Puente et al. Porcine Health Management (2023) 9:29 Porcine Health Management https://doi.org/10.1186/s40813-023-00326-w RESEARCH Open Access Detection and genetic characterization of enteric viruses in diarrhoea outbreaks from swine farms in Spain Héctor Puente1*, Héctor Arguello1,2, Martí Cortey3, Manuel Gómez‑García1, Oscar Mencía‑Ares1, Lucía Pérez‑Perez1, Ivan Díaz4,5,6 and Ana Carvajal1,2 Abstract Background The aim of this work was to study the prevalence and distribution of Porcine astrovirus (PAstV), Porcine kobuvirus (PKoV), Porcine torovirus (PToV), Mammalian orthoreovirus (MRV) and Porcine mastadenovirus (PAdV) as well as their association with widely recognized virus that cause diarrhoea in swine such as coronavirus (CoVs) and rotavi‑ rus (RVs) in diarrhoea outbreaks from Spanish swine farms. Furthermore, a selection of the viral strains was genetically characterized. Results PAstV, PKoV, PToV, MRV and PAdV were frequently detected. Particularly, PAstV and PKoV were detected in almost 50% and 30% of the investigated farms, respectively, with an age-dependent distribution; PAstV was mainly detected in postweaning and fattening pigs, while PKoV was more frequent in sucking piglets. Viral co-infections were detected in almost half of the outbreaks, combining CoVs, RVs and the viruses studied, with a maximum of 5 dif‑ ferent viral species reported in three investigated farms. Using a next generation sequencing approach, we obtained a total of 24 ARN viral genomes (> 90% genome sequence), characterizing for first time the full genome of circulating strains of PAstV2, PAstV4, PAstV5 and PToV on Spanish farms. Phylogenetic analyses showed that PAstV, PKoV and PToV from Spanish swine farms clustered together with isolates of the same viral species from neighboring pig producing countries. Conclusions Although further studies to evaluate the role of these enteric viruses in diarrhoea outbreaks are required, their wide distribution and frequent association in co-infections cannot be disregard. Hence, their inclusion into routine diagnostic panels for diarrhoea in swine should be considered. Keywords Swine, Multiplex RT-PCR, Phylogenetic analysis, Porcine astrovirus, Porcine kobuvirus, Porcine torovirus, Mammalian orthoreovirus, Porcine mastadenovirus *Correspondence: Héctor Puente hpuef@unileon.es Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Puente et al. Porcine Health Management (2023) 9:29 Page 2 of 14 Background Mammalian orthoreovirus (MRV) [30–34] or Porcine Enteric diseases caused by viruses are highly prevalent mastadenovirus (PAdV) [1, 35–37] have been detected and negatively impact productivity in pig production [1]. in pigs with diarrhoea worldwide (Table 1), although Coronaviruses (CoVs) and rotaviruses (RVs) are the most their etiological role remains unclear. These viruses are common and well-recognized viruses responsible for frequently identified in co-infections with CoVs and RVs diarrhoea in pigs [2–4]. Among CoVs, Transmissible gas- [4, 38–45], and are also commonly detected in healthy troenteritis virus (TGEV) and Porcine epidemic diarrhoea animals [39, 46–50]. The enteropathogenicity of PAstV, virus (PEDV), two Alphacoronavirus, have swept farms MRV and PAdV has been demonstrated on experimen- all over the world since their emergence in the last cen- tally infected conventional piglets [51–55], but to the best tury [5, 6]. More recently, a recombinant virus between of the authors’ knowledge, no experimental challenge has TGEV and PEDV, known as Swine enteric coronavirus been reported for PKoV and PToV. (SeCoV), has been identified on European pig farms [7– The aim of this work was to study the prevalence and 10]. These three CoVs target the small intestine in pigs of distribution of PAstV, PKoV, PToV, MRV and PAdV in all ages, shortening villi and inducing an undistinguish- diarrhoea outbreaks on swine farms in Spain, as well as able diarrhoeic disease [3, 11]. Other two porcine CoVs, to identify their association with widely recognized virus Porcine delta coronavirus (PDCoV) [12] and Swine acute that cause diarrhoea, such as CoV and RV. In addition, diarrhoea syndrome coronavirus (SADS-CoV) [13, 14], a selection of the viral strains was genetically character- an Alphacoronavirus, have been also recently described ized. Altogether, our results provide information on the in diarrhoea outbreaks, although they have never been aetiology of enteric disease in swine that can be used to detected on European swine farms. RVs are also a major devise and implement appropriate diagnostic and control cause of acute gastroenteritis in swine worldwide with strategies. Rotavirus A (RVA) and Rotavirus C (RVC) as the most relevant species [3]. They are mainly involved in diar- Results rhoea outbreaks in both nursing and weaned piglets Prevalence and distribution of individual swine enteric although they can be detected in all stages in porcine viruses in diarrhoea outbreaks in Spain production [3]. Globally, 160 out of 206 (77.7%) farms were positive In addition, several other swine enteric viruses such as to at least one of the enteric viruses tested. PAstV was Porcine astrovirus (PAstV) [15–20], Porcine kobuvirus the most frequently detected virus (48.5%; n = 100) fol- (PKoV) [16, 21–24], Porcine torovirus (PToV) [25–29], lowed by PKoV (27.2%; n = 56), PEDV (19.9%; n = 41), Table 1 Main characteristics of porcine enteric viruses with well-recognized or unclear involvement in the etiology of diarrhoea outbreaks on swine farms Virus Genus Family Enveloped Viral genome structure Genome size (Kb) Identified virus in swine samples Well-recognized viruses responsible for diarrhoea outbreaks on swine farms PEDV Alphacoronavirus Coronaviridae Yes RNA single-stranded positive sense 28–30 [2, 58, 72] TGEV Alphacoronavirus Coronaviridae Yes RNA single-stranded positive sense 28–30 [72–74] SeCoV Alphacoronavirus Coronaviridae Yes RNA single-stranded positive sense 28–30 [7–10] PDCoV Deltacoronavirus Coronaviridae Yes RNA single-stranded positive sense 28–30 [12, 75, 76] SADS Alphacoronavirus Coronaviridae Yes RNA single-stranded positive sense 28–30 [13, 14, 77–79] RVA Rotavirus Sedoreoviridae No RNA double-stranded segmented (11) 18–19 [3, 80, 81] RVC Rotavirus Sedoreoviridae No RNA double-stranded segmented (11) 18–19 [3, 80, 82] Enteric viruses with an unclear role in diarrhoea outbreaks on swine farms PAstV Astrovirus Astroviridae No RNA single-stranded positive sense 6.4–7.3 [15–20] PKoV Kobuvirus Picornaviridae No RNA single-stranded positive sense 8.2–8.3 [16, 21–24] PToV Torovirus Tobaniviridae Yes RNA single-stranded positive sense 25–30 [25–29] MRV Orthoreovirus Spinareoviridae No RNA double-stranded segmented (10) 23–24 [30–34] PAdV Mastadenovirus Adenoviridae No DNA double-stranded lineal 32–34 [1, 35–37] MRV: Mammalian orthoreovirus, PAdV: Porcine mastadenovirus, PAstV: Porcine astrovirus, PEDV: Porcine epidemic diarrhoea virus, PDCoV: Porcine deltacoronavirus, PKoV: Porcine kobuvirus, PToV: Porcine torovirus, RVA: Rotavirus A, RVC: Rotavirus C, SADS: Swine acute diarrhoea syndrome coronavirus, SeCoV: Swine enteric coronavirus, TGEV: Transmissible gastroenteritis virus Puente et al. Porcine Health Management (2023) 9:29 Page 3 of 14 PAdV (14.1%; n = 29), RVA (13.6%; n = 28), PToV (11.2%; suspected outbreaks (OR = 2.52, CI 95% 1.27–5.04) as n = 23), RVC (6.3%; n = 13) and MRV (4.4%; n = 9). TGEV compared with those without viral suspicion (85.4% vs. or SeCoV were not detected in any of the investigated 69.9%). Accordingly, the prevalence of each of the inves- farms. tigated viruses was higher in outbreaks with suspected The prevalence of positive diarrhoea outbreaks (a viral aetiology, with the exception of PKoV, RVA and RVC positive result for at least one of the tested viruses) was (Fig. 2), although statistical differences were only demon- similar (p = 0.787) among nursing (75.8%, 50 out of 66), strated for PEDV (p < 0.001) and PAdV (p = 0.002). postweaning (76.6%, 49 out of 64) and fattening periods (80.3%, 61 out of 76). However, different age distribu- Prevalence and distribution of viral co‑infections tion patterns were observed for each of the investigated in diarrhoea outbreaks in Spain viruses (Fig. 1). Detection of PAstV and RVA was signifi- In 69 out of the 206 analysed outbreaks (33.5%) a single cantly lower in nursing diarrhoea outbreaks as compared viral aetiological agent was detected, while 56 (27.2%) with postweaning (p < 0.001 and p = 0.025, respectively) presented a co-infection of two viruses at the same time and fattening outbreaks (p < 0.001 and p = 0.036, respec- and 35 (17.0%) were positive for more than two (up to tively). In contrast, PKoV were more prevalent in nurs- 5 different virus species simultaneously detected on ing and postweaning than in fattening (p < 0.001), while three different farms). Detection of a single viral agent RVC were only detected in diarrhoea outbreaks affecting was more frequent in diarrhoea outbreaks in lactation lactating piglets. Finally, PToV and PAdV were more fre- as compared with postweaning (26.6%, 17 out of 64, quently detected in fattening outbreaks as compared to p = 0.019) and fattening (28.9%, 22 out of 76, p = 0.031). nursing (p = 0.016 and p = 0.005, respectively). A similar On the contrary, outbreaks with two or more co-infect- trend was observed for PEDV and MRV although with- ing viruses were more prevalent in postweaning (50.0%, out statistical significance. 32 out of 64, p = 0.017) and fattening (51.3%, 39 out of In addition, it was evidenced that most of the investi- 76, p = 0.009) as compared with nursing (30.3%, 20 out of gated outbreaks (50.0%; 103 out of 206) occurred during 66). Besides, outbreaks presenting two or more co-infect- the first trimester as compared with the rest of the year ing viruses were more frequent when viral aetiology was (p < 0.001). PEDV was more commonly detected during suspected according to the criteria of the veterinarian the winter (January to March) (p = 0.011) while no asso- responsible for the farm (55.3% vs. 33.0%, p = 0.001). ciated seasonality could be confirmed for any of the other As shown in Additional file 2, PAstV and PKoV were investigated enteric viruses (Additional file 1). detected in almost 30% of the positive outbreaks as a sin- Based on clinical criteria, the diarrhoea outbreaks gle infection while PToV, MRV and PAdV were detected included in this research were classified by the veteri- in over 90% of positive outbreaks co-infecting with PEDV, narian responsible for the farm into outbreaks with or RVs or other enteric viruses. The most frequent viral without a suspected viral aetiology. The risk of a positive combinations (top 8) are shown in Table 2. Once again, result to at least one the tested viruses was higher in viral a clear age-dependent distribution was observed with Fig. 1 Prevalence of positive diarrhoea outbreaks to each of the different investigated enteric viruses according to the age of the affected animals. Out of the 206 investigated diarrhoea outbreaks, 66 affected nursing piglets (< 21 days), 64 postweaning-growing piglets (21–70 days) and 76 fattening pigs (> 70 days) Puente et al. Porcine Health Management (2023) 9:29 Page 4 of 14 Fig. 2 Prevalence of positive diarrhoea outbreaks to each of the different investigated enteric viruses according to the epidemiological and clinical appearance of the outbreaks (non-suspected—n = 103- or suspected—n = 103-viral involvement) co-infections including PAstV more prevalent in post- for PAstV3, 73.4–91.8% for PAstV4 and 83.3–94.4% for weaning and fattening outbreaks and co-infections with PAstV5. Most of the Spanish PAstV sequences clustered PKoV recorded among the top 3 in nursing outbreaks. together with North American strains of the same line- The viruses most frequently associated with PEDV, age, although PAstV4 clustered with sequences from RVA and RVC are shown in Fig. 3. PAstV was identi- Japan and some PAstV2 with sequences from Europe and fied in more than 50% of the PEDV and RVA outbreaks China (Fig. 4). while RVC mainly co-infects with PKoV, probably as a Regarding the phylogenetic analysis of PKoV (Fig. 5), consequence of the age dependent distribution of these the three sequences obtained in our study clustered infections. Comparison of the prevalence of each of the together with previously reported Spanish and Hun- investigated viruses between PEDV, RVA and RVC posi- garian isolates. The nucleotide sequence identity was tive and negative outbreaks reveal that PToV were more 90.3–91.4% among the Spanish isolates identified in this commonly detected in PEDV and RVA positive outbreaks study and 87.2–91.9% with the previously reported whole (p = 0.021 and p = 0.005, respectively) while PAstV detec- genome PKoV sequences. tion was more common in RVA positive farms (p = 0.027). Finally, a single PToV complete genome was obtained. When compared with previously described PToV its Sequencing and phylogenetic analysis of PAstV, PKoV nucleotide identity was 88.6–94.1%, but was lower when and PToV compared with torovirus sequences from other animal By using a next generation sequencing (NGS) in 14 species (77.3–87.9%). This Spanish PToV sequence clus- pooled faecal samples, we identified 10 RNA virus spe- tered together with viral sequences from Europe, par- cies from six different genera: PAstV2 (6), PAstV3 (7), ticularly Germany, but also with sequences from the USA PAstV4 (10), PAstV5 (7), PkoV (8), PToV (5), PEDV (10), and Japan (Fig. 6). RV (2·RVA and 2 RVC) and MRV (1) (Additional file 3). From this array of viruses, the whole or almost whole Discussion genome was sequenced in 24, including PAstV2 (5), The enteropathogenic role of PAstV, PKoV, PToV, MRV PAstV3 (1), PAstV4 (8), PAstV5 (2), PKoV (3), PToV (1) and PAdV is still controversial and has not been evi- and PEDV (4). denced in a number of case–control studies compar- Four species of PAstV (PAstV2 to PAstV5) were ing detection ratios in diarrhoeic and healthy pigs [39, detected in the investigated outbreaks on Spanish farms, 46–50]. Taking this fact into account, we investigated the being PAstV4 and PAstV2 the most represented. The prevalence of these enteric viral species focusing on diar- nucleotide sequence identities between PAstV2 and rhoea outbreaks and looking into their association with PAstV4 strains recovered in our research were 77.2– well-recognized viral enteropathogens as CoVs (PEDV, 95.2% and 87.2–93.7% respectively, while its comparison TGEV and SeCoV) and RVs (RVA and RVC). The design with previously described sequences provided nucleo- of our study does not allow to draw conclusions about the tide identities of 68.7–91.0% for PAstV2, 87.6–89.1% role of these viruses in the aetiology of enteric disease, Puente et al. Porcine Health Management Table 2 Most common viral co-infections reported (top 8) in the investigated diarrhoea outbreaks according to the age of the affected animals Age of the affected animals (number of investigated outbreaks) (2023) 9:29 Nursing (n = 66) Postweaning (n = 64) Fattening (n = 76) Total (n = 206) Rank Viruses Prevalence (%) Rank Viruses Prevalence (%) Rank Viruses Prevalence (%) Rank Viruses Prevalence (%) 1 PKoV + PEDV 7.6 1 PAstV + PKoV 32.8 1 PAstV + PAdV 18.4 1 PAstV + PKoV 12.6 2 PKoV + RVC 7.6 2 PAstV + PEDV 15.6 2 PAstV + PToV 13.2 2 PAstV + PEDV 11.2 3 PKoV + RVA 4.5 3 PKoV + RVA 12.5 3 PAstV + PEDV 13.2 3 PAstV + PAdV 10.7 4 PAstV + PEDV 4.5 4 PAstV + RVA 10.9 4 PAstV + RVA 7.9 4 PAstV + RVA 9.7 5 PAstV + PKoV 4.5 5 PAstV + PToV 10.9 5 PAstV + MRV 7.9 5 PAstV + PToV 8.7 6 PAstV + RVA 3.0 6 PAstV + PAdV 9.4 6 PToV + PEDV 5.3 6 PKoV + RVA 5.3 7 PKoV + PToV 3.0 7 PKoV + PEDV 7.8 7 PToV + RVA 5.3 7 PKoV + PEDV 4.9 8 PToV + PEDV 3.0 8 PAstV + PToV 7.8 8 PToV + PAdV 3.9 8 PToV + PEDV 4.4 PAdV: Porcine mastadenovirus, PAstV: Porcine astrovirus, PEDV: Porcine epidemic diarrhoea virus, PKoV: Porcine kobuvirus, PToV: Porcine torovirus, RVA: Rotavirus A and RVC: Rotavirus C Page 5 of 14 Puente et al. Porcine Health Management (2023) 9:29 Page 6 of 14 Fig. 3 Detection of each of the different investigated enteric viruses (Porcine astrovirus or PAstV, Porcine kobuvirus or PKoV, Porcine torovirus or PToV, Mammalian orthoreovirus or MRV and Porcine adenovirus or PAdV) in positive outbreaks involving well-recognized viruses that cause diarrhoea (Porcine epidemic diarrhoea virus or PEDV—n = 41-, Rotavirus A or RVA—n = 28- and Rotavirus C or RVC—n = 13-) but it aims to increase knowledge about their frequency, reports on the prevalence of MRV on European swine distribution and co-infections on farms with enteric farms. Regarding well-recognized enteropathogenic problems. viruses, neither TGEV nor SeCoV were detected. PEDV Our results confirmed that PAstV and PKoV infec- was identified in 19.9% of the investigated diarrhoea out- tions are highly prevalent on European farms, as previ- breaks and the prevalence of RVA and RVC positive out- ously proposed by a number of studies [19, 46, 49, 50, breaks was 13.6 and 6.3%, respectively. 56]. PAstV was the most frequent enteric virus in our Three age distribution patterns were observed among research, detected in almost half of the outbreaks, fol- the investigated enteric viruses. Similarly to previous lowed by PKoV, detected in more than 25% of the inves- studies in China [16, 47], Canada [44] and several Euro- tigated farms. In agreement with a previous report in pean countries [38, 49, 50], PKoV were more prevalent in Hungary [49] PAdV was less common (14.1% of posi- nursing and postweaning piglets as compared with fat- tive outbreaks) while PToV was only identified in 11.2% tening stages. A similar distribution has been reported for of the investigated farms in spite of the broad distribu- RVC [3] and was confirmed in our research, with no pos- tion of this virus, evidenced by another Spanish study itive RVC outbreak occurring after weaning. In contrast, which estimated over 99% of seropositive animals among PAstV and RVA showed a significant increase in their pigs older than 11 weeks of age [57]. Finally, MRV was prevalence in weaned pigs as compared to nursing pigs. the least frequently detected enteric virus (4.4%) among Previous studies on European swine farms have reported those with an unclear aetiological role in diarrhoea out- a similar trend for PAstV [46, 49, 50]. As described for breaks. To the authors knowledge there are no previous endemic RVA on swine farms [1], the decline of specific Puente et al. Porcine Health Management (2023) 9:29 Page 7 of 14 Fig. 4 Neighbor-joining phylogenetic tree based on the p-distance among the nucleotide sequences of the complete genomes for PAstV. Along the branches, percentage of bootstrap values based on 10,000 replicates (only values equal or larger than 70% are shown). The filled circles identified the isolates sequenced in this research. GenBank accession number, country and year of the outbreak are also shown below the strains. The PAstV lineages referred in the text are included on the right of the tree. Scale bars indicate nucleotide substitutions per site Puente et al. Porcine Health Management (2023) 9:29 Page 8 of 14 Fig. 5 Neighbor-joining phylogenetic tree based on the p-distance among the nucleotide sequences of the complete genomes for PKoV. Along the branches, percentage of bootstrap values based on 10,000 replicates (only values equal or larger than 70% are shown). The filled circles identified the isolates sequenced in this research. GenBank accession number, country and year of the outbreak are also shown below the strains. Scale bars indicate nucleotide substitutions per site Puente et al. Porcine Health Management (2023) 9:29 Page 9 of 14 Fig. 6 Neighbor-joining phylogenetic tree based on the p-distance among the nucleotide sequences of the complete genomes for PToV. Along the branches, percentage of bootstrap values based on 10,000 replicates (only values equal or larger than 70% are shown). The filled circles identified the isolates sequenced in this research. GenBank accession number, country and year of the outbreak are also shown below the strains. Scale bars indicate nucleotide substitutions per site maternal antibodies could explain the increase in preva- it has been proposed that the exposure to low ambient lence of PAstV in the postweaning period. Finally and in temperatures may enhance PEDV replication through line with previous studies [28, 49], PToV and PAdV were up-regulated heat shock protein Hsp70 in the pig intes- identified more frequently in diarrhoea outbreaks during tine, contributing to PEDV seasonality [60]. In contrast, the fattening period. no seasonality has been described for porcine RV [3]. A seasonal distribution was only demonstrated for Interestingly, veterinary clinical criteria suggesting a PEDV positive outbreaks which were more common dur- presumptive viral aetiology was associated with the viral ing the coldest months of the year (January–March). Low detection using molecular methods. This was particu- temperatures allowing for CoV survival in the environ- larly evident for PEDV positive outbreaks but also for ment and facilitating indirect transmission may explain PAdV and to a lesser extent for PAstV, PToV or MRV. In seasonal variation in PEDV outbreaks [58, 59]. Moreover, contrast, RVA, RVC and PKoV were detected mainly in Puente et al. Porcine Health Management (2023) 9:29 Page 10 of 14 non-viral suspected outbreaks. The fact that RV infec- is a major limitation of our research. Quantitative tions are endemic in swine populations and frequently approaches based on qPCR or next generation sequenc- associated with other diarrhea-producing agents such as ing (NGS) would have allowed for viral load estimations. enterotoxigenic Escherichia coli or Clostridium perfrin- As proposed by Cortey et al. [63] the determination of gens type A [3, 61, 62] could complicate its recognition by the relative abundance of different viral species in co- practitioners. infections makes it possible to identify the predominant Co-infections were relatively frequent with two or virus which can most likely be proposed as the primary more viruses detected in almost 40% of the investigated etiological agent. outbreaks. This result is in line with previous studies on Finally, the study involved the fine tune genomic char- European farms [16, 49, 63] reporting a high frequency acterization of a selection of samples to obtain the com- of co-infections involving several enteric viruses in pigs plete sequence of part of these viruses. Phylogenetic with diarrhoea. A higher proportion of viral enteric co- analysis revealed that the majority of the PAstV strains infections, close to 70%, has been described on swine were classified as PAstV4 (50.0%, 8/16) and PAstV2 farms from Asia [4, 40]. In agreement with previous (31.2%, 5/16), indicating that these two PAstV species are studies conducted in Europe [49], China [4, 47] or Can- the most prevalent in swine with diarrhoea in Spain, as ada [44], the number of different virus species detected it has been described in several European countries [46, within an outbreak increased with the age of the affected 50, 67], in North America [68] and in Asia [39, 69, 70]. pigs. Hence, single infections were more common in PKoV phylogenetic analyses revealed that all available nursing piglets while simultaneous co-infections with Spanish complete sequences (including the three identi- up to five viruses were observed in postweaning and fat- fied in our study) were included in a single local cluster tening pigs. Many factors associated with weaning such together with one Hungarian strain and well differenti- as dietary changes, placement into a new and more con- ated from other European isolates [38]. Finally, the single taminated environment, mixing of pigs or loss of pas- PToV whole genome sequence obtained in this research sive immunity can favor the infection by different viral clustered together with other PToV isolates from Europe, species. Also, it has been proposed that the maturation America or Asia [25, 26]. of the gut associated immune system after weaning may prevent the development of enteric disease associated to Conclusions single infections for those enteric viruses with a moder- Although the design of our study does not allow for the ate enteropathogenicity [64]. evaluation of the role of PAstV, PKoV, PToV, MRV or The most common co-infections varied depending on PAdV as causative agents of diarrhoea in pigs, we have the age or life stage of the affected pigs. As expected from demonstrated a high prevalence of these enteric viruses the observed patterns of age distribution, co-infections in diarrhoea outbreaks, particularly for PAstV and involving PKoV were the most prevalent in lactating PKoV detected in almost 50% and 30% of the investi- piglets (27.2%) while co-infections with PAstV predomi- gated farms. The fact that these enteric viruses are fre- nated in postweaning and fattening outbreaks (87.4% quently involved in co-infections among them and also and 60.6%, respectively). Although it has been recently with well-recognized viral enteropathogens as CoVs or proposed that PEDV and PKoV might play some syner- RVs make necessary further studies for surveillance and getic role [65], no significant association between both characterization as well as investigations to evaluate their viruses was observed in our research. The fact that most pathogenic potential in both experimental and natural of the PEDV outbreaks occurred in postweaning or fat- infections. Once these studies are available, the inclusion tening pigs when PKoV is less prevalent may explain this of these viruses into the routine diagnostic panels for result. In contrast, PToV shedding was more frequent diarrhoea in swine should be considered. in PEDV and RVA positive outbreaks. Although there is very little information known about PToV infections, it Methods is worth mentioning that the first report of PToV in the Sample collection and nucleic acid extraction United States was in a PEDV positive pig [66]. In addi- The study was conducted between January 2017 and tion, an increase in the prevalence of PAstV shedding was October 2020 on 206 Spanish swine farms with diarrhoea observed in RVA positive outbreaks. Further research outbreaks affecting nursing piglets (< 21 days) (66 farms), is required to decipher the interactions between enteric postweaning-growing (21–70 days) (64 farms) or fatten- viruses which may play a primary or secondary role in ing pigs (> 70 days) (76 farms). From each farm, two to six the aetiology of enteric disease in pigs. individual fecal samples were submitted for diagnostic Given the high prevalence of viral co-infections purposes to the Infectious Diseases Unit of the Animal detected, the use of conventional PCR for viral detection Health Department of the University of León (Spain). The Puente et al. Porcine Health Management (2023) 9:29 Page 11 of 14 veterinarian responsible for the farm classified the out- The RT-PCR products were visualized on a 1.5% aga- break as viral or non-viral suspicion, according to criteria rose gel containing RedSafe Nucleic Acid Staining Solu- such as speed of progression of the disease or response to tion (iNtRON Biotechnology, Inc.) and compared with antimicrobials. expected lengths (Table 3). Individual fecal samples were mixed to prepare one pooled sample per farm that was diluted 1:2 (v/v) in ster- Sequencing and phylogenetic analysis ile phosphate buffered saline (PBS), homogenized by vor- A total of 14 pooled faecal samples were chosen for tex mixing and centrifuged for 10 min at 20,000 g. The sequencing based on their RT-PCR results (samples nucleic acid was extracted from 140 µl of the supernatant with the highest number of different viruses detected). using QIAMP Viral RNA and DNA Mini Kit (QIAGEN), From each pooled sample, total RNA was extracted following the manufacturer’s instructions, and stored at using a TRIzol LS reagent (Thermo Scientific) proto- − 80 °C until use. col. The total RNA extraction was directly sequenced at the Genomics Bioinformatics Service (SGB) of the Molecular detection of porcine enteric viruses Autonomous University of Barcelona (UAB), without Three duplex and one triplex RT-PCRs were carried out using any primer or amplification step. NGS was car- using Verso 1-Step RT-PCR ReddyMix kit (Thermo Sci- ried out using an Illumina Miseq Platform. RNA virus entific) for the detection of PEDV, TGEV, SeCoV (simul- sequences were obtained from NGS outputs applying taneous detection of S gene of PEDV and N gene of a tailor-made, virus-specific script developed by Cortey TGEV), RVA, RVC, PToV, PAstV, PKoV, MRV and PAdV et al. [63]. Subsequently, a database for every RNA virus (Table 3). The reactions were conducted under the fol- species identified was constructed by downloading the lowing conditions: 50 °C for 30 min, 95 °C for 2 min, 45 available complete genome sequences from GenBank. cycles at 95 °C for 20 s, 50 °C for 30 s, and 72 °C for 1 min Whole genome (> 90%) sequences were deposited in and 30 s, followed by a final extension step at 72 °C for NCBI GenBank with the accession numbers ON649880, 10 min. ON714996–ON715001 and ON792965–ON792977. Four Table 3 Primer sets used for the detection of porcine enteric viruses by multiplex RT-PCR Virus Primer Sequence (5’–3’) Target Size (bp) References RT-PCR multiplex 1 TGEV TGEV-TA GAT​GGC​GAC​CAG​ATA​GAA​GT Nucleocapsid gene 612 [83] TGEV-TB GCA​ATA​GGG​T TG​C TT​GTA​CC PToV ToV-F ATT​GCT​TAT​TGG​TGG​C TT​CC Spike gene 464 This study ToV-R GGC​TAC​TCA​AAC​T TA​ACA​C T PAstV AstV-F ATG​TCT​T TG​GGA​TGTGG​ RdRp gene 334 This study AstV-R TTT​GGT​CCT​CCC​C TC​CAA​AG RT-PCR multiplex 2 PEDV PEDV-F TTC​TGA​GTC​ACG​AAC​AGC​CA Spike gene 651 [84] PEDV-R CAT​ATG​CAG​CCT​GCT​C TG​AA PKoV KV-F1 CGG​GAC​TGG​T TT​GGA​GGA​ACA​ Leader gene 446 [24] KV-R CTC​TAT​CAA​GCA​GTA​CAT​GG This study RT-PCR multiplex 3 RVC RVC-VP7-F GCT​GTC​TGA​CAA​ACT​GGT​C Viral protein 7 gene 1061 [85] RVC-VP7-R GCC​ACA​TGA​TCT​TGT​T TA​CGC​ MRV MRV-L1-F CTC​GTG​AAA​TGG​CGA​ATG​TG RdRp Lambda 3 gene 638 This study MRV-L1-R TAG​ACT​CAC​GCT​GAC​CGT​CC RT-PCR multiplex 4 RVA RVA-NSP2-F GGC​T TT​TAA​AGC​GTC​TCA​GTC​ Non-structural protein 2 gene 1059 [86] RVA-NSP2-R GGT​CAC​ATA​AGC​GCT​T TC​TATTC​ PAdV AdV-PALF GAT​GTC​ATG​GAY​AAC​GTC​AAC​ Hexon gene 612 [36] AdV-PARF CAC​GGA​GGA​GTC​RAA​C TG​GATG​ MRV: Mammalian orthoreovirus, PAdV: Porcine mastadenovirus, PAstV: Porcine astrovirus, PEDV: Porcine epidemic diarrhoea virus, PKoV: Porcine kobuvirus, PToV: Porcine torovirus, RVA: Rotavirus A, RVC: Rotavirus C and TGEV: Transmissible gastroenteritis virus Puente et al. Porcine Health Management (2023) 9:29 Page 12 of 14 whole genome sequences of PEDV also recovered in this Acknowledgements We acknowledge the excellent technique assistance provided by Diana work were already deposited with accession numbers Molina, as well as the help and willingness of veterinary practitioners and MN692786, MN692787, MN692789 and MN692791 [8]. farmers. Finally, the sequences obtained were aligned and phy- Author contributions logenetically compared with the datasets constructed Study design and direction: HP and AC. In vitro experiments: HP, LPP, MGG and for every complete or near-complete genome identified. OM. Analysis of the results and writing of the manuscript: HP, MC, ID, HA and The phylogenetic relationships among sequences were AC. All authors read and approved the final manuscript. analyzed using the software MEGA11 [71], by means of Funding a Neighbor-joining (NJ) algorithm, using the model of This work was supported by the program of the National Institute of Agricul‑ maximum composite likelihood and 10,000 bootstrap tural and Food Research and Technology (INIA project E-RTA2015-0003-C02-01 and E-RTA2015-0003-C02-02) of Spanish Government. H. Puente, O. Mencía- replicates to estimate the confidence of the internal Ares, L. Pérez-Pérez, M. Cortey and H. Argüello were supported by Spanish branches of the trees. Government (FPU17/00466, FPU16/03485, PRE2020-093762, RYC-2015-1715-4 and BEAGAL- 18-106, respectively) and M. Gómez-García by Junta de Castilla & León (LE131-18). Statistical analysis Availability of data and materials exact and ANOVA tests using Epi Info™. A two-sided Statistical analyses of data were performed by Fisher’s Data are available in the GenBank database and by direct contact with the correspondence author. p value of ≤ 0.05 was considered to indicate statistical significance. Declarations Ethics approval and consent to participate Not applicable. Abbreviations CI Confidence interval Consent for publication CoV Coronavirus Not applicable. MRV  Mammalian orthoreovirus NGS Next generation sequencing Competing interests PAdV  Porcine mastadenovirus The authors declare that they have no competing interests. PAstV  Porcine astrovirus PBS Phosphate-buffered saline Author details PEDV  Porcine epidemic diarrhoea virus 1 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de PDCoV  Porcine deltacoronavirus León, León, Spain. 2 INDEGSAL, Universidad de León, León, Spain. 3 Departa‑ PKoV  Porcine kobuvirus ment de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat PToV  Porcine torovirus Autònoma de Barcelona, Bellaterra, Spain. 4 IRTA, Programa de Sanitat Animal, RNA Ribonucleic acid Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat RT-PCR Reverse transcription-polymerase chain reaction Autònoma de Barcelona, Bellaterra, Spain. 5 Unitat Mixta d’investigació RV Rotavirus IRTA‑UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), RVA  Rotavirus A Campus de la Universitat Autònoma de Barcelona, Bellaterra, Spain. 6 WOAH RVC  Rotavirus C Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Bellaterra, Spain. SADS  Swine acute diarrhoea syndrome coronaviruS SeCoV  Swine enteric coronavirus Received: 3 May 2023 Accepted: 18 June 2023 TGEV  Transmissible gastroenteritis virus Supplementary Information The online version contains supplementary material available at https://​doi.​ References org/​10.​1186/​s40813-​023-​00326-w. 1. Thomson JR, Friendship RM. Digestive system. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz K, Stevenson GW, Zhang J, editors. Dis swine. Additional file 1: Percentage of positive detection to each of the different 11th ed. Hoboken: Wiley; 2019. p. 234–63. investigated enteric viruses in diarrhoea outbreaks occurring between 2. Pensaert MB, Martelli P. Porcine epidemic diarrhea: a retrospect from January 2017 and December 2019. Europe and matters of debate. Virus Res. 2016;226:1–6. 3. Vlasova AN, Amimo JO, Saif LJ. Porcine rotaviruses: epidemiology, Additional file 2: Distribution of single infections and co-infections for immune responses and control strategies. 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Arch Virol. 2013;158:549–58. Ready to submit your research ? Choose BMC and benefit from: 68. Mor SK, Chander Y, Marthaler D, Patnayak DP, Goyal SM. Detection and molecular characterization of Porcine astrovirus strains associated with • fast, convenient online submission swine diarrhea. J Vet Diagn Investig. 2012;24:1064–7. 69. Cai Y, Yin W, Zhou Y, Li B, Ai L, Pan M, et al. Molecular detection of Porcine • thorough peer review by experienced researchers in your field astrovirus in Sichuan Province, China. Virol J. 2016;13:1–5. • rapid publication on acceptance 70. Lee MH, Jeoung HY, Park HR, Lim JA, Song JY, An DJ. Phylogenetic analysis • support for research data, including large and complex data types of porcine astrovirus in domestic pigs and wild boars in South Korea. Virus Genes. 2013;46:175–81. • gold Open Access which fosters wider collaboration and increased citations 71. Tamura K, Stecher G, Kumar S. 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