Taxonomically, porcine circoviruses (PCV) can be classified in the genus Circovirus in the family Circoviriedae. PCVs are small, non-enveloped, single stranded circular DNA viruses [8]. So far, three PCV types, namely PCV1, PCV2, and PCV3 have been documented [3]. PCV1 is a cell culture-derived virus and is considered nonpathogenic for pigs. In contrast, PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVAD) which causes severe economic losses in the swine industry worldwide. Recently, PCV3 was reported in the United States of America, associated with porcine dermatitis, nephropathy syndrome and reproductive failure as well as cardiac and multisystemic inflammation [3, 4]. As a brand new porcine circovirus, PCV3 has been found to widely exist on pig farms in the United States, Poland, South Korea, and China using conventional PCR or real-time PCR techniques [1,2,3,4, 6]. However, a serological detection method, such as ELISA, has never been reported for PCV3 surveillance in China. In this study we established and validated an indirect enzyme-linked immunosorbent assay (ELISA) for PCV3. A national PCV3 serological survey was then conducted using the established ELISA.

An optimized PCV3 Cap gene (Genbank: KX778720.1) was synthesized and cloned into a PET-28a plasmid vector. The primers were designed to remove the nuclear signal sequence from the PCV3 Cap gene in order to generate a 205aa truncated, recombinant Cap protein (Supplementary Table 1). The recombinant plasmid was introduced into E. coli BL21 (DE3) cells by transformation according to the manufacturer’s instructions, and PCV3 Cap was expressed in E. coli, as previously described [7]. Soluble recombinant Cap protein in the supernatant was assessed by SDS-PAGE, followed by Western-blot using PCV3-specific positive porcine sera. The expected size of the PCV3 Cap recombinant protein, based on its calculated molecular weight, is 24kDa. The SDS-PAGE analysis of the recombinant Cap protein showed that a protein of expected molecular weight was being expressed (Fig. 1). The expression of the recombinant protein in the supernatant was further confirmed by Western-blot analysis with PCV3-specific positive serum (Fig. 1). After purification using Ni2+-NTA agorose beads, the purity of the recombinant Cap protein was shown to be > 90%. Based on the results of the checkerboard titration, the optimal antigen concentration for plate coating was 1µg/mL, with a serum dilution of 1:200 in a maximal P/N ratio (Table 1). Under these optimization conditions, the best secondary antibody dilution was found to be 1:5000.

Fig. 1
figure 1

SDS-PAGE (A) and western-blot (B) analysis of the PCV3 capsid protein expressed in E. coli. (A) SDS-PAGE for PCV3 capsid protein expression in E. coli. M, protein marker; lane 1, total protein without IPTG induction; lane 2, total protein after IPTG induction; lane 3, purified protein. (B) Western-blot for the PCV3 capsid protein expression without purification. M, protein marker; lane 1, total protein from E.coli transformed with the PET28a-vector; line 2, total protein for E.coli transformed with the PET28a-PCV3-Cap vector

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Table 1 Optical dilutions for coating antigen and antibody for ELISA
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To establish an indirect enzyme-linked immunosorbent assay (ELISA), the recombinant PCV3 Cap protein was purified using Ni2+-NTA agarose before being used as an antigen to detect anti-PCV3 Cap antibodies in sera. Briefly, 96-well microtitre plates were coated with recombinant PCV3 Cap protein in carbonate-bicarbonate buffer (pH-9.6) and incubated overnight at 4 °C. The plate was washed 3 times with PBST (PBS containing 0.05% of Tween 20) and blocked with 5% BSA for two hours at 37 °C. Serum samples were incubated for 30min at 37 °C. The wells were washed three times with PBST, followed by incubation for 15 min at 37 °C with HRP-labeled goat anti-pig IgG (Sigma-Aldrich, Shanghai, China). Wells were again washed 5 times with PBST and the reaction was developed by adding 100 µl of TMB in each well. After 15 min of incubation at room temperature, the reaction was stopped using 100 µl of 2N H2SO4 and the OD was recorded at 450 nm using an ELISA plate reader. To optimize the PCV3 Cap ELISA, various concentrations of recombinant Cap protein and serially-diluted sera were determined by checkerboard titration. HRP-labeled secondary antibody was diluted to 1:2500, 1:5000, and 1:10000 in PBST and the best concentration was determined based on OD450 values and P/N ratios.

To determine the cutoff value for the established ELISA, 20 serum samples from cesarean-derived, colostrum-deprived (CD/CD) pigs were used as negative controls; they had an average absorbance of 0.292. The cutoff value differentiating positive and negative serum samples was determined as 3 standard deviations above the mean of the negative controls. Therefore, the threshold of the ELISA was 0.292 + 3 × 0.006 = 0.31 and any serum sample with OD values at or above this cutoff was consider positive. To test the sensitivity of the PCV3 ELISA, two fold PCV3-postive serum dilutions starting at 1:100 were tested and P/N values were calculated. All serum samples were verified to be positive at a maximum dilution of 1:3200 which indicated the high sensitivity of the established ELISA. To determine specificity of the PCV3 ELISA, positive sera against PCV1, PCV2, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, as well as classical swine fever virus was tested with the established ELISA. The results showed that all of the above serum samples were negative with OD450 values below the cutoff value 0.31. This indicated that there were no serological cross-reactions with the aforementioned pathogens (Supplementary Table 2).

A retrospective serological survey was next conducted using this established ELISA for 1688 serum samples collected from 2013 to 2017 which were originally used for pseudorabies gB antibody evaluation. Detailed information for these serum samples is summarized in Supplementary Table 3. Eight hundred and forty out of the 1688 serum samples tested were PCV3-positive using this ELISA with absorbances ranging from 0.34 to 1.12. Positive serum samples were detected in 16 different provinces as shown in Fig. 2. According to the year of sera collection, PCV3 antibodies were detected from clinical serum samples as early as 2015 with a 22.35% positive rates (76/370 clinical samples). The PCV3 serological positive rates increased to 46.54% (215/462 clinical samples) in 2016 and 51.88% positive rates (152/293 clinical samples) in the first half of 2017.

Fig. 2
figure 2

Distribution of PCV3 antibody-positive provinces in China. The positive provinces are indicated with dark shading

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Since the first report in the United States, PCV3 has subsequently been reported in Poland, South Korea, and China using conventional or real-time PCR targeting viral genes [1,2,3,4, 6]. The first Chinese PCV3 isolated, designated PCV3-China/GD 2016 (Access No. KY418606, isolated in Guangdong province), was reported in March 2017 and shared 97.4%-98.5% genome similarity with US PCV3 strains [5]. It is imperative to conduct a nationwide survey to establish the current status of PCV3 infection in China. As a newly-emerging swine virus, positive results either through PCR or ELISA, are required since they indicate recent infection with PCV3. When compared to PCR, ELISA has more advantages because it requires less operation time/labor and has higher throughput and therefore capability to rapidly confirm the PCV3 infection status in pig populations. Therefore, in this study, we established an indirect ELISA using recombinant PCV3 Cap protein. The PCV3 ELISA showed good sensitivity to positive serum samples and had no cross-reaction with other swine viral pathogens including PCV1 and PCV2. A nationwide retrospective serological survey indicated PCV3 was first detected in 2015 and has spread widely in China since then.

PCV3 has been associated with porcine dermatitis, a nephropathy syndrome and reproductive failure as well as cardiac and multisystemic inflammation [3, 4]. However, PCV3 was also detected in healthy pigs without any clinical signs. It is imperative to establish an PCV3 infection model in pigs to evaluate its significance on the swine industry. So far, any efforts to isolate PCV3 on primary cells and cell lines have failed [3]. Therefore, successful virus isolation on cells will be the top priority for future PCV3 studies.