Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

Abstract

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

Importance: Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

FIG 1

TAstV-2 capsid protein induces time- and dose-dependent diarrhea. Turkey poults (n = 5 to 9 per group) were orally inoculated with PBS alone or 50 µg of purified recombinant TAstV-2 capsid in PBS. (A) Percentage of TAstV-2 capsid-treated animals with a clinical score of 3 or higher over time. (B) At 6 h postinoculation, poults were sacrificed and intestinal sections were stained for TAstV-2 capsid (green), actin (red), and nuclei (blue) and examined by immunofluorescence microscopy. Bar, 20 µm. (C) TAstV-2 capsid and virally inoculated poults were sacrificed at the indicated time points, and intestinal sections were stained for TAstV-2 capsid (green) and nuclei (blue) and examined by immunofluorescence assay. Bar, 20 µm. (D) Turkey poults (n = 3 to 7 per group) were inoculated with PBS, 50 µg TAstV-2 capsid, 50 µg of proteinase K (ProtK)-digested TAstV-2 capsid, or 50 µg HAstV-1 capsid and scored for diarrhea at 24 h posttreatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Administration of TAstV-2 capsid induces in vivo barrier permeability and disrupts NHE3 localization. (A and B) At 6 h postinoculation (hpi), intestines (n = 6) were harvested from treatment and control groups and mounted in Ussing chambers. Transepithelial electrical resistance (TER) (A) and mucosal-to-serosal flux of [³H]mannitol (B) were assayed as detailed in Text S1 in the supplemental material. (C) Turkey poults were inoculated with capsid protein or TAstV-2. At the indicated times, intestines were isolated and separated into membrane (M) or cytosolic (C) fractions. Proteins (5 µg) were separated by SDS-PAGE and probed for NHE3, Na⁺K⁺-ATPase, or β-actin. Samples were run in duplicate. (D) NHE3 localized in each fraction was quantified using ImageJ from six independent blots. NHE3 expression was normalized to β-actin. Na⁺K⁺-ATPase expression was used as a fractionation control. (E) PBS- and TAstV-2 capsid-inoculated turkey poults (n = 2 to 4 per group) were sacrificed at 24 hpi, and intestinal sections were stained for SGLT-1 (green) and nuclei (blue) and examined by immunofluorescence microscopy. Bar, 20 µm. Asterisks represent statistically significant differences compared to controls. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.