Dual Recognition of Sialic Acid and αGal Epitopes by the VP8* Domains of the Bovine Rotavirus G6P[5] WC3 and of Its Mono-reassortant G4P[5] RotaTeq Vaccine Strains

Figures

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  FIG 1. Full-length VP8* amino acid sequence and phylogenetic analyses. (A) Structural features of the VP8* domain of the spike protein VP4 shows the VP8* (left) and VP5* (right) domains with trypsin cleavage sites. The VP8* domains of the original GenBank-deposited WC3 (G6P[5] WC3 marked by a superscript 1) and RotaTeq (G4P[5] RotaTeq marked by a superscript 3) strains, and G6P[5] (marked by a superscript 2) and G4P[5] RotaTeq (marked by a superscript 4) strains used in this study, and control G3P[3] RRV and G8P[14] HAL1166 strains were 231 amino acids in length. The VP8* hemagglutination (HA) domain (amino acids 93 to 208) is shown in the schematic diagram. In the multiple sequence alignment, the amino acids at the sialic acid (SA) binding site of P[I] strains (see panel B) are marked with red asterisks (amino acids 101, 144, 146, 155, and 187 to 190). Amino acids at the blood group A binding site of P[III] strains are marked with blue asterisks (amino acids 101 and 187 to 190). The information regarding the SA and HBGA binding sites is from Isa et al. (26) and Hu et al. (27). The full-length VP8* amino acid sequences of the bovine G6P[5] WC3 strain and bovine-human mono-reassortant…

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  FIG 2. Full-length VP7 amino acid sequence. The VP7 amino acids of the original GenBank-deposited WC3 (G6P[5] WC3 marked by a superscript 1) and RotaTeq (G4P[5] RotaTeq marked by a superscript 3) strains, and G6P[5] (marked by a superscript 2) and G4P[5] RotaTeq (marked by a superscript 4) strains used in this study were 326 amino acids in length. Comparison of deduced amino acid sequences of VP7 gene between the current WC3 and original GenBank database-deposited WC3 strains showed that three amino acid substitutions were found at amino acids 192 (N192T), 295 (C296V), and 310 (V310M). The full-length VP7 amino acid sequences of both current and original GenBank database-deposited G4P[5] strains were identical.

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  FIG 3. Binding of the recombinant VP8* domain of bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains to synthetic histo-blood group antigens (HBGAs) and CHO cells after GGTA1 transfection. (A) The binding abilities of the recombinant GST- VP8* domains corresponding to P[5], P[1], P[3], and P[25] RVA strains and the GST-P domain of human norovirus strain VA387 (GII.4) were determined using horseradish peroxidase (HRP)-conjugated streptavidin. HBGA binding was visualized using TMB, which was measured at 450 nm in three independent experiments. Error bars represent means ± the standard deviations (SD). (B) The αGal epitope was removed from the synthetic Galα3Galβ4GlcNAcβ HBGA using α-galactosidase. The binding specificity to the VP8* domain of the bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains and to P particles of the human norovirus VA387 strain was determined using an HBGA binding assay. (C) Mock-transfected parent CHO cells or GGTA1-transfected CHO cells were coincubated with FITC-conjugated anti-αGal MAb and AF594-labeled VP8* proteins of either G6P[5] WC3 or G4P[5] RotaTeq vaccine strains. No labeling was visible on mock-transfected CHO cells that…

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  FIG 4. Binding of recombinant VP8* domain of bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains to saliva. (A) Binding of the GST-VP8* domains was tested on a panel of human, bovine, and porcine saliva samples, using an anti-GST antibody (1:1,000 dilution), followed by the addition of an HRP-conjugated goat anti-mouse IgG antibody. The binding results to individual saliva samples were sorted by the αGal signals of individual saliva samples. A trend for correlation between the salivary αGal signals and VP8* binding levels for both P[5]-bearing strains was observed. Capital letters H, P, and B on the x axis identifies human (indicated by yellow bars), pig (indicated by blue bars), and cow (indicated by green bars) samples, respectively. (B) GST-P domain of norovirus strain VP387 (GII0.4) was tested as a positive control for binding to a panel of saliva samples from 54 human individuals. The A and B type signals of each individual saliva sample were sorted particularly based on the stronger intensity of A type signal, and the binding activity of the P domain to each individual saliva sample was plotted on a graph. A trend for correlation with the salivary A and B signals…

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  FIG 5. Binding activity of P[5] VP8* domains and the amount of Lewisy, H, and A HBGAs in saliva. Binding of the GST-VP8* domains was tested on a panel of human (capital letter H on the x axis, indicated by yellow bars), bovine (capital letter B on the x axis, indicated by green bars), and porcine (capital letter P on the x axis, indicated by blue bars) saliva samples, using an anti-GST antibody (1:1,000 dilution), followed by the addition of an HRP-conjugated goat anti-mouse IgG antibody. (A to C) The binding results for individual saliva samples were sorted by their Ley (A), H (B), and A (C) signals. No correlation was observed between the salivary Ley, H, and A signals and VP8* binding levels in either P[5]-bearing strain. Binding of saliva samples was visualized using TMB, which was measured at 450 nm in three independent experiments. Error bars represent means ± the SD.

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  FIG 6. Expression of the αGal epitope and infectivity of the bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains in various cell lines. (A) The expression level of the αGal epitope in Old World monkey (MA104), bovine (MDBK), human (Caco-2), and porcine (LLC-PK) cells was determined using an immunofluorescence assay with MAbs specific to the αGal epitope. Scale bars, 20 μm. (B) The infectivity of both P[5]-bearing strains (MOI, 1 FFU/cell) in the cell lines was assessed using an immunofluorescence assay with MAbs specific to the RVA VP6 protein at 15 h postinfection. All experiments were performed on three independent occasions. Scale bars, 200 μm. Panels A and B show representative images.

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  FIG 7. Infection of the bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains requires a terminal SA. (A) The effect of pretreatment of permissive MA104 cells with neuraminidase from Vibrio cholerae on the infectivity of both P[5]-bearing RVA strains (MOI, 1 FFU/cell) was assessed using an immunofluorescence assay with MAbs specific to the RVA VP6 protein. Panel A shows one representative set of results. (B) The number of RVA antigen-positive cells following NA treatment, expressed as a percentage of mock-treated virus-infected cells, was quantified from three independent fields of view. All experiments were performed on three independent occasions. Scale bars, 200 μm. Error bars represent means ± the SD from triplicate experiments. ***, P < 0.001.

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  FIG 8. Infectivity of the bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains is blocked by NANA, NGNA, or αGal. (A) The effect of preincubation of MA-104 cells with NANA, NGNA, or αGal on the infectivity of the bovine G6P[5] WC3 strain (MOI, 1 FFU/cell) was assessed using an immunofluorescence assay with MAbs specific for the RVA VP6 protein. (B) The effect of preincubation of MA-104 cells with NANA, NGNA, or αGal on the infectivity of the bovine-human mono-reassortant G4P[5] strain (MOI, 1 FFU/cell) was assessed using an immunofluorescence assay with MAbs specific for the RVA VP6 protein. (C) The number of infected MA-104 cells with both P[5]-bearing RVA strains was expressed as a percentage of the mock-treated, virus-infected control. All experiments were performed on three independent occasions. Panels A and B show representative sets of results. The scale bars in panels A and B indicate 200 μm. Error bars in panel C represent the means ± the SD from triplicate experiments. ***, P < 0.001.

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  FIG 9. Characterization of bovine ileal enteroids. (A) Representative images of sequential bovine ileal enteroids from intestinal crypts that were grown for 1, 3, or 5 days in complete medium with growth factors. (B) After 10 days of growth in complete medium with growth factors, enteroids typically had two major morphologies: cystic (left) and multilobular (right). (C and D) Differentiated bovine ileal enteroids (3D) grown in Matrigel with a differentiation medium lacking Wnt3A, SB202190, and nicotinamide and with half the normal concentration of Noggin and R-spondin, were fixed with 4% paraformaldehyde and embedded in paraffin. (C) Thin sections were stained with H&E (upper panel), and periodic acid-Schiff (PAS, lower panel). (D) Thin sections were stained using immunohistochemistry to determine the expression levels of villin for enterocytes, chromogranin A for enteroendocrine cells, and mucin 2 for goblet cells. Nuclei were stained with DAPI.

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  FIG 10. Expression of α2,6-linked SA and αGal in differentiated human and bovine jejunal enteroids and human small intestinal sections. (A) Differentiated human jejunal and bovine ileal enteroids grown in Matrigel with a differentiation medium were fixed with 4% paraformaldehyde. Thin sections of differentiated HIEs and BIEs were stained using immunohistochemistry to determine the expression levels of α2,6-linked sialic acids and the αGal epitope. Nuclei were stained with DAPI. Representative images are shown. (B) Detection of α2,6-linked SAs in the human small intestine. Frozen human duodenal tissue sections were fixed with 10% formalin and incubated with biotin-labeled SNL at 10 μg/ml in PBS, followed by incubation with DyLight 488-conjugated streptavidin (green) in the same buffer (α2,6-SA). Nuclei were stained with DAPI (blue). To determine the SA dependence of the labeling, tissue sections were pretreated with neuraminidase for 4 h (+NA). Positive control sections were preincubated with the manufacturer’s enzyme buffer only (–NA). The right panel shows higher-magnification images of the inlets from the left panel. The data are representative from two individuals.

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  FIG 11. Replication of the bovine G6P[5] WC3 and bovine-human mono-reassortant G4P[5] strains in differentiated human and bovine intestinal enteroids. (A) Differentiated human jejunal and bovine ileal enteroids grown in Matrigel with a differentiation medium were infected with the bovine G6P[5] WC3 strain or the bovine and human mono-reassortant G4P[5] strain at an MOI of 10 FFU/cell for the indicated times. The infected cells were assessed using flow cytometry with an MAb against the rotavirus VP6 protein and then FITC-conjugated secondary antibodies. The data are representative from three independent experiments. (B) Differentiated HIEs and BIEs infected with the bovine G6P[5] WC3 strain or the bovine and human mono-reassortant G4P[5] strain, at an MOI of 10 FFU/cell for the indicated times, were harvested and used to determine total viral RNA by real-time RT-PCR. All experiments were performed on three independent occasions. Error bars represent means ± the SD from triplicate experiments. (C) Differentiated HIEs and BIEs grown in Matrigel with a differentiation medium were infected with the bovine G6P[5] WC3 strain or the bovine-human mono-reassortant G4P[5] strain at an MOI of 10…