Genetic And Variation Analyses On CSFV E0 & E2 Gene In Parts Of China And Sequence & Analysis Of The Complete Genome Of Epidemic Strains

Classical swine fever (CSF) is a highly infectious disease caused by Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, caused huge economic lost every year to pigs breeding industry. some scholars consider the reason for immunization failure is that recent isolates have shifted away from HCLV isolates.In order to get hold of the genetic variation of CSFV and indicate CSFV prevailing strains molecular variety rule and molecular epidemiology background,we developed a study on CSFV E0 and E2 genetic and variation and determined the whole genomes of two epidemic strains of CSFV.The results followed as below:The major antigen coding regions of E2 genes were amplificated from 22 pathologic materials collected in 2008~2009 by RT-PCR, 22 gene fragments were acquired.Comparison and analysis of the epidemic strain with referenced strains and 56 sequences, which saved by China Animal Health and Epidemiology Center among 2005~2009, was performed and analyzed by using software such as DNAStar, Clustalx, and phylogenetic tree was drawn by Mega4.1.The results showing: the nucleartide and amino acid homology of E2 main antigen coding regions between 22 epidemic strain and HCLV stain is 78.4%~83.2% and 81.0%~88.9%, separately.Among the 78 epidemic strains, only one belongs to group 1.1 within group 1, 77 strains belong to group 2. Among the group 2 epidemic strains,74 strains belong to subgroup 2.1,accounting for 94.87% of the analysis;3 strains belong to subgroup 2.2,accounting for 3.85% of the analysis;None belongs to subgroup 2.3. There are some variations occur on the major antigen coding regions of E2 genes:(1) There are some variations occur on the sites related to immune evasion (aa729 and aa734). (2) There is a variation in the critical site of B cell epitope(aa772) of Strain SX10-08.(3) There is a variation in the Cys site (aa737),which is critical for maintaining the conformation of B/C antigen domaine.Using RT-PCR, the E0 gene sequences of 11 epidemic strains were acquired from the samples of infecting pigs tissue from 8 provinces in China, the result after analysising the sequence of the 11 strains display all of them belong to the subgroup 2.1,of which four strain belong to subgroup 2.1a.The homology of nucleotide and amino acid between epidemic strain and HCLV strain are 82.3%～84.3% and 86.4%～89.5%, between epidemic strain and Shimen stain are 83.3%～84.6%and 86.4%～91.2%. There is a substitute between DKNR (Q)G and DRNQG in four epidemic strains, former is the B cell epitope recognized by monoclonal antibody B4-22 and 24/16, whether this substitute may affect the antigenicity of these epidemic strains to be further explored in the research.According to the genome sequences of classical swine fever virus (CSFV) published in GenBank and the reference, eleven pairs of specific primers were synthesized.By using RT-PCT, the genome of a CSFV isolate SXCDK and HEBZ is gained after eleven fragments had been successfully amplified. The genome of CSFV isolate SXCDK or HEBZ, is 12 296nt in length.It is composed of a 373nt 5'terminal non-translated region (NTR), a 11 697nt open reading frame (ORF) encoding a polyprotein of 3 898 amino acids (aa), and a 226nt 3'NTR. From an evolutionary point of view, isolate SXCDK is closely related to Taiwan isolate 96TD, with a pairwise distance of 0.038, isolate HEBZ is closely related to Taiwan isolate TWN2004,with a pairwise distance of 0.035。Among the nine precisely located epitopes, four and five have difference in isolates SXCDK and HEBZ.Conclusion: Gene clustering results showed that prevailing strains of subgroup 2.1 in the group 2 are prevailing, group 1 gradually subsided in our country; there are some acid amino variations in some epitopes in the epidemic stains, whether these variations may affect the antigenicity of these epidemic strains to be further explored in the research. This research deteminated the whole genomes of two epidemic strains of 2009, SXCDK and HEBZ, isolate SXCDK is the first CSFV subgroup 2.1 strain whose genome sequence has been determined in the mainland of China.