Molecular Mechanisms Of Cell Entry Of Classical Swine Fever Virus And Molecular Epidemiological Investigation

Classical swine fever virus(CSFV)belongs to the genus Pestivirus within the family Flaviviridae.It is a small,enveloped virus with a positive,single-stranded RNA genome.The pig is the only natural host of this virus.The infected pigs are characteristic of classical swine fever(CSF),a highly contagious infectious disease which is a huge threat to the pig industry in China,and it is listed as Class A infectious diseases in our country.Even though immune prevention can be effective in controlling infection,there is no specific antiviral drugs against CSFV and culling causes huge economic losses.Under the pressure of the vaccine immune,mutation and evolution of CSFV often occur.The viral life cycle includes adsorb with host cell,internalization,intracellular localization,strip shell,copy,and packaging.Any step of the process is interfered,the proliferation of the virus will be inhibited.It is a hot point in the research of antiviral drugs designed according to the mechanism of virus entry.The purpose of this study is to discuss cell entry of CSFV through a series of experiments.It will be contribute to control CSF.The main work of this study is as follow:1.Small molecule inhibitors influence cell entry of CSFVAcridine orange staining showed 10 mM NH4C1 and 100 μM chloroquine could significantly increased the acidity of the endosomes.The copy numbers of CSFV genome reduced to 6.6%and 31.6%in 25 μM chloroquine and 10 mM NH4C1 treated cells respectively,suggesting that CSFV entered PK-15 cells in a low pH dependent manner.The copy numbers of CSFV genome in the supernatant and the cell culture reduced to 0.14%and 39.6%in 5 mM MβCD and 5 μg/ml of filipin pretreated cells,respectively.The copy numbers of CSFV genome reduced to 9.0%and 6.7%after treatment of 10 mM MβCD for 12 h and 36 h on cells infected by CSFV for 24 h,respectively.This result suggested that cholesterol on the cell membrane probably played a role in CSFV endocytosis and budding.The copy numbers of CSFV genome reduced to 4.97%and 32.2%in cells pretreated with 50 μM of chlorpromazine and 160μM dynasore,respectively.The results of JEV as the positive control were consistent with those in former studies which indicated that CSFV requires clathrin and dynamin to enter the PK-15 cells.2.CSFV entry into the PK-15 cell through the clathrin-mediated endocytosisIFA showed overexpression of wild type Eps 15 protein(DIII△2)and wild-type dynamin II(dynamin WT)did not affect the CSFV infection,but overexpression of dominant negative mutant of both Eps 15 protein(E△95-295)and dynamin II(dynamin DN)caused CSFV infection failure in cells.Compared to those in cells that overexpressed dynamin WT,the genome copy numbers reduced to 51.3%for CSFV and 42.8%JEV in cells that overexpressed dynamin DN.Compared to that in the control group,the gene copy numbers of CSFV were reduced to 61.2%in the supernant and 67.8%in the whole cells after being interfered by clathrin.Through the confocal laser scanning microscope,it was observed that the virus infection reduced with lower expression of the clathrin.In the early stage of virus entry,clathrin could be colocalized with both CSFV and JEV.Those results showed that CSFV enters PK-15 cells through the clathrin-mediated endocytosis.3.Molecular epidemiological investigation of classical swine fever virus in some areas of Eastern ChinaPhylogenetic analysis of partial classical genes:5’-NTR,E2,NS5B and whole length of E2indicated that 3 epidemic strains belonged to the 2.1b subgroup.Whole length homology analysis of gene E2 among the 3 prevalent strains and the vaccine strain(HCLV,GenBank,No:AF531433)showed 81.9-82.1%nucleotide homology and 88.5-88.7%amino acids homology with 49 amino acid mutations including 25 of them located in the antigenic determinant region(690-866);there were no mutations in 15 cysteine sites deciding protein space structure and 4 conserved sites for B cell epitopes which decision protein space structure and four conserved B cell epitope;among 7 glycosylation sites,only the 90th amino acid had mutation.All the 88th amino acid mutated to Ser.Our investigation provides theoretical data for molecular epidemics in Eastern China.