Development of an RT-PCR-RFLP assay for the detection and differentiation of wild-type and vaccine strains of classical swine fever virus

Abstract

An RT-PCR-RFLP assay was developed to detect and differentiate between wild-type classical swine fever virus (CSFV) and rabbit attenuated vaccine CSFV. A pair of specific primers was designed based on the Shimen strain and was used to verify the efficacy of this assay. Eight epidemic wild-type strains isolated from 20 pathology samples obtained from pigs that were suspected to have swine fever and two vaccine strains were amplified by RT-PCR and were cloned and sequenced. These experiments produced a fragment of 825bp that was amplified from the genomic RNA of wild-type CSFV, and this fragment could be digested with Apa I into two fragments, of 322 and 503bp, when analyzed with RFLP. In contrast, the vaccine strain could not be digested with Apa I. The lowest concentration of RNA that could be detected using this assay was 0.02μg/ml. The eight epidemic wild-type strains contained the Apa I recognition sequence GGGCCC, whereas the two vaccine strains contained the sequence GAGCCC. The eight epidemic wild-type strains belonged to Group 2. In contrast, the two vaccine strains with a close genetic relationship to HCLV belonged to Group 1. Eight common wild-type strains and 22 reference strains carry a highly conserved sequence in the E0 protein, providing its RNase activity (EWNKHGWC). In addition, the B-cell epitope TWFGAYA is highly conserved across these strains, and the B-cell epitope DKN of SD05 becomes DNN. The RT-PCR-RFLP assay developed in the present study can be used to detect and differentiate wild-type CSFV from the attenuated vaccine strain in pigs suspected of being infected with CSFV.