Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses

Abstract

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.

FIG. 1.

MNV-1.CW3 replicates more efficiently in STAT1^−/− and immunocompetent hosts than does MNV-1.CW1. Mice deficient in the STAT-1 molecule (A) or immunocompetent 129 mice (B) were inoculated perorally with 1 × 10⁷ PFU of either MNV-1.CW3 or MNV-1.CW1. Organs were harvested at 24 hpi, and viral burdens in the proximal small intestine, spleen, liver, and lung were determined. The limit of detection was 10 PFU/ml. The P values comparing virus titers following infection of STAT1^−/− mice with MNV-1.CW3 or MNV-1.CW1 are as follows: intestine, P = 0.0095; spleen, P = 0.0095; liver, P = 0.0095; lung, P = 0.17. The P values comparing virus titers following infection of 129 mice with MNV-1.CW3 or MNV-1.CW1 are as follows: intestine, P = 0.61; spleen, P = 0.61; liver, P = 0.038; lung, P = 0.91.

FIG. 2.

MNV-1 disseminates rapidly in immunocompetent hosts from the proximal small intestine and is cleared from most tissues by 7 days postinfection. 129 mice were inoculated perorally with 1 × 10⁷ PFU of MNV-1.CW3, and organs were harvested at 3 and 12 hpi and at 1, 2, 3, 5, and 7 dpi. Viral burdens in the proximal small intestine (A), spleen (B), liver (C), and lung (D) were determined. At least three mice were analyzed at each time point. In tissues in which no plaques were observed, animals were assigned a titer at the limit of detection, 10 PFU, as depicted by a dashed line.

FIG. 3.

MNV-1 replicates and disseminates more efficiently in STAT1^−/− mice following oral inoculation. (A to D) 129 and STAT1^−/− mice were inoculated perorally with 1 × 10⁷ PFU of MNV-1.CW3 or mock infected, and organs were harvested at 3, 12, 24, 48, and 72 hpi. Virus titers in the proximal small intestine (A), ) spleen and liver (B), lung and axillary lymph nodes (C), and serum (D) were determined. Tissues from at least three mice of each strain were analyzed for viral burden at each time point. Viral loads are reported as the average titer for all animals in a particular group. Data for 129 mice infected at 3 to 72 hpi that were reported in Fig. 2 are repeated here for clarity. In tissues in which no plaques were observed, animals were assigned a titer at the limit of detection, 10 PFU for tissues and 30 PFU/ml for serum. When virus was detected in only a proportion of a group of mice, the number of mice with detectable virus is reported as a ratio over the total number analyzed. The limit of detection is depicted by a dashed black line in each graph. All mock-infected tissues were negative. Statistical analysis of intestinal titer comparison between 129 and STAT1^−/− mice is as follows: 3 hpi, P = 0.20; 12 hpi, P = 0.032; 24 hpi, P = 0.0087; 48 hpi, P = 0.0095; and 72 hpi, P < 0.0001. The P values for peripheral tissues at 24 hpi are as follows: spleen, P = 0.0043; liver, P = 0.0022; lung, P = 0.093; axillary lymph nodes, P = 0.79. The P values for peripheral tissue at 48 hpi are as follows: spleen, P = 0.029; liver, P = 0.029; lung, P = 0.0095; axillary lymph nodes, P = 0.10. The P values for peripheral tissue at 72 hpi are as follows: spleen, P = 0.0022; liver, P = 0.0022; lung, P = 0.0022; axillary lymph nodes, P = 0.0022. (E) The total viral burden in the five tissues analyzed (proximal small intestine, spleen, liver, lung, and axillary lymph nodes) per mouse was determined additively, and the averaged total for all mice analyzed per time point is reported. The fold increase in titers in STAT1^−/− compared to 129 tissues was determined by dividing the average viral burden in STAT1^−/− mice by the average burden in 129 mice.

FIG. 4.

MNV-1 replicates and disseminates more efficiently in the absence of IFN signaling. (A to D) 129, STAT1^−/−, and IFN-αβγR^−/− mice were inoculated perorally with 1 × 10⁷ PFU of MNV-1.CW3. At 48 hpi, animals were perfused and organs were harvested and weighed. Virus titers, reported as PFU per gram of tissue, in the proximal small intestine (A), mesenteric lymph nodes (B), spleen (C), and liver (D) were determined. The P values comparing viral loads in STAT1^−/− and IFN-αβγR^−/− mice are as follows: intestine, P = 1.0; mesenteric lymph nodes, P = 0.53; spleen, P = 0.0059; liver, P = 0.0007. (E and F) Intestinal homogenates (E) and sera (F) from 129 and STAT1^−/− mice infected perorally with 10⁷ PFU MNV-1.CW3 for 12, 24, and 72 hpi were tested in IFN-β-specific ELISA. Mock-infected control mice harvested at 24 hpi were also tested. At least four mice were analyzed for each group.

FIG. 5.

Clinical disease induced by MNV-1 infection is prevented by STAT1-dependent responses. 129 and STAT1^−/− mice were inoculated perorally with 1 × 10⁷ PFU of MNV-1.CW3 or were mock infected. (A) Individual mice were weighed at approximately the same time of day every 1 to 2 days beginning on the day of infection (day 0), and the data are presented as the averaged weight of all mice in a particular group. (B to E) Groups of mock-infected and MNV-1-infected mice were sacrificed at 24 and 72 hpi, and the stomachs and small intestines were harvested as described in Materials and Methods. Feces from each mouse was also collected, weighed, and scored. (B) Weight of stomach contents. The P values comparing the weights of stomach contents in mock-infected and MNV-1-infected mice are as follows: 129 mice at 24 hpi, P = 0.18; 129 mice at 72 hpi, P = 0.40; STAT1^−/− mice at 24 hpi, P = 0.025; STAT1^−/− mice at 72 hpi, P < 0.0001. (C) Intestinal fluid accumulation. The P values comparing intestinal fluid accumulation in mock-infected and MNV-1-infected mice are as follows: 129 mice at 24 hpi, P = 0.73; 129 mice at 72 hpi, P = 0.47; STAT1^−/− mice at 24 hpi, P = 0.93; STAT1^−/− mice at 72 hpi, P = 0.74. (D) Weight of fecal contents. The P values comparing the weights of fecal contents in mock-infected and MNV-1-infected mice are as follows: 129 mice at 24 hpi, P = 0.77; 129 mice at 72 hpi, P = 0.019; STAT1^−/− mice at 24 hpi, P = 0.41; STAT1^−/− mice at 72 hpi, P < 0.0001. (E) Diarrheal score. The P values comparing the diarrheal scores in mock-infected and MNV-1-infected mice are as follows: 129 mice at 24 hpi, P = 1.0; 129 mice at 72 hpi, P = 0.22; STAT1^−/− mice at 24 hpi, P = 0.012; STAT1^−/− mice at 72 hpi, P < 0.0001. (F) Weight of suckling mice. Suckling 6-day-old 129 mice were inoculated with 1 × 10⁷ PFU of MNV-1.CW3 by oral gavage or mock infected, and individual mice were weighed each day following infection beginning on the day of infection (day 0). The data are presented as the averaged weight of all mice in a particular group.

FIG. 6.

MNV-1 infection is associated with histopathological changes in the intestines of 129 and STAT1^−/− mice. H&E-stained intestinal sections from 129 and STAT1^−/− mice were scored for the number of inflammatory cells (A) and the number of apoptotic cells (B). For each animal, the inflammatory and apoptotic cells from 10 random high-power (40×) fields were counted and averaged. The number of animals analyzed for each time point is reported above each bar, and data represents the average for all animals per group. Mock-infected 129 mice were analyzed at both 24 and 72 hpi, and mock-infected STAT1^−/− animals were analyzed at 48 hpi. The P values comparing the numbers of inflammatory cells in mock-infected and MNV-1-infected 129 mice are as follows: 24 hpi, P = 0.043; 72 hpi, P = 0.37. The P value comparing the numbers of inflammatory cells in mock-infected and MNV-1-infected STAT1^−/− mice at 48 hpi is 0.0080. The P values comparing the numbers of apoptotic cells in mock-infected and MNV-1-infected 129 mice are as follows: 24 hpi, P = 0.013; 72 hpi, P = 0.11. The P value comparing the numbers of apoptotic cells in mock-infected and MNV-1-infected STAT1^−/− mice at 48 hpi is 0.00070.

FIG. 7.

MNV-1 replicates in lamina propria cells of 129 mice and multiple cell types in the intestines of STAT1^−/− mice. Intestinal sections prepared from MNV-1.CW3-infected or mock-infected 129 or STAT1^−/− mice were stained with guinea pig polyclonal antibody raised against the MNV-1 ProPol nonstructural intermediate (42). Fluorescently conjugated secondary antibody was then used to visualize viral protein through immunofluorescence, in which viral protein is pseudocolored in green and nuclei in blue. (A to C) ProPol antigen was detected in lamina propria cells of 129 mice at 24 hpi. Panels A, B, and C represent serial sections of the same intestinal villous of an infected 129 mouse; two were incubated with anti-ProPol antibody (A and B), and one was incubated with normal guinea pig serum (C). (G to I) ProPol antigen was detected in epithelial cells (G and H) and lamina propria cells (I) of STAT1^−/− mice at 12 hpi. (J to L) ProPol antigen was detected in lamina propria cells (J and K) and Peyer's patch cells (L) of STAT1^−/− mice at 48 hpi. (D to F and M to O) No ProPol antigen was detected in control sections of mock-infected 129 or STAT1^−/− mice. Magnifications, ×60 (A to F, H, I, N, and O); ×40 (G, J, K, and M; and ×20 (L). At least three infected and three control mock-infected mice were analyzed in independent experiments for each condition and showed similar staining patterns. No antigen was detected in serial sections of intestines from the MNV-1.CW3-infected mice of either strain stained with normal guinea pig serum.

FIG. 8.

MNV-1 infection induces red pulp hypertrophy and white pulp activation in 129 mice and splenocyte apoptosis in STAT1^−/− mice. 129 and STAT1^−/− mice were inoculated perorally with 1 × 10⁷ PFU MNV-1.CW3 or mock infected, and organs were harvested at 72 hpi. H&E-stained spleen sections of at least three mice per group were analyzed for signs of pathology. Representative histology is shown for one mock-infected control 129 mouse, one MNV-1.CW3-infected 129 mouse, one mock-infected control STAT1^−/− mouse, and one MNV-1.CW3-infected STAT1^−/− mouse at 72 hpi. Splenic red pulp hypertrophy and activated lymphocytes in the white pulp are apparent in infected 129 mice, while significant pycnotic nuclear debris is observed in these regions of infected STAT1^−/− mice.

FIG. 9.

Significant increases in the macrophage marker F4/80 and the B-cell marker B220 are induced on splenocytes of MNV-1-infected wild-type mice. 129 mice were inoculated perorally with 1 × 10⁷ PFU MNV-1.CW3 or mock infected, and spleens were harvested at 72 hpi. Noninoculated mice (untreated) were also analyzed. Cells (1 × 10⁶) from spleen homogenates prepared from these animals were stained separately with fluorescently conjugated antibodies specific for F4/80, CD11b, CD11c, B220, CD4, and CD8. Data are reported as the averaged percentage of total splenocytes positive for the respective surface marker from four independent experiments for splenocytes from untreated mice and six independent experiments for splenocytes from mock-infected and MNV-1.CW3-infected mice for F4/80, CD11b, CD11c, and B220 analysis. Three independent experiments were performed for mock-infected and MNV-1.CW3-infected groups for CD4 and CD8 analysis. The number of animals tested per group is shown above each bar. The numbers of positive cells in mock-infected homogenates did not differ statistically from those in untreated mice for any marker. When indicated by asterisks, the increase in the MNV-1.CW3-infected group was statistically significant compared to the mock-infected group (F4/80, P < 0.0001; B220, P = 0.0031; CD11b, P = 0.075; CD11c, P = 0.020; CD4, P = 0.30; CD8, P = 0.92). Similar statistical differences were observed between untreated and MNV-1.CW3-infected groups (F4/80, P < 0.0001; B220, P = 0.041; CD11b, P = 0.58; CD11c, P = 0.054). No staining was observed with matched isotype control antibodies.