Genomic Sequencing Analysis And Pathogenicity Test Of Csfv In Guangxi

Clinical tissue samples of suspected classical swine fever (CSF) locally collected from Guangxi districts were primarily detected using RT-PCR techniques and positive samples were inoculated in PK-15and cultured for virus isolation. Two strains respectively named GXGP03and GXLC15were isolated and their genomes were cloned and sequenced resulting in12295bp and12296bp in length. Analysis of two isolates compared with HCLV, Shi men, Paderborn, GXWZ02strains, revealed that the homology was84.2%～85.5%with HCLV and Shimen,94.2%～94.9%with Paderborn and GXWZ02, respectively. Phylogenetic analysis revealed that GXGP03and GXLC15belonged to subgroup2.1and kept up a close relationship with Paberbron and GXWZ02but not HCLV and Shimen strains. Phylogeneties made by Erns gene, NS3gene, NS4B gene and NS5A gene were similar to phylogeneties of three genes (full-length sequences, E2and NS5B). This suggests that these genes may be the basis of the potential gene for genetic sub-grouping. Analysis of the3’UTR sequences of GXGP03and GXLC15compared with HCLV, India vaccine strain, Shimen, Shimen-HVRI, Paderborn and GXWZ02revealed14bases deletion in GXGP03and GXLC15likely virulent strains and moderate virulent strains.Comparison of amino acid sequences of Erns and E2showed that Erns and E2glycosylation were different in number. The relationship which may exist between this glycosylation and the virulence need further more researches. The results found at the position of476amino acid in GXGP03and GXLC15were Ser which means that GXLC15and GXGP03growth in PK-15cells didn’t depend on the acetyl heparan sulfate.Animal experiment proved that the GXLC15and GXGP03strain had an obvious pathogenicity in pigs. Hemorrhages of the skin, diarrhea and high fever were observed on the challenged pigs. In postmortem examination we could observe pathological changes in lymph nodes with swollen, edematous and hemorrhages; spleen and kidney with hemorrhages; petechiae could also be observed in the urinary bladder. Secretions, excretions and serum of the challenged pigs were later analyzed by real-time quantitative PCR in which viremia could be detected at day6post infections (d. p. i), but in the secretions and excretions virus could be detected at6-10d. p. i. The animal experimental suggested a closely relationship of the virulence with the changes in leukocyte, body temperature, clinical symptoms and virus excretion. Leukopenia started at4d.p. i and the period of rapid increasing in antibody titers was at4-10d. p.i with exceeding antibody titer of1:1024. Leukopenia and increased antibody titers were observed before fever, viremia and hemorrhages occur. Histopathological changes were more severe in spleen and lung of the challenged pigs. According to the clinical symptoms, pathological changes observed in animal experiment joint with molecular biological characteristics, we could conclude that GXLC15and GXGP03isolates were moderate virulent strains causing a subacute disease in pigs.