Enteric bacteria promote human and mouse norovirus infection of B cells

Abstract

The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual's histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

Fig. 1. MuNoVs infect B cells in culture

(A) The indicated mouse cell lines were infected with MNV-1 (left) or MNV-3 (right) at multiplicity of infection (MOI) 5 and virus growth curves determined by using standard TCID₅₀ assay. The limit of detection is indicated by a dashed line. (B) The indicated cell lines were mock-inoculated (left) or infected with MNV-1 (middle) or MNV-3 (right) at MOI 5, and cell viability was determined at various times after infection by using propidium iodide staining. (C) M12 or WEHI-231 cells were infected with MNV-1 (black bars) or MNV-3 (gray bars) at MOI 20 for M12 cells or MOI 5 for WEHI-231 cells. At the indicated dpi on the x axis, cells were stained with antibody to ProPol and 4′,6-diamidino-2-phenylindole (DAPI) and imaged on a fluorescent microscope. The percentage of virally infected cells in each cell line was then quantified as the average ratio of ProPol⁺ cells per total cells. (D) Duplicate wells of M12 cells were infected with MNV-1 (black line) or MNV-3 (gray line) at MOI 5 and passaged every 2 days. At the first passage and every fifth passage, the virus titers in the supernatants were determined by using a standard TCID₅₀ assay (left). A portion of these cultures were analyzed by means of immunofluorescence assay for infectivity rates (middle). (Inset) Representative images merging the viral ProPol signal (red) and the DAPI staining of nuclei (blue) are shown from passage 10 (P10) cultures. A representative Western blot of cell lysates from persistently MNV-1– or MNV-3– infected M12 cultures (two independent cultures per virus strain) generated at passage 23 (P23) is shown. The MNV-1 virus stock used for initial infections was also tested (labeled as “+”). The blot was probed with antibody to VP1 and reprobed for actin as a loading control. For all, n = 3 to 5 experimental repeats. Error bars denote mean ± SD; Student’s t test in (C), *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. MuNoVs target Peyer’s patch B cells

(A and B) Groups (n = 5 mice) of B6 mice (black bars) and μMT mice (white bars) were infected with 10⁷ TCID₅₀ units (A) MNV-1 or (B) MNV-3 and harvested at 0.5 or 1 dpi. Virus titers were determined by performing plaque assay on homogenates of the indicated tissues. The data are presented as plaque-forming units (PFU) per gram of tissue on a logarithmic scale, and data for all mice in each group were averaged (n = 2 experiments). Limits of detection are indicated by dashed lines. Error bars denote mean ± SD; Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Groups of B6 mice (n = 8 mice) were inoculated with either mock inoculum or 10⁷ TCID₅₀ units MNV-1 or MNV-3. At 1 dpi, Peyer’s patches were harvested, and quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed on bulk cells (black bars) and purified CD19⁺ cells (white bars) by using virus ORF1-specific primers (n = 5 experiments). Data are reported as viral genomes per cell on a logarithmic scale. The limit of detection is indicated by a dashed line. (D) Stat1^−/− mice (n = 2 mice) were inoculated with mock inoculum (gray bars) or 10⁷ TCID₅₀ units MNV-1 (black bars). Intracellular staining for the MNV-1 nonstructural N-term protein was performed on B cells isolated from Peyer’s patches. CD19 or B220 are markers of B cells. Portions of cells were stained with preimmune sera in place of antibody to N-term as a background control. Data are presented as the percentage of B cells stained by N-term subtracted by the percentage of B cells stained by preimmune sera (n = 3 experiments).

Fig. 3. HuNoVs productively infect B cells in culture

(A) A GII.4-Sydney HuNoV-positive stool sample was inoculated onto human BJAB B cells (black line) or filtered through a 0.2- μm filter before application (gray line). The inoculum contained 1 × 10⁶ genome copy numbers, indicated by a dashed line. Viral genome copy numbers per well were determined by means of genogroup II-specific quantitative RT-PCR (n = 12 experiments). The 3- and 5-dpi genome copy numbers were compared with 0 dpi under each condition for statistical purposes, indicated by asterisks. The unfiltered and filtered data sets were statistically different from each other at 3 and 5 dpi, as indicated by the gray pound sign, but not at 0 dpi. (B) 1 × 10⁶ genome copy numbers of unfiltered (black bars) or filtered (gray bars) stool inoculum was untreated (solid bars) or UV-treated (hatched bars) before inoculation onto B cells. Samples were analyzed as described above, and data were reported as the fold-increase in viral genome copy numbers from 0 to 3 or 5 dpi (n = 3 experiments). (C and D) Mock inoculum or 5 × 10⁵ genome copy numbers of unfiltered GII.4-Sydney HuNoV-positive stool was applied to BJAB cells, and the cells were washed after 2 hours. (C) Cell lysates were tested in Western blotting by using a polyclonal antibody to NS6. The asterisk indicates a band of the expected size for the HuNoV NS5-NS6 processing intermediate (35 kD) that was only observed in infected cells at 3 to 5 dpi. No mature NS6 protein was detected, which is consistent with a report demonstrating that the NS5-NS6 cleavage site of a HuNoV is processed very inefficiently by the viral protease (33). (D) Cells were stained with antibody to VP1 (red) and DAPI (blue) and imaged on a fluorescent microscope. No VP1 signal was detected in mock-inoculated cells at 5 dpi, nor infected cells stained with an isotype control antibody. (E) 5 × 10⁵ genome copy numbers of a P0 inoculum was passaged onto naïve BJABs. At 0, 3, and 5 dpi, wells were collected and analyzed by means of genogroup II-specific quantitative RT-PCR (n = 4 experiments). The data are presented as the fold-increase in genomes from 0 to 3 or 5 dpi. The genome copy numbers detected at each time point were compared with 0 dpi for statistical purposes, indicated by black asterisks. (F) 1 × 10⁶ genome equivalents of the unfiltered (black bars) or filtered (gray bars) GII.4-Sydney HuNoV-positive stool sample were applied to the apical side of a transwell with polarized HT-29 IECs grown on the membrane and BJAB B cells cultured in the basal compartment. At 0 and 3 dpi, the basal compartment was collected for viral genome analysis by means of quantitative RT-PCR (n = 5 experiments). The data are presented as the fold-increase in genomes from 0 to 3 dpi. In two experiments, unfiltered stool was applied to a coculture with no cells in the basal chamber as a control (white bars). The 3-dpi genome copy numbers were compared with 0 dpi under each condition for statistical purposes, indicated by black asterisks. Similar coculture results were obtained by using another GII.4-Sydney HuNoV-positive stool sample. For all, error bars denote mean ± SD; Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. Intestinal bacteria facilitate NoV infections

(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.