Abstract
Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome (PDNS), reproductive failure, and multisystemic inflammation. Capsid protein (Cap) encoded by PCV3 ORF2 gene has been identified as an immunogenic protein. Currently, there is no immunofluorescence assay (IFA) available for serological diagnosis. Here, the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal (NLS). Two types of recombinant plasmids were constructed for recombinant protein expression in Sf9 cells by using a baculovirus expression system: plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence. Expression of the nuclear localization sequences was further analyzed by IFA. Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells. No cross-reactivity was observed with porcine circovirus type 2, porcine pseudorabies virus, classical swine fever virus, or porcine reproductive and respiratory syndrome virus. In summary, we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies. This method is suitable for the diagnosis and epidemiological investigation of PCV3. This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.
Highlights
Growth stage PigletsPorcine circovirus type 3 (PCV3) antibody positivity rateNursery 37.31% 25/67Fattening 37.62% 38/101Sow 59.35% 73/123Total 45.83% 143/312 was synthesized (GenScript Biotech Corp, Nanjing, China) and cloned into the pFastBac1 vector (Invitrogen, Thermo Fisher Scientific, Inc., Carlsbad, USA)
ELISA has been used to test for positive porcine serum samples (Deng et al 2018; Xu et al 2018) while PCV3-Capsid protein (Cap) protein-based indirect ELISA is the only method for PCV3 antibody detection (Deng et al 2018)
Verification of baculovirus-expressed PCV3 Cap proteins A previous study established indirect immunofluorescence assay (IFA) to detect PCV2 serum antibodies by cloning PCV2 ORF2 gene into the eukaryotic expression vector pCEP5, and results were determined by observing the presence or absence of a green fluorescence signal in nucleus or perinucleus (Racine et al 2004)
Summary
Growth stage PigletsPCV3 antibody positivity rateNursery 37.31% 25/67Fattening 37.62% 38/101Sow 59.35% 73/123Total 45.83% 143/312 was synthesized (GenScript Biotech Corp, Nanjing, China) and cloned into the pFastBac1 vector (Invitrogen, Thermo Fisher Scientific, Inc., Carlsbad, USA). ELISA has been used to test for positive porcine serum samples (Deng et al 2018; Xu et al 2018) while PCV3-Cap protein-based indirect ELISA is the only method for PCV3 antibody detection (Deng et al 2018).
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