Rescue and identification of recombinant Porcine Circovirus Type 3

Abstract PCV3 is prevalent and causes many forms of swine diseases worldwide. To date, PCV3 isolation has been unsuccessful. Therefore, obtaining PCV3 and studying its biological traits are urgently needed. In the present study, recombinant PCV3 (rPCV3) was successfully generated, and it’s biologically characterization was performed. The genome sequence of PCV3 was optimized, cloned and inserted into the pBluescript SK vector. PK-15 cells transfected with the recombinant plasmid were serially passaged and characterized. The obtained rPCV3 was purified through sucrose density gradient centrifugation and ion exchange chromatography and observed via Transmission Electron Microscope (TEM). Absolute qPCR was used to determine PCV3 viral load. PK-15 cells were treated with nocodazole to determine the relationship between rPCV3 proliferation and mitosis. Especially, PK-15 cell infected with rPCV3 was compared with that infected with PCV3 positive tissues (wPCV3). Specific fluorescence in the nuclei, brownish-red puncta on cell monolayers, and target bands in NC membrane were observed in transfected PK-15 cells. TEM showed that the particle diameter of rPCV3 was approximately 20 nm. rPCV3 was continuously passaged for up to 25 passages with a progressive decrease in viral load. Nocodazole experiments demonstrated that rPCV3 proliferation was dependent on cell mitosis. Cytoplasmic fluorescence was observed whether the cells were infected with rPCV3 or wPCV3; importantly, copy numbers decreased in a time-dependent manner. Our study is the first to observe PCV3 particles via TEM. We revealed that both rPCV3 and wPCV3 cannot enter the PK-15 cell nucleus, which may explain why virus isolation was unsuccessful in these cells.