DNA constructs and transformation of rice plants. ARP1 was selected from a library of VHH fragments generated from llamas immunized with RRV (strain MMU18006, P5B[3], G3) (61), as previously described (29). Briefly, a phage display library was constructed from B lymphocyte cDNA encoding VHHs and selected by biopanning on RRV at a low pH (2.3). ARP1 was previously referred to as 2B10 (29) or VHH1 (30). The gene encoding ARP1 was synthesized with an optimized codon usage for plants and inserted into a binary T-DNA vector (pZH2B/35SNos). This vector contains a cassette for overexpression of ARP1 and a combination cassette for RNAi suppression of production of the major rice endogenous storage proteins, prolamin (13 kDa) and glutelin (Figure 1A and ref. 35). The plasmid was transformed into a japonica variety of rice plants, Nippon-Bare, using a Agrobacterium-mediated method described previously (32).

Protein analyses. Total protein was extracted from transgenic rice plant seeds using a buffer containing 2% (wt/vol) SDS, 8 M urea, 5% (wt/vol) β-mercaptoethanol, 50 mM Tris-HCl (pH 6.8), and 20% (wt/vol) glycerol as previously described (62). The level of ARP1 was determined by Western blot densitometry analysis using purified yeast-derived ARP1 (BAC BV) as a standard. For detection of ARP1, a rabbit anti-ARP1 polyclonal antibody was prepared in our laboratory.

ARP1 was also extracted from MucoRice-ARP1 powder using PBS (rice water) or 8 M urea in PBS at room temperature. After centrifugation, the supernatants were analyzed by SDS-PAGE and Western blot. Purified ARP1 from MucoRice-ARP1 was produced from MucoRice-ARP1 containing rice water, using gel filtration on Sephadex G100 columns (GE Healthcare).

Mass spectrometry. Mass spectrometric analysis was performed as previously described (63). Samples were injected into a nanoflow LC system (Dina; KYA Technologies) and sprayed into a quadrupole time-of-flight tandem mass spectrometer (QSTAR Elite; AB SCIEX). The QSTAR analysis failed to detect 39 amino acids of the C-terminal peptide generated by trypsin because of the large mass (4256 Da) (Supplemental Table 1). To obviate this problem, a linear ion trap Orbitrap mass spectrometer (LTQ Orbitrap Velos; Thermo Fisher Scientific), which enables shotgun proteomics analysis with high resolution and high mass accuracy, was used. The data were analyzed using a Mascot Search Server.

Immune electron microscopy. The distribution of ARP1 in rice seeds was analyzed using immunoelectron microscopy as previously described, with some modification (32). Rice caryopses sections stained with a rabbit anti-ARP1 polyclonal antibody and gold particle–conjugated (18 nm) goat anti-rabbit IgG (Jackson) were examined using transmission electron microscopy (HITACHI). For localization of ARP1 in the seed, a frozen section was stained with rabbit anti-ARP1 polyclonal antibody and visualized using 3,3′-diaminobenzidine.

Binding to RRV by ELISA. ELISA plates were coated with HBC anti-RRV (38) as a capture antibody, followed by RRV (2 × 106 ffu ml–1) and ARP1 (2-fold dilutions between 100 and 12.5 ng ml–1). Biotinylated rabbit anti-VHH K492 antibody (BAC BV), followed by AP conjugated streptavidin (BD Pharmingen), was used for quantification of ARP1 bound to RRV. The assay was developed using para-nitrophenol phosphate (pNPP) (Sigma-Aldrich) as a substrate, and the optical density was read at 405 nm using a Varioskan Flash (Thermo Electron Corporation). HBC was produced by vaccination of pregnant cows in a Swiss dairy farm with human strains of RV, i.e., Wa, RV3, RV5, and ST3, representing serotypes G1 to G4 (38).

In vitro heat stability test. To test the heat stability, samples with 100 ng ml–1 of different ARP1 preparations (MucoRice-ARP1 containing rice water, ARP1 purified from MucoRice-ARP1, and ARP1 purified from yeast) and HBC containing 100 ng ml–1 of total protein were boiled at 100°C for 10, 20, and 30 minutes. After cooling, 2-fold dilutions of each sample were tested in ELISA as described above for the ARP1-containing samples. For the HBC samples, a rabbit anti-RV K230 antiserum (a gift from Lennart Svensson, University of Linköping, Linköping, Sweden) was used as a capture antibody for RRV. The functional anti-RV IgG antibodies contained in the HBC samples were detected using AP-conjugated goat anti-bovine IgG (H+L) (Jackson ImmunoResearch Laboratories). The percentage of binding activity was calculated in relation to nonboiled samples in the same ELISA plate at a particular concentration before reaching binding saturation (25 ng ml–1 ARP1 for ARP1-containing samples and 50 ng ml–1 total protein for HBC samples).

In vitro neutralization assay. In vitro neutralization assays were carried out using MA104 cells and the human RV strains Wa G1P[8], ST-3 G4P[6], 69M G8P[10], F45 G9P[8] and Va70 G4P[8] as previously described (64). Briefly, 105 MA104 cells ml–1 were seeded in 96-well plates. Forty-eight hours later, 2-fold dilutions of antibodies or rice protein preparations were incubated in duplicates with 200 ffu of RVs, and the mixture was subsequently used for infection of the seeded cells. Infected cells were detected by immunofluorescence staining using a monoclonal mouse anti-VP6 antibody (Austral Biologicals) and FITC-conjugated rabbit anti mouse IgG antibodies (Dako). Significant neutralization was defined by a reduction of the infected cells higher than 60% in relation to the control wells.

In vivo assays. Pregnant BALB/c mice were purchased from Japan SLC. Each dam was housed individually with her litter in cages in the same room under negative pressure in the animal facility on a 12-hour light/12-hour dark cycle. Food and deionized water were autoclaved and provided ad libitum.

To determine the level of protection against RRV infection conferred by MucoRice-ARP1 in immunocompetent mice (BALB/c), 4 day-old pups were infected orally using 2 × 107 ffu trypsin-activated RRV (n = 6 to 10 per group). Rice water derived from the supernatant of a mixture of MucoRice-ARP1 powder and PBS, containing a total of 8.5 μg of ARP1, was intragastrically administered to the pups 9 hours prior to RRV inoculation and subsequently given twice daily for 4 consecutive days. In order to examine the heat stability and long-term stability at room temperature, MucoRice-ARP1 heat -treated at 94°C for 10 and 30 minutes or stored at room temperature over 1 year was intragastrically applied to the pups prior to RRV inoculation and subsequently given twice daily for 4 consecutive days. In order to examine the therapeutic effects of MucoRice-ARP1, MucoRice-ARP1 was intragastrically given to the pups 9 hours after RRV inoculation and subsequently given twice daily for 4 consecutive days. In the control groups, PBS or MucoRice-ARP1 was intragastrically administered twice daily for 4 consecutive days without RRV inoculation. The pups were examined daily for evidence of RV-induced diarrhea by gentle abdominal palpation. Diarrhea was recorded and scored blindly from 1 to 4 based on stool color, amount, and consistency as described previously, with a minor modification (65). Normal feces or absence of feces were given a score of 1. Exceptionally loose feces were given a score of 2. Loose yellow-green feces were given a score of 3. Watery feces were given a score of 4. A score of 2 or greater was considered diarrhea, as described previously (65). The percentage of mice with diarrhea for each group was calculated by dividing the number of diarrheic samples by the total number of mice scored for diarrhea each day. The diarrhea severity was determined by dividing the sum of all scores by the number of total number of mice scored for diarrhea each day. Finally, we calculated percentage of mice with diarrhea on a daily basis in each group based on accumulated data after repeated experiments and compared the percentage of diarrhea and the mean diarrhea severity on day 2 after RRV inoculation among groups using the Kruskal-Wallis test followed by the Mann-Whitney test. Samples of small intestine were collected 3 days after infection for histopathological analysis and viral RNA quantification by real-time PCR against VP7 RNA.

Evaluation of histopathology. To evaluate histopathology, small intestinal samples were collected at 3 days after RRV inoculation (n = 3 for each group). Samples from duodenum, jejunum, and ileum were fixed in 4% paraformaldehyde for 12 hours. Subsequently, the samples were transferred to graded ethanol for dehydration, embedded in paraffin wax, and sectioned at 4 μm using a microtome. Sections were stained with H&E (65) and visualized under light microscope. Duodenal, jejunal, or ileal villi were examined for presence of enterocyte injury, inflammation, and vacuolization by a person blinded to the treatment given to the mice.

Quantification of viral RNA. Total RNA was isolated from small intestines of neonatal pups, using TRIzol reagent (Life Technologies) and treated with RNase-free DNase (QIAGEN) following the manufacture’s protocol. To evaluate viral shedding in the chronic RRV infection SCID mouse model, fecal specimens were prepared as 10% suspensions with PBS, and total RNA was isolated using TRIzol following the manufacturer’s protocol. RV VP7 mRNA or viral genomic RNA was amplified at 58°C in the presence of 600 nmol l–1 primers, 300 nmol l–1 probe, and 5 mmol l–1 Mn to generate a 121-bp–long amplicon. The sense primer (VP7 forward, 5′-CCAAGGGAAAATGTAGCAGTAATTC-3′; nt 791-815), the antisense primer (VP7 reverse, 5′-TGCCACCATTCTTTCCAATTAA-3′; nt 891-912), and the probe (5′-6FAMTAACGGCTGATCCAACCACAGCACCTAMRA-3′; nt 843-867) were designed on the basis of the VP7 gene sequence of RRV (GenBank AF295303). Reverse transcription reactions were carried out in a final volume of 20 μl using Superscript III Reverse Transcriptase (Invitrogen) following the manufacturer’s protocol. Each RT reaction sample was analyzed by the Light-Cycler 480 System II (Roche Applied Science) following the manufacturer’s protocol. A standard curve was generated using a plasmid that contained a RRV VP7 gene, and the lowest level of detection of the PCR was 10 viral RNA copies. The presence of less than 10 copies of VP7 RNA per weight (mg) was defined as clearance of infection. The ratio of VP7 gene copy number to the weight of the stool sample (mg) or to the weight of the small intestine segment (mg) was compared among the groups.

In vivo assays in SCID mice. The efficacy of MucoRice-ARP1 against chronic RRV infection was determined in SCID mice. Pregnant C.B-17 SCID/SCID mice were purchased from Nihon Clea Inc. Four-day-old pups were infected orally with 2 × 107 ffu RRV. All of the mice developed diarrhea and chronic infection. The litters were weaned by removing each mouse from the dam at 21 days of age. After inoculation, all mice were confirmed to have low serum immunoglobulin levels by ELISA and examined for pathogen surveillance, but no specific pathogen, including murine norovirus, was found at 5 weeks of age. When mice were 6 weeks old (i.e., 6 weeks after RRV infection), 200 mg of MucoRice-ARP1 powder (containing 1.7 mg of ARP1) or nontransformed WT rice powder was intragastrically administered twice daily for 7 consecutive days. Mice were examined on days –1, 1, 3, 9, and 14 for diarrhea and viral shedding. Diarrhea scores, percentage of mice with diarrhea, and disease severity were measured as described above. Viral shedding was measured by VP7-specific real-time PCR using fecal samples. In order to examine the therapeutic effects of MucoRice-ARP1 in SCID mouse pups, MucoRice-ARP1 was intragastrically given to the pups 9 hours after RRV inoculation and subsequently given twice daily for 4 consecutive days. Diarrhea scores, percentage of animals with diarrhea, and disease severity were measured as described above.

Statistics. Individual data for the percentage of mice with diarrhea, the disease severity scores, and differences in the intestinal virus load as assessed by real-time PCR were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney test. P < 0.05 was considered statistically significant.

Study approval. All mouse experiments were approved by the local ethics committee of the Institute of the Medical Science at the University of Tokyo.