Received: 14 December 2019 | Revised: 21 March 2020 | Accepted: 16 April 2020 DOI: 10.1111/tbed.13596 ORIGINAL ARTICLE Detection and genotyping of Porcine circovirus 2 (PCV-2) and detection of Porcine circovirus 3 (PCV-3) in sera from fattening pigs of different European countries Viviane Saporiti1,2 | Eva Huerta1,2 | Florencia Correa-Fiz1,2 | Bernd Grosse Liesner3 | Oliver Duran3 | Joaquim Segalés2,4,5 | Marina Sibila1,2 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de Abstract la Universitat Autònoma de Barcelona, PCV-2 is considered one of the most economically important viral agents in swine Bellaterra, Spain 2 worldwide. Recently, PCV-3 has been found in pigs affected by different disorders OIE Collaborating Centre for the Research and Control of Emerging and Re-emerging and in healthy animals. The objective of this epidemiological work was to describe Swine Diseases in Europe (IRTA-CReSA), the frequency of detection of PCV-2 and PCV-3 in pig farms of 9 European countries. Bellaterra, Spain 3 Boehringer Ingelheim Vetmedica GmbH AH Moreover, a second aim was to assess the most frequent PCV-2 genotypes found in Swine, Ingelheim, Germany the studied farms. Sera from 5 to 10 pigs per farm were collected from 2 to 11 farms 4 Departament de Sanitat i Anatomia per studied country. A total of 624 sera of fattening pigs (10–25 week old) from 64 Animals, Facultat de Veterinària, UAB, Bellaterra, Spain farms from Spain (n = 11), Belgium (n = 10), France (n = 8), Germany (n = 8), Italy 5 UAB, Centre de Recerca en Sanitat (n = 7), Denmark (n = 8), the Netherlands (n = 5), Ireland (n = 5) and Sweden (n = 2) Animal (CReSA, IRTA-UAB), Campus de were analysed by conventional PCR. In addition, one or two PCV-2-positive samples la Universitat Autònoma de Barcelona, Bellaterra, Spain per farm were genotyped by sequencing the ORF2 gene. PCV-3 PCR-positive sam- ples with relatively low Ct values were also sequenced and phylogenetically analysed. Correspondence Joaquim Segalés, UAB, Centre de Recerca en PCV-2 DNA was detected in pig sera from all European tested countries, but Sweden. Sanitat Animal (CReSA, IRTA-UAB), Campus A total of 132 out of 624 (21%) sera were positive for PCV-2 PCR, corresponding de la Universitat Autònoma de Barcelona, Bellaterra, Spain. to 30 out of the 64 (47%) tested farms. PCV-3 DNA was detected in 52 out of 624 Email: joaquim.segales@irta.cat (8%) sera, corresponding also to 30 out of the 64 (47%) studied farms from all tested Funding information countries. A total of 48 PCV-2 PCR-positive samples were successfully sequenced Instituto Nacional de Investigación y and genotyped, being PCV-2d the most frequently genotype found (n = 28), followed Tecnologia Agraria y Alimentaria, Grant/ Award Number: E-RTA2017-00007-00-00 by PCV-2b (n = 11) and PCV-2a (n = 9). These results pointed out PCV-2d as the most prevalent genotype currently in Europe. The PCV-3 phylogenetic analysis showed high identity (>98%) among sequences from all the analysed countries. The relatively low co-infection (3%), likely suggest an independent circulation patterns of PCV-2 and PCV-3. KEYWORDS genotype, phylogenetic analysis, porcine circovirus type 2 (PCV-2), porcine circovirus type 3 (PCV-3), swine Segalés and Sibila equally contributed to this study. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH Transbound Emerg Dis. 2020;67:2521–2531.  wileyonlinelibrary.com/journal/tbed | 2521 2522 | SAPORITI et al. 1 | I NTRO D U C TI O N between genotypes, bootstrap higher than 70% and at least 15 se- quences in each cluster (Franzo & Segalés, 2018). In contrast, PCV-3 Porcine circoviruses (PCVs) are small and non-enveloped vi- shares high similarity among available sequences, which makes diffi- ruses represented by three recognized species (PCV-1, PCV-2 cult to establish further classification of this virus species into geno- and PCV-3), and a tentative novel one designated as PCV-4. The types (Klaumann, Correa-Fiz, et al., 2018). Despite it is too early for a PCV-1 is considered non-pathogenic for swine and was firstly de- definitive classification, two (PCV-3a and PCV-3b) (Fux et al., 2018; scribed in 1982 as a contaminant of a pig kidney cell line (Tischer, Li et al., 2018) or three main clades (PCV-3a, PCV-3b and PCV-3c) Gelderblom, Vettermann, & Koch, 1982). PCV-2 was discovered as (Fu et al., 2018) have been proposed for this virus. Even though there the cause of a sporadic systemic disease in the 1990s in Canada are some different proposed groups, most authors agreed that the (Clark, 1996; Harding, 1996), which were followed by severe out- marker positions that determine the clade divisions are located in breaks all over the world (Segalés, Kekarainen, & Cortey, 2013). the amino acids (aa) sites S122A (ORF1), and A24V and R27K (ORF2) PCV-2 is the causative agent of systemic and reproductive dis- (Chen et al., 2017; Fux et al., 2018; Li et al., 2018). eases (named collectively as porcine circovirus diseases, PCVDs), PCV-2 is frequently found in pigs co-infected with other resulting in important losses to the pig production (Segalés, 2012). pathogens, such as Porcine reproductive and respiratory syn- In 2015, PCV-3 was firstly described in pigs with different clini- drome virus (PRRSV), Porcine parvovirus (PPV), Influenza A virus cal–pathological outcomes (Palinski et al., 2017; Phan et al., 2016). (IAV) and Mycoplasma hyopneumoniae, among others (Opriessnig & Subsequently, this virus has been detected by molecular methods Halbur, 2012; Segalés et al., 2013). These co-infections and the in- in pigs displaying a number of different conditions such as porcine teraction of the mentioned pathogens with PCV-2 have been widely dermatitis and nephropathy syndrome (PDNS) (Arruda et al., 2019; studied (Allan et al., 1999; Grau-Roma, Fraile, & Segalés, 2011; Palinski et al., 2017), respiratory disease (Kedkovid et al., 2018; Qi Opriessnig, Fenaux, et al., 2004; Opriessnig, Thacker, et al., 2004). et al., 2019; Shen et al., 2018; Zhai et al., 2017), enteric disease On the contrary, PCV-2 and PCV-3 co-infections have been only as- (Qi et al., 2019; Zhai et al., 2017), reproductive problems (Arruda sessed in studies from Asia (Kim et al., 2018; Xia et al., 2019) and only et al., 2019; Deim et al., 2019; Faccini et al., 2017), multisystemic one in Europe (Woźniak, Miłek, Matyba, & Stadejek, 2019). affection (Phan et al., 2016) and in healthy animals (Klaumann, Taking into account the abovementioned premises, the present Franzo, et al., 2018; Stadejek, Woźniak, Miłek, & Biernacka, 2017). work aimed to assess the frequency of detection of PCV-2 and PCV- The PCV-4 is the most recently described PCV species, and it has 3, as well as their co-infection in serum samples of fattening pigs been detected in animals with respiratory disease, diarrhoea and coming from 64 farms located in 9 European countries. In addition, PDNS-like lesions (Zhang et al., 2019). PCV-1, PCV-2 and PCV-3 the PCV-2 genotype dominance in these herds and the phylogenetic have been found infecting pigs all around the world, whereas variability of PCV-3 were also investigated. PCV-4 has been only described in China so far. Porcine circoviruses have a circular and single-stranded DNA genome, with a length around 1,759, 1,768 and 2,000 nt in PCV-1, 2 | M ATE R I A L A N D M E TH O DS PCV-2 and PCV-3 species, respectively. The genome of PCVs has three major open reading frames (ORFs). ORF1 encodes the repli- 2.1 | Samples cation proteins (Rep and Rep’), ORF2 encodes the structural protein (cap), and ORF3 encodes non-structural proteins with apoptotic A total of 624 serum samples from pigs ranging from 10 to 25 weeks capacity in PCV-1 and PCV-2 (Cheung, 2012). PCV-2 has also an of age were collected from randomly selected clinically healthy ani- ORF4, which codifies for a protein with anti-apoptotic effects (He mals. All available information regarding the sampled animals and et al., 2013; Lin et al., 2018). So far, ORF3 or ORF4 have not been farms are provided in Table S1. Sera from 5 to 10 pigs per farm were described in the PCV-3 genome. collected from 2 to 11 farms per country. The farms under study The highest genome variation among PCVs has been found in were from Spain (n = 11), Belgium (n = 10) France (n = 8), Germany PCV-2, which prompted for different proposals of sub-classifications (n = 8), Denmark (n = 8), Italy (n = 7), the Netherlands (n = 5), Ireland beyond the species level (Segalés et al., 2008). The most accepted (n = 5) and Sweden (n = 2). PCV-2 classification includes four different genotypes (from PCV-2a to PCV-2d) (Segalés et al., 2008). Taking into account that PCV-2 has a high mutation rate, close to single-stranded RNA viruses (Firth, 2.2 | DNA extraction, and conventional and Charleston, Duffy, Shapiro, & Holmes, 2009), it was expectable to quantitative PCR methods find new sequences representing novel genotypes, and thus, PCV-2e (Davies, Wang, Dvorak, Marthaler, & Murtaugh, 2016; Harmon DNA was extracted from 200 μl of serum using MagMAx™ Pathogen et al., 2015) and PCV-2f (Bao et al., 2018) have also been suggested. RNA/DNA Kit (Applied Biosystems®) according to the manufactur- Moreover, a recently new proposed classification re-arranged the er's protocol. A PCV-2 field isolate was used as positive extraction classification into eight genotypes in total, from PCV-2a to PCV-2h control. A PCR-negative serum sample for both PCV-2 and PCV-3 based on ORF2 gene analysis with maximum p-distance of 13% was used as negative extraction control. SAPORITI et al. | 2523 The PCV-2 PCR targeted the cap gene region (ORF-2) and was were reported, the sample was discarded to avoid error in performed using the primers PCV2all_F and PCV2all_R previously genotyping. described by Oliver-Ferrando et al. (2016) with slight modifica- The PCV-2 sequences obtained were phylogenetically anal- tions. The PCR mix included 2.5 μl of extracted DNA, 1.25 μl of ysed following the proposed classification by Franzo and Segalés each mentioned primer at 10 μM, 5 μl of 5 × PCR buffer, 2.5 μl (2018). The amplicons of PCV-2 ORF2 gene obtained herein ® of MgCl2 at 25 mM, 0.75 U of GoTaq Flexi DNA Polymerase were aligned against the representative strains of the proposed (Promega), 1 μl of dNTP stock solution at 5 mM, and DEPC-treated PCV-2 genotypes using MAFFT software (Katoh, Rozewicki, & water up to 25 μl of final volume. The thermal conditions initiated Yamada, 2019). A neighbour joining method using the p-distance with one cycle at 94°C for 5 min, followed by 35 cycles at 95°C for model was used to build the phylogenetic tree with 1,000 boot- 30 s, 53°C for 30 s and 72°C for 40 s. The final elongation was at straps. The phylogenetic tree was further edited using the iTOL 72°C for 7 min. software (Letunic & Bork, 2019). The PCV-3 PCR targeted the rep gene region (ORF1) of the virus The consensus sequences of PCV-2 ORF2 were deposited at the and was performed as previously described (Saporiti et al., 2019). NCBI GenBank with accession numbers MN653165 - MN653212. Briefly, 2.5 µl of extracted DNA, 12.5 µl of Go Taq® G2 Green Master Mix (Promega) and 0.4 µM of each primer were added to the reaction, as well as sterile water to reach final volume of 25 µl. 2.3.2 | PCV-3 Amplification was also performed at the same thermal conditions as previous described (Saporiti et al., 2019). For PCV-3 phylogenetic analyses, the full genome of the virus was The PCR products of PCV-2 and PCV-3 were checked by elec- attempted from those samples with the lowest qPCR Ct values trophoresis on 1.5% TAE agarose gel. Considering that PCV-3 se- per country. Three PCRs aiming the complete genome were per- quencing was demonstrated to be possible only at low Ct values (Fux formed using previously described primers and thermal conditions et al., 2018; Saporiti et al., 2019), a real-time qPCR was performed (Fux et al., 2018) with slight modifications. Briefly, 2 µl of extracted (Saporiti et al., 2019) in order to select those samples with the lowest DNA, 1 µl of each primer (10 µM), 10 µl of 5× SuperFi Buffer, 2 µl Ct values. The qPCR results were expressed in log10 of PCV-3 DNA of dNTP Mix (10 µM, Roche), 1 Units of DNA polymerase Platinum copies/µl of serum. SuperFi (Invitrogen) and sterile water were mixed to bring the final volume up to 50 µl. Thermal conditions were at 98°C for 5 min, 40 cycles of 98°C for 30 s, 55°C for 1 min and 72°C for 2 min, followed 2.3 | Sequencing and genotyping/ by a final extension at 72°C for 7 min. The amplicons were purified phylogenetic studies with a NucleoSpin® Gel extraction (Macherey-Nagel) Kit accord- ing to the manufacturer's protocol and sequenced by the Sanger 2.3.1 | PCV-2 method (ABI 3730XL - Macrogen Europe, Madrid, Spain). The qual- ity of the sequences was analysed by the Finch TV software and The PCR-positive samples per farm showing the strongest band in- trimmed in BioEdit versus 7.2.6 (Hall, 1999). After eliminating low- tensity in the gel (one or two per farm) were selected for genotyp- quality sequences, the three different amplicons obtained per sam- ing. PCV-2 genotyping was based on the ORF2 sequence (Franzo ple were assembled using the reference mapped-based strategy (Li et al., 2015). Amplification was performed by PCR using the same & Durbin, 2010) to achieve PCV-3 genomes with a maximum length primers targeting the ORF2 region mentioned above (Oliver- of 1.902 bp. The Integrative Genomics Viewer software (Robinson, Ferrando et al., 2016) with slight modifications. The reaction was Thorvaldsdóttir, Wenger, Zehir, & Mesirov, 2017) was used for visu- carried out on a mixture containing 3 µl of extracted DNA, 1× PCR alizing the assembly. buffer, 0.25 mM of dNTPs, 0.2 μM of each mentioned primers, one For the PCV-3 genome analysis, the sequences were then unit of DNA polymerase Platinum™ SuperFi™ (Invitrogen™) kit and trimmed and a gap was included in the nucleotide positions missing sterile water to final volume of 50 μl. The PCR thermal conditions (1,056–1,153). The partial genome sequences obtained were aligned were 98°C for five minutes followed by 40 cycles of 94°C for 30 s, against the 44 sequences within the same genomic region previously 53°C for 30 s and 72°C for 1 min, and a final elongation at 72°C used and classified by Fux et al. (2018). The obtained sequences were for 7 min. translated using MEGA 7 (Kumar, Stecher, & Tamura, 2016), and the PCV-2 amplicons were purified with ExoSAP-IT™ (Thermo amino acids were inspected manually to assess the previously de- Fisher Scientific) kit and sequenced by the Sanger method scribed group-specific motifs (Fux et al., 2018). A neighbour joining (BigDye ® Terminator v3.1 Cycle Sequencing Kit). The sequenc- (NJ) tree was constructed using the p-distance model. The tree was ing reactions were analysed using an ABI PRISM 3130xl Genetic evaluated with 1,000 bootstrap replicates to analyse the robustness ® Analyzer (Applied Biosystem ) at Servei de Genòmica, Universitat of the clustering (MEGA7). The nucleotide identity matrix comparing Autònoma de Barcelona (Spain). The quality of the sequences was the obtained sequences was done using Clustal Omega (Sievers & analysed by the Finch TV programme and trimmed in BioEdit soft- Higgins, 2014). The phylogenetic trees were edited using the iTOL ware 7.2.6 (Hall, 1999). When double or bad quality sequences software (Letunic & Bork, 2019). 2524 | SAPORITI et al. F I G U R E 1 Neighbour joining tree with p-distance model and bootstrapping at 1,000 replicates. Tree constructed based on ORF-2 gene of 48 PCV-2-positive samples following the Franzo and Segalés (2018) genotyping method. The samples were labelled using the ‘Country_ Farm_Sample’ code and coloured by genotype (PCV-2a in purple, PCV-2b in orange, PCV-2c in blue, PCV-2d in green, PCV-2e in pink, PCV-2f in yellow, PCV-2g in red and PCV-2h in light blue) [Colour figure can be viewed at wileyonlinelibrary.com] Sequences of PCV-3 were deposited at the NCBI GenBank with 3.2 | Sequencing and phylogenetic analysis the accession numbers MN583564 - MN583578. The cap gene region was sequenced and genotyped from 48 out of the 60 PCV-2 PCR-positive selected samples. Among the 12 non- 3 | R E S U LT S genotyped cases, 2 of them corresponded to two samples show- ing double sequences and the remaining 10 displayed bad quality. 3.1 | PCV-2 and PCV-3 detection Globally, 9 sequences corresponded to genotype PCV-2a (18.7%), 11 to PCV-2b (22.9%) and 28 to PCV-2d (58.3%) (Figure 1 and Table 2). PCV-2 DNA was detected in 132 out of 624 analysed sera (21%), corre- Remarkably, one French farm (FR4) had co-infection with two differ- sponding to 30 out of 64 farms (47%). The virus genome was detected ent genotypes, PCV-2a and PCV-2b. in a variable proportion of pigs from all tested countries, ranging from Regarding PCV-3, a qPCR was performed as a screening to se- 3 positive samples out of 50 (6%) in the Netherlands to 47 out of 67 lect the samples (n = 16) with the highest viral load from the differ- (70%) in France. Sweden was the only country where PCV-2 DNA was ent selected countries. The Ct values obtained were in the range not detected in any of the two analysed farms (Table 1). 32–27 which corresponded to 1.6–3 Log 10 PCV-3 DNA copies/ PCV-3 DNA was found in 52 out of 624 studied sera (8%) coming µl, respectively. Due to the low viral load in the analysed samples, also from 30 farms out of the 64 tested farms (47%); the frequency it was not possible to obtain the complete genome sequence; how- of PCV-3 PCR positivity ranged from 4% in Ireland (2/50) and Italy ever, 1,902 nt length sequences were recovered from 15 out of the (3/67) to 14% (7/50) and 15% (3/20) in the Netherlands and Sweden, 16 selected samples and used to perform the phylogenetic analysis. respectively (Table 1). The analysed sequences were from all countries but Denmark and Only 3% (16/624) of the tested sera was positive to both viruses Ireland, because all the positive samples from these countries had by PCR, which corresponded to 20% (13/64) of the studied farms. a very low PCV-3 load (from 0.1 to 1.3 Log10 copies/µl and one There was no evidence of co-infection in samples from three coun- sample has the amount below the quantification limit). The iden- tries: Germany, the Netherlands and Sweden (Table 1). tity matrix between the PCV-3 sequences obtained in this study The amount of virus in the PCV-3-positive samples was low and ranged from 98.9% to 100%, and when compared with all the 255 ranged from 0.97–3 Log10 copies/µl, being 18 out of 51 positive available references at GenBank (July 2019) with the same length, tested samples (one positive sample was not able to be tested quan- the similarity ranged from 96.7% to 100%. The NJ phylogenetic tree titatively as no more serum sample nor DNA was available) below was built including the references from Fux et al. (2018). Based in the quantification limit of the technique (between one and ten cop- the proposed clade classification and amino acid marker positions ies of DNA/μl). described by Fux (2018), eight samples clustered in the proposed TA B L E 1 Results of PCV-2 and PCV-3 PCR positivity per country, farm and sample levels FARMS† SAMPLES‡ Co-infection Co-infection Countries n PCV-2 (%) PCV-3 (%) (%) n PCV-2 (%) PCV-3 (%) (%) Spain (SP) 11 4 (36.6) 4 (36.4) 1 (9.0) 110 18 (16.3) 7 (6.4) 1 (0.9) Belgium (BG) 10 8 (80.0) 7 (70.0) 4 (40.0) 100 29 (29.0) 13 (13.0) 4 (4.0) France (FR) 8 7 (87.5) 5 (62.5) 5 (62.5) 67 47 (70.1) 7 (10.4) 7 (10.4) Germany (GER) 8 1 (12.5) 3 (37.5) 0 (0.0) 80 8 (10.0) 5 (6.3) 0 (0.0) Italy (IT) 7 3 (42.8) 1 (14.3) 1 (14.2) 67 10 (14.9) 3 (4.5) 1 (1.5) Denmark (DK) 8 3 (37.5) 3 (37.5) 1 (12.5) 80 11 (13.7) 5 (6.3) 2 (2.5) The Netherlands (NE) 5 1 (20.0) 3 (60.0) 0 (0.0) 50 3 (6.0) 7 (14.0) 0 (0.0) Ireland (IR) 5 3(60.0) 2 (40.0) 1 (20.0) 50 6 (12.0) 2 (4.0) 1 (2.0) Sweden (SW) 2 0 (0.0) 2 (100.0) 0 (0.0) 20 0 (0.0) 3 (15.0) 0 (0.0) TOTAL 64 30 (46.8) 30 (46.9) 13 (20.3) 624 132 (21.1) 52 (8.3) 16 (2.6) † Number of farms positive by PCV-2 and/or PCV-3 PCR (percentage of positive farms among total investigated ones). ‡ Number of serum samples positive for PCV-2 and/or PCV-3 PCR (percentage of positive samples among total investigated ones). SAPORITI et al. | 2525 2526 | SAPORITI et al. TA B L E 2 Number of PCV-2 genotypes per country and percentage of each genotype found among the sequenced samples No of Countries sequences PCV-2a PCV-2b PCV-2d Spain (SP) 6 SP11-2, SP11-3 SP4-8, SP5-7, SP8-5, SP8-7 n=2 n=4 Belgium (BG) 12 BG1-1, BG1-2, BG8-3, BG8-5 BG4-3, BG4-4 BG2-5, BG2-6, BG5-5, BG9-9, n=4 n=2 BG9-10, BG10-6 n=6 France (FR) 11 FR4-1, FR5-5, FR5-8 FR3-4, FR3-8, FR4-3, FR7-5, FR7-6 FR1-3, FR1-5, FR2-5 n=3 n=5 n=3 Germany (GER) 2 GER2-3, GER2-4 n=2 Italy (IT) 5 IT1-1, IT1-3 IT2-5, I7-1, IT7-2 n=5 Denmark (DK) 5 DK10-9- DK10-10 DK2-3, DK2-9, Dk6-8 n=2 n=3 The Netherlands (NE) 3 NE3-1, NE3-3, NE3-9 n=3 Ireland (IR) 4 IR3-9, IR1-4, IR5-2, IR5-10 n=4 Sweden (SW) 0 Total 48 9 (18.7%) 11 (22.9%) 28 (58.3%) Note: Each sequence genotype is coded using the country followed by the number of farm and the number of animal of the corresponding farm (‘Country_Farm_Sample’). subgroup a1, two in the subgroup a2, and five that were classified and 14.3% and 40.2% of pigs at 10 and 16 weeks old, respectively as b1 (Figure 2). in a study conducted in 2011 by Fablet et al. (2011). In the island of Sardinia, in Italy, a 10% (12/120) of PCV-2 frequency was found (Dei Giudici et al., 2019). Although the mentioned study just referred to 4 | D I S CU S S I O N a specific region of Italy, it was close to the prevalence found in the present study (14.9%, 10/67) in the same country. Pigs from Poland Porcine circoviruses are considered ubiquitous in the swine pro- were not screened in the present study, but two different studies duction worldwide as indicated in many studies (Klaumann, Franzo, recently described a higher prevalence of PCV-2 in serum from an- et al., 2018; Segalés et al., 2013). Therefore, it was expected to find imals from non-vaccinated farms (58.3%, 35/60 and 63,5%, 33/52) PCV-2 and PCV-3 in most, if not all studied European countries compared to vaccinated ones (2.8%, 3/106 and 17,6%, 54/307) (PCV-2 was not found in the two investigated Swedish herds), even (Woźniak, Miłek, Bąska, Miłek, Bąska, & Stadejek, 2019; Woźniak, the sampling per country was limited to 10 fattening pigs per farm. Miłek, Matyba, et al., 2019). Unfortunately, the information on vacci- In consequence, it is likely that the present study underestimates nation status was not available for 5 tested farms in this study; how- the real prevalence of both viruses in these countries, but provides ever, PCV-2 vaccines were used in most of them (n = 44), and hence, further evidence of PCV-3 distribution in Europe, the frequency of it was expectable to find a relatively low percentage of infected an- PCV-2/PCV-3 co-infections in fattening pigs and an updated PCV-2 imals overall. The case of Sweden was interesting since both farms genotyping in this geographic area. By consulting at the GenBank were negative for PCV-2 detection. In fact, a previous study of database (October 2nd, 2019), among the last 1,000 PCV-2 se- PCV-2 detection in Sweden performed on stool samples yielded also quences uploaded, only 47 were from Europe, indicating the need a low prevalence (2.9%, 3/104) (Zhou et al., 2016). Anyway, the most of continuous surveillance and update current genotypes circulation likely reason for the low prevalence detected could be the low num- in this continent. ber of herds screened, as well as the sample matrix used (Woźniak, In spite of ubiquity, the prevalence of PCV-2 in Europe can vary Miłek, Matyba, et al., 2019). In the study of Zhou and collaborators between countries. A recent study from Belgium indicated a 15.7% (2016), five European Countries (Austria, Germany, Hungary, Spain (50/319) frequency of PCV-2 detection in pigs displaying PCVD-like and Sweden) were screened and showed a higher percentage of disease (Wei, Xie, Theuns, & Nauwynck, 2019), while in the present PCV-2 DNA (42.2%, 176/417) (Zhou et al., 2016) when compared study in the same country was slightly higher, showing a frequency of to the present work (21.1%, 132/624). It is very likely that differ- 29% (29/100) in farms without major clinical diseases. In France, the ent countries involved in each study, sampling ages, number of age difference was even higher, with a 70% (47/67) of positivity herein groups studied, epidemiological situation at each time, health status, SAPORITI et al. | 2527 F I G U R E 2 PCV-3 phylogenetic tree constructed using the neighbour joining (NJ) method and p-distance model. The 15 PCV-3 sequenced samples are labelled in red, and following the ‘Country_Farm_Sample’ code. The genomes used as reference for the comparison analysis are labelled in black. The blue, orange, green and pink colours indicate the subtypes proposed by Fux et al. (2018) [Colour figure can be viewed at wileyonlinelibrary.com] sample matrix, PCR method used and number of animals per farm fully supported since it was found in farms from all studied coun- may account for the differences among published studies. tries. Also, the present work reports the presence of PCV-3 in the Regarding PCV-3, several studies have explored the prevalence Netherlands, France and Belgium for the first time. of infection in European countries (Denmark, Germany, Ireland, The percentage of co-infection of PCV-2 and PCV-3 in the pig Italy, Poland, Spain and Sweden), obtaining relatively comparable serum samples was fairly low (3%, 16/624), suggesting an inde- results ranging from 10% to 30% (Collins, McKillen, & Allan, 2017; pendent circulation pattern of both viruses. The results are in ac- Franzo, Legnardi, et al., 2018; Fux et al., 2018; Klaumann, Franzo, cordance with the low frequency of co-infection found in Poland, et al., 2018; Stadejek et al., 2017; Ye, Berg, Fossum, Wallgren, & in pigs vaccinated (0.9%, 1/106) and non-vaccinated (11.7%, 7/60) Blomström, 2018). The results of the present study offered slightly against PCV-2 (Woźniak, Miłek, Bąska, et al., 2019), which is so far lower frequency of detection (8.3% overall), but this was expectable the only study investigating the concomitant presence of both vi- since from each farm only one age group was investigated. In all ruses in Europe. Such difference among PCV-2 vaccinated and cases, the belief that PCV-3 is a ubiquitous virus across Europe was non-vaccinated farms is logical, since the vaccine is very effective in 2528 | SAPORITI et al. decreasing the prevalence of infection of this virus (Segalés, 2015). & Kekarainen, 2012; Vidigal et al., 2012). The high identity of Therefore, PCV-2 vaccinated farms should have lower percentage PCV-3 between available sequences (>98%) makes difficult to es- of co-infected animals. On the other hand, studies performed in tablish a potential genotype classification (Klaumann, Correa-Fiz, Asia have described the presence of both viruses to a variable fre- et al., 2018). However, taking into account the proposed marker quency such as 1.5% (3/200) (Sun et al., 2018), 10.3% (28/272) (Zhao positions determining clade divisions located in the amino acid et al., 2018), 12.5% (1/8) (Kedkovid et al., 2018), 15.7% (35/222) (Ku (aa) sites S122A for ORF1 and, A24V and R27K for ORF2 (Fux et al., 2017) and 28.3% (13/46) (Kim et al., 2017). Again, differences et al., 2018), the 15 obtained sequences clustered in 3 different among countries and studies may depend on the epidemiology of subgroups. At this stage, it is difficult to elucidate any potential each region, but many other factors as sampling strategy and PCR clade distribution by country, since PCV-3 sequences found in methods used may account for variability. different farms from some countries (Belgium, France, Germany Since PCV-2 has a high mutation rate (Firth et al., 2009), vari- and Italy) were represented in more than one clade, while the se- ation in its genome is constant and the generation of novel se- quences from the Netherlands were found in one single group. quences classified in novel genotypes is likely (Franzo & Segalés, Despite previous classification proposals, the high identity among 2018). Although PCV-2a seemed to be the most frequent genotype different PCV-3 sequences obtained here fits with the very recent before the advent of PCV-2-systemic disease outbreaks over the publication suggesting that one single PCV-3 genotype does exist world, genotype PCV-2b was the most spread one during them and so far (Franzo et al., 2020). Moreover, since sera samples were afterwards (Segalés et al., 2013). Curiously, in the last 5–7 years, obtained from farms with no overt disease, it cannot be stated PCV-2d has become the most prevalent genotype worldwide (Dei any apparent relationship between PCV-3 classification and virus Giudici et al., 2019; Franzo & Segalés, 2018; Grierson, Werling, pathogenicity or any specific disease. Bidewell, & Williamson, 2018; Xiao, Halbur, & Opriessnig, 2015). In conclusion, PCV-2 and PCV-3 are spread all over Europe, but This finding has been recognized mainly in Asia (Franzo & Segalés, with a very low rate of co-infection, suggesting independent cir- 2018), where most of the sequences available on GenBank comes culation patterns of both viruses. The most common genotype of from in the last 5 years. In the same line, the present study rein- PCV-2 found in this study was PCV-2d. This finding would reinforce forces the domination of PCV-2d genotype also in Europe, since the global trend shift from PCV-2a to PCV-2b first and then from it was found in 58.3% (28/48) of the genotyped samples. In con- PCV-2b to PCV-2d (Franzo & Segalés, 2018). The obtained PCV-3 trast, PCV-2-b and PCV-2a accounted for 22.9% (11/48) and 18.7% sequences were closely related, with >98% of identity, further sug- (9/48) of the sequenced cases. PCV-2a genotype was detected in gesting the existence of one single genotype circulating in Europe. three countries (Spain, Belgium and France). Noteworthy, these countries had the highest frequency of PCV-2 detection in general, AC K N OW L E D G E M E N T S with 16% in Spain, 29% in Belgium and 70% in France, which could The authors would like to acknowledge the financial support from increase the probability to detect this PCV-2a genotype in spite Boehringer Ingelheim Vetmedica GmbH AH Swine (Germany) and of the decreasing trend globally (Dei Giudici et al., 2019; Franzo E-RTA2017-00007-00-00 INIA Project from the Instituto Nacional de & Segalés, 2018; Grierson et al., 2018; Xiao et al., 2015). Taken Investigación y Tecnologia Agraria y Alimentaria (Spanish Government) altogether, all three genotypes seem to be circulating in Europe and CERCA Programme/Generalitat de Catalunya. We also thank all nowadays, although the real prevalence of each of them should the veterinarians involved in the present work that allowed the study be elucidated by means of larger epidemiological studies in each to be carried out by collecting samples from all mentioned countries. country. Curiously, PCV-2 sequences from all genotypes tended to cluster within a same country, which probably reflects that most C O N FL I C T O F I N T E R E S T isolates are circulating at a local level rather than at a continental All authors have declared no conflict of interest. level. Almost all samples from the same farm clustered close one to another, which they also belonged to the same genotype, ex- E T H I C A L A P P R OVA L cept for two samples from France (FR_4_1 and FR4_3), which were The authors confirm that the ethical policies of the journal, as noted genotyped as PCV-2a and PCV-2b, respectively. Infection by more on the journal's author guidelines page, have been adhered to. No than one genotype in the same farm and even in the same pig has ethical approval was required as this is a research data from general already been widely documented (Grau-Roma et al., 2008; Hesse, samples obtained in particular surveillance programmes performed Kerrigan, & Rowland, 2008). Although the genotypes obtained in in the different countries. the present study were subjected to the new classification pro- posed by Franzo and Segalés (2018), with eight different geno- DATA AVA I L A B I L I T Y S TAT E M E N T types, only the three major ones were detected. The data that support the findings of this study are openly available It must be emphasized that obtained results may prompt the in the GenBank repository (https://www.ncbi.nlm.nih.gov/genba​ future interest to investigate the phylogeography of PCV-3 to de- nk/) with Accession numbers: from MN653165 to MN653212 for termine its epidemiology at a global level, as it has been performed PCV-2 sequences, and from MN583564 to MN583578 for PCV-3 for other single-stranded DNA pig viruses (Cortey, Pileri, Segalés, sequences. SAPORITI et al. | 2529 ORCID circovirus 3 field strains from Denmark, Italy and Spain demon- strates a high within-Europe genetic heterogeneity. Transboundary Joaquim Segalés https://orcid.org/0000-0002-1539-7261 and Emerging Diseases, 65(3), 602–606. https://doi.org/10.1111/ tbed.12836 REFERENCES Franzo, G., & Segalés, J. (2018). Porcine circovirus 2 (PCV-2) geno- Allan, G. M., Kennedy, S., McNeilly, F., Foster, J. C., Ellis, J. 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