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. 2019 Feb 5;93(4):e01774-18. doi: 10.1128/JVI.01774-18

FIG 6.

FIG 6

Screening for neutralizing monoclonal antibodies specific for SA11. (A) rsSA11-NLuc-Δ332 (1.0 × 102 PFU) was incubated at 37°C for 1 h with hybridoma culture supernatant (1:5 dilution). The virus-antibody mixture was inoculated onto MA104 cells, and NLuc titers in cell lysates were measured at 24 h postinfection. PBS was used as a negative control. Candidate NT-positive hybridoma clones are indicated by asterisks. Murine anti-SA11 serum raised after oral infection with SA11 virus was used as a positive control. (B) rsSA11-NLuc-Δ332 (1.0 × 102 PFU) was incubated with different concentrations of purified MAb clones 26, 29, 47, and 11. The virus-MAb mixture was inoculated onto MA104 cells, and NLuc titers in the cell lysates were measured at 24 h postinfection. MAb clone 11 was used as a negative control. (C) Hemagglutinin (HA)-tagged recombinant SA11 VP4 protein expressed in 293T cells was detected in an indirect immunofluorescence assay using MAb clone 29 and a rabbit anti-hemagglutinin peptide antibody, followed by anti-mouse IgG-CF594 and anti-rabbit IgG-CF488 secondary antibodies, respectively. (D) rsSA11-NLuc-Δ332, rsSA11-NLuc-esc29, and rsSA11-R441G-NLuc were incubated with various concentrations of MAb clone 29 and then inoculated onto MA104 cells. NLuc titers in the cell lysates were measured at 24 h postinfection. (E) MA104 cells were infected with rsSA11-NLuc-Δ332 or rsSA11-R441G-NLuc, and viral antigens were detected in an indirect immunofluorescence assay using a rabbit anti-NSP4 antibody and MAb clone 29, followed by an anti-rabbit IgG-CF488 conjugate or an anti-mouse IgG-CF594 conjugate, respectively. (F) Crystal structure of the trimeric VP5*CT (VP4 residues N252-L523) from a rhesus rotavirus (RRV) strain (PDB accession number 1SLQ). R441 is indicated in red. (G) Alignment of amino acid residues 428 to 455 of the VP4 proteins of RV reference strains. The P genotypes of the VP4 gene of each strain are shown. Amino acids identical to the consensus sequence (SA11) are indicated by dots. R441 is underlined.