Mosquito Wars: an Overview of Dengue Fever Virus

Six-thousand genetically modified sterile, non-biting male mosquitoes were released in a remote area in eastern Malaysia one month ago as part of a study, which ultimately aims to cut the number of cases of dengue fever, the most prevalent mosquito-borne viral diseases in the world.  A similar study was previously conducted in Grand Cayman Island.  This study reported an 80% drop in the mosquito population.  The female mosquitoes spread the disease, which killed 134 people in Malaysia alone last year.

In 2010, 1.6 million people in the Americas were affected with dengue, of which 49,000 cases were Severe Dengue fever (SD), also known as Dengue Hemorrhagic Fever.  The release of sterile mosquitoes into the wild populations is an attempt by the government to reduce the malevolent mosquito population, thus reducing the number of cases of dengue fever.

Dengue fever is caused by an RNA virus that is spread by Aedes aegypti mosquitos.  Dengue fever is categorized into classical dengue fever and SD, which is very dangerous and potentially deadly.  Researchers are still investigating the Dengue virus to learn exactly what makes it so virulent.  Akey et al. have just just released a new finding in Science which may explain how it works to evade the immune system by a phenomenon known as viral mimicry.  See Viral Mimicry below for more details on this finding.

Genome & Structure  The dengue virus (DENV) genome consists of a positive sense single RNA strand.  The genome encodes ten proteins, which are translated as one continuous polypeptide.  This polypeptide is then cleaved into ten proteins.  These proteins include three structural proteins: the capsid(C), envelope (E), and pre-membrane (preM) protein and seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, which are involved in viral pathogenesis.

Guzman, M. G. et al. Dengue: A continuing global threat. Nature Reviews Microbiology 8, S7–S16 (2010). doi:10.1038/nrmicro2460

The dengue virus has a roughly spherical shape. Inside the virus is the nucleocapsid, which is made of the viral genome and C proteins. The nucleocapsid is surrounded by a membrane called the viral envelope, a lipid bilayer that is taken from the host. Embedded in the viral envelope are E and M proteins that span through the lipid bilayer. These proteins form a protective outer layer that controls the entry of the virus into human cells.
© 2011 Nature Education

Entry & Replication  The virus enters human leukocytes of monocyte lineage, and epithelial cells are pemissive to infection via endocytosis mediated by interaction of the viral E protein with a yet unidentified receptor.  When it reaches the acidic environment of the inner cytoplasm, the virus membrane fuses with the endosomal membrane and releases the nucleocapsid into the cytoplasm.  This is mediated by a conformational change in the E protein on the viral membrane.

Top) Dengie Virus Structure pre-infection Bottom) Dengue Virus inside the endosome. Hydrophobic amino acids which fuse with endosome mebrane shown in red

The viral RNA (vRNA) is then replicated and translated.  Intermediate negative sense copies of the vRNA, which serve as templates for new positive-sense RNA strands, are synthesized via DENV NS5 protein.  The NS5 protein has two functional domains: a methyltransferase, which is required for recognition of the vRNA by the host cell translational apparatus, and a polymease domain, which can catalyze synthesis of new vRNA de novo without any primer.  The contact with the ssRNA and the polymerase is sufficient to initiate and continue nucleic acid synthesis.  The vRNA is directly translated using the host cell’s rough endoplasmic reticulum (ER) into the long polypeptide, as mentioned earlier.  The newly synthesized vRNA is enclosed by capsid proteins.  This nucleocapsid enters the rough ER and is enveloped by the rough ER membrane.  After gaining its envelope, it is coated with preM and E proteins, its protective outer layer. Dengue virus infection induces changes in the intracellular membrane in the cytosol.  These changes produce viral packets where the replication complexes accumulate.  C-terminal regions of C, preM, and E contain hydrophobic residues, which serve to anchor the polypeptide into the ER membraine.  An ER peptidase along with the viral NS3-NS2B heterodimer complex cleave the polypeptide into the remaining proteins.  The assembled virus is exported via the trans-golgi network, where the virus is matured.  Maturation occurs when the host protease furin cleaves the bond between viral proteins preM and M.  This allows for structural rearrangements, such as the ones seen after endocytosis of the virus particle.

The dengue virus attaches to the surface of a host cell and enters the cell by a process called endocytosis. Once deep inside the cell, the virus fuses with the endosomal membrane and is released into the cytoplasm. The virus particle comes apart, releasing the viral genome. The viral RNA (vRNA) is translated into a single polypeptide that is cut into ten proteins, and the viral genome is replicated. Virus assembly occurs on the surface of the endoplasmic reticulum (ER) when the structural proteins and newly synthesized RNA bud out from the ER. The immature viral particles are transported through the trans-Golgi network (TGN), where they mature and convert to their infectious form. The mature viruses are then released from the cell and can go on to infect other cells.
© 2005 Nature Publishing Group Mukhopadhyay, S., Kuhn, R. J., & Rossmann M. G. A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3, 13–22 (2005). doi:10.1038/nrmicro1067

Autoimmune responses  Antibodies against DENV can cross react with several host proteins and endothelial cells.   This could be responsible for the endothelial dysfunction and leaking seen in severe dengue fever.  Antibodies against the viral E protein cross-react with and mature plasminogen into plasmin; plasmin is a protease responsible for dissolving blood clots.  It has also been shown that certain DENV serotypes can bind to platelets in the presence of anti-DENV antibodies resulting in antibody mediated lysis of platelets, and that IgM anti-platelet antibody titers are elevated in cases of severe dengue.  The presence of these antibodies not only induces complement mediated lysis if platelets, but also inhibits ADP-dependent platelet aggregation.  This results in bleeding in acute dengue cases.

Antibody-dependent enhancement  A controversial feature of dengue secondary infections by a different serotype than the primary infection is antibody-dependent enhancement.  After the primary infection, cross-reactive antibodies provide immunity against heterologous serotypes for up to four months post-primary infection.  After this time frame, the antibodies are in sub-neutralizing concentrations.  This facilitates dengue virus infection of FCγ receptor bearing cells, such as monocytes.  This leads to a larger number of infected cells, as compared to the primary infection, when no antibodes were present or when antibodies were in high enough concentration to neutralize the heterologous virus.  In vitro studies suggest that the antibodies may play a role in maturing the virus preM protein, which is required for the virus to become mature and most infectious.

a | The dengue virus life cycle and sources of antigens are shown. Dengue virions bind to cell surface receptors (these have not been completely characterized), and the virions are internalized through endocytosis. Acidification of the endocytic vescicle leads to rearrangement of the surface envelope (E) glycoprotein, fusion of the viral and vesicle membranes and release of viral RNA into the cytoplasm. Viral genomic RNA is then translated to produce viral proteins in endoplasmic reticulum (ER)-derived membrane structures, and the viral proteins and newly synthesized viral RNA assemble into immature virions within the ER lumen. Cleavage of the viral precursor membrane (pre-M) protein by the host cell enzyme furin leads to the formation of mature virions, which are secreted from the cell. In addition, some of the synthesized non-structural protein 1 (NS1) is expressed on the plasma membrane of the cell or secreted, and some virions are secreted in an immature form. Mature and immature virions induce antibody responses to the E protein, and these antibodies can function in neutralization or in antibody-dependent enhancement of infection. Immature virions also induce antibody responses to the pre-M protein. Antibodies specific for NS1 can interact with membrane-bound NS1 and cause complement-dependent lysis of virus-infected cells. b | The structure of the dengue virus E glycoprotein ectodomain and characteristics of E protein-specific antibodies are shown. The three domains of the E protein are coloured in red (domain I), yellow (domain II) and blue (domain III). c | The mechanisms of neutralization and enhancement by dengue virus-specific antibodies are shown. At high levels of epitope occupancy, antibodies can block the binding of virions to the cellular receptor or can block fusion at a post-binding stage. At lower epitope occupancy levels, antibodies can enhance the uptake of virions into cells by interacting with immunoglobulin (Fc) receptors.

Humoral Immune Response   The humoral response is responsible for controlling the infection and dissemination of the dengue virus in the body.  The cross-reactive nature of the antibodies which lead to temporary heterotypic immunity is thought to occur in the viral E protein specific antibodies.  The other main targets of the antibody response are the viral M and NS1 proteins.  The neutralizing antibodies are directed against the viral E protein.  This inhibits viral attachment and entry into host cells.  Antibodies may also bind to complement proteins and promote activation of the complement cascade.  Complement activation is also a feature of severe Dengue and is associated with plasma leakage; this may be a major factor in the pathogenesis of severe dengue.

Multiple theories of dengue immune pathogenesis.
“Original antigenic sin” has the potential to occur during a DENV secondary infection with a heterologous serotype of DENV. For example, this begins when (1) primary infection occurs with Serotype 1 of DENV, resulting in adaptive immune responses where (2) Serotype 1–specific T cells are selected, activated, and (3) clonally expanded to combat infection. During the resolution of primary infection, memory Serotype 1–specific T cells are formed and are retained in higher frequency in the T cell repertoire than other T naïve cells. (4) A secondary challenge Serotype 1 would evoke a memory recall response and (5) effective containment of infection by highly specific T cells. (6) A secondary challenge with a heterologous strain, Serotype 2, has the potential to reactivate memory T cells that are of greater specificity for Serotype 1 than for Serotype 2. (7) These memory Serotype 1–specific T cells outcompete naïve T cells that would be more specific for Serotype 2, resulting in an expanded memory T cell pool that is low specificity for Serotype 2 and (8) poor viral clearance in vivo. Antibody-dependent enhanced replication also has the potential to occur during a secondary, heterologous infection. During primary infection (9), B cell selection occurs, promoting Serotype 1–specific antibody production. (10) These preexisting antibodies are present during the secondary challenge. (12) If the secondary challenge is with Serotype 1 again, (13) antibody-mediated neutralization of DENV occurs, (14) limiting infection. (15) If the secondary challenge is heterologous, as with Serotype 2, antibody specificity may be low (16) and weakly neutralizing antibodies can promote Fc receptor–mediated uptake of virus-antibody complexes. (17) Increased uptake of virus into the cell without efficient antibody-mediated neutralization results in production of higher viral titers and increases activation of pro-inflammatory intracellular signaling pathways. (18) Cytokine storm can occur during either primary or secondary infection when infected cells produce high levels of cytokines or (19) may also be derived from noninfected immune cells, such as activated T cells. (20) Cytokines act directly on the host vasculature and promote vascular leakage when they reach pathological levels. (21) MCs also release cytokines and additional de novo synthesized and pre-stored vasoactive mediators when they are activated by DENV. (22) Prior to secondary infection, MCs may also be sensitized by binding DENV-specific antibodies, which can also mediate MC activation in response to DENV. (23) MC-derived mediators act directly on the host vasculature to promote vascular leakage.
doi:10.1371/journal.ppat.1003783.g002

Cellular Immune Response DENV can infect both natural killer and helper T cells.  The cellular immune response to these infections can be both beneficial and harmful.  Serotype specific T-cell responses include proliferation, target cell lysis, and cytokine production.  Helper T-cells produce IFNγ, TNFα, TNFβ, IL-2, and CCL4 cytokines. The immune system memory of the primary infection alters the response to the secondary infection.  This influences the clinical outcome.  Activated memory T cells recognize conserved and altered peptide epitopes.  The most effective responses are elicited by the most highly conserved epitopes, and this modifies the cellular immune response.  Plasma leakage occurs depending on this immune response.  A conserved antigen will lead to a complete immune response terminating in target cell lysis, whereas a partially conserved antigen will lead to release of few cytokines and inefficient cell lysis. This phenomenon of low affinity for the heterologous serotype and high affinity for the primary infection serotype is referred to as “Original Antigenic Sin”.

a | The dengue virus life cycle and sources of antigens are shown. Viral attachment, internalization, fusion and translation proceed as described in Fig. 1. Newly synthesized viral proteins enter the MHC class I and II presentation pathways and viral peptide epitopes are presented on the cell surface within the binding groove of MHC molecules. MHC class II molecules present peptides to CD4+ T cells, which principally produce cytokines but are also capable of lysing infected cells. MHC class I molecules present peptides to CD8+ T cells, which principally lyse infected cells but also produce cytokines. b | A schematic of the dengue virus polyprotein is shown at the top and the locations of well-defined epitopes that are recognized by human T cells are marked by arrows. c | Three of the well-defined T cell epitopes are shown to demonstrate the incomplete sequence conservation of typical T cell epitopes. The location of the epitope, its recognition by CD4+ or CD8+ T cells and its HLA restriction are indicated at the top. The predominant sequences for each of the four serotypes are shown. Residues that are completely conserved are shown in black and residues that are not completely conserved are shown in red. d | Variant epitopes alter the T cell functional response, and the figure shows two examples. The full agonist peptide (top) induces a full range of T cell responses — production of multiple cytokines (for example, IFNγ, TNF and CCL4) and lysis of the infected cell. A partial agonist peptide varying at one residue (bottom; altered residue in red) induces a skewed functional response, involving production of some cytokines (CCL4 in this example) but little production of other cytokines (such as IFNγ) and inefficient cell lysis. C, capsid protein; CCL4, CC-chemokine ligand 4; E, envelope protein; ER, endoplasmic reticulum; IFNγ, interferon-γ; NS, non-structural protein; pre-M, precursor membrane protein; TNF, tumour necrosis factor

Cytokines   Immediately upon entry of the virus into a host cell, the virus is recognized and the appropriate antiviral response is raised.  The Toll-Like Receptor (TLR) and the cytoplasmic receptor families are the two main mediators of dengue virus sensing.  Binding to TLR leads to activation of interferon regulating factors and the nuclear factor kappa B (NFkB).  These cascades activate production of IFN and proinflammatory cytokines.  These cytokines stimulate dendritic cell maturation and antiviral response. Dengue virus is believed to mainly infect dendritic cells, macrophages, and monocytes by the antibody-virus complexes-FCγ receptor mediated endocytosis.  While not completely understood, it is generally agreed that these infected cells and activated endothelial cells produce TNFα and NO, which increase vascular permeability.  Several IL-family cytokines are also produced.  Enhanced levels of pro-inflammatory and vasoactive cytokines before and at the time of plasma leakage in patients with severe Dengue suggest that excessive cytokine production induces vascular permeability.  This is proposed to be the cause of hemorrhages and edema in these patients. However it is not fully understood how these cytokines cause dysfunction of vascular endothelial cells.

Viral Mimicry is a phenomenon by which viral proteins evade the host immune system.  This is achieved by having high structural similarity to host proteins.  When an immune system cell encounters it, the immune cell is tricked into believing it is a host cell, and thus it leaves it alone.  Viral mimicry is a well-established mechanism to thwart the immune system by several viruses.

In the recent study by Akey et al. published on Science on Februyary 21, 2014, the authors found that the α/β subdomain of the DENV protein NS1 wing has a high structural similarity to the helicase domains of human RIG-1 (retinoic acid inducible gene -1) and MDA5 (melanoma differentiation antigen 5).  These cytoplasmic helicase domains recognize viral RNAs and trigger antiviral responses.  The question that arises is why the virus has adapted this particular immune evasion system because, as far as is know, the the DENV NS1 protein does not localize to the cytoplasm where RIG-1 or MDA5 detect RNA.

Similarity of the NS1 wing α/β subdomain to the RIG-I family of innate immune proteins. An SF2 helicase domain of RIG-I [blue; PDB accession code 3TBK (25)] is superimposed on the WNV NS1 wing domain (yellow). Orange, connector subdomain.
Akey, David L., et al. “Flavivirus NS1 Structures Reveal Surfaces for Associations with Membranes and the Immune System.” Science (New York, NY) (2014).

 

 

 

 

 

 

 

 

 

Symptoms

• High fever and at least two of the following:
  • Severe headache
  • Severe eye pain (behind eyes)
  • Joint pain
  • Muscle and/or bone pain
  • Rash
  • Mild bleeding manifestation (e.g., nose or gum bleed, petechiae, or easy bruising)
  • Low white cell count

Laboratory Diagnosis of suspected dengue specimens involves isolation of the virus, using serological tests, or molecular methods.

1. Immunological response to dengue infection
2. Classical testing algorithms of dengue:
   1. MAC Elisa – measures IgM antibody titers
   2. IgG Elisa – measures IgG antibody titers
   3. NS1 Elisa – detects viral NS1 antigen

Vaccines are being developed by several companies around the world.  The major complication with dengue vaccines is that due to secondary infections, they must be effective against all four serotypes.  Most vaccines under study are against the immature viral preM protein and the E protein.  While there are some vaccines in development against the viral RNA, as well.  Sanofi-Pasteur is leading the race in dengue vaccine development.  It currently has a live attenuated chimeric tetravalent vaccine in stage 3 clinical trials.

Treatments & Therapies There is no specific medication for treatment of a dengue infection. Persons who think they have dengue should use analgesics (pain relievers) with acetaminophen and avoid those containing aspirin. They should also rest, drink plenty of fluids, and consult a physician. If they feel worse (e.g., develop vomiting and severe abdominal pain) in the first 24 hours after the fever declines, they should go immediately to the hospital for evaluation.

Risk Factors  for all Dengue serotype infections include location and ethnicity.  Dengue is mainly reserved to the sub-tropical regions around the globe.  All four serotypes are found together.  People of Afro-Cuban ancestry are less prone to dengue infections.

See this map for recent reports around the world http://www.healthmap.org/dengue/en/

All the images from this blog post can be found here in full-screen size  Dengue Virus Figures

Acknowledgements

I thank Dr. Wouter Schul, Associate Director Research Project Management at Novartis Institutes for Biomedical Sciences, for critical reading and suggestions, which led to further revision of this post.

References

Akey, David L., et al. “Flavivirus NS1 Structures Reveal Surfaces for Associations with Membranes and the Immune System.” Science (New York, NY) (2014).

Bäck, Anne Tuiskunen, and Åke Lundkvist. “Dengue viruses–an overview.”Infection ecology & epidemiology 3 (2013).

Lei, Huan-Yao, et al. “Immunopathogenesis of dengue virus infection.” Journal of biomedical science 8.5 (2001): 377-388.

Mukhopadhyay, S., Kuhn, R. J., & Rossmann M. G. A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3, 13–22 (2005). doi:10.1038/nrmicro1067

Rothman, Alan L. “Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms.” Nature Reviews Immunology 11.8 (2011): 532-543.

Szymanski, Michal R., et al. “Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes STOICHIOMETRIES, INTRINSIC AFFINITIES, COOPERATIVITIES, BASE, AND CONFORMATIONAL SPECIFICITIES.” Journal of Biological Chemistry 286.38 (2011): 33095-33108.

World Health Organization  Dengue Health Topic

Centers for Disease Control and Prevention Dengue Fact Sheet

Tsamsa Virus: A New Anthrax Treatment

Lately, there has been a lot of commotion regarding a newly discovered bacteriophage that kills anthrax bacteria.  However, the implications of this phage are not new, and, furthermore, therapies derived from it may not be efficacious for treating advanced stage anthrax infections.

What is anthrax?  Anthrax is a serious infection caused by the gram-positive, rod-shaped Bacillus anthracis bacterium.  It is found naturally in the soil, and affects domestic animals as well as wildlife around the world.  Humans can also become infected with the bacteria.

Colored Scanning Electron Microscope (SEM) image of B. anthracis spores.

How Does Infection Occur?  Infection occurs when the spores, the dormant form of the bacteria, are ingest, in haled, or come into contact with a lesion on the skin.  These infections can occur by eating raw or undercooked meat, drinking contaminated water, or while working with infected animals or animal products, such as wool, hides, or hair.  However, anthrax is not contagious.  The Centers for Disease Control and Prevention have reported rare cases where anthrax has been transmitted from person-to-person via infectious discharges from skin lesions.  Also, a new mode of infection has been reported.  This new mode involves infection following use of contaminated intra-venous needles by drug users.

Diagram courtesy of the Centers for Disease Control and Prevention

Symptoms