Rescue Of The Infectious Virus From PCV2CC12Cloned Genomic DNA And Mouse Infection Model

Porcine circovirus diseases（PCVD）is a PCV2-associated disease entities and oneof the severe infectious diseases affecting pig industry worldwide. This infection iscaused by porcine circovirus type2, a member of genus Circovirus within the familyof Circoviridae, and characterized by weight loss or wasting, pallor of the skin,respiratory distress, diarrhea, icterus, reproductive failure and enlarged subcutaneouslymph nodes.In this study, a novel porcine circovirus2(PCV2) strain, designated as PCV2CC12,was detected and isolated from pigs with clinical signs of postweaning multisystemicwasting syndrome(PMWS) in Changchun. Sequence alignment analysis revealed aunique mutation at position of nucleotide607(607A>G), leading to a transition ofamino acid from Asparagine to Serine at position of186(N186S) in the Rep protein.In addition, two rare mutations were found in the Cap protein, where Arginine issubstituted by Lysine (R59K) and Alanine is replaced by Threonine (A190T)compared with known PCV2genomic sequences in GenBank. Phylogenetic analysisclustered the CC12strain to a clade of PCV2b, a predominant genotype consisting ofthe majority of PCV2strains from Northeast provinces in China.Recombinant plasmids with one copy or two tandem repeat of the full-lengthgenome sequences of CC12were generated by inserting PCV2genome sequence intopBluescript II SK vector and used to transfect PK15cells in an attempt to rescue theinfectious virion. After8thblind passages, PCV2CC12genome or virus antigen wasdetected by PCR, immunoperoxidase monolayer assay (IPMA), immunofluorescentassay (IFA) and Western blot, indicating the CC12virus was successfully rescued.The rescued CC12was named rCC12. Characterization of TCID50and biological property of rCC12showed no significant difference with the parental CC12virus,thus establishing a stable CC12infectious clones system with the potentials for futureinvestigation on the mechanism of virus replication, viral pathogenesis and interactionbetween virus and host cells.Mouse model for PCV2CC12infection was established by inoculating virusintraperitoneally. Virus distribution, histopathological lesions and antibody changewere determined at different time points postinoculation. Immunohistochemistryrevealed PCV2antigens were mainly distributed in the parachymal organs like liver,kidney, spleen, lymph node, heart, lung and thymus during early infection, while theantigens were detected mainly in lymph nodes at35days postinoculation (DPI).Infiltration of inflammationary cells were found in liver, lung, spleen, lymph node andthymus. Enzyme-linked immunosorbent assay showed PCV2-specific antibody wasdetected as early as7DPI, reached peak level at28DPI, and began to decline at35DPI. The mouse model established will be used for future investigation on themechanism of virus pathogenesis and immune response.