Molecular Epidemiological Studies of Classical Swine Fever Viruses in Taiwan

Classical swine fever (CSF) is a highly contagious viral disease of swine caused by classical swine fever virus (CSFV). Phylogenetic analysis of CSFV field isolates are useful to trace the geographic origins of the disease and to understand the distribution of CSFV genotypes. Two envelope glycoprotein (Erns and E2) regions of CSFV were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced directly from 167 specimens collected between 1989 and 2007 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and, furthermore, the Erns region provided better discrimination of CSFV genotypes than the E2 region. Of the 167 isolates collected, 124 were clustered within subgroups 2.1 and 2.2, which were considered to be potential exotic strains, whereas the remaining 43 isolates were clustered within subgroup 3.4, which is considered to contain the historical strains. Since the subgroup 2.1 could be further separated into two different clusters with high bootstrap values of 98% and 85% in the Erns tree, we proposed that subgroup 2.1 should be further classified as 2.1a and 2.1b. The subgroup 2.1a viruses were introduced to Taiwan in 1994 and caused CSF outbreaks in 1995, and then predominated in the field onwards. However, the subgroup 3.4 viruses were prevalent in Taiwan prior to 1996 and seemed to disappear from the field since it could not be isolated from the field thereafter. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a in the last two decades. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (containing 2,385 bp) displayed better resolution than that based on the complete open reading frame gene (containing 11,691 bp). The ALD strain showed cluster together with the GPE- vaccine virus only in the complete envelope glycoprotein gene tree. Two exotic viruses with the genotypes 2.1a and 2.1b were selected as challenging candidates to evaluate the protective efficacy of the LPC vaccine. SPF pigs were vaccinated with various dosages (1, 1/10, and 1/100 doses) of LPC vaccine and challenged with a CSFV exotic strains, either from genotype 2.1a or from genotype 2.1b. The results demonstrated that the LPC vaccine that is currently used in Taiwan could provide full protection against these two exotic CSFVs. Live attenuated vaccine strains of CSFV can persist in the tonsils and lymph nodes of piglets for a long period of time after immunization. Routinely, RT-PCR followed by DNA sequencing has been the method used to detect CSFV and excludes the interference of vaccine viruses in field cases. Herein, a simple one-step RT-PCR method was developed, based on T-rich insertions in the viral genome, for simultaneous detection and differentiation of wild-type and vaccine strains of CSFV. The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3'' nontranslated regions (3'' NTR) of the genome of lapinized CSFV vaccine strains. Using a one-step RT-PCR or a semi-nested RT-PCR followed by agarose gel or multi-capillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least three attenuated lapinized vaccine strains, LPC (lapinized Philippines Coronel), HCLV (hog cholera lapinized virus), and C (Chinese)-strain. The detection limit for the wild-type virus was 6.3 TCID50 (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID50/ml for semi-nested RT-PCR. In previous studies, notable T-rich insertions of 12–13 nucleotides (nts) were found in the 3'' NTR of the genome of CSFV lapinized vaccine strains. However, this study discovered that two T-rich insertions of 42 and 36 nts are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nts increases the size of PCR fragments, which are thus simple genetic markers for the rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV. This study also developed a DNA chip to enable simultaneous detection, genotyping, and differentiation between wild-type and vaccine-type CSFV. One-step RT-PCR amplification was performed with biotin-labeled primers, followed by hybridization to the DNA probe immobilized on the plastic chips. The DNA chip can not only accurately differentiate between the three major genotypes of CSFV, but can also discriminate between the wild-type and vaccine-type CSFV. The limit of detection for the wild-type virus was 10 TCID50/ml for RT-PCR and 1 TCID50/ml for the DNA chips. The sensitivity of the visual DNA chip was 10 times higher than that of the RT-PCR, as confirmed by agarose gel. The RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping CSFV and discriminating between vaccine and wild-type CSFV in clinical samples.