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Genetic characterization and phylogenetic analysis of porcine circovirus type 2 strains present in Cuban swine herds
Research in Veterinary Science 89 (2010) 301–305
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Genetic characterization and phylogenetic analysis of porcine circovirus type 2 strains present in Cuban swine herds Lester J. Pérez 1, Heidy Díaz de Arce *,1, María T. Frías Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba
a r t i c l e
i n f o
Article history: Accepted 8 March 2010
Keywords: Porcine circovirus 2 Genetic characterization Phylogeny Genotype
a b s t r a c t Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Porcine circoviruses (PCVs) are small non-enveloped viruses with a single-stranded circular DNA genome which belong to the Circoviridae family (Allan and Ellis, 2000). PCVs are classified into two species, porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) (Todd et al., 2000). PCV1 is not regarded as a pathogen for pigs (Allan et al., 1995; Tischer et al., 1986), whereas PCV2 is accepted as the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) (Harding and Clark, 1998) and has been also associated to other diseases such as porcine dermatitis and nephropathy syndrome (PDNS) (Smith et al., 1993; Rosell et al., 2000), reproductive failure (West et al., 1999; O’Connor et al., 2001; Ohlinger et al., 2000) and proliferative and necrotizing pneumonia (PNP) (Allan and Ellis, 2000). All these syndromes have been collectively grouped as porcine circovirus diseases (PCVD) (Segalés et al., 2005) or porcine circovirosis (Almeida et al., 2004). PCV2 is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) in pigs, a multifactorial disease, * Corresponding author. Tel.: +53 47 863206; fax: +53 47 861104. E-mail addresses: [email protected], [email protected] (H.D. de Arce). 1 These authors contributed equally to this work. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.013
considered as one of the most important swine diseases worldwide. PMWS causes severe economic losses due to increased mortality rates and reduced feed conversion efficiency in weaning and fattening pigs (Segalés et al., 2005). Additional factors besides the presence of PCV2 are necessary to the development of this clinical disease (Segalés et al., 2005). The main factors reported for the development of PMWS comprise the immune system activation (Darwich et al., 2004), given by pig vaccination (Krakowka et al., 2001, 2002), viral co-infections and the genotype of PCV2 involved (Lohse et al., 2008; Tomás et al., 2008). PCV2 consists of a single-stranded circular DNA genome which contains about 1767–1768 nucleotides (Hamel et al., 1998). The genome contains three open reading frames (ORFs) (Niagro et al., 1998). The ORF1 encodes the replication-related proteins: Rep (helicase) and Rep0 (nickase) which is the result of Rep-transcript alternative splicing process (Mankertz and Hillenbrand, 2001; Mankertz et al., 2003). The ORF2 encodes the capsid protein, the only structural viral protein which is also the most variable PCV2 protein (Nawagitgul et al., 2000; Olvera et al., 2007). Finally the ORF3, embedded within ORF1, encodes a protein which is not essential for viral replication but fundamental to the development of the viral pathogenesis (An et al., 2008; Liu et al., 2005). To date, a high number of PCV2 whole genome sequences has been submitted to the GenBank database and even when high
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levels of sequence identity among different PCV2 strains and isolates have been shown, PCV2 can be divided into different clonal lineages (Olvera et al., 2007). Thus, three viral genotypes have been identified (Dupont et al., 2007). Some evidences revealed that PCV2 from genotype 2 prevailed in both PMWS-affected herds and nonaffected herds, from 1997 to 2003, as long as PCV2 strains from genotype 1 have been more commonly found in PMWS outbreaks since 2004 (Carman et al., 2008; Cheung et al., 2007). PCV2 strains belonging to genotype 3 have been recovered only from Danish pig herds during the 80s when PMWS was not described or at least had not been yet indentified (Dupont et al., 2007). Recent studies have shown that different PCV2 genotypes differ in pathogenicity according to both in vitro (Meerts et al., 2005) and in vivo experiments (Opriessnig et al., 2006). Thus, PCV2 from genotype 1 has been suggested as potentially more virulent than PCV2 from genotype 2 and may constitute a crucial factor for triggering the switch from PCV2 infection to PMWS disease as proposed by Timmusk et al. (2005), Cheung et al. (2007) and Lohse et al. (2008). The presence of PCV2 in Cuban pig population has been reported recently (Pérez et al., 2009). However, genetic information about these viruses is not still available. Therefore, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. 2. Materials and methods 2.1. Viral sequences Six PCV2 sequences that were obtained from infected tissues of pigs with clinical signs, including respiratory and wasting disorders, from six different swine herds of four Cuban geographic regions (Pinar del Río (PR) (1), La Habana (HA) (1), Villa Clara (VC) (3) and Ciego de Avila (CA) (1)) during 2007 and 2008 were investigated. 2.2. Nucleic acid isolation Total DNA was extracted from 100 lL of each sample (10% tissue homogenate) using WizardÒ Genomic DNA Purification Kit,
(Promega, Madison, WI, USA) following the manufacturer’s instructions. DNA was diluted in 10 lL of nuclease free water (Promega, Madison, WI, USA). 2.3. Polymerase chain reaction and sequencing In order to determine the whole PCV2 genome, the DNA extracted was amplified by PCR using two sets of oligonucleotides: (1) SEQ PCV-1NF: 50 -GGACCCCAACCCCATAAAA-30 , SEQ PCV1NR: 50 -CCCTCACCTATGACCCCTATGT-30 ; (2) SEQ PCV-2NF: 50 -TGTTTTCGAACGCAGTGCC-30 , SEQ PCV2NR: 50 -CCGTTG TCCCTGAGATCTAGGA-30 which produced two amplicons overlapping at both ends of 1254 nucleotides (nt) and 1045 nt, respectively (Gagnon et al., 2007). The amplification reaction was carried out in a volume of 50 lL comprised of 2 lL of DNA sample (extracted as described above), 1 GoTaqÒ Flexi DNA Polymerase (Promega, Madison, WI, USA) [200 lM of each dNTP, 1.5 mM MgCl2 (pH 8.5)] and 1 lM of each primer. The PCR reaction was done in a thermal cycler (Eppendorf, Mastercycler) under the following conditions: one cycle of 5 min at 95 °C followed by 40 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 56 °C, and elongation for 1 min 30 s at 72 °C and finally one cycle of 10 min at 72 °C. The amplified products were analyzed by electrophoresis in a 2% agarose gel, stained with ethidium bromide (0.5 g/mL) in TBE buffer pH 8.4 (89 mM Tris; 89 mM Boric Acid; 2 mM EDTA) and visualized under UV light. At least two DNA fragments of the expected size from each PCV2 positive tissue samples, amplified each one by primer forward or reverse, were directly purified by WizardÒ PCR Preps DNA Purification System (Promega, Madison, WI, USA) and submitted for sequencing that was conducted under BigDyeTM terminator cycling conditions by an external laboratory (Macrogen, Korea). 2.4. Sequence analyses The quality of the DNA sequences was checked using Chromas 1.56 program (http://www.technelysium.com.au/chromas.html). Overlapping fragments were assembled applying the BioEdit package 5.0.9 (Hall, 1999). Complete PCV2 sequences were submitted
Fig. 1. Alignment of amino acids predicted from the ORF2 nucleotide sequences of PCV2 sequences obtained from infected tissues of pigs belonging to six different swine herds of four Cuban geographic regions (Ciego de Avila, Villa Clara, La Habana and Pinar del Río). Regions reported by Grau-Roma et al. (2008) as more heterogenic are highlighted in black lines.
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to the EMBL/GenBank database under the accession numbers of FN398022–FN398027. The degree of identity between sequences was obtained by performing pairwise sequence alignments using the BioEdit package 5.0.9 (Hall, 1999). The amino acids predicted from the ORF2 nucleotide sequences were compared by aligning the six PCV2 sequences using the algorithm ClustalW method including in the program MEGA version 4 (Tamura et al., 2007). The alignment of the sequences was visually inspected using BioEdit version 5.0.9 program (Hall, 1999). 2.5. Phylogenetic analyses A nucleotide distance matrix between sequences was computed to infer phylogenies by a Neighbor-Joining (NJ) tree using MEGA 4 program (Tamura et al., 2007). Confidence in the NJ tree was estimated by 1000 bootstrap replicates. A phylogenetic tree was constructed including the total viral genomes from the six Cuban PCV2 sequences of this study, as well as 85 entire sequences, from GenBank database, representative of all PCV2 genotypes which comprised virus considered prototypes (accession numbers: AF055394, AF055392 and EU148503) of PCV2 genotypes 1, 2 and 3, respectively. The tree was rooted with a PCV1 sequence (accession number FJ475129). 3. Results 3.1. Genetic characterization All the PCV2 sequences showed a genome length of 1767 nt except one of them with a genome of 1768 nt in length (accession number FN398026). The six full PCV2 sequences, pairwise compared, shared a nucleotide identity of 99.15–100% indicating a homogeneous PCV2 population in Cuba. The variation was mainly due to the variability within the ORF2 (98.91–100% identity) while the ORF1 was highly conserved (99.50–100%). Ten nucleotide substitutions were observed in the ORF2 gene but only two led to amino acid replacements: (i) 63-R K, (ii) 190-A T. These amino acid changes were located between amino acid positions 57–91 and 185–191, respectively (Fig. 1). 3.2. Phylogenetic analyses The phylogenetic analysis of the six PCV2 sequences of this study along with 85 entire sequences published in GenBank database which are representative of all PCV2 genotypes, is shown in Fig. 2. The phylogenetic tree reproduces all the PCV2 clusters described by Olvera et al. (2007) (Fig. 2). Five Cuban PCV2 sequences were grouped with those described as the PCV2 emergent variants associated with severe porcine circovirosis reported in Canada from late 2004 to 2006 (Carman et al., 2008) in a defined cluster within the genotype 1, according to the nomenclature established by Grau-Roma et al. (2008), supported by high confidence values (Fig. 2). The remaining PCV2 sequence was clustered within the genotype 1 closed related to published sequences from China, Denmark, Austria, Netherlands, France, Brazil and Romania (Fig. 2). All Cuban viruses were located within the Cluster 1A, according to Olvera et al. (2007). 4. Discussion In the present study, we characterized and performed phylogenetic analysis of PCV2 sequences that were obtained from infected tissues of pigs with clinical signs, including respiratory
Fig. 2. Neighbor-joining phylogenetic condensed tree derived from the 85 whole PCV2 sequences. Numbers indicate percentage bootstrap values calculated on 1000 bootstraps, non-significant branch values were omitted. *Prototype viruses of PCV2 genotypes. Black lines denote Cuban PCV2 sequences.
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and wasting disorders, from six different swine herds of four geographic regions. The Cuban PCV2 sequences appeared to be close related when an analysis of the entire viral genome sequences was performed, yielding values of 99.15–100%. The main variations on amino acid sequences of the capsid protein were found within immunoreactive areas described by Larochelle et al. (2002) and Grau-Roma et al. (2008) (Fig. 1). The amino acid replacements between PCV2 sequences were located in the heterogenic regions (amino acid positions 59–91 and 185–191) which is in accordance with previous studies where these regions have been stressed as two of the three immunogenic domains revealing polymorphism in PCV2 (Larochelle et al., 2002; Grau-Roma et al., 2008). One the main objectives of this work was to classify the Cuban PCV2 sequences taking into account that several case-control studies provides evidence of increased virulence of PCV2 strains from genotype 1 in pigs (Lohse et al., 2008; Carman et al., 2008). Thus, Carman et al. (2008) found that pigs infected with PCV2 RFLP type 321 variant (genotype 1) showed more histological lesions and significantly more viral antigen into lymphoid tissues than pigs infected with previously reported PCV2 RFLP type 422 strains (genotype 2) (Carman et al., 2008). In addition, first outbreaks of PMWS in Denmark in 2001 were only related to PCV2 genotype 1 sequences (Dupont et al., 2007). Interestingly, in Australia only PCV2 belonging to genotype 2 has been found and this country remains free from PMWS (Muhling et al., 2006). This study revealed that all the Cuban PCV2 sequences analyzed belonged to genotype 1, according to the classification proposed by Grau-Roma et al. (2008) and they were located within the same Cluster (1A) according to the cluster classification of Olvera et al. (2007); but interestingly, four of the them were grouped with high confident values with those described as the PCV2 variants associated with severe porcine circovirosis reported in Canada during a 2004–2006 period (Carman et al., 2008). Since a major importation of pigs from Canada was accomplished in 2005, PCV2 infected pigs from this source appeared to be the most likely origin of the viruses circulating in Cuban swine herds currently. On the other hand, since the PCV2 variant obtained from a swine herd in Pinar del Río province in 2008, was not clustered with any other group of PCV2 within genotype 1, this might suggest that different introductions of the agent from unknown sources have occurred in the country. The occurrence of PMWS in Cuba has not been yet investigated. However, the presence of PCV2 sequences from genotype 1 which are considered the potentially most virulent viruses and the recent report of pig co-infection with PCV2 and some porcine viruses, described as potential triggers of this disease in Cuban swine herds, (Pérez et al., 2009) highlights the importance of undertake such studies. In conclusion, this is the first genetic study of Cuban PCV2 strains and provides a characterization of the viruses circulating in the country. However, a more extensive sequence analysis is required to better understand the epidemiology of PCV2 infections in Cuban pig population. Acknowledgement Work at CENSA was supported by the project 00300248 from the Cuban Ministry of Science, Technology and Environment. References Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. J. Vet. Diagn. Invest. 12, 3–14. Allan, G.M., McNeilly, F., Cassidy, J.P., Reilly, G.A., Adair, B., Ellis, W.A., McNulty, M.S., 1995. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet. Microbiol. 44, 49–64.
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Meerts, P., Van Gucht, S., Cox, E., Vandebosch, A., Nauwynck, H.J., 2005. Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication. Viral Immunol. 18, 333–341. Muhling, J., Raye, W.S., Buddle, J.R., Wilcox, G.E., 2006. Genetic characterisation of Australian strains of porcine circovirus types 1 and 2. Aust. Vet. J. 84, 421–425. Nawagitgul, P., Morozov, I., Bolin, S.R., Harms, P.A., Sorden, S.D., Paul, P.S., 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 81, 2281–2287. Niagro, F.D., Forsthoefel, A.N., Lawther, R.P., Kamalanathan, L., Ritchie, B.W., Latimer, K.S., Lukert, P.D., 1998. Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses. Arch. Virol. 143 (9), 1723–1744. O’Connor, B., Gauvreau, H., West, K., Bogdan, J., Ayroud, M., Clark, E., Konoby, C., Allan, G., Ellis, J.A., 2001. 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L.J. Pérez et al. / Research in Veterinary Science 89 (2010) 301–305 Segalés, J., Allan, G.M., Domingo, M., 2005. Porcine circovirus diseases. Anim. Health Res. Rev. 6, 119–142. Smith, W.J., Thompson, J.R., Done, S., 1993. Dermatitis/nephropathy syndrome of pigs. Vet. Rec. 132, 47. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Timmusk, S., Wallgren, P., Be´lak, K., Berg, M., Fossum, C., 2005. Genetic analysis of PCV2 capsid protein sequences reveals two main groups of Swedish isolates. In: International Conference on Animal Circoviruses and Associated Diseases, Belfast, N. Ireland, UK, p. 82. Tischer, I., Mields, W., Wolff, D., Vagt, M., Griem, W., 1986. Studies on epidemiology and pathogenicity of porcine circovirus. Arch. Virol. 91, 271–276.
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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Genetic characterization and phylogenetic analysis of porcine circovirus type 2 strains present in Cuban swine herds Lester J. Pérez 1, Heidy Díaz de Arce *,1, María T. Frías Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba
a r t i c l e
i n f o
Article history: Accepted 8 March 2010
Keywords: Porcine circovirus 2 Genetic characterization Phylogeny Genotype
a b s t r a c t Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Porcine circoviruses (PCVs) are small non-enveloped viruses with a single-stranded circular DNA genome which belong to the Circoviridae family (Allan and Ellis, 2000). PCVs are classified into two species, porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) (Todd et al., 2000). PCV1 is not regarded as a pathogen for pigs (Allan et al., 1995; Tischer et al., 1986), whereas PCV2 is accepted as the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) (Harding and Clark, 1998) and has been also associated to other diseases such as porcine dermatitis and nephropathy syndrome (PDNS) (Smith et al., 1993; Rosell et al., 2000), reproductive failure (West et al., 1999; O’Connor et al., 2001; Ohlinger et al., 2000) and proliferative and necrotizing pneumonia (PNP) (Allan and Ellis, 2000). All these syndromes have been collectively grouped as porcine circovirus diseases (PCVD) (Segalés et al., 2005) or porcine circovirosis (Almeida et al., 2004). PCV2 is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) in pigs, a multifactorial disease, * Corresponding author. Tel.: +53 47 863206; fax: +53 47 861104. E-mail addresses: [email protected], [email protected] (H.D. de Arce). 1 These authors contributed equally to this work. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.013
considered as one of the most important swine diseases worldwide. PMWS causes severe economic losses due to increased mortality rates and reduced feed conversion efficiency in weaning and fattening pigs (Segalés et al., 2005). Additional factors besides the presence of PCV2 are necessary to the development of this clinical disease (Segalés et al., 2005). The main factors reported for the development of PMWS comprise the immune system activation (Darwich et al., 2004), given by pig vaccination (Krakowka et al., 2001, 2002), viral co-infections and the genotype of PCV2 involved (Lohse et al., 2008; Tomás et al., 2008). PCV2 consists of a single-stranded circular DNA genome which contains about 1767–1768 nucleotides (Hamel et al., 1998). The genome contains three open reading frames (ORFs) (Niagro et al., 1998). The ORF1 encodes the replication-related proteins: Rep (helicase) and Rep0 (nickase) which is the result of Rep-transcript alternative splicing process (Mankertz and Hillenbrand, 2001; Mankertz et al., 2003). The ORF2 encodes the capsid protein, the only structural viral protein which is also the most variable PCV2 protein (Nawagitgul et al., 2000; Olvera et al., 2007). Finally the ORF3, embedded within ORF1, encodes a protein which is not essential for viral replication but fundamental to the development of the viral pathogenesis (An et al., 2008; Liu et al., 2005). To date, a high number of PCV2 whole genome sequences has been submitted to the GenBank database and even when high
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levels of sequence identity among different PCV2 strains and isolates have been shown, PCV2 can be divided into different clonal lineages (Olvera et al., 2007). Thus, three viral genotypes have been identified (Dupont et al., 2007). Some evidences revealed that PCV2 from genotype 2 prevailed in both PMWS-affected herds and nonaffected herds, from 1997 to 2003, as long as PCV2 strains from genotype 1 have been more commonly found in PMWS outbreaks since 2004 (Carman et al., 2008; Cheung et al., 2007). PCV2 strains belonging to genotype 3 have been recovered only from Danish pig herds during the 80s when PMWS was not described or at least had not been yet indentified (Dupont et al., 2007). Recent studies have shown that different PCV2 genotypes differ in pathogenicity according to both in vitro (Meerts et al., 2005) and in vivo experiments (Opriessnig et al., 2006). Thus, PCV2 from genotype 1 has been suggested as potentially more virulent than PCV2 from genotype 2 and may constitute a crucial factor for triggering the switch from PCV2 infection to PMWS disease as proposed by Timmusk et al. (2005), Cheung et al. (2007) and Lohse et al. (2008). The presence of PCV2 in Cuban pig population has been reported recently (Pérez et al., 2009). However, genetic information about these viruses is not still available. Therefore, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. 2. Materials and methods 2.1. Viral sequences Six PCV2 sequences that were obtained from infected tissues of pigs with clinical signs, including respiratory and wasting disorders, from six different swine herds of four Cuban geographic regions (Pinar del Río (PR) (1), La Habana (HA) (1), Villa Clara (VC) (3) and Ciego de Avila (CA) (1)) during 2007 and 2008 were investigated. 2.2. Nucleic acid isolation Total DNA was extracted from 100 lL of each sample (10% tissue homogenate) using WizardÒ Genomic DNA Purification Kit,
(Promega, Madison, WI, USA) following the manufacturer’s instructions. DNA was diluted in 10 lL of nuclease free water (Promega, Madison, WI, USA). 2.3. Polymerase chain reaction and sequencing In order to determine the whole PCV2 genome, the DNA extracted was amplified by PCR using two sets of oligonucleotides: (1) SEQ PCV-1NF: 50 -GGACCCCAACCCCATAAAA-30 , SEQ PCV1NR: 50 -CCCTCACCTATGACCCCTATGT-30 ; (2) SEQ PCV-2NF: 50 -TGTTTTCGAACGCAGTGCC-30 , SEQ PCV2NR: 50 -CCGTTG TCCCTGAGATCTAGGA-30 which produced two amplicons overlapping at both ends of 1254 nucleotides (nt) and 1045 nt, respectively (Gagnon et al., 2007). The amplification reaction was carried out in a volume of 50 lL comprised of 2 lL of DNA sample (extracted as described above), 1 GoTaqÒ Flexi DNA Polymerase (Promega, Madison, WI, USA) [200 lM of each dNTP, 1.5 mM MgCl2 (pH 8.5)] and 1 lM of each primer. The PCR reaction was done in a thermal cycler (Eppendorf, Mastercycler) under the following conditions: one cycle of 5 min at 95 °C followed by 40 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 56 °C, and elongation for 1 min 30 s at 72 °C and finally one cycle of 10 min at 72 °C. The amplified products were analyzed by electrophoresis in a 2% agarose gel, stained with ethidium bromide (0.5 g/mL) in TBE buffer pH 8.4 (89 mM Tris; 89 mM Boric Acid; 2 mM EDTA) and visualized under UV light. At least two DNA fragments of the expected size from each PCV2 positive tissue samples, amplified each one by primer forward or reverse, were directly purified by WizardÒ PCR Preps DNA Purification System (Promega, Madison, WI, USA) and submitted for sequencing that was conducted under BigDyeTM terminator cycling conditions by an external laboratory (Macrogen, Korea). 2.4. Sequence analyses The quality of the DNA sequences was checked using Chromas 1.56 program (http://www.technelysium.com.au/chromas.html). Overlapping fragments were assembled applying the BioEdit package 5.0.9 (Hall, 1999). Complete PCV2 sequences were submitted
Fig. 1. Alignment of amino acids predicted from the ORF2 nucleotide sequences of PCV2 sequences obtained from infected tissues of pigs belonging to six different swine herds of four Cuban geographic regions (Ciego de Avila, Villa Clara, La Habana and Pinar del Río). Regions reported by Grau-Roma et al. (2008) as more heterogenic are highlighted in black lines.
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to the EMBL/GenBank database under the accession numbers of FN398022–FN398027. The degree of identity between sequences was obtained by performing pairwise sequence alignments using the BioEdit package 5.0.9 (Hall, 1999). The amino acids predicted from the ORF2 nucleotide sequences were compared by aligning the six PCV2 sequences using the algorithm ClustalW method including in the program MEGA version 4 (Tamura et al., 2007). The alignment of the sequences was visually inspected using BioEdit version 5.0.9 program (Hall, 1999). 2.5. Phylogenetic analyses A nucleotide distance matrix between sequences was computed to infer phylogenies by a Neighbor-Joining (NJ) tree using MEGA 4 program (Tamura et al., 2007). Confidence in the NJ tree was estimated by 1000 bootstrap replicates. A phylogenetic tree was constructed including the total viral genomes from the six Cuban PCV2 sequences of this study, as well as 85 entire sequences, from GenBank database, representative of all PCV2 genotypes which comprised virus considered prototypes (accession numbers: AF055394, AF055392 and EU148503) of PCV2 genotypes 1, 2 and 3, respectively. The tree was rooted with a PCV1 sequence (accession number FJ475129). 3. Results 3.1. Genetic characterization All the PCV2 sequences showed a genome length of 1767 nt except one of them with a genome of 1768 nt in length (accession number FN398026). The six full PCV2 sequences, pairwise compared, shared a nucleotide identity of 99.15–100% indicating a homogeneous PCV2 population in Cuba. The variation was mainly due to the variability within the ORF2 (98.91–100% identity) while the ORF1 was highly conserved (99.50–100%). Ten nucleotide substitutions were observed in the ORF2 gene but only two led to amino acid replacements: (i) 63-R K, (ii) 190-A T. These amino acid changes were located between amino acid positions 57–91 and 185–191, respectively (Fig. 1). 3.2. Phylogenetic analyses The phylogenetic analysis of the six PCV2 sequences of this study along with 85 entire sequences published in GenBank database which are representative of all PCV2 genotypes, is shown in Fig. 2. The phylogenetic tree reproduces all the PCV2 clusters described by Olvera et al. (2007) (Fig. 2). Five Cuban PCV2 sequences were grouped with those described as the PCV2 emergent variants associated with severe porcine circovirosis reported in Canada from late 2004 to 2006 (Carman et al., 2008) in a defined cluster within the genotype 1, according to the nomenclature established by Grau-Roma et al. (2008), supported by high confidence values (Fig. 2). The remaining PCV2 sequence was clustered within the genotype 1 closed related to published sequences from China, Denmark, Austria, Netherlands, France, Brazil and Romania (Fig. 2). All Cuban viruses were located within the Cluster 1A, according to Olvera et al. (2007). 4. Discussion In the present study, we characterized and performed phylogenetic analysis of PCV2 sequences that were obtained from infected tissues of pigs with clinical signs, including respiratory
Fig. 2. Neighbor-joining phylogenetic condensed tree derived from the 85 whole PCV2 sequences. Numbers indicate percentage bootstrap values calculated on 1000 bootstraps, non-significant branch values were omitted. *Prototype viruses of PCV2 genotypes. Black lines denote Cuban PCV2 sequences.
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and wasting disorders, from six different swine herds of four geographic regions. The Cuban PCV2 sequences appeared to be close related when an analysis of the entire viral genome sequences was performed, yielding values of 99.15–100%. The main variations on amino acid sequences of the capsid protein were found within immunoreactive areas described by Larochelle et al. (2002) and Grau-Roma et al. (2008) (Fig. 1). The amino acid replacements between PCV2 sequences were located in the heterogenic regions (amino acid positions 59–91 and 185–191) which is in accordance with previous studies where these regions have been stressed as two of the three immunogenic domains revealing polymorphism in PCV2 (Larochelle et al., 2002; Grau-Roma et al., 2008). One the main objectives of this work was to classify the Cuban PCV2 sequences taking into account that several case-control studies provides evidence of increased virulence of PCV2 strains from genotype 1 in pigs (Lohse et al., 2008; Carman et al., 2008). Thus, Carman et al. (2008) found that pigs infected with PCV2 RFLP type 321 variant (genotype 1) showed more histological lesions and significantly more viral antigen into lymphoid tissues than pigs infected with previously reported PCV2 RFLP type 422 strains (genotype 2) (Carman et al., 2008). In addition, first outbreaks of PMWS in Denmark in 2001 were only related to PCV2 genotype 1 sequences (Dupont et al., 2007). Interestingly, in Australia only PCV2 belonging to genotype 2 has been found and this country remains free from PMWS (Muhling et al., 2006). This study revealed that all the Cuban PCV2 sequences analyzed belonged to genotype 1, according to the classification proposed by Grau-Roma et al. (2008) and they were located within the same Cluster (1A) according to the cluster classification of Olvera et al. (2007); but interestingly, four of the them were grouped with high confident values with those described as the PCV2 variants associated with severe porcine circovirosis reported in Canada during a 2004–2006 period (Carman et al., 2008). Since a major importation of pigs from Canada was accomplished in 2005, PCV2 infected pigs from this source appeared to be the most likely origin of the viruses circulating in Cuban swine herds currently. On the other hand, since the PCV2 variant obtained from a swine herd in Pinar del Río province in 2008, was not clustered with any other group of PCV2 within genotype 1, this might suggest that different introductions of the agent from unknown sources have occurred in the country. The occurrence of PMWS in Cuba has not been yet investigated. However, the presence of PCV2 sequences from genotype 1 which are considered the potentially most virulent viruses and the recent report of pig co-infection with PCV2 and some porcine viruses, described as potential triggers of this disease in Cuban swine herds, (Pérez et al., 2009) highlights the importance of undertake such studies. In conclusion, this is the first genetic study of Cuban PCV2 strains and provides a characterization of the viruses circulating in the country. However, a more extensive sequence analysis is required to better understand the epidemiology of PCV2 infections in Cuban pig population. Acknowledgement Work at CENSA was supported by the project 00300248 from the Cuban Ministry of Science, Technology and Environment. References Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. J. Vet. Diagn. Invest. 12, 3–14. Allan, G.M., McNeilly, F., Cassidy, J.P., Reilly, G.A., Adair, B., Ellis, W.A., McNulty, M.S., 1995. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet. Microbiol. 44, 49–64.
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