Genetic characterization of porcine circovirus type 2 ( PCV 2 ) from wild boars detected in different regions of Ukraine

L. V. Dudar, I. G. Budzanivska, V. P. Polishchuk © 2018 L. V. Dudar et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDС 578


Introduction
Porcine circovirus type 2 (PCV2) impacts global swine production, is economically important, and is associated with multiple disease entities including multisystemic disease, wasting, pneumonia, diarrhea and reproductive failure.Porcine circovirus (PCV), which belongs to the family Circoviridae, genus Circovirus [1], is one of the smallest animal viruses with unenveloped, singlestranded circular genome and a size of 17 nm in diameter [2,3].Two species of PCV, PCV1 and PCV2, have been characterized [4].PCV1 is considered to be nonpathogenic to pigs by experimental inoculation [1], and has originally been identified as a persistent tissue culture contaminant in pig kidney cell lines [5].PCV2 has been shown to be the causative agent of the post-weaning multi- ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2018. Vol. 34.N 1. P 41-48 doi: http://dx.doi.org/10.7124/bc.00096F systemic wasting syndrome (PMWS) of pigs [6,7].At least, five Open Reading Frames (ORF) have been reported to be effectively transcribed, but the most studied and widely sequenced region is the ORF2, which encodes for the Cap protein.This protein represents the only component of the viral capsid and has been proven to be the major target of the host immune response.The ORF2-based classification criteria have been collectively adopted to define the PCV2 genotypes because of its higher phylogenetic signal and lower tendency to recombine.The PCV2 geographical distribution and its wide presence have been undoubtedly linked to livestock movements and trade routes leading to the rapid spread of new strains in various countries.The PCV2 genotype 2a is considered to be prevalent until 2003, when a change in genotype prevalence (commonly known as a genotype shift) occurred in favour of the genotype 2b, with a parallel enhancement of the outbreak severity.A similar situation happened again in 2010 when the genotype 2d appeared and rapidly spreaded to the detriment of PCV2b prevalence.The aforementioned high evolutionary rate (about 10 −3 -10 −4 substitutions/site/year) and the huge viral population size provide optimal conditions for natural selection to act.One of the suggested reasons for its wide acquired genetic variability could reside in a selective pressure promoted also by the vaccination-induced immunity escape [8].
Consequently, the wild swine population represents a major source of genetic variability and/or simply of viral exchange, but it is not concerned by the vaccination burden (Porcine circovirus type 2, PCV2) evolution before and after the vaccination introduction.
The aim of this study was to bring together the available genomic information on the wild and domestic pig PCV2 strains from different regions of Ukraine to explore their evolutionary pathways.

Materials and Methods
Field samples: clinical samples (serum samples and lymph nodes) from the different farms in high pig density provinces of Ukraine submitted to Molecular Diagnostic Laboratory at CVD (Center of Veterinary Diagnostics) du ring 2014-2015 were included in this study.These samples were kept at -80°C until performing DNA extraction and PCR.Viral DNA was extracted from lymphoid tissue homogenates and serum samples using NucleoSpin Extract Viral DNA Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions.
PCR amplification: A full-length ORF2 gene of PCV2 was amplified in 50 µl of reaction mixture by PCR with forward primer, PCV2-f1 (5'-CCA TGC CCT GAA TTT CCA TA-3') and reverse primer PCV2-r1 (5'-ACA GCG CAC TTC TTT CGT TT-3') according to Takahagi et al. (2008).The amplification reaction was performed with an initial step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min and a final extension step at 72°C for 7 min.The PCV2 positive samples of 702 nt were used for DNA sequencing.
Sequencing and phylogenetic analysis: the PCR products were separated by 1.5% agarose gel electrophoresis and purified with Nucleo-Spin Extract II (Macherey-Nagel, Düren, Germany) for the sequences.DNA sequencing was carried out with primers used in the previ-ous PCR reaction.A total of 4 sequences from Ukrainian pigs were obtained and translated into amino acid sequences andanalyzed together with the representative complete genome sequences reported in GenBank.A phylogenetic tree was constructed by MEGA 6 software (Tamura et al., 2007) using the neighbor-joining (NJ) method with 1000 bootstrapping replicates (Saitou and Nei, 1987).

Results and Discussion
All four PCV2 sequences from wild boars detected in Ukraine in this study had a genome length of 1768 nt and revealed nucleotide identities ranged between 99-91.5% (Tab.1), indicating no significant difference between PCV2 genotype of wild boars (complete sequences) from different countries.
Ukrainian strains from the Chernigiv, Cherkassy and Zaporigga regions have a common origin with the strains from Brazil and Germany.The strain from Kharkiv has a common origin with the strain from Croatia that was allocated in 2009.
The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [9].The tree with the highest log likelihood (-3225.2487) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained by applying the BioNJ method to a matrix of pairwise distances estimated using a JTT model.A discrete Gamma distribution was used to model evolutionary rate differences among sites (2 ca tego ries (+G, parameter = 1.7649)).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 17 amino acid sequences.The coding data were translated assuming a Standard genetic code table.All positions containing gaps and missing data were eliminated.There were a total of 539 positions in the final dataset.Evolutionary analyses were conducted in MEGA6 [10].
The evolutionary history was inferred by using the Maximum Likelihood method based   on the Kimura 2-parameter model [11].The tree with the highest log likelihood (-4739.0701) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained by applying the BioNJ method to a matrix of pairwise distances estimated using the Maximum Composite Like lihood (MCL) approach.A discrete Gamma distribution was used to mo del evolutionary rate differences among sites (2 categories (+G, parameter = 0.0500)).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 17 nucleotide sequences.Codon positions included were 1st+2nd+3rd.All positions containing gaps and missing data were eliminated.There were a total of 1743 positions in the final dataset.Evolutionary analyses were conducted in MEGA6 [10].
The tree topology based on the nucleotide and amino acid sequence data coincides which confirms the validity of our conclusions (Fig. 1, 2).
Comparison of the circulating strains of wild and domestic animals in Ukraine showed that most of them are quite different and belong to different subgroups.The same situation was described in other European countries.However, the strains isolated from the pigs of the Zaporigga and Chernigiv regions have a common origin with the strain isolated from wild boars in the Zaporigga region.The strains isolated from the pigs from the Cherkasy and Kharkiv regions have a common origin with the strain isolated from wild boars from the Kharkiv region (Fig. .3).This fact demonstrates a common origin of described isolates and could be caused by direct transmission of PCV-2 between wild boars and farm animals, that is not allowed by the biosecurity policy.
The evolutionary history was inferred using the Neighbor-Joining method [12].The optimal tree with the sum of branch length = 0.28887349 is shown.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [13].The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.The analysis involved 41 amino acid sequences.The coding data were translated assuming a Standard genetic code table.All positions containing gaps and missing data were eliminated.There were a total of 124 positions in the final dataset.Evolutionary analyses were conducted in MEGA6 [10].
The phylogenetic tree analyses classified the isolates of this study in two subgroups according to the classification proposed by Grau-Roma et al. (2008) [14].Based on the subgroup terminology described previously (Olvera et al., 2007) [15], nucleotides 262-267 and amino acids 88-89 of ORF2 were compared and classified.The nucleotide sequen ces "CCCCGC", "CCCCTC" and "AAAATC" are the signatures motif for PCV2b subgroup 1A/B, 1C and PCV2a, respectively.The amino acid "PR" was enclosed with subgroup1A/B, while the PL and KI were related with subgroup 1C and PCV2a (Cheung et al., 2007) [16].The isolates from Zaporigga, Chernigiv and Cherkasy 1 were divided into 1A/ B subgroups together with PCV2 sequences of strains from wild boars from Brazil and Germany.The isolates from Charkiv 1 were divided into 2 subgroups together with PCV2 sequences of strain from wild boars from Croatia (Fig. 1-3).
Genetically, the characteristics of Ukrainian isolates of PCV2 showed a high level of diversity.As described, the detected isolates belong to both subgroups (1A\B and 2) of PCV strains.Additionally to the interesting model that could be applied to other human and animal diseases, this rouses the interest to a continuous monitoring of viral epidemiology, particularly for rapidly evolving viruses like PCV2, and the necessity to share the related information to prevent or promptly act in response to the potential emergence of actual vaccine-immunity escape mutants [17][18][19].