PCV3感染性克隆的构建与病毒拯救OA

Porcine circovirus type 3(PCV3)can cause porcine dermatitis and nephropathy syndrome and is closely related to various diseases such as reproductive disorders and multi-systemic inflammation in pigs,which hinders the healthy development of the pig breeding industry.Due to the difficulty in isolating PCV3,its pathogenic mechanism is poorly understood.In this study,PCV3 was rescued by reverse genetics technology and its replication and proliferation characteristics were analyzed.A pair of reverse primers were designed around the single internal restriction site HindⅢ of the PCV3 genome.The full-length PCV3 ge-nome with HindⅢ restriction sites and protective bases at both ends was obtained by PCR using the PCV3 genome extracted from positive tissues as a template.After treatment with restriction enzymes and DNA ligase,the linear genome was circularized to obtain the PCV3 infectious clone.After transfection into PK-15 cells for 48 hours,the cells were passaged and the replication and proliferation of PCV3 in the correspond-ing generations of cells were identified by PCR,Western blot and IFA.The PCR results showed that the full-length PCV3 genome was 2 000 bp and belonged to the 3a subtype.The rescued PCV3 could be pas-saged at least 45 times in the PK-15 cell line(P45),and no genomic sequence variation was detected from P0 to P40.Western blot and IFA results showed that the Cap protein was successfully detected in PK-15 cells infected with PCV3-P40.The virus proliferation curve showed that PCV3-P40 reached the peak titer(108 TCID50/mL)at 48 hours after infection,and then showed a significant downward trend.With the in-crease in the number of passages,the adhesion ability of PK-15 cells infected with PCV3 decreased signifi-cantly,accompanied by the abnormal accumulation of a large number of cell-like fragment-like particles.In conclusion,this study successfully established an in vitro culture system for PCV3,providing basic materi-als for in-depth exploration of the pathogenic molecular mechanism of PCV3 and the development of antivi-ral strategies.