porcine circovirus ( pcv ) is a small nonenveloped virus that contains a single - stranded circular dna of about 1.76  kb .
pcv was originally isolated as a noncytopathic contaminant of the pk-15 cell line   and was classified as a member of circoviridae family , genus circovirus , based on its morphological and genomic characteristics [ 2 , 3 ] .  
pcv-1 was first detected as a contaminant of the porcine kidney pk-15 cell line   whereas pcv-2 has been associated with postweaning multisystemic wasting syndrome ( pmws ) [ 5 , 6 ] , porcine dermatitis and nephropathy syndrome ( pdns ) , proliferative necrotizing pneumonia , and reproductive disorders , all of them included by the term porcine circovirus associated diseases ( pcvad ) .
pmws is an emerging disease in pigs first described in a swine herd in canada in 1991   and also endemic in many swine - producing countries . in argentina ,
diagnosis of pmws is based on the presence of compatible clinical signs , characteristic histopathological lesions , and detection of pcv-2 antigen within typical lesions [ 10 , 13 ] .
the genomic structure of pcv-2 consists of two intergenic regions flanked by three open reading frames ( orfs ) : orf-1 , which encodes two replication proteins ( rep and rep ) , orf2 , which encodes the cap protein containing the immunoreactive epitopes and is more variable at nucleotide sequence than orf1 , and orf3 , which encodes a proapoptotic protein .
currently , pcv-2 genotype definition and nomenclature has been proposed according to the genome sequence of the whole genome and/or orf2 [ 15 , 16 ] .
five genotypes have been identified to date [ 17 , 18 ] : pcv-2a and pcv-2b , which correspond to the main phylogenetic groups , pcv-2c , which has been described only in denmark , and pcv-2d and 2e , which have been described in china .
pcv-2a has been further subdivided into five clusters ( 2a2e )   and pcv-2b into three clusters ( 1a1c ) .
since 2005 , high mortality outbreaks of pmws reported in north america have been associated with pcv-2b [ 19 , 20 ] . in south
phylogenetic studies have shown that pcv-2 isolates belong to pcv-2b and pcv-2a [ 21 , 22 ] . the objectives of this study were to compare the nucleotide and amino acid sequences of the pcv-2 orf2 identified in lymph nodes from pigs with pcvad from different herds of argentina between 2003 and 2008 .
immunohistochemistry was performed with a polyclonal anti - pcv-2 antibody ( vmrd , inc . , wa , usa , 210 - 70 pcrv ) .
slides were flooded for 15  min with 3% h2o2 to remove endogenous peroxidase activity .
tissues were rinsed for 5  min in 0.1  m pbs ( ph 7.5 ) and then incubated with preheated 0.05% protease xiv for 40 minutes .
tissue sections were rinsed in pbs and flooded with 0.5% skim milk in pbs for 20  min at room temperature .
pcv-2 antibody was used at a 1/200 dilution in 0.1  m pbs , and incubated for 1 hour at 37c .
biotinylated g protein ( 1/500 ) was used as a secondary antibody and was incubated for 40  min at room temperature .
streptavidin - peroxidase ( lsab2 system hrp k0673 , dako laboratories co. , ca , usa ) was applied for 15  min at room temperature .
sections were finally incubated in diaminobenzidine - hydrogen peroxide solution for 8  min and counterstained with harris ' haematoxylin .
immunohistochemistry were graded based on the intensity of immunolabelling as follows : + = slight , + + = moderate , and + + + = abundant .
the samples used in this study corresponded to pig submissions to the diagnostic pathology service , faculty of veterinary science , la plata national university , argentina , from 2003 to 2005 , and to the virology laboratory from the faculty of veterinary science of buenos aires from 2007 to 2008 .
these samples originated from 101  lymph nodes from pigs ( 36 to 140 days old ) with confirmatory pcvad diagnosis by immunohistochemistry ( ihc ) . samples from 2003 to 2005 were pooled in nine groups ( 610  samples / pool ) according to the farm , province , and year of collection .
samples were homogenized with a mortar and pestle , in some cases after deparaffinization with xylene , and kept at 80c until processed .
dna extraction was carried out using the qiaamp dna mini kit ( qiagen , valencia , usa ) in accordance with the manufacturer 's instructions .
pcv-2 specific dna was amplified by pcr with gotaq ( promega ) in a mycycler thermocycler ( biorad , uk ) .
the orf2 from positive samples was further amplified from nucleotides 998 to 1757 ( pcv-2b genome ; genbank accession number , af112862 ) , using the protocol reported by fort et al . .
in this protocol , two internal primers ( caparv 5-accctttgaatactacaga-3 and capbfw 5-gggaggagtagtttacata-3 ) were added to make the sequencing process more reliable , since it amplifies the whole orf2 in two partially overlapping fragments .
cycling conditions were as follows : 94c 5  min , 35  cycles of 45  sec at 94c , 45  sec at 42c and 1  min at 72c , and a final extension cycle of 7  min at 72c .
orf2 amplified fragments were sequenced with the bigdye terminator kit ( applied biosystems , foster city , ca , usa ) on an abi 3500xl genetic analyzer ( applied biosystems , foster city , ca , usa ) .
then , all available isolates obtained were included in a multiple alignment using clustal x version 1.8.3 program , and the percent identities were calculated .
the phylogenetic trees , calculated by the neighbor - joining method , were computed with the dnadist and neighbor modules of the phylip package .
bootstrapping values ( 1000 replicates ) were calculated with the seqboot , dnadist , neighbor , and consense modules .
branches with bootstrapping values 70 were considered significant , corresponding to a confidence interval 95% . for visualization and printing of the trees , the treeview program ,
, we used the sequence of a porcine circovirus 1 ( genbank accession number ay184287 ) .
the signs that predominated in the affected pigs were principally wasting in which the animals presented slow growth , lethargy , anorexia , cachexia , diarrhea , and dyspnea ( table 1 ) . from those animals , 101 lymph nodes were processed and 58 ( 57.4% ) were positive for pcv-2 by ihc .
samples from 2003 to 2005 were grouped in 9 pools and 8 ( 88.8% ) were positive for pcv-2 by pcr .
seven samples from 2007 and 2008 were processed individually and all resulted positive by pcr .
the entire cap coding region of 16 argentinean samples was amplified and sequenced ( table 1 ) .
the sequence analysis showed that they contained 699  nt , and no gaps were detected .
all sequences presented the typical pcv-2 1486 motif tcaaacccc / cgc . at the amino acid level , only two polymorphic sites were detected in the following samples : in 7109 we found k63r and in 7013 , 7108 , and 7198 we found t190a . only the k63r mutation was localized in an immunogenic domain , specifically at the domain a.   these sequences were aligned with cap gene sequences from pcv-2 strains available in the genbank database from different countries .
all argentinean amino acid sequences showed high identity with the sequences grouped in genotype 2b . the phylogenetic tree obtained with the argentinean and reference pcv-2 strain sequences showed two main clusters wellsupported by bootstrap analysis ( figure 1 ) .
all the argentinean sequences analyzed grouped together in one cluster and were closely related to sequences from genotype pcv-2b isolates , and more specifically to cluster 1a - b . in this last case , the low bootstrap values made the differentiation of the two clusters uncertain .
this cluster grouped also isolates from europe ( hungary , france , austria , and the netherlands ) , asia ( china ) , and north america ( usa and canada ) . in the other cluster , named 1c , only grouped sequences from asia ( china ) .
recent phylogenetic analysis of complete pcv-2 genomic sequences obtained from genbank   and full - length orf2 sequences   resulted in two major genogroups : 2a and 2b , which are circulating worldwide , 2c , which have been sporadically reported in the 80s , and 2d and 2e , recently described in china .
the significance of these differences in terms of disease and/or geographic location is currently unknown .
however , the severe re - emergence of pcvad in north america in 2005 coincided with the identification of the pcv-2b genotype .
in addition , an epizootic of pmws in switzerland was found to be associated with pcv-2b while pcv-2a was identified in single cases of pmws prior to 2003 .
furthermore , experimental studies in gnotobiotic pigs with or without immunostimulation using pcv-2b alone have shown only moderate lesions in various organs , thus indicating that pcv-2b - stimulated pigs became infected but did not progress to a disease state . however , another study using gnotobiotic pigs and cell culture - derived pcv-2a and pcv-2b clones showed that both were able to induce severe disease in this model .
a study has highlighted the importance of the order of infection in which gnotobiotic pigs inoculated with pcv-2b seven days after a pcv-2a infection , but not vice versa , cause pmws .
unfortunately , sequence studies with pcv-2a followed with pcv-2b in conventional pigs are still lacking .
the above results are indicative of sometimes contradicting information regarding pcv-2 genotyping and its relationship with disease condition .
although it is generally believed that the causes of illness depend on unknown factors , more information related to pcv-2 genotyping could help for a better understanding of the pathogenic mechanisms of pcv-2 .   in the present study
, pcv-2 sequences obtained from 16 different farms belonging to the main pig - producing provinces of argentina were analyzed to determine the pcv-2 genotype . only samples from 2003 to 2008 with confirmatory diagnosis of pcvad
sequence and phylogenetic analyses clustered the pcv-2b group with a common a - b subgroup , with an identity of 99.8% .
the reason we named a new cluster a - b is because bootstrap values were not suitable enough , based on the sequences used in this study , to support the differentiation between clusters a and b. the pcv-2b sequences identified were very similar to an isolate previously reported in argentina in 2005 ( genbank ef458306 ) .
the argentinean pcv-2b genotype isolates were found to be closely related to some isolates from brazil 2004 , north america 2005 , europe 2003 - 2004 , and china 2002 .
in addition , all sequences present the 1486 motif tca / aac / ccc / cgc belonging to the pcv-2b genotype described by cheung et al . 2007 , and contain amino acid substitutions similar to those described by grau - roma et al . 2008 . in canada , pcv-2b isolates from pmws outbreaks in 2005 share an interval identity between 99% and 100%
. however , other studies have identified distinctive genotypes from pcvad samples [ 28 , 32 ] and a variable identity within each genotype . in argentina , the first outbreak of pmws was reported in 2002   and thereafter clinical pcvad emerged as emerging diseases .
our study comprised pcvad samples taken from 2003 to 2008 and the results suggest that pcv-2b is the predominant genotype in argentina and that it could be associated with a systemic form of pcvad , namely , pmws . in argentina , serological or immunohistochemical retrospective studies on pcv-2 have not been carried out and , therefore it remains unknown how and when pcv-2b was introduced in the argentinean herds .
other sources of infection such as inappropriate used of vaccines can be ruledout because only inactivated pcv-2 and pcv-2 orf2 protein vaccines have been licensed .   in summary
, the present study contributes to the knowledge on the distribution of pcv-2 genotypes circulating in argentina .
our findings may also help to establish a base of information to study the emergence of new viral variants in this region .
 porcine circovirus type 2 ( pcv-2 ) has been associated with syndromes grouped by the term porcine circovirus associated diseases ( pcvad ) . 
 the pcv-2 isolates have been grouped into two major groups or genotypes according to their nucleotide sequence of whole genomes and/or orf-2 : pcv-2b , which have , in turn , been subdivided into three clusters ( 1a1c ) , and pcv-2a , which has been subdivided into five clusters ( 2a2e ) . in the present study , we obtained 16 sequences of pcv-2 from different farms from 2003 to 2008 , from animals with confirmatory diagnosis of pcvad . 
 since results showed an identity of 99.8% among them , they were grouped within a common cluster 1a - b . 
 this preliminary study suggests a stable circulation of pcv-2b among the argentinean pig population . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion