Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses

Cell lines.RAW264.7 cells (ATCC) were maintained in Dulbecco modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (HyClone), 100 U penicillin/ml, 100 μg/ml streptomycin, 10 mM HEPES, and 2 mM l-glutamine (complete DMEM).

Viruses and virion purification.Two independent triply plaque-purified virus isolates were used for in vivo infections, MNV-1.CW1 (46) and a previously unpublished plaque isolate clonally selected from the same tissue homogenate as MNV-1.CW1 (referred to as MNV-1.CW3) (GenBank accession no. EF014462). The stock of MNV-1.CW1 used for experiments was generated from the fourth passage (P4) of this isolate in cell culture, and the stock of MNV-1.CW3 was generated from P5, unless otherwise indicated. Virions were purified as described previously (46). Briefly, RAW264.7 cells were infected at a multiplicity of infection of 0.05 for approximately 40 h and frozen and thawed two times, and cell debris was removed by centrifugation at 3,000 rpm for 20 min. The supernatant fluid was layered onto a 30% sucrose cushion and centrifuged in an SW32 rotor at 90,000 × g for 3 h at 4°C. The pellet was resuspended in 10% Sarkosyl, the tubes washed with phosphate-buffered saline (PBS), and the total volume mixed with cesium chloride to reach a refractive index of 1.3665. The mixture was then centrifuged in an SW55 rotor at 115,000 × g for 40 h at 12°C. The gradients were fractionated into four fractions and individually dialyzed against PBS overnight at 4°C; the virus titer of each fraction was determined by plaque assay. Mock preparations were generated in the same manner, beginning with RAW264.7 cell lysates from uninfected cultures.

Mice, inoculations, and tissue processing.129S6/SvEvTac (Taconic no. 129SVE; referred to as 129 hereafter), 129S6/SvEvTac-Stat^tm1Rds (Taconic no. 002045-M; referred to as STAT1^−/− hereafter), and IFNαBγR^−/− (kindly provided by Kate Ryman, Louisiana State University Health Sciences Center, Shreveport) mice were bred and housed at Louisiana State University Health Sciences Center, Shreveport (LSUHSC-S), under specific-pathogen-free conditions in accordance with federal and university guidelines. Four- to 5-week-old sex-matched mice were inoculated by the peroral route in a volume of 25 μl. Suckling 6-day-old 129 mice were inoculated by oral gavage in a volume of 50 μl. At various times after infection, tissue samples were aseptically removed, weighed when indicated, snap-frozen in a dry ice-ethanol bath, and stored at −80°C prior to analysis. Blood was collected by cardiac puncture and centrifuged through a serum separator tube for serum collection. Perfusions were performed with PBS when indicated by making a small incision in the left ventricle, inserting a 23-gauge needle into the incision, and pumping PBS through the circulatory system until the lungs and liver turned pale. Tissue samples for histological analysis were collected aseptically and fixed for a minimum of 48 h in 10% buffered formalin. Paraffin embedding, sectioning, and hematoxylin-eosin (H&E) staining were performed by either the Histology Core at LSUHSC-S or Wax-it Histology Services (Vancouver, British Columbia, Canada). Intestinal and spleen sections were scored blindly by a gastrointestinal pathologist for signs of histopathology.

MNV-1 plaque assay.Tissue samples for viral burden analysis were homogenized in 1 ml complete DMEM by bead beating with 1.0-mm zirconia/silica beads (BioSpec Products, Inc.). Tissue homogenates were diluted 1:5, and serum samples were diluted 1:15 in complete DMEM and tested for virus titers by using a plaque assay that has been previously described (46). Briefly, 2 × 10⁶ RAW264.7 cells were seeded into each well of six-well plates, and infections were performed the following day with 10-fold dilutions of virus samples in duplicate. After a 1-h infection at room temperature, the inoculum was removed and the cells were overlaid with 1.5% SeaPlaque agarose (Cambridge Biosciences) in complete minimal essential medium. Following 48 h of incubation at 37°C, plaques were visualized 3 to 4 h after being overlaid with 1.5% SeaKem agarose (Cambridge Biosciences) in complete minimal essential medium containing 0.01% neutral red.

MNV-1 ELISA.Antigen for indirect enzyme-linked immunosorbent assay (ELISA) detection of MNV-1-specific antibodies was prepared as described previously for an animal lentivirus (41). Briefly, RAW264.7 cells were infected with MNV-1 at a multiplicity of infection of 0.05 for 3 days and frozen and thawed three times, and cell debris was removed by centrifugation at 1,000 × g for 10 min at 4°C. The supernatant fluid was then centrifuged at 106,000 × g for 90 min at 4°C, and the pellet was resuspended in 1/10 of the original volume in TN buffer (50 mM Tris [pH 7.5], 0.15 M NaCl). This centrifugation was repeated, and the resultant pellet was resuspended in 1/100 of the original volume in TN buffer. The sample was then treated with 0.1% sodium dodecyl sulfate for 10 min at 25°C and centrifuged at 1,000 × g for 15 min at 4°C, and the aqueous phase containing antigen was collected. Negative control antigen was prepared from RAW264.7 cells that were mock infected. Indirect ELISA was performed using standard procedures. Antigen was diluted 1:4 prior to coating, and serum samples were diluted 1:10 prior to testing. Positive control serum (harvested from 129 mice infected with MNV-1 for 7 weeks) and negative control serum (harvested from naïve 129 mice) were tested with both positive and negative control antigens on each plate. All samples were tested in duplicate. Experimental samples were considered positive for MNV-1-specific antibodies if their absorbance was more than three standard deviations (standard deviation = 0.033, calculated from five independent negative control samples) above the mean for negative controls tested on each plate.

IFN detection.The Mouse Interferon Beta ELISA kit from PBL Biomedical Laboratories was used to quantify IFN-β levels in intestinal homogenates and sera of infected and control mice. A 1:2 dilution of intestinal homogenates and a 1:20 dilution of serum samples were used in ELISA.

Gastroenteritis analyses.For all gastroenteritis studies, mice were deprived of food for 18 to 20 h prior to harvesting. For measuring gastric emptying, stomachs were ligated at both the esophageal and the intestinal junctions, dissected, and weighed. Stomachs were then opened, rinsed, and reweighed. For measuring intestinal fluid accumulation, intestines were ligated at the pyloric and the cecal junctions, dissected, and immediately weighed. The lengths were also measured. For measuring stool consistency and volume, all feces below the cecum was collected, scored, and weighed. The following scoring system was used: 0, normal feces; 1, mixed stool samples containing both solid and pasty feces; 2, pasty feces; 3, semiliquid feces; 4, liquid feces.

Immunofluorescence.Sections from paraffin-embedded tissues were deparaffinized in xylene and dehydrated, and antigen retrieval was performed by boiling slides in citrate buffer (8.2 mM sodium citrate, 1.8 mM citric acid) for 20 min. Sections were blocked for 15 min at 25°C in 1% bovine serum albumin, 0.2% nonfat milk, 0.3% Triton X, and 5% normal mouse serum in PBS and then incubated with previously described polyclonal guinea pig antibody to MNV-1 protease-polymerase (ProPol) (42) at a 1:50 dilution for 2 h at 25°C. Negative controls were incubated with normal guinea pig serum (Biomeda) at a 1:50 dilution. All slides were subsequently incubated with 10 μg/ml Alexa Fluor 488-conjugated anti-guinea pig antibody (Invitrogen). Nuclei were visualized with DAPI (4′,6′-diamidino-2-phenylindole)-containing VectaShield mounting medium (Vector Laboratories Inc.). A Nikon Eclipse TE300 inverted fluorescence microscope was used for imaging.

Flow cytometry.Spleens were dissected from mock-infected and MNV-1-infected 129 mice at various times postinfection, and single cell suspensions were prepared. After treatment with red blood cell lysis buffer (Sigma), splenocytes were incubated with Fcγ receptor block (BD PharMingen). Cells were then stained with one of the following fluorescently conjugated antibodies in PBS containing 1% fetal calf serum: anti-B220, anti-CD4, anti-CD8, anti-CD11c, or anti-CD11b (BD PharMingen) or anti-F4/80 (Serotec Inc.). Matched isotype controls were used for all antibodies. All incubations were performed at 4°C. After staining, cells were washed and then fixed for 15 min with 2% ultrapure methanol-free formaldehyde (Polyscience Inc.). Flow cytometric analysis was performed on a FACSCalibur instrument (Becton Dickinson), and data were analyzed using Cell Quest software (BD PharMingen).

Statistical analysis.GraphPad Prism was used for data analysis and graphical representations. In all graphs, error bars indicate standard errors of the mean. To determine statistical differences in viral burden and histological scoring analysis, P values were determined by unpaired two-tailed Mann-Whitney tests. To determine statistical differences in all other analyses, P values were determined by unpaired two-tailed t tests. In figures, one asterisk represents P values of 0.01 to 0.05, two asterisks represent P values of 0.001 to 0.01, and three asterisks represent P values of less than 0.001.

Kinetics of MNV-1 replication in 129 mice.To further examine the extent of MNV-1 replication in wild-type mice, we determined the levels of infectious virus in various tissues of 129 mice over the course of infection. Based on our previous work demonstrating that MNV-1 replicates in 6 to 12 h in vitro (46), we analyzed titers in 129 mice at a time point that should precede new virus replication (3 hpi) and at time points that should correspond to new virus production (12 hpi to 7 dpi). After peroral inoculation with 10⁷ PFU MNV-1.CW3, we detected infectious virus in the proximal small intestines at between 3 hpi and 5 dpi, with only a modest cycling in the level of virus over this time course (Fig. 2A). The peak titer was 240 PFU, at 3 dpi. Infectious virus was undetectable in the proximal small intestines by 7 dpi. Viral burden was first detected in the spleens and livers of 129 mice at 1 dpi (Fig. 2B and C). Virus in the spleens of these animals peaked at 3 dpi (830 PFU), and infectious virions were undetectable in half (two of four) of the animals by 7 dpi (Fig. 2B). Interestingly, there was detectable virus in the spleens of a proportion of 129 mice at both 7 (two of four mice) and 14 (one of five mice) dpi that was at the limit of detection (10 PFU) of our assay (data not shown), suggesting a low-level persistent infection in this tissue. Similar to intestinal titers, the level of infectious virus detected in the livers of 129 mice cycled modestly over time but never increased significantly over the amount first detected at 24 hpi (33 PFU) (Fig. 2C). Infectious virus was detected in the lungs of wild-type mice only at 3 dpi (37 PFU) (Fig. 2D). These data confirm that MNV-1 can seed the intestines of wild-type mice and spread within 24 h to the spleen and liver, and later to the lung, and that infectious virus is undetectable in most tissues by 7 dpi. In previous work, we were unable to detect viral RNA in tissues of 129 mice infected with parental virus at 3 dpi (23). The kinetic discrepancy between these two sets of data may be due to dosage, since approximately 1,000-fold less virus was used in earlier studies compared to the present infections, or it may be due to the stock of virus, since parental virus was used in earlier work and MNV-1.CW3 was used here.

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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.

STAT1-dependent responses rapidly inhibit MNV-1 replication in the proximal small intestine.To analyze the mechanisms by which STAT-1 restricts MNV-1 infection in vivo, we compared viral loads in the proximal small intestines of 129 mice versus STAT1^−/− mice through the first 72 h of infection. The level of infectious virus in this primary site of infection was 8.7-fold higher in STAT1^−/− mice than in 129 mice at 3 hpi (Fig. 3A). The increased amount of virus in STAT1^−/− mice compared to wild-type mice at 3 hpi suggests that either basal IFN in the intestine or IFN produced rapidly following infection (but preceding virus replication) inhibits MNV-1 seeding. While viral loads did not increase significantly over the course of infection in the intestines of 129 mice, viral loads steadily increased over the first 48 h of infection in STAT1^−/− mice (2,985-fold increase from 3 hpi to 48 hpi). These data provide evidence that STAT1-dependent responses rapidly control MNV-1 infection at the initial site of infection.

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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.

STAT1-dependent responses limit MNV-1 dissemination.To determine whether STAT-1 prevents viral dissemination, we also analyzed viral loads in various peripheral tissues over the 72-h course of infection. In addition to spleens, livers, and lungs, we also quantified the viral burden in axillary lymph nodes because we noted that they were inflamed macroscopically in STAT1^−/− mice by 24 hpi (data not shown). The quantities of virus in the spleens and livers of STAT1^−/− mice were significantly higher than those in 129 mice throughout the course of infection (Fig. 3B). At 24 hpi, the time point at which virus was first detected in these tissues, the levels of virus were 220-fold higher in the spleens and 180-fold higher in the livers of STAT1^−/− mice compared to 129 mice. The increased viral burden in STAT1^−/− spleen and liver tissues compared to 129 tissues was even more pronounced at 48 and 72 hpi. These data suggest either that MNV-1 dissemination is severely limited by STAT1-dependent responses or that dissemination occurs normally in the presence of these responses but viral replication is suppressed in peripheral tissues. Notably, while spleen and liver viral loads were identical to one another at all time points in STAT1^−/− mice, viral loads in 129 mice were significantly lower in the livers than in the spleens at 48 and 72 hpi (48 hpi, P = 0.029; 72 hpi, P = 0.0022). Thus, in the presence of STAT-1, MNV-1 replicated more efficiently in spleen tissue. This observation suggests the presence of a tissue-specific STAT1-dependent response that is more potent at controlling MNV-1 infection in the liver than in the spleen. Low levels of virus were first detected in the lungs and axillary lymph nodes at 24 hpi, and peak levels occurred at 48 hpi in these distal peripheral tissues (Fig. 3C). In contrast, much lower levels of virus were detected in the axillary lymph nodes and lungs of a small proportion of 129 animals, suggesting that dissemination to these sites or viral replication in these tissues was greatly diminished by STAT1-dependent responses.

We observed two kinetic patterns of virus production in peripheral tissues of STAT1^−/− mice, one in which there was a drastic increase in infectious virus between 12 and 24 hpi (Fig. 3B) (spleen and liver) and another in which there was a low level of virus at 24 hpi but a strong increase between 24 and 48 hpi (Fig. 3C) (lung and axillary lymph nodes). The kinetics of these amplifications of virus at peripheral sites of STAT1^−/− mice is consistent with rapid hematogenous dissemination of virus in the absence of STAT1-dependent responses. These findings are consistent with previous reports demonstrating the detection of viral RNA in the blood of experimentally infected STAT1^−/− mice (23) and the detection of MNV-1 antigen in blood cells of a naturally infected OTI Rag1^−/−/IFN-γR^−/− animal (44). To directly analyze whether mice were viremic following MNV-1 infection, we determined the viral burdens in sera of 129 and STAT1^−/− mice perorally inoculated with 10⁷ PFU MNV-1.CW3 (Fig. 3D). No virus was detectable in the sera of 129 mice throughout the 72-h course of infection. In contrast, in STAT1^−/− mice, virus was detectable by 24 hpi (1.4 × 10³ PFU/ml) and peaked by 48 hpi (1.3 × 10⁶ PFU/ml). Thus, these data demonstrate that STAT1-dependent responses suppress viremia, an effect that is likely responsible for directly limiting viral dissemination. Notably, viral loads in the intestines and spleens of 129 mice were above the limit of detection (30 PFU/ml) for infectious virus in the serum. Thus, blood-borne virus cannot account for the amount of infectious virus detected in these tissues, a finding that is further supported by the detection of infectious virus in proximal intestines, mesenteric lymph nodes, spleens, and livers of perfused 129 mice (Fig. 4A to D).

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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.

The additive value of titers in the tissues analyzed (proximal small intestine, spleen, liver, lung, and axillary lymph nodes) at each time point provides insight into the extent of virus infection in wild-type mice (Fig. 3E). In 129 mice, the cumulative titers increased 20-fold (from 60 to 1,200 PFU) between 3 and 72 hpi. The viral loads reported in this work represent the amount of virus in approximately 1 cm of proximal small intestine, one-fourth of the spleen, one-fourth of a lobe of liver, one-fourth of a lung, and four axillary lymph nodes; as such, they represent an underestimation of total infectious virus in the animals.

Analysis of the cumulative titer data also reveals the extent of STAT1-dependent suppression of MNV-1 replication. At 3 hpi, virus was detected in the proximal small intestines of both strains of mice, but the level of virus was 8.7-fold higher in STAT1^−/− mice than in 129 mice. At 12 hpi (presumably indicative of the first round of in vivo replication), the level of infectious virus was 16-fold higher in STAT1^−/− mice than in 129 mice, and it increased to 5,100-fold by 48 hpi. Thus, these data demonstrate the critical importance of STAT-1 in controlling viral replication and spread.

While STAT1-independent IFN responses (17, 33, 34, 40) and IFN-independent roles for STAT-1 (24) have both been reported, STAT1-dependent IFN responses are critical to the control of viral infections, and deficiencies in the absence of STAT-1 are most likely indicative of a loss of IFN signaling (9, 12, 29). Recent observations further suggest that STAT1-dependent IFN responses are critical in controlling viral replication early after infection, while STAT1-independent IFN responses are required for later viral clearance (40). To further dissect whether increased MNV-1 susceptibility in STAT1^−/− mice correlates with deficient IFN signaling, we infected 129 and STAT1^−/− mice, and mice lacking both type I and type II IFN receptors (IFN-αβγR^−/− mice) with 10⁷ PFU MNV-1.CW3 and compared viral loads in intestines, mesenteric lymph nodes (as these are expected to drain the intestines), spleens, and livers. Supporting the hypothesis that increased MNV-1 susceptibility in STAT1^−/− mice compared to wild-type mice is due to a loss of IFN signaling, the viral burdens in the intestines and mesenteric lymph nodes of IFN-αβγR^−/− mice parallel those observed in STAT1^−/− mice at 48 hpi (Fig. 4A and B). Furthermore, significant levels of type I IFN-β were detected in both intestinal homogenates and sera of 129 mice, but not STAT1^−/− mice, by 12 hpi, and these peaked at 24 hpi (Fig. 4E and F). Interestingly, we observed increased infectious virus in the spleens and livers of IFN-αβγR^−/− mice compared to STAT1^−/− mice (Fig. 4C and D), suggesting a possible tissue-specific role for STAT1-independent effects of IFN as well.

Overall, these data support two mechanisms by which STAT1-dependent IFN signaling limits MNV-1 infection in immunocompetent hosts: (i) The decreased viral loads in the intestinal tissue of 129 mice compared to STAT1^−/− and IFN-αβγR^−/− mice suggests that IFN-induced host factors directly inhibit viral replication at the primary site of entry, and (ii) the decreased levels of virus in peripheral tissues suggest that IFN responses limit MNV-1 dissemination to and replication in secondary tissues.

MNV-1 infection induces inflammation in the intestines of 129 mice, but clinical disease is prevented by STAT1-dependent responses.Common symptoms of human norovirus infection include delayed gastric emptying, vomiting, and diarrhea. To determine whether MNV-1 infection also correlates with the development of gastroenteritis, we monitored 129, STAT1^−/−, and IFN-αβγR^−/− mice for the development of gastric bloating (as a sign of delayed gastric emptying) and diarrhea. Upon macroscopic examination, 129 mice infected with MNV-1 did not display clinical signs of gastroenteritis. In contrast, STAT1^−/− and IFN-αβγR^−/− mice presented with distended stomachs and severe diarrhea by 48 hpi (data not shown). To determine whether MNV-1 infection caused more subtle symptoms of gastroenteritis in wild-type hosts and to quantify the severity of gastroenteritis induced in STAT1^−/− mice, we analyzed mice for weight loss (Fig. 5A), decreased gastric emptying (Fig. 5B), increased intestinal fluid accumulation (Fig. 5C), and changes in internal stool volume (Fig. 5D) and consistency (Fig. 5E) following MNV-1 infection. There were no statistically significant differences between mock-infected and MNV-1-infected 129 mice except for decreased stool contents in infected mice at 72 hpi (Fig. 5D). While the difference was not statistically significant, we did note a reduction in the rate of weight gain in infected 129 mice beginning at 4 dpi (Fig. 5A). It is thus possible that MNV-1 infection of 129 hosts induces a very mild gastroenteritis or anorexia; the latter symptom is caused by calicivirus infection of other animal hosts (5, 37, 43). Because rotavirus infection causes diarrhea in suckling but not adult mice (16, 35), we determined whether symptomatic MNV-1 infection of wild-type hosts was restricted to suckling mice. Specifically, 6-day-old 129 pups were inoculated intragastrically with 10⁷ PFU MNV-1.CW3 or mock infected and were observed daily for diarrhea (data not shown) and weight gain (Fig. 5F). MNV-1 infection did not induce diarrhea or weight loss in suckling mice.

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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.

In contrast to 129 mice, STAT1^−/− mice began losing weight quickly after MNV-1 infection (Fig. 5A). The duration of this experiment was determined by the survival of infected mice, since two of eight mice succumbed to lethal disease at 3 dpi and the remaining six mice succumbed at 4 dpi. The gastric bloating and diarrhea observed visually in infected STAT1^−/− mice were confirmed by a striking increase in the stomach contents of infected mice (Fig. 5B) and by statistically significant changes in diarrheal scoring index (Fig. 5E), respectively. As observed in 129 mice, infected STAT1^−/− mice also had decreased stool contents by 72 hpi (Fig. 5D). There was no change in intestinal fluid accumulation (Fig. 5C). Interestingly, the delayed gastric emptying observed after human norovirus infection is considered the likely cause of the high incidence of vomiting episodes (28), but mice do not vomit because they lack an emetic reflex. It is therefore possible that mouse and human viruses in this genus cause similar intestinal pathologies but that there is no visible clinical outcome in the murine setting. We are presently attempting to determine the basis of gastric bloating in MNV-1-infected STAT1^−/− mice, with possible explanations including decreased gastric motor function, as proposed for human norovirus infection (28), and pyloric inflammation. A more detailed understanding of how MNV-1 causes specific intestinal pathologies even in an immunodeficient setting may offer clues as to the prevention of human norovirus-induced disease.

To determine whether MNV-1 infection of the intestine is accompanied by histopathological changes in wild-type mice or mice lacking STAT-1, we infected 129 and STAT1^−/− mice perorally with 10⁷ PFU MNV-1.CW3 and analyzed tissue sections for histopathology and MNV-1 protein expression. Upon histopathological examination of the entire small intestine, we observed that intestines from infected 129 mice contained higher numbers of granulocytes at 24 hpi (Fig. 6A) than intestines from mock-infected 129 mice. No increase in the number of apoptotic cells was observed in the intestines of MNV-1-infected mice (Fig. 6B). This inflammation is consistent with histological changes observed in intestinal biopsy samples of human volunteers after exposure to noroviruses (1, 10, 38, 39): Volunteers who become ill retain an intact intestinal mucosa but display broadening and blunting of the villi coordinate with monocyte and neutrophil infiltration of the lamina propria. Thus, both human and murine norovirus infections are associated with a mild inflammatory infiltration of the lamina propria in immunocompetent hosts.

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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.

In contrast to the mild inflammation induced by MNV-1 infection of 129 hosts, infection of STAT1^−/− mice was associated with a decreased number of inflammatory cells in the intestinal tissue compared to mock-infected STAT1^−/− mice (Fig. 6A) and with significant numbers of apoptotic cells defined by pycnotic nuclei (Fig. 6B) by 48 hpi. While the decrease in inflammatory cells was surprising, it is possible that there is a correlative relationship between inflammatory cell number and infection-induced apoptosis in these mice. Overall, MNV-1 infection of wild-type mice results in mild intestinal inflammation, but infection-induced apoptosis is prevented by STAT1-dependent responses.

To define the site(s) of MNV-1 replication in the intestine, we performed immunofluorescence assays on sections of the entire small intestine by using polyclonal antibody to the nonstructural ProPol processing intermediate (42). In 129 mice, viral antigen was detected in cells of the lamina propria (which include macrophages and dendritic cells) in rare villi at 24 hpi (Fig. 7A and B). As detection of nonstructural proteins is indicative of ongoing viral translation, these data suggest low-level viral replication in the intestines of immunocompetent hosts. In STAT1^−/− mice, we detected different patterns of ProPol staining at early (12 hpi) and later (24 to 72 hpi) time points: a majority of the ProPol detected at 12 hpi was in the cytoplasm of epithelial cells lining the villi, on both the apical and basolateral sides (Fig. 7G and H). Consistent with this finding, a recent study by Ward et al. also reported detection of MNV-1 ProPol in intestinal epithelial cells in a naturally infected Rag1^−/−/Stat1^−/− mouse (44). Viral protein was also detected in lamina propria cells at 12 hpi (Fig. 7I), although much less frequently than in epithelial cells. Conversely, at later times, ProPol localized to lamina propria cells (Fig. 7J and K) and cells in the Peyer's patches (Fig. 7L) but very infrequently to epithelial cells (data not shown). Antigen was not detected in intestinal sections of mock-infected control mice (Fig. 7D to F and M to O) or in MNV-1-infected intestinal sections probed with preimmune sera (Fig. 7C and data not shown). These data demonstrate that MNV-1 initially seeds and replicates in intestinal epithelial cells in STAT1^−/− mice but replicates predominantly in lamina propria and Peyer's patch cells at later times postinfection. We did not detect ProPol in the intestines of 129 mice at time points earlier than 24 hpi. It is unclear whether epithelial cell infection is prevented in normal hosts or whether the level of antigen was below the limit of detection.

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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.

MNV-1 infection causes histopathological changes in the spleens of wild-type mice, but tissue destruction is prevented by STAT1-dependent responses.To determine whether MNV-1 infection also induced pathological changes in peripheral organs, we examined spleens, livers, and lungs from 129 and STAT1^−/− mice. Infected 129 mice displayed increased nuclear staining in the red pulp, suggesting either hyperplasia or cellular hypertrophy of the splenic red pulp, and activated white pulp by 72 hpi compared to mock-infected mice (Fig. 8). There was no significant difference in overall splenocyte numbers upon infection (data not shown), suggesting that the observed red pulp changes were due to increased cell size. Further supporting this conclusion, direct counting of cells in the red pulp in random fields of H&E-stained spleen sections revealed no hyperplasia in infected mice compared to control mice (data not shown).

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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.

In contrast to the red pulp hypertrophy and white pulp activation observed in infected 129 mice, the spleen tissue of infected STAT1^−/− mice displayed significant pycnotic nuclear debris indicative of apoptosis in both the red and white pulp at 72 hpi (Fig. 8). Further, STAT1^−/− mice but not 129 mice developed severe pneumonia, as observed previously with parental virus infections (23). Neither 129 nor STAT1^−/− mice showed histopathological changes in the liver through 72 hpi (data not shown). Thus, MNV-1 induces splenic white pulp activation and hypertrophy of red pulp cells, but STAT1-dependent responses prevent splenic apoptosis and lung inflammation.

To determine whether the types of cells in the spleens of 129 mice were altered upon MNV-1 infection, we performed fluorescence-activated cell sorter analysis of the levels of macrophage, dendritic cell, B-cell, and T-cell markers in this tissue by using antibodies to F4/80 or CD11b (macrophages), CD11c (dendritic cells), B220 (B cells), and CD4 and CD8 (T cells). In infected 129 mice at 72 hpi, total F4/80⁺ splenocytes increased 7.6% and total B220⁺ splenocytes increased 5.7% over those in mock-infected mice (Fig. 9). While F4/80 is expressed on subpopulations of dendritic cells in addition to macrophages and B220 is expressed on plasmacytoid dendritic cells in addition to B cells, there was only a modest 0.57% increase in CD11c⁺ (the pan-murine dendritic cell marker) splenocyte numbers upon infection, suggesting that the increased numbers of F4/80⁺ and B220⁺ splenocytes correspond to increases in macrophage and B cell numbers, respectively. Although CD11b is expressed on macrophages, we did not observe a statistically significant increase in CD11b⁺ cells in the spleen upon infection. The discrepancy between F4/80 and CD11b staining may be due to the additional expression of CD11b on numerous other cell types, including granulocytes, myeloid-derived dendritic cells, NK cells, and B-1 cells. No difference was observed in the percentage of T cells expressing CD4 or CD8. Thus, MNV-1 infection induced an increase in macrophage and B-cell numbers, or an increased expression of particular markers, in the spleens of wild-type mice by 72 hpi.

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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.

Significance of MNV-1 infection of immunocompetent hosts.In the work described here, we demonstrated that MNV-1 seeds the proximal small intestine and may replicate in cells of the lamina propria, rapidly disseminates to multiple peripheral tissues, and increases in vivo at least 20-fold over a 72-h course of infection in wild-type 129 mice. Infection is associated with histopathological changes in both the intestine and spleen but does not result in clinical disease. In future studies, it will be critical to define the mechanisms by which murine noroviruses induce these specific changes and the basis of STAT1-dependent protection from infection-induced apoptosis.

Infectious virus was consistently detected in the proximal small intestines of 129 mice between 3 hpi and 5 dpi, and viral nonstructural protein was detected in lamina propria cells, indicating a low level of viral replication in the intestine. While infectious virus was detected in intestines at 3 and 12 hpi, no virus was detected in peripheral organs until 24 hpi. Thus, the intestine represents the primary site of MNV-1 seeding and likely facilitates systemic spread. This is notable in light of the current paradigm that human noroviruses are associated only with gastroenteritis and not with peripheral disease. Interestingly, though, the human viruses are infectious after respiratory exposure, and it is possible that very mild or sporadic pathologies associated with human norovirus infection have not been observed due to the difficulties in their detection (32). Supporting increased pathogenic potential of human norovirus infection, a recent case report detected norovirus RNA in the serum and cerebrospinal fluid of a child with encephalopathy (21). In addition, during a norovirus outbreak among military personnel in Afghanistan in May 2002, three infected patients presented with diminished alertness, headache, neck stiffness, and light sensitivity in addition to gastroenteritis; one of these patients also displayed disseminated intravascular coagulation (6). Similarly, an accumulating body of work shows that rotavirus infection, which has long been assumed to cause infection of only the gastrointestinal tract, is associated with extraintestinal viremic dissemination and histopathological changes in additional tissues (3, 4, 7, 8, 15). We believe that the present study warrants a similar consideration of potential extraintestinal spread of human noroviruses.

The finding that a low level of infectious virus remains in the spleens of a proportion of 129 mice as late as 14 dpi is in accordance with the recently reported observation that MNV-1 RNA is present in the spleens, mesenteric lymph nodes, and jejunums of a small proportion of outbred CD1 mice at 5 weeks following infection (20). In addition, the viral genome has been detected in the spleens, mesenteric lymph nodes, and jejunums of CD1 mice as late as 8 weeks following infection with three newly identified murine noroviruses, MNV-2, MNV-3, and MNV-4 (19). Taken together, these data demonstrate that MNVs can persist asymptomatically in lymphoid compartments of immunocompetent hosts, a pathogenic outcome that could affect the development and maintenance of a memory immune response. Consistent with this finding, members of the Caliciviridae Vesivirus genus cause persistent asymptomatic infection of cats (31, 45). There is also evidence that human noroviruses continue to shed for weeks after infection, suggesting that they too are maintained persistently in some hosts (18, 36) and that human norovirus infection does not induce lasting protective immunity (2, 13, 22, 30). Thus, it is possible that memory immune responses do not develop properly in the face of prolonged low-level norovirus replication in lymph nodes and splenic tissue. The effect of persistent replication on the immune response to norovirus infection will be critical to define and may offer important insight into vaccination strategies and novel antiviral approaches.

In summary, we have presented data demonstrating that STAT-1 is critical in (i) controlling murine norovirus replication at the primary site of infection, (ii) preventing high levels of viral dissemination to and/or replication in peripheral tissues, and (iii) protecting lymphoid tissue from pathological cell death. Thus, STAT1-dependent IFN responses play a key role in limiting the severity of norovirus pathogenesis. Based on the seeming inability of memory immune responses to control secondary norovirus infections and the potential emergence of human norovirus strains with increased virulence and/or pathogenic potential (6, 21, 25), it will be important to continue to define the mechanisms of IFN control and to identify IFN-based treatment options for norovirus infections.