Fig. 4: effect of the frequency of dose application and post-inoculation time of application on the inhibition of ECwt infection by N-acetylcysteine (NAC). A: following the first diarrhoeal episode, ECwt-infected mice (n = 9 mice for each experimental group) were treated separately with one application of NAC (6 mg/kg/day) for one day or three applications (18 mg/kg/day) for one, two, three or four days, which corresponded to a total number of applications of one, three, six, nine or 12, respectively. Intestinal villi were isolated and analysed by immunochemistry using rabbit polyclonal antibodies (Abs) against rotavirus structural proteins (SP), horseradish peroxidase (HRP)-conjugate goat anti-rabbit Ab and aminoethylcarbazole substrate. Radio immunoprecipitation assay lysates from villi were analysed by ELISA using guinea pig Abs against rotavirus SP for capturing rotavirus antigen and rabbit Abs against rotavirus SP for its detection. Reaction was revealed with HRP conjugated goat anti-rabbit Ab and o-phenylenediamine dihydrochloride substrate before reading at optical density (OD) 492 nm . Graph shows significant NAC inhibitory effect from three NAC applications (p < 0.001); B: ECwt-infected mice (n = 9 mice for each experimental group) were treated with NAC (18 mg/kg/day distributed in 3 daily applications) after 24, 48, 72 and 96 h post-inoculation (h.p.i.) which corresponded to a total number of 12, nine, six and three applications, respectively. Rotavirus SP accumulated in villus cells was analysed by immunochemistry and ELISA as indicated in A. Data are from three separate experiments using three mice per experiment. Data are expressed as mean percentage ± standard deviation (SD) of infected cells or mean OD 492 nm ± SD of rotavirus antigen. Graph shows significant inhibitory effect when NAC treatment started before 72 h following the first diarrhoeal episode (p < 0.01).