Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanger expression

Abstract

Astroviruses are known to be a leading cause of diarrhea in infants and the immunocompromised; however, our understanding of this endemic pathogen is limited. Histological analyses of astrovirus pathogenesis demonstrate clinical disease is not associated with changes to intestinal architecture, inflammation, or cell death. Recent studies in vitro have suggested that astroviruses induce actin rearrangement leading to loss of barrier function. The current study used the type-2 turkey astrovirus (TAstV-2) and turkey poult model of astrovirus disease to examine how astrovirus infection affects the ultrastructure and electrophysiology of the intestinal epithelium. These data demonstrate that infection results in changes to the epithelial ultrastructure, rearrangement of F-actin, decreased absorption of sodium, as well as redistribution of the sodium/hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm. Collectively, these data suggest astrovirus infection induces sodium malabsorption, possibly through redistribution of specific sodium transporters, which results in the development of an osmotic diarrhea.

Fig 1. No evidence of histological changes following infection with TAstV-2

Sections of jejunum were collected from control and TAstV-2 infected poults at 4 dpi. Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, stained using H&E, and examined using light microscopy. White bar = 100µM. Panels are representative fields from 4 individuals per group.

Fig 2. TAstV-2 infection mediated changes to the apical ultrastructure of epithelial cells

Jejunum from control and TAstV-2 infected animals were collected at 4 dpi and analyzed by transmission electronic microscopy at the tip of villi, middle of villi, and the crypts. Normal tight junctions (TJ) are demarcated in the control micrographs, while electron dense aggregations (arrows) were observed in the TAstV-2-infected jejunum. White bar represents 0.5 µM

Fig 3. Actin reorganization following TAstV-2 infection

(A) Control and TAstV-2-infected jejunum sections collected at 4 dpi were stained for F-actin using phalloidin conjugated with Alexa fluor 546 (red), TAstV-2 nonstructural protein (green) and nuclei (blue), as detailed in methods. Arrow heads denote normal apical F-actin fluorescence, while arrows denote areas of rearrangement in actin. F-actin reorganization was prominent in the areas of virus localization (yellow color in merged panel). White bar represents 50µm. (B) Fluorescence images from TAstV-2 infected tissues were overlaid with a 25 µm grid. Individual grids were scored for actin rearrangement (normal/comparable with control tissues, moderate changes, severe changes). Grids were then assessed for the presence or absence of astrovirus fluorescence within the infected epithelium. Three villi, from 3 different samples were analyzed. The percent distribution of both parameters was calculated and means represented by bars. * denotes significant difference p < 0.001 between changes in actin in grids with TAstV-2 and grids without.

Fig 4. Cellular expression of NHE protein changes following TAstV-2 infection

Expression of sodium hydrogen exchangers (NHEs) in control and TAstV-2-infected jejunum at 4 days post infection was examined in whole tissue lysates (A and D) or detergent soluble (DS) and detergent insoluble (DIS) fractions (B, C, E, and F) as detailed in materials and methods section. Both NHE2 and NHE3 were identified as a single band of approximately 87 kd mass on western blots. β-actin bands are shown as loading controls for respective lanes in all blots. The densitometric data for NHE3 and NHE2 expression (NHE/β-actin) in whole tissue lysates is presented as mean ± SE in panel A and D. The densitometric data for NHE3 and NHE2 expression (NHE/β-actin) in detergent fractions is presented in panel C and F, respectively. All blots are representative of 3 or more experiments.