Dengue virus (DENV) is the cause of dengue fever. It is a mosquito-borne, single-stranded, positive-sense RNA virus of the family Flaviviridae and genus Orthoflavivirus. Four serotypes of the virus have been found, and a reported fifth has yet to be confirmed,Dwivedi, V. D., Tripathi, I. P., Tripathi, R. C., Bharadwaj, S., & Mishra, S. K. (2017). Genomics, proteomics and evolution of Dengue virus. Briefings in functional genomics.16(4): 217–227, https://doi.org/10.1093/bfgp/elw040 all of which can cause the full spectrum of disease. Nevertheless, the mainstream scientific community's understanding of dengue virus may be simplistic as, rather than distinct antigenic groups, a continuum appears to exist. This same study identified 47 strains of dengue virus. Additionally, coinfection with and lack of rapid tests for Zika virus and chikungunya complicate matters in real-world infections.
The Incidence of Dengue virus has increased dramatically within the last 20 years, becoming one of the worst mosquito-borne human pathogens that tropical countries have to deal with. Estimates published in 2013 indicate that as many as 390 million infections occur each year, and many dengue infections are increasingly understood to be asymptomatic or subclinical.
Evolution Dengue virus is classified into four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). A single report of a fifth serotype, DENV-5, isolated from an outbreak in October 2013 has not been replicated or further reported on. The rate of nucleotide substitution for the DENV-1 envelope gene has been estimated to be 6.5 per nucleotide per year, a rate similar to other RNA viruses. The American African genotype has been estimated to have evolved between 1907 and 1949. This period includes World War I and World War II, which were associated with considerable movement of populations and environmental disturbance, factors known to promote the evolution of new vector-borne viral species.
An analysis of all four serotypes estimated that their most recent common ancestor existed about 340 AD (95% confidence interval: 280 BC–850 AD).Costa RL, Voloch CM, Schrago CG (2012) Comparative evolutionary epidemiology of dengue virus serotypes. Infect Genet Evol 12(2):309-314
Lifecycle
Until a few hundred years ago, Dengue virus was transmitted in sylvatic cycles in Africa, Southeast Asia and South Asia between mosquitoes of the genus Aedes and nonhuman primates, with rare emergences into human populations. The global spread of Dengue virus, however, has followed its emergence from sylvatic cycles and the primary lifecycle now exclusively involves transmission between humans and Aedes mosquitoes. Vertical transmission from mosquito to mosquito has also been observed in some vector species. Dogs have been found to be infected by the virus, but more research is needed to determine if dogs or other animals can serve as reservoirs or are just incidental hosts.
When a mosquito feeds on infected human blood, the virus establishes itself in the mosquito's gut and subsequently infects other tissues. The infected mosquito then seeks blood, biting a different human. At this moment, the human's skin cells (keratinocytes) get infected first. Subsequently, dendritic cells (DC) take up the virus and get infected too. The DC's mobility disperses virus in the body where some other cell types can get infected, as well (macrophages, hepatocytes and bone marrow cells).
Dengue virus lifecycle in cells, both in arthropod and mammalian cells, is typical for single-stranded (+)-RNA viruses. After binding to specific receptors on the cell the virion gets endocytosed. Endosomes usually get acidified and become lysosomes which digest their content. But the acidic condition triggers a conformational change in the envelope of the virion, making its membrane fuse with the endosome membrane, releasing the viral capsid with the viral RNA genome into the cell's cytosol.
Viral RNA is capped and, so, cannot be distinguished from human mRNA. It gets translated by the cell's ribosomes into a polyprotein with 3,391 amino acids. This polyprotein cleaves itself to produce, after several steps, three structural (C, E, prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). All non-structural proteins are necessary to together create the viral replication complex (RC) which makes copies of capped viral RNA to produce more polyprotein and new virions. New virions move to exocytic vesicles via the secretory pathway and, there, get their mature form before they are released from the cell. One infected cell may release up to between 1,000 and 10,000 new virions.
Recent findings suggest that as the virus infects human cells, host homeostatic processes such as autophagy and ER stress response, not to mention apoptosis, are triggered depending on the infected cell type. The activation of autophagy and ER stress during infection enhances virus reproduction. Attempts to provide detailed summaries of the life cycle of dengue at the cellular level are published in review articles from different research groups.
Genome The DENV genome is about 11000 bases of positive-sense, single-stranded RNA (ssRNA) that codes for three structural proteins (capsid protein C, membrane protein M, envelope protein E) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). It also includes short noncoding regions on both the 5' and 3' ends.
Structural and enzymatic proteins
E protein The DENV E (envelope) protein, found as a dimer on the surface of the mature viral particle, is important in the initial attachment of this particle to the host cell. Each E protein monomer comprises three ectodomains, ED1 to ED3, and a transmembrane segment. ED2 includes the dimerization interface, two glycosylation sites, and the peptide of fusion with the cellular membrane. ED3 is a continuous polypeptide segment; its fold is compact and immunoglobulin-like. Several molecules that interact with the viral E protein (ICAM3-grabbing nonintegrin, CD209, Rab 5, GRP 78, and the mannose receptor) have been shown to be important factors mediating attachment and viral entry. The membrane form of ribosomal protein SA may also be involved in the attachment. E protein is known to contain physicochemically conserved B cells and T cells specific epitopes, which can be exploited to design vaccine. Recombinant domains of the E protein are used as well-defined antigens in the serological detection of antibodies directed against Dengue virus and as immunogens in vaccine candidates.
prM/M protein The DENV prM (membrane) protein, which is important in the formation and maturation of the viral particle, consists of seven antiparallel β-strands stabilized by three disulfide bonds.
The glycoprotein shell of the mature DENV virion consists of 180 copies each of the E and M proteins. The immature virion starts out with the E and prM proteins forming 90 heterodimers that give a spiky exterior to the viral particle. This immature viral particle buds into the endoplasmic reticulum and eventually travels via the secretory pathway to the Golgi apparatus. As the virion passes through the trans-Golgi network, it is exposed to low pH. This acidic environment causes a conformational change in the E protein, which disassociates it from the prM protein and causes it to form E homodimers, which lie flat against the viral surface, giving the maturing virion a smooth appearance. During this maturation, pr peptide is cleaved from the M peptide by the host protease, furin. The M protein then acts as a transmembrane protein under the E-protein shell of the mature virion. The pr peptide stays associated with the E protein until the viral particle is released into the extracellular environment. This pr peptide acts like a cap, covering the hydrophobic fusion loop of the E protein until the viral particle has exited the cell.
NS3 protein The DENV NS3 is a serine protease, as well as an RNA helicase and RTPase/NTPase. The protease domain consists of six β-strands arranged into two β-barrels formed by residues 1–180 of the protein. The catalytic triad (His-51, Asp-75 and Ser-135) is found between these two β-barrels, and activity is dependent on the presence of a 43 amino acid segment of the NS2B cofactor. This cofactor wraps around the NS3 protease domain and becomes part of the active site. The remaining NS2B residues before and after the cofactor region contain helical domains involved in membrane binding. The remaining NS3 residues (180–618) form the three subdomains of the DENV helicase. A six-stranded parallel β-sheet surrounded by four α-helices makes up subdomains I and II, and subdomain III is composed of four α-helices surrounded by three shorter α-helices and two antiparallel β-strands.
NS4A protein DENV NS4A is a nonstructural protein involved in altering cell membrane curvature and induction of autophagy. In addition to its membrane altering property, NS4A is a scaffold for the virus replication complex and undergoes oligomerization. Mutations of NS4A that affect interaction with NS4B abolished or severely reduced virus replication indicating the importance of NS4A and its interaction with NS4B in dengue reproduction.
NS5 protein The DENV NS5 protein is a 900-residue peptide with a methyltransferase domain at its N-terminal end (residues 1–296) and a RNA-dependent RNA polymerase (RdRp) at its C-terminal end (residues 320–900). The methyltransferase domain consists of an α/β/β sandwich flanked by N-and C-terminal subdomains. The DENV RdRp is similar to other RdRps containing palm, finger, and thumb subdomains and a GDD motif for incorporating nucleotides.
Complexes between the E protein and neutralizing antibodies Crystal structures of complexes between antibodies and either the ectodomain (sE) of the viral E protein or its domain 3 (ED3) have helped understand the molecular bases of the virus recognition and neutralization. Some of the epitopes are partially or totally inaccessible in the known structure of the mature virion. The corresponding antibodies are, therefore, assumed to bind to alternate or transitional conformations of the virus at 37 °C. The murine antibody E111 neutralizes DENV1. Its Fab and scFv fragments were crystallized in complex with the ED3 domain of DENV1. Its epitope is located around β-strands C and C' of ED3, and the intervening loop. The murine antibody 1A1D-2 strongly neutralizes DENV1, DENV2, and DENV3. Its Fab fragment was crystallized in complex with the ED3 domain of DENV2. Its epitope straddles β-strands A and G of ED3. The murine antibody 2H12 cross-reacts with all four DENV serotypes. It neutralizes the corresponding viruses, except DENV2. Its Fab fragment was crystallized in complex with the ED3 domains of DENV1, DENV3 and DENV4. Its epitope is located around the conserved AB loop of ED3. The murine antibody 4E11 neutralizes all four DENV serotypes with varying efficacies. Its scFv fragment was crystallized in complex with the ED3 domains of the four DENV serotypes. Its epitope straddles β-strands A and G of ED3 as does the epitope of 1A1D-2. The structures at 2.0 Å resolution have enabled one to analyze the roles of water molecules within the protein interfaces and the roles of somatic hypermutations outside of these interfaces in the interactions and cross-recognitions. The chimpanzee antibody 5H2 potently neutralizes DENV4. Its Fab fragment was crystallized in complex with the sE protein of DENV4. Its epitope is included in domain 1 (ED1) of the E protein. The human antibodies Ede1-C10, Ede2-A11, and Ede2-B7 potently neutralize all four DENV serotypes. Their Fab or scFv fragments were crystallized in complex with the sE protein of DENV2. The recognition determinants of these antibodies are at a serotype-invariant site in the E dimer interface and include the exposed side chains of the E fusion loop and the two conserved glycan side-chains.
Disease