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The epidemiology and severity of dengue infection continues to expand in endemic regions. The four dengue viruses circle the globe through transmission from tropical countries, effecting nearly a million people each year with children bearing the greatest burden. Certain preventive measures are needed to be considered however innovative techniques are required for complete treatment and to provide a cure against the disease. Several methods are currently in use, but have a less effectiveness due to the severity of the infection and incomplete knowledge of the viral pathogenesis. In this article, I have discussed certain pioneering methods such as the flightless female technique, the development of terminator mosquitoes, the targeting of viral envelope protein for the design of an effective tetravalent vaccine and mainly focused towards the development of RNA interference mechanism as a highly effectual tool against the virus providing a complete therapy for the elimination of dengue disease.
Dengue is a viral disease that is transmitted through arthropod vectors, having an impact spread through a wide geographical distribution. The disease is a mosquito-transmitted viral infection that results in fever, pain in muscles/joints, headache, nausea, vomiting and rashes. Some infections cause Dengue Hemorrhagic Fever or DHF, a life threatening form of disease that increases vascular permeability and shocks caused by the attack of T cells over dengue infected cells after the release of cytokines. The most bearing patients of this disease are children due to their low circulating maternal dengue antibodies.
Dengue virus is a part of the Flavivirus genus belonging to the Flaviviradae group. The dengue virus can be grouped into four types that are the YFV, yellow fever virus, WNV, west Nile virus, JEV, Japanese encephalitis virus and TBEV, tick-borne encephalitis virus. These four types of dengue viruses can lead to the threatening DENV infection which range from DF, asymptomatic infection, to DHF and DSS (dengue shock syndrome). DENV has caused most human disease among the arbovirus and has lead to about 50 to 100 million DENV infected patients every year.
DENV is spread to humans by a bite of an infected mosquito of the Aedes species. The most predominant carrier of the DENV virus is the Aedes aegypti mosquito. This specie of mosquito survives mainly in the tropical and subtropical areas in an urban transmission cycle. Species like Ae albopictus and Ae polynesiensis are often involved in the transmission of this disease. Infected humans with any of the four mentioned viruses can develop the two main syndromes; DHF and DSS. This disease results in capillary leakage along with thrombocytopenia, damaged liver with increase in aspirate aminotransferase, and alanine amino-transferase, and haemostasis. Apart from this, dengue shock can arise in patients and can cause increase peripheral vascular resistance and increased diastolic blood pressure.
DHF is an acute febrile disease that results in bleeding, plasma leakage, thrombocytopenia, and pleural. It usually starts with sudden fever where the body temperature reaches up to 38-40 decree Celsius and remains for 2-7 days. Symptoms like hemorrhagic feature, petechiae and bruised skin are seen in most patients. Patients with DHF are warned of increased chances of shock that may lead to loss of platelet count and abnormal permeability of the capillary.
DSS results in patient with low pulse pressure, shock and hypotension. The liver of the infected person is expected to be tender, palpable and cause secretion of abnormal liver enzymes. The symptoms of DSS include pain in the abdominal, vomiting, lethargy and unexpected change in body temperature with sweating. Any signs of these symptoms require the patient to be hospitalized to control shock.
Current researches have focused on secondary dengue infections to find out the mechanism that cause vascular permeability and hemorrhaging. Results have shown high concentrations of interferon α to persist after few days of fever during symptomatic infections. In secondary infection of the disease, immune enhancement is considered as the most believed hypothesis for the pathogenesis of DHF. The infected virus complexes with the non neutralizing antibodies by a process known as Antibody dependant enhancement or ADE or the virus, and thus phagocytosis is enhanced by mono nuclear cells which is considered as the primary site of viral replication. The replication of virus induces the release of vasoactive mediators by infected monocytes, which results in hemorrhagic manifestations and vascular permeability characterized as DHF and DSS.
The dengue virus (DENV) has a single stranded RNA genome. The genome consists of Untranslated regions or UTRs that regulates the translation of RNA. Translation of the RNA gives a polypeptide that then processed by cellular and viral proteins to give 3 structural and 7 non structural proteins. The virus has a spherical enveloped structure, consisting of 3 major proteins; the envelope protein (most abundant), the membrane, and the capsid protein. The mature DENV virus consists of envelope glycoprotein that is aligned parallel in pairs. This E protein mediates the cell attachment (attachment of virus to the cell) and fusion of virus into the cell. The envelope glycoprotein consists of 3 functional domains, the receptor binding domain, the dimerisation domain and the central domain. Under acidic conditions the virus enters the cells by receptor mediated endocytosis which E protein causes the fusion of the viral and cellular membranes. Ones the cell is burst open the virus ejects its RNA from the viral nucleocapsid and releases it into the cells cytoplasm where it is translated. The virus matures inside the cells (makes copies) and then releases itself to infect the other cells repeating the cycle again.
The diagram below shows the structure of the virus:
Source: Emil V, 2008. Science Centric
Reports in medical history to record the first dengue fever case started in 1779 and 1780. Since then, for every 10 to 30 years DF pandemics have occurred infrequently in any
given location. South-East Asia had first seen the multiple serotypes of the dengue virus and after uncontrolled of population and city growth, DHF had become a major threat to public health.
Through 1953-70s, the first signs of DHF were seen in Manila and remained in south-east Asia. In 1980s through 1990s, the epidemic continued to spread in India, Sri Lanka, Maldives Island, Pakistan and east China. Most hospitalized patients had syndromes of DHH and death among children was common in Asia.
In 1971, DEN-2 had begun to spread in few Islands and had caused severe spread of DF/DHH. In 1977, more than 40% of the population surveyed was found to be infected with the dengue virus DENV-1. In 4 years time, DENV-2 had begun to infect the same percent of the population, mainly to adults aged 55 years. In 1981, children were seen infected and cases of death among children and adults were continuing to rise.
The main reasons that lead to the spread of the epidemic were uncontrolled growth in population, poorly planned urbanization, poor mosquito control, frequent air travelling, and deterioration.
In 2006, dengue transmission began to decrease as effective mosquito control methods were adapted in Singapore and Cuba. Before this, by 1973, an island-wide control method for aedes aegypti had begun implementation and had shown to reduce the epidemic by great figures.
Several control measures have been discovered that help prevent the spread of dengue viruses. A vector control method of the dengue virus disease is one that involves the control of infected mosquitoes. This can be done by either the treatment or destruction of the infected containers. The vectors DF and DHF usually breed around domestic homes and can be controlled both individually and as community. Several measures can be used, for instance, anti-adult and anti-larval measures. Community actions like sharing knowledge and discussing practices against the vector species and the disease can be one step to reduce the epidemic. As a community and an individual with interests in human health, one must look into methods to control sites of mosquito growth and manage disposal of domestic waste.
Production of vaccine need to be tetravalent as an already existing dengue antibody has shown to be a risk factor for DHF. Several developments have been made in the production of second generation recombinant dengue virus. Another vaccine is under process that involves the clone of DEN-2 PDK-53 vaccine and chimaeras viruses that include the insertion of capsid, premembrane and genes of DEN 1, 3, 4 in the DEN-2 PDK-53. These are expected to be stronger and more immunogenic control methods of the future.
Dengue virus diagnosis can be categorized in two major steps. Stage I, viraemia and fever including NS1 antigens in blood; Stage 2, early post-febrile period (excess of IgM and IgG antibodies). Patients with primary infection show less amounts of viraemia or fever, however, secondary infections results in longer duration of viraemia with extended presence of NS1 antigens in blood. In early stages of the disease, diagnosis is possible by detecting virus RNA or virus proteins in the blood. Serological tests are undertaken, but will only show positive results until effervescences.
An affordable, fast and accurate diagnosis of dengue fever can be taken at the febrile stage. This form if test uses NS1 monoclonal antibody in ELISA (enzyme-linked immunosorbent assay) format to find the dengue NS1 antigen in the blood. This test has shown 85% of PCR-positive results. This method of diagnosis was advanced when a combination of NS1 antigen-capture with NS1 antibody test was made. This allowed for bedside diagnosis of dengue virus disease in patient. Furthermore, NS1 could be used as ELISA (IgM capture) based diagnosis which replaces the detection of viral antigen and allowing more accurate results for primary and secondary detection of dengue infection.
Dengue fever can be managed by symptomatic and supportive measures. Antipyretics are required to maintain the body temperature. In any case of dengue fever, aspirin must be avoided, as it may cause gastritis, acidosis and bleeding. Oral fluids must be taken by patients that have excessive vomits, analgesics, sweating or diarrhea. Records of blood pressure, urinary outputs, plasma leakage, hemorrhagic and volume replacement must be maintained. As patients lose a lot of plasma and platelet counts, isotonic solutions and plasma expanders must be given. Fluid replacements are made in 24 hours. Apart from these, bolus of body weight, colloidal fluid, and blood transfusion must be undertaken.
Management strategies to control the disease
Flightless mosquitoes and sterile insect technique
Sterile insect technique involves irradiation to produce sterile male mosquitoes. It is a form of biological control that involves the male only release of sterile mosquitoes that would compete with wild males to mate with wild type females, but will not be able to produce any offspring thus reducing the population of the next generation of mosquitoes. Although this technique has provided control over various different diseases, it has long proven to be difficult with mosquitoes due to the fragility of the males. In order to overcome this problem a recent research has been published involving the use of flightless mosquitoes.
The transgenic strains of aedes aegypti were announced in 2010 by a team of UCI and British scientist producing a new strain of mosquitoes in which the females cannot fly. The transgenic males are allowed to mate with the wild type female producing next generation of flightless female mosquitoes that are unable to fly due to miss development of their wings and so they can neither mate nor bite. These strains eliminate the requirement of sterilization, permit the male only release and also enable the release of eggs instead of the adult mosquitoes. The technique is expected to facilitate an area wide control for elimination of dengue in near future.
Scientists altered the insect's genes to disrupt development of their wing muscles
Terminator mosquitoes for dengue control
Terminator technology involves the use to transgenic male mosquitoes that are called as Released Insects with a Dominant Lethal. These transgenic males when released in the environment mate with the wild type females resulting in death of the progeny in its late larval stages, this way the larvae can compete for the food with the normal ones reducing the natural population. The mathematical model for the technique predicts that it will effectively eradicate dengue within a period of one year. The RIDL trait was created using piggyBac transposon with the transposase gene removed for restricted multiplication of the gene. RIDL is a tetracycline repressible lethal system which consists of the key dominant lethal gene (tTAV) and a red fluorescent market gene. Thus in absence of tetracycline a positive feedback loop causes enhanced expression of lethal gene that is toxic and kills the insect. The information currently available doesn't tell us what is killing the target animal thus the safety of the technique is further needed to be considered. Another hazard is that horizontal gene transfer occurs in piggyBac inserts. Within a single eukaryotic genome extensive crossing of tranposon jumping gene occurs among distinct but related families due to the presence of similar transposase coding genes. In addition to this the problem of transgene escape is also needed to be considered, artificial transposons are considered as aggressive genome invaders, transgenic insects carrying them would act as efficient delivery system (Atkinson MP et al, 2007). Current studies are focused on finding more about the transposable elements such as piggyBac and their effects when used in preclinical therapy.
Strategies for the production of an effective vaccine
An ideal vaccine would focus on the development of a tetravalent vaccine that must be effective against all the 4 serotypes of dengue virus in order to preclude the development of antigen disease enhancement, DHF and DSS caused by vaccination. The vaccine should also be cost effective, efficient, and should provide long term immunity against the virus.
For the development of an effective vaccine the understanding of the viral surface 3D structure is highly crucial. Recent studies involving the determination of the overall 3D structure of the viral surface glycoprotein has provided information about the fusion of membranes and receptor recognition pathway. E protein is identified to be the major envelope protein, thus regions of E protein are identified as a target for the development of an effective vaccine. Along with this the structure also allows to design a potent immunogen to block the binding of the specific receptors preventing the entry of virus into the cells. The success of the live attenuated virus vaccine of yellow fever has provided a clear guidance of the development of a successful dengue vaccine. Current vaccine approach is mainly focused on live attenuated virus vaccine, inactive virus vaccine and subunit vaccine, which are in their early clinical trials.
Model for antibody dependant enhancement of dengue virus replication:
Source: Stephen S. Whitehead et al, 2007 Nature.
List of dengue vaccine under development:
Source: Stephen S. Whitehead et al, 2007 Nature.
RNA interference mechanism
RNAi is been used as an innovative research tool to study various therapeutic and genetic functions. The mechanism is studied in many organisms, initially in C. elegans, and later in plants, mice, chicken embryo, fungi and many insect species including mosquitoes.
The overall mechanism is divided into two broad phases, the initiation and the effector phase. In the initiation phase, the DICER complex binds to the long double stranded RNA and breaks it down into short RNA. These small interfering RNA or siRNA will now assemble with special proteins to form a complex which triggers the RNAi pathway. The second phase that is the effector phase, involves the formation of RNA induced silencing complex or RISC (containing the siRNA with DICER). The key proteins that are essential for the RISC formation are DICER protein, that is found during initiation phase and the Argonaute or Ago protein found in the effector phase. The Ago is an essential protein in effector phase since it helps in directing the RNAi complex to the target mRNA (to be degraded). The Function of Ago protein have been studied in several mosquito species, the result of this study showed that silencing of Ago gene made mosquitoes more prone to the viral infection. The Ago gene in aedes aegypti was
characterized to see that the injection of double stranded RNA which was complementary to ago mRNA reduced the ago protein levels and increased the levels of DENV2.
Once the RISC complex with siRNA binds to target mRNA at the cleavage site (that is located at the center), it cuts the mRNA which is then degraded by the cellular mRNase (Seokyoung K and Young S. H, 2008).
Stages of gene silencing with dsRNA:
Source: J. GateHouse, 2008. Trends in Biotech
RNAi mechanism is currently been used as an application for the vector control. Arthropods such as mosquitoes encode a functional RNA silencing pathway (Rui lu et al, 2004), the RNAi mechanism in aedes aegypti is important in controlling the levels of mRNA of the virus in the vector. In order to target the DENV2, the mosquito cells transcribe an inverted repeat RNA thus causing the RNA genome of the virus to be unable to support the replication of the virus (Adelman et al, 2002). The expression of premembrane coding region of the dengue virus in adult aedes aegypti caused activation of a resistance pathway against the type 2 dengue virus.
The RNAi effect in insect is studied to be both inducible and heritable. The injection of dsRNA in insect is inducible and the expression of gene in later stages of development is not altered. Recent studies showed that both inheritable and stable RNAi suppression was observed in transgenic insects consisting of gene specific dsRNA.
These mechanisms direct to the understanding and focus to an innovative approach towards the development of a strategy for the control of dengue. Aedes aegypti is considered as a 'dirty syringe' spreading the pathogen while feeding on a host for a blood meal. The mosquito releases saliva that contains the virus which causes infection in the host body. RNAi, which is described above acts as the innate immune response of the vector preventing it from getting the infection by keeping the virus to low levels as to not cause pathology. If the virus is not affecting the mosquito's fitness then why would it invest energy to fully eliminate it. This phenomenon was proved by constructing knockdown of genes responsible for turn on the RNAi pathway; reduction in RNAi caused an increased viral replication. Keeping this in mind on could hypothesis what if the RNAi gene expression is increased to levels that virus would be able to replicate, and thus not be able to transfer from the vector to the host. Example of therapy based on this concept is mentioned in detail below.
Therapy based on RNAi gene silencing
The RNAi response to DENV can be possibly induced in the mosquito's midgut when the mosquito feeds onto an infected blood containing the virus, thus by increasing the RNAi response the viral genome can be destroyed before the virus is able to replicate and evade the mosquito's innate immune response. This research is focused on midgut as the target for the destruction of the virus because it is the first tissue the virus encounter inside the mosquito and it determines the competence of the vector. This hypothesis is tested using a molecular transgenic technique to engineer strains of aedes aegypti that form double stranded RNA in mosquito cells and thus cause triggering of RNAi response due to the transcription of an anti DEN effector molecule. The effector molecule contains RNA fragments from the premembrane containing regions of DENV2, sialokinin intron sequences and also antisense RNA complementary to the sense RNA of premembrane. An inducible promoter cardoxypeptidase is used that is expressed in all midgut cells after the ingestion of a blood meal. Also a PAX-eGFP gene is used as an eye marker in order to facilitate screening of the transformed mosquitoes.
Transformation of the transgene is conducted using a binary system that involves co injection of 2 plasmids into the embryo of mosquitoes. One of the plasmid contains the market gene along with the anti DENV2 gene and the second plasmid contains the transposase coding gene. The transposase gene prevents mobilization of transposon and cause stable integration of the anti DENV gene. Many such mosquito families have been established containing stable integration of anti DENV gene into the genome and production of anti DENV RNA to eliminate the dengue virus (Emily A Travanty et al, 2004).
Table showing the effector gene constructs:
Source: Emily A T et al, 2004
Map of transforming construct with the position of northern and southern probe:
Source: Emily A T et al, 2004
Southern and Northern analysis of midgut specific carboxypeptidase transgenic families:
Source: Emily A T et al, 2004
A similar research was conducted recently using RNAi mechanism that involved genetic modification of aedes aegypti to impair vector competence for DENV 2. The transgenic aedes aegypti contained the same inverted repeat sequence from premembrance along with a carboxypeptidase promoter. The promoter and anti DENV sequence with the effector gene was inserted into the genome of a white eye mosquito by using a non autonomous Mosl transformation system.
The transgenic mosquito contained a reduced viral envelope antigen in midgut and salivary gland after the ingestion of a blood meal. Most of the transgenic mosquitoes were tested by DENV2 titration method to show that they poorly support viral replication and well as significantly reducing the transmission of virus in vitro.
Engineering RNAi in genetically modified mosquito (injection of dsRNA and antigen in midgut):
This method showed that a high level of resistance against DENV provide a powerful tool to control transmission of dengue virus by a population replacement strategy, (Alexander W. E. Franz, et al, 2006). Considering the effectiveness RNAi mechanism we can conclude that development of transgenic mosquitoes will act as a highly effectual tool against the virus providing a complete therapy for the elimination of dengue disease in the near future.