The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.
Animals ; Swine ; Circovirus/genetics* ; Vaccines, DNA/genetics* ; Replicon/genetics* ; Genetic Vectors/genetics* ; Plasmids/genetics*

Porcine circovirus type 2 (PCV2) can cause immunosuppression on herds. PCV2, as an essential pathogen of PCV2-systemic disease (PCV2-SD), has caused considerable economic losses in pig industry worldwide. Here we review and address the evolution, viral protein and immunolesion of PCV2 and preventive techniques of PCV2-SD.
Animals ; Circoviridae Infections ; veterinary ; Circovirus ; genetics ; Phylogeny ; Swine ; Swine Diseases ; virology ; Viral Proteins ; genetics

A pair of primers with BamH I restriction site were designed to amplify the complete genome of goose circovirus. Two copies of the genome were ligated in tandem and cloned into pGEM-T Easy vector to construct an infectious clone named as pGEMT-2GoCV. The pGEMT-2GoCV linearized with EcoR I was transfected to negative embryos and gosling with Lipfectamine. PCR detection verified the proliferation of GoCV in geese. Some sera of the embryo transfected group were detected to be positive at 2 and 4 weeks after hatching and one bursa was detected to be positive at 4 weeks. Some sera of the gosling transfected group were also detected to be positive at 2 weeks after transfection. Furthermore, the mark in the PCR products were identified by BamH I digestion and the GoCV in positive tissue and sera were quantitated by Real-time PCR. The results showed that the virus load in positive bursa was 1.57 x 10(6) copies/mg, the virus load in positive sera were 3.52 x 10(4)-5.92 x 10(5) copies/microL. In conclusion, the infectious DNA clone constructed with two copies of full-length GoCV genome in tandem can transfect embryo and gosling and propagate the marked goose circovirus.
Animals ; Circovirus ; genetics ; Geese ; virology ; Real-Time Polymerase Chain Reaction ; Transfection

To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.
Animals ; Baculoviridae ; genetics ; metabolism ; Circovirus ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Insecta ; cytology ; metabolism ; Open Reading Frames ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Swine ; Viral Proteins ; genetics ; metabolism ; Virion

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals ; Cells, Cultured ; Circovirus ; Interferon Type I/genetics* ; Macrophages, Alveolar/virology* ; Membrane Proteins/metabolism* ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

ORF1 and ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 (AY035820). Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector plECMV (deleted partial gE and gI of pseudorabies virus) to generate recombinant transfer plasmid pIEORF1-ORF2. The genomic DNA of PRV TK-/gE- /LacZ+ strain and pIEORF1-ORF2 were co-transfected into IBRS-2 cells with lipofectin, and recombinant virus TK- /gE- /gI- /ORF1-ORF2+ was selected by PCR with ORF1 gene and ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF1 and ORF2 gene of PCV2 had been inserted into the genome of TK- /gE- /LacZ+ strain and the expressed ORF1-ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed the insertion of ORF1 and ORF2 gene did not influence propagation of recombinant virus.
Animals ; Cell Line ; Circovirus ; classification ; genetics ; Gene Transfer Techniques ; Genes, Viral ; Herpesvirus 1, Suid ; genetics ; Open Reading Frames ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Swine

Pigeon circovrius (PiCV) is a member of circovirus, which is usually regarded as an immunosuppression agent. There were reports that pigeons infected by PiCV showed symptoms of lethargy, weight loss, vomiting, diarrhea, respiratory distress, etc. In this study, we established a PCR method for the detection of PiCV DNA. Samples from 5 different farms in Zhejiang Province were examined and samples from a farm in Hangzhou were positive. Furthermore, the genomic segments of 2 strains of PiCV were amplified, cloned and sequenced using designed primers and the complete genomes of the strains were then assembled and named as PiCV-zj1 and PiCV-zj2, respectively. The sequences were deposited in GenBank under the GenBank Accession number of DQ090945 and DQ090944, respectively. Sequence Analysis had shown that the complete genomes of 2 strains of PiCV from Zhejiang Province had 2 039 nucleotides totally in length and common characters of circovirus such as a stem-loop structure and conserved motifs for Rep protein, which were supposed to be related to the replication of the virus. Pairwise comparisons showed that the nucleotide sequence of the genome of PiCV strains from Zhejiang Province had 86%-89.1% identities to that of 11 published PiCV strains, and that the amino acid identities of the replication-associated protein (Rep) and capsid protein (Cap) displayed 92.1%-94.7% and 76.6%-81.4%, respectively. A phylogenic tree was built using PHYLIP with bootstrap support for 1 000 replicates. The result showed that 10 strains from Europe and America formed one big branch and the others from Zhejiang Province and Australia formed the other two, respectively. This was the first report on the detection and full genome sequencing of PiCV in China.
Animals ; Base Sequence ; Circoviridae Infections ; virology ; Circovirus ; classification ; genetics ; Cloning, Molecular ; Columbidae ; virology ; Genome, Viral ; genetics ; Models, Genetic ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Viral Proteins ; genetics

The Cap gene of antisense strand of circovirus has the most variation of the genome, and encodes a capsid protein which has the main immunogenicity. The N-terminal of capsid protein makes up of nuclear localization signal which is involved with virus location. This review summarizes the research advance of Cap gene of circovirus in the sequence characteristics, its encoding capsid protein, basic functions of the capsid protein and its interaction with MKRN1 protein, Hsp40 protein, receptor protein gClqR and complement factor C1qB protein. This paper lays a theory foundation for the further study of the capsid protein in the aspects of viral attachment, replication and transportation.
Animals ; Capsid Proteins ; genetics ; immunology ; metabolism ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; genetics ; immunology ; Genetic Variation ; Genome, Viral ; genetics ; Nuclear Localization Signals ; Protein Binding ; Virus Replication

To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up to 10(8.5) TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 10(7) TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.
Adenoviruses, Human ; genetics ; Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Circovirus ; immunology ; Defective Viruses ; genetics ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; Virus Replication

Infectious clone is a useful tool in exploring viral replication and pathogenesis. In order to prevent linear PCV2 cyclization, PCR mutagenesis was used to construct the first molecular clone (pSK-2PCV2) by ligating two copies of the complete PCV2 genome with the pBluescript SK (pSK) vector. In addition, pSK-PCV2 and ds-PCV2 were constructed. PK-15 cells were transfected with above three infectious clones. Indirect immunofluorescence assay (IFA) revealed that the virus antigen mainly localized in infected cell nucleolus and cytoplasm. PCV2 specific nucleotide fragment in cell culture was amplified by RT-PCR. Typical porcine circovirus particles with diameter about 17 nm were also observed by transmission electron microscope (TEM) in the infected cells. The rescued virus sequences from the cultures had 100% homology with the inserting PCV2 genome. The rescued virus shared similar properties with that of the parental virus. The study establishes a platform for further research on the virus molecular biology and pathogenicity.
Animals ; Cell Line ; Circoviridae Infections ; virology ; Circovirus ; genetics ; growth & development ; pathogenicity ; physiology ; Cloning, Molecular ; DNA, Viral ; genetics ; physiology ; Recombination, Genetic ; genetics ; Swine ; Transfection ; Virulence ; Virus Replication