Abstract
Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection. We show that rhesus macaques are susceptible to oral infection with human noroviruses from two different genogroups. Variation in duration of virus shedding (days to weeks) between animals, evolution of the virus over the time of infection, induction of virus-specific adaptive immune responses, susceptibility to reinfection and preferential replication of norovirus in the jejunum of rhesus macaques was similar to infection reported in humans. We found minor pathological signs and changes in epithelial cell surface glycosylation patterns in the small intestine during infection. Detection of viral protein and RNA in intestinal biopsies confirmed the presence of the virus in chromogranin A-expressing epithelial cells, as it does in humans. Thus, rhesus macaques are a promising non-human primate model to evaluate vaccines and therapeutics against norovirus disease.
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Data availability
All data supporting the findings of this study are available within the paper, its Extended Data or Source Data files. Representative microscopy images are included in the main or extended data figures. Additional microscopy image files are available from the corresponding author upon request. Sequence data for human norovirus isolated from rhesus macaques have been deposited in GenBank with accession numbers OR397766–OR397788. Source data are provided with this paper.
Change history
15 February 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41564-024-01636-7
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Acknowledgements
This project was funded by the Intramural Research Programs of the VRC and of NIAID, NIH. We acknowledge the outstanding work of veterinary (A. Cook and K. Guerriero) and research technicians at DVR and Bioqual, Inc., as well as animal programme coordinators C. N. Miller Dulan, J. Graves and A. Silvious, and past coordinators E. McCarthy and H. Bao at the VRC, DVR and BioQual, Inc. for expert animal care. We thank VRC Sequencing Core members J. Roberts-Torres and F. Laboune for technical assistance with sequencing procedures. We thank Roederer and Green laboratory members for discussions.
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M.R. and I.R. conceived and designed experiments. I.R. performed experiments, analysed data and prepared figures. K.D.W. assisted with experiments. B.M.N. prepared biopsies for microscopy. D.A.A. evaluated biopsies by microscopy and assisted with necropsy. A.R.H. and D.C.D. assisted with sequencing support. S.D. and S.G. analysed sequencing data. N.C., S.V.S., K.Y.G., A.S.O., R.V. and P.D.K. provided reagents. J.-P.T. and R.W. provided technical support. K.B., K.Y.G. and M.R. provided conceptual advice. I.R., M.R., K.Y.G., B.M.N., D.A.A., A.R.H., S.G. and A.S.O. wrote, and all authors reviewed the manuscript.
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P.D.K., R.V., M.R. and I.R. have applied for a patent on norovirus stabilized VLPs (application no. PCT/US2021/055018), which were used in ELISA assays of this paper. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Serum responses against a mix of norovirus inoculum in monkeys.
IgM (top panel) and IgG (bottom panel) titers in serum of inoculated monkeys were measured by ELISA against GI.1, GII.3, GII.4 and GII.17 VLPs up to eleven months after the challenge. Data points represent a mean of three technical replicates.
Extended Data Fig. 2 Serum IgA responses to GII.1 norovirus challenge in NHPs.
Serum IgA antibody responses measured to norovirus GII.1 Hawaii virus-like particle in ELISA. Data points represent a mean of two technical replicates.
Extended Data Fig. 3 Histo-blood group antigen phenotype in saliva of challenged animals.
ELISA plates were coated with saliva from different monkeys and HBGA phenotype was characterized using HBGA-specific antibodies. Each color represents a single animal. Leb detection was carried out using two clones, SPM194 and T218, where the latter has cross-reactivity to H-1 HBGA. Data points represent three technical replicates.
Extended Data Fig. 4 Necropsy of norovirus challenged animals.
a, Viral titers per g of stool from animals challenged with NVS3 at 1 × 108 genome copies (gc) per animal. On x axis shown days after inoculation. Animals were euthanized on the following day of the first positive stool sample. NHP #12 was euthanized on day 4, and NHP #13 on day 5 after the challenge. Dotted line indicates viral RNA detection limit at 104 gc/g of stool. Results represent a mean of two technical replicates. b, Detection of viral genome per mg of tissue collected from small and large intestines after the necropsy. Each data point summarizes a mean of two technical replicates.
Extended Data Fig. 5 H-1 reduction after norovirus challenge.
Representative H-1 immunostaining (brown) of jejunal biopsies. Diffuse and strong immunostaining along the brush border of the intestinal mucosal epithelial cells and within the cytoplasm apical to nucleus before the challenge (pre-). At 2 days post-challenge, there is a decrease in H-1 immunostaining intensity along the brush border, but the cytoplasmic immunostaining remains. The immunostaining at 30 days is similar in distribution and intensity to that seen in the pre-challenge biopsy. H-1 IHC, 40x.
Extended Data Fig. 6 Norovirus synonymous mutations in host.
Synonymous mutations found in different regions of human norovirus isolated from several animals during the virus shedding. Each block of rows represents frequency of single nucleotide polymorphisms (snp) found in the virus collected from individual animals. The source of each challenge (ch) is indicated in bold at the top of each block. Isolates that were used in passaging experiments of the shown animals are represented in italic bold. Grey gradient color designates frequency of identified mutations and orange gradient indicates viral titers per g of stool measured at each represented timepoint. Asterisk indicates samples that were amplified before sequencing. Only mutations with >10% detection frequency are shown. ORF1, open reading frame 1; RdRp, RNA-dependent RNA polymerase; VP1/2, viral protein 1/2; nt, nucleotide.
Extended Data Fig. 7 GI.1 norovirus challenge in pre-selected macaques.
a, Norovirus GI.1 genome copies detected in stool collected from animals over the following time after the challenge. Animals received GI.1 norovirus orally at 1 × 108 gc/animal dose. Dotted line indicates viral RNA detection limit at 104 gc/g of stool. Results represent a mean of two technical replicates run twice, and the error bars indicate a standard deviation. b, c, Serum responses of IgM (b) and IgG (c) against GI.1 VLP in ELISA post inoculation (p.i.). Data points represent a mean of two technical replicates. d, e, Chromogenic immunohistochemistry of jejunal biopsies indicating H-1 HBGA presence in brown. Biopsies were collected two weeks before (d) and two days after the challenge (e). f, saliva HBGA phenotype of GI.1 challenged animals. Each color represents a single HBGA. Leb detection was performed using two clones, SPM194 and T218, where the latter has cross-reactivity to H-1 HBGA. Data points represent three technical replicates. Horizontal lines indicate a mean and error bars one standard deviation.
Extended Data Fig. 8 Virus and serum titers in NHP #6.
Data collected from NHP #6 after two challenges (virus stock and dose indicated by orange arrows) is characterized by viral titers found in their stool and by serum antibody titers of IgM and IgG (EP – endpoint). The animal was removed from the study due to observed active H. pylori infection after a challenge. Results represent a mean of three technical replicates, and error bars indicate one standard deviation. Viral RNA detection limit at 104 gc/g of stool.
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Rimkute, I., Chaimongkol, N., Woods, K.D. et al. A non-human primate model for human norovirus infection. Nat Microbiol 9, 776–786 (2024). https://doi.org/10.1038/s41564-023-01585-7
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DOI: https://doi.org/10.1038/s41564-023-01585-7