Abstract
BackgroundPorcine circovirus type 2 (PCV2) 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.ResultsIn this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 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 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.ConclusionsWe have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 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 PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.
Highlights
Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases
Three open reading frame 2 (ORF2) variants amplified by PCR from native PCV2 genomes: variant 622 (GenBank: KJ128269), variant 521 (GenBank: KJ128273) and variant 113 (GenBank: KJ128274) as well as ORF2 of the most representative PCV2-Cap gene variant 622 which was codon-optimized by S. cerevisiae codon usage (GenBank: KJ128275) were cloned into pFX7 expression vector [28] and used for the expression in yeast S. cerevisiae
We purified three slightly different PCV2 Cap proteins variants encoded by native Cap-encoding sequences and confirmed that they all formed homogenous in size and shape virus-like particles (VLPs) similar to native virions or VLPs produced in baculovirus expression system [14]
Summary
Porcine circovirus type 2 (PCV2) 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. It was shown that PCV2 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 are responsible for developing of PCVAD [9]
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