Although chronic gastrointestinal dysmotility syndromes are a common worldwide health problem, underlying causes for these disorders are poorly understood. We show that flavivirus infection of enteric neurons leads to acute neuronal injury and cell death, inflammation, bowel dilation, and slowing of intestinal transit in mice. Flavivirus-primed CD8 + T cells promote these phenotypes, as their absence diminished enteric neuron injury and intestinal transit delays, and their adoptive transfer reestablished dysmotility after flavivirus infection. Remarkably, mice surviving acute flavivirus infection developed chronic gastrointestinal dysmotility that was exacerbated by immunization with an unrelated alphavirus vaccine or exposure to a non-infectious inflammatory stimulus. This model of chronic post-infectious gastrointestinal dysmotility in mice suggests that viral infections with tropism for enteric neurons and the ensuing immune response might contribute to the development of bowel motility disorders in humans. These results suggest an opportunity for unique approaches to diagnosis and therapy of gastrointestinal dysmotility syndromes.

Given that neurotropic flaviviruses reportedly can target cells of the GI tract, we evaluated the consequences of infection. In mice, WNV, ZIKV, and POWV infection of the GI tract acutely resulted in dilation of segments of the small and large intestines. High levels of viral RNA were detected in these tissues, which persisted for weeks in surviving mice. Functional studies revealed that flavivirus-infected mice developed delayed GI transit during the acute phase of infection. Immunohistochemical and flow cytometry analysis of intestinal tissues from WNV-infected mice showed viral antigens localized to enteric ganglia and infiltration of CD3 + lymphocytes and monocytes into the myenteric plexus and muscularis propria within 6 days of infection. This phase of GI dysmotility was in part immune mediated, as it was absent in WNV-infected mice lacking CD8 + T cells and could be re-established by adoptive transfer of WNV-primed, but not naive, CD8 + T cells. Remarkably, survivors of WNV infection sustained chronic delayed GI transit for months, exhibited neuroplastic changes, and were vulnerable to exacerbations of GI dysmotility after immunization with an unrelated alphavirus vaccine or exposure to poly(I:C), a non-infectious inflammatory stimulus. These results suggest that systemic infection by neurotropic viruses can damage the ENS, which leads to an intestinal dysmotility disorder in mice that models features of acute intestinal pseudo-obstruction and chronic IBS in humans.

Bacterial (), protozoan () and enteric viral () agents that cause infectious gastroenteritis have been implicated in the development of GI dysmotility disorders (). Multiple herpesviruses (e.g., herpes simplex virus 1 (HSV-1), varicella-zoster virus, and Epstein-Barr virus) also can infect the ENS of humans or experimentally infected animals (). Analysis of GI tract tissues from human patients with achalasia () and experimentally infected animals () showed infection-induced inflammation of enteric ganglia and immune cell infiltration of the myenteric plexus ganglia and muscularis propria, suggesting a possible immune-mediated pathogenesis of GI dysmotility disorders.

Less is known about flavivirus infection of enteric neurons. Mice infected with some neurotropic flaviviruses develop gastrointestinal (GI) tract pathology (). Analysis of selected GI tissues from infected rodents showed viral antigens by immunohistochemistry, viral RNA by qRT-PCR (), inflammatory lesions of the myenteric plexus, villus blunting, and enterocyte necrosis (). Consistent with these findings, pathological lesions and WNV antigens have been observed in the GI tract of infected birds (). Moreover, multiple human case reports describe GI symptoms (e.g., vomiting, diarrhea, and/or abdominal pain) in people acutely infected with WNV (), and WNV antigens have been detected in the intestine (). However, the consequences of WNV infection on GI tract function have not been explored.

West Nile virus (WNV) is a mosquito-transmitted flavivirus that causes an acute febrile illness with a subset of cases progressing to meningitis, encephalitis, and death (). WNV is neurotropic, and infection results in injury to neurons in the cerebral cortex, brain stem, and spinal cord (). WNV is related genetically to several other neurotropic flaviviruses, including Zika virus (ZIKV), which causes congenital malformations in developing fetuses during pregnancy (), and Powassan virus (POWV), an emerging tick-transmitted flavivirus that causes neuroinvasive disease and long-term neurological sequelae in 50% of survivors ().

The enteric nervous system (ENS) is comprised of complex neural and glial networks. The myenteric plexus is situated between inner circular and outer longitudinal muscle layers of the bowel (muscularis propria) and primarily controls gut motility. The submucosal plexus is located between the muscularis propria and the mucosa, where it regulates intestinal epithelial function and repair, intestinal blood flow, and responses to sensory stimuli (). ENS dysfunction or degeneration causes several intestinal dysmotility disorders, which present a considerable burden on human health. It is estimated that 10% to 30% of the population of Western countries suffers from some form of intestinal dysmotility (). One major diagnostic classification, irritable bowel syndrome (IBS), affects 10% of the population (), causing abdominal pain and diarrhea or constipation. Rare disorders, including chronic intestinal pseudo-obstructive syndrome, Hirschsprung disease, achalasia, and gastroparesis, also cause substantial morbidity and mortality. Dysmotility disorders with established organic causes, like Hirschsprung disease, typically manifest in childhood (), whereas acquired dysmotility disorders are idiopathic but often appear to follow infections or inflammatory events (reviewed in).

To determine if the secondary inflammatory stimulus resulted in immune cell infiltration of the muscularis propria, we performed flow cytometry analysis of T cell and myeloid cell populations ( Figures 7 F–7K) on mid- and distal small intestine tissue 11 dpi with VEEV TC-83. Despite confirming that GI transit was still delayed 10 days after VEEV TC-83 infection in mice previously infected with WNV ( Figure S6 D), we did not observe an increase in number of CD45leukocytes, CD8T cells, WNV-specific CD8T cells, or Ly6Cmonocytes compared to age-matched, sham-infected controls ( Figures 7 F–7K and S6 E). This suggests that the exacerbated slowing of GI transit induced by VEEV TC-83 in the chronic phase after WNV infection is not caused by new immune cell infiltration.

A hallmark of some GI dysmotility disorders in humans is the intermittent nature of symptoms with diarrhea and/or constipation and abdominal pain occurring episodically (). Moreover, symptom exacerbation may be associated with intercurrent infection or inflammation (). We tested if mice surviving WNV infection were sensitized to inflammation-induced bowel dysmotility using stimuli that do not slow transit in sham-infected mice ( Figure 7 C). We inoculated mice in the convalescent stage with either a live-attenuated viral vaccine (Venezuelan equine encephalitis [VEEV], TC-83 strain; Figure 7 D) at 4 weeks post WNV infection or a non-infectious immune stimulus (poly(I:C); Figure 7 E) at 8 weeks post WNV infection. Bowel transit was assessed by gavage of carmine red dye on days 3 and 10 after VEEV TC-83 infection or poly(I:C) treatment. Remarkably, at both 4 and 8 weeks post infection, surviving WNV-infected mice showed slower GI transit in response to VEEV TC-83 or poly(I:C). In comparison, sham-infected mice administered VEEV TC-83 or poly(I:C) did not show altered GI transit times. Analysis of intestinal tissues collected on day 3 after VEEV TC-83 infection showed VEEV RNA levels that were predominantly below the limit of detection ( Figure S6 C), suggesting that delayed transit did not result from infection of the GI tract by this attenuated virus. Thus, systemic infection by WNV induces chronically delayed GI transit that is exacerbated by unrelated inflammatory stimuli.

We performed longitudinal studies of GI transit on cohorts of WNV- and sham-infected mice using passage of carmine red dye in feces at multiple times post infection. A defect in GI transit secondary to KUNV and WNV infection was maintained as far out as 4 and 7 weeks post infection, respectively ( Figures 7 A and 7B ). Most WNV-infected mice showed delayed transit of dye during the acute stages of infection. Animals surviving WNV infection sustained persistent dysmotility in the convalescent phase (days 21–49). Mice that ultimately succumbed to WNV developed the most severe delays in GI transit during the acute phase of infection (transit time > 5 hr post gavage) ( Figure 7 A, blue symbols). We tested if this clinical phenotype could be mitigated by treating WNV-infected mice with hE16, a humanized anti-WNV mAb (). Therapy was initiated at 4 dpi, when high levels of WNV RNA already were present in all segments of the GI tract (see Figures 2 A–2E). Compared to isotype control mAb-treated mice, hE16-treated animals did not develop GI dysmotility by 10 dpi ( Figure S6 A) or succumb to infection through 21 dpi ( Figure S6 B).

Lines indicate median values (A, D, and E), and dotted lines indicate the limit of detection (A and C–E). Results are from three experiments with n = 11 (6 dpi, isotype control), n = 10 (10 dpi, isotype control) and n = 11 (6 and 10 dpi, hE16) (A); n = 11 (WNV + isotype control and WNV + hE16) (B); two experiments with n = 10 (day 3, VEEV TC-83) (C) and three experiments with n = 5 (sham + PBS), n = 8 (WNV + PBS), n = 6 (sham + VEEV TC-83) and n = 9 (WNV + VEEV TC-83) (D and E) and mice per group. Carmine red dye transit time and survival of hE16-treated mice were compared to isotype control mAb-treated mice by Mann-Whitney and log-rank tests, respectively. VEEV TC-83-infected animals were compared to age matched sham-infected controls by Mann-Whitney test (ns, P > 0.05; ∗ , P < 0.05; ∗∗ , P < 0.01).

(D and E) (E) WNV-infected mice secondarily infected with VEEV TC-83 were assessed for GI dysmotility by carmine red dye gavage at 10 dpi, and CD45 + leukocytes were analyzed by flow cytometry (E) from the distal small intestine on 11 dpi. Symbols show results from individual animals.

(C) Indicated GI tract tissues were collected from naïve mice immunized with VEEV TC-83 on day 3 post-inoculation, and VEEV RNA in tissue homogenates was quantified by qRT-PCR.

(A and B) WNV-infected mice treated with a single 500 mg dose of isotype control or humanized E16 (hE16) mAb at 4 dpi were gavaged orally with carmine dye to assess GI transit (A) at indicated time points and then monitored for mortality (B) through 21 dpi.

Results are from three (A, B, E, and F–K) or five (D) experiments with n = 15 (sham) and n = 25 (WNV) (A); n = 13 (day 28 sham) and n = 14 (28 dpi, KUNV) (B); n = 15 (day 28 sham + VEEV TC-83) and n = 15 (28 dpi, WNV + VEEV TC-83) (D); n = 15 (day 56 sham + poly(I:C)) and n = 16 (57 dpi, WNV + poly(I:C)) (E); and n = 5 (sham + PBS), n = 8 (WNV + PBS), n = 6 (sham + VEEV TC-83), and n = 9 (WNV + VEEV TC-83) (F–K) mice per group. (A and B and D and E) Dotted lines correspond to twice the baseline transit time; if no fecal pellets were produced, the experiment was stopped at 360 min. Infected mice were compared to sham-infected controls by a one-way ANOVA Kruskal-Wallis with Dunn’s test (ns (not significant); p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

(F–K) Black lines show median time when carmine red appears in fecal pellets. Total numbers and proportions of CD8 + T cells (F and I), WNV-specific CD8 + T cells (NS4B tetramer + ; G and J) and Ly6C hi Ly6G − monocytes (H and K) were assessed by flow cytometry from the distal small intestines of mice at 11 dpi with VEEV TC-83 or PBS treatment. Data are presented as total (F–H), percent of CD45 + cells (I and K), and percent of CD8 + T cells (J).

(D–E) Surviving mice were monitored for GI tract dysmotility at 28 and 56 dpi (C [scheme]) and subsequently immunized subcutaneously with live-attenuated VEEV TC-83 vaccine at 32 dpi (D) or administered via an intraperitoneal route 100 μg of poly(I:C) at 60 dpi (E).

(A and B) GI tract transit was evaluated by oral gavage of carmine red dye prior to and during the acute and convalescent phases of WNV (A) and KUNV (B) infection. Blue data points represent individual mice that succumbed to WNV infection.

Analysis of the distal small intestine from CD8amice at 6 dpi showed an absence of cellular infiltration of the myenteric ganglia ( Figure 6 D; dashed lines, muscularis propria; solid lines, myenteric ganglia) and few detectable muscularis- and ganglia-associated TUNEL-positive cells ( Figure 6 E). Blinded quantitation of the muscularis and myenteric ganglia revealed no differences between sham and WNV-infected CD8amice at 6 dpi in the number of TUNEL-positive cells ( Figure 6 F) or proportion of TUNEL-positive myenteric ganglia ( Figure 6 G). These data support the hypothesis that CD8T cells contribute to the early phase of GI dysmotility in WNV-infected mice. We did not evaluate whether CD8T cells modulated the later phase of acute GI transit dysfunction in WNV- or ZIKV-infected mice because by that time, greater than 95% of CD8amice succumb to uncontrolled infection in the brain and spinal cord ().

We hypothesized that infiltrating WNV-specific CD8T cells caused immune-mediated injury of enteric ganglia that led to bowel dilation and delayed GI transit. To test this hypothesis, we inoculated Rag1, Tcrβtm, and CD8amice, which respectively lack B and T cells, αβ and γδ T cells, or only CD8T cells. At day 6 after WNV infection, all three mouse strains had FITC-dextran transit patterns that were comparable to sham-infected controls ( Figure 6 A), even though viral RNA levels in their GI tract were equivalent to or higher than WT mice ( Figures S5 D–S5H). To confirm their contribution to WNV-induced acute GI dysmotility, we adoptively transferred CD8T cells (5 × 10cells per mouse) harvested from lymphoid tissues ( Figure S5 I) of sham (naive) and WNV-infected (day +7) donor mice to WNV-infected recipient CD8amice ( Figure 6 B) and analyzed FITC-dextran transit. Animals injected with PBS or administered naive T cells did not display delayed GI transit, whereas those given CD8T cells from WNV-infected donors exhibited delayed GI transit ( Figure 6 C).

Black boxes (D) represent areas shown at higher magnification in panels beneath each image. Dashed lines show muscularis propria; solid lines show myenteric ganglia. Scale bars: 100 μm (E [top row]), 50 μm (D [top row] and E [bottom row]), and 20 μm (D [bottom row]). The results are from three experiments with n = 11 (Rag1 −/− sham), n = 11 (Rag1 −/− + WNV), n = 17 (Tcrβtm −/− sham), n = 11 (Tcrβtm −/− + WNV), n = 9 (CD8a −/− sham), and n = 11 (CD8a −/− + WNV) (A); n = 8 (CD8a −/− mock), n = 9 (CD8a −/− + sham naive donor), and n = 12 (CD8a −/− + WNV donor) (C); and n = 5 (CD8a −/− sham) and n = 8 (CD8a −/− WNV) (D–G) mice per group. Motility in infected mice was compared to sham-infected mice using a Kruskal-Wallis one-way ANOVA test, and total numbers of muscularis and ganglia-associated TUNEL-positive cells were compared by Mann-Whitney test (ns (not significant); p > 0.05, ∗ p < 0.05).

(D and E) Sections of distal small intestine were collected on 6 dpi from CD8a −/− mice and stained with H&E (D) or with TUNEL and Tuj1 antibody (E).

(A–C) FITC-dextran fluorescence GI transit measurements in Rag1 −/− , Tcrβtm −/− , and CD8a −/− (A) or in recipient CD8a −/− (B and C) mice that were adoptively transferred at 2 dpi CD8 + T cells from sham (naive)- or WNV-infected WT donors harvested at day 7. Data are mean ± SD.

To determine if WNV infection of the myenteric plexus resulted in death of ENS ganglion cells, we performed TUNEL staining on sections of distal small intestine collected at 6 and 10 dpi ( Figure 5 A). We observed TUNEL-positive cells in the muscularis layer ( Figure 5 B) and enteric ganglia of the intestines of WNV-infected mice on both days ( Figures 5 A [white arrow] and 5 C). Blinded quantification showed that a significant fraction of myenteric ganglia was TUNEL-positive after WNV infection ( Figure S5 A). Additionally, whole-mount preparations of the distal small intestine at 6 dpi showed colocalization of WNV antigens and cleaved caspase 3 in enteric ganglia ( Figure 5 D). In comparison, staining for cleaved caspase 3 in sham-infected animals was not observed. By 28 dpi, the density of Phox2bneurons in whole-mount sections of the distal small intestine was reduced compared to sham-infected controls ( Figures S5 B and S5C). Thus, WNV infection of the GI tract results in structural modification of the ENS, including death of myenteric neurons.

(I) Flow cytometry analysis of splenocytes from WNV-infected mice before or after enrichment for CD8 + T cells. Bars indicate median values, and dotted lines indicate the limit of detection of the qRT-PCR assay. Data are from three (A and D–I) or four (B and C) experiments with n = 8 (day 6, sham), n = 11 (WNV 6 dpi), n = 7 (day 10, sham), n = 14 (WNV 10 dpi) (A); n = 7 (day 28, sham), n = 11 (WNV 28 dpi) (B and C); and n = 11 (Rag1 −/− + WNV), n = 20 (Tcrbtm −/− + WNV), and n = 10 (CD8a −/− + WNV) (D–H) mice per group. WNV-infected animals were compared to sham-infected controls (A and C) by Mann-Whitney or WNV-infected WT mice (D–H) by one way ANOVA with a multiple comparisons correction ( ∗ , P < 0.05; ∗∗ , P < 0.01; ∗∗∗∗ , P < 0.0001).

(D–H) Portions of stomach (D), proximal (E), mid (F), and distal small intestine (G), and proximal colon (H) from WNV-infected Rag1, Tcrbtm, and CD8amice were collected at indicated times, and WNV RNA levels were quantified by qRT-PCR analysis from tissue homogenates. Data from congenic WT mice are from Figure 2 and included for reference.

(B and C) (B) Whole mount sections of terminal ileum from sham and 28 dpi WNV-infected WT mice were stained for Phox2b (neuronal nuclei; red), Tuj1 (neurites; green) and nuclei (DAPI; blue) to assess myenteric neuron density (C).

(A) Proportion of TUNEL-positive myenteric ganglia from WT mice at indicated times post-infection were quantified in a blinded manner in distal small intestinal sections for a distance corresponding to 100 crypts.

Images are representative of three experiments with n = 8 (day 6, sham), n = 11 (day 6, WNV), n = 7 (day 10, sham), n = 14 (day 10, WNV) (A–C), and n = 6 (sham) and n = 5 (day 6, WNV) (D) mice per group. Scale bars: 100 μm (A [top row] and D) and 50 μm (A [bottom row]). Results in (B) and (C) are from three experiments (Mann-Whitney test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

(A) Sections of distal small intestine from sham- and WNV-infected mice (6 and 10 dpi) were stained with TUNEL (red) for neurons and nerve fibers (Tuj1, green) and cell nuclei (Hoechst, blue). White boxes represent areas shown at higher magnification in panels beneath each image. White arrows represent ganglia-associated apoptotic cells.

Flow cytometry of muscularis propria cells obtained from tissue during the acute (6 dpi) and chronic (28 dpi) phases of WNV infection ( Figure S4 E) did not show significant increases in the total number of CD45or CD8cells ( Figures 4 I and S4 F). However, the proportion of CD8T cells was elevated in infected mice at 6 dpi ( Figure 4 L). Moreover, the number of WNV-specific, D-NS4B peptide tetramerCD8T cells was elevated at 6 dpi ( Figure 4 J) and represented a significant fraction of CD8T cells in the muscularis layer at both time points ( Figure 4 M). Similar to the T cell data, numbers of CD11bF4/80CD64CX3CR1macrophages in the muscular layer were not different between sham- and WNV-infected mice at either time point (data not shown), although the proportion of monocytes (CD11bLy6CLy6G) was higher at 6 dpi ( Figure 4 N). We did not observe differences in proportions or numbers of CD4T cells or neutrophils (CD11bLy6CLy6G) at either time point post infection (data not shown). These data show that WNV infection of the GI tract induces an immune cell infiltration that includes an influx of antigen-specific CD8T cells.

We evaluated the composition of the immune cell infiltrate in the intestine associated with WNV infection in more detail. Immunohistochemical staining showed CD3T cell infiltration of the muscularis and myenteric ganglia of the proximal, mid-, and distal small intestine ( Figures 4 A, S4 A, and S4C; white arrows). At 6 dpi, the numbers of muscularis-infiltrating ( Figure 4 C) and ganglia-associated ( Figure 4 F) CD3T cells were higher throughout the length of the GI tract compared to sections from sham-infected animals and were statistically significant in the mid- and distal small intestine. By 10 and 28 dpi, the numbers of CD3T cells were diminished in the muscularis and myenteric plexus ganglia ( Figures 4 B, S4 B, and S4D) of all intestinal segments ( Figures 4 D, 4E, 4G, and 4H), and this finding correlated with decreased levels of WNV RNA ( Figure 2 ).

Data are presented as total number of cells per gram of tissue. Images are representative of three experiments with n = 8 (day 6, sham), n = 11 (day 6, WNV), n = 7 (day 10, sham), n = 14 (day 10, WNV), and two experiments with n = 4 (day 28, sham) and n = 8 (day 28, WNV) mice per group. White arrows denote CD3 + T cells. Scale bars: 100 µm (A–D, top row) and 50 µm (A–D, bottom row). WNV-infected animals were compared to age matched sham-infected controls (Mann-Whitney test; ns, P > 0.05).

(A–D) Sections of proximal and mid small intestine from sham- and WNV-infected mice were stained for enteric glia (GFAP; green), T cells (CD3; red) and cell nuclei (Hoechst; blue) at 6 and 10 dpi (A, C) and 28 dpi (B, D).

(I–N) Flow cytometry quantification of cells per gram of tissue (I–K) and proportions (L–N) of CD8 + T cells, WNV NS4B-specific CD8 + T cells, and Ly6C hi Ly6G − monocytes per gram of small intestinal tissue from sham, 6 dpi, and 28 dpi. Flow cytometry data are from three experiments with n = 6 (day 6 sham), n = 9 (6 dpi), n = 6 (day 28 sham), and n = 8 (28 dpi).

(C–H) The number of CD3 + T cells within the muscularis layer (C–E) or GFAP + myenteric ganglia (F–H) was quantified along a length of indicated GI tissues of 100 crypts. Data are from three experiments with n = 8 (day 6 sham), n = 11 (6 dpi), n = 7 (day 10 sham), n = 14 (10 dpi), n = 4 (day 28 sham), and n = 8 (28 dpi) mice per group. White arrows in (A) represent CD3 + T cells. Scale bars: 100 μm and 50 μm (A and B, top and bottom rows, respectively).

(A and B) Sections of the distal small intestine from sham- and WNV-infected mice were stained for enteric glia (GFAP, green), infiltrating T cells (CD3, red), and cell nuclei (Hoechst, blue) at 6 and 10 dpi (A) and 28 dpi (B).

Despite the severe phenotype in the muscularis propria, high levels of viral RNA, and altered GI transit, WNV infection at 6 and 10 dpi did not result in villus blunting, gross or microscopic hemorrhage, or increased cell death of the epithelium ( Figures 3 D and S3 C) compared to naive mice. However, leukocyte infiltration of the muscularis propria and myenteric plexus ganglia was apparent in sections from WNV-infected, but not sham-infected, animals. Immune cell infiltration was most pronounced at 6 dpi ( Figures 3 D and S3 C; dashed lines, muscularis propria; solid lines, myenteric ganglia) with reduced numbers of immune cells seen at 10 and 28 dpi ( Figures 3 D and S3 C; dashed lines, muscularis propria; solid lines, myenteric ganglia). Neurons within the myenteric ganglia displayed nuclear hypertrophy ( Figure S3 D) at 28 dpi, suggesting a compensatory response to viral- or immune-mediated damage to the ENS ().

As histological sections sample only a portion of a ganglion, we stained whole mount preparations of the distal small intestinal muscularis propria collected on 6 dpi for Tuj1, glial fibrillary acidic protein (GFAP), and WNV antigens ( Figure 3 C). While no WNV antigen staining was detected in sham-infected mice ( Figure 3 C, top row), we observed costaining for WNV antigens and Tuj1enteric neurons with a range of viral antigen-positive cells among infected animals. Some ganglia showed dispersed clusters of infected neurons ( Figure 3 C, middle row, white arrows), whereas others had WNV infection in most neuronal bodies within the ganglia ( Figure 3 C, bottom row, white arrows) and in their axons ( Figure 3 C, bottom row, white arrow heads). However, WNV antigen staining did not colocalize with glial cell or macrophage markers ( Figure 3 C; data not shown).

To determine the cellular targets of flavivirus infection in the intestine, we analyzed WNV tropism in the mid- and distal small intestines, the areas of highest penetrance of the phenotype. Intestinal sections of WNV- or sham-infected mice were stained for viral antigens and the marker Tuj1 (an antibody to neuron-specific beta-3 tubulin). We detected WNV antigens that were confined to the ganglia of the myenteric plexus of the distal small intestine ( Figure 3 A; white arrow) and throughout the remainder of the small intestine ( Figures S3 A–S3B; white arrows), as well as in isolated neurons of the submucosal plexus ( Figure S3 B; white arrowhead) on 6 dpi. WNV and Tuj1 double-positive myenteric ganglia were quantified in a blinded manner from tissues collected at 6 dpi ( Figure 3 B). Sections of myenteric ganglia contained detectable WNV antigens throughout the small intestine. We did not observe viral antigens in the GI tract epithelium. These data support the conclusion that WNV preferentially infects enteric neurons of the myenteric and submucosal plexus.

Images are representative from three experiments with n = 8 (day 6, sham), n = 11 (day 6, WNV), n = 7 (day 10, sham), n = 14 (day 10, WNV) (A–C); and two experiments with n = 4 (day 28, sham) and n = 8 (day 28, WNV) (D) mice per group. Scale bars: 100 µm (A and B, top row), 50 µm (A and B, bottom row, C, top row), and 20 µm (C, bottom row). For assessment of neuronal nuclei area, the area of 100 nuclei per tissue sample was quantified. WNV-infected animals were compared to age matched sham-infected controls (Mann-Whitney test; ∗∗∗∗ , P < 0.0001).

(C) H & E stained sections of proximal and mid small intestine from naïve, and 6, 10, or 28 dpi. Black boxes: areas at higher magnification in panels beneath. Red and black arrows: myenteric ganglia with and without immune cell infiltration, respectively.

(A and B) Sections of proximal (A) and mid small intestine (B) from WNV infected mice at day 6 were stained for neurons and nerve fibers (Tuj1; green), WNV antigen (red), and cell nuclei (Hoechst; blue). White boxes: areas shown at higher magnification in panels beneath each image. White arrows: WNV-infected ganglia.

Dashed lines represent muscularis propria; solid lines represent myenteric ganglia. Images are from three experiments with n = 8 (day 6, sham), n = 11 (day 6, WNV), n = 7 (day 10, sham), n = 14 (day 10, WNV), two experiments with n = 4 (day 28, sham), and n = 8 (day 28, WNV) (A and D) and three experiments with n = 6 (sham) and n = 5 (day 6, WNV) (C) mice per group. Scale bars: 100 μm (A [top row] and C), 50 μm (A [bottom row] and D [top row]), and 20 μm (D [bottom row]). Results in (B) are from three experiments (Mann-Whitney test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

(B) Total and WNV-antigen-positive myenteric ganglia were quantified in indicated tissue sections for a distance of 100 crypt units and expressed as percentage of ganglia infected with WNV.

(A) Sham- and WNV-infected sections from distal small intestine at 6 dpi were stained for neuronal cell bodies and nerve fibers (Tuj1, green), WNV antigen (red), and cell nuclei (Hoechst, blue). White boxes represent areas shown at higher magnification in panels beneath each image. White arrows show representative WNV-infected ganglia.

Sections of paraffin embedded bowel histoloigic sections (A and B) and muscularis propria whole-mount preparations (C) from distal small intestine were analyzed by immunofluorescence microscopy for WNV antigens or stained with H&E (D).

To test whether systemic viral infections more generally cause changes in GI transit, we inoculated 5-week-old WT mice with a pathogenic strain of chikungunya virus (La Reunion, 2006), an unrelated arthritogenic alphavirus that rarely can spread to the central nervous system (CNS) (). We then measured viral RNA levels and GI transit after FITC-dextran gavage at 6 dpi. Chikungunya virus (CHIKV) accumulated in the spleen ( Figure S2 H) at 8 and 10 dpi as expected (), but levels in GI tract tissues were much lower ( Figures S2 I–S2M). Moreover, CHIKV-infected mice did not develop intestinal dilation at 6 or 10 dpi ( Figures 1 A and 1D). Consistent with this observation, FITC-dextran transit was not altered at 6 dpi in CHIKV-infected as compared to sham-infected mice ( Figure S2 N). Thus, systemic RNA virus infections do not generally dysregulate GI tract motility.

As some flavivirus infections are associated acutely with GI symptoms () and delayed gastric emptying (), we hypothesized that the dilated bowel observed after WNV infection might occur because of defects in bowel motility. To test this hypothesis, we measured the luminal transit of 70-kDa fluorescein isothiocyanate (FITC)-labeled dextran during the acute phase of WNV ( Figure 2 F), ZIKV ( Figure 2 G), POWV ( Figure 2 H), and KUNV ( Figure 2 I) infections. Since 70-kDa FITC-dextran is poorly absorbed by the intact intestine, luminal concentrations in each bowel region after oral gavage provide a measure of GI transit. FITC-dextran transit was slowed in many of the WNV-infected mice compared to sham-infected controls at 6 and 10 dpi ( Figure 2 F). Analogous results were obtained in WT mice treated with anti-Ifnar1 mAb and inoculated with ZIKV ( Figure 2 G) or in WT mice infected with POWV ( Figure 2 H), although the effect was delayed compared to WNV-infected animals. In comparison, mice administered anti-Ifnar1 mAb alone or isotype control mAb and ZIKV showed no evidence of delayed GI transit ( Figure 2 G). Even mice infected with the attenuated KUNV variant displayed delays in GI transit time that were comparable to those observed in WNV-infected animals at 6 dpi, although the effect waned in most mice by 10 dpi ( Figure 2 I).

We next assessed infection of the GI tract with other neurotropic flaviviruses. Mice treated with anti-Ifnar1-blocking mAb and inoculated with ZIKV had viral RNA detected in all sections of the GI tract in the majority of animals ( Figures S2 B–S2F). WT mice inoculated with POWV, a tick-borne flavivirus, also sustained high levels of viral RNA in all GI tract tissues tested by 6 dpi ( Figure S2 G).

To determine if the GI pathology was a result of direct intestinal infection, defined segments of the GI tract were analyzed after infection to quantify flavivirus RNA ( Figures 2 A–2E and S2 A–S2G). After extensive tissue perfusion, we measured viral RNA levels, because infectious virus is inactivated by bile acids in the tissue homogenates. As early as day 2 after WNV infection, viral RNA was detected in regions of the GI tract of a subset of animals, and by day 4, all mice showed WNV infection in virtually all regions ( Figures 2 A–2E). Viral RNA levels peaked at 6–8 days post infection (dpi), with the highest viral titers observed in the mid- and distal small intestine before declining at 21 dpi. Viral RNA remained detectable through 56 dpi in all segments of the GI tract but was more prevalent in the colon at this time. We also inoculated mice with Kunjin virus (KUNV), a naturally attenuated variant of WNV (), and found more variable infection of the GI tract at day 6, although viral RNA was present in the small intestine in most animals ( Figure S2 A).

Results are from three experiments with n = 10 (KUNV 6 dpi) (A); n = 15 (anti-Ifnar1 mAb + ZIKV) mice per time point (B–F); n = 10 (POWV 6 dpi) (G); n = 11 (day 8 and 10, CHIKV) (H–M) and n = 10 (day 6, sham or CHIKV) (N). CHIKV-infected mice were compared to sham infected mice (Mann-Whitney test; (ns, P > 0.05)).

(H–M) Portions of spleen (H), stomach (I) proximal (J), mid (K), distal (L) small intestine, and proximal colon (M) from CHIKV-infected WT mice were collected at indicated times, and CHIKV RNA in tissue homogenates was quantified by qRT-PCR.

(A–G) Sections of stomach, proximal, mid, distal small intestine and proximal colon were collected at indicated times from KUNV (A), ZIKV (B–F) or POWV (G) infected mice at indicated time points, and viral RNA was quantified by qRT-PCR from tissue homogenates.

Results are from three experiments with n = 11–20 animals per time point (A–E) and with n = 10 (day 6, sham), n = 15 (day 6, WNV), n = 10 (day 10, sham), and n = 14 (day 10, WNV) (F); n = 12 (day 6 isotype mAb + ZIKV), n = 14 (day 6 anti-Ifnar1 mAb + ZIKV), n = 8 (day 10 isotype mAb + ZIKV), n = 14 (day 10 anti-Ifnar1 mAb + ZIKV), and n = 15 (day 10 anti-Ifnar1 mAb only) (G); n = 7 (day 6, sham), n = 8 (day 6, POWV), n = 9 (day 10, sham), and n = 11 (day 10, POWV) (H); and n = 10 (day 6, sham), n = 20 (day 6, KUNV), n = 12 (day 10, sham) and n = 13 (day 10, KUNV) (I). Motility was compared using a Kruskal-Wallis one-way ANOVA with Dunn’s test (F–I) (not significant [ns]; p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01).

(F–I) GI tract motility in WNV-infected (F), isotype- and anti-Ifnar1-mAb treated, ZIKV-infected (G), POWV-infected (H), and KUNV-infected (I) WT mice was measured by FITC-dextran fluorescence transit 2 hr post-oral gavage at 6 or 10 dpi. Data are mean ± SD of FITC-dextran distribution (see STAR Methods ).

(A–E) Sections of stomach (A), proximal (B), mid (C)-, and distal (D) small intestine and proximal colon (E) were collected after WNV infection. Viral RNA was quantified by qRT-PCR from tissue homogenates. Dotted lines represent limit of detection of assay.

To define the consequences of flavivirus infection in the GI tract, we inoculated wild-type (WT) C57BL/6 mice with WNV (New York, 1999) or ZIKV (Dakar, 1984). WNV-infected mice displayed dilatation of intestinal segments that was readily apparent by day 8 and showed increased penetrance by day 10 ( Figures 1 A and 1B ). Comparable penetrance of a similar intestinal phenotype occurred in ZIKV-infected mice that were additionally treated with anti-Ifnar1 monoclonal antibody (mAb) ( Figures 1 A and 1C); blockade of type I interferon (IFN) signaling was necessary to facilitate systemic ZIKV infection (). We noted some degree of regional specificity of intestinal dilation, as the greatest frequency of lesions occurred in the mid- and distal small intestines of WNV ( Figures S1 A–S1E)- and ZIKV ( Figures S1 F–S1J)-infected mice. None of the sham-infected littermate mice showed evidence of bowel dilation.

Data are from three experiments with n = 13 and 12 (WNV: 8 and 10 dpi, respectively), n = 10 and 13 (8 and 10 dpi, isotype mAb + ZIKV), and n = 15 (8 and 10 dpi, anti-Ifnar1 mAb + ZIKV) mice per group. Proportions of diseased tissues were compared to sham or isotype control mAb-treated mice (Chi-squared test; ns, P > 0.05; ∗∗ , P < 0.01; ∗∗∗∗ , P < 0.0001).

(A–J) Segments of GI tract were assessed for macroscopic pathology at indicated times after WNV infection ((A) stomach, (B) proximal, (C) mid, and (D) distal small intestine and (E) colon) or after ZIKV infection ((F) stomach, (G) proximal, (H) mid, (I) distal small intestine, and (J) colon).

(B–D) Proportions of mice with dilated bowel after WNV (B), ZIKV (C), or CHIKV (D) infection. Results are from three experiments with n = 10 (both day 8 and 10, sham), n = 13 and n = 12 (day 8 and day 10, WNV, respectively), n = 10 and n = 13 (day 8 and day 10 isotype mAb, ZIKV), n = 15 (day 8 and day 10 anti-Ifnar1 mAb, ZIKV), and n = 10 (day 10 sham and CHIKV) mice per group. Proportions were compared to sham-infected or isotype control-treated animals (Chi-squared test; ∗∗∗∗ p < 0.001; ns, not significant).

(A) Macroscopic GI tract pathology exhibited at day 10 after CHIKV, WNV, or ZIKV infection. GI tracts from naive and uninfected anti-Ifnar1-mAb-injected mice are shown as controls. Black and red arrows represent regions of intestinal dilation and adjacent more normal-appearing regions, respectively. Black boxes represent regions magnified in images to the right. Data are representative of three experiments.

Discussion

Klem et al., 2017 Klem F.

Wadhwa A.

Prokop L.J.

Sundt W.J.

Farrugia G.

Camilleri M.

Singh S.

Grover M. Prevalence, Risk Factors, and Outcomes of Irritable Bowel Syndrome After Infectious Enteritis: A Systematic Review and Meta-analysis. Brun et al., 2010 Brun P.

Giron M.C.

Zoppellaro C.

Bin A.

Porzionato A.

De Caro R.

Barbara G.

Stanghellini V.

Corinaldesi R.

Zaninotto G.

et al. Herpes simplex virus type 1 infection of the rat enteric nervous system evokes small-bowel neuromuscular abnormalities. Facco et al., 2008 Facco M.

Brun P.

Baesso I.

Costantini M.

Rizzetto C.

Berto A.

Baldan N.

Palù G.

Semenzato G.

Castagliuolo I.

Zaninotto G. T cells in the myenteric plexus of achalasia patients show a skewed TCR repertoire and react to HSV-1 antigens. Khoury-Hanold et al., 2016 Khoury-Hanold W.

Yordy B.

Kong P.

Kong Y.

Ge W.

Szigeti-Buck K.

Ralevski A.

Horvath T.L.

Iwasaki A. Viral Spread to Enteric Neurons Links Genital HSV-1 Infection to Toxic Megacolon and Lethality. Brun et al., 2018 Brun P.

Qesari M.

Marconi P.C.

Kotsafti A.

Porzionato A.

Macchi V.

Schwendener R.A.

Scarpa M.

Giron M.C.

Palù G.

et al. Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment. Khoury-Hanold et al., 2016 Khoury-Hanold W.

Yordy B.

Kong P.

Kong Y.

Ge W.

Szigeti-Buck K.

Ralevski A.

Horvath T.L.

Iwasaki A. Viral Spread to Enteric Neurons Links Genital HSV-1 Infection to Toxic Megacolon and Lethality. + T cell infiltration of the myenteric plexus, which occurred throughout the length of the small intestine but was more prominent in the mid- and distal segments and correlated with increased cell death within the muscularis propria and ganglia of the myenteric plexus. GI motility disorders in humans often occur following episodes of acute protozoan, bacterial, and viral gastroenteritis, termed post-infectious IBS (). Prior studies have demonstrated that viruses can infect the cells of the ENS and induce immune cell infiltration into the myenteric plexus (). Although viral infection is a postulated cause of human dysmotility disorders, causality has not been proven. Our findings are consistent with recent studies in mice () demonstrating that infection of the ENS by HSV can induce acute GI dysmotility, although Khoury-Hanold et al. described toxic megacolon, and Brun et al. showed that intestinal dysmotility was due to infiltrating macrophages. In comparison, WNV RNA persisted in the bowel of surviving animals, and this was associated with chronic delays in GI transit times. WNV infection of neurons of the ENS induced CD3T cell infiltration of the myenteric plexus, which occurred throughout the length of the small intestine but was more prominent in the mid- and distal segments and correlated with increased cell death within the muscularis propria and ganglia of the myenteric plexus.

Klem et al., 2017 Klem F.

Wadhwa A.

Prokop L.J.

Sundt W.J.

Farrugia G.

Camilleri M.

Singh S.

Grover M. Prevalence, Risk Factors, and Outcomes of Irritable Bowel Syndrome After Infectious Enteritis: A Systematic Review and Meta-analysis. The dysregulated GI transit that we observed following multiple flavivirus infections in mice shares features of acquired intestinal dysmotility syndromes in humans, including post-infectious IBS (). Animals developed a chronic motility disorder that lasted for weeks to months after acute infection with WNV or even the attenuated variant, KUNV. Remarkably, WNV-infected animals became vulnerable to exacerbated delays in GI transit time in response to unrelated secondary systemic inflammatory stimuli, including administration of a live-attenuated vaccine or non-infectious poly(I:C). Thus, infections by WNV and possibly other neurotropic viruses targeting the ENS can induce an acute, chronic, and potentially relapsing motility disorder, which might serve as a model for study and evaluation of therapies for acquired human GI disorders, including IBS. Indeed, treatment of WNV-infected mice at 4 dpi with a single dose of hE16 mAb prevented the acute dysmotility syndrome. As several neurotropic flaviviruses can infect non-human primates productively, confirmatory studies are planned to test whether similar GI transit disorders occur in animals more closely related to humans.

+ T cells contribute to the acute phase of GI tract dysmotility. At 6 dpi, when transit defects were seen in WT WNV-infected mice, intestinal transit was normal in WNV-infected Rag1−/−, Tcrβtm−/−, and CD8a−/− mice, all of which lack CD8+ T cells. Consistent with these data, adoptive transfer of WNV-primed, but not naive, CD8+ T cells into recipient CD8a−/− mice resulted in delayed GI transit in the context of WNV infection. WNV-infected CD8a−/− mice did not show increased TUNEL-positive staining within the muscularis propria and myenteric ganglia of the distal small intestine, indicating that CD8+ T cell-mediated cell death of ENS neurons correlates with development of acute GI tract dysmotility. The specific CD8+ T cell effector functions (e.g., perforin and granzyme secretion, Fas ligand expression, or cytokine production) required to mediate ENS damage remain to be elucidated. We were unable to evaluate the impact of CD8+ T cells on the chronic phase of GI tract transit dysfunction, because mice lacking CD8+ T cells succumb too rapidly to WNV and ZIKV ( Elong Ngono et al., 2017 Elong Ngono A.

Vizcarra E.A.

Tang W.W.

Sheets N.

Joo Y.

Kim K.

Gorman M.J.

Diamond M.S.

Shresta S. Mapping and Role of the CD8+ T Cell Response During Primary Zika Virus Infection in Mice. Shrestha and Diamond, 2004 Shrestha B.

Diamond M.S. Role of CD8+ T cells in control of West Nile virus infection. Our experiments in immune-deficient mice suggest that CD8T cells contribute to the acute phase of GI tract dysmotility. At 6 dpi, when transit defects were seen in WT WNV-infected mice, intestinal transit was normal in WNV-infected Rag1, Tcrβtm, and CD8amice, all of which lack CD8T cells. Consistent with these data, adoptive transfer of WNV-primed, but not naive, CD8T cells into recipient CD8amice resulted in delayed GI transit in the context of WNV infection. WNV-infected CD8amice did not show increased TUNEL-positive staining within the muscularis propria and myenteric ganglia of the distal small intestine, indicating that CD8T cell-mediated cell death of ENS neurons correlates with development of acute GI tract dysmotility. The specific CD8T cell effector functions (e.g., perforin and granzyme secretion, Fas ligand expression, or cytokine production) required to mediate ENS damage remain to be elucidated. We were unable to evaluate the impact of CD8T cells on the chronic phase of GI tract transit dysfunction, because mice lacking CD8T cells succumb too rapidly to WNV and ZIKV ().

High levels of WNV RNA were measured within all GI tract regions tested during the acute phase, and this was associated with bowel dilation, injury to ENS neurons, and infiltration of antigen-specific CD8+ T cells. Gradual clearance of WNV RNA from the intestines in surviving animals correlated with improved GI transit times and diminished immune cell infiltration. Notwithstanding this improvement, surviving mice still exhibited delayed GI tract transit at 49 dpi and did not return to baseline relative to sham-infected animals. As some of these animals had persistent WNV RNA throughout their intestines even at these late time points, the long-term presence of viral pathogen-associated molecular patterns (PAMPs), antigens, or immunogenic peptides could sustain inflammation and chronic GI dysmotility. Although we did not observe persistent T cell infiltration of the muscularis or myenteric plexus in surviving animals at 28 dpi by microscopy, we did detect WNV-specific CD8+ T cells within the small intestine by flow cytometry. However, upon administration of secondary, systemic inflammatory stimulus that exacerbated GI transit delays, the number and proportion of WNV-specific CD8+ T cells remained comparable to control animals, suggesting that the secondary bouts of GI dysmotility may not be caused by further CD8+ T cell infiltration of the myenteric plexus. The possible role of WNV persistence in maintenance of chronic GI dysfunction remains unclear. Viral and T cell-mediated structural changes to the ENS also could lead to a distinct response by myenteric neurons to inflammatory or neuroendocrine signals that exacerbate GI dysfunction even after viral infection is cleared.

Flavivirus infection in the GI tract of mice provides a possible model for defining mechanisms that cause persistent and relapsing GI motility disorders in humans. Whereas microbial agents that cause acute viral gastroenteritis (e.g., noroviruses and rotaviruses) infect cells in the mucosa of the GI tract, systemic infection with neurotropic viruses that target the neurons of the ENS may be a source of acute and persistent GI pathology and dysmotility. We wish to highlight that neurotropic flaviviruses are unlikely to be the major cause of common GI dysmotility syndromes in humans like IBS because of their low incidence. Rather, our data suggest that systemic infection by viruses (e.g., enteroviruses, herpesviruses, astroviruses, and flaviviruses) targeting neurons of the ENS may be an underappreciated cause of acquired bowel motility disorders or could cause some cases of more serious but less common GI motility disorders. A limitation of the current study is the exclusive reliance of experiments in mice. Ultimately, establishing the roles of neurotropic viral infections in the development of human GI transit disorders will require a rigorous, multi-institutional, and likely large serological study with well-characterized affected cohorts and matched controls.