title: Construction and biological characterisation of recombinant porcine circovirus type 2 expressing the V5 epitope tag.
abstract: Porcine circovirus type 2 (PCV2) is a major causal agent of post-weaning multisystemic wasting syndrome in piglets. To investigate the feasibility of PCV2 expressing an exogenous epitope, a 14-amino-acid V5 epitope derived from simian parainfluenza virus type 5, was inserted into the C terminus of the capsid protein. Recombinant PCV2 expressing the V5 epitope, recPCV2/CL-V5, was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA), a serum neutralisation assay (SNA), a capture enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The V5 epitope was detected in the recombinant marker virus by IPMA and capture ELISA. Furthermore, there was no detectable difference in the antigenicity of the recombinant marker virus compared with the parental virus by IPMA and SNA using PCV2-positive serum and the neutralising monoclonal antibody 1D2. However, recPCV2/CL-V5 marker virus could be differentiated from the parental virus by PCR, IPMA and capture ELISA. The recombinant marker virus was stable on multiplication through 10 passages in PK-15 cells, with a maximum titre of 10(6.25) 50% tissue culture infective dose (TCID(50))/ml. BALB/c mice were inoculated with the recombinant or parental virus via the intranasal and intraperitoneal routes. The parental and recombinant viruses both could replicate in mice, cause microscopic pathological changes, and induce mice to generate anti-PCV2 antibodies. Furthermore, the recombinant marker virus could also induce anti-V5 epitope tag antibodies. These results indicated that V5 epitope could be displayed on the surface of the capsid protein by inserting its gene just before stop codon of open reading frame 2. More importantly, insertion of the V5 epitope did not seem to interfere with biological characterisation of the recPCV2/CL-V5 marker virus.
authors.affiliation: Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Huang
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Lu
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Wei
authors.forename: Longjun
authors.initials: L
authors.lastname: Guo
authors.forename: Hongli
authors.initials: H
authors.lastname: Wu
authors.forename: Feiyan
authors.initials: F
authors.lastname: Zhang
authors.forename: Yujie
authors.initials: Y
authors.lastname: Fu
authors.forename: Changming
authors.initials: C
authors.lastname: Liu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology; D017925:Circovirus / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005260:Female; D015870:Gene Expression; D051379:Mice; D008807:Mice, Inbred BALB C; D018184:Paramyxoviridae Infections / Q000235:genetics / Q000276:immunology; D053570:Porcine Postweaning Multisystemic Wasting Syndrome / Q000276:immunology / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013552:Swine; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins