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 reverse-transcription polymerase chain reaction (RT-PCR) was performed on the original sample using the QIAGEN OneStep RT-PCR kit (Qiagen) using primer sequences shown in Table 1 (primers were designed to cover the breakpoint between the enterovirus and the torovirus-like sequence). The reaction was performed as follows: 50 C for 30 min followed by a PCR activation step at 95 C for 15 min, 40 cycles of amplification: 30 s at 94 C, 30 s at 55 C, and 2 min at 72 C, and a final extension step for 10 min at 72 C in a Biometra T3000 thermocycler (Biometra). PCR products were run on a polyacrylamide gel, stained with ethidium bromide, and visualized under UV light. Samples were then purified with ExoSAP-IT (Affymetrix), and positive products were Sanger sequenced with the ABI PRISM BigDye Terminator cycle sequencing reaction kit (Applied Biosystems). The raw chromatograms are provided in Supplementary Files S1 and S2, and the alignments used to generate Fig. 1 are also provided as Supplementary Data.