Identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs

Conceição-Neto, Nádia; Theuns, Sebastiaan; Cui, Tingting; Zeller, Mark; Yinda, Claude Kwe; Christiaens, Isaura; Heylen, Elisabeth; Van Ranst, Marc; Carpentier, Sebastien; Nauwynck, Hans J.; Matthijnssens, Jelle

Extraction of proteins from the fecal sample was carried out as previously described by Carpentier et al. (2005) and Buts et al. (2014) . In short, 350 ll of fecal suspension were resuspended in 350 ll of ice-cold extraction buffer [50 mM Tris-HCl pH 8.5, 5 mM EDTA, 100 mM KCl, 1% w/v DTT, 30% w/v sucrose; complete protease inhibitor cocktail (Roche Applied Science)] and vortexed for 30 s. Seven hundred microliters of ice-cold Tris buffered phenol (pH 8.0) were added and the sample was vortexed for 10 min at 4 C. After centrifugation (10 min, 12,000Âg, 4 C), the phenolic phase was collected, re-extracted with 350 ll of extraction buffer and vortexed for 30 s. After centrifugation (5 min, 12,000Âg, 4 C), the phenolic phase was collected and precipitated overnight with five volumes 100 mM ammonium acetate in methanol at À20 C. After centrifugation at 16,000Âg for 30 min at 4 C, the supernatant was removed and the pellet was rinsed twice in ice-cold acetone/0.2% DTT. Between the two rinsing steps, the sample was incubated for 60 min at À20 C. The pellet was airdried, resuspended in 75 ll of lysis buffer (8 M urea, 5 mM DDT, 30 mM Tris DTT), and vortexed for 5 min at room temperature. Then the protein concentration was determined using the 2-Dquant kit from Amersham Biosciences.