Abstract
Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the HindIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.
Highlights
Porcine circovirus has long harmed the sound development of pig husbandry, and especially the Porcine circovirus type 3 (PCV3) that has appeared in recent years has caused tremendous economic losses for pig husbandry (Li and Tian, 2017; Ouyang et al, 2019)
The PCV3 infectious clone was achieved by self-cyclization of the genomic DNA followed digestion of recombinant genomic DNA by HindIII, gel extraction and ligation at the created HindIII site by T4 DNA ligase (Figure 2A)
The identified cyclized PCV3 DNA was transfected into the 3D4/21 cell line with Lipofectamine 3000 (Invitrogen) in accordance with the manufacturer’s protocol
Summary
Porcine circovirus has long harmed the sound development of pig husbandry, and especially the PCV3 that has appeared in recent years has caused tremendous economic losses for pig husbandry (Li and Tian, 2017; Ouyang et al, 2019). Current research indicates that PCV is mainly divided into three genotypes (Ge et al, 2018). PCV1 is generally considered non-pathogenic (Tischer et al, 1986). PCV2 is widely prevalent worldwide, previous studies have shown that PCV2 is the main pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) and swine dermatitis and nephrotic syndrome (PDNS) (An et al, 2007; Welti et al, 2012). In recent years, some researchers have detected PCV3 from PDNS piglets (Palinski et al, 2017; Kedkovid et al, 2018).
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