Expression Of Food-and-mouth Disease Virus VPI Antigenic Epitope Using Porcine Circovirus Type2as Vector

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is considered asone of the most important diseases of cloven-hoofed animals, this disease can quickly spread in avariety of species between animals. There are seven serotypes of FMDV (O, A, C, Asia1, South AfricaTerritories SAT1-3). Serotype O is the most prevalent serotype of FMDV in China. Vaccination is oneof the most valuable measures to control and eradicate this disease. FMDV VP1protein is one of themain protective antigens of the virus; a synthetic peptide of VP1protein can induce host protectiveimmuno-response. Porcine circovirus type2(PCV2) is associated with a number of diseases includepost-weaning multisystemic wasting syndrome (PMWS) collectively known as porcine circovirusdisease (PCVD). PCV2infection can destroy the immunity system of the pig, resulting in theimmuno-suppression. The genome of PCV2has two major open reading frames (ORFs), ORF2encodesthe capsid (Cap) protein is the only structural protein of this virus. PCV2Cap protein can self-assemblyinto virus-like particles (VLP); PCV2can be vectored as to show the exogenous epitope on virionsurface.In this paper, a recombinant porcine circovirus type2(PCV2) was constructed to expressing theneutralization epitope of type O FMDV (HM229661) of swine by fusion PCR, the neutralizationepitope region (141-160aa) of FMDV VP1protein was inserted into the C-terminus of the PCV2Capprotein to construct a recombinant PCV2in this study. The cloned virus strain termed asrecPCV2-CL-VP1was rescued by transfecting an infectious clone into PK-15cells. The recombinantvirus of recPCV2-CL-VP1was detected by an immonoperoxidase monolayer assay (IPMA), a captureenzyme linked immunosorbent assay (ELISA) and immuno-electron microscope. The VP1epitope wasdetermined in the recombinant virus by capture ELISA. Furthermore, there was no detectable differencein the antigenicity of the recombinant virus compared with the parental virus of PCV2-CL strain byIPMA. However, the recombinant virus recPCV2-CL-VP1could be differentiated from the parentalvirus by PCR and capture ELISA. The recombinant virus was similar to parental virus in morphologicalfeatures.The virus propagation was lower than that of the parental virus. The cloned virus couldpropagate stably in PK-15cells for ten passages with103.050%tissue culture infective dose(TCID50)/mL. BALB/c mice were inoculated with the recombinant or parental virus via the intranasaland intraperitoneal routes, then the propagation of the virus was detected and virus DNA in the internalorgans of the mice were quantitated by real-time quantitative PCR, furthermore, the anti-VP1antibodiesin serum of the BALB/c mice after repeating immunization live and inactivated recombinant virusrecPCV2-CL-VP1was also detected. IPMA was used to detect the level of PCV2-positve antibody inthe sera of BALB/c mice infected with the recombinant and parental virusesrespectively. During theinfection experiment, the viral load in lung, spleen and heart of BALB/c mice infected with therecombinant virus is significantly lower than that of the parental virus (PCV2-CL); however the viralload in the liver and kidney was obviously higher than the parental virus. Both of the parental andrecombinant viruses could induce higher titer anti-Cap antibodies, the maximum of IPMA antibody titer of the recPCV2-CL-VP1mice after a challenge was1:1600, while that was1:800in the parental viruschallenged group. Furthermore, repeated immunization of the recombinant virus mixed with Freund’sadjuvant could also induce anti-VP1epitope tag antibodieswhich was not detected in the parental virus.These results indicated that VP1epitope could be displayed on the surface of the capsid protein byinserting its gene just before stop codon of open reading frame2. More importantly, insertion of theVP1epitope did seem to interfere with biological characterization of the recPCV2-CL-VP1virus.In conclusion, the recombinant recPCV2-CL-VP1strain expressing FMDV VP1neutralizationepitope was successfully established, and the VP1epitope was displaying on the surface of the capsidprotein, providing foundation for the further development of engineering vaccine against both PCV2and FMDV.