BACKGROUND
circoviruses belong to the circoviridae family and infect a variety of animal and plant species.
they are named after their circular single-stranded dna  genome and represent the smallest dna viruses infecting mammals.
porcine circovirus  <dig>  is found all over the world in the domestic pigs and the wild boars which seldom display the clinical condition.
pcv <dig> serological prevalence in the wild boars is almost as high as in domestic pigs  <cit> .
pcv <dig> is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as pcv2-associated diseases .
overall, pcvad includes clinical syndromes and diseases such as postweaning multisystemic wasting syndrome , porcine dermatitis and nephropathy syndrome , porcine reproductive disorders, respiratory disease complex, granulomatous enteritis, congenital tremor, necrotizing tracheitis and lymphadenitis and exudative epidermitis  <cit> .
pcv <dig> has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production  <cit> .
the ubiquitous nature of the virus and the retrospective evidence of this infection long before disease appearance provided the reason for debate about the real capabilities of this virus in its association with the disease.
how the virus causes disease is still not fully understood.
infection with pcv <dig> in combination with still unknown co-factors can cause immunopathological disorders such as pmws, or induce protective immunity.
it might be that pcv2-induced diseases are the result of pcv2-caused immune disorder.
it was demonstrated that pigs with pmws display a marked depletion of t- and b-lymphocytes, and their lymphoid organs are infiltrated by histiocytes and multinucleated giant cells and might cause deficiency of total and neutralizing antibody response  <cit> .
the interaction of pcv <dig> with the innate immune system have been mostly studied in vitro looking for the effects of the virus on dendritic cells , macrophages and peripheral blood mononuclear cells  <cit> .
it was shown that pcv <dig> infection of plasmacytoid dcs impairs the induction of interferon-α and tumor necrosis factor-α, which silences responsiveness to pathogen-associated recognition signals by compromising immune defense development against other pathogens.
as a result this may lead to a host susceptibility to secondary infections which actually are responsible for developing of pcvad  <cit> .
pcv <dig> is a non-enveloped virus with circular single stranded dna molecule of approximately  <dig>  bases packed in icosahedral 12–23 nm diameter virion particle.
virus genome sequences with nucleotide diversity cut-off of  <dig> % were divided into four genotypic groups  <cit> .
the two major groups designated as pcv2a, pcv2b are found worldwide and have been demonstrated to be virulent enough to trigger pcvad  <cit> .
the two more genotype types - pcv2c reported from denmark in the 1980s and pcv2d found in china are not distributed so widely  <cit> .
the pcv <dig> genome contains two major open reading frames .
the replicase  proteins responsible for initiation of viral replication are encoded by orf <dig> located on the viral plus strand.
a sole structural protein of the viral coat encoded by orf <dig> on complementary strand is the unique structural capsid  protein  <cit> .
cap protein is the major immunogenic protein containing specific epitopes of pvc <dig>  <cit> .
the cap protein reacts strongly with the serum of pcv2-infected pigs and therefore, it is a major candidate antigen for the development of serological diagnostic methods.
there were multiple attempts to produce recombinant cap protein in different expression systems including e. coli, lactococcus lactis, pichia pastoris, baculovirus or pseudorabies virus expression systems  <cit> .
in most cases the expression of orf <dig> gene of pcv <dig> was achieved by optimizing codon composition, removing n terminal part of the protein rich of arginine residues or by adding different tags useful for purification.
these approaches have been shown to be successful to yield truncated or full length monomeric cap protein.
expression of pcv <dig> cap in insect cells resulted in recombinant protein self-assembled into virus-like particles  that were structurally and antigenically indistinguishable from regular pcv <dig> capsids  <cit> .
recombinant vlps due to their repetitive surface epitopes, the virus-like structure and the capability to induce humoral and/or ctl responses are more suitable for the development of immunogenic vaccines and sensitive diagnostic tests as compared to monomeric viral antigens  <cit> .
at present, few baculovirus-derived pcv2a subunit vaccines are available  <cit> .
pcv <dig> cap vpls produced in baculovirus expression systems were shown to be a suitable antigen for detection of pcv <dig> infection by the indirect igg pcv <dig> elisa  <cit> .
however, the possibilities to employ cheaper antigens for diagnostic purposes are considered mostly by using monomerus pcv <dig> cap protein  <cit> .
in the current study we demonstrate that pcv2b-derived cap vlps can be efficiently produced in yeast sacharomyces cerevisiae and are excellent antigens for generation of monoclonal antibodies  reactive with virus-infected cells.
moreover, we demonstrate that yeast-derived recombinant pcv <dig> cap vlps represent a promising antigen for a highly sensitive and specific immunoassay for serologic diagnosis of pcv <dig> infection.
RESULTS
production of pcv <dig> vlps in yeast
pcv2-positive samples collected between  <dig> and  <dig> from domestic pigs from  <dig> regions of lithuania were used for the amplification of full-length pcv <dig> genomic dna with overlapping primers.
dna sequence alignment of the  <dig> cloned and sequenced pcv <dig> genomes circulating in lithuania revealed six slightly different genome variants .
all six pcv <dig> genome variants belonged to genotype pcv2b according to the previously proposed criteria  <cit> .
three open reading frame  <dig>  variants amplified by pcr from native pcv <dig> genomes: variant  <dig> , variant  <dig>  and variant  <dig>  as well as orf <dig> of the most representative pcv2-cap gene variant  <dig> which was codon-optimized by s. cerevisiae codon usage  were cloned into pfx <dig> expression vector  <cit>  and used for the expression in yeast s. cerevisiae.
the expression of four cloned genes was detected in sds-page and confirmed by western blot using pig serum .
all recombinant pcv2-cap proteins were found in the soluble fraction of yeast cell lysate which allowed their efficient purification .
all four variants of pcv2-cap proteins generated in yeast were purified by density gradient centrifugation and examined using electron microscope .
despite the slight differences in expression levels of pcv2-cap gene variants, all four purified cap proteins were assembling into regular homogenous in size vlps about 17–22 nm in diameter similar to native pcv <dig> virions .
pcv-cap vlps encoded by the most representative pcv2-cap gene variant  <dig> were used for generation of mabs and development of an indirect elisa for detection of pcv2-specific igg.figure  <dig>
analysis of recombinant pcv <dig> cap protein-derived vlps produced in yeast.
coomassie blue-stained sds-page;  western blot with pig serum.
: in lanes:  <dig> - whole crude lysate of yeast transformed with pfx7-pcv2cap- <dig> plasmid,  <dig> - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- <dig> plasmid;  <dig> - whole crude lysate of yeast transformed with pfx7- pfx7-pcv2cap- <dig> plasmid;  <dig> - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- <dig> plasmid;  <dig> - whole crude lysate of yeast transformed with pfx7-pcv2cap- <dig> plasmid;  <dig> - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- <dig> plasmid;  <dig> - whole crude lysate of yeast transformed with pfx7-pcv2cap-622s plasmid;  <dig> - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap-622s plasmid;  <dig> - whole crude lysate of yeast transformed with pfx <dig> plasmid, m- pre-stained protein weight marker ;  <dig> - cap <dig> protein vlps purified by density gradient centrifugation;  <dig> - cap <dig> protein vlps purified by density gradient centrifugation;  <dig> – cap <dig> protein vlps purified by density gradient centrifugation;  <dig> - cap622s protein vlps  purified by density gradient centrifugation.
: electron microscopy pictures of vlps formed by pcv2-cap <dig>  protein pcv2-cap <dig>  protein pcv2-cap <dig> and protein pcv2-cap622s protein stained with 2% aqueous uranyl acetate solution and examined by morgagni  <dig> electron microscope.
generation and characterization of monoclonal antibodies against pcv2b-cap vlps
to generate the mabs, mice were immunized with recombinant pcv <dig> cap  <dig> vlps and tested for the presence of cap-specific serum antibodies.
spleen cells of a mouse showing the highest titer of cap-specific antibodies were used to generate hybridomas.
after fusion, all hybrid clones were screened for the reactivity with pcv <dig> cap vlps and specific ones were subjected to isotyping and recloning.
after these procedures, three stable hybridoma cell lines producing pcv <dig> cap-specific mabs of igg isotype were obtained.
two of them  produced mabs of igg2a subtype and one clone  of igg <dig> subtype.
the specificity of all three mabs was proven by elisa using all three variants of yeast-expressed cap proteins: pcv <dig> cap <dig> protein, pcv <dig> cap <dig> protein, and pcv <dig> cap <dig> protein.
no reactivity of the mabs with hantaan  and tioman nucleocapsid proteins  <cit>  used as a negative control was observed, which confirmed the specificity of these mabs to the pcv <dig> cap protein .
western blot analysis of mab specificity using the lysate of yeast expressing pcv <dig> cap protein revealed that only one mab  recognized sds-denaturated pcv <dig> cap protein .
meanwhile, the mabs #7c <dig> and #24c <dig> did not react with pcv <dig> cap protein in western blot thus indicating that they are directed against conformational epitopes .
to evaluate the diagnostic potential of the generated mabs, their reactivity with pcv2-infected  swine testicle cells  and pcv1-infected porcine kidney  cells was tested by an indirect immunofluorescence assay  using commercially available slides .
the mabs #7c <dig> and #14g <dig> were reactive with pcv2-infected swine testicular cells but did not recognize pcv1-infected pk cells thus confirming their specificity to pcv <dig> cap protein .
meanwhile, the mab #24c <dig> was not reactive in both ifa tests.figure  <dig>
indirect immunofluorescence staining of pcv <dig> infected swine testicle  cells and pcv1-infected pk cells with mabs #7c <dig>  #14g <dig> and #24c <dig>  neg - mab #4 f <dig> directed against parainfluenza type 3 n protein  <cit> .
development of an indirect elisa for detection of pcv <dig> cap-specific igg
as a first step in the development of pcv2-cap vlp-based elisa, checkerboard titrations were performed to determine the optimal concentration of the cap antigen and the test serum.
to optimize plate coating, the recombinant pcv2-cap  <dig> protein was immobilized on elisa plates at five different concentrations: 2; 1;  <dig> ;  <dig> ;  <dig>  μg/ml.
the optimal antigen concentration for plate coating as determined by a checkerboard titration was  <dig>  μg/ml.
to determine the optimal serum sample dilution, serum samples identified by the commercial serelisa test  as high positive, low positive and negative were diluted in serial two fold dilutions ranging from 1: <dig> to 1: <dig>  the od <dig> values obtained in an indirect elisa were plotted against the dilutions .
high od readings with positive control serum and low background signals with negative sera were obtained at serum dilution 1: <dig>  the absorbance values declined at serum dilution 1: <dig>  as the commercial serelisa test uses serum dilution 1: <dig> and the differences between od values at serum dilutions 1: <dig> and 1: <dig> were insignificant in our newly developed elisa, for convenience the appropriate dilution  was selected for the indirect igg pcv <dig> cap-based elisa.
control antigen – yeast expressed hamster polyomavirus  vp <dig> protein vlps  <cit>  was included in the test to determine the specificity of the assay.
the positive samples revealed significantly higher od values  with pcv <dig> cap protein as compared to the control hapyv-vp <dig> antigen  .figure  <dig>
titration curves of swine serum samples.
either positive or negative for pcv <dig> antibodies swine serum samples were titrated by the newly developed indirect igg pcv <dig> cap vlp-based elisa using recombinant pcv <dig> cap vlps at a concentration of  <dig>  μg/ml  and control antigen  at a concentration of 2 μg/ml .
sera were tested in serial duplicate twofold dilutions ranging from 1: <dig> to 1: <dig>  the results are expressed as the od <dig> and correspond to the average of the values obtained in at least two different analyses.
optical density results of serum samples in the newly developed indirect igg pcv <dig> cap vlp-based elisa.
four hundred and nine serum samples previously characterized by the commercial serelisa test were analyzed by the newly developed indirect pcv2-cap vlp igg elisa.
the absorbance at 450 nm obtained for the serum samples are shown.
the dotted line indicates the assay cut-off, which was established to be  <dig> .
for the indirect pcv <dig> cap vlp igg elisa.
opened circles represent discrepant results, filled circles indicate matching results with commercial serelisa test.
id sera, serum sample number.
the cut-off value was calculated as the mean od <dig> value of  <dig> serum specimens negative by serelisa plus  <dig> standard deviations .
this calculation gives 99% confidence.
the mean od <dig> value and sd were  <dig>  and  <dig> , respectively.
therefore, the positive threshold determined for the newly developed indirect igg pcv <dig> cap vlp-based elisa was  <dig> .
a serum sample was considered positive when its od <dig> value was greater than the positive threshold .
four hundred-nine serum samples were tested to determine the sensitivity and specificity of the newly developed elisa.
the sensitivity and specificity were calculated for the indirect igg pcv2-cap vlp-based elisa using serelisa test as a gold standard.
all serum samples were tested in parallel with a commercial serelisa kit.
from  <dig> serum samples tested,  <dig> samples were positive by both assays.
thus, the calculated sensitivity of the new elisa test was 100% .
one hundred-twelve serum samples negative by serelisa were tested to determine the specificity of the indirect igg pcv2-cap vlp-based elisa.
seventy-three sera were below and  <dig> serum samples were above the cut-off by indirect igg pcv2-cap vlp-based elisa .
indirect igg pcv2-cap vlp-based elisa identified these samples as positive , although in the commercial serelisa test these samples were found to be negative.
discrepant serum samples were re-tested by pcr.
overall, all samples of the suspected pcv <dig> cases were pcv <dig> dna positive.
thus, samples positive by the newly developed indirect igg pcv2-cap vlp-based elisa were confirmed by pcr but missed by commercial serelisa igg test.
all serum samples were tested in duplicate and the correlation coefficient  between two measurements was calculated.
the r value was shown to be  <dig> , which indicates very high reproducibility between tests.table  <dig>
comparative evaluation of the indirect igg pcv <dig> cap vlp-based elisa with commercial serelisa kit for detection of anti-pcv <dig> antibodies
indirect igg elisa
commercial serelisa test
positive
negative
total
336
73
total
297
112
the values represent the numbers of sera tested.
these results indicate that the newly developed indirect igg pcv2-cap vlp-based elisa had higher sensitivity as compared to the commercial serelisa test, which failed to detect some pcv2-positive samples as positives.
discussion
virus-like particles present viral antigens in a more authentic conformation than monomeric structural proteins.
therefore, recombinant vlps are more preferable for vaccine or diagnostic purposes  <cit> .
vlps mimic the structure of authentic virus particles they are derived from and are easily recognized by the immune system and able to stimulate both b-cell and t-cell immune responses.
multiple expression systems such as e. coli, l. lactis, p. pastoris allowed generation of monomeric pcv <dig> cap protein but not vlps  <cit> .
different modifications of the gene encoding pcv <dig> protein were also necessary for generation of recombinant pcv <dig> cap protein including removal of the n terminal part with nuclear localization signal and fusing it with different tags or yeast signal peptide as well as the optimization of codon usage according to the host  <cit> .
even for the investigation of the structure of pcv <dig> cap-derived vlps, both the full length and an n-terminally truncated monomeric cap protein was purified from e.coli and reassembled into vlps in vitro  <cit> .
on the other hand, baculovirus/insect cell expression system supported pcv2-cap protein self-assembly into vlps  <cit> .
yeast saccharomyces cerevisiae might provide an alternative expression system for pcv <dig> cap protein production as there are several reports which confirm that yeast can support vlp auto-assembly of viral proteins  <cit> .
in a bucarey’s study, the failure of pcv <dig> cap protein production in s.cerevisiae using native pcv2-cap coding sequence was reported  <cit> .
it was demonstrated that only codon optimized gene allowed generation of pcv <dig> cap vlps in yeast.
moreover, in bucarey’s study generated vlps were not homogenous in size and shape  <cit> .
this is opposite to our results presented in the current study .
we demonstrate that pcv2b-cap vlps were efficiently produced in yeast s.cerevisiae using native virus orf <dig> versions of cap-encoding genes as well as the codon-optimized gene.
we purified three slightly different pcv <dig> cap proteins variants encoded by native cap-encoding sequences and confirmed that they all formed homogenous in size and shape vlps similar to native virions or vlps produced in baculovirus expression system  <cit> .
yeast-produced pcv <dig> cap vlps were highly immunogenic and induced pcv2-specific immune response in mice.
we generated three mabs with different capabilities to recognize both the recombinant pcv <dig> cap protein and virus-infected cells.
the mab #14g <dig> demonstrated exclusive and very specific reactivity with linear pcv <dig> cap protein epitope in western blot, elisa and ifa implying the localization of this epitope on the surface of vlps.
the mab #24c <dig> was non-reactive with virus-infected cells and did not recognize sds-denaturated pcv <dig> cap protein, which suggests that its epitope is accessible only in native pcv <dig> cap vlps.
the mab #7c <dig> also recognized conformational pcv <dig> epitope but it was accessible on virus-infected cells as demonstrated in ifa .
all three mabs were highly specific and did not show any cross-reactivity with pcv <dig> cap protein in all assays despite a significant homology between pcv <dig> and pcv <dig>  in terms of amino-acid sequences.
therefore, the newly generated mab #7c <dig> and especially the mab #14g <dig> could be used as highly specific reagents for pcv <dig> detection by ifa or monoclonal antibody-based blocking elisa.
the cap protein is a major candidate antigen for the development of serologic diagnostic methods as it is the major immunogenic protein containing specific epitopes of pvc <dig>  <cit> .
in the previous studies recombinant pcv <dig> cap protein expressed in either prokaryotes or eukaryotes was used in an indirect elisa as coating antigen.
it was shown that pcv <dig> cap vlps or cap protein expressed in insect cells as well as cap protein expressed in e. coli showed high immunoreactivity with serum antibodies  <cit> .
the diagnostic sensitivity, specificity, and accuracy of elisas performed with pcv <dig> cap vlps and pcv <dig> were similar  to ifa  <cit> .
however the sensitivity of elisa’s developed using e. coli-expressed recombinant proteins were lower.
this is likely because natural virus particles are perfect in structure whereas recombinant e. coli-expressed proteins are not always refolded completely.
sun and colleagues developed protein-based elisa for pcv <dig> specific diagnosis using e. coli-expressed antigenic domain of pcv <dig> cap protein  <cit> .
the developed assay failed to detect some pcv2-positive serum samples identified by ifa.
the specificity was lower also in a monoclonal antibody-based blocking elisa developed by huang and colleagues  <cit> .
its sensitivity and specificity were determined to be  <dig> % and  <dig> %, respectively.
higher sensitivity was achieved in a quantitative immunofluorescence assay  using the recombinant pcv <dig> nucleocapsid protein expressed in vero cells by a lentivirus system, but the specificity was lower when compared to elisa  <cit> .
in the current study, the newly developed indirect igg pcv2-cap vlp-based elisa proved to be a highly sensitive and specific tool for the serologic diagnosis of pcv <dig> infection and was even more sensitive than the commercial serelisa test.
it might be that not only vlp structure of yeast-expressed cap protein influenced the higher sensitivity of the newly developed indirect igg pcv2-cap vlp-based elisa.
based on the genbank database and several studies published in different countries a shift on genotype prevalence from pcv2a to b have been observed  <cit> .
our analysis of pcv <dig> genotype spread in lithuania pig farms in the samples collected between  <dig> and  <dig> confirmed that all isolated virus genomes belonged to the pcv2b genotype.
most of commercial diagnostic tests are based on pcv2a genotype-derived cap proteins.
our preliminary data showed that some pcv2-negative serum samples identified by a commercial serelisa test were negative in an indirect igg cap vlp-based elisa when pcv2a genotype-derived cap vpls were used but were positive when pcv2b genotype-derived cap vlps were used as a coating antigen.
however to prove the hypothesis more serological assays should be performed.
CONCLUSIONS
recombinant vlps are more preferable for vaccine development or diagnostic purposes as they present viral antigens in a proper conformation.
in this study we have demonstrated that s.cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape pcv <dig> cap protein-based vlps similar to native virions.
yeast cultures are easy to maintain, which makes them faster and less expensive to use than other eukaryotic expression systems.
we showed that yeast expression system tolerated native virus genes encoding pcv <dig> cap protein variants as well as the codon-optimized gene.
yeast-derived pcv <dig> cap vlps were capable to induce the generation of pcv2-specific mabs that did not show any cross-reactivity with pcv1-infected cells.
moreover, the employment of pcv <dig> cap vlps as an antigen in the indirect igg pcv <dig> cap vlp-based elisa demonstrated that this newly developed pcv <dig> diagnostic test was more sensitive and specific than the commercial serelisa test and is potentially useful as a diagnostic assay in screening pcv2-suspected swine samples.

 circoviruses belong to the circoviridae family and infect a variety of animal and plant species. they are named after their circular single-stranded dna genome and represent the smallest dna viruses infecting mammals. porcine circovirus is found all over the world in the domestic pigs and the wild boars which seldom display the clinical condition. pcv serological prevalence in the wild boars is almost as high as in domestic pigs. pcv is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as pcv2-associated diseases. overall, pcvad includes clinical syndromes and diseases such as postweaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, porcine reproductive disorders, respiratory disease complex, granulomatous enteritis, congenital tremor, necrotizing tracheitis and lymphadenitis and exudative epidermitis. pcv has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production. the ubiquitous nature of the virus and the retrospective evidence of this infection long before disease appearance provided the reason for debate about the real capabilities of this virus in its association with the disease. how the virus causes disease is still not fully understood. infection with pcv in combination with still unknown co-factors can cause immunopathological disorders such as pmws, or induce protective immunity. it might be that pcv2-induced diseases are the result of pcv2-caused immune disorder. it was demonstrated that pigs with pmws display a marked depletion of t- and b-lymphocytes, and their lymphoid organs are infiltrated by histiocytes and multinucleated giant cells and might cause deficiency of total and neutralizing antibody response. the interaction of pcv with the innate immune system have been mostly studied in vitro looking for the effects of the virus on dendritic cells, macrophages and peripheral blood mononuclear cells. it was shown that pcv infection of plasmacytoid dcs impairs the induction of interferon-α and tumor necrosis factor-α, which silences responsiveness to pathogen-associated recognition signals by compromising immune defense development against other pathogens. as a result this may lead to a host susceptibility to secondary infections which actually are responsible for developing of pcvad. pcv is a non-enveloped virus with circular single stranded dna molecule of approximately bases packed in icosahedral 12–23 nm diameter virion particle. virus genome sequences with nucleotide diversity cut-off of % were divided into four genotypic groups. the two major groups designated as pcv2a, pcv2b are found worldwide and have been demonstrated to be virulent enough to trigger pcvad. the two more genotype types - pcv2c reported from denmark in the 1980s and pcv2d found in china are not distributed so widely. the pcv genome contains two major open reading frames. the replicase proteins responsible for initiation of viral replication are encoded by orf located on the viral plus strand. a sole structural protein of the viral coat encoded by orf on complementary strand is the unique structural capsid protein. cap protein is the major immunogenic protein containing specific epitopes of pvc. the cap protein reacts strongly with the serum of pcv2-infected pigs and therefore, it is a major candidate antigen for the development of serological diagnostic methods. there were multiple attempts to produce recombinant cap protein in different expression systems including e. coli, lactococcus lactis, pichia pastoris, baculovirus or pseudorabies virus expression systems. in most cases the expression of orf gene of pcv was achieved by optimizing codon composition, removing n terminal part of the protein rich of arginine residues or by adding different tags useful for purification. these approaches have been shown to be successful to yield truncated or full length monomeric cap protein. expression of pcv cap in insect cells resulted in recombinant protein self-assembled into virus-like particles that were structurally and antigenically indistinguishable from regular pcv capsids. recombinant vlps due to their repetitive surface epitopes, the virus-like structure and the capability to induce humoral and/or ctl responses are more suitable for the development of immunogenic vaccines and sensitive diagnostic tests as compared to monomeric viral antigens. at present, few baculovirus-derived pcv2a subunit vaccines are available. pcv cap vpls produced in baculovirus expression systems were shown to be a suitable antigen for detection of pcv infection by the indirect igg pcv elisa. however, the possibilities to employ cheaper antigens for diagnostic purposes are considered mostly by using monomerus pcv cap protein. in the current study we demonstrate that pcv2b-derived cap vlps can be efficiently produced in yeast sacharomyces cerevisiae and are excellent antigens for generation of monoclonal antibodies reactive with virus-infected cells. moreover, we demonstrate that yeast-derived recombinant pcv cap vlps represent a promising antigen for a highly sensitive and specific immunoassay for serologic diagnosis of pcv infection.  production of pcv vlps in yeast pcv2-positive samples collected between and from domestic pigs from regions of lithuania were used for the amplification of full-length pcv genomic dna with overlapping primers. dna sequence alignment of the cloned and sequenced pcv genomes circulating in lithuania revealed six slightly different genome variants. all six pcv genome variants belonged to genotype pcv2b according to the previously proposed criteria. three open reading frame variants amplified by pcr from native pcv genomes: variant, variant and variant as well as orf of the most representative pcv2-cap gene variant which was codon-optimized by s. cerevisiae codon usage were cloned into pfx expression vector and used for the expression in yeast s. cerevisiae. the expression of four cloned genes was detected in sds-page and confirmed by western blot using pig serum. all recombinant pcv2-cap proteins were found in the soluble fraction of yeast cell lysate which allowed their efficient purification. all four variants of pcv2-cap proteins generated in yeast were purified by density gradient centrifugation and examined using electron microscope. despite the slight differences in expression levels of pcv2-cap gene variants, all four purified cap proteins were assembling into regular homogenous in size vlps about 17–22 nm in diameter similar to native pcv virions. pcv-cap vlps encoded by the most representative pcv2-cap gene variant were used for generation of mabs and development of an indirect elisa for detection of pcv2-specific igg.figure analysis of recombinant pcv cap protein-derived vlps produced in yeast. coomassie blue-stained sds-page; western blot with pig serum.: in lanes: - whole crude lysate of yeast transformed with pfx7-pcv2cap- plasmid, - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- plasmid; - whole crude lysate of yeast transformed with pfx7- pfx7-pcv2cap- plasmid; - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- plasmid; - whole crude lysate of yeast transformed with pfx7-pcv2cap- plasmid; - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap- plasmid; - whole crude lysate of yeast transformed with pfx7-pcv2cap-622s plasmid; - the soluble fraction of the lysate of yeast transformed with pfx7-pcv2cap-622s plasmid; - whole crude lysate of yeast transformed with pfx plasmid, m- pre-stained protein weight marker; - cap protein vlps purified by density gradient centrifugation; - cap protein vlps purified by density gradient centrifugation; – cap protein vlps purified by density gradient centrifugation; - cap622s protein vlps purified by density gradient centrifugation.: electron microscopy pictures of vlps formed by pcv2-cap protein pcv2-cap protein pcv2-cap and protein pcv2-cap622s protein stained with 2% aqueous uranyl acetate solution and examined by morgagni electron microscope. generation and characterization of monoclonal antibodies against pcv2b-cap vlps to generate the mabs, mice were immunized with recombinant pcv cap vlps and tested for the presence of cap-specific serum antibodies. spleen cells of a mouse showing the highest titer of cap-specific antibodies were used to generate hybridomas. after fusion, all hybrid clones were screened for the reactivity with pcv cap vlps and specific ones were subjected to isotyping and recloning. after these procedures, three stable hybridoma cell lines producing pcv cap-specific mabs of igg isotype were obtained. two of them produced mabs of igg2a subtype and one clone of igg subtype. the specificity of all three mabs was proven by elisa using all three variants of yeast-expressed cap proteins: pcv cap protein, pcv cap protein, and pcv cap protein. no reactivity of the mabs with hantaan and tioman nucleocapsid proteins used as a negative control was observed, which confirmed the specificity of these mabs to the pcv cap protein. western blot analysis of mab specificity using the lysate of yeast expressing pcv cap protein revealed that only one mab recognized sds-denaturated pcv cap protein. meanwhile, the mabs #7c and #24c did not react with pcv cap protein in western blot thus indicating that they are directed against conformational epitopes. to evaluate the diagnostic potential of the generated mabs, their reactivity with pcv2-infected swine testicle cells and pcv1-infected porcine kidney cells was tested by an indirect immunofluorescence assay using commercially available slides. the mabs #7c and #14g were reactive with pcv2-infected swine testicular cells but did not recognize pcv1-infected pk cells thus confirming their specificity to pcv cap protein. meanwhile, the mab #24c was not reactive in both ifa tests.figure indirect immunofluorescence staining of pcv infected swine testicle cells and pcv1-infected pk cells with mabs #7c #14g and #24c neg - mab #4 f directed against parainfluenza type 3 n protein. development of an indirect elisa for detection of pcv cap-specific igg as a first step in the development of pcv2-cap vlp-based elisa, checkerboard titrations were performed to determine the optimal concentration of the cap antigen and the test serum. to optimize plate coating, the recombinant pcv2-cap protein was immobilized on elisa plates at five different concentrations: 2; 1;;;  μg/ml. the optimal antigen concentration for plate coating as determined by a checkerboard titration was  μg/ml. to determine the optimal serum sample dilution, serum samples identified by the commercial serelisa test as high positive, low positive and negative were diluted in serial two fold dilutions ranging from 1: to 1: the od values obtained in an indirect elisa were plotted against the dilutions. high od readings with positive control serum and low background signals with negative sera were obtained at serum dilution 1: the absorbance values declined at serum dilution 1: as the commercial serelisa test uses serum dilution 1: and the differences between od values at serum dilutions 1: and 1: were insignificant in our newly developed elisa, for convenience the appropriate dilution was selected for the indirect igg pcv cap-based elisa. control antigen – yeast expressed hamster polyomavirus vp protein vlps was included in the test to determine the specificity of the assay. the positive samples revealed significantly higher od values with pcv cap protein as compared to the control hapyv-vp antigen.figure titration curves of swine serum samples. either positive or negative for pcv antibodies swine serum samples were titrated by the newly developed indirect igg pcv cap vlp-based elisa using recombinant pcv cap vlps at a concentration of  μg/ml and control antigen at a concentration of 2 μg/ml. sera were tested in serial duplicate twofold dilutions ranging from 1: to 1: the results are expressed as the od and correspond to the average of the values obtained in at least two different analyses. optical density results of serum samples in the newly developed indirect igg pcv cap vlp-based elisa. four hundred and nine serum samples previously characterized by the commercial serelisa test were analyzed by the newly developed indirect pcv2-cap vlp igg elisa. the absorbance at 450 nm obtained for the serum samples are shown. the dotted line indicates the assay cut-off, which was established to be. for the indirect pcv cap vlp igg elisa. opened circles represent discrepant results, filled circles indicate matching results with commercial serelisa test. id sera, serum sample number. the cut-off value was calculated as the mean od value of serum specimens negative by serelisa plus standard deviations. this calculation gives 99% confidence. the mean od value and sd were and, respectively. therefore, the positive threshold determined for the newly developed indirect igg pcv cap vlp-based elisa was. a serum sample was considered positive when its od value was greater than the positive threshold. four hundred-nine serum samples were tested to determine the sensitivity and specificity of the newly developed elisa. the sensitivity and specificity were calculated for the indirect igg pcv2-cap vlp-based elisa using serelisa test as a gold standard. all serum samples were tested in parallel with a commercial serelisa kit. from serum samples tested, samples were positive by both assays. thus, the calculated sensitivity of the new elisa test was 100%. one hundred-twelve serum samples negative by serelisa were tested to determine the specificity of the indirect igg pcv2-cap vlp-based elisa. seventy-three sera were below and serum samples were above the cut-off by indirect igg pcv2-cap vlp-based elisa. indirect igg pcv2-cap vlp-based elisa identified these samples as positive, although in the commercial serelisa test these samples were found to be negative. discrepant serum samples were re-tested by pcr. overall, all samples of the suspected pcv cases were pcv dna positive. thus, samples positive by the newly developed indirect igg pcv2-cap vlp-based elisa were confirmed by pcr but missed by commercial serelisa igg test. all serum samples were tested in duplicate and the correlation coefficient between two measurements was calculated. the r value was shown to be, which indicates very high reproducibility between tests.table comparative evaluation of the indirect igg pcv cap vlp-based elisa with commercial serelisa kit for detection of anti-pcv antibodies indirect igg elisa commercial serelisa test positive negative total 336 73 total 297 112 the values represent the numbers of sera tested. these results indicate that the newly developed indirect igg pcv2-cap vlp-based elisa had higher sensitivity as compared to the commercial serelisa test, which failed to detect some pcv2-positive samples as positives. discussion virus-like particles present viral antigens in a more authentic conformation than monomeric structural proteins. therefore, recombinant vlps are more preferable for vaccine or diagnostic purposes. vlps mimic the structure of authentic virus particles they are derived from and are easily recognized by the immune system and able to stimulate both b-cell and t-cell immune responses. multiple expression systems such as e. coli, l. lactis, p. pastoris allowed generation of monomeric pcv cap protein but not vlps. different modifications of the gene encoding pcv protein were also necessary for generation of recombinant pcv cap protein including removal of the n terminal part with nuclear localization signal and fusing it with different tags or yeast signal peptide as well as the optimization of codon usage according to the host. even for the investigation of the structure of pcv cap-derived vlps, both the full length and an n-terminally truncated monomeric cap protein was purified from e.coli and reassembled into vlps in vitro. on the other hand, baculovirus/insect cell expression system supported pcv2-cap protein self-assembly into vlps. yeast saccharomyces cerevisiae might provide an alternative expression system for pcv cap protein production as there are several reports which confirm that yeast can support vlp auto-assembly of viral proteins. in a bucarey’s study, the failure of pcv cap protein production in s.cerevisiae using native pcv2-cap coding sequence was reported. it was demonstrated that only codon optimized gene allowed generation of pcv cap vlps in yeast. moreover, in bucarey’s study generated vlps were not homogenous in size and shape. this is opposite to our results presented in the current study. we demonstrate that pcv2b-cap vlps were efficiently produced in yeast s.cerevisiae using native virus orf versions of cap-encoding genes as well as the codon-optimized gene. we purified three slightly different pcv cap proteins variants encoded by native cap-encoding sequences and confirmed that they all formed homogenous in size and shape vlps similar to native virions or vlps produced in baculovirus expression system. yeast-produced pcv cap vlps were highly immunogenic and induced pcv2-specific immune response in mice. we generated three mabs with different capabilities to recognize both the recombinant pcv cap protein and virus-infected cells. the mab #14g demonstrated exclusive and very specific reactivity with linear pcv cap protein epitope in western blot, elisa and ifa implying the localization of this epitope on the surface of vlps. the mab #24c was non-reactive with virus-infected cells and did not recognize sds-denaturated pcv cap protein, which suggests that its epitope is accessible only in native pcv cap vlps. the mab #7c also recognized conformational pcv epitope but it was accessible on virus-infected cells as demonstrated in ifa. all three mabs were highly specific and did not show any cross-reactivity with pcv cap protein in all assays despite a significant homology between pcv and pcv in terms of amino-acid sequences. therefore, the newly generated mab #7c and especially the mab #14g could be used as highly specific reagents for pcv detection by ifa or monoclonal antibody-based blocking elisa. the cap protein is a major candidate antigen for the development of serologic diagnostic methods as it is the major immunogenic protein containing specific epitopes of pvc. in the previous studies recombinant pcv cap protein expressed in either prokaryotes or eukaryotes was used in an indirect elisa as coating antigen. it was shown that pcv cap vlps or cap protein expressed in insect cells as well as cap protein expressed in e. coli showed high immunoreactivity with serum antibodies. the diagnostic sensitivity, specificity, and accuracy of elisas performed with pcv cap vlps and pcv were similar to ifa. however the sensitivity of elisa’s developed using e. coli-expressed recombinant proteins were lower. this is likely because natural virus particles are perfect in structure whereas recombinant e. coli-expressed proteins are not always refolded completely. sun and colleagues developed protein-based elisa for pcv specific diagnosis using e. coli-expressed antigenic domain of pcv cap protein. the developed assay failed to detect some pcv2-positive serum samples identified by ifa. the specificity was lower also in a monoclonal antibody-based blocking elisa developed by huang and colleagues. its sensitivity and specificity were determined to be % and %, respectively. higher sensitivity was achieved in a quantitative immunofluorescence assay using the recombinant pcv nucleocapsid protein expressed in vero cells by a lentivirus system, but the specificity was lower when compared to elisa. in the current study, the newly developed indirect igg pcv2-cap vlp-based elisa proved to be a highly sensitive and specific tool for the serologic diagnosis of pcv infection and was even more sensitive than the commercial serelisa test. it might be that not only vlp structure of yeast-expressed cap protein influenced the higher sensitivity of the newly developed indirect igg pcv2-cap vlp-based elisa. based on the genbank database and several studies published in different countries a shift on genotype prevalence from pcv2a to b have been observed. our analysis of pcv genotype spread in lithuania pig farms in the samples collected between and confirmed that all isolated virus genomes belonged to the pcv2b genotype. most of commercial diagnostic tests are based on pcv2a genotype-derived cap proteins. our preliminary data showed that some pcv2-negative serum samples identified by a commercial serelisa test were negative in an indirect igg cap vlp-based elisa when pcv2a genotype-derived cap vpls were used but were positive when pcv2b genotype-derived cap vlps were used as a coating antigen. however to prove the hypothesis more serological assays should be performed.  recombinant vlps are more preferable for vaccine development or diagnostic purposes as they present viral antigens in a proper conformation. in this study we have demonstrated that s.cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape pcv cap protein-based vlps similar to native virions. yeast cultures are easy to maintain, which makes them faster and less expensive to use than other eukaryotic expression systems. we showed that yeast expression system tolerated native virus genes encoding pcv cap protein variants as well as the codon-optimized gene. yeast-derived pcv cap vlps were capable to induce the generation of pcv2-specific mabs that did not show any cross-reactivity with pcv1-infected cells. moreover, the employment of pcv cap vlps as an antigen in the indirect igg pcv cap vlp-based elisa demonstrated that this newly developed pcv diagnostic test was more sensitive and specific than the commercial serelisa test and is potentially useful as a diagnostic assay in screening pcv2-suspected swine samples. 
four hundred-nine serum samples collected from pigs in lithuania were tested for pcv2-specific igg to determine the sensitivity and specificity of the newly developed elisa in parallel using a commercial serelisa test as a gold standard. in this study we have demonstrated that pcv capsid protein based virus-like particles were efficiently produced in yeast s. cerevisiae and induced production of monoclonal antibodies reactive with virus-infected cells. virus-like particlesporcine circovirus 2monoclonal antibodiesissue-copyright-statement© the author 2014 it has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production. we have demonstrated that s. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape pcv cap protein-based vlps similar to native virions. moreover, yeast-derived pcv cap vlps were capable to induce the generation of pcv2-specific mabs that did not show any cross-reactivity with pcv1-infected cells. yeast expression system tolerated native virus genes encoding pcv cap protein variants as well as the codon-optimized gene. from tested serum samples, samples were positive by both assays. thirty-nine sera from serum samples were determined as negative by serelisa but were found to be positive both in the newly developed indirect igg pcv cap vlp-based elisa and the pcr test. porcine circovirus type is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as pcv2-associated diseases.
 porcine circovirus type is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as pcv2-associated diseases. it has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.  in this study we have demonstrated that pcv capsid protein based virus-like particles were efficiently produced in yeast s. cerevisiae and induced production of monoclonal antibodies reactive with virus-infected cells. moreover, pcv cap vlps served as a highly specific recombinant antigen for the development of an indirect igg pcv cap vlp-based elisa for the detection of virus-specific igg antibodies in swine sera. four hundred-nine serum samples collected from pigs in lithuania were tested for pcv2-specific igg to determine the sensitivity and specificity of the newly developed elisa in parallel using a commercial serelisa test as a gold standard. from tested serum samples, samples were positive by both assays. thirty-nine sera from serum samples were determined as negative by serelisa but were found to be positive both in the newly developed indirect igg pcv cap vlp-based elisa and the pcr test.  we have demonstrated that s. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape pcv cap protein-based vlps similar to native virions. yeast expression system tolerated native virus genes encoding pcv cap protein variants as well as the codon-optimized gene. moreover, yeast-derived pcv cap vlps were capable to induce the generation of pcv2-specific mabs that did not show any cross-reactivity with pcv1-infected cells. the high sensitivity and specificity of the indirect igg pcv cap vlp-based elisa clearly suggested that this assay is potentially useful diagnostic tool for screening pcv2–suspected samples. keywords virus-like particlesporcine circovirus 2monoclonal antibodiesissue-copyright-statement© the author 2014 