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Paper
Paper Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia D. Novosel, T. Tuboly, A. Csagola, M. Lorincz, V. Cubric-Curik, A. Jungic, I. Curik, J. Segalés, M. Cortey, Z. Lipej Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.
Introduction
Porcine circoviruses (PCVs) are non-enveloped viruses of 17 nm in diameter that belong to the genus Circovirus, family Circoviridae. They contain a circular, single-stranded DNA genome of approximately 1.76 kb (Allan and others 1994, Meehan and others 1997, Hamel and others 1998, Liu and others 2001). Two types of PCV have been described: the non-pathogenic PCV type 1 (PCV1), first described in 1974 as a contaminant of PK-15 cells (Tischer and others 1974, 1982), and the pathogenic PCV type 2 (PCV2), which has been linked to several diseases (Chae 2004). The PCV genome contains few ORFs. The ORF1 or rep gene encodes the full-length replicase protein Rep and its truncated form Rep′ (Mankertz and others 1998). The ORF2 Veterinary Record (2014) D. Novosel, PhD, DVM Z. Lipej, DVM, PhD Department of Pathology, Croatian Veterinary Institute, Zagreb 10000, Croatia T. Tuboly, Prof, PhD, DVM A. Csagola, PhD, DVM M. Lorincz, DVM Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary V. Cubric-Curik, Prof, PhD I. Curik, Prof, PhD Department of Animal Science, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia
doi: 10.1136/vr.102064 A. Jungic, DVM Virology Department, Croatian Veterinary Institute, Zagreb, Croatia J. Segalés, Prof, PhD, DVM, Dipl. ECPHM, Dipl. ESVP Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Barcelona, Spain M. Cortey, PhD, MSc, BSc Unité de Virologie Moléculaire, Emergence et Co-évolution Virale, Marseille, France E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted February 3, 2014
or Cap gene encodes the viral capsid protein (Nawagitgul and others 2000). Finally, the ORF3, which overlaps with ORF1 in the opposite transcriptional orientation, encodes a protein shown in vitro to be apparently involved in virus-induced apoptosis (Liu and others 2006). Phylogenetic trees based on PCV2 ORF2 sequences are similar to those constructed from complete PCV2 genomes (Olvera and others 2007), which has led several epidemiological studies to focus on ORF2 analysis. ORF1, in contrast, is longer than ORF2 and under greater selective pressure, so it has been used as a phylogenetic marker less often than ORF2 (Olvera and others 2007). Four PCV2 genotypes are recognised: PCV2a, PCV2b, PCV2c and PCV2d (Segales and others 2008, Guo and others 2010). In 2010, a new type of PCV, chimeric PCV1/2a, was detected in domestic pigs. This virus contained the complete PCV1 ORF1 and the complete PCV2 ORF2 (Gagnon and others 2010). It is technically possible that this strain resulted from natural recombination between PCV1 and PCV2, since both viruses are continuously present in pig populations. However, it was considered more likely to have originated from a commercially available chimeric vaccine strain (Gagnon and others 2010). Nevertheless, several researchers have suggested the possibility of genetic recombination among PCV2 genotypes (Csagola and others 2006, Lefebvre and others 2008, Cadar and others 2012). This would make epidemiological studies based on phylogenetic analyses of the ORF2 region alone misleading. Furthermore, classical phylogenetic trees may not properly evaluate the exchange of genetic material among strains (Olvera and others 2010), while phylogenetic networks could represent better the evolutionary process in viruses (Morrison 2005), and they have been proposed for viruses in which recombination events occur frequently such as PCV2 (Olvera and others 2010). Moreover, haplotype networks were used in a Cuban study that revealed the best agreement between epidemiological data and phylogenetic inferences (Perez and others 2011). April 26, 2014 | Veterinary Record
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Paper PCV2 has been present in pig populations around the world for several decades (Allan and others 2012). However, it was not associated with any particular disease until the mid-1990s, when postweaning multisystemic wasting syndrome, now referred to as PCV2 systemic disease (PCV2-SD) (Segales 2012), was reported in a Canadian high-health pig farm (Clark 1997, Harding and Clark 1997, Ellis and others 1998). A retrospective investigation revealed that PCV2 had existed in domestic pigs much earlier. The earliest available sample where PCV2 was confirmed was from 1962 (Jacobsen and others 2009). The disease is currently present on all continents, including Oceania (Allan and others 2012). After being associated with PCV2-SD, PCV2 has been linked to reproductive disorders (West and others 1999, O’Connor and others 2001), now referred to collectively as PCV2-reproductive disease (PCV2-RD) (Segales 2012), porcine dermatitis and nephropathy syndrome (Rosell and others 2000), the so-called porcine respiratory disease complex (Thacker and Thacker 2000), and proliferative and necrotising pneumonia (PNP) (Pesch and others 2000). Together, all these diseases are commonly referred to as PCV2-associated diseases (PCVDs). How PCV2 is involved in the pathogenesis of PCVDs remains unclear. Koch’s postulates establishing PCV2 as the causative agent have been partially satisfied for only PCV2-SD and PCV2-RD (Ellis and others 1998, Krakowka and others 2001, Saha and others 2011). PCV2-SD was detected in Croatia in 2001 during health monitoring at a large pig production facility (Lipej and others 2002, 2005) and subsequently was detected in wild boars (Lipej and others 2007). Nevertheless, the precise strain(s) of PCV2 present in Croatia, how the virus arrived in the country and whether one or multiple strains of PCV2 are associated with observed PCVD outbreaks remain unknown. To address these latter questions, PCV2 sequences were obtained from tissue samples of naturally infected fetuses and growing domestic pigs and a few wild boars in Croatia. The sequences were used to construct phylogenetic networks and perform amino acid analysis and principal coordinate analysis in order to study PCV2 origins, evolution and associations with disease in wild boar and domestic pigs.
Materials and methods
Case selection and sampling
Various tissues were collected at postmortem examination from 85 dead pigs or aborted fetuses from different pig herds submitted to the Croatian Veterinary Institute for routine herd health monitoring. In this study, all examined samples originated form eight large farrow-tofinish pig production units located in north-western and north-eastern Croatia, the most important pig-rearing areas of the country. Seven of these farms imported gilts between 2004 and 2007. Data from EU-COMTRADE (http://comtrade.un.org/, Accessed October 18, 2012) and the Croatian Agriculture Agency (www.hpa.hr/, Accessed October 18, 2012) indicated that these reproductive gilts came from the Netherlands, Denmark and Austria. Breeding herd size varied from 1000 to 12,000 sows, and globally represented around 30 per cent of total sow inventory of Croatia. Of the 85 samples, 63 were tissue pools containing heart, liver, spleen and lungs of fetuses from eight farms where prolonged gestation, mummification, late-term abortions, stillbirths and poordoing piglets were observed. Fetuses had been collected from June to September 2007. All fetuses were larger than 18 mm and were in the last third of gestation. These 63 samples had been previously shown by PCR to contain PCV2 (Novosel 2010). However, none of the fetuses showed heart or liver pathology associated with PCV2 infection and all were negative for PCV2, both by immunohistochemistry and in situ hybridisation (ISH) (Novosel 2010). Possible PCV2 contamination was excluded since sampling was performed with sterile instruments with time distance. Same samples were further investigated for the presence of porcine reproductive and respiratory syndrome virus (PRRSV), torque teno sus virus (TTSuV) 1 and 2 and porcine parvovirus (PPV) 1, 2, 3 and 4. PPV3 was absent, TTSuV1 and 2 and PPV1 and 2 were detected to a low prevalence (2/63), and PRRSV and PPV4 to a moderate prevalence (6/63). Of the remaining 22 samples, 14 corresponded to lymph nodes from domestic pigs with severe lymphoid Veterinary Record | April 26, 2014
and renal lesions characteristic of PCV2-SD; such samples were collected from May to August 2007. ISH of these 14 samples revealed abundant PCV2 genome in renal and lymphoid tissues. Mycotoxins and Leptospira spp. were ruled out as potential causes of the kidney lesions in these animals. Three more samples originated from three wild boars found dead in 2009 in forests of eastern Croatia, displaying severe cachexia. All boars weighed around 15 kg and were submitted as suspected cases of classical swine fever. Classical swine fever was excluded by ELISA and PCR, and histopathology and ISH revealed abundant PCV2 genome in characteristic PCV2-SD lymphoid lesions. No renal lesions were observed in wild boars. Finally, five samples were lung tissue from animals affected by PNP; all five animals were from different farms, submitted from June 2008 to January 2010. All of these animals were also diagnosed as having porcine reproductive and respiratory syndrome (PRRS), in addition to having a strong ISH signal for PCV2 genome in alveolar macrophages, pneumocytes and epithelial cells (Novosel and Lipej 2009). These pigs with PNP also had characteristic lymphoid lesions of PCV-SD and absence of renal lesions.
Polymerase chain reaction
DNA was extracted from the samples using the QIAamp DNA Mini Kit (Qiagen). Two PCR assays were utilised, a first one to confirm the detection of PCV2 genome (partial sequence) and a second one for complete genome sequence retrieval. First, PCV2 genome was amplified in the first-round PCR using MCV1 and MCV2 primers as described previously (Fenaux and others 2000). Subsequently, complete PCV2 genomes were amplified using the CBB1–CBB2 and CBB3– CSZ2 primer pairs as described previously (Csagola and others 2006). The length of CBB1-2 amplicons was 1295 nucleotides and the length of CBB2-CSZ2 PCR amplicons was 907 nucleotides. The CBB1–2 and CBB2–CSZ2 amplicons were overlapping PCR products. Amplification reactions were prepared by mixing 1 µl of each DNA sample with 5 µl GreenTaq Buffer (Fermentas), 20 µM deoxynucleotide triphosphates (dNTPs; Fermentas), 2.5 µM of each oligonucleotide primer and 1 U/μl GreenTaq DNA polymerase (Fermentas) in a total volume of 50 µl. Amplifications were performed in a TGradient Thermocycler (Biometra). Amplicons were detected by electrophoresis in 2 per cent agarose gels in the presence of 1 µl/100 ml GR Safe DNA Stain I.
Sequence analyses
Amplicons of PCV2 genomes were purified and sequenced by Macrogen. The sequencing reactions were repeated twice, in opposite directions, from the same PCR products. Sequences were aligned using the ClustalW program in MEGA V.5 software (Tamura and others 2011) and the following reference sequences (GenBank accession numbers): Imp 1010-Stoon PCV2a (AF055392) (Meehan and others 1998), Imp 1011-48285 PCV2b (AF055394), DK1980PMWSfree PCV2c (EU148503) (Dupont and others 2008), MDJ PCV2d (HM038031) (Guo and others 2010), and FMV09-1134568 PCV1/2a (FJ790425) (Gagnon and others 2010). Phylogenetic analyses were performed using the following PCV2 genome regions: the complete genome (nucleotides, nt, 1–1767), the complete ORF1 (nt 51–995), a 320 nt partial ORF1 sequence (nt 60–379), the complete ORF2 (nt 1033–1734) and a partial ORF2 sequence (nt 1118–1569). Sequence polymorphism and haplotype diversity were computed using DnaSP V.5 software (Librado and Rozas 2009). In order to visualise the relationship between haplotypes, haplotype median-joining (MJ) networks were constructed. The analysis was performed using the MJ algorithm was implemented into the Network V.4.6.0.0. software (Fluxus Technology). Amino acid alignment analysis was carried out using MEGA 5. Nucleotide and peptide comparisons among the PCV2 sequences identified were performed using the specific motifs 1485-TCA/AAC/ CCC/CG-1473 (86-SNPRSV) for PCV2b and 1485-ACC/AAC/AAA/ AT-1486 (86-TNKISI) for PCV2a (Trible and Rowland 2012). The pairwise distance matrix between sequences was computed using the Tamura-Nei model (Tamura and Nei 1993) implemented in MEGA V.5 and was used as input to perform principle coordinate analysis (PCA) using SAS software. Phylogenetic relationships among the PCV2 sequences obtained in this study and other PCV2 isolates
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Paper deposited in GenBank were analysed. Online supplementary Table S1 shows the GenBank accession numbers of sequences used to construct the MJ networks and amino acid alignments as well as carry out the PCA. These latter analyses were carried out using the complete PCV2 genome, the complete ORF1, a partial ORF1 sequence, the complete ORF2 and a partial ORF2 sequence. Partial ORF1 and ORF2 analyses were performed since only partial ORF1 and ORF2 sequences were recovered from previous Croatian and neighbour countries (Slovenia and Serbia) sequences (Toplak and others 2012).
Results
Sequence polymorphism
A total of 85 PCR products were retrieved in this study. From these, 17 corresponded to PCV2 complete sequences and the remaining 66 were partial sequences. These 17 complete Croatian PCV2 strains exhibited significant sequence polymorphism and corresponded to 16 new haplotypes. Haplotype diversity (Hd) was higher in the ORF1 region (Hd 0.9756) than in the ORF2 region (Hd 0.9579).
Phylogenetic analysis MJ networks
Fourteen (Cro1–6, 8–10 and 12–16) out of 17 complete PCV2 (Cro1– 16 and Cro22) sequences belonged to genotype PCV2b (Fig 1a–e). Such PCV2b strains were highly homologous based on alignments of the ORF2 region or the complete genome (Fig 1a,c); 11 (Cro1–3, 5–6, 8–10, 12–13 and 16) of them had 99 per cent nucleotide identity in the ORF2 region. Two (Cro7 and Cro11) out of the 17 sequences belonged to genotype PCV2a. The remaining sequence (Cro22) did not fit with PCV2a and PCV2b genotype definition. According to the MJ network of Cro22 ORF1 and ORF2 sequences (Fig 1b,c), such a strain showed a phylogenetic relationship with Chinese strain Chi7. According to the ORF1 MJ network (Fig 1b), Cro22 and Chi7 had the same ancestor nods as those of Spa2, Spa3 and Aus3, and they were related with genotype a. According to the ORF2 MJ network (Fig 1c), Cro22 and Cro15 had ancestry nods in genotype b while Chi7 had them in genotype a.
Principal coordinate analysis
PCA was carried out with all 17 strains and the results were similar to ORF2 and complete genome alignment. From these, 14 clustered within genotype PCV2b. In contrast, the two homologous strains Cro7 and Cro11 clustered within genotype PCV2a. Cro14 and Cro15 clustered to some extent within PCV2b, while Cro22 appeared not to cluster with either PCV2a or PCV2b (Fig 2). PCA replicated the results of the sequence polymorphism.
Amino acid alignment analysis
The 14 Croatian strains clustering within PCV2b showed high amino acid sequence identity in the Cap protein. The same was true for Cro7 and Cro11, which clustered within PCV2a, whereas the Cap sequence for Cro22 was divergent. Sequences that clustered with PCV2a possessed the characteristic PCV2a motif 1485-ACC/AAC/AAA/AT-1486 (86-TNKISI), while sequences clustering with PCV2b shared the PCV2b-specific motif 1485-TCA/AAC/CCC/CG-1473 (86-SNPRSV). The divergent novel Croatian strain Cro22 possessed the motif 86-SNPLTV, which is shared by the PCV2c reference strain Den5 and the PCV2d reference strain Chi4, but is absent from PCV2a and PCV2b genotypes (Fig 3). The residues at positions 3, 16, 54 of Cap protein in Cro22 were identical with Chi7; also at 3 was identical to genotype b strains, at 16 to genotype c and at 54 to genotype a. At position 125-126-128 Chi7 is like Spa7 and had F-P-S dissimilar from all other strains, while Cro22 was like genotype b strains. At residues positions 205-210-212-218, Cro22 was identical with Cro15; also, Cro22 at residue position 205 was identical to subtype A and D strains, at 210 to subtype D, and at 212 and 218 dissimilar to all other strains.
Discussion
Croatia is a relatively small country with modest pig production of approximately 85,000 breeding sows, though this number has been increasing annually. The majority of PCV2 strains circulating in Croatia showed high homology but some of them were
s ignificantly genetically divergent. Comparison of the novel strains identified here and those already reported in GenBank based on MJ analysis, PCA and amino acid sequence alignments provides insight into the genetic diversity of PCV2. A total of 23 PCV2 sequences are available to date from pigs in Croatia (Jemersic and others 2004, Lipej and others 2005, 2007); among these, 18 (Cro1– 6; 8–10; 12–18; 21; 23) clusters within the PCV2b genotype are highly homologous, with nine belonging to the same haplotype. As expected, diversity was higher in ORF1 than in ORF2. The ORF1 region, which encodes the Rep protein, is under greater selective pressure than the ORF2 region, which encodes the Cap protein (Olvera and others 2007). These results indicate that the PCV2b genotype is more common in Croatia, similar to the situation globally (Dupont and others 2008). The MJ networks revealed direct relationship between Croatian PCV2b sequences and Dutch, Danish and Austrian sequences, indicating that those sequences are genetically and evolutionary connected. Similarly PCA results showed a close relationship between Croatian sequences clustered in PCV2b and sequences from centralnorthern Europe (the Netherlands, Denmark and France). Therefore, epidemiological and viral sequence data linked the origin of PCV2b strains in Croatia with those countries. Slovenian and Serbian strains had the same ancestral nodes with Croatian ones, but according to phylogenetic networks, none of them were on the evolutionary way of Croatian strains. Altogether, compiled data fits with the possibility that international trade accounts for the arrival of the novel strains in Croatia and further emphasises the hypothesis that the swine trade is responsible for global spread of PCV2 (Firth and others 2009, Vidigal and others 2012). Consistent with the abovementioned hypothesis, Cro7 and Cro11 sequences clustering within the PCV2a genotype came from a farm whose production relied on its own breeding line rather than purchases from external breeding stock. This farm reported significant losses due to PCV2-SD and is located close to the border between Croatia and Hungary. PCA indicated that these sequences were related to Hungarian ones; in fact, the MJ network analysis suggested that Cro7 was a mutated form of Cro11. The third Croatian sequence clustering within the PCV2a genotype, the previously known Cro19, was different from Cro7 and Cro11, but also phylogenetically related to one Hungarian sequence. These results indicate that all three Croatian PCV2a genotypes are more likely to be longstanding endemic strains transported locally rather than ‘new arrivals’. Nevertheless, recent airborne transmission cannot be ruled out based on the data available. The majority of the available Croatian PCV2 sequences clustered together closely and showed evident phylogenetic relationships, but the novel Croatian strains Cro14, Cro15 and Cro22 behaved differently. MJ analysis, PCA and amino acid sequence alignments based on the complete genomes and on ORF2 sequences alone showed that Cro14 and Cro15 clustered within the PCV2b genotype, whereas Cro22 remained unclustered. The Cro14 ORF1 and Cro15 ORF1 and ORF2 regions were divergent but still clustered in the PCV2b genotype while the Cro14 ORF 1 region clustered in genotype PCV2a. Cro22 had a close phylogenetic relationship with Chinese Chi7 and differences in the ORF1 and ORF2 regions suggest a possible recombination, as previously indicated (Csagola and others 2006, Cadar and others 2012). Cro22 was isolated from a wild boar affected by PCV2SD and may represent an isolated PCV2 viral population. Further analysis is needed to determine whether Cro22 is an entirely novel genotype. The present results indicate that PCV2b is the most prevalent genotype in Croatia, followed by PCV2a. PCV2a was the predominant genotype in Europe before the 2000s. A shift from PCV2a to PCV2b occurred in the pig population worldwide between 2000 and 2005 (Dupont and others 2008, Segales and others 2013). As apparently no prior information is available in Croatia indicating a higher prevalence of PCV2a, the conclusion about occurrence of such genetic shift is still a hypothesis, although the present results may also reflect that the global shift might have occurred also in Croatia. Our data are consistent with the notion that importation of r eproductive gilts infected with PCV2b may cause this genetic April 26, 2014 | Veterinary Record
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Paper (a)
(b) Chi3
Cro1,Cro2,Cro3,Cro4,Cro5 Cro6,Cro8,Cro9,Cro10,Cro12 Cro13,Cro16,Net1,Net2,Net3 Den1,Den2,Aus1,Spa1, Fra1, Chi3 Fra2,Fra4,Fra5,Fra6,Fra7,SIk1 Chi4 SIk2,SIk4,Hun1,Rom1,Chi10 Chi5
d
Chi2 Hun2,Hun4,Hun5 Can2,SIK3,Fra3 Ger1,Ger2
Hum3 Spa1 Spa2
Aus2
Chi2
PCV1 Chi6
PCV1/2a
Den5
Can3
Can1,Can2 USA2
Den3 USA1,USA3 Hun2
a
Chi1 Chi7
C
Den4
Cro11 Cro07
Cro14
Cro22
d
Chi1
b
Cro15
Chi4
Chi5
PCV1 Chi6
a
SIk3
ORF1
Hun5 Net1 Fra3 Cro7
Cro11
Chi7
Spa1
Cro2
Spa3
PCV1/2a
(c)
Cro15
SIk1
Aus2 Complete genome
Cro16
Fra7
Cro1
Cro8,Net3,Fra1 Fra5,Fra6,SIk2 SIK4, Chip,Chi10
Spa2 Can3
Chi8
Hun1
Cro22
b
Den1
Cro6
Ger2 Den3 Den4 Den5
Fra2
Hun4 Can2 Cro14
Ger1
c
Cro9,Cro10,Aus1 Den2,Net2 Fra4
Cro5 Cro13
Cro4,Cro12 Rom1
Hun3
(d) Den4
Den3
Cro4
Den5
c
d
d Chi3 Chi4 Chi5
Chi10 Cro14 Fra5
Fra2
Cro1,Cro4,Cro5,Cro8,Cro8,Cro12 Cro16,Net3,Chi8,Chi9,Chi10 Fra1,Fra5,Fra6,Fra7,Rom1 SIK1,SIK2,SIk4
Chi5
Chi4
b ORF2
Cro3
b
Cro23
Cro22
Den1
a
Chi1
c
Can1,Can4 USA1, UsA2,USA3
Cro13 Cro14
Fra4 Chi7
a
Hun3
Partial ORF1
Cro15 Spa2
Chi2
Spa3
Aus2 Can2
Spa2
PCV1/2a Can3
Can4
Can1 USA2
Cro22
Hun3
Chi7 Cro7,Cro11,Hum2 Hun4,Hun5,Sil3 Ger1,Ger2
PCV1/2a
Den5
Spa1
Cor2,Cor6,Cor9,Cor10 Aus1,Den2,Net2,Spa1
PCV1 Chi6
Cro1,Cro2,Cro3,Cro5,Cro8,Cro9 Cro10,Cro12,Cro13,Cro16,Cro16,Net1 Net2,Net3,Den1,Den2,Aus1,Rom1 Hun1,Fra1,Fra6,Fra7,Spa1,SIK1 SIK2,SIK4,SIK6,Chi8,Chi9,
Den3
Chi1 Chi2
Fra4
Cro15
PCV1 Chi6 Can3
Den4
Chi3
Spa3 USA1 USA3
Aus2
Hun2
Hun4
Ger2 Cro11 Ger1 Can2,Fra3,Hun5 Net1,SIk3 Cro7
(e) Chi3 Chi4 Chi5
d
Srb4
Spa1 Fra5
Cro8
Cro1,Cro3,Cro12, Cro20,Slo4,Srb2,Srb3 SIK1,Fra6,Chi10
Slo5
Cro14
Spa2
a
PCV1 Chi6
Cro9 Cro15
Slo3
b
Den1
Cro14
Cro22
Chi8
Fra4 Fra2
Spa3
Chi2
Can4
Chi1
Can1
USA1 USA3
USA2
Chi7
Cro2,Cro4,Cro5,Cro6,Cro13, Cro16,Cro17,Cro18,Cro21 Den2,Net1,Net2,Net3,us1 Hun1,Rom1,SIK2,SIK4,Fea1 Fra7,Chi9
Can3
Can2
Srb1 Cro11
Hun5 Ger1 SIK1
Aus2
c Den4 Den5
Hun4 Ger3 Fra3
Den3
Cro19 Hun3
Partial ORF2
Slo1 Hun2 Cro7
Slo2
FIG 1: Median-joining (MJ) network of (a) complete porcine circovirus (PCV2) genomes, (b) complete ORF1, (c) complete ORF2, (d) partial ORF1 and (e) partial ORF2 sequences showing the phylogenetic relationships among Croatian PCV2 isolates and related isolates in GenBank (see online supplementary Table S1). Taxonomic names of Croatian isolates are written in red. Coloured dots indicate the source of Croatian isolates as follows: blue dots indicate strains from animals with PCV2-reproductive disease (RD); green dots, strains from wild boars with PCV2-systemic disease (SD); pink dots, strains from animals with proliferative and necrotising pneumonia (PNP); light yellow dots, strains from animals with PCV2-SD with severe interstitial nephritis; red dots, strains from domestic pigs with PCV2-SD from previous studies. Taxonomic names of non-Croatian strains from GenBank are written in black and labelled with grey dots. Dot size is proportional to the frequencies of isolates belonging to the given haplotype
shift. However, data available for Croatia have originated from 2003 onwards and importation of such gilts did not begin extensively until 2004, so no previous data do exist. Further studies are therefore needed to determine whether and how these extensive imports have affected PCV2 epidemiology in the country. For at least the last 10 years PCVDs have been the most costly health problem for large pig producers around the world. Research and management of PCVDs is hindered by the lack of detailed information Veterinary Record | April 26, 2014
about their aetiology and progression. It is tempting to suggest that the specific clinical and pathological manifestations of different PCVDs may reflect sequence variations in the infecting genotype and that small differences in Cap protein may confer different target organ tropism (Mankertz and others 2000). However, proof for this hypothesis is still lacking and not supported by the results of this study, in which PCV2 sequences were retrieved from different PCVD clinical manifestations.
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Paper PCA2 0.28 0.26 0.24 0.20 0.18 0.16 0.14 0.12 0.08 0.06 0.04 0.02 0.00 –0.02 –0.04 –0.02 –0.04 –0.06 –0.08 –0.10 –0.12 –0.14 –0.16 –0.18 –0.20 –0.22 –0.11
Cro1 Fra3 Aus2 Can2
Cro7,Cro11,Hun2,Hun4,Hun5 Slk3,Aus2,Slk3,Ger1
a Can1 GRU3 Spa3 Can4 Chi1 Chi7 Spa2 Chi2 Hun3
USA1,USA3
c
Cro22 GRU2 d Chi4
Cro14 b Cro15 Fra2 Fra4 Fra5 Chi10 GRU1Chi9 Cro4 –0.06
–0.01
0.04
Den5 Den3 Den4
Chi3,Chi5
Cro1,Cro2,Cro3,Cro5,Cro6,Cro8,Cro9,Cro10,Cro13,Cro16 Net1,Net2,Den1,Den2,Fra1,Fra5,Fra6,Fra7,Slk1,Slk2,Slk4 Rom1,Hun1,Spa1,Chi8,Chi9,Chi10 0.09
0.14 0.19 PCA1
0.24
0.29
0.34
0.39
0.44
FIG 2: Nucleotide diversity. Two-dimensional plot illustrating genetic relationships among complete porcine circovirus type 2 (PCV2) ORF2 sequences. * Sequences showing difference values of 0.000 were grouped into GRU1 (Cro2, Cro5, Cro6, Cro13, Cro16, Net1, Net2, Net3, Den2, Fra1, Fra7, Rom1, Slk2, Slk4), GRU2 (Chi3, Chi5) and GRU3 (USA1, USA3)
FIG 3: Alignment of porcine circovirus type 2 (PCV2) capsid (Cap) protein sequences (encoded by ORF2) among Croatian sequences and reference sequences for PCV2a (Can4), PCV2b (Fra6), PCV2c (Den5) and PCV2d (Chi4). Motifs specific for PCV2b and PCV2a are labelled. Dots indicate where the amino acid sequence matches the sequence at the top
April 26, 2014 | Veterinary Record
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Paper Conclusions
This study identified 17 new, complete PCV2 genome sequences in naturally infected fetuses and growing domestic pigs and a few wild boar affected by various PCVDs in Croatia. Of the 17 sequences, 14 belonged to genotype PCV2b and two to PCV2a; one strain remained unclustered and may represent a novel yet undiscovered genotype. PCV2b is the predominant genotype and phylogenetic studies suggest that this genotype may have been introduced in Croatia through international trade, while PCV2a genotypes are more likely to be longstanding endemic strains transported locally rather than ‘new arrivals’. Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/vr-2013-102064)
Acknowledgements
The skilled technical and laboratory work of Dr Eszter Kovács is acknowledged. Research studies of AC were funded by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Funding This study was funded by the Ministry of Science, Education and Sport of the Republic of Croatia (Project 0481153) and the Sixth Framework Programme (Project 513928, ‘The Control of Porcine Circovirus Diseases (PCVDs): Towards Improved Food Quality and Safety’). Competing interests None.
References
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Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia D. Novosel, T. Tuboly, A. Csagola, et al. Veterinary Record 2014 174: 431 originally published online March 3, 2014
doi: 10.1136/vr.102064
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Paper Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia D. Novosel, T. Tuboly, A. Csagola, M. Lorincz, V. Cubric-Curik, A. Jungic, I. Curik, J. Segalés, M. Cortey, Z. Lipej Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.
Introduction
Porcine circoviruses (PCVs) are non-enveloped viruses of 17 nm in diameter that belong to the genus Circovirus, family Circoviridae. They contain a circular, single-stranded DNA genome of approximately 1.76 kb (Allan and others 1994, Meehan and others 1997, Hamel and others 1998, Liu and others 2001). Two types of PCV have been described: the non-pathogenic PCV type 1 (PCV1), first described in 1974 as a contaminant of PK-15 cells (Tischer and others 1974, 1982), and the pathogenic PCV type 2 (PCV2), which has been linked to several diseases (Chae 2004). The PCV genome contains few ORFs. The ORF1 or rep gene encodes the full-length replicase protein Rep and its truncated form Rep′ (Mankertz and others 1998). The ORF2 Veterinary Record (2014) D. Novosel, PhD, DVM Z. Lipej, DVM, PhD Department of Pathology, Croatian Veterinary Institute, Zagreb 10000, Croatia T. Tuboly, Prof, PhD, DVM A. Csagola, PhD, DVM M. Lorincz, DVM Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary V. Cubric-Curik, Prof, PhD I. Curik, Prof, PhD Department of Animal Science, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia
doi: 10.1136/vr.102064 A. Jungic, DVM Virology Department, Croatian Veterinary Institute, Zagreb, Croatia J. Segalés, Prof, PhD, DVM, Dipl. ECPHM, Dipl. ESVP Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Barcelona, Spain M. Cortey, PhD, MSc, BSc Unité de Virologie Moléculaire, Emergence et Co-évolution Virale, Marseille, France E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted February 3, 2014
or Cap gene encodes the viral capsid protein (Nawagitgul and others 2000). Finally, the ORF3, which overlaps with ORF1 in the opposite transcriptional orientation, encodes a protein shown in vitro to be apparently involved in virus-induced apoptosis (Liu and others 2006). Phylogenetic trees based on PCV2 ORF2 sequences are similar to those constructed from complete PCV2 genomes (Olvera and others 2007), which has led several epidemiological studies to focus on ORF2 analysis. ORF1, in contrast, is longer than ORF2 and under greater selective pressure, so it has been used as a phylogenetic marker less often than ORF2 (Olvera and others 2007). Four PCV2 genotypes are recognised: PCV2a, PCV2b, PCV2c and PCV2d (Segales and others 2008, Guo and others 2010). In 2010, a new type of PCV, chimeric PCV1/2a, was detected in domestic pigs. This virus contained the complete PCV1 ORF1 and the complete PCV2 ORF2 (Gagnon and others 2010). It is technically possible that this strain resulted from natural recombination between PCV1 and PCV2, since both viruses are continuously present in pig populations. However, it was considered more likely to have originated from a commercially available chimeric vaccine strain (Gagnon and others 2010). Nevertheless, several researchers have suggested the possibility of genetic recombination among PCV2 genotypes (Csagola and others 2006, Lefebvre and others 2008, Cadar and others 2012). This would make epidemiological studies based on phylogenetic analyses of the ORF2 region alone misleading. Furthermore, classical phylogenetic trees may not properly evaluate the exchange of genetic material among strains (Olvera and others 2010), while phylogenetic networks could represent better the evolutionary process in viruses (Morrison 2005), and they have been proposed for viruses in which recombination events occur frequently such as PCV2 (Olvera and others 2010). Moreover, haplotype networks were used in a Cuban study that revealed the best agreement between epidemiological data and phylogenetic inferences (Perez and others 2011). April 26, 2014 | Veterinary Record
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Paper PCV2 has been present in pig populations around the world for several decades (Allan and others 2012). However, it was not associated with any particular disease until the mid-1990s, when postweaning multisystemic wasting syndrome, now referred to as PCV2 systemic disease (PCV2-SD) (Segales 2012), was reported in a Canadian high-health pig farm (Clark 1997, Harding and Clark 1997, Ellis and others 1998). A retrospective investigation revealed that PCV2 had existed in domestic pigs much earlier. The earliest available sample where PCV2 was confirmed was from 1962 (Jacobsen and others 2009). The disease is currently present on all continents, including Oceania (Allan and others 2012). After being associated with PCV2-SD, PCV2 has been linked to reproductive disorders (West and others 1999, O’Connor and others 2001), now referred to collectively as PCV2-reproductive disease (PCV2-RD) (Segales 2012), porcine dermatitis and nephropathy syndrome (Rosell and others 2000), the so-called porcine respiratory disease complex (Thacker and Thacker 2000), and proliferative and necrotising pneumonia (PNP) (Pesch and others 2000). Together, all these diseases are commonly referred to as PCV2-associated diseases (PCVDs). How PCV2 is involved in the pathogenesis of PCVDs remains unclear. Koch’s postulates establishing PCV2 as the causative agent have been partially satisfied for only PCV2-SD and PCV2-RD (Ellis and others 1998, Krakowka and others 2001, Saha and others 2011). PCV2-SD was detected in Croatia in 2001 during health monitoring at a large pig production facility (Lipej and others 2002, 2005) and subsequently was detected in wild boars (Lipej and others 2007). Nevertheless, the precise strain(s) of PCV2 present in Croatia, how the virus arrived in the country and whether one or multiple strains of PCV2 are associated with observed PCVD outbreaks remain unknown. To address these latter questions, PCV2 sequences were obtained from tissue samples of naturally infected fetuses and growing domestic pigs and a few wild boars in Croatia. The sequences were used to construct phylogenetic networks and perform amino acid analysis and principal coordinate analysis in order to study PCV2 origins, evolution and associations with disease in wild boar and domestic pigs.
Materials and methods
Case selection and sampling
Various tissues were collected at postmortem examination from 85 dead pigs or aborted fetuses from different pig herds submitted to the Croatian Veterinary Institute for routine herd health monitoring. In this study, all examined samples originated form eight large farrow-tofinish pig production units located in north-western and north-eastern Croatia, the most important pig-rearing areas of the country. Seven of these farms imported gilts between 2004 and 2007. Data from EU-COMTRADE (http://comtrade.un.org/, Accessed October 18, 2012) and the Croatian Agriculture Agency (www.hpa.hr/, Accessed October 18, 2012) indicated that these reproductive gilts came from the Netherlands, Denmark and Austria. Breeding herd size varied from 1000 to 12,000 sows, and globally represented around 30 per cent of total sow inventory of Croatia. Of the 85 samples, 63 were tissue pools containing heart, liver, spleen and lungs of fetuses from eight farms where prolonged gestation, mummification, late-term abortions, stillbirths and poordoing piglets were observed. Fetuses had been collected from June to September 2007. All fetuses were larger than 18 mm and were in the last third of gestation. These 63 samples had been previously shown by PCR to contain PCV2 (Novosel 2010). However, none of the fetuses showed heart or liver pathology associated with PCV2 infection and all were negative for PCV2, both by immunohistochemistry and in situ hybridisation (ISH) (Novosel 2010). Possible PCV2 contamination was excluded since sampling was performed with sterile instruments with time distance. Same samples were further investigated for the presence of porcine reproductive and respiratory syndrome virus (PRRSV), torque teno sus virus (TTSuV) 1 and 2 and porcine parvovirus (PPV) 1, 2, 3 and 4. PPV3 was absent, TTSuV1 and 2 and PPV1 and 2 were detected to a low prevalence (2/63), and PRRSV and PPV4 to a moderate prevalence (6/63). Of the remaining 22 samples, 14 corresponded to lymph nodes from domestic pigs with severe lymphoid Veterinary Record | April 26, 2014
and renal lesions characteristic of PCV2-SD; such samples were collected from May to August 2007. ISH of these 14 samples revealed abundant PCV2 genome in renal and lymphoid tissues. Mycotoxins and Leptospira spp. were ruled out as potential causes of the kidney lesions in these animals. Three more samples originated from three wild boars found dead in 2009 in forests of eastern Croatia, displaying severe cachexia. All boars weighed around 15 kg and were submitted as suspected cases of classical swine fever. Classical swine fever was excluded by ELISA and PCR, and histopathology and ISH revealed abundant PCV2 genome in characteristic PCV2-SD lymphoid lesions. No renal lesions were observed in wild boars. Finally, five samples were lung tissue from animals affected by PNP; all five animals were from different farms, submitted from June 2008 to January 2010. All of these animals were also diagnosed as having porcine reproductive and respiratory syndrome (PRRS), in addition to having a strong ISH signal for PCV2 genome in alveolar macrophages, pneumocytes and epithelial cells (Novosel and Lipej 2009). These pigs with PNP also had characteristic lymphoid lesions of PCV-SD and absence of renal lesions.
Polymerase chain reaction
DNA was extracted from the samples using the QIAamp DNA Mini Kit (Qiagen). Two PCR assays were utilised, a first one to confirm the detection of PCV2 genome (partial sequence) and a second one for complete genome sequence retrieval. First, PCV2 genome was amplified in the first-round PCR using MCV1 and MCV2 primers as described previously (Fenaux and others 2000). Subsequently, complete PCV2 genomes were amplified using the CBB1–CBB2 and CBB3– CSZ2 primer pairs as described previously (Csagola and others 2006). The length of CBB1-2 amplicons was 1295 nucleotides and the length of CBB2-CSZ2 PCR amplicons was 907 nucleotides. The CBB1–2 and CBB2–CSZ2 amplicons were overlapping PCR products. Amplification reactions were prepared by mixing 1 µl of each DNA sample with 5 µl GreenTaq Buffer (Fermentas), 20 µM deoxynucleotide triphosphates (dNTPs; Fermentas), 2.5 µM of each oligonucleotide primer and 1 U/μl GreenTaq DNA polymerase (Fermentas) in a total volume of 50 µl. Amplifications were performed in a TGradient Thermocycler (Biometra). Amplicons were detected by electrophoresis in 2 per cent agarose gels in the presence of 1 µl/100 ml GR Safe DNA Stain I.
Sequence analyses
Amplicons of PCV2 genomes were purified and sequenced by Macrogen. The sequencing reactions were repeated twice, in opposite directions, from the same PCR products. Sequences were aligned using the ClustalW program in MEGA V.5 software (Tamura and others 2011) and the following reference sequences (GenBank accession numbers): Imp 1010-Stoon PCV2a (AF055392) (Meehan and others 1998), Imp 1011-48285 PCV2b (AF055394), DK1980PMWSfree PCV2c (EU148503) (Dupont and others 2008), MDJ PCV2d (HM038031) (Guo and others 2010), and FMV09-1134568 PCV1/2a (FJ790425) (Gagnon and others 2010). Phylogenetic analyses were performed using the following PCV2 genome regions: the complete genome (nucleotides, nt, 1–1767), the complete ORF1 (nt 51–995), a 320 nt partial ORF1 sequence (nt 60–379), the complete ORF2 (nt 1033–1734) and a partial ORF2 sequence (nt 1118–1569). Sequence polymorphism and haplotype diversity were computed using DnaSP V.5 software (Librado and Rozas 2009). In order to visualise the relationship between haplotypes, haplotype median-joining (MJ) networks were constructed. The analysis was performed using the MJ algorithm was implemented into the Network V.4.6.0.0. software (Fluxus Technology). Amino acid alignment analysis was carried out using MEGA 5. Nucleotide and peptide comparisons among the PCV2 sequences identified were performed using the specific motifs 1485-TCA/AAC/ CCC/CG-1473 (86-SNPRSV) for PCV2b and 1485-ACC/AAC/AAA/ AT-1486 (86-TNKISI) for PCV2a (Trible and Rowland 2012). The pairwise distance matrix between sequences was computed using the Tamura-Nei model (Tamura and Nei 1993) implemented in MEGA V.5 and was used as input to perform principle coordinate analysis (PCA) using SAS software. Phylogenetic relationships among the PCV2 sequences obtained in this study and other PCV2 isolates
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Paper deposited in GenBank were analysed. Online supplementary Table S1 shows the GenBank accession numbers of sequences used to construct the MJ networks and amino acid alignments as well as carry out the PCA. These latter analyses were carried out using the complete PCV2 genome, the complete ORF1, a partial ORF1 sequence, the complete ORF2 and a partial ORF2 sequence. Partial ORF1 and ORF2 analyses were performed since only partial ORF1 and ORF2 sequences were recovered from previous Croatian and neighbour countries (Slovenia and Serbia) sequences (Toplak and others 2012).
Results
Sequence polymorphism
A total of 85 PCR products were retrieved in this study. From these, 17 corresponded to PCV2 complete sequences and the remaining 66 were partial sequences. These 17 complete Croatian PCV2 strains exhibited significant sequence polymorphism and corresponded to 16 new haplotypes. Haplotype diversity (Hd) was higher in the ORF1 region (Hd 0.9756) than in the ORF2 region (Hd 0.9579).
Phylogenetic analysis MJ networks
Fourteen (Cro1–6, 8–10 and 12–16) out of 17 complete PCV2 (Cro1– 16 and Cro22) sequences belonged to genotype PCV2b (Fig 1a–e). Such PCV2b strains were highly homologous based on alignments of the ORF2 region or the complete genome (Fig 1a,c); 11 (Cro1–3, 5–6, 8–10, 12–13 and 16) of them had 99 per cent nucleotide identity in the ORF2 region. Two (Cro7 and Cro11) out of the 17 sequences belonged to genotype PCV2a. The remaining sequence (Cro22) did not fit with PCV2a and PCV2b genotype definition. According to the MJ network of Cro22 ORF1 and ORF2 sequences (Fig 1b,c), such a strain showed a phylogenetic relationship with Chinese strain Chi7. According to the ORF1 MJ network (Fig 1b), Cro22 and Chi7 had the same ancestor nods as those of Spa2, Spa3 and Aus3, and they were related with genotype a. According to the ORF2 MJ network (Fig 1c), Cro22 and Cro15 had ancestry nods in genotype b while Chi7 had them in genotype a.
Principal coordinate analysis
PCA was carried out with all 17 strains and the results were similar to ORF2 and complete genome alignment. From these, 14 clustered within genotype PCV2b. In contrast, the two homologous strains Cro7 and Cro11 clustered within genotype PCV2a. Cro14 and Cro15 clustered to some extent within PCV2b, while Cro22 appeared not to cluster with either PCV2a or PCV2b (Fig 2). PCA replicated the results of the sequence polymorphism.
Amino acid alignment analysis
The 14 Croatian strains clustering within PCV2b showed high amino acid sequence identity in the Cap protein. The same was true for Cro7 and Cro11, which clustered within PCV2a, whereas the Cap sequence for Cro22 was divergent. Sequences that clustered with PCV2a possessed the characteristic PCV2a motif 1485-ACC/AAC/AAA/AT-1486 (86-TNKISI), while sequences clustering with PCV2b shared the PCV2b-specific motif 1485-TCA/AAC/CCC/CG-1473 (86-SNPRSV). The divergent novel Croatian strain Cro22 possessed the motif 86-SNPLTV, which is shared by the PCV2c reference strain Den5 and the PCV2d reference strain Chi4, but is absent from PCV2a and PCV2b genotypes (Fig 3). The residues at positions 3, 16, 54 of Cap protein in Cro22 were identical with Chi7; also at 3 was identical to genotype b strains, at 16 to genotype c and at 54 to genotype a. At position 125-126-128 Chi7 is like Spa7 and had F-P-S dissimilar from all other strains, while Cro22 was like genotype b strains. At residues positions 205-210-212-218, Cro22 was identical with Cro15; also, Cro22 at residue position 205 was identical to subtype A and D strains, at 210 to subtype D, and at 212 and 218 dissimilar to all other strains.
Discussion
Croatia is a relatively small country with modest pig production of approximately 85,000 breeding sows, though this number has been increasing annually. The majority of PCV2 strains circulating in Croatia showed high homology but some of them were
s ignificantly genetically divergent. Comparison of the novel strains identified here and those already reported in GenBank based on MJ analysis, PCA and amino acid sequence alignments provides insight into the genetic diversity of PCV2. A total of 23 PCV2 sequences are available to date from pigs in Croatia (Jemersic and others 2004, Lipej and others 2005, 2007); among these, 18 (Cro1– 6; 8–10; 12–18; 21; 23) clusters within the PCV2b genotype are highly homologous, with nine belonging to the same haplotype. As expected, diversity was higher in ORF1 than in ORF2. The ORF1 region, which encodes the Rep protein, is under greater selective pressure than the ORF2 region, which encodes the Cap protein (Olvera and others 2007). These results indicate that the PCV2b genotype is more common in Croatia, similar to the situation globally (Dupont and others 2008). The MJ networks revealed direct relationship between Croatian PCV2b sequences and Dutch, Danish and Austrian sequences, indicating that those sequences are genetically and evolutionary connected. Similarly PCA results showed a close relationship between Croatian sequences clustered in PCV2b and sequences from centralnorthern Europe (the Netherlands, Denmark and France). Therefore, epidemiological and viral sequence data linked the origin of PCV2b strains in Croatia with those countries. Slovenian and Serbian strains had the same ancestral nodes with Croatian ones, but according to phylogenetic networks, none of them were on the evolutionary way of Croatian strains. Altogether, compiled data fits with the possibility that international trade accounts for the arrival of the novel strains in Croatia and further emphasises the hypothesis that the swine trade is responsible for global spread of PCV2 (Firth and others 2009, Vidigal and others 2012). Consistent with the abovementioned hypothesis, Cro7 and Cro11 sequences clustering within the PCV2a genotype came from a farm whose production relied on its own breeding line rather than purchases from external breeding stock. This farm reported significant losses due to PCV2-SD and is located close to the border between Croatia and Hungary. PCA indicated that these sequences were related to Hungarian ones; in fact, the MJ network analysis suggested that Cro7 was a mutated form of Cro11. The third Croatian sequence clustering within the PCV2a genotype, the previously known Cro19, was different from Cro7 and Cro11, but also phylogenetically related to one Hungarian sequence. These results indicate that all three Croatian PCV2a genotypes are more likely to be longstanding endemic strains transported locally rather than ‘new arrivals’. Nevertheless, recent airborne transmission cannot be ruled out based on the data available. The majority of the available Croatian PCV2 sequences clustered together closely and showed evident phylogenetic relationships, but the novel Croatian strains Cro14, Cro15 and Cro22 behaved differently. MJ analysis, PCA and amino acid sequence alignments based on the complete genomes and on ORF2 sequences alone showed that Cro14 and Cro15 clustered within the PCV2b genotype, whereas Cro22 remained unclustered. The Cro14 ORF1 and Cro15 ORF1 and ORF2 regions were divergent but still clustered in the PCV2b genotype while the Cro14 ORF 1 region clustered in genotype PCV2a. Cro22 had a close phylogenetic relationship with Chinese Chi7 and differences in the ORF1 and ORF2 regions suggest a possible recombination, as previously indicated (Csagola and others 2006, Cadar and others 2012). Cro22 was isolated from a wild boar affected by PCV2SD and may represent an isolated PCV2 viral population. Further analysis is needed to determine whether Cro22 is an entirely novel genotype. The present results indicate that PCV2b is the most prevalent genotype in Croatia, followed by PCV2a. PCV2a was the predominant genotype in Europe before the 2000s. A shift from PCV2a to PCV2b occurred in the pig population worldwide between 2000 and 2005 (Dupont and others 2008, Segales and others 2013). As apparently no prior information is available in Croatia indicating a higher prevalence of PCV2a, the conclusion about occurrence of such genetic shift is still a hypothesis, although the present results may also reflect that the global shift might have occurred also in Croatia. Our data are consistent with the notion that importation of r eproductive gilts infected with PCV2b may cause this genetic April 26, 2014 | Veterinary Record
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Paper (a)
(b) Chi3
Cro1,Cro2,Cro3,Cro4,Cro5 Cro6,Cro8,Cro9,Cro10,Cro12 Cro13,Cro16,Net1,Net2,Net3 Den1,Den2,Aus1,Spa1, Fra1, Chi3 Fra2,Fra4,Fra5,Fra6,Fra7,SIk1 Chi4 SIk2,SIk4,Hun1,Rom1,Chi10 Chi5
d
Chi2 Hun2,Hun4,Hun5 Can2,SIK3,Fra3 Ger1,Ger2
Hum3 Spa1 Spa2
Aus2
Chi2
PCV1 Chi6
PCV1/2a
Den5
Can3
Can1,Can2 USA2
Den3 USA1,USA3 Hun2
a
Chi1 Chi7
C
Den4
Cro11 Cro07
Cro14
Cro22
d
Chi1
b
Cro15
Chi4
Chi5
PCV1 Chi6
a
SIk3
ORF1
Hun5 Net1 Fra3 Cro7
Cro11
Chi7
Spa1
Cro2
Spa3
PCV1/2a
(c)
Cro15
SIk1
Aus2 Complete genome
Cro16
Fra7
Cro1
Cro8,Net3,Fra1 Fra5,Fra6,SIk2 SIK4, Chip,Chi10
Spa2 Can3
Chi8
Hun1
Cro22
b
Den1
Cro6
Ger2 Den3 Den4 Den5
Fra2
Hun4 Can2 Cro14
Ger1
c
Cro9,Cro10,Aus1 Den2,Net2 Fra4
Cro5 Cro13
Cro4,Cro12 Rom1
Hun3
(d) Den4
Den3
Cro4
Den5
c
d
d Chi3 Chi4 Chi5
Chi10 Cro14 Fra5
Fra2
Cro1,Cro4,Cro5,Cro8,Cro8,Cro12 Cro16,Net3,Chi8,Chi9,Chi10 Fra1,Fra5,Fra6,Fra7,Rom1 SIK1,SIK2,SIk4
Chi5
Chi4
b ORF2
Cro3
b
Cro23
Cro22
Den1
a
Chi1
c
Can1,Can4 USA1, UsA2,USA3
Cro13 Cro14
Fra4 Chi7
a
Hun3
Partial ORF1
Cro15 Spa2
Chi2
Spa3
Aus2 Can2
Spa2
PCV1/2a Can3
Can4
Can1 USA2
Cro22
Hun3
Chi7 Cro7,Cro11,Hum2 Hun4,Hun5,Sil3 Ger1,Ger2
PCV1/2a
Den5
Spa1
Cor2,Cor6,Cor9,Cor10 Aus1,Den2,Net2,Spa1
PCV1 Chi6
Cro1,Cro2,Cro3,Cro5,Cro8,Cro9 Cro10,Cro12,Cro13,Cro16,Cro16,Net1 Net2,Net3,Den1,Den2,Aus1,Rom1 Hun1,Fra1,Fra6,Fra7,Spa1,SIK1 SIK2,SIK4,SIK6,Chi8,Chi9,
Den3
Chi1 Chi2
Fra4
Cro15
PCV1 Chi6 Can3
Den4
Chi3
Spa3 USA1 USA3
Aus2
Hun2
Hun4
Ger2 Cro11 Ger1 Can2,Fra3,Hun5 Net1,SIk3 Cro7
(e) Chi3 Chi4 Chi5
d
Srb4
Spa1 Fra5
Cro8
Cro1,Cro3,Cro12, Cro20,Slo4,Srb2,Srb3 SIK1,Fra6,Chi10
Slo5
Cro14
Spa2
a
PCV1 Chi6
Cro9 Cro15
Slo3
b
Den1
Cro14
Cro22
Chi8
Fra4 Fra2
Spa3
Chi2
Can4
Chi1
Can1
USA1 USA3
USA2
Chi7
Cro2,Cro4,Cro5,Cro6,Cro13, Cro16,Cro17,Cro18,Cro21 Den2,Net1,Net2,Net3,us1 Hun1,Rom1,SIK2,SIK4,Fea1 Fra7,Chi9
Can3
Can2
Srb1 Cro11
Hun5 Ger1 SIK1
Aus2
c Den4 Den5
Hun4 Ger3 Fra3
Den3
Cro19 Hun3
Partial ORF2
Slo1 Hun2 Cro7
Slo2
FIG 1: Median-joining (MJ) network of (a) complete porcine circovirus (PCV2) genomes, (b) complete ORF1, (c) complete ORF2, (d) partial ORF1 and (e) partial ORF2 sequences showing the phylogenetic relationships among Croatian PCV2 isolates and related isolates in GenBank (see online supplementary Table S1). Taxonomic names of Croatian isolates are written in red. Coloured dots indicate the source of Croatian isolates as follows: blue dots indicate strains from animals with PCV2-reproductive disease (RD); green dots, strains from wild boars with PCV2-systemic disease (SD); pink dots, strains from animals with proliferative and necrotising pneumonia (PNP); light yellow dots, strains from animals with PCV2-SD with severe interstitial nephritis; red dots, strains from domestic pigs with PCV2-SD from previous studies. Taxonomic names of non-Croatian strains from GenBank are written in black and labelled with grey dots. Dot size is proportional to the frequencies of isolates belonging to the given haplotype
shift. However, data available for Croatia have originated from 2003 onwards and importation of such gilts did not begin extensively until 2004, so no previous data do exist. Further studies are therefore needed to determine whether and how these extensive imports have affected PCV2 epidemiology in the country. For at least the last 10 years PCVDs have been the most costly health problem for large pig producers around the world. Research and management of PCVDs is hindered by the lack of detailed information Veterinary Record | April 26, 2014
about their aetiology and progression. It is tempting to suggest that the specific clinical and pathological manifestations of different PCVDs may reflect sequence variations in the infecting genotype and that small differences in Cap protein may confer different target organ tropism (Mankertz and others 2000). However, proof for this hypothesis is still lacking and not supported by the results of this study, in which PCV2 sequences were retrieved from different PCVD clinical manifestations.
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Paper PCA2 0.28 0.26 0.24 0.20 0.18 0.16 0.14 0.12 0.08 0.06 0.04 0.02 0.00 –0.02 –0.04 –0.02 –0.04 –0.06 –0.08 –0.10 –0.12 –0.14 –0.16 –0.18 –0.20 –0.22 –0.11
Cro1 Fra3 Aus2 Can2
Cro7,Cro11,Hun2,Hun4,Hun5 Slk3,Aus2,Slk3,Ger1
a Can1 GRU3 Spa3 Can4 Chi1 Chi7 Spa2 Chi2 Hun3
USA1,USA3
c
Cro22 GRU2 d Chi4
Cro14 b Cro15 Fra2 Fra4 Fra5 Chi10 GRU1Chi9 Cro4 –0.06
–0.01
0.04
Den5 Den3 Den4
Chi3,Chi5
Cro1,Cro2,Cro3,Cro5,Cro6,Cro8,Cro9,Cro10,Cro13,Cro16 Net1,Net2,Den1,Den2,Fra1,Fra5,Fra6,Fra7,Slk1,Slk2,Slk4 Rom1,Hun1,Spa1,Chi8,Chi9,Chi10 0.09
0.14 0.19 PCA1
0.24
0.29
0.34
0.39
0.44
FIG 2: Nucleotide diversity. Two-dimensional plot illustrating genetic relationships among complete porcine circovirus type 2 (PCV2) ORF2 sequences. * Sequences showing difference values of 0.000 were grouped into GRU1 (Cro2, Cro5, Cro6, Cro13, Cro16, Net1, Net2, Net3, Den2, Fra1, Fra7, Rom1, Slk2, Slk4), GRU2 (Chi3, Chi5) and GRU3 (USA1, USA3)
FIG 3: Alignment of porcine circovirus type 2 (PCV2) capsid (Cap) protein sequences (encoded by ORF2) among Croatian sequences and reference sequences for PCV2a (Can4), PCV2b (Fra6), PCV2c (Den5) and PCV2d (Chi4). Motifs specific for PCV2b and PCV2a are labelled. Dots indicate where the amino acid sequence matches the sequence at the top
April 26, 2014 | Veterinary Record
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Paper Conclusions
This study identified 17 new, complete PCV2 genome sequences in naturally infected fetuses and growing domestic pigs and a few wild boar affected by various PCVDs in Croatia. Of the 17 sequences, 14 belonged to genotype PCV2b and two to PCV2a; one strain remained unclustered and may represent a novel yet undiscovered genotype. PCV2b is the predominant genotype and phylogenetic studies suggest that this genotype may have been introduced in Croatia through international trade, while PCV2a genotypes are more likely to be longstanding endemic strains transported locally rather than ‘new arrivals’. Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/vr-2013-102064)
Acknowledgements
The skilled technical and laboratory work of Dr Eszter Kovács is acknowledged. Research studies of AC were funded by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Funding This study was funded by the Ministry of Science, Education and Sport of the Republic of Croatia (Project 0481153) and the Sixth Framework Programme (Project 513928, ‘The Control of Porcine Circovirus Diseases (PCVDs): Towards Improved Food Quality and Safety’). Competing interests None.
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April 26, 2014 | Veterinary Record
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Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia D. Novosel, T. Tuboly, A. Csagola, et al. Veterinary Record 2014 174: 431 originally published online March 3, 2014
doi: 10.1136/vr.102064
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