Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

Abstract

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003-2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.

Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China

“…Sequence data is available in GenBank as listed in Table 3. Details of their molecular phylogenetic relationships have been described elsewhere [10,26]. …”

“…The plasmids containing full-length E2 gene of the vaccine C-strain and eight subgroup 2.1 strains used in this study were previously described [10,26]. The C-strain specific primer sets C-E2-AD-f/C-E2-AD-r and C-E2-BC-f/C-E2-BC-r were used to amplify the fragment covering the two antigenic units (B/C+A/D) and the fragment only containing antigenic unit B/C, respectively.…”

“…Genetic diversity of E2 among different groups has been extensively studied [4,10,26-29]. The N-terminal half of E2 is more variable than the C-terminal half [10], suggesting that the antigenic units could be under positive selection apparently due to constant exposure to high immunologic pressure.…”

Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences

“…CSFV can be divided into three genotypes (1, 2 and 3), each comprising three or four subgenotypes (1.1-1.3; 2.1-2.3; 3.1-3.4) [7,8]. To assign a newly identified CSFV isolate to a genotype and to describe its phylogenetic relations to other known isolates, nucleotide sequencing is mandatory as other techniques like restriction enzyme analysis may allow segregation on genotype level, but resolution on subgenotype level is often insufficient [7,9-11]. Within the highly variable genus Pestivirus (single stranded positive-sense RNA viruses) CSFV is the least variable member [7].…”

Complete Genome Sequences of Classical Swine Fever Virus Isolates Belonging to a New Subgenotype, 2.1c, from Hunan Province, China

“…CSFV subgenotype 2.1 has been further divided into 2.1a and 2.1b (3, 4). In mainland China, several genotypes of CSFV strains (1.1, 2.1, 2.2 and 2.3) are largely responsible for CSF outbreaks (5), and CSFVs of subgenotype 2.1b have predominated since the 1990s (6, 7, 8). During 1989–2003 in Taiwan, a dramatic switch from subgroup 3.4 to 2.1 was observed, with subgroup 2.1a viruses predominating from 1995 onwards (4).…”