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Background: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). Methods: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. Results: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. Conclusion: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine. [ABSTRACT FROM AUTHOR]

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance. [ABSTRACT FROM AUTHOR]

• We obtained PK-15 cell clones with different permissivity to PCV2 infection. • We constructed a transposon vector overexpressing pig interferon-gamma fused with IgG Fc domain. • We generated two PK-15 cell lines stably expressing IFNg-Fc fusion protein. • We demonstrated that the two cell lines can be used for PCV2 vaccine production. Porcine circovirus 2 (PCV2)-associated diseases affect the swine industry worldwide. Vaccination is the major tool for the disease control, but the vaccine production is hindered by lower propagation rate of PCV2 in vitro. Previous studies showed that interferons (IFNs) can increase PCV2 yield in PK-15 cells. In the present study, we constructed a Sleepy Beauty (SB) transposon vector expressing porcine IFNg gene fused with the coding sequence for immunoglobulin G Fc domain. After dilution cloning, the transposon and transposase vectors were co-transfected into PK-15 cell clones with higher permissivity to PCV2 infection. Two transgenic PK-15 cell lines, namely PK15-IFNgRan and PK15-IFNgSB which contained randomly integrated transfer vector or SB cassette without selection marker, were screened by PCR analysis. The characterization results demonstrated that the two transgenic cell lines can stably express IFNg-Fc fusion protein with potent antiviral activities. Both viral titration and quantitative PCR analyses showed that the two transgenic cell lines are highly permissive to PCV2 infection with significantly increased viral yields. These results indicate that the two transgenic PK-15 cell lines, PK15-IFNgSB in particular, can be used for PCV2 vaccine development. [ABSTRACT FROM AUTHOR]