NSP4 PLAYS A MAJOR ROLE IN THE EARLY SECRETORY DIARRHEA INDUCED BY ROTAVIRUS IN A CACO-2 EXPERIMENTAL MODEL

Abstract

Aim: Among Rotavirus' (RV) multiple mechanisms of diarrhea a major role is played by viral enterotoxin NSP4. RV infection induces an early anion secretion and subsequent cell damage in a Caco-2 cell model. Aim of the study was to address the role of NSP4 in a model of RV infection in human-derived intestinal cell line. Methods: Caco-2 cells were infected with RV strain SA-11 (5PFU/cell) and mounted in Ussing chambers at different times after infection. Production of NSP4 was assessed by western blotting in immunoprecipitates of supernatants and cell lysates. Short circuit current (Isc) and trans-epithelial resistance (TEER), reflecting transepithelial ion transport and tissue integrity were recorded. In parallel experiments, non-specific human immunoglobulins (Ig) preparations for parenteral use and specific anti-NSP4 Ig were added to enterocyte monolayers. Results: RV induced a biphasic response with a significant increase in Isc after 1-3 hours of infection, consistent with active anion secretion. TEER started to fall progressively 24 hours after infection, indicating monolayer loss of integrity. Starting from 1 hour after infection, NSP4 was detected in the supernatant of infected cells at increasing concentrations up to 48 hours post-infection. Addition of anti-NSP4 Ig, as well as no-specific human Ig, blocked the RV-induced secretion but not TEER modifications. Discussion: RV induces a dual, chronologically distinct, pathogenic effect in human enterocytes: 1) an early anion secretion which is NSP4-dependent and inhibited by specific Ig and by non-specific human Ig; 2) a subsequent cytopathic damage which is not prevented by anti-NSP4 Ig but blocked by human Ig.