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

A mixed diagnostic fecal sample of fattening pigs (n ¼ 12) from Belgium was sent to the Laboratory of Virology (Ghent University, Merelbeke, Belgium) in January 2015. No healthy controls were collected at the moment of the outbreak. The sample was prepared using a slightly modified version of the NetoVIR protocol (Conceição-Neto et al. 2015) . A 10% weight/volume fecal suspension in viral transport medium (DMEM, 10% P/S, 5% gentamycin, and 0.01% fungizone) was prepared from sample 15V010 and filtered through 0.45 lm membrane filters (Millipore). The filtrate was treated with a cocktail of Benzonase (Novagen) and Micrococcal Nuclease (New England Biolabs) at 37 C for 2 h to digest free-floating nucleic acids, in homemade buffer (1 M Tris, 100 mM CaCl 2 , and 30 mM MgCl 2 , pH ¼ 8). RNA and DNA were extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions but without addition of carrier RNA to the lysis buffer.