Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay

Abstract

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

Induction of a Cellular DNA Damage Response by Porcine Circovirus Type 2 Facilitates Viral Replication and Mediates Apoptotic Responses

“…3A). We also used PCV2 virus-like particles (VLPs)37 (Fig. 3B) to inoculate into PK15 monolayer cells and collected the 48 h PCV2 VLP-inoculated lysates for determination of the DDR activation.…”

“…PCV2 VLPs were prepared as described by Zhang et al 37. and inoculated into PK15 monolayer cells for 48 h for determination of DNA damage responses activation.…”

Self-assembly into virus–like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

“…The enzyme-linked immunosorbent assay (ELISA) based on VLPs is widely used to measure antibodies or the neutralizing epitopes (Almanza et al 2008;Chao et al 2019). PCV2 VLPs as coating antigens can detect serum neutralizing antibodies (SNAbs) in ELISA (Nainys et al 2014;Zhang et al 2016). Some reports show that PCV3 infection in pigs do not present any significant clinical signs or symptoms and wild boars may also have susceptibility (Franzo et al 2018b;Klaumann et al 2019;Zheng et al 2017), thus it is particularly important to establish an effective antibody detection method to assess the PCV3 infection in pigs.…”

“…The enzyme-linked immunosorbent assay (ELISA) based on VLPs is widely used to measure antibodies or neutralizing epitopes (Almanza et al 2008;Chao et al 2019). For example, PCV2 VLPs are used as coating antigens to be able to detect serum neutralizing antibodies (SNAbs) in ELISA (Nainys et al 2014;Zhang et al 2016). Currently, PCV3 Cap proteins harvested from insect cells or E. coli have been used as antigens to detect PCV3-specific antibodies in an ELISA (Deng et al 2018;Zhang et al 2019).…”

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome

“…Sf9 cells were cultured in Sf-900 II SFM insect media (Thermo Fisher Scientific, USA) at 27 °C and the Bac-to-Bac ® Baculovirus Expression System was used to prepare recombinant baculovirus according to the manufacturer's instructions (Thermo Fisher Scientific, USA). PCV2 VLPs were prepared from E. coli in our laboratory (Zhang et al, 2016), and monoclonal antibody was produced as described previously (Walker et al, 2000).…”