Phylogenetic tree based on the complete VP1 amino acid sequences of 74 sapovirus strains.

<p>These strains represent all reported 14 genogroups (GI-GXIV) and the newly reported rat SaV strains using MEGA 6. The 10 animal SaV strains with additional sequences determined in this study are boxed with dotted lines. Among these, the three SaV strains that were newly identified in this study are also indicated with arrows. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).</p

Genomic characteristics of 50 representative sapovirus strains in 15 genogroups.

<p>Genomic characteristics of 50 representative sapovirus strains in 15 genogroups.</p

Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.

<p>Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.</p

Phylogenetic tree of the putative NS7 amino acid sequences (approx. 500 aa) of sapovirus strains.

<p>These 44 strains represent 12 genogroups (GI-GV, GVI, GVII, GVIII, GIX, GXII, GXIII, and GXIV) using MEGA 6. The amino acid sequences covering from “WKGL” sequence to the end of the putative NS7, “XXME”, were used. Only 44 of the 74 SaV strains in the VP1-tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156373#pone.0156373.g003" target="_blank">Fig 3</a>) have sequences for this region. The 10 strains analyzed in this study are boxed with dotted lines. Among these, the eight SaV strains whose corresponding sequences were determined in this study are indicated with arrows. The NS7 sequences of porcine GIX and GX SaVs, and rat GII and GXV SaVs are not yet available. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).</p

Phylogenetic tree of the full length genomic sequences of 34 sapovirus strains using MEGA 6.

<p>The five strains whose complete genomes were determined in this study are boxed with dotted lines. Among these, the two SaV strains that were newly identified in this study are also indicated with arrows. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).</p

Sapovirus genomic organization: non-structural (NS) and structural proteins VP1 and VP2, and the conserved motifs.

<p>Genomic organization of a common sapovirus, including two open reading frames (ORF1 and ORF2). ORF1 encodes the predicted viral NS proteins (NS1, NS2, NS3 [NTPase], NS4, NS5 [VPg], NS6-NS7 [protease-RNA-dependent RNA polymerase (RdRp)], and the major capsid protein (VP1). ORF2 encodes the minor capsid protein VP2. The following amino acid or nucleotide motifs are conserved among all available sequenced SaVs: GXPGXGKT, PL (N / D) CD, and WDE (F / Y) D of NS3, KGKXX and XDEYXX of NS5, GDCG of NS6, WKGL, KDEL, DYSXWDST, GLPSG, and YGDD of NS7, PPG and WGS of VP1, and the first three nucleotides (GTG) at the 5’–end. Also shown are the first five amino acids of NS1 (M [A / V] S [K / R] P) and around the NS7-VP1 cleavage site (FEME / G, the slash indicates the putative cleavage site by viral protease NS6) that are conserved among GI-GV, GVIII, and GXIII SaVs.</p

Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.

<p>Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.</p

Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.

<p>Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.</p

NoV VLPs were colocalized with each HBGA on the surface of epithelial and goblet cells.

<p>Fresh human ileum biopsy specimens from a single individual (individual B) were incubated with 2.5 µg/ml of NoV VLPs in PBS(-) for 1 h at 4°C and subjected to immunofluorescence microscopy. Cryostat sections were incubated with rabbit anti-Ueno 7k VLP serum and mouse anti-type H1, H2 or Le^b HBGA antibody and stained with Alexa dye–conjugated secondary antibodies and DAPI. Immunofluorescence images for type H1, H2 or Le^b HBGA are shown in panels A, B and C, respectively. Panels d–f and g–i are high magnification views of areas in boxes in panels a–c, respectively. Green, NoV VLPs; red, type H1 HBGA, type H2 HBGA or type Le^b HBGA. Scale bars in panel c = 100 µm, and in panel f and i = 20 µm.</p

Antibodies used in this study.

1<p>rabbit serum immunized with Ueno 7k VLPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066534#pone.0066534-Hansman1" target="_blank">[19]</a>.</p>2<p>rabbit serum immunized with 485 VLPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066534#pone.0066534-Hansman1" target="_blank">[19]</a>.</p