Spatiotemporal Dynamics and Epistatic Interaction Sites in Dengue Virus Type 1: A Comprehensive Sequence-Based Analysis

<div><p>The continuing threat of dengue fever necessitates a comprehensive characterisation of its epidemiological trends. Phylogenetic and recombination events were reconstructed based on 100 worldwide dengue virus (DENV) type 1 genome sequences with an outgroup (prototypes of DENV2-4). The phylodynamic characteristics and site-specific variation were then analysed using data without the outgroup. Five genotypes (GI-GV) and a ladder-like structure with short terminal branch topology were observed in this study. Apparently, the transmission of DENV1 was geographically random before gradual localising with human activity as GI-GIII in South Asia, GIV in the South Pacific, and GV in the Americas. Genotypes IV and V have recently shown higher population densities compared to older genotypes. All codon regions and all tree branches were skewed toward a negative selection, which indicated that their variation was restricted by protein function. Notably, multi-epistatic interaction sites were found in both PrM 221 and NS3 1730. Recombination events accumulated in regions E, NS3-NS4A, and particularly in region NS5. The estimated coevolution pattern also highlights the need for further study of the biological role of protein PrM 221 and NS3 1730. The recent transmission of emergent GV sublineages into Central America and Europe mandates closely monitoring of genotype interaction and succession.</p> </div

Estimated site-specific variation.

<p>The entropy-based variability over sites of (A) 10217 nucleotides and (B) 3392 amino acid residues was analysed using the DAMBE program. X axis: (A) nucleotide position; (B) amino acid position. Y axis: entropy value. Window sizes of 20 and 1 were used in the nucleotide and aa analyses, respectively. (C) Site-specific selection detection. Normalised dN-dS values were plotted for each codon site. (D) Epistatic interaction among DENV-1 codon sequences. In the above graph depicting the codon regions, each square node represents a residue position in the DENV1 codon sequence that participates in at least one interaction. The arrows (edges) representing those interactions are annotated with the fraction of graphs sampled in the chain sample that contains the edge. The analysis reports edges with marginal posterior probabilities (PP) exceeding a default cutoff of 0.5. These data are shown as PP{→}/PP{↔}/PP{←} beside the arrow, which indicates the direction. Both (C) and (D) were performed using the HyPhy software package. (E) Comparison of predicted three-dimensional residue substitution structures in (Left) PrM and (Right) NS3 proteins. The Mochizuki strain, a prototype DENV-1 strain, was used as a template for structural comparison. The wild-type (green) and substitution variant (blue) structures in the figure are aligned and oriented such that the substitution sites are positioned at the top. The substitution sites are highlighted in darker colours. The side chains of substitutions in the PrM¹⁰⁷ and NS3²⁵⁵ are shown. For each substitution, the magnified view (closed box on the right) is oriented for a clear depiction of conformational change due to substitution. An additional magnified surface view of NS3²⁵⁵ is shown on the lower right.</p

Phylodynamic analysis based on 100 DENV1 genome sequences.

<p>(A) Maximum clade credibility (MCC) tree based on 10316 nt gap-stripped MSA, which was constructed using the BMCMC method with BEAST program. The tree shows the proportional relationship between branch length and time; the dashed line below is the scale bar for genetic distance. Each branch thickness indicates the state probability and is colour coded to indicate the most probable locations. Blue bars at nodes indicate 95% highest probability density (HPD). For major lineages, the bootstrap (BS) values and posterior probability (PP) values for the key nodes are indicated as in BS-NJ/BS-ML above and as in PP-MrBayes/PP-BEAST below. Genotypes and subgenotypes are indicated on the right. The thick solid line indicates the median estimates, and the grey area displays the 95% HPD. (B) Overview of geographic dispersal of DENV-1 obtained by SPREAD software (worldmap available at <u>Central </u><u>Intelligence </u><u>Agency</u>). (C) Comparison of Bayesian skyline plot between DENV-1 and each genotype. The <i>x</i>-axis is the time-scale in years, and the <i>y</i>-axis is a logarithmic scale of <i>N</i>e<i>τ</i> (where Ne is the effective population size and τ is the generation time).</p

Additional file 1: of AIDS-related opportunistic illnesses and early initiation of HIV care remain critical in the contemporary HAART era: a retrospective cohort study in Taiwan

Table S1. The spectrum of 394 AIDS-related opportunistic illnesses and the distribution of the median CD4+ lymphocyte. (DOCX 20 kb

Probability of Survival in Patients on ART who Transfer Out and who do not Transfer Out.

<p>Probability of Survival in Patients on ART who Transfer Out and who do not Transfer Out.</p

Specificity and epitope of flavivirus GR mAb DEN2-12.

<p>(A) Layout of the dot blot assay. (B) Binding specificity was examined by Western blot analysis as described in Methods. Lysates of 293T cells transfected with pCB-D1 (D1 tr) were also included. (C) Dot blot assay using lysates from 293T cells transfected with the WT pCB-D1 or each of the 67 alanine E mutants. Each membrane was probed with mAb DEN2-12 or mixed mAbs (a pool of mAbs recognizing different epitopes). The dots containing mutations in domains I, II and III are underlined by red, yellow and blue lines, respectively; the ID of each dot is shown in panel A. Two-fold dilutions of the WT lysates were dotted on row 1 to assess the exposure of each membrane. Arrows indicate mutants of epitope residues, which showed severe reduction (R.I.≤0.3) in binding by dot blot assay. One representative experiment of two was shown. (D) The relative intensities of WT dots in row 1 showed a linear decrease from 1× to 1∶16 dilution for membranes probed with mixed mAbs (open bar) and mAb DEN2-12 (closed bar). (E) The intensities of each dot were quantified to determine the R.I. as described in Methods. Data are means and standard errors of two experiments. (F) Capture ELISA was performed by using WT or mutant VLPs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Crill3" target="_blank">[28]</a>, which had severe reduction in binding by dot blot and Western blot analyses. Data are means and standard errors of duplicates from one representative experiment of two. Lower graph shows the amounts of mutant VLPs added, which were not less than that of WT. (G) Structure-based analysis by UCSF chimera program to determine the locations of and distance (°A) between epitope residues from the same (shaded) or adjacent monomer.</p

Summary of the specificity, binding to alanine E mutants and PRNT₅₀ of 12 mAbs.

<p>*Classes of mAbs include GR (group-reactive), CR (complex-reactive), sCR (subcomplex-reactive) and TS (type-specific). Specificity was determined by Western blot (WB) analysis using lysates derived form DENV1, 2, 3, 4 or JEV-infected C6/36 cells as described in Methods.</p><p>**Alanine E mutants with consistent reduction in binding by dot blot and WB analyses are shown. ++: severe reduction in binding (reduction in R.I.≥70%);</p><p>+: moderate reduction in binding (50%≤reduction in R.I.<70%); underlined: reduced binding tested by VLP-capture ELISA.</p>†<p>Different regions (strand, loop, etc.) of domains II and III are defined based on the X-ray structure of DENV E protein <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Modis1" target="_blank">[7]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Modis2" target="_blank">[24]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Shrestha1" target="_blank">[33]</a>.</p>‡<p>PRNT₅₀ was presented as the lowest concentration that inhibited ≥50% of plaques <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Chen1" target="_blank">[38]</a>.</p

Location of epitope residues on E protein recognized by potent neutralizing mAbs.

<p>Epitope residues of mAbs against DENV1 (A) and DENV2 (B). Epitope residues are highlighted with dark blue (DENV1 residues), purple (DENV2 residues), or magenta (DENV1 and DENV2 residues at the same position). Top view of E-E dimers (upper) and side view of domain III (lower right) with ribbon presentation of ß-strands and loops (lower left) are shown by the program UCSF chimera.</p

Epitope mapping of GR mAb DEN4-4.

<p>The results of (A, B, C) dot blot assay, (D) VLP-capture ELISA, and (E) structure based analysis of the locations of and distance (°A) between epitope residues from the same or adjacent monomer are presented as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd-0001447-g001" target="_blank">Figure 1</a>.</p

Summary of predominant epitopes recognized by anti-E Abs in human sera after DENV infection.

<p>*Primary or secondary infection was determined by PRNT₅₀ as described in Methods.</p><p>**DF, dengue fever according to WHO case definition <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-World1" target="_blank">[2]</a>.</p>†<p>Predominant epitopes recognized by anti-E Abs in polyclonal human sera were identified by dot blot assay and residues with severe (reduction in R.I.≥70%) or moderate (50%≤reduction in R.I.<70%, shown in parenthesis) impairment in binding were shown.</p