title: Dengue type 4 live-attenuated vaccine viruses passaged in vero cells affect genetic stability and dengue-induced hemorrhaging in mice.
abstract: Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3' NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q(438)H, E-V(463)L, NS2B-Q(78)H, and NS2B-A(113)T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Hsiang-Chi
authors: HC
authors: Lee
authors: Yu-Ting
authors: YT
authors: Yen
authors: Wen-Yu
authors: WY
authors: Chen
authors: Betty A
authors: BA
authors: Wu-Hsieh
authors: Suh-Chin
authors: SC
authors: Wu
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D005814:Genes, Viral / Q000235:genetics; D006470:Hemorrhage / Q000276:immunology* / Q000821:virology*; D006801:Humans; D007121:Immunocompetence / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000235:genetics; D012692:Serial Passage; D019595:Severe Dengue / Q000276:immunology* / Q000517:prevention & control / Q000821:virology*; D014613:Vaccines, Attenuated / Q000276:immunology*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014774:Virulence / Q000235:genetics / Q000276:immunology
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D009711:Nucleotides; D014613:Vaccines, Attenuated; D017361:Viral Nonstructural Proteins