Inhibitory Activities of Bovine Macromolecular Whey Proteins on Rotavirus Infections In Vitro and In Vivo

Dose–response effects of macromolecular whey protein (MMWP) on WA, RRV, YM, and RF rotavirus infectivity of Caco-2 and FHs 74 Int cells in relation to controls (no protein). Logarithmic presentation: Wa infection of Caco-2 cells (open circles); RRV infection of Caco-2 cells (solid circles); YM infection of Caco-2 cells (open squares); RF infection of Caco-2 cells (solid squares); Wa infection of FHs 74 Int cells (open triangles); RRV infection of FHs 74 Int cells (solid triangles). Results are shown as mean±standard deviation of at least 2 experiments performed in triplicate. The first points with statistically significant effects (P<0.05) are indicated with an asterisk (*).

Separation of proteins in macromolecular whey protein (MMWP) by cation-exchange chromatography. (A) Purification was performed in 50mM ammonium acetate (pH 5.0) with 8 M urea on a 255-mL CM-Sepharose FF column. Proteins were eluted with a gradient of 0.05 to 1 Mammonium acetate (dotted line) and monitored at 280nm (solid line). After separation, the indicated fractions were selected for 5 pools, named CM1 to CM5. Insert: SDS-PAGE analysis of the obtained protein pools. Gels (18% acylamide with a Tris-glycine buffer system) were stained with Coomassie Brilliant Blue R-250. Lanes 1 to 5: CM1 to CM5, respectively. Positions of molecular mass standards are indicated. (B) Analyses of the effects on the infectivity of the Wa rotavirus onto Caco-2 cells of collected CM pools given as the mean±standard deviation of at least 2 experiments performed in triplicate. The first points with a statistically significant effect (P<0.05) are indicated with an asterisk (*).

Separation of proteins in CM-Sepharose column fraction 3 (CM3) by anion-exchange chromatography. (A) Purification was performed on an 88-mL Q-Sepharose FF column and the proteins were eluted with a gradient (dotted line) of ammonium bicarbonate (0.02 to 1 M). Absorbance at 280nm is shown (solid line). After separation, the indicated fractions were selected for 7 pools, named CM3Q1 to CM3Q7. Insert: SDS-PAGE (18%) analysis of the obtained CM3Q pools (Coomassie stained). Lanes 1 to 7: CM3Q1 to CM3Q7, respectively. Mobility of the molecular mass standards is indicated. (B) Ability of the collected CM3Q pools to affect Wa infectivity (Caco-2), given as mean±standard deviation of at least 2 experiments performed in triplicate. The first points with statistically significant effects (P<0.05) are indicated with an asterisk (*).

Effect of reducing agents on the isolated CM3Q3 fraction. Reduction was performed by adding 1,4-dithioerythritol to a final concentration at 18mM. Lanes 1 and 2: SDS-PAGE analysis of CM3Q3 (18%, Coomassie stained). Lanes 3 and 4: Western blot analysis of CM3Q3 probed with antibovine IgG antibodies. Reductive status of the sample buffer is indicated (+/−).

Mice (BALB/c) were challenged orally with 100 50% shedding doses of the murine EMcN rotavirus. (A) Rotavirus antigen shedding in stools. Results are given as the mean value±standard deviation of triplicate absorbance values at 492nm, which reflect the viral shedding for each mouse group (n=10 mice per group). Groups of mice were given the virus alone (control mice, open circles), or the virus preincubated with either BSA (solid circles), lactadherin (open squares), MUC1 (solid squares), the CM3Q3 fraction (open triangles), or macromolecular whey protein (MMWP; solid triangles). Serum samples were collected 3 wk after virus inoculation. (B) Rotavirus-specific serum IgG antibodies determined by ELISA. Values for absorbance at 492nm correspond to the 1:100 serum dilutions.

Effect on BALB/c mice (n=10 per group) of oral administration of a reduced amount of the murine EMcN rotavirus (10 50% shedding doses). (A) Rotavirus antigen shedding in stools of BALB/c mice (mean value±standard deviation). Groups of mice were given the virus alone (control mice, open circles), or the virus preincubated with either BSA (solid circles), lactadherin (open squares), MUC1 (solid squares), the CM3Q3 fraction (open triangles), or macromolecular whey protein (MMWP; solid triangles). Serum samples were collected 3 wk after virus inoculation. (B) Test for rotavirus-specific serum IgG antibodies in the challenged mice.