Table 1.
Summary of the patient and infection characteristics.
Fig 1.
Phylogenetic analysis of DENV-2 study sequences.
The maximum-likelihood tree was constructed based on the complete coding sequences generated during this study and those retrieved from GenBank. Sequences of only 37 isolates, mainly comprising of three groups of isolates (NS2A-V83I, n = 15; NS2A-T119N, n = 6; E-V164I+NS2A-L153S, n = 9), are shown in the tree. Viral sequences obtained from patients with DF (mild) and DHF/DSS (severe) manifestations are highlighted in green and red respectively. An enlarged view has been inserted to illustrate the two mutant groups (NS2A-V83I and NS2A-T119N) that were exclusively observed in patients with mild disease (DF) (highlighted in green boxes). Figures on branches are bootstrap values. Only bootstrap values more than 90% are shown on the major nodes.
Table 2.
Distribution of amino acid substitutions within DENV-2 cosmopolitan clade III cohort.
Fig 2.
Median joining network analysis and amino acid substitution map of study sequences.
The median joining network was drawn in Network version 4.6.1.2 [28] using complete coding sequences (10173 nucleotides) of DENV-2 cosmopolitan clade III sequences (n = 89). Circles represent either individual isolates or clusters. The diameter of each circle is proportional to the number of isolates within each circle. The length of lines linking circles is not proportional to the mutational distance between them. The empty nodes represent hypothetical ancestral strains or strains present in the population but not sampled. Different colors indicate clinical categories as shown in the figure legend. As the analysis only included clade III sequences, mutations were stated as compared to wild-type clade III sequences (Table 2). Only amino acid substitutions associated with groups of isolates (n>6) are mapped. DF = dengue fever; DHF = dengue hemorrhagic fever; DSS = dengue shock syndrome
Table 3.
Association between substitutions and the severe clinical outcome (DHF/DSS).
Fig 3.
Comparison of RNA secondary structures of the variable region of 3’UTR between wild type (NS2A-V83) and mutant (NS2A-V83I) variants.
The first (5’ end) 120 nucleotides of 3’UTR was used to draw the secondary structure as predicted by the MFOLD web server [31]. Position 1 indicates the first nucleotide of 3’UTR. (A). Wild type (NS2A-V83) DENV-2 cosmopolitan clade III (B). Mutant (NS2A-V83I) DENV-2 cosmopolitan clade III. The mutated position (19T) in Stem Loop-I (SL-I) is indicated with an asterisk. Stem Loop structures (I-III) were named as illustrated elsewhere [68].
Fig 4.
Comparison of RNA secondary structures of the 5’UTR between wild type (NS2A-V83) and mutant (NS2A-V83I) variants.
The complete sequence (96 nucleotides) of 5’UTR was used to draw the secondary structure as predicted by the MFOLD web server [31]. Position 1 indicates the first nucleotide of 5’UTR. (A). Wild type (NS2A-V83) DENV-2 cosmopolitan clade III (B). Mutant (NS2A-V83I) DENV-2 cosmopolitan clade III. The mutated position (34C) in the top loop of Stem Loop A (SLA) is indicated with an asterisk. Stem Loop structures were named as illustrated elsewhere [64].