porcine parvovirus ( ppv ) , first isolated from sows in germany , has been found to occur worldwide [ 24 ] .
this syndrome is characterized by stillbirth , mummified fetuses , early embryonic and fetal death , delayed return to estrus , and infertility ( abbreviated as smedi ) [ 5 , 6 ] .
ppv is also shown to be an agent able to increase the effects of porcine circovirus type 2 infection in the clinical course of postweaning multisystemic wasting syndrome , which is a significant disease in global swine production .
five different groups of porcine parvoviruses ( ppv ) have been identified : classic ppv ( ppv1 ) , ppv2 , ppv3 ( known as porcine parv4 , hokovirus , or partetravirus ) , and ppv4 and porcine bocaviruses , which all have substantial genetic divergence [ 912 ] .
recently , a new parvovirus provisionally proposed to be named as ppv5 was discovered in the united states .
classic ppv has one serotype subdivided into four clinical genotypes ( biotypes ) according to their pathogenicity . the nadl-8 strain can cause viremia and crosses the placenta to infect fetuses , leading to fetus death .
in contrast , nonpathogenic nadl-2 strain is currently widely used as an attenuated vaccine and causes only limited viremia without crossing the placental barrier in experimental infections .
the other two groups are the kresse and iaf - a83 strains , which are associated with dermatitis and enteric diseases , respectively .
this group of viruses also infects rodents and carnivores and belongs to the parvoviridae subfamily within the parvoviridae family .
ppv has a negative , single - stranded dna of about 5  kb with distinct hairpin termini .
the genome contains two major open reading frames ( orfs ) , each located in the same frame of the complementary strand .
, vp2 , and vp3 are encoded in orf2 . vp1 and vp2 are translated from differently spliced rnas , whereas vp3 is formed by proteolytic cleavage of vp2 [ 17 , 18 ] .
vp2 consists of an eight - stranded antiparallel -barrel motif with 4 large loops between -strands .
these loops are shown to possess many b - cell epitopes and can tolerate insertions [ 19 , 20 ] that make ppv vp2 a potential antigen carrier and play a key role in ppv diagnosis and immune prophylaxis .
the structure of ppv capsid composed of baculovirus system generated recombinant vp2 is available in 3.5   resolution ( pdb accession number 1k3v ) .
immunogenic major capsid protein vp2 of ppv has been synthesized in several expression systems including bacteria [ 21 , 23 ] .
ppv vp2 protein expressed using the baculovirus expression vector system was shown to assemble into virus - like particles ( vlps ) similar in size and morphology to the original virions .
vlps generated in baculovirus system exhibit positive immunoreactivity for ppv and are used in most commercial elisa tests .
most recently , immunogenic ppv vp2 protein was synthesized in yeast pichia pastoris   the formation of recombinant antigenic human parvovirus capsid protein vlps in s. cerevisiae has been recently demonstrated [ 28 , 29 ] .
regarding costs , yield , and ease of handling , vlp production in yeast represents an alternative to the recombinant baculovirus expression system , which is so far the dominating source of vp2-derived vlps of parvoviruses [ 27 , 28 ] . in the current study
, we have generated the ppv vp2 protein as vlps in s. cerevisiae expression system , demonstated their structural and antigenic similarity with viral capsids and developed a new indirect igg elisa based on the use of ppv vp2-derived vlps .
moreover , we have developed a panel of ppv vp2 protein - specific monoclonal antibodies and demonstrated their reactivity with ppv - infected cells .
one hundred and eighty - three swine serum samples from farms in lithuania ( n = 160 ) , romania ( n = 14 ) , and ukraine ( n = 13 ) were collected in years 20082010 and used in this study .
viral nucleic acids ( nas ) were extracted from porcine kidney cell culture pk-15 ( atcc ccl-33 ) infected with porcine parvovirus strain nadl-2 .
nas were extracted using commercial qiaamp ultrasens virus kit ( qiagen gmbh , hilden , germany ) following the manufacturer 's manual and stored at 70c until use .
the ppv vp2 gene was amplified using high fidelity enzyme mix ( fermentas / thermo fisher scientific , vilnius , lithuania ) directly from extracted nas using the following pair of primers ( idt , munich , germany ) :  ppv - vp2-f 5-tctactagtacaatgagtgaaaatgtggaacaa-3  ppv - vp2-r 5-gagactagtctagtataattttcttggtataagt-3 ppv - vp2-f 5-tctactagtacaatgagtgaaaatgtggaacaa-3 ppv - vp2-r 5-gagactagtctagtataattttcttggtataagt-3 the primers used for amplification incorporated bcui site ( underlined ) for subcloning into the yeast vector pfx7 .
the thermal cycle conditions were the following : initial denaturation for 3  min at 95c , followed by 30 cycles of 94c for 1  min , 55c for 1  min , and 72c for 2  min , and then the final elongation at 72c for 10  min .
the pcr amplification product was digested with bcui and inserted into xbai - linearized and dephosphorylated yeast expression plasmid pfx7 under control of yeast gal1 - 10 promoter   and confirmed by pcr and subsequent dna sequence analysis .
the nucleotide sequence of the amplified ppv vp2 was compared with those in genbank using the basic local alignment search tool ( blast ) .
all dna manipulations were performed according to standard procedures   using enzymes and kits from fermentas / thermo fisher scientific .
recombinant constructs were screened in escherichia coli dh5 f. recombinant construct containing ppv vp2 sequence was screened in e. coli dh5f cells .
saccharomyces cerevisiae haploid strain ah22 mata ( leu2 his4 pep4 ) was used for the expression of ppv vp2 protein .
selection of yeast transformants resistant to formaldehyde was carried out on the yepd ( 1% yeast extract , 2% peptone , 2% dextrose , difco , sparks , md , usa ) agar supplemented with 5  mm formaldehyde .
s. cerevisiae transformants were grown in yepd medium supplemented with 5  mm formaldehyde or in yepg induction medium ( 1% yeast extract , 2% peptone , 2% galactose , difco ) .
cultivation of transformed yeast cells , expression and purification of ppv vp2 was performed as previously described [ 32 , 33 ] .
after purification , the total protein concentration was determined by the bradford assay ( roth , karlsruhe , germany ) with bovine serum albumin ( bsa ) used as a standard .
the samples were boiled in a reducing sample buffer and separated in gel electrophoresis in sds - tris - glycine buffer .
proteins were visualized by staining with coomassie brilliant blue ( sigma - aldrich co. , st .
blotting , proteins were electrotransferred to immobilon p membrane ( millipore , bedford , ma , usa ) as described by sambrook and russell .
the membranes were blocked with 5% milk in phosphate buffered saline ( pbs ) for 2  h. the blocking solution was removed and the blots were incubated with the mabs against ppv vp2 protein ( undiluted hybridoma supernatants ) .
secondary antibodies conjugated to horseradish peroxidase ( hrp ) ( bio - rad , hercules , ca , usa ) were used for detection of specific antibody binding .
the blots were stained with 3,3,5,5-tetramethylbenzidine ( tmb ) ready - to - use chromogenic substrate ( clinical science products inc . , mansfield , ma , usa ) .
after purification by cscl ultracentrifugation , suspension of the recombinant ppv vp2 protein was placed on 400-mesh carbon coated copper grids ( agar scientific , stansted , uk ) .
the protein samples were stained with 2% aqueous uranyl acetate solution ( reachim , moscow , russia ) and examined with a morgagni-268 electron microscope ( fei , eindhoven , the netherlands ) .
porcine serum samples were assayed for the presence of anti - ppv antibodies using a commercial ingezim ppv compact kit ( ingenasa , madrid , spain ) .
this is an enzymatic assay based on the blocking elisa technique which uses mab specific for porcine parvovirus vp2 protein , and baculovirus expression systems generated recombinant capsid of vp2 .
two blocking percentage ( bp ) values were used for result interpretation : samples with bp higher than 30% were considered as positive and samples with bp lower than 25% were considered as negative .
polystyrene microtiter plates ( nerbe plus , winsen / luhe , germany ) were coated with 50  ng per well of recombinant ppv vp2 protein , diluted in 100  l of 0.05  m carbonate - bicarbonate coating buffer ( ph 9.6 ) and incubated overnight at 4c .
plates were washed three times with pbst ( phosphate buffered saline with 0.05% ( v / v ) tween 20 , ( bio - rad , richmond , ca , usa ) ) and then blocked by the addition of 150  l of blocking buffer per well ( 1x roti - block , carl roth gmbh & co. ) and incubation at room temperature for 1 hour . after blocking , the plates were washed three times with pbst and 100  l aliquots of serum specimens , diluted 1  :  400 in pbst with 1% bsa , were added to the wells .
antigen concentration and serum dilution level for this assay were determined by titration to reach optimal conditions for sensitivity and specificity ( data not shown ) .
after 2  h of incubation at 37c , the plates were rinsed three times with pbst .
hrp - conjugated rabbit anti - pig igg ( sigma - aldrich biosciences , seattle , usa ) diluted 1  :  30 000 in pbst , containing 1% bsa , were added to the wells in 100  l aliquots and incubated for 1  h at 37c .
binding of specific antibodies was visualized by the addition of 100  l / well of tmb substrate ( clinical science products inc . , mansfield , ma , usa ) .
after 10  min of incubation at the room temperature , the reaction was stopped by adding 100  l / well of 10% sulphuric acid and the optical density ( od ) was measured at 450  nm ( reference filter 620  nm ) .
eight - week - old female balb / c mice ( obtained from a breeding colony at the center for innovative medicine , vilnius , lithuania ) were immunized at days 0 , 28 , and 56 by a subcutaneous injection of 50  g of recombinant ppv vp2 protein . for an initial immunization ,
three days after the final injection , mouse spleen cells were fused with sp2/0-ag 14 mouse myeloma cells using polyethylene glycol 1500 ( peg / dmso solution , hybri - max , sigma - aldrich ) .
hybrid cells were selected in growth medium supplemented with hypoxanthine , aminopterin , and thymidine ( 50hat media supplement , sigma - aldrich ) .
samples of supernatant from wells with viable clones were screened by an indirect elisa ( as described above ) using goat anti - mouse igg ( bio - rad ) diluted 1  :  5000 to detect specific antibodies to ppv vp2 .
hybridomas secreting specific antibodies to ppv vp2 protein were subcloned twice by a limiting dilution assay .
hybridoma cells were maintained in complete dulbecco 's modified eagle 's medium ( dmem , biochrom , berlin , germany ) containing 15% fetal calf serum ( biochrom ) and antibiotics .
antibodies in hybridoma culture supernatants were isotyped using the mouse monoclonal antibody isotyping kit ( pierce , thermo scientific ) in accordance with the manufacturer 's protocol .
the reactivity of the mabs with ppv - infected cells was analyzed by immunofluorescence assay ( ifa ) using porcine parvovirus fa substrate slides ( vmrd , inc .
, pullman , usa ) containing fixed swine testicle cells infected and noninfected with porcine parvovirus strain ky-11 .
the slides were treated according to the manufacturer 's protocol , incubated with undiluted hybridoma supernatants and developed with fluorescein isothiocyanate- ( fitc- ) labelled secondary antibody ( bd biosciences , franklin lakes , nj , usa ) .
the immunostained slides were observed by fluorescent microscope olympus ix-70 ( olympus , tokyo , japan ) .
the gene encoding ppv vp2 was derived from the nucleic acids extract obtained from ppv - infected cell culture .
the 1700  bp sequence amplified by pcr was sequenced and confirmed to be identical to vp2 gene from porcine parvovirus strain nadl-2 ( genbank entry number nc001718 ) .
the gene was cloned into s. cerevisiae expression vector pfx7 under the galactose - inducible promoter .
a sds - page analysis of the lysate of induced yeast biomass revealed a major protein band of approximately 64  kda ( figure 1 , lane 2 ) .
no additional protein bands were observed in crude lysates of s. cerevisiae harboring empty yeast vector pfx7 ( figure 1 , lane 1 ) .
after centrifugation of lysates through 30% sucrose cushion , the pellets harboring recombinant proteins were subjected to cscl - gradient centrifugation .
cscl gradients revealed recombinant ppv vp2 protein ( figure 1 , lane 3 ) in fractions with buoyant density of 1.2861.308  g / ml .
in several preparative procedures , the yield of purified recombinant ppv vp2 protein was found to be 8 - 9  mg per liter of induced yeast culture .
after cscl gradient purification , the recombinant vp2 protein was dialyzed against pbs and stored at 20c in pbs containing 50% glycerol .
typical icosahedral structures of parvoviruses with a diameter of approximately 2530  nm were observed indicating that ppv vp2 protein is self - assembled to vlps ( figure 2(a ) ) .
vlps of ppv produced in s. cerevisiae expression system were similar to those previously generated in insect cells   or native ppv particles .
treatment with 25  mm edta or 10  mm egta did not cause the dissociation of recombinant vlps indicating that the assembled structures do not require divalent ions ( data not shown ) .
the recombinant ppv vp2-derived vlps were found to be stable when lyophilized and stored at 20c longer than a year and vlps remained intact when resolubilized in pbs as no pentamers or disrupted particles were observed by electron microscopy ( figure 2(b ) ) .
moreover , the elisa results using freshly prepared and resolubilized ppv vp2 antigen were fully concordant ( data not shown ) .
the stability of vlps is crucial to ensure their successful transportation and possible application in the point - of - care tests .
therefore , it is successfully employed for the serodiagnostics as well as epidemiological studies of ppv infection .
moreover , vp2 protein is the major agent for developing vaccines . to meet the need for stable recombinant vlps of ppv
, several expression systems were tested as an alternative to baculovirus expression system that is a major source of the antigen for the market .
e. coli , lactobacillus casei , and recently yeast pichia pastoris   were reported to have been successfully used for producing ppv vp2 protein , but vlp formation in these expression systems has not been confirmed . to our knowledge , our study provides the first evidence of stable recombinant ppv vp2 vlps not produced in baculovirus expression system . 

s. cerevisiae expression system has been shown to be efficient in producing antigenic vlps of diagnostic relevance of human parvoviruses [ 28 , 29 ] and porcine circovirus .
therefore , it represents a potential system to meet the need for vlp - forming antigens to detect and differentiate a number of newly discovered porcine .
ppv vp2 protein - derived vlps generated in s. cerevisiae were used to develop an indirect elisa for the detection of ppv - specific igg antibodies in swine serum specimens . in order to test the antigenic properties of yeast - derived vlps
, 187 serum samples were tested using ingezim ppv compact test as a gold standard and further retested with the newly developed indirect igg ppv elisa .
both assays were performed in parallel for every serum sample to determine the sensitivity and specificity of the new indirect igg ppv elisa .
the cut - off value for the new assay was calculated as the mean od value of the 39 negative sera ( identified with the commercial kit ) plus 2 standard deviations ( x-+2sd ) resulting in 95% confidence .
therefore , sera with od values above 0.330 were considered positive ( n = 129 ) and those with od value below this cut - off were assessed as negative ( n = 58 ) in the newly developed indirect igg ppv elisa .
thirty - eight out of the 39 sera tested as negative with a commercial kit were assessed as negative by the indirect igg ppv elisa .
nine out of 137 positive and all 11 doubtful serum samples by ingezim assay showed the od value below the cut - off in the indirect igg ppv elisa and were considered as negative ( table 1 ) .
thus , the calculated specificity and sensitivity for the new indirect igg ppv elisa were 97.4% ( 38/39 ) and 93.4% ( 128/137 ) , respectively .
all 9 false - negative samples of the new assay were weak positive in ingezim kit showing blocking percentage in the 3345% range .
all samples above bp equal to 30% were considered positive in this commercial kit . the only false - positive sample in the indirect igg ppv elisa showed od = 0.354 that is just above the cut - off od of 0.330 .
to obtain more precise estimation of the sensitivity and specificity of the new assay , additional evaluation with more serum samples and alternative assays must be done in the future .
alternatively , the precision of the test can be improved using other formats of elisa . in summary ,
results of the current study are promising to the use of ppv vp2 antigen synthesized in yeast s. cerevisiae in diagnostic kits .
purified recombinant ppv vp2 protein was used to immunize mice and generate ppv vp2-specific mabs . after screening and cloning of positive hybridoma clones ,
six mabs produced by hybridoma clones were of igg1 subtype and the remaining three were found to be of igg2a subtype .
all mabs reacted specifically with recombinant ppv vp2 protein in elisa and did not react with other yeast - expressed proteins used as a negative control ( table 2 ) . to characterize the nature of the epitopes recognized by the mabs , their reactivity in western blotting was analyzed .
the mabs 4f11 , 16g11 , 25c5 , 6d1 , and 10a7 recognized sds - denatured ppv vp2 protein in western blotting assay ( figure 3 , lanes 2 ) .
this result indicates that these mabs recognize sds - denatured epitopes of the ppv vp2 protein .
the other four mabs ( clones 1f8 , 16a1 , 22g2 , and 23a7 ) did not recognize sds - denatured ppv vp2 protein in western blotting assay ( data not shown ) , suggesting that these mabs recognize conformation - dependent epitopes .
the specificities of the mabs were further analyzed by ifa to verify the ability of the mabs to recognize native virion .
for this purpose , commercial porcine parvovirus control slides containing virus - infected and noninfected fixed cells were used .
none of the mabs reacted with noninfected cells , which confirms the specificity of the assay ( figure 4 , negative control ) .
both groups of mabs recognizing linear or conformational epitopes reacted with infected cells ; however , only the latter ones produced images with sharp nucleus - shaped patterns . in contrast , the mabs recognizing linear epitopes produced signal outside the nuclei but in lesser intensity ( figure 4 ) .
this difference could be explained by the possibility that ppv vlps finish their assembly in the nucleus forming conformational epitopes .
taking into consideration trimer translocation model for other parvoviruses , conformational epitopes might be available only in intact capsid but not in trimmers or pentamers formed by vp2 protein .
this possibility emphasizes the importance of properly assembled vlps to elicit strong immune responses when using recombinant antigens as potential vaccines .
further epitope mapping needs to be done to answer if linear epitopes remain accessible on the intact vlp surface or are hidden within the structure .
however , our generated mabs represent an attractive tool for studying intracellular ppv infection and capsid formation process .
the ppv vp2-derived vlps generated in s. cerevisiae have not been yet tested for a possible use as a vaccine in pigs ; however , considering results on the antigenic structure and the immunogenicity in mice described in this study , this is an attractive alternative to the currently used recombinant ppv vaccines . in previous studies ,
ppv vp2-derived vlps have been shown to be effective epitope carriers to elicit a strong immune response in mice [ 41 , 42 ] .
furthermore , yeast expression system does not require additional contaminant elimination procedure as described for baculovirus expression system   for such recombinant subunit vaccine preparation .
therefore , ppv vp2-derived vlps generated in yeast s. cerevisiae are a promising platform for new ppv vaccine development .
in this study , we have demonstrated that yeast s. cerevisiae is a suitable host for the production of recombinant ppv vp2 protein as stable immunogenic vlps .
the recombinant yeast - derived ppv vp2 protein can be employed in an indirect elisa for detection of ppv - specific igg antibodies in swine sera with high specificity and sensitivity .
the mabs raised against yeast - derived ppv vp2 vlps recognize virus - infected cells and differentiate conformational and linear epitopes of ppv vp2 protein .
 porcine parvovirus ( ppv ) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets . 
 the gene coding for the major capsid protein vp2 of ppv was amplified using viral nucleic acid extract from swine serum and inserted into yeast saccharomyces cerevisiae expression plasmid . 
 recombinant ppv vp2 protein was efficiently expressed in yeast and purified using density gradient centrifugation . 
 electron microscopy analysis of purified ppv vp2 protein revealed the self - assembly of virus - like particles ( vlps ) . 
 nine monoclonal antibodies ( mabs ) against the recombinant ppv vp2 protein were generated . 
 the specificity of the newly generated mabs was proven by immunofluorescence analysis of ppv - infected cells . 
 indirect igg elisa based on the recombinant vlps for detection of ppv - specific antibodies in swine sera was developed and evaluated . 
 the sensitivity and specificity of the new assay were found to be 93.4% and 97.4% , respectively . in conclusion 
 , yeast s. cerevisiae represents a promising expression system for generating recombinant ppv vp2 protein vlps of diagnostic relevance . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions