Content

Dengue virus biology
How to use the Dengue virus resource
Data overview

Dengue virus biology

Classification

positive strand ssRNA virus

Ecology and epidemiology

The only known hosts for Dengue virus are humans and non-human primates as well as the mosquito transmission vector ( Aedes species). An estimated 2.5 billion people living in tropical and sub-tropical regions, mostly in small and large cities, are at risk of contracting Dengue virus infections. Of these, about 50 to 100 million are estimated to become infected every year, up to 500,000 develop dengue hemorrhagic fever and about 20,000 deaths occur, with children bearing a disproportionate amount of the disease burden [WHO estimates]. The number of reported cases and affected countries has been rising for the past 50 years (see graph below based on WHO statistics for 1951 - 2005 obtained from WHO DengueNet). Dengue cases reported worldwide 1951-2005

Disease

Dengue virus can cause dengue fever (DF) and dengue hemorrhagic fever (DHF). DF typically has a incubation time of 4 to 7 days, after which there is a rapid onset of fever and other non- specific symptoms such as headaches. The fever may be short (1 to 2 days), prolonged (6 to 7 days), or biphasic. Initial symptoms are followed by severe muscle and joint pain (break-bone fever), nausea, vomiting and weakness. About half of the patients show some skin involvement such as flushed faces early in infection or a petechial or maculopapular rash during defervesence.

DHF is a more severe form of disease in which there is increased vascular leakage and thrombocytopenia. DHF cases are graded as I-IV based on severity and grades III and IV are dengue shock syndrome (DSS). DHF case fatality is less than 0.5% under appropriate care in a hospital, but may be as high as 10% under suboptimal treatment conditions.

Immunity and vaccines

Dengue virus infection results in life-long immunity against re-infection with the same serotype and short-live protection against heterotypic infections. However, the immune response also appears to play a major role in the pathogenesis of DHF and DSS, which are more common in heterotypic secondary infections (antibody-dependent enhancement of disease). Therefore, a vaccine has to induce immunity against all 4 serotypes. Clinical and pre-clinical tests of vaccine candidates are ongoing, but none have finished development yet.

Variability

There are four Dengue virus serotypes (1, 2, 3, 4) and each appears to be capable of causing the full spectrum of Dengue virus disease. However, there are some indications that Dengue virus strains may vary in pathogenicity.

Virion structure

Small enveloped virions (approx. 50 nm) with two membrane proteins (M, E) surrounding a (+)ssRNA genome of ~11kb complexed with capsid proteins (C).

Replication

Enveloped viruses enter via receptor-mediated endocytosis. Low pH induced fusion leads to the release of the genomic RNA into the cytoplasm where it is translated into a polyprotein that is processed into mature structural and non-structural (NS) proteins. Replication proceeds via a (-) strand full length intermediate synthesized by the RNA dependent RNA polymerase (RdRp)-containing replicase in cytoplasmic replication complexes near perinuclear membranes. Progeny virions assemble at intracellular membranes through budding and then proceed through the secretory pathway.

Proteins

Structural proteins

Capsid (C): Basic protein involved in packaging the viral genome into a nucleocapsid core.Capsid is found in two forms: membrane anchord capsid (anchC) can be cleaved by the host cell peptidase to yield the mature capsid protein.
M protein (M): Membrane glycoprotein expressed as a precursor (prM). prM is cleaved during particle maturation.
E protein (E): Membrane glycoprotein containing receptor binding site and fusion peptide.

Non-structural proteins

NS1: Unknown function; may play a role in RNA replication. Cell surface and secreted forms exists as well.
NS2A: May be part of replication complex.
NS2B: Serine protease co-factor.
NS3: Serine protease; RNA helicase.
NS4A: Unknown function.
2K: Unknown function; this fragment is removed from the N terminus of NS4B by the host signal peptidase.
NS4B: Unknown function.
NS5: RNA dependent RNA polymerase, methyl transferase, guanylyl transferase.

Using the Dengue Virus Resource

Query Builder

Select the sequence type [Protein, Coding region, or Nucleotide]
Select a single value from each list or fill in the optional text boxes to build your query. Note that not all fields are populated for all sequences. Available fields:
1. Type [1,2,3,4]
2. Disease severity [Dengue fever, Dengue hemorrhagic fever, Dengue shock syndrome]
3. Country/Region of sampling
4. Region of the genome that the sequences should span [From, To]
5. Collection year [From, To]
6. Sequence substring
7. Complete sequence only. These are sequences that have been selected as complete or nearly complete genomes. This selection applies to all queries in the Query Builder table.
Hit "Add to Query Builder". All fields will be combined by "AND". For example "Type 1 AND Disease DF AND From UTR5 to M". A line with the details of your query as well as the number of sequences matching it will be added to the Query Builder. Multiple queries can be build in this way.
Queries can be (de)selected with the checkmark at the beginning of each Query Builder line. The [X] button deletes selected queries, while the "Get sequences" button will open a new window with a detailed view of the sequence results.

Results view

Sequences in the results view can be sorted by up to three fields in decending or ascending order.
Add additional sequences in fasta format with the "Add your own sequences"
Sequences can be (de)selected with the checkmark in the first column of the table
For the selected sequences one can
1. retrieve sequences in fasta format (either the nucleotide, CDS, or protein sequence). Fasta format headers include the sequence identifier and a string identifying type and isolation country.
2. retrieve a list of accessions
3. Calculate a multiple alignment (with MUSCLE) or a tree based on the multiple alignment. This step is only allowed if all sequences span the same region of the genome. Due to the amount of data, alignments are pre-calculated and are currently only available for protein sequences. Alignments are clipped to the genome region selected in the Query Builder.

Multiple alignment view

The consensus and variability of the alignment is shown on the top
Residues identical to the consensus are denoted by dots. Gaps are denoted by dashes.
Invariant positions are highlighted in blue
Clicking on any sequence shows a popup that provides links to
1. view the selected sequence in GenBank.
2. select the sequence for a pairwise alignment with BLAST 2 seq.
3. select the sequence as the new anchor sequence, replacing the consensus.
Alignments can be viewed in a print friendly version or downloaded in fasta format
A tree can be built by selecting "Build Tree"

Sequence clustering and phylogenetic analysis

Scope: Interactive tool DatasetExplorer is a part of the NCBI Virus Variation Resource that provides an easy way to perform preliminary analysis on nucleotide and protein sequences from the NCBI Virus Variation Sequence Database. Datasets are visually represented using phylogenetic/clustering trees. Users can select an algorithm to be used for building a tree as well as similarity criterion.
Overview of the Methodology: Construction of clustering/phylogenetic trees can be started either from the Results view or from the Alignment view. Either way, the tree is based on the pre-calculated alignment (see above) of the selected region of the query results.
Sequence Region Selection: After selecting "Build Tree", the graphic view of the multiple alignments of sequences selected from the previous step is displayed. The black and red colors in the graphics represent the presence and absence of amino acid residues at the corresponding positions. The positions in the longest sequence of the selected set for the first and last amino acid of each sequence are shown. A histogram showing the total number of amino acid residues at each position is displayed at the top of the page. The program automatically selects the sequence region to be analyzed so that the majority of the sequences in the set will be included. The sequence region can also be defined by users by first selecting all sequences in the set, and then entering the start and end positions in the boxes provided. When clicking the "Select sequences" button, the region from sequences that have complete coverage between the two positions will be selected, and sequences excluded from the selection will be highlighted with a background color in the graphic view.
Phylogenetic/Clustering Tree: A clustering or phylogenetic tree can be built by selecting one of the clustering algorithms and a distance calculating method from the list, and clicking the "Next step" button.
Protein and Nucleotide Distances: We offer different distance measures for calculating nucleotide and protein pairwise sequence distances, such as those based on Felsenstein F84 distance and Hammering distance for nucleotide sequences; the Dayhoff PAM matrix, the JTT matrix model, the PBM model, and Kimura's approximation for protein sequences implemented in the PHYLIP package, as well as the mPAM weight matrix for protein sequences.
Tree Modification: An adaptive approach is used to visualize the tree in an aggregated form adapted to the user's screen, allowing users to interactively refine or aggregate visualization of different parts of the tree (see a paper for details). A branch on the tree can be selected by clicking the root node, and the resolution of the selected branch can be changed by moving along the scale bar. Sequences on the tree can be searched by the fields in the database, and the resulting sequences or groups will be highlighted in green color.
Tree Export: The complete tree can be exported in the Newick format by clicking the "Download full tree" button. The downloaded tree can be displayed by many tree-viewing programs.