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

Here, an outbreak of diarrhea in a Belgian pig farm was reported, 2 days after the arrival of new pigs, suggesting that these novel pigs were the source of infection. Since PEDV was being reported in Europe at the time, it was first diagnostically tested for. Even though the sample was positive for PEDV (RT-qPCR), the viral loads were rather low (Cq > 30). Our study does not provide evidence for direct causality of disease by a single viral agent, and the high number of viral reads attributed to a porcine bocavirus, the presence of other possible causative agents of diarrhea such as astroviruses, enteroviruses, and picobirnaviruses, merely indicate that more work needs to be performed to elucidate the role of coinfections in gastrointestinal disease. Moreover, none of these viral species are routinely tested for at diagnostic laboratories in cases of diarrhea on pig farms. Alongside these findings, an enterovirus with an insertion of a torovirus was described. Due to the large number of viral reads attributed to bocavirus and the enterovirus-torovirus recombinant, we attempted to isolate them using primary porcine kidney epithelial cells and ST (swine testis) cells (data not shown). Although CPE was observed 2 days after inoculation on porcine kidney epithelial cells, neither bocavirus nor the enterovirus-torovirus recombinant could be detected using PCR assays on any of the passaged cells. This is in contrast with the recently described recombinant virus from North Carolina, USA, where Shang et al. (2017) could isolate the virus using the ST cell line. We speculate that due to the large amount of additional non-recombinant enteroviruses in the sample, this might have put the recombinant enterovirus in an in vitro disadvantage. Using, Pfam and Hmmer searches for motifs, the insertion was predicted to enclose an L-protease from the C28 family. This protein has been best studied for foot-and-mouth disease virus (FMDV), a member of the Picornaviridae and is known to cleave host cell proteins, namely the p220 subunit of eukaryotic initiation factor 4 F (eIF-4 F). The cleavage of this initiation factor 4 F results in the shutoff of cap-dependent host cell protein synthesis, without affecting viral protein synthesis which can occur in the presence of cleaved p220 (Piccone et al. 1995) . Moreover, our structural analysis confirmed that the function prediction from the structure was a cysteine proteinase (for both our insertion and L pro of FMDV), even though their relatedness in sequence identity is low (Fig. 1E ). Structure prediction is often important to determine function when sequence similarities are lower. The predicted cleavage sites for the virus could be identified, suggesting that the inserted gene is producing a separate protein. Even though a small contig of 256 bp of Torovirus from the same insertion region could be detected in our pooled fecal sample, it clustered very distantly from the gene inserted (Fig. 1A) . Since the fecal sample resulted of a pool of 12 pig feces, this is not surprising and likely has a different host origin. In addition to confirming the presence of the enterovirus-torovirus recombinant using Sanger sequencing, we used proteomics to infer whether the protein of the insertion could be found in the sample. This is specially challenging since fecal samples are a very complex matrix. In addition, the torovirus insertion codes for a non-structural protein, which are only present inside infected cells, which m