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. Author manuscript; available in PMC: 2015 Nov 7.
Published in final edited form as: Science. 2014 Nov 7;346(6210):755–759. doi: 10.1126/science.1257147

Fig. 4. Intestinal bacteria facilitate NoV infections.

Fig. 4

(A) Lysates were prepared from the indicated colony-forming units (CFU) of heat-killed E. cloacae for the purpose of Western blotting. Membranes were probed with antibody to H antigen. Synthetic H-type HBGA (synth. H) was tested as a positive control. No H-type HBGA was detected in E. coli lysates or BJAB cell lysates. (B) Filtered GII.4-Sydney HuNoV-positive stool inoculum was incubated with the indicated dose of heat-killed E. cloacae, 106 CFU heat-killed E. coli, 1 mg/mL E. coli LPS, or 500 ng/mL synthetic H-type HBGA before inoculation onto B cells. To test for antibody-mediated neutralization of virus infectivity, unfiltered stool inoculum was incubated with 10 μg/mL antibody to VP1 before B cell inoculation. Viral genome copy numbers were determined at 0 and 3 dpi under each condition (n = 3 to 4 experiments). Data are reported as the fold-increase in copy numbers over time. Each condition was compared with the untreated filtered inoculum data set for statistical purposes. (C) Unfiltered stool (black bars), filtered stool (gray bars), or filtered stool preincubated with 500 ng/mL H antigen (white bars) was inoculated onto BJAB cells for the indicated times at 4°C (n = 4 experiments). Viral genome copy numbers were quantified from unwashed cells to determine the amount of input virus and from washed cells to determine the amount of cell-attached virus. Data are reported as the percent of viral genomes remaining cell-associated compared with input. (D) Groups of phosphate-buffered saline (PBS)–treated (black bars) and Abx-treated (white bars) B6 mice (n = 3 to 4 mice) were infected with 107 TCID50 units MNV-1 (left) or MNV-3 (right). At 1 dpi, virus was titered from the distal ileum (DI), colon, and mesenteric lymph nodes (MLNs) by using a standard virus plaque assay (n = 3 experiments). PBS-treated and Abx-treated groups under each condition were compared for statistical purposes. For all, error bars denote mean ± SD; Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.