Study On The Characteristics Of Chinese Dengue Type â…¢ And Type â…£ Virus Isolates And Dengue â…£ Virus Attenuated

Ban18strain was isolated in1981in the region of Xishuangbanna. Ban18strains and theinternational standard strain H241were passaged in Vero cells and suckling mouse brain respectivelyand virus titer was determined using plaque assay in Vero cell monolayer. The results showed thatboth two virus isolates had damaging effects on the Vero cells and visible plaques were detected. Thevirus titers were2.0×105PFU/mL and5.1×107PFU/mL respectively. Primers were designedaccording to the whole genome sequences that have been submitted to GenBank. Complete genomesequence of the two isolates were determined and analyzed. Results indicated that the whole genomesequences of the two strains were both10664nt in length and contained one open reading frame,which encoded3387amino acids. Nucleotide homology and amino acid homology between Ban18strain and H241strain were99.7%and99.4%, with6nucleotide and3amino acid differences. E genesequences were used for genotyping, and the two strains both belonged to genotype Ⅰ. Dengue type4virus Ban18strain and its primary hamster kidney cell passagedattenuated strain, Ban18HK20strain and Ban18HK30strain, are passaged in Vero cell.Both biological and molecular characteristics are studied in this paper. Results showedthat three viruse strains can be effectively neutralized by dengue-4specific neutralizingantibody, the neutralization index>1000, indicating the three virus strains were denguetype4virus. The Ban18strain produced small and clear plaque while Ban18HK20strainand Ban18HK30strain produced larger but fuzzy plaques in Vero cell.Virulence of thethree strains were tested in9~11g mice and the lg(PFU/LD50) value of the two passagedstrains were>4.5, significantly higher than that of Ban18strain. And both the passagedstrains were proved to be attenuated in2~4day suckling mice, showed that the Ban18strain was gradually attenuated during passaging. Mice were vaccinated with106PFU/mLand challenged with H241strain two weeks after immunization. The protection index ofBan18and Ban18HK20strains were about100, while Ban18HK30had no protect effect,indicating that the immunogenicity of Ban18strain and Ban18HK20strain was higherthan that of Ban18HK30strain. Ban18HK20strain and Ban18HK30strain were plaquepurified and then selected9and5plaque clone respectively. Plaque morphology andvirulence in suckling mice of all the plaque clones were evaluated. The results indicatedthat3of9clones of Ban18HK20strain were less virulent, while all Ban18HK30plaqueclones showed no difference in virulent compared with their uncloned parental viruses.The full length sequence of Ban18strain, Ban18HK20strain and Ban18HK30strain were determined. Results showed that there existed8nucleotide differences,3ofwhich induced amino acid mutation, between Ban18and Ban18HK20strains. Theamino acid changes at C111, E155, E369and3’UTR219are considered to be closelyrelated to virulence. Two dengue type3virus strains, GD-1strain and GD-11strain, were isolated fromHepu county, Guangxi province in1980. Two strains were passaged twice in NIHsuckling mice brain and virus titer were both3×103PFU/mL. The DEN-3internationalstandard strain H87strain was passaged in suckling mice brain and virus titer was2.6×105PFU/mL. Plaque morphology showed obvious difference between GD-1, GD-11andH87strain. Complete genome were sequenced and analyzed, results showed that thecomplete genome of the two strain were both10707nt. Nucleotide homology betweenH87strain and the two isolates were both96.0%, while amino acid homology were96.0%. There existed4different amino acid sites between GD-1and GD-11strain.Different amino acid sites between GD-1, GD-11and H87strain were39and41respectively. Secondary structures of3’UTR were predicted and the differences weresignificant. Phylogenetic tree were constructed according to E protein sequences.Phylogenetic tree showed that GD-1strain and GD-11strain both belonged to subtypeⅡ, and were both closest with two Thailand isolates.