice orally inoculated with CR6 were treated 21 days later with a single 25-µg dose of IFN-λ. Shedding was monitored for 35 days postinjection. Dashed lines in (B) to (D) represent limit of detection. Statistical significance was determined by one-way (B) or two-way (A, C, D) ANOVA. n.s., not significant (P > 0.05); *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001. Error bars in (A) denote SEM and in (D) denote SD.