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

In this sample, 71,088 reads were assigned to the genus Enterovirus, of which 2 complete genomes could be retrieved. Interestingly, a contig of 8,043 nt in length was identified and assigned to enterovirus, with a partial in-frame torovirus-like gene insertion. Remapping of curated reads against this recombinant sequence using BWA (Li and Durbin 2009 ) as well as resequencing the insertion and breakpoints using Sanger sequencing on the original sample, confirmed that the obtained sequence was genuine and neither an artifact of the de novo assembly nor a result of random amplification. In Fig. 1B , a description of the genome organization of this novel recombinant enterovirus genome (Porcine enterovirus b 15V010/BEL/2015) is shown. As depicted in Fig. 1B , the torovirus-like gene insertion of 636 nt was present between the enterovirus 2 C and 3 A non-structural proteins. The inserted region formed a phylogenetic outgroup most closely related to several toroviruses. In the sample, we observed the presence of a small Torovirus contig of 256 bp, which was included in the phylogenetic tree (Fig. 1A , Torovirus/BEL/2015), showing very low similarity with the gene insertion found in the recombinant virus. Initial BLASTp searches attributed the highest aa similarity of this torovirus-like insertion to a porcine torovirus (66% aa similarity with NC_022787), but with the most recent reports of 2 similar recombinant viruses, it now shares 92.0% aa identity with the insertion of the recombinant enterovirus G from North Carolina (08/NC_USA/2015) (Shang et al. 2017 ) and 90.7% aa identity with the strain from Texas (EVG/Porcine/USA/Texas1/ 2014) (Knutson et al. 2017) . To infer whether the insertion was produced as a separate moiety or fused with the upstream or downstream enterovirus protein, we identified potential polyprotein cleavage sites, which were present up-and downstream of the insertion (Fig. 1B) . Pfam searches for motifs using hidden Markov models, revealed that the insertion seems to enclose an L-protease from the C28 family (e-value ¼ 1.3e À05 ). We also attempted to infer the function of the insertion using Phyre2 (Kelley et al. 2015) , which predicted a cysteine proteinase function (99.1% confidence, Fig. 1E ). The same function was also predicted for the FMDV L pro , but not for the porcine torovirus that showed the highest sequence homology with the insertion (Fig. 1E) .