Figure 3A The five-year occurrence frequency of residues in 226th and 228th sites from <1995 to 2016-2020.Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT-PCR. Data information: , n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the bone marrow (C) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the spleen (D), and footpad (E) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The survival rates of mice (F) were recorded daily. Data information: In (F), n = 10 biological replicates. In (F), the data were analyzed statistically using the log-rank (Mantel-Cox) test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquito infectivity was determined by qRT-PCR at 8 days after the blood meal. The number at the top of each column represents the infected number/total number (H). Data information: In (H one dot represents one mosquito, and the horizontal lines represent the median of the results. In (H, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and blood samples were collected for DENV2 viremia detection by qRT-PCR (J). Data information: In (J), n = 4 biological replicates. In J), the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test J). The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 (K Data information: In K, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 L). The number at the top of each column represents the infected number/total number (L). Data information: In L), one dot represents one mosquito, and the horizontal lines represent the median of the results. In , L), the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).