Construction of a novel porcine circovirus type 2 infectious clone as a basis for the development of a PCV2 iDNA vaccine

Wang, Weicheng; Zeng, Zhiyong; Tang, Deyuan; Hai-ying, Liang; Zhao, Liang; ZhenJiang, Dai

doi:10.1016/j.jviromet.2015.04.005

Cited by 3 publications

(3 citation statements)

References 26 publications

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“…1), is replicated in a rolling circle manner [33], early studies constructed infectious clone of it through the ligation of two copies of PCV2 genomic DNA, thereby facilitating to enhance the replication feasibility and form continuous genome for progeny viruses [34]. In this study we used 1.1 copy of PCV2 genome including two in tandem PCV2 origins to generate infectious clones, as consistent with a recent report [35]. Importantly, rescued viruses were obtained and confirmed, as indicated by TEM observation and IFA assay.…”

Section: Discussionsupporting

confidence: 62%

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

Cai

Zhan³

et al. 2019

BMC Vet Res

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Background Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G 710 to A 710 ) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. Results Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID 50 ), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. Conclusions These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

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Section: Discussionsupporting

confidence: 62%

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

Cai

Zhan³

et al. 2019

BMC Vet Res

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“…DNA vaccine delivers immunogenic antigens to the host’s cells by using DNA plasmids as a vector, which is more stable and cheaper to obtain than RNA vaccine. There are several studies have tried to research PCV2 DNA vaccines, they inserted the PCV2 ORF2 gene into the eukaryotic expression vector pEGFP-N1 or pcDNA3.1, and then found the recombinant plasmids could induce an anti-PCV2 immune response and protect BALB/c mice or Kunming mice from PCV2 infection ( Sylla et al, 2014 ; Wang et al, 2015a ). However, the vaccine efficacy was limited, and more importantly, the pathogenicity of Cap was not been fully considered.…”

Section: Introductionmentioning

confidence: 99%

The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice

Shi²,

Wang³

et al. 2023

Front. Microbiol.

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Nowadays, vaccines are broadly used to prevent porcine circovirus type 2 (PCV2) infection-induced expenditures, but the virus is still spreading among pigs. The current PCV2 vaccines all rely on the immunogenicity of Cap, yet our previous studies found that Cap is also the major component mediating the PCV2 infection-induced immune suppression through its interaction with host gC1qR. Thereby, new vaccines are still necessary for PCV2 prevention and control. In this study, we constructed a new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap. We introduced the Intron A and WPRE elements into the vector to improve the Cap expression level, and fused the IL-2 secretory signal peptides to the N-terminal of Cap to mediate the secretion of Cap. We also screened and selected chemokines CXCL12, CCL22, and CCL25 to migrate dendritic cells. In addition, we contained the vectors with PEI and then ultrasonic them into nano size to enhance the entrance of the vectors. Finally, the animal experiments showed that the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap could induce stronger humoral and cellular immune responses than the PCV2 DNA vaccine expressing the wild-type Cap and the non-ultrasonic treated PCV2 DNA vaccine in mice, and protect the mice from PCV2 infection and lung lesions. The results indicate the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap has a certain development value, and provide new insight into the development of novel PCV2 vaccines.

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“…With the development of genetic engineering technology, a variety of virus strains have been constructed through reverse genetics (Yoo et al, 2004;Huang et al, 2011). With the continuous analysis of the genome structure and function of PCV2 and PCV3, researchers have obtained infectious clones by constructing eukaryotic expression vectors of PCV2 and PCV3 (Wang et al, 2015;Jiang H. et al, 2019). According to the genome structure of PCV, the porcine circovirus genome is a single-stranded negativestranded DNA, the virions are only 14-17 nm, and there is no capsule on the surface of the virus capsid (Mankertz et al, 2004;Xiao et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

A Novel Technique for Constructing Infectious Cloning of Type 3 Porcine Circovirus

Jiang

et al. 2020

Front. Microbiol.

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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.

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Construction of a novel porcine circovirus type 2 infectious clone as a basis for the development of a PCV2 iDNA vaccine

Cited by 3 publications

References 26 publications

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice

A Novel Technique for Constructing Infectious Cloning of Type 3 Porcine Circovirus

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