Development of an indirect immunofluorescence assay for PCV3 antibody detection based on capsid protein

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.

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis

“…PCV-2 is often diagnosed based on the assessment of clinical signs in association with antigen or nucleic acid identification through tests-based immunofluorescence, hybridization in situ, immunoperoxidase, and PCR or quantitative PCR (qPCR), which are the gold-standard methods used for porcine circovirus 2 detection purposes [ 6 , 7 , 8 , 9 , 10 ]. A TaqMan rtPCR assay was recently developed to help detecting porcine circovirus 4.…”

Revisiting Porcine Circovirus Infection: Recent Insights and Its Significance in the Piggery Sector

“…Serological techniques such as indirect immunofluorescence assay (IIFA) [ 235 , 236 ] and enzyme-linked immunosorbent assay (ELISA) [ 235 , 237 ] have been developed and utilized for the detection of anti-PCV antibodies in pigs. However, such tests cannot be used for the diagnosis of a clinical disease, as antibodies against PCV2 or PCV3 may also be present in clinically healthy pigs.…”