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Discriminating the Eight Genotypes of the Porcine Circovirus Type 2 With Link et al. Virol J (2021) 18:70 https://doi.org/10.1186/s12985-021-01541-z RESEARCH Open Access Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR Ellen Kathrin Link1, Matthias Eddicks2, Liangliang Nan1, Mathias Ritzmann2, Gerd Sutter1 and Robert Fux1* Abstract Background: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an impor- tant task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associ- ated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods: Based on the analysis of several hundred PCV2 full genome sequences, we identifed PCV2 genotype specifc sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specifc for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/ PCV2c). To improve specifc binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplifcation efciency, diagnostic sensitivity and tested assay specifcity for the respective genotypes. Results: All eight PCV2 genotype specifc qPCRs demonstrated appropriate amplifcation efciencies between 91 and 97%. Testing samples from an epidemiological feld study demonstrated a diagnostic sensitivity of the respective genotype specifc qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specifcity of most qPCRs was excellent. Limited unspecifc signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specifc qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The intro- duced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers. Keywords: PCV2, Genotype, Real-time PCR, QPCR, TaqMan PCR, Locked nucleic acid Background One of the interesting features of the porcine circovi- *Correspondence: [email protected] rus type 2 (PCV2), a member of the family Circoviridae 1 Division of Virology, Department of Veterinary Sciences, LMU Munich, within the realm Monodnaviria, is the high nucleotide Veterinärstrasse 13, 80539 Munich, Germany substitution rate, which leads to an evolutionary dynamic Full list of author information is available at the end of the article that is comparable to that of RNA viruses and lesser to © The Author(s) 2021. 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. Link et al. Virol J (2021) 18:70 Page 2 of 12 that of double stranded DNA viruses [1]. Recent studies, time caused by the genotype PCV2b, which replaced analyzing appropriate big data sets, estimated the rate PCV2a as the dominant virus variant in PCVD out- –3 –4 at 1.4 × ­10 to 7.8 × ­10 substitutions per site per year breaks [8, 11–13]. Tis change is commonly known as [2], with a higher rate for the genotype PCV2a compared “PCV2 genotype shift”. For several years PCV2b domi- to PCV2b and PCV2d, and a slightly higher rate for the nated the world as causative agent of PCVD [3, 14–16] open reading frame 2 (ORF2; capsid protein gene) com- and caused severe economic losses. In 2008, Dupont pared to ORF1 (replicase protein gene) [3]. Although and colleagues published a retrospective study of PCV2 has a very small genome (1767–1777 bp), the high PCV2 isolates from Danish archives. Within this col- evolutionary rate is the cause of a great abundance of lection, three genome sequences obtained from sam- PCV2 variants in the feld. Terefore, the precise classi- ples collected in 1980, 1987 and 1990 did not cluster fcation of strains within the species PCV2 has been the with known PCV2a or 2b strains, but were assigned source for controversial discussions. Recently, Franzo and to a new branch of the PCV2 phylogenetic tree, later Segales proposed a new genotyping methodology based referred to as genotype PCV2c [8]. Interestingly, after on a phylogeny-grounded genotype defnition includ- their discovery, PCV2c strains were not detected in ing three requirements: (1) a maximum intra-genotype swine populations worldwide for a long period. More p-distance of the ORF2 sequence of 13%, (2) a bootstrap recently, PCV2c was detected in feral pigs in the Brazil- support at the corresponding internal node > 70% and ian Pantanal [17] and a Chinese group reported poten- (3) at least ffteen available sequences. Tis classifcation tial recombinants between PCV2b and PCV2c strains scheme allowed the defnition of eight PCV2 genotypes, [18]. However, for global swine industry PCV2c is still including the previously described genotypes PCV2a to of minor interest today. In contrast, genotype PCV2d PCV2f and the two new genotypes PCV2g and PCV2h is a challenge and a serious economic problem in pig [4] (see Table 1). production systems all over the world. It has become Up to now, the earliest proof of PCV2 in swine was the most prevalent PCV2 genotype (“second genotype reported from a German sample from 1962 [5]. In this shift”), at least in clinically afected animals. About a specimen, PCV2 detection was positive using DNA decade ago, Guo and colleagues reported an emerging in situ hybridization and PCR. However, since only a “mutant” PCV2 (strain BDH, HM038017) character- 66 bp sequence of ORF1 (GenBank EU158775) is avail- ized by a mutation in the stop codon of ORF2 resulting able in this case, the genotype cannot be specifed. in a capsid protein of 234 amino acid (aa) and which One can speculate whether this is the frst evidence of showed higher levels of virulence compared to classi- PCV2a, as it is believed that this genotype is the frst cal PCV2a or 2b strains [19, 20]. Remarkably, other to circulate in the pig population worldwide and has groups could not confrm a particular virulence of this been linked to the onset of the postweaning multisys- virus variant [21]. However, BDH-like viruses spread temic wasting syndrome (PMWS) [6–9]. In the frst quickly to North America [22], Europe [23] and the period, PCV2a was the most prevalent PCV2 geno- rest of the world [24]. In the phylogenetic tree of PCV2, type in clinically afected pigs. Tis changed with the these viruses formed a separate cluster together with rise of a second cluster of PCV2 variants, eventually virus strains originally described by Olvera and col- called PCV2b [10]. Initially in Europe, later in North leagues as subgroup 1C (= PCV2b, subgroup 1C) [13]. America, an increased incidence of PCV2 associated Terefore, diferent names were used for this group for diseases (PCVD) was observed in the early 2000s, this some time (mutant PCV2, mPCV2b, PCV2b-1C), but Table 1 Typical features of PCV2 genotypes Genotype Full genome ORF2 Occurrence PCV2a 1768 bp 702 bp/233 aa worldwide PCV2b 1767 bp 702 bp/233 aa worldwide PCV2c 1767 bp 705 bp/234 aa Brazil, China?, Denmarka PCV2d 1767 bp 705 bp/234 aa worldwide PCV2e 1777 bp 717 bp/238 aa China, Japan, Mexico, USA PCV2f 1767 bp 705 bp/234 aa Asia (China, India, Indonesia), Brazil, Croatia PCV2g 1767 bp or 1768 bp 702 bp/233 aa or 705 bp/234 aa Asia, Europe (Germany, Romania, Ukraine) PCV2h 1767 bp 705 bp/234aa Asia (China, India, Indonesia, Thailand, Viet Nam) a Historical samples Link et al. Virol J (2021) 18:70 Page 3 of 12 eventually this cluster was accepted as the genotype later it was integrated into a putative new genotype PCV2d [2]. Although the PCV2d reference strain BDH PCV2d [19], but alternative names (e.g. PCV2b-1C, was isolated in 2008 [19], the oldest published PCV2d mPCV2b) were still used [22, 23, 33] until the revision sequence (AY484410) was obtained from a Dutch study of PCV2 genotyping by Franzo and colleagues in 2015, carried out in 2001/2002 [25]. Furthermore, Xiao and which exemplifed the classifcation to genotype PCV2d colleagues demonstrated a signifcant heterogene- [2]. In the same year Xiao and colleagues introduced ity within the genotype PCV2d and subdivided it into the virus as one of the PCV2d-1 reference strains [24], the major clades PCV2d-1 (containing mainly “old” until recently it became one of the reference strains for sequences, obtained before 2008) and PCV2d-2 (BDH- genotype PCV2g [4].
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