CHARACTERIZATION OF PORCINE PARVOVIRUS TYPE 3 AND PORCINE CIRCOVIRUS TYPE 2 IN WILD BOARS (SUS SCROFA) IN SLOVAKIA

In recent years, various novel parvoviruses have been identified in pigs (Sus scrofa) by using modern molecular techniques, revealing new clades within the family Parvoviridae. In addition to the well-known porcine parvovirus (PPV) type 1 (PPV1), divergent sequences of PPV2–PPV5 have been characterized (Xiao et al. 2013). One of these emerging viruses, also known as porcine parvovirus type 3 (PPV3; Cheung et al. 2010), was discovered in Hong Kong and initially named porcine hokovirus (Lau et al. 2008). In a recently proposed taxonomy, the virus represents a species within the genus Tetraparvovirus, termed ungulate tetraparvovirus 2 (Cotmore et al. 2014). Porcine parvovirus type 3 has a single-stranded, linear DNA genome of approximately 5.4 kilobases (kb; Bellehumeur et al. 2013). The viral genome contains two major nonoverlapping open reading frames (ORFs), which encode nonstructural protein NS1 and overlapping VP1/VP2 proteins of the capsid. To date, PPV3 has been confirmed in domestic pigs in several parts of the world, including Europe (Streck et al. 2013), Asia (Li et al. 2013), North America (Xiao et al. 2012), and Africa (Adlhoch et al. 2013). The overall prevalence has been estimated to be between 3.1% and 58.6% (Szelei et al. 2010; Li et al. 2012). Furthermore, recent detection of the virus in beef suggests that it can infect both swine and cattle (Bos taurus; Zhang et al. 2014). The occurrence of PPV3 in European wild boar (Sus scrofa L.) has been investigated only in wild boars in Germany (Adlhoch et al. 2010) and Romania (Cadar et al. 2011).

In addition to PPV3, a wide spectrum of pathogens shared among Suidae has been confirmed in wild boars (Ruiz-Fons et al. 2008). One of them, porcine circovirus type 2 (PCV2) has been the object of intensive research in the last decade. Porcine circovirus type 2 is an icosahedral, nonenveloped virus containing an approximately 1.76-kb circular DNA genome. This small virus of the family Circoviridae has been associated with several pathologic conditions in domestic pigs that were collectively described under the name of porcine circovirus- or porcine circovirus-associated diseases (PCVD or PCVAD; Allan et al. 2002; Opriessnig et al. 2007). Among them, postweaning multisystemic wasting syndrome (PMWS) is considered the most significant in terms of mortality and consequent economic impact on the swine industry. Whereas PCV2 is considered to be ubiquitous in domestic pigs (Allan and Ellis 2000), knowledge of the PCV2 infection rates in wild boars is still insufficient.

In general, information on newly identified porcine viruses in Slovakia is very limited. Considering the possible role of wild boars as a reservoir for viruses that can infect domestic pigs, investigations in this field can contribute to a better understanding of the epidemiology of such viral infections. Herein, we determine the prevalence of PPV3 and PCV2 in wild boars in Slovakia and genetically characterize Slovak isolates.

The samples were obtained from hunted wild boars in Slovakia. There are approximately 50,000 wild boars with the highest density in the southern part of central Slovakia. Wild boars are hunted in Slovakia by battue or one-man hunting methods.

We used 194 tissue homogenates prepared from pooled samples of three organs (tonsil, spleen, and kidney). Wild boars were hunted in March–May 2012 in 64 of 79 districts representing all eight administrative regions of Slovakia. No more than four animals from each district were selected for study to ensure proper spatial distribution of data. The selection was composed of 118 samples from wild boars <1 yr old and 76 from the animals >1 yr. Age was determined by experienced hunters based on dentition, wear pattern, and morphology. Clinical samples of tonsil, spleen, and kidney were collected by the Veterinary Institute in Zvolen, Slovakia, as a part of a regular national classical swine fever virus (CSFV) surveillance program.

We prepared 20% (w/v) homogenates of pooled organ (tonsil, spleen, and kidney) samples in 0.01 M phosphate-buffered saline (pH 7.4; Sigma-Aldrich Co., St. Louis, Missouri, USA). Total DNA was extracted from 200 μL of homogenates by using Chelex 100 Molecular Biology Grade Resin (Bio-Rad Laboratories, Inc., Hercules, California, USA) according to the manufacturer's recommendations. The extracted DNA was stored at −20 C until examination.

DNA samples were primarily assessed for PPV3, whose genome was later characterized in two regions. In a single PPV3 diagnostic PCR, the primers PPV3DF/PPV3DR flanking 392-base pair (bp) fragments from the predicted VP1 gene were used (Cadar et al. 2011). The PCR mixture consisted of 1× ThermoPol Reaction Buffer (New England Biolabs, Inc., Ipswich, Massachusetts, USA), 200 μM deoxynucleoside triphosphates (dNTPs; Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA), 0.3 μM of each primer, 0.5 U of Taq DNA polymerase (New England Biolabs, Inc.), and 2 μL of template and ultrapure H₂O for molecular biology (EMD Millipore Corporation, Billerica, Massachusetts, USA) added to the final volume of 25 μL. The reaction was performed under the following conditions: one step at 95 C for 2 min followed by 37 cycles of 95 C for 1 min, 56 C for 1 min, and 68 C for 1 min, with the final step at 68 C for 5 min.

For phylogenetic analyses, the partial sequence of the predicted NS1 gene was chosen. The seminested PCR was employed for amplification of a 1,036-bp fragment. The first round of PCR was carried out in a 50-μL mixture with PPV3F1040 and PPV3DR primer pair (Cadar et al. 2011) together with 4 μL of template and other components added in the same concentration as in the diagnostic PCR. After an initial denaturation at 95 C for 2 min, 30 cycles of 95 C for 1 min, 52 C for 1 min and 68 C for 1 min, and extension at 68 C for 5 min were performed. A 50-μL seminested PCR differed from the first-round PCR only by adding 2 μL of the PCR product as a template, employing PPV3R2075 reverse primer (Cadar et al. 2011) and performing 35 cycles of amplification.

For PCV2 diagnostics in the same animals, the CF8 and CR8 primer pair flanking a 264-bp fragment from ORF2 was used (Larochelle et al. 1999). The single PCR protocol was the same as for PPV3 detection, except the annealing temperature was set to 62 C.

For phylogenetic analysis, the entire ORF2 region in length 702 or 705 bp was chosen. To obtain the complete ORF2 sequences, a nested PCR for amplification of a 759-bp fragment was performed. Both amplification reactions consisted of 1× Phusion HF Buffer (Thermo Fisher Scientific), 200 μM dNTPs (Thermo Fisher Scientific), 0.5 μM primers described previously (Vlasakova et al. 2011), 0.02 U/μL Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), 2 μL of template and ultrapure H₂O (EMD Millipore Corporation) added to the final volume of 25 μl in the first round and 50 μL in the second round of PCR. Cycling conditions were set up according to the polymerase manufacturer's recommendations.

All PCR products were visualized under ultraviolet light on 2% agarose gel (SERVA Electrophoresis GmbH, Heidelberg, Germany) after staining with GelRed^TM Nucleic Acid Gel Stain (Biotium, Inc., Hayward, California, USA).

Selected amplicons from VP1 and NS1 genomic regions of PPV3, as well as amplicons from ORF2 of PCV2 isolates, were sequenced in both directions by the Sanger method by using PCR primers and fluorescently labeled dideoxynucleoside triphosphates (Microsynth Austria GmbH, Vienna, Austria). Sequences were submitted to the GenBank database under Accession No. KP768467–KP768526. The chromatograms were proofread in Lasergene's SeqMan Pro, and nucleotide sequences were aligned by ClustalW method implemented in Lasergene's MegAlign, where sequence distances were also calculated (DNASTAR, Inc., Madison, Wisconsin, USA). Phylogenetic trees were constructed by the neighbor-joining method with bootstrap value for 1,000 replicates, while the evolutionary distances were computed according to the Kimura 2-parameter model by using the MEGA6 software (Tamura et al. 2013).

The differences in PCR screening results between selected groups were evaluated by using the chi-square test. Values of P<0.05 were considered statistically significant. All data analyses were carried out by using GraphPad Prism version 5.00 for Windows (GraphPad Software, Inc., La Jolla, California, USA).

We detected PPV3 in 37 of 194 (19.1%) animals sampled throughout the monitored area. Differences in geographic distribution of virus among eight regions were observed (Fig. 1A). While the highest prevalences were detected in the southwest of the country (50% in Bratislava and 33% in Trnava region), the presence of PPV3-positive animals was lower in the remaining parts of Slovakia (0–13%), with an exception of 32% in Kosice region located in the southeast (Table 1). PPV3 was detected only in 25 of 64 districts, suggesting that among the districts sampled, 61% were virus free. The presence of PPV3 was demonstrated similarly in both age categories, 18.6% of animals under 1 yr old and 19.7% for the adults (Table 1).

The overall PCV2 prevalence was 43.8%. Contrary to PPV3 results, these findings indicated moderate regional differences in its distribution as PCV2 prevalence varied from 31% to 56% (Table 1). The virus was detected widely in the study area excluding 12 randomly distributed districts (Fig. 1B). Among young wild boars, 48.3% of animals were infected compared with 36.8% in animals >1 yr (Table 1), although the difference was not statistically significant (P = 0.116).

The percentage of coinfection with both viruses was 11.3% and seemed to be not influenced by the age, as similar rates of coinfection in young (11.0%) and adult wild boars (11.8%) were observed (Table 1).

For phylogenetic studies, 20 of 37 PPV3-positive samples were selected based on location. Comparison of partial VP1 sequences (356 bp) showed that PPV3 isolates from Slovakia were at least 98% identical and possessed 96–100% nucleotide identity to 110 sequences retrieved from GenBank. Phylogenetic analysis of this genomic region also confirmed high sequence similarity among viral isolates originating from all over the world (data not shown).

Partial NS1 sequences (895 bp) of the same PPV3 isolates shared 96–100% identity. The isolates sharing 100% identity in NS1 region were also 100% identical in the VP1 region. The comparison of NS1 obtained to 79 deposited sequences from different continents revealed 93–100% nucleotide identity. The phylogenetic tree constructed from nucleotide sequences of virus detected in Slovakia, as well as in other parts of the world, showed close evolutionary distances between members of all tree branches. In spite of this, obtained sequences were located in two clusters whose reliability was supported by bootstrap resampling (arrow in Fig. 2). The majority of PPV3 isolates from Slovakia (n = 15) were similar to those from Europe, Africa, and North America. Five sequences were grouped in the other cluster mostly formed by isolates from Asia (Fig. 2).

Among 20 PCV2 nucleotide sequences of complete ORF2, 91–100% identity was observed. They were 91–100% identical to domestic pig isolates from Slovakia. The analyzed sequences shared at least 87% nucleotide identity with 569 PCV2 sequences deposited in GenBank. The phylogenetic tree was constructed on the basis of alignment of 702-bp ORF2 sequences from 99 PCV2 isolates. Most of the PCV2 variants analyzed in Slovak wild boars (n = 17) belonged to the 1A/1B cluster of PCV2b genotype. Three sequences were clustered into PCV2a, cluster 2D (Fig. 3).

Circulation of PPV3 in domestic pigs and wild boars has been found by using molecular detection methods in several parts of the world, suggesting a possible global distribution of this virus (Lau et al. 2008; Cadar et al. 2011; Xiao et al. 2012; Adlhoch et al. 2013). We documented 19.1% overall prevalence of PPV3 in the wild boar population in Slovakia. This prevalence is lower than reports in wild boars in Germany (32.7%; Adlhoch et al. 2010) and Romania (22.8% and 50.5%; Cadar et al. 2011). The distribution of virus showed geographic variations among Slovak regions; PPV3 was spread predominantly across south Slovakia, while the lowest rates of prevalence were found in the northern regions, with the Zilina region apparently free of infection. The number of PPV3-infected animals in adults was not significantly higher than in juveniles, which contrasts with reports of increasing PPV3 prevalence with age of wild boars (Adlhoch et al. 2010; Cadar et al. 2011) and domestic pigs (Xiao et al. 2012). It is not clear if this discrepancy reflects different types of specimens used for virus detection. In our study, all samples originally used in the national CSFV surveillance program were prepared by the same procedure, resulting in homogenates of three pooled organs (tonsil, spleen, and kidney). The use of these homogenates of lymphoid and parenchymal tissues increased the probability of virus detection in infected wild boars.

In the PPV3 genome, the ORF1 encoding NS1 has been recognized as the region suitable for studies on genetic variability (Cadar et al. 2011). The phylogenetic tree generated for this genomic region displayed high overall similarity between Slovak viral sequences and others collected worldwide. Slovak sequences were grouped into two known clusters (Streck et al. 2013). Five of them were located in the cluster predominantly formed by isolates from Chinese swine together with several isolates from Romanian wild boars and German domestic pigs. These isolates were distributed across Slovakia with no apparent association with a specific geographic area. The remaining sequences belonged to the second cluster, mostly consisting of isolates from Europe (Germany, Romania, and the UK), as well as from the US, Canada, and Cameroon. Several Slovak sequences included in this cluster showed single nucleotide substitutions characteristic for isolates originating from geographically close districts. Taken together, although the common PPV3 genotype is widespread worldwide, the viral genome is subject to diversification, which is reflected by sequence variability associated with geographic origin, as has been recently observed in isolates from animals from Eastern Europe (Cadar et al. 2013).

In general, PCV2 was more prevalent than PPV3 in selected animals since the overall frequency of PCV2 infection in Slovakia reached 43.8%. Other data on wild boar populations obtained by molecular methods detecting viral DNA from Slovenia (25.0%; Toplak et al. 2004), Hungary (20.5%; Cságola et al. 2006), Germany (63.1%; Reiner et al. 2010), Romania (43.9%, 8.3% in Transylvania; Turcitu et al. 2011; Cadar et al. 2012), and Poland (75.6%; Fabisiak et al. 2012), indicate that PCV2 prevalence in Europe varies broadly. Fifty-seven of 85 infected wild boars analyzed (67%) were <1 yr old, suggesting viral infection in early life. Despite the absence of evidence of poor health in the animals tested, this observation is in accordance with sporadic reports of PMWS-affected young wild boars (Vicente et al. 2004; Morandi et al. 2010).

The ORF2 encoding capsid protein is considered a suitable region for genotyping and epidemiologic analyses of PCV2 (Olvera et al. 2007; Segalés et al. 2008; Cortey et al. 2011). The alignment analysis did not confirm any association of Slovak sequences with their geographic origin. Phylogenetic analysis of this genomic region grouped Slovak isolates from wild boars and domestic pigs (Vlasakova et al. 2011) into two clusters, PCV2b-1A/1B and PCV2a-2D. Furthermore, virus prevalence in wild boars was significantly lower (P = 0.001) compared with swine in Slovakia, where 64.2% prevalence was observed (Vlasakova et al. 2011). Similar findings have been observed in other countries (Reiner et al. 2010; Cadar et al. 2012). Additionally, a certain independence of PCV2 circulation in domestic pigs and wild boars with only limited cross-over infections between the two populations has been proposed (Reiner et al. 2011; Cadar et al. 2012). Considering that intensive pig farming, which allows little or no direct contact between pigs and wild boars, is predominant in Slovakia, the role of PCV2 exchange between farmed and free-living animals in virus distribution and its genetic diversity requires further elucidation.

Coinfection with PPV3 and PCV2 in wild boars has not been reported in the scientific literature. The proportion of wild boars simultaneously infected with PPV3 and PCV2 in this study (11.3%) was higher than that in Hungarian (3.3%; Cságola et al. 2012) and North American domestic pigs (2.6% in serum, 7.3% in lung samples), where no significant association of PPV3 prevalence with PCV2 infection and PCVAD status of animals was reported (Opriessnig et al. 2014). Nevertheless, a 20.2% coinfection rate was reported in dead pigs in China (Li et al. 2013).

We described the distribution and genetic characterization of PPV3 in wild boars in Slovakia representing new data from the region of all neighboring countries. Coinfection of PPV3 and PCV2 in wild boars is reported for the first time. Whereas PPV3 was unevenly distributed, PCV2 was detected in animals from almost all districts of Slovakia. Prevalence of coinfection with both viruses was low and similar among young and adult wild boars.

This work was supported by Slovak Research and Development Agency (APVV-0379-10), Scientific Grant Agency of Slovak Republic (VEGA 1/0704/11), INFEKTZOON (The Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic for the Structural Funds of EU – ITMS 26220120002), and MediPark Kosice (ITMS 26220220185). The authors are grateful to Miroslav Mojzis from the Veterinary Institute in Zvolen, Slovakia, for providing the samples. We thank Peter Nettleton (Edinburgh, UK) for critical reading of the manuscript and English grammar corrections.