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Age-associated features of norovirus infection analysed in mice

Nature Microbiology volume 8, pages 1095–1107 (2023)Cite this article

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Abstract

Norovirus (NoV) is the leading global cause of viral gastroenteritis. Young children bear the highest burden of disease and play a key role in viral transmission throughout the population. However, which host factors contribute to age-associated variability in NoV severity and shedding are not well-defined. The murine NoV (MNoV) strain CR6 causes persistent infection in adult mice and targets intestinal tuft cells. Here we find that natural transmission of CR6 from infected dams occurred only in juvenile mice. Direct oral CR6 inoculation of wild-type neonatal mice led to accumulation of viral RNA in the ileum and prolonged shedding in the stool that was replication-independent. This viral exposure induced both innate and adaptive immune responses including interferon-stimulated gene expression and MNoV-specific antibody responses. Interestingly, viral uptake depended on passive ileal absorption of luminal virus, a process blocked by cortisone acetate administration, which prevented ileal viral RNA accumulation. Neonates lacking interferon signalling in haematopoietic cells were susceptible to productive infection, viral dissemination and lethality, which depended on the canonical MNoV receptor CD300LF. Together, our findings reveal developmentally associated aspects of persistent MNoV infection, including distinct tissue and cellular tropism, mechanisms of interferon regulation and severity of infection in the absence of interferon signalling. These emphasize the importance of defining viral pathogenesis phenotypes across the developmental spectrum and highlight passive viral uptake as an important contributor to enteric infections in early life.

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Fig. 1: Natural transmission of persistent MNoV from infected dams occurs after P16.
Fig. 2: Inoculation of pups with persistent MNoV is associated with distinct localization.
Fig. 3: Tuft cells and CD300 family viral receptors have a limited role in neonatal viral RNA shedding.
Fig. 4: STAT1 signalling protects against lethality and extraintestinal CR6 spread in neonatal mice.
Fig. 5: Persistent MNoV replicates in neonates in the absence of STAT1 signalling.
Fig. 6: Non-specific uptake leads to the accumulation of persistent MNoV RNA in neonatal small intestines.

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Materials availability

All reagents are available from M.T.B. under a material transfer agreement with Washington University.

Data availability

Data from this study are included in the main paper and in Extended Data. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Baldridge laboratory for helpful discussions, and specifically H. Deng and L. Foster for assistance with mouse colony maintenance; and R. Orchard (University of Texas Southwestern Medical Center) for providing Fc-CD300lf complexes. We are grateful to Kim Green (NIAID, NIH) for providing anti-NS6/7 antibody. We are grateful to Michael Aguet (ISREC - School of Life Sciences Ecole Polytechnique Fédérale de Lausanne) for providing Ifnar1−/− mice. This work was supported by the National Institutes of Health (NIH) grants R01AI141478 (S.M.K., M.T.B.), R01AI139314 and R01AI127552 (M.T.B.), R01A1148467 (C.B.W.) and F31AI167499-01 (E.A.K.), as well as the Burroughs Wellcome Fund (C.B.W.), National Science Foundation DGE-1745038/DGE-2139839 (E.A.K.), and the Pew Biomedical Scholars Program of the Pew Charitable Trusts (M.T.B.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Division of Infectious Diseases, Department of Medicine, Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St Louis, MO, USA

    Elizabeth A. Kennedy, Somya Aggarwal, Arko Dhar & Megan T. Baldridge

  2. Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA

    Stephanie M. Karst

  3. Departments of Laboratory Medicine and Immunobiology, Yale School of Medicine, New Haven, CT, USA

    Craig B. Wilen

  4. Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA

    Megan T. Baldridge

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  1. Elizabeth A. Kennedy
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Contributions

E.A.K., A.D. and S.A. performed the experiments. E.A.K. and M.T.B. analysed the results. E.A.K., S.M.K., C.B.W. and M.T.B. designed the project. E.A.K. and M.T.B. wrote the manuscript. All authors read and edited the manuscript.

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Correspondence to Megan T. Baldridge.

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Nature Microbiology thanks Nihal Altan-Bonnet, Jason Mackenzie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Source and genetic background of mice influences persistent MNoV shedding as neonates and adults.

(A) C57BL/6 neonates bred at Washington University in St. Louis (WUSTL), Charles River (CR), or Jackson Laboratories (JAX) were orally inoculated with CR6 at P6 and MNoV genome copies detected in 7dpi stool samples by RT-qPCR. (B) Neonates on the indicated backgrounds, all sourced from JAX, were orally inoculated with CR6 at P6 and MNoV genome copies detected in 7dpi stool samples by RT-qPCR. (C, D) Neonates on the indicated background, sourced from CR, were orally inoculated with CR6 at P6 and MNoV genome copies detected in 7dpi samples by RT-qPCR in stool (C) and tissues (D). (E, F) Adult mice on the indicated background, sourced from CR, were orally inoculated with CR6 and MNoV genome copies detected in 10dpi samples by RT-qPCR in stool (E) and tissues (F). Analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test (A, B), two-tailed t-test (C) two-tailed Mann-Whitney test (D-F) corrected with the Holm-Šídák method (D, F). (A) WUSTL (n = 15, 4 litters), CR (n = 16, 3 litters), JAX (n = 10, 2 litters), *p = 0.0427, **p = 0.0037. (B) C57Bl/6 (n = 10, 2 litters), BALB/c (n = 4, 2 litters), A/J (n = 10, 3 litters), NOD (n = 10, 3 litters), 129S1 (n = 9, 2 litters), PWK/PhJ (n = 6, 2 litters), **p = 0.0023 (C57BL/6 vs. A/J), **p = 0.0012 (C57BL/6 vs. 129S1), ***p = 0.0003. (C) C57Bl/6 (n = 16, 3 litters), BALB/c (n = 8, 2 litters). (D) C57Bl/6 (n = 12, 2 litters), BALB/c (n = 10, 2 litters). ***p = 0.0001. (E) N = 10 mice per group from 2 independent experiments, **p = 0.0015. (F) N = 4 mice per group from one experiment, p = 0.0563 (ileum), p = 0.0571 (colon). Dashed lines indicate limit of detection for assays. ns, not significant (p > 0.05).

Source data

Extended Data Fig. 2 Tuft cells and Cd300lf expression in early life.

(A) Representative images of tuft cells quantified by immunofluorescent staining of DCLK1 in naïve C57BL/6 mice (quantified data shown in Fig. 3C). (B) Cd300lf was quantified in intestinal samples from naïve C57BL/6 mice by RT-qPCR at the indicated timepoints. P6 (n = 6, 1 litter), P13 (n = 4, 2 litters), P21 (n = 4, 2 litters), adult (n = 7, 2 experiments), analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test. *p = 0.0183, **p = 0.0087. Dashed lines indicate limit of detection for assay. ns, not significant (p > 0.05).

Source data

Extended Data Fig. 3 Mouse but not human Fc-CD300LF blocks CR6 infection in vitro.

CR6 was incubated with Fc-fusion proteins with either the human or mouse CD300LF ectodomains for 1 hour at 37 C, prior to infection of BV2 cells at an MOI of 0.05. Cell viability was measured by CellTiter Glo 48 hours post-infection. 3 technical replicates from a single experiment.

Source data

Extended Data Fig. 4 CR6 induces ISG expression in the ileum.

Wild-type neonates were orally inoculated with CR6 at P6. Mx2 was quantified by RT-qPCR from ilea collected at 1 dpi (A) or 7 dpi (B) and compared to naïve neonates. (C) Ifit1 expression was quantified in the ileum and colon of CR6-inoculated neonates at 7dpi. (A) Naïve (n = 5, 1 litter), infected (n = 6, 3 litters), analyzed by Welch’s two-tailed t-test, *p = 0.0289. (B) Naïve (n = 5, 2 litters), infected (n = 13, 4 litters), analyzed by two-tailed t-test, **p = 0.0188. (C) N = 12 pups from 2 litters sourced from Charles River, analyzed by two-tailed Mann-Whitney test, ****p < 0.0001.

Source data

Extended Data Fig. 5 2-CMC blocks CR6 shedding in wild-type adult mice.

Wild-type adult mice were orally inoculated with CR6. 100 mg/kg 2-CMC or PBS were injected subcutaneously daily at 7-9dpi. Stool was collected from 7-11dpi (A) and tissues at 11dpi (B), and MNoV genome copies quantified by RT-qPCR. (A) PBS (n = 4), 2-CMC (n = 6), from two experiments. Analyzed by two-tailed Mann-Whitney test corrected with the Holm-Šídák method, *p = 0.0376 (9dpi, 10dpi), *p = 0.0236 (11dpi). (B) PBS (n = 2), 2-CMC (n = 4), from one experiment. Dashed lines indicate limit of detection for assays. ns, not significant.

Source data

Extended Data Fig. 6 CR6 replicates in the spleens of Stat1-/- neonates.

(A) Wild-type neonates (P6) were orally inoculated with CR6. Infectious virus from 1-2dpi stool was quantified by plaque assay on BV2 cells. n = 2 stools collected per time point. (B) Additional replicates of spleens from CR6-inoculated Stat1-/- neonates, stained as in Fig. 5J. (C) Spleen from a naïve neonate stained as in Fig. 5J. (D) 2’ only antibody control for spleen in Fig. 5J.

Source data

Extended Data Fig. 7 Evans blue dye is retained in the distal small intestine of neonatal mice.

(A) Adult mice were gavaged with 400ul Evans blue dye and stool collected at 4- and 24-hours post-gavage. (B) Neonates were gavaged at P6 with 50ul Evans blue dye and stool collected at 24- and 72-hours post-gavage. Stool was resuspended in PBS for assessment of blue colour. Representative of 3 adults from one experiment and 8 neonates from three litters. (C) Neonates were gavaged at P6 with 50ul Evans blue dye and treated with 0.5 mg/g cortisone acetate or vehicle. Intestines were collected at 7dpi post-gavage and assessed for blue colour across the length of the intestines. Representative of 3 cortisone acetate and 3 vehicle-treated neonates from two litters. (D-F) Ileal samples were collected from naïve mice at P6, P13, P21, and as adults (left) or at 7dpi (P13) from littermates inoculated with CR6 and treated subcutaneously with 0.5 mg/g cortisone acetate or vehicle at P6 (right). Naga (D), Prdm1 (E), and Sis (F) expression was quantified by RT-qPCR. P6 (n = 4,1. litter), P13 (n = 4, 2 litters), P21 (n = 3, 2 litters), adult (n = 3, 1 experiment), 7dpi cortisone acetate (n = 5, 2 litters), 7dpi vehicle (n = 5, 2 litters). Naïve time course analyzed by Welch’s ANOVA test with Dunnett’s T3 multiple comparisons test (D, E, time course), ANOVA with Dunnett’s multiple comparisons test (F, time course), two-tailed Mann-Whitney test (D, F, vehicle vs. cortisone acetate), or Welch’s two-tailed t-test (E, vehicle vs cortisone acetate). (D) *p = 0.0295 (P6 vs. P21), *p = 0.0298 (P6 vs. adult), **p = 0.0079 (vehicle vs. cortisone acetate). (E) *p = 0.0399 (P6 vs. P21), *p = 0.0245 (P6 vs. adult), ***p = 0.0003 (vehicle vs. cortisone acetate). (F) ****p < 0.0001 (P6 vs. P21, P6 vs. adult), **p = 0.0079 (vehicle vs. cortisone acetate). ns, not significant (p > 0.05).

Source data

Extended Data Fig. 8 CR6 uptake is localized to the distal small intestine.

Ilea stained for Epcam and CR6 RNA. All replicates collected are shown. (A) Additional replicates of distal small intestinal sections from neonates inoculated with CR6 at P6, collected at 4 or 8hpi. (B) Proximal small intestinal sections collected at 8hpi. (C) Distal small intestinal sections from naïve neonates. Scale bars are 100μm long.

Extended Data Fig. 9 Cortisone acetate treatment does not affect CR6 infection in adult mice.

Wild-type adult mice were orally inoculated with CR6 and treated subcutaneously with 0.5 mg/g cortisone acetate or vehicle. Stool (A) and tissue (B) virus levels were quantified by RT-qPCR at 7dpi. Vehicle (n = 5) and cortisone acetate (n = 5), from two independent experiments. Analyzed by Welch’s two-tailed t-test (A) and two-tailed Mann-Whitney test corrected with the Holm-Šídák method (B). Dashed lines indicate limit of detection for assays. ns, not significant.

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Kennedy, E.A., Aggarwal, S., Dhar, A. et al. Age-associated features of norovirus infection analysed in mice. Nat Microbiol 8, 1095–1107 (2023). https://doi.org/10.1038/s41564-023-01383-1

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