First identification of porcine parvovirus 6 in Poland

Porcine parvovirus type 1 is a major causative agent of swine reproductive failure. During the past decade, several new parvoviruses have been discovered in pigs. Porcine parvovirus type 6 (PPV6), recently identified, has been reported in pigs in China and in the USA while the PPV6 status in the European pig population remains undetermined. In the present study, PPV6 DNA was identified in serum samples collected from domestic pigs in Poland. In investigated herds, the prevalence of PPV6 was 14.9 % (15/101 samples). Sequencing was conducted, and 11 nearly complete PPV6 genomes were obtained. Phylogenetic analysis indicated that PPV6 sequences cluster into four distinct groups, and the Polish PPV6 strains from three individual farms were present in three of these four groups. In addition, the Polish PPV6 strain P15-1 was identified as a putative recombination of an ORF1 from US stains and an ORF2 from Chinese strains. This is the first identification of PPV6 in Europe, and this finding will encourage future epidemiological studies on parvoviruses in European pigs. Electronic supplementary material The online version of this article (doi:10.1007/s11262-016-1386-y) contains supplementary material, which is available to authorized users.


Introduction
Parvoviruses are small, non-enveloped DNA viruses with a single-stranded linear genome of approximately 4.0-6.3 kb in size containing two to three main open reading frames (ORFs) [1,2]. The family Parvoviridae consists of two subfamilies: the Parvovirinae infecting vertebrates and the Densovirinae infecting arthropods. The subfamily Parvovirinae can be further divided into eight genera [3] of which Protoparvovirus, Bocaparvovirus, Copiparvovirus, and Tetraparvovirus contain viruses that infect pigs.
Porcine parvovirus 1 (PPV1) is a well-known pathogen in pigs and frequently associated with reproductive failure in breeding herds, resulting in widespread breeding herd vaccination in an effort to control this virus [4]. PPV1 was first isolated in Germany in 1965 as a contaminant of cell cultures used for classical swine fever virus propagation [5]. During the past decade, several new species of parvoviruses have been identified in pigs commonly known as PPV2 through PPV6. Unlike PPV1 which belongs to the genus Protoparvovirus, the emerging PPV species belong to the genera Tetraparvovirus (PPV2, PPV3) and Copiparvovirus (PPV4, PPV5, and PPV6) [3].
PPV6 shares only 20.5-42.6 % nucleotide similarity with other members of the subfamily Parvovirinae and is most closely related to PPV4 [6]. PPV6 was first identified in aborted pig fetuses in China in 2014 and subsequently was also reported in the USA [6,7]. The distribution of PPV6 in the global pig population and possible clinical signs associated with PPV6 infection remain to be determined. In this study, PPV6 DNA was identified in serum samples collected from domestic Polish pigs and further characterized by genomic sequencing. Eleven nearly complete PPV6 sequences were obtained. To our knowledge, this study is the first to describe PPV6 in European pigs.

Materials and methods
Serum samples (n = 101) were collected from 2-to 18-week-old commercial cross-bred pigs located on three commercial Polish pig farms designated as U, K, and P. The herds ranged in size from 600 to 1000 pigs. Total nucleic acids were extracted from serum samples using the MagMAX TM Viral RNA Extraction Kit (Thermo Scientific) on an automated extraction system (Thermo Scientific Kingfisher Flex) according to the manufacturer's instructions. The extracted samples were stored at -20°C until testing. Initial detection of PPV6 DNA in the sample set was done by a real-time PCR developed based on the alignments of PPV6 available in the GenBank. A pair of primers (PPV6F3250:5 0 -GGCTTCATAATCCCTCCAAAACCT-3 0 , PPV6R3404:5 0 -GCTCATCTTCCTCTTGTTTCTCCTG-3 0 ) targeting 154 bp in ORF2 and a specific TaqMan Ò probe (PPV6prob: Cal Fluor Orange 560-5 0 -CCTCCTCCTCCTC CCTCTCCAATTCCT-3 0 -BHQ1) within the region of the detection primers were designed using Primer Express software (Version 3.0; Applied Biosystems). The PCRs were carried out in 96-well plates, with each reaction containing a total volume of 25 ll, 12.5 ll of the TaqMan Ò -Universal PCR Master Mix (Applied Biosystems Ò ), 2.5 ll of the sample or standard DNA, 1 ll of 10 lM of each of the two primers, 0.5 ll of 10 lM probe, and 7.5 ll nuclease-free water. Amplification reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems Ò ) under universal conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. Samples with no cycle threshold (C T ) at 37 cycles were considered negative. The specificity of the probe was confirmed by BLAST analysis and using DNA samples positive for other DNA viruses including PPV1 through PPV5, porcine circoviruses 1 and 2, and Torque teno sus virus. The sensitivity of the PPV6 real-time PCR assay was determined by testing 10-fold serial dilutions of the PPV6 DNA standard in triplicate. The generated standard curve with a regression value of R 2 = 0.989 was linear and showed a reaction efficiency of 114.092. The detection limit was 50 genome copies per reaction with Ct values around 36 in all three replicates. Real-time positive samples were also tested by a conventional PCR assay as previously described [6]. To further characterize PPV6 DNA positive samples, a set of overlapping primers was designed to amplify the nearly complete PPV6 genome (Table S1). The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 ll volume containing 25 ll of Q5 High-Fidelity 29 mix, 2 ll of a 10 lM forward primer, 2 ll of a 10 lM reverse primer, 2 ll of the extracted DNA, and 19 ll of ddH 2 O under the following thermocycler conditions: denaturation for 30 s at 98°C followed by 30 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 60 s, and a final extension step at 72°C for 10 min. The PCR products were purified by the PureLink TM Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Life Technologies) according to the manufacturer's instructions and were sequenced at the Edinburgh Genomics facility, University of Edinburgh, Edinburgh, United Kingdom. The sequences were assembled by the SeqMan program of Lasergene 7.0 software (DNASTAR Inc., Madison, Wisconsin, USA) and compared with other available PPV6 sequences using the BLAST web-based program (http://www.ncbi.nlm.nih.gov/ BLAST). PPV6 sequences were then aligned with available reference sequences downloaded from GenBank by Clustal Omega (http://www.ebi.ac.uk/). Phylogenetic trees were inferred by maximum-likelihood (ML) method (amino acid sequences) or neighbor-joining method (nucleotide sequences) implemented in MEGA v7.0 [8]. Support for individual nodes was determined by 1000 bootstrap replicates.

Results and discussion
In the present study, among the 101 serum samples from three different Polish farms, PPV6 DNA was detected in 14.9 % (15/101) of the samples. The individual PPV6 farm prevalence rate ranged from 3.8 % for farm P to 22.2 % for farm K (Table S2). Eleven of the PCR positive samples were sequenced (Table S2) the NS1 protein which is commonly used to differentiate members of the subfamily Parvovirinae, all PPV6 strains identified in Poland were closely related to PPV6 reference strains and located in the genus Copiparvovirus together with PPV4 and PPV5 (Fig. 1a) as described previously [6,7]. The complete coding regions of the identified PPV6 strains shared 98.0-99.8 % nucleotide sequence identity with US PPV6 strains and 96.4-98.9 % nucleotide sequence identity with Chinese PPV6 strains. Sequences isolated from the same farm showed a high nucleotide identity (98.6-99.9 %) to each other. The motifs of the calcium binding loop (YXGXR) and the catalytic residues (HDXXY) of PLA2 were found to be conserved among PPV6 strains. Compared with NS1, VP1 resembles the variable protein in the PPV6 genome. The VP1 sequences of the investigated Polish PPV6 strains showed 94.3-100 % nucleotide identity (95.0-100 % amino acid identity) with reference sequences. A previous study indicated that there may be three conserved regions in the PPV6 VP1 protein [7]. However, compared with reference PPV6 sequences identified in China or in the USA, the first two regions were not conserved (Table 1). In the first region (aa 164-256), four amino acid mutations were observed, three amino acid changes were present in Farm U PPV6 strains (U18-1, U18-4, U18-5, U18-8 and U18-9), and the other one was present in a Farm K PPV6 strain (K17-3). Furthermore, Farm U strains contained a total of 28 mutations in the second region (aa 414-787). Phylogenetic analysis of the VP1 gene showed that PPV6 strains clustered in four distinct groups (Fig. 1b). While the PPV6 sequences from Farm K were closely related to the three US strains, Farm U sequences, which shared most mutations in the putative conserved regions, formed a distinct cluster. Interestingly, strain P15-1 from Farm P shared 99.9 % homology (one nucleotide difference) with the US strain KSU7-SD-2014 (GenBank accession no. KR709268) in the ORF1 region. In contrast, in the ORF2 region, P15-1 showed 99.9 % similarity (one nucleotide difference) with the Chinese strain TJ (GenBank accession no. KF999685). Moreover, phylogenetic analysis showed that P15-1 clustered with different strains in NS1

Conclusions
Our findings demonstrate that PPV6 circulates in swine herds in Poland. This study represents the first identification of PPV6 in European pigs, indicating that PPV6 is not restricted to Asia and North America. Phylogenetic analysis suggests that the PPV6 P15-1 strain might be a chimeric virus containing an ORF1 gene from the USA and an ORF2 gene from China. Further studies are ongoing to investigate the epidemiology and evolution of parvovirus in European pigs. To date, the exact relatedness of pathogenesis and occurrence of novel species of PPV strains in pigs remains unknown.

Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
Serum samples All serum samples used in this study were collected as part of routine surveillance studies.
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