Supplemental Methods s3

1 Supplemental Methods

2 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 Laboratory

16 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 mesenteric

37 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 and

44 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 on

46 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