Pools of samples including stool and bowel from litter 1 (advanced clinical signs), and litter 2 (initial clinical signs) were macerated by adding 5 ml of D-MEM (Gibco Cat. 10313-021 Lot. 1374740) in the presence of antibiotics (Penicillin 10.00 U/ml, streptomycin 10,000 mcg/ml Gibco Cat. 15140-122 and 200 mM L-glutamine, Gibco 25030-081), and centrifuged at 1500 rpm for 10 min. The supernatant was filtered using a Millex GP filter unit, 0.22 μm (Millipore Express PES membrane, Cat. SLGP033RB).
Vero cells were grown in six-well plates (Corning Inc. COSTAR 3527) previously washed with D-MEM (5 times) to remove the fetal bovine serum. These plates were then inoculated with filtered supernatants in D-MEM supplemented with different concentrations of trypsin (2.5, 5, 10, and 20 μg/ml, and 2 mg/ml) (DifcoTripsine 250 Cat. 215240, Lot. 4181462). After 2 h, this material was removed and the plates were filled with fresh complete medium containing trypsin.
At 48 h post-infection the cells were resuspended, collected, sonicated for 5 min at 37 °C (BransonicSonifier, 5510 Ultrasonic cleaner), and centrifuged at 5000 rpm. An aliquot of 500 μl was collected for posterior RNA extraction. The remaining supernatant was used to infect serial passages in Vero cells in the above conditions (Passage 2).
Supernatant titrations were performed with TCID50/ml, using Reed & Münch formula [16 ].
Vero cells were grown in six-well plates (Corning Inc. COSTAR 3527) previously washed with D-MEM (5 times) to remove the fetal bovine serum. These plates were then inoculated with filtered supernatants in D-MEM supplemented with different concentrations of trypsin (2.5, 5, 10, and 20 μg/ml, and 2 mg/ml) (DifcoTripsine 250 Cat. 215240, Lot. 4181462). After 2 h, this material was removed and the plates were filled with fresh complete medium containing trypsin.
At 48 h post-infection the cells were resuspended, collected, sonicated for 5 min at 37 °C (BransonicSonifier, 5510 Ultrasonic cleaner), and centrifuged at 5000 rpm. An aliquot of 500 μl was collected for posterior RNA extraction. The remaining supernatant was used to infect serial passages in Vero cells in the above conditions (Passage 2).
Supernatant titrations were performed with TCID50/ml, using Reed & Münch formula [16 ].