Fig 3. Dengue MTase mutants are attenuated and immunogenic in mice.

A) Kinetics of MTase mutants and WT viremia in vivo. Mice were infected i.p. with 1×10⁴ PFU of WT virus or a mix of 1×10⁴ PFU of each MTase mutant (total of 4×10⁴ PFU). Viral titers in the plasma were measured at indicated time points by serotype-specific real-time RT-PCR. No virus was detected for WT and MTase mutant for DENV-3 and DENV-4, respectively. B) IgG titers of immunized mice. Blood was taken 30 days post immunization and total IgG antibody titers against DENV-1-4 were measured by UV-inactivated DENV particle ELISA. C) Neutralizing antibody (nAB) titers of imunized mice. Blood was taken 30 days post immunization and nAB against all four DENV serotypes were measured by flow cytometry-based neutralization assay. D) 30 days post immunization mice were challenged with DENV-1 (1x10⁶ PFU of strain 08K3126), DENV-2 (1x10⁷ PFU of strain D2Y98P), DENV-3 (1.5x10⁶ PFU of strain VN32/96) or DENV-4 (3x10⁶ PFU of strain TVP360). Viremia after challenge was measured by real-time PCR on days 1, 3, 5, and 7 post challenge. Naïve mice challenged with DENV-2 succumbed at day 4 post infection. Data are representative of three experiments with a total of 9–13 mice (A, B) or two experiments with a total of 6–9 mice per group (C, D). Shown are means with SD for all panels. Statistical analysis was performed using student's t-test (B, C, D), **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05.