[An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2]

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.