Supplemental Methods s3

1 Supplemental Methods2 HuNoV samples. Ten human stool samples from confirmed HuNoV outbreaks 3 were obtained from the Michigan Department of Community Health. Samples were 4 stored at -80˚C. HuNoV genomes titers in individual human stool samples were 5 measured by a multiplex quantitative Real-Time PCR (qRT-PCR) specific for GI and 6 GII, and virus strains were genotyped by sequencing regions in the RdRp (ORF1) and 7 the ORF1/2 junction [1]. Human stool suspensions (10 % w/v) were prepared in 8 phosphate buffered saline (PBS, pH 7.4; Gibco Life Technologies) and clarified by 9 centrifugation. Inocula for mouse infections were prepared by mixing equal volumes of 10 GI- and GII-positive human stool suspensions (i.e., GI+II mix), or GII-positive human 11 stool suspensions (i.e., GII mix). HuNoV genomes in the pooled inoculum were then 12 back-titered by qRT-PCR each time an experiment was performed to determine the 13 genome titer used to infect animals.14 Mice. All animal studies described herein were performed in accordance with 15 local and federal guidelines as outlined in the “Guide for the Care and Use of Laboratory16 Animals” of the National Institutes of Health. Protocols were approved by the University 17 of Michigan Committee on Use and Care of Animals (UCUCA Number: 09710) and by 18 Colorado State University's Institutional Animal Care and Use Committee (IACUC 19 Number: 12-3829A). Six to seven week old wild-type Balb/c (000651) and four to eight 20 week old Rag-/-c-/- mice on a B6/B10 background (4111) were purchased from Jackson 21 Laboratories and Taconic Farms, respectively. Rag-/-c-/- mice on a Balb/c background 22 were bred and housed at Colorado State University and University of Michigan. 23 Humanized mice were generated as previously described [2] at Colorado State 24 University. For the original experiments, humanized and non-humanized mice were 25 shipped to University of Michigan for infection experiments. For later experiments, a 26 colony of Rag-/-c-/- mice on a Balb/c background was established at the University of 27 Michigan. Tissues and feces from mice in this colony were tested by qRT-PCR for MNV 28 as described [3] and shown to be negative.29 Infection of mice. Mice were housed individually in wire bottom cages. All 30 murine feces was collected throughout the 12 hours before and at 12 - 24 h intervals 31 after infection and frozen in 10 ml PBS. Mice were infected intraperitoneally with a 0.2 32 ml sterile-filtered human stool suspension and/or orally with a 0.05 ml clarified, 33 unfiltered human stool suspension.34 Mice were humanely euthanized according to approved protocol. The following tissues 35 were harvested: half of the heart, brain, spleen, stomach, one kidney, 36 duodenum/jejunum, proximal ileum, distal ileum, cecum, and colon, all of the mesenteric37 lymph nodes and kidney, 1 lobe of the lung, a quarter of liver with the gall bladder, and 38 1 femur to extract bone marrow. Samples were homogenized in 0.5 ml PBS with 500 39 mg of 1.0 mm silica beads (BioSpec) using a MagNA Lyser (Roche) for 1 min at 6,000 40 rpm, centrifuged at 12,000 rpm using tabletop centrifuge 5424 (Eppendorf) for 10 min, 41 and stored at -80˚C. Remaining tissue pieces were fixed for histopathology.42 Quantification/typing of HuNoV genomes by qRT-PCR. Total genomic RNA 43 was extracted from 0.14 ml clarified murine fecal suspensions, tissue homogenates and44 inoculum samples (clarified and mixed human stool suspensions) using the QIAamp 45 Viral RNA Mini Kit (Qiagen). Quantitative RT-PCR of HuNoV genome was performed on46 fecal and tissue samples with 2 μl RNA extract using QuantiTect Probe RT-PCR Kit 47 (Qiagen). Genome quantification was also performed under certified diagnostic 48 conditions at the Consultant Laboratory for Noroviruses at the Robert Koch-Institute 49 using primers and probes specific to the ORF1/2 junction of GI and GII [1]. The limit of 50 detection of the Taqman is between 1-10 genomes per reaction volume or 214-2140 51 genomes/ml. The qRT-PCR procedure was certified according to DIN EN ISO 15189 52 and DIN EN ISO/IEC 17025 for diagnostic purposes and exhibits an intra-assay 53 coefficient of variation of 0.15 - 1.94 % and an inter-assay variation of 0.41 - 2.34 %. 54 Sensitivity and specificity was 100% in an inter-laboratory test using blinded samples. 55 For genotyping of fecal samples, region A and C [4] were amplified by OneStep 56 RT-PCR and HotStarTaq Master Mix Kit (Qiagen), sequenced directly using the BigDye 57 terminator cycle sequencing kit (Applied Biosystems), and analyzed using the norovirus 58 typing tool [5]. Tissue samples were not subjected to genotyping.59 Determination of fold increase. Inoculum titers were determined for each 60 experiment after back-titration of the human stool suspensions by qRT-PCR. For qRT-61 PCR titers, a factor of 214.29 was multiplied per tissue to obtain genomes per ml 62 homogenate. This value was then multiplied by 1 for mesenteric lymph nodes and 63 kidney, by 4 for liver, and by 2 for the remaining tissues to obtain total genomes in each 64 tissue. For murine feces the value was multiplied by 10. Total genome copies per 65 mouse were calculated by adding genome copies in all tissues and feces. Fold change 66 was determined by dividing total genome copies by inoculum genome copies.67 Nucleic acid preparation and 454 pyrosequencing. Total nucleic acid was 68 isolated from 0.2 ml of clarified human stool filtrate (6 % w/v) using Ampliprep DNA 69 extraction machine (Roche) according to manufacturer's instructions. Total nucleic acid 70 from each sample was reverse transcribed and amplified as described [6]. Amplification 71 products were pooled, adaptor-ligated and sequenced at the Washington University 72 Genome Sequencing Center on the 454 GS-FLX platform (454 Life Sciences).73 A genome sequence of HuNoV GII was obtained from mouse feces 48 hours post-74 infection using Sanger sequencing of PCR amplicons (Genbank# KC631815). This 75 genome sequence was used as a reference for mapping of reads obtained from the 76 human stools of patient #9 and #10 (Table S1) using the extraction and amplification 77 protocol described above. Read mapping was performed using gsMapper (Roche). 78 Default settings were used to map and identify high-confidence variants. Sequences 79 spanning the entire genome were obtained for human stool #9, while human stool #10 80 was incomplete. Capsid genes from human stools #9 and #10 were also sequenced by 81 the Sanger method and alignments indicated they were identical.82 Production and purification of HuNoV VLPs. Recombinant capsid monomers 83 of GII.7 (Genbank# KC832474) were produced by the Gateway® Technology and 84 expressed using the BaculoDirect™ Baculovirus Expression System (Invitrogen, 85 Carlsbad, USA) according to the manufacturer’s instruction. Briefly, amplification 86 products of complete open reading frame 2 and 3 of GII.7 HuNoV was cloned into the 87 Gateway® pENTR 1A vector and subsequently transferred to BaculoDirect linear DNA 88 by LR recombination. High titer viral stocks were generated in recombinant baculovirus-89 transformed Spodoptera frugiperda ovarian cells (Sf9) using complete Grace’s Insect 90 Medium (Invitrogen). For expression of capsid monomers, High Five™ cells maintained 91 in serum-free Express Five® SFM medium (Invitrogen) in suspension culture were 92 infected with recombinant baculovirus at a multiplicity of infection (MOI) of 10, and cell 93 culture supernatant and infected cells were harvested at day 7 post inoculation. VLPs 94 were purified from supernatant and cell lysate by ultracentrifugation through a 30 % 95 (w/v) sucrose cushion in TEN buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 0.1 M NaCl), 96 followed by isopycnic potassium tartrate-glycerol density gradient ultracentrifugation [7]. 97 VLP purity was analyzed by SDS-PAGE, Western blotting, and electron microscopy. 98 Protein concentration was determined using Bradford reagent (Sigma Chemical, USA) 99 and photometry at 280 nm.100 Generation of antibodies. Anti-VLP antibodies were made at Cocalico 101 Biologicals, Inc., Reamstown, PA, USA, in rabbits following standard protocols. Non-102 structural antibodies were generated against conserved antigenic peptide sequences of 103 all non-structural proteins NS1 – 7 at GenScript USA Inc., Piscataway, NJ, USA, in 104 rabbits following standard protocols. Rabbit polyclonal anti-nonstructural peptide 105 antibodies were then affinity purified at GenScript USA Inc. Only sera that were raised 106 against peptides from NS4 (RVGRQLKDVRTMPEC) and NS6 (CSNAKSMDLGTTPGD)107 showed low non-specific staining and thus were used in the experiments. Pre-immune 108 sera were collected from each rabbit used to generate each antibody.109 Histopathology and Immunohistochemistry. Mouse tissues were harvested 110 and fixed in 10 % buffered formalin (Fisher Scientific) for 24 h. Tissues were embedded 111 in paraffin and processed at the University of Michigan Pathology Core for Animal 112 Research following standard histological procedures. Tissues were subjected to 113 hematoxylin and eosin stain for histopathological examinations. Sodium citrate buffer 114 was used for antigen retrieval prior to performing immunohistochemistry as described 115 previously [8] with the following modifications. Tissue sections were stained with a 116 1:5000 dilution of anti-HuNoV GII.7 VLP rabbit polyclonal antibody or the corresponding 117 pre-bleed rabbit serum, 1:1000 dilutions of the GII.4 NS4- or NS6-specific antisera or 118 the corresponding pre-immune sera, and 1:1000 dilution of anti-MNV VLP (strain S99) 119 rabbit polyclonal antibody or the corresponding pre-bleed rabbit serum.120 121 122 References: 123 124 1. Hoehne, M. and E. Schreier, Detection of Norovirus genogroup I and II by 125 multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe. BMC 126 Infect Dis, 2006. 6: p. 69. 127 2. Berges, B.K., et al., HIV-1 infection and CD4 T cell depletion in the humanized 128 Rag2-/-gamma c-/- (RAG-hu) mouse model. Retrovirology, 2006. 3: p. 76. 129 3. Taube, S., et al., Ganglioside-linked terminal sialic acid moieties on murine 130 macrophages function as attachment receptors for murine noroviruses. J Virol, 131 2009. 83(9): p. 4092-101. 132 4. Mattison, K., et al., Multicenter comparison of two norovirus ORF2-based 133 genotyping protocols. J Clin Microbiol, 2009. 47(12): p. 3927-32. 134 5. Kroneman, A., et al., An automated genotyping tool for enteroviruses and 135 noroviruses. J Clin Virol, 2011. 51(2): p. 121-5. 136 6. Wang, D., et al., Viral discovery and sequence recovery using DNA microarrays. 137 PLoS Biol, 2003. 1(2): p. E2. 138 7. Ashley, C.R. and E.O. Caul, Potassium tartrate-glycerol as a density gradient 139 substrate for separation of small, round viruses from human feces. J Clin 140 Microbiol, 1982. 16(2): p. 377-81. 141 8. Zhang, J., et al., Expression and sub-cellular localization of the CCAAT/enhancer 142 binding protein alpha in relation to postnatal development and malignancy of the 143 prostate. Prostate, 2008. 68(11): p. 1206-14. 144 145

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