title: Dengue type four viruses with E-Glu345Lys adaptive mutation from MRC-5 cells induce low viremia but elicit potent neutralizing antibodies in rhesus monkeys.
abstract: Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Hsiao-Han
authors: HH
authors: Lin
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Hsiang-Chi
authors: HC
authors: Lee
authors: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors: Xiao-Feng
authors: XF
authors: Li
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Meng-Ju
authors: MJ
authors: Tsai
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Hung-Ju
authors: HJ
authors: Hsiao
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Jia-Guan
authors: JG
authors: Peng
authors: Institute of Bioinformatics and Structural Biology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Shih-Che
authors: SC
authors: Sue
authors: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors: Cheng-Feng
authors: CF
authors: Qin
authors: Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan.
authors: Suh-Chin
authors: SC
authors: Wu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D003715:Dengue / Q000276:immunology* / Q000401:mortality / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology*; D004195:Disease Models, Animal; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D007114:Immunization; D008253:Macaca mulatta; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D014766:Viremia; D014779:Virus Replication / Q000187:drug effects
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D014759:Viral Envelope Proteins; C466981:glycoprotein E, dengue virus type 4; D006493:Heparin