Construction And Biological Study Of Chimeric Porcine Circovirus 1-2 With A KT3 Signature

The primary causative agent of porcine circovirus associated disease (PCVAD) is porcine circovirus 2 (PCV2), which is associated with postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproductive failure, and others. PCV2 have had a serious economic impact on the global swine industry along with its pandemic in the world.It has been shown that vaccination is an effective method to reduce PCVAD losses. The antibody level activited by vaccine carrying a tractable marker could be availably monitored by immuno-serological method in swine populations with subclinical infection of PCV2, to examine whether effective immune response have been stimulated.In this study, infectious DNA clone of recombinant PCVI-2 KT3 was constructed, and generated the rescued virus. Then, we preliminarily evaluate its safety and immunogenicity in susceptible piglets.1. Construction of infectious DNA clones of chimeric porcine circovirus (PCV) 1-2 KT3In this study, short epitope tags were inserted into the C terminus of the Cap of the PCV1-2 genome by overlap extension PCR, generating the plasmid pBSK(+)-PCV1-2 KT3 Kpn Ⅰ. Then the plasmid T-PCV1-2 KT3 BstE Ⅱ and the infectious DNA clones pBSK(+)-PCV1-2 KT3 EKS (EKS, including two ori of PCV) and pBKS(+)-PCV1-2 KT3 PEK (PEK) (including one ori of PCV) were constructed.The virus PCV 1-2 KT3 could be rescued by the transfection of EKS 、 PEK and self-ligated DNA of PCV 1-2 KT3 genome into PCVs-free Dulac cells, or by the cotransfection of pBSK(+)-PCV1-2 KT3 Kpn I and T-PCV1-2 KT3 BstE II into cells mentioned above, although the efficiency varies transfection efficiency varies with different methods developed above, chimeric porcine circoviruses with a KT3 signature were eventually rescued. After subsequent serial passages, the replication titers of the rescued viruses ranged from 1048 to 105.0 TCIDso/mL.2. Preliminary study on the feasibility of the KT3-tagged PCV1-2 viruses as a vaccine to distinguish infected from vaccinated animals (DIVA vaccine)A total of 8 pigs were randomly assigned into three groups,3 pigs in group 1 were vaccinated with 105.0 50% tissue culture infective dose (TCID50) of chimeric PCV1-2 KT3 virus.3 pigs in group 2 were similarly vaccinated with 105 5.0 TCID50 of chimeric PCV1-2 live vaccine. And 2 pigs in group 3 were not vaccinated and served as a mock group. Blood, nasal swabs, rectal swabs were collected weekly before and after vaccination. On 49 days post inoculation (dpi), necropsies were performed. The result showed that pigs vaccinated with the chimeric PCV1-2 KT3 virus developed high titers of PCV2 fluorescent antibody and neutralizing antibody, with which could be coincided by anti-KT3 antibody within the whole period of the experiment. PCV1-2 KT3 virus was detected at low levels of viral load in vaccinated piglets, and did not cause histopathological lesions in these animals. In conclusion, PCV1-2 KT3 virus shows satisfactory safety and immunogenicity, preliminarily proving the feasibility of the KT3-tagged viruse as a live-attenuated vaccine to distinguish infected from vaccinated animals (DIVA vaccine).