Detection of porcine enteric viruses (Kobuvirus, Mamastrovirus and Sapelovirus) in domestic pigs in Corsica, France

Many enteric viruses are found in pig farms around the world and can cause death of animals or important production losses for breeders. Among the wide spectrum of enteric viral species, porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV) and porcine Astrovirus (PAstV) are frequently found in pig feces. In this study we investigated sixteen pig farms in Corsica, France, to evaluate the circulation of three enteric viruses (PKoV, PAstV-1 and PSV). In addition to the three viruses studied by RT–qPCR (908 pig feces samples), 26 stool samples were tested using the Next Generation Sequencing method (NGS). Our results showed viral RNA detection rates (i) of 62.0% [58.7–65.1] (n = 563/908) for PSV, (ii) of 44.8% [41.5–48.1] (n = 407/908) for PKoV and (iii) of 8.6% [6.8–10.6] (n = 78/908) for PAstV-1. Significant differences were observed for all three viruses according to age (P-value = 2.4e–13 for PAstV-1; 2.4e–12 for PKoV and 0.005 for PSV). The type of breeding was significantly associated with RNA detection only for PAstV-1 (P-value = 9.6e–6). Among the 26 samples tested with NGS method, consensus sequences corresponding to 10 different species of virus were detected. This study provides first insight on the presence of three common porcine enteric viruses in France. We also showed that they are frequently encountered in pigs born and bred in Corsica, which demonstrates endemic local circulation.


Introduction
Pig farms make an important contribution to the economy of world agriculture and are an important source of food. Porcine diarrhea can cause mortality in animals, especially in piglets, and cause economic losses to the pig farmers; many of the pathogens responsible can also infect humans. A very broad spectrum of viruses that can cause porcine diarrhea has been found in pig feces, including porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV), porcine Sapovirus, porcine Astrovirus (PAstV), porcine Bocavirus and porcine Rotavirus [1][2][3][4][5][6][7]. The

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PLOS ONE | https://doi.org/10.1371/journal.pone.0260161 January 14, 2022 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 types of breeding system operated in Corsica were included: seven outdoor extensive farms (Efarms), six outdoor semi-extensive farms (SE-farms) and three indoor closed farms (C-farms); (iii). for each stool sample, the township, anonymous breeder code, breeding type, age and breed of pig and nature of the sample ("rectal" or "on the ground" feces) were recorded; (iv). Four age categories were defined among the young pigs: 1-3 months, 3-4 months, 4-6 months and adults (older than 6 months). Samples from plots where post-weaning pigs were held together with older pigs (age mixed) were classified as the "Herd" group. Information on the individual health status of the pigs was not collected by a case report form. However, there is no apparent disease in the farms during the collect according to breeders. All the samples were collected between April and September 2017.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
One gram of fecal sample was resuspended in 9 mL of phosphate-buffered saline and then centrifuged at 5,000 × g for 10 min. The resulting supernatant was collected and stored at −80˚C until processed. Viral RNA was extracted from 200 μL of supernatant using QIAamp Cador Pathogen on a QIAcube HT (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Samples were spiked with an internal control (T4 and MS2 phages) before extraction, to monitor the extraction and subsequent steps, as described previously. Each pool was spiked before extraction with a predefined amount of MS2 bacteriophage in order to monitor the subsequent steps (nucleic acid purification, reverse transcription and PCR amplification) and to detect the presence of inhibitors and enzymatic reactions as described previously [35]. All extractions and RT-qPCRs were checked with the presence of a curve for phage and positive and negative controls respectively. Nucleic acids were eluted in 100 μL of RE buffer and stored at -80˚C. Samples initially collected for the detection of HEV RNA were analyzed by RT-qPCR for PKoV, PSV and PAstV-1. Of the 919 samples initially collected, 908 were available for this study. Details of the three molecular assay [8,36] are presented in Table 1 using an Applied Quant Studio 3 (Applied Biosystems, CA, USA). For PKoV RNA and PAstV-1 RNA detection, a RT-qPCR test was considered positive if negative controls were negative, positive controls were positive and an exponential curve was observed before a 35-Ct threshold.
For PSV RNA detection, the qPCR machine was programmed to perform a melt-curve analysis at the end of the run to ensure assay specificity; RT-qPCR results were considered positive if a melt curve was detected at between 83˚C and 85˚C and an amplification curve was observed before a 35-Ct threshold. The QuantiTect SYBR1 Green PCR Kit was used for this biomolecular detection (Qiagen, Hilden, Germany).

Statistical analyses
The detection rate of RNA viruses (PKoV, PAstV-1 and PSV) in pig fecal samples was calculated at the individual level and the pig farm level. Positivity rate was also estimated in each subgroup, and a two-sided 95% confidence interval [95% CI] was calculated. Categorical variables were expressed as the number of cases (percentages). Frequencies were compared using the χ 2 test or Fisher's exact test (P < 0.05). A bivariate analysis was carried out to identify the variables that were related to infection with each virus. The multivariate logistic regression analysis included variables that were related to outcome variables in the bivariate analysis with a P-value < 0.2 or a possible association. Odds ratios (ORs), including their 95% CIs, were calculated for the logistic regression models. As in Capai, F. [34], samples with no associated age (Herd group) were excluded from the multivariate analysis, and previous results for the detection rate of HEV RNA among pig feces were included in the analysis to estimate a possible association between coinfections with different viruses [37]. All statistical analyses were performed using the R program (http://www.r-project.org).

Virus genome sequencing
Virus genome sequencing was performed for 26 stool samples as described previously [38]. Only, 26 analyses could be realized for financial reason and these 26 samples were randomly selected among the overall samples. A random RT-qPCR was performed using tagged random primers. A ProtoScript1 II Reverse Transcriptase kit (New England Biolabs) was used for reverse transcription with random tagged primers, and Platinum1 Taq High Fidelity polymerase enzyme (Thermo Fisher Scientific) with specific primers for amplification. After Qubit quantification using Qubit1 dsDNA HS Assay Kit and Qubit 2.0 fluorometer (ThermoFisher Scientific), amplicons were fragmented (sonication) into fragments of 200 bp length. Libraries were built by adding barcodes for sample identification, and primers for amplification using the AB Library Builder System (ThermoFisher Scientific). To pool equimolar amounts of the barcoded samples, a quantification by quantitative PCR using an Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific) was performed. An automated Ion Chef instrument (ThermoFisher) was used for emulsion PCR of the pools and loading them on a 520 chip. Sequencing was performed using S5 Ion torrent technology (Thermo Fisher Scientific) following the manufacturer's instructions. Reads were trimmed (reads with quality score < 0.99 and length < 100 bp were removed, and the 30 first and 30 last nucleotides were removed from the reads), and de novo contigs were produced. These contigs were submitted to Blastn to determine the best reference sequences(s). A consensus sequence was obtained after mapping of the reads on the previously determined reference using CLC genomics workbench software 20.0.4 (Qiagen). The de novo contig was compared with the consensus sequence to ensure that the reference sequence did not affect the consensus sequence. Only sequences corresponding to enteric viruses found in pigs were selected for analysis.

Results
As suggested by Arya, Antonisamy [39], we previously calculated the minimum sample size required to achieve the objectives related to the HEV (n = 176 stool specimens) [34]. Overall, we collected 919 pig feces samples from 16 pig farms selected according to location and breeding system. For PSV RNA detection, using the SYBR green A range of temperatures around 84˚C (83-85˚C) was tolerated. Indeed, a one-nucleotide difference within the amplified sequence can impact the melt-curve dissociation temperature [40,41].

Viral RNA detection rate for the three viruses in feces from domestic pigs and univariate analysis
Overall, 908 samples were available for the detection of the three virus of interest, 310 were from E-farms, 396 from SE-farm and 201 from C-farm.
For PSV and PKoV, there was no statistical association with the type of breeding system; in contrast, PAstV-1 was detected more frequently in C-farms compared with SE-and E-farms (P-value = 9.6e-6) ( Table 2).
Significant differences were observed for all three viruses according to age (P-value = 2.4e-13 for PAstV-1; 2.4e-12 for PKoV and 0.005 for PSV) ( Table 2). RNA virus detection by age group showed a significant decrease in the rate of positive cases after three months for PAstV-1 (30.6% vs. 6.0%; P-value = 3.87e-7) and between 1 and 3 months (69.4%) and in adults (28.9%) for PKoV (P-value = 6.37e-12). For PSV, the detection rates by age group were between 61.0% and 77.5% (Fig 1). However, the positivity rate among pigs under six months of age was significantly lower than that in pigs older than 6 months (71.4% vs. 61.1%; Pvalue = 0.014; OR = 1.59, CI 95% 1.09-2.32). Table 3 lists all infections and coinfections detected in pig feces samples. Of the 908 samples tested, 697 samples were positive for at least one virus (76.8%). A total of 344 samples contained at least two distinct viral RNA (37.9%), of which 259 specimens (28.5%) were coinfected by two viruses, 78 specimens (8.6%) were coinfected by three viruses and seven specimens (0.8%) were positive for all four viruses (Table 3).

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Detection of porcine enteric viruses among pig farms Detection rate of viral RNA at the farm level. At least one pig was detected positive for PKoV RNA in each of the 16 pig farms sampled (100%), 62.5% for PAstV-1 infection (n = 10/16) and 93.8% for PSV infection (n = 15/16). The positivity rate at farm level ranged between 22.5% and 80.8%, 0.0% and 34.0%, and 0.0% and 87.5% for PKoV, PAstV-1 and PSV, respectively. The detail of the detection rates of the three viruses for each farm were presented on the S1 Table.
For the other results, Table 5 summarizes all the sequences obtained, their length, the reference sequence to which each corresponds and the percentage of nucleotide identity with this sequence. Six of the recovered sequences had a quality score required for submission to Genbank and the accession numbers range from OL739527 to OL739532.

Discussion
This study investigated the detection rate of three enteric viral infections, PKoV, Astrovirus-1 and Sapelovirus, in 908 pigs of different ages and breeding systems. To our knowledge, this work is the first to study factors associated with presence of viral RNA of PSV, PKoV and PAstV-1 in stools of domestic pigs in France.

RNA detection rate of the three enteric viruses
PSV was the most prevalent (62%) and observed rates are higher than those described in the US, Brazil and India (7-31%) [9,17,20]. However, the existing results for PSV are not all

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Detection of porcine enteric viruses among pig farms  performed with the same method (SYBR, classical RT-qPCR, NGS) so it is complicated to compare the results in the literature. PKoV RNA was detected in almost half of samples, which is similar to data reported from pig farms in China (45.7%) [42] and Japan (45.4%) [43]. In five European countries, PKoV infections have been described previously as highly prevalent in both diarrheic and healthy pigs, 54.5% and 58.2%, respectively [8].

Very early exposure of pigs
The analysis of the detection of viral RNA according to pig age showed that for all viruses tested, pigs < 4 months old were consistently the age group exhibiting the highest rate. This association between age and rate of infection was confirmed for PKoV and PAstV-1 in the multivariate analysis. It may correlate with high exposure after weaning (about 2 months after birth) but also suggest persisting presence of viruses in the breeding environment and loss of maternal immunity. Piglet passive immunity is derived from colostrum and not from breastfeeding [47], with a decrease in immunoglobulins A, G and M after farrowing [48]. This trend was also observed during our previous study of HEV) [34]. Correlation of decreasing positivity rates and increasing age of pigs is described for Kobuviruses in Italy [49] and East Africa [50]. A higher PKoV detection rate in young piglets has also been reported in other studies [10,51,52]. However, the kinetics of infection were different in each study and may depend on the organization of each farm and other environmental characteristics. Study in different age-group 0-1months, 1-2months, etc. or follow-up of individual animals could help confirming this hypothesis.
In traditional Corsican farms, the average slaughter age is higher (12-18 months) compared with industrial farms. Therefore, at the time of slaughtering the majority of Nustrale Corsican pigs will have cleared replicative viral infection. The same finding was already reported for hepatitis E virus [34]. In contrast, the situation is different for Sapeloviruses with rates of replicating infection at 60% in adult pigs. Farming type does not seem to influence the detection rates of studied viruses but still need confirmation from future studies.

A potentially very broad spectrum of viruses in pig feces
In this study, 37.9% of pigs were coinfected with at least two different viruses (including HEV). Strong trends were observed in the multivariate analysis regarding the associations between different viral infections (PKoV/PSV, PKoV/PAstV-1, PSV/HEV and PSV/PKoV). This wide variety of viruses in the feces was confirmed with the NGS method, which showed the presence of other viruses frequently found in pigs: Posavirus, other Astroviruses, Bocavirus, Enterovirus G, Circovirus, Pasivirus and Rotavirus. Coinfections were also evidenced in the NGS results, with more than half of the samples containing consensus sequences corresponding to at least two different viruses. These results are in line with previous studies reporting multiple coinfections of farmed pigs with porcine enteric viruses [6,37], especially in pigs with diarrhea [53][54][55]. Our results are also interesting concerning the epidemiological cycle of HEV because co-infections can diminish the immune system of pigs, change the symptomatology of infections, [56,57]. For example, experimental co-infection of HEV with Porcine Reproductive and Respiratory Syndrome Virus significantly prolongated HEV shedding [58]. Our study has several limitations. First, it was not initially designed to study these three viruses but to determine the prevalence of the HEV in pig farms in Corsica. Differences in detection could exist depending on criteria such as sampling time and place, age of pigs and clinical background of the tested animal population (diarrheic or healthy pigs). The lack of collection of clinical information (diarrhea or other symptoms) for the pigs included may have led to a bias in the analysis of the data; these data could have been essential for the improvement of knowledge concerning the studied viruses. The number of viruses studied could also have been larger to better assess the presence of major enteric viruses in Corsican pig farms. Finally, the lack of phylogenetic analysis of the different strains found is also a major limitation of our study. Indeed, the sequences obtained being in very varied zones of the genome of the various viruses, the phylogenetic analyses could not be realized.
In future studies, information about possible symptoms in pigs should be collected. It would also be useful to evaluate the phylogeny of the different strains found and to set up an RT-qPCR primer system to distinguish the different strains of Sapelovirus. Moreover, in view of the large variety of viruses present in the pig feces and the availability of microfluidic PCR technology, it would be helpful to set up microarrays that can detect all the principal known porcine enteric viruses. Moreover, the real impact of Kobuvirus, Astroviruses, Sapelovirus and other enteric viruses on animal health and breeding systems remains largely unknown and needs further epidemiological studies.
In conclusion, this study provides first insight on the presence of three common porcine enteric viruses in France and showed that they are frequently encountered in Corsica in pig farms using the traditional extensive breeding. The three viruses studied were found on almost all the farms, indicating widespread distribution. Moreover, the pigs tested were born and bred in Corsica, which demonstrates endemic local circulation. Whether such infections and co-infections can affect the productivity, impact the growth of pigs or cause immune weakness remains to be established. So far, this study has to be considered as a first step in the study of enteric viruses in Corsican pig farms.
Supporting information S1