DENGUE VIRUS: NO ONE IS SAFE Caitlin Reed Smith College April 29, 2005
OVERVIEW OF THEMES <ul><li>Background Information </li></ul><ul><li>Clinical Presentation & Diagnosis </li></ul><ul><li>Bi...
WHAT IS DENGUE? <ul><li>Flavivirus (type of arbovirus) </li></ul><ul><li>Transmitted from  Aedes aegypti  and  Aedes albop...
DENGUE (cont’d) <ul><li>Three Manifestations: </li></ul><ul><li>1.  Dengue Fever </li></ul><ul><li>2.  Dengue Hemorrhagic ...
WHY DO WE CARE ABOUT DENGUE? <ul><li>CDC Category A Infectious Disease </li></ul><ul><li>Infects 50-100 million people eve...
WHY NOW? <ul><li>Failed eradication attempt in the Americas in 1970 </li></ul><ul><li>Previously unestablished serotypes a...
<ul><li>Mosquitoes transmit </li></ul><ul><li>dengue to human dendritic </li></ul><ul><li>cells </li></ul><ul><li>2.  Deng...
SYMPTOMS  OF DHF GRADE I:  Fever with other symptoms such as vomiting, headache, muscle and joint pain: positive tournique...
BASIC BIOLOGY <ul><li>Single, positive-stranded RNA surrounded by an icosahedral core </li></ul><ul><li>90 glycoprotein E ...
FUSION PROTEIN E Modis, Ogata, et. al., 2004 .
FIRST INFECTION <ul><li>Humoral and cellular immune response </li></ul><ul><li>- Ab serum neutralizing levels increase </l...
SECOND INFECTION <ul><li>Antibody dependent enhancement </li></ul><ul><ul><li>- Enhancing immunoglobulin G (IgG)  antibodi...
ENTRY INTO CELL <ul><li>Dengue infection </li></ul><ul><li>Endosome entry & pH change </li></ul><ul><li>E protein conforma...
PROTEIN E INSERTION INTO PM Modis, Ogata, et. al., 2004.
DENGUE IN THE CELL <ul><li>Dendritic cell infection    T-cell activation </li></ul><ul><li>Previous infection = increase ...
PATHOGENIC STRATEGIES OF DENGUE  <ul><li>Invades circulatory system, causing: </li></ul><ul><li>-  vascular permeability <...
LABORATORY DIAGNOSIS OF DENGUE <ul><li>METHODS: </li></ul><ul><li>1.  Viral Isolation & Characterization </li></ul><ul><li...
VIRAL ISOLATION & CHARACTERIZATION <ul><li>Old “Gold Standard” </li></ul><ul><li>Cell Culture (mammals & mosquitoes) </li>...
GENOMIC SEQUENCING <ul><li>Quicker, more reliable means of diagnosis </li></ul><ul><li>NASBA method (RNA-specific amplific...
ANTIBODY DETECTION <ul><li>Most common methods </li></ul><ul><li>1.  Hemagglutinin inhibition test (HI test) </li></ul><ul...
MOST PROMISING VACCINE <ul><li>ChimeriVax-Dengue </li></ul><ul><li>- Tetravalent </li></ul><ul><li>- Uses yellow fever    ...
WHAT ABOUT HUMANS? <ul><li>Tetravalent vaccine </li></ul><ul><ul><li>ChimeriVax-Dengue? </li></ul></ul><ul><ul><li>20% ser...
PUBLIC HEALTH STRATEGIES  <ul><li>Vector Control </li></ul><ul><li>Surveillance </li></ul><ul><li>Preparation for outbreak...
MOSQUITO NETS images/Bed%20net.jpg
NO MORE MOSQUITOES! images/tincan.jpg images/unsanitary.jpg  ABOUT THAT STANDING WATER…
“ Children play in sewage in Nairobi's sprawling Mukuru Kaiyaba slum.”
IMPEDIMENTS <ul><li>Still lack complete understanding of dengue virus virulence </li></ul><ul><li>Social/socioeconomic </l...
REFERENCES <ul><li>“ Arthropod-borne Viruses Infection”  </li></ul><ul><ul><li>
REFERENCES <ul><li>“ Dengue Virus Profile.” (2000). .  (accesse...
REFERENCES <ul><li>Kao, C., C. King, D. Chao, H. Wu, and G. Chang. (2005). Laboratory diagnosis of dengue virus  infection...
REFERENCES <ul><li>Shu, P. and J. Huang. (2004).  Current advances in dengue diagnosis.  Clinical and Diagnostic  Laborato...
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Caitlin Reed- "Dengue Virus: No One is Safe"


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  • Dengue is a flavivirus like malaria and yellow fever. Flaviviruses are part of the larger group of arboviruses, which are viruses transmitted between arthropods and mammals. Dengue’s vectors are two types of Aedes mosquitoes; Aedes aegypti and Aedes albopictus. However, it is the Aedes aegypti species that is most notorious for transmitting dengue. In fact, there was very little information about Aedes albopictus besides that it is one of dengue’s two vectors (, 2005). There are four different strains of dengue, classified simply as dengue 1-4. These serotypes are genetically quite similar, but different enough to represent different virulence (Chamers, Liang, Droll, et. al., 2003).
  • Once infected, a person can present with three different manifestations of dengue: Dengue fever (DF) – DF is the mildest form of a dengue infection, as the virus never invades the body. The symptoms are simply fever, headache, and potentially a rash. After about a week of symptoms, it will go away and the person will feel normal again. Dengue fever is the result of a person’s first infection with the dengue virus (WHO, 2002). Dengue hemorrhagic fever (DHF) – DHF is a more severe infection, as it occurs when the virus actually enters the circulatory system and infects the entire body. DHF occurs after a second exposure to dengue, though this second exposure must be to a serotype different than the first infection. DHF can be fatal if left untreated (WHO, 2002). Dengue shock syndrome (DSS) – DSS is the most serious dengue infection. It is an extreme case of DHF, and results in death 5% of the time. However, the fatality rate is dependent upon which part of the world one lives in, as the supplies available in the Americas make the fatality rate much lower than other less fortunate countries who do not have access to simple IV equipment or a blood bank in case of the need for transfusions (WHO, 2002). This was exemplified in 1998 when there were 1.3 million cases of DHF in the world, 55% of which occurred in the Americas. There were 3600 deaths that year, but only 2% of those were in the Americas (Stephenson, 2005). All a hospital or clinic needs is clean IVs, rehydrating solution, and a supply of blood for transfusions when necessary. However, in many part of the world, these are all luxuries (WHO, 2002). People only need to worry about DHF and DSS if they have already had DF. DF is simply a fever, and the body will fight it off naturally. However, it predisposes people to DHF if there is a subsequent infection with one of the other dengue serotypes (, 2000).
  • Dengue, like all hemorrhagic viruses, is considered a Category A infectious disease by the CDC. This classification of most dangerous is due to the following characteristics of dengue: Has potential to cause panic and social disruption Greatly affects the public health in areas where it is endemic Easily transmitted Preparation to combat it demands special action (, 2004) There are an estimated 50-100 million cases of some form of dengue every year! Of those, hundreds of thousands are dengue hemorrhagic fever. Not only that, but 2.5 billion people, almost half of the world’s 6 billion + population, live in areas where dengue is endemic (WHO, 2002). Because of its ability to exploit the immune system of those it infects, dengue virus has proven very difficult to find a vaccine for, though other flaviviruses, such as yellow fever, have vaccines (Shu and Huang, 2004). There have been many recent outbreaks in the past few years, and in 1998 alone there were 1.3 million cases of DHF (WHO, 2002). In the last five years, there have been the worst outbreaks that the world has ever recorded, and in the Americas dengue has especially begun to spread. Though right now only Texas and Hawaii have the Aedes aegypti that spreads dengue, people fear that it will soon evolve to slightly cooler climates and move further north, therefore extending its range and its threat (, 2000). Global warming may also have an effect on dengue’s main vector. It may be the case that parts of the world will essentially adapt to the mosquito as the temperatures increase. Like mosquito evolution, the result would be the same; Aedes aegypti could far extend its range and therefore dengue could become endemic in many more places than it already is.
  • In the 1950s &amp; 1960s, there was a huge Aedes aegypti eradication attempt in the Americas. Though those behind this attempt were not looking to eradicate dengue, what they were looking to eradicate – urban yellow fever – has the same mosquito vector as dengue. However, after they thought their attempts were successful, they stopped what they were doing and figured nature taking its course would do no harm because there were no Aedes aegypti to spread. They were wrong. In the 1970s, these mosquitoes appeared and became more abundant than they had been pre-eradication attempts (, 2005). There has been no mention of further eradication attempts and since 1970, and the incidence of dengue has quadrupled from around 12 million cases per year to a conservative estimate of 50 million cases per year (WHO, 2002). Another problem facing people today is that previously unestablished serotypes are establishing themselves where they have never been before. For example, there was a dengue-1 outbreak in India in 1996. The more recent 2003 outbreak was of dengue-3. This could be very problematic because two stable serotype populations in the same area will lead to a large increase in the occurence of DHF and DSS, as it is the second infection by a different serotype that is the most dangerous (Shrivastava, 2004). Hawaii’s 2001 outbreak was a wake-up call for Americans. Dengue had not appeared in North America in over 20 years until the Hawaii outbreak, so the outbreak acted as a reminder that all tropical areas are at risk, not just the tropics in developing areas (Wilson &amp; Chen, 2002). Taiwan’s “outbreak” consisted of 25 people with no deaths resulting. It was used as an opportunity for data collection and further exploration into the dengue virus. It made clinicians and physicians in Taiwan more adept at recognizing DHF before it was too late to treat, and helped them understand the cyclical nature of dengue outbreaks. It also gave them demographic data as to who is most likely to be infected (30-50 year-old males, 50-70 year-old females) and when most infections occur (between October and December). Finally, it enabled them to record how long it took for those infected to be admitted to hospitals (4.3 days) and how long they usually had to stay in the hospital before the symptoms abated (one week). This information may decrease the time it takes for admittance into hospitals in the future, and allow physicians to recognize the threat of dengue hemorrhagic fever early on in their patients (Lai, Lee, Kao, et. al. 2004). Puerto Rico experienced the worst dengue outbreak in recent year. In just 11 months, there were 24,700 cases of dengue reported there between 1994 and 1995. Outbreaks like this in such a small area pose a great economic danger to that particular area, especially when it is dependent on tourism. The same applies for Hawaii. When there are dangerous, deadly outbreaks of a disease, tourists are not going to be inclined to visit those places. This could have serious detrimental effects on the economy and therefore on the people of that area (Rigau-Perez, Vorndam, and Clark, 2001).
  • Present in most tropical and sub-tropical (less humid) climates Africa Southeast Asia and China India Middle East Caribbean and Central and South America Australia and the South and Central Pacific Some parts of the U.S., namely Texas and Hawaii Dengue is found anywhere that Aedes aegypti can live, and that means any tropical and sub-tropical climate. This includes Central &amp; South America, parts of Africa, South Asia, and countries in the Pacific like Australia, New Zealand, Fiji, etc. It is also present in parts of North America, though so far the only outbreaks have been recorded in Hawaii and Texas (Stephenson, 2005). With the impending threat of mosquito evolution and global warming, however, Ae. aegypti could vastly increase its range.
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  • People contract dengue from mosquitoes, as has been established. Once dengue enters one’s body, it enters the dendritic cells (specialized cells found in most tissues) which migrate to the lymphatic system. Once in the lymphatic system, where white blood cells are produced, they target all areas where there is an abundance of WBCs, including the spleen, liver, and glands. Entering the white blood cells and lymphatic tissue gives dengue access to the circulatory system and therefore the entire body (, 1999).
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  • The first symptoms that dengue virus has entered one’s body include fever, a rash, and a headache (the dengue triad). If this is all that ever presents, then the person is safe for the time being, experiencing only dengue fever. However, if the problems become more widespread to include muscle and joint pain and a positive tourniquet test, then the person probably has DHF, a great cause for concern. This would be considered a Grade I infection (WHO, 1999). Grade II infections include the aforementioned symptoms, as well as spontaneous bleeding. This is more blatant DHF as the hemorrhaging occurs on its own and not via a tourniquet test (WHO, 1999). Grade III symptoms are indicate of DSS, and include the failure of the circulatory system, clammy skin, rapid but weak pulse, and restlessness (WHO, 1999). Grade IV symptoms are severe shock; no measurable blood pressure or pulse. Unless a patient with Grade IV symptoms receives treatment, they will die (WHO, 1999).
  • Here is just a schematic of the gradation of the dengue virus. You can see the Grade I symptoms at the top, and they get progressively worse as you read down the chart, ending at death due to shock (WHO, 1999). The first symptom is a fever. Other symptoms that might accompany the fever are a rash and a headache. These three together are known as the dengue triad, and they occur with regular dengue fever (DF). They are not necessarily cause for alarm because dengue fever often abates after a week without progression to dengue hemorrhagic fever (DHF). However, if the patient has experienced DF before, and suspect they have it again because of the dengue triad, they should consult a physician immediately because second infections are where the danger lies (, 2005). A “tourniquet test” (a.k.a. capillary fragility test) is the first test physicians do when checking to see if DF has become DHF. This entails putting a blood pressure cuff on the patient’s arm for two minutes, applying enough pressure to cut off the veinous return of blood. At the end of the two minutes, the cuff is removed and the number of burst capillaries (petechiae) are counted in a two inch area. More than 11 burst capillaries is too many, and indicates that the capillaries are either weak or there is a low platelet count, both indicative of DHF (, 1998). Hepatomegaly is the medical term for a swollen liver. This is caused by the dengue virus attacking the liver via the circulatory system. The immune response is what causes the swelling (WHO, 1999). Thrombocytopenia is a condition of a person having too few platelets. Average platelet count is 150,000-400,000/mm 3 . Dengue-infected people could have as low as 20,000! Everyone needs platelets to prevent over-bleeding when bruised. They activate clogging factor VIII and also clump together to close small holes in blood vessels throughout the body. When they do not do this, such as when a person’s vascular permeability is increased due to DHF, the person will bleed out of their blood vessels (, 2005). Hemoconcentration occurs when blood vessels lose plasma volume, a result of thrombocytopenia. Hemoconcentration is an increase in the concentration of red blood cells in the blood, which is a predecessor to shock (Shepherd, Hinfrey, and Shoff, 2002). Shock occurs when the circulatory system starts malfunctioning. The person may become unconscious, develop a lowered body temperature, and may have a weak pulse. The infection of the circulatory system by dengue may induce shock, primarily because the thrombocytopenia causes extensive internal bleeding that, left untreated, can easily kill someone (Hadinegoro, Purwanto, and Chatab, 1999).
  • This picture is an example of early hemorrhaging, known as petechiae. Petechiae are tiny dots of blood underneath the skin. They are caused by individual capillaries bursting, which is why they are so small. They are early indicators of subcutaneous hemorrhaging, and therefore a cause of concern for people who have recently been bitten by a mosquito in a dengue-endemic area, especially if they have experienced the symptoms of dengue fever before (, 2001).
  • This picture is of a condition called purpura. Purpura is indicative of more advanced hemorrhaging, as it is a sign of not just single capillary bursting but extensive blood vessel leakage. It is a result of thrombocytopenia (low platelet count) due to the dengue virus and should indicate to the person infected that they must seek medical attention immediately (, 2005).
  • This picture is perhaps the most painful looking, and illustrates advanced internal bleeding, called ecchymosis. Ecchymosis presents itself as large bruises, indicative of severe internal bleeding (hemorrhaging). Like purpura, it is caused by clotting disorders like thrombocytopenia induced by a second infection of the dengue virus (, 2003).
  • This final picture is an example of someone in late stages of DHF, bleeding from their nose. Orifice bleeding is one of the last kinds of bleeding to occur in DHF, and can happen from the ears, anus, and vagina, as well as the nose. Left untreated, this kind of hemorrhaging will inevitably lead to death (, 2005).
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  • Dengue is made up of a single positive strand of RNA surrounded by an icosahedral core. This icosahedral core is made up of 90 glycoprotein E dimers which overly protein M (Kuhn, Zhang, Rossmann, et. al., 2002). The above picture is of dengue, but all you can see are the 90 protein E dimers because they are what overlie the rest of the virus. The different colors indicate the three different domains of protein E; domain I is in red, II is in yellow, and III is in blue (Modis, Ogata, Clements, et. al., 2004). An enlarged picture of a single protein E will be shown on a coming slide. Dengue’s envelope protein E dimers are what determine a serotype’s virulence. There are different amino acids on protein E depending on which serotype it is that appear to be the difference between the four serotypes. One study conducted on dengue-2 and dengue-4 yielded results that suggested residues 155, and 402 are where the differences that affect virulence lie (there are other differences, but these are the two that the researchers believe have bearing on virulence). The following substitutions on dengue-4 occur; isoleucine for threonine at residue 155 and leucine for phenylalanine at residue 402. There was no mention of dengue-1 or dengue-3 in this study (Chambers, Liang, Droll, et. al., 2003).
  • This diagram is of dengue’s genome, split into structural proteins (C, M, and E) and nonstructural proteins, of which there are seven. The structural proteins are the capsid proteins, membrane proteins, and, most importantly, envelope proteins (the prM portion is just the premembrane) (, 2005). Dengue has an RNA polymerase encoded in it that enables it to replicate. When it does, first a precursor protein is formed and then cleavage into the 10 different protein regions above occurs. When replication is complete, there are between 10,000 and 11,000 ribonucleotides that make up the virus (, 2005).
  • This is a picture of the E protein dimer, pre-fusion with the cell membrane. Note the three domains (I, II, and III) and the fusion loops that are part of domain II, the yellow domain. These loops are responsible for membrane fusion once dengue enters the cell, a process that will explained in the coming slides (Modis, Ogata, Clements, et. al., 2004).
  • How exactly does dengue enter the cell one might ask? Well, it’s a complicated but exciting process. The first time a person is infected with dengue, it is fought off by the immune systems. The second time, however, the results of the immune response are not as successful. The following slides will explain immune response to primary and secondary infections, as well as membrane fusion.
  • The first time a person is infected with dengue, it is fought off by both the humoral and cellular immune systems. The humoral response increases the antibody (Ab) serum neutralizing levels, which prevents dengue from ever fusing with the cell membrane. The cellular response involves dendritic cells (the cells dengue enters in order to get to the white blood cells to infect the person) migrate to the lymph nodes and activate T-lymphocytes (CD4+ and CD8+). These T-cells are able to fight off the first infection and enable development of memory cells for the serotype that tried to attack the body. These memory cells will enable the person to fight off any future dengue infection of the same serotype (Ho, Wang, Shaio, et. al., 2001). The only time there is an exception to this rule is when an infant has inherited immunity from her mother. For her first year of life, she will be susceptible to DHF upon first infection with a different serotype of dengue. After a year, however, the baby will no longer have these antibodies and she will therefore not be susceptible to DHF upon first infection of the dengue virus (Halstead, 1998; Stephenson, 2005).
  • However, the problem arises upon the second infection, but only if it is of a different serotype than the first infection. What happens then is a process known as Antibody Dependent Enhancement, ADE. When someone already has antibodies for one dengue serotype and they get infected by a second serotype, those first type’s antibodies (immunoglobulin-G) try to combat it. However, when they attach themselves to the new strain, they cross-react with it and actually increase the virus-binding efficiency and disable the immune system from fighting it off as it normally would. The antibodies bind with the virus and form a complex which then binds to the Fc receptor (Mady, Erbe, Kurane, et. al., 1991). Because the antibodies are cross-reactive, though, and do not kill the virus, it can then enter the cell (usually a phagocyte), fuse with the membrane, and begin causing extensive damage because it triggers the immune system to release excessive amounts of proinflammatory cytokines. The excessive release of these cytokines puts pressure on the blood vessels and increase their permeability, which is what initially causes the hemorrhaging characteristic of dengue hemorrhagic fever (Stephenson, 2005).
  • Like all flaviviruses, the E protein of dengue is responsible for fusion to the host cell (a macrophage). After fusion with the Fc receptor, it enters an endosome where the pH is low enough to cause a conformational change of the E protein which enables the virus to merge with the endosomal membrane and release the viral RNA into the target cell. From here, the virus can replicate and ultimately leave the cell to infect other cells (, 2004). The following slides show the process of the E protein fusion and viral replication.
  • Inside the endosome of the animal cell, the pH is lower than outside. This causes a conformational change of the E protein of dengue, which ultimately enables membrane fusion of the virus and the endosome. When the pH becomes more acidic, the E protein transforms from dimeric form into trimeric form. Domain III shifts 70 degrees so it is closer to the hydrophobic loops of domain II, and thereby forcing the target membrane and viral membrane together and enabling the virus to release its RNA into the cell and begin the replication process (Modis, Ogata, Clements, and Harrison, 2004).
  • These pictures illustrate protein E after membrane fusion. There are three residues in dengue (and in all flaviviruses) on the fusion loops that are exposed to the cell membrane post-conformational change. These are Tryptophan-101, Leucine-107, and Phenylalanine-108. Together they form a hydrophobic rim that enables the protein to insert itself into the membrane. To stay in the membrane, there is an aromatic (hydrophobic) anchor made up of Trp 101 and Phe 108. The black dot you see near the fusion loops is a chloride ion (Modis, Ogata, Clements, and Harrison, 2004). As with all single-stranded RNA viruses, it is at this point that dengue can release its RNA into the cell, replicate, and spread.
  • This slide is just a reminder of how viruses act once inside the cell. After the RNA is released into the cell, the replication process can occur and then the disease can proliferate and spread throughout the body.
  • To summarize, dendritic cells are the first cells dengue enters when a person is infected by the virus. When dengue attacks dendritic cells, they mature and migrate to the lymphatic system. This migration activates CD4+ and CD8+ T-lymphocytes and, if it is a first infection, these lymphocytes are enough to kill the virus. If it is a second infection by a different serotype though, the antibodies for the first serotype actually increase the speed of infection and onset of hemorrhagic fever by increasing the efficiency of viral uptake. The antibodies for a different strain try to fight off the new strain, but actually cross react with it and form a complex with it that enables it to enter the macrophage via the Fc receptor. The antibodies’ attempt to fight it off means they seek it out and attach themselves to it quickly, so more of it may be taken up, therefore an increased viral load and decreased incubation period often occur with second infections of a different strain. Another thing that dengue infection prompts is the excessive secretion of proinflammatory cytokines. The excessive release of these cytokines actually make the disease worse rather than combat by putting too much pressure on the endothelial cells in the vascular system, which can cause increased permeability and therefore plasma leakage, a symptom indicative of DHF (Ho, Wang, Shaio, et. al., 2001). The aforementioned immune system response of secondary infections of a different strain is known as antibody dependent enhancement, ADE. However, ADE only lasts for 20 years. If someone goes 20 years without ever getting DHF, they should not develop the condition if they get bitten later on by a mosquito infected with a different strain from their original infection. This means that a one-time infection does not require a person to live in fear forever if they are in areas where dengue exists, or if they want to travel to these areas. However, they will have to be extremely careful for 20 years, a fate not so bad if the person is infected as a child (Stephenson, 2005).
  • This slide summarizes the pathogenic strategies of dengue. Though much is known about the virus’s entry into the host cell, not much is understood about its exact mechanism of action. What is know is that once inside the circulatory and lymphatic systems, it is able to increase vascular permeability by causing excessive cytokine release, which subsequently causes plasma leakage. When this internal bleeding happens, the person’s platelets aggregate to clog the holes in the blood vessels. However, continuous bleeding requires many platelets, and after a while a phenomena called disseminated intravascular coagulation occurs in which the clotting factors are all used. This leads to extensive internal bleeding (hemorrhaging) and a malfunction of the circulatory system. When this occurs, the person may go into shock and internally bleed to death if left untreated by blood transfusion (WHO, 1999). The above picture is of a blood clot (
  • The following slides will explain the three main ways physicians are able to identify dengue.
  • This slide lists the primary means of dengue identification. Viral isolation and characterization, genomic sequencing, and antibody detection are the most commonly used methods in laboratories dealing with dengue. All will be explained in the following three slides (Shu and Huang, 2004).
  • Currently, labs use mosquito cells more often than human cells when culturing the cells they need for virus isolation. However, cell culture is not as sensitive as RT-PCR (Reverse Transcriptase Polymerase Chain Reaction), so researchers have begun using RT-PCR for quicker results. Cell culture and recognition by means of indirect immunofluorescence (glows apple green as shown above) requires four days for results, whereas RT-PCR combined with the molecular techniques of isolation and identification takes just one day. This large discrepancy in time it takes to diagnose means that more labs are turning to a molecular method, especially when they are working with patients who potentially have DHF. When studying dengue solely to understand its virology, epidemiology, and pathogenesis, using serum from mosquitoes or blood samples from people known to have DHF, virus isolation by means of cell culture is a useful technique. However, when outbreaks occur, it is integral to have quicker methods of recognition and diagnosis, and that is when genomic sequencing proves very useful (Shu and Huang, 2004).
  • Genomic sequencing is quickly becoming a highly used and reliable source of virus identification. NASBA (Nucleic Acid Sequence Based Amplification) is one of the more recent techniques, and it uses an isothermal RNA-specific amplification assay. This enables researchers to find viral and bacterial RNA in the clinical samples. Because of its temperature of conduction (41 degrees Celsius), NASBA is a practical, efficient, and sensitive way to conduct epidemiological studies with dengue virus (Shu and Huang, 2004). However, an even more accurate technique is RT-PCR (picture above). Its many benefits include its quickness, reliability, sensitivity, and ease at which it can be conducted. It is used with acute serum samples, and though there are five options for detection, one is used most often because it seems to provide the most accuracy, and that is the 5’-3’ oligonucleotide probe (aka TaqMan assay). Its accuracy lies in its sequence specificity. Researchers are currently working on developing a multiplex TaqMan assay that would enable them to detect all four serotypes of dengue. Right now, TaqMan can recognize the dengue virus, but not differentiate among the serotypes because of their very similar genomes. By using TaqMan in addition to other chemical formats, researchers are trying to advance their ability to detect the different serotypes. These other chemical formats are DNA binding fluorophores, 5’ nuclease, and self-fluorescing amplicons (Shu and Huang, 2004).
  • The hemagglutinin inhibition test (HI test) has been the most commonly used test for diagnosing dengue. It has the ability of differentiating between primary and secondary infections and is a relatively easy test to carry out. However, HI tests are not as sensitive as other techniques, and are being replaced by ELISA and rapid immunochromatography tests (Shu and Huang, 2004). ELISA (Enzyme-Linked Immunosorbent Assay) is quickly replacing HI tests because it is more sensitive, specific, and simple than the HI test, and there are even commercial kits available. There are two types of ELISA used to detect dengue; IgM and IgG. The IgM ELISA is most effective with primary infections, as IgM antibodies become detectable soon after the first infection (about 3 days). These antibodies are at their peak two weeks after infection, but then become undetectable after a couple of months. It is at this time that IgG antibodies form and remain in the body, which is why IgG ELISA is useful for secondary infections. If a person gets bit by a mosquito and they fear a dengue infection, they can go to a hospital almost immediately and have their IgG antibody level measured. If it is higher than it would be normally, the physician can deduce that they have probably again been infected by the dengue virus. This kind of expediency is very important when it comes to diagnosis (Shu and Huang, 2004). Finally, there are very quick immunochromatography test kits available commercially (as shown above) that can detect both IgM and IgG antibodies, and claim to be able to differentiate between primary and secondary infections. Immunochromatography works by passing capillaries through a membrane to separate out different components (such as antibodies). Testing has suggested that though these tests can definitely determine the presence of dengue, their accuracy in testing for IgM antibodies is lower than ELISA. However, their accuracy in testing for IgG antibodies seems to be greater than ELISA, which suggests they would be good for secondary infections but not great for primary ones. Researchers recommend these be used by clinicians and physicians for a quick, easy, early detection of dengue, as they only take 5-30 minutes, but not necessarily the only test used. They would be a good way of screening for dengue, as they are indeed great at detecting antibodies, and they are more likely to present with false-positives than false-negatives. When it comes to definitively diagnosing the dengue virus though, more accurate tests should be used (Shu and Huang, 2004). It is important to note here that though all these tests can detect antibodies, they do not differentiate among the serotypes. This is an ongoing challenge vaccine developers face, and will be described in the coming slides.
  • Vaccine development for the dengue virus is extremely difficult because there is not just one dengue virus, but rather four. To make vaccine development even harder, each serotype only provides immunity for itself and actually INCREASES susceptibility to a more severe form of dengue if infection by a different serotype occurs (Chambers, Liang, Droll, et. al., 2003). This challenges vaccine developers because a vaccine suitable for all four serotypes would have to have enough of each serotype to create antibodies, but not so much that one of them would cross-react with another’s antibodies and actually cause DHF. Also, being made of single-stranded RNA means that mutations can happen rather easily. This could cause a reversion from non-virulent to virulent strains and subsequently the onset of DHF (Stephenson, 2005). Public health strategies could be implemented that could decrease the number of yearly infections, but are extremely hard to enforce in many parts of the world where dengue is endemic. I will explore potential public health strategies that could quell dengue while vaccines are being developed. Dengue is too big of a problem to put all one’s hope in vaccines, and any decrease in prevalence would be progress, as outbreaks have only been increasing in recent years.
  • The most promising vaccine to date is being worked on in the U.S. and it uses the same base as the yellow fever virus vaccine, but replaces the premembrane and virion envelope genes for yellow fever with the ones for each dengue serotype. Current studies are largely successful because the genomes remain stable even after 20 passages through cells. The mode of experimentation required inoculating 24 monkeys with the tetravalent ChimeriVax-Dengue vaccine one time (a subcutaneous shot) and then exposing them to a “virulent virus challenge” (exposing them to the dengue virus) six months after the vaccination. All but two of the monkeys were resistant to the disease at that six month mark, an encouraging result for vaccine developers and all those who live in dengue-endemic areas. Every single monkey in the trial successfully seroconverted (created antibodies) for each dengue serotype, and none of the subjects experienced any cross-reactivity. The side effects (brain lesions) of the dengue vaccine in the test monkeys is substantially less than those found with the yellow fever vaccine that is used as this one’s backbone, and there were no problems with reversion to virulence at the time of publication (Guirakoo, Pugachev, and Zhang, 2004). The picture on this slide shows how scientists removed portions of the dengue virus (using cloned plasmids) and inserted them into the yellow fever vaccine doing a three-fragment ligation, after the premembrane and envelope portion of the yellow fever vaccine were removed. Creating three plasmids was necessary because the 5’/3’Den1 plasmid came out with a mutation at the M-39 position during the original cloning process. It required the researchers to conduct oligonucleotide-directed mutagenesis to rid it of the mutation and create the mutation-free clone (and “extra” plasmid). The final plasmid was able to be cloned without mutations. After the two mutation-free plasmids were created, the ligation could occur and vaccine be completed. This had to be done four times, one for each dengue serotype, and then the four monovalent vaccines could be put together into the tetravalent formula that is called tetravalent ChimeriVax-Dengue vaccine (Guirakoo, Pugachev, and Zhang, 2004).
  • There were trials done on 10 human subjects using a tetravalent dengue vaccination, and the results were not as promising as they were in the monkeys. However, the paper citing this experiment does not refer to their vaccine as the aforementioned ChimeriVax-Dengue vaccine, so it is possible that a different tetravalent dengue vaccine was administered. What these scientists found was that only 20% of the subjects (just two out of the ten) seroconverted for all four serotypes (Sun, Edelman, Kanesa-Thasan, et. al, 2003). This is dangerous because of the problem with cross-reactivity and dengue. Incomplete seroconversion could lead to cross-reactivity of one of the serotypes that has no antibodies with one of the other serotype’s antibodies, and hemorrhagic fever could result. Clearly more research on humans will be necessary to creating a more successful vaccine.
  • Currently WHO has the above plan in order to combat dengue, though there is little information about their implementation. The available information suggests that doing these things would decrease the prevalence and fatality rates of those infected with the dengue virus, but it is not clear how they would carry each step out (WHO, 2002). However, it can be assumed that they would try and control Aedes aegypti the same way they try to control all other mosquito vectors, which requires decreasing the amount of standing water where the mosquitoes could breed. The use of insecticides is also an option. Surveillance would require people reporting sightings of Aedes aegypti and researchers observing areas where they might be present. Preparation for outbreaks would involve providing clinics and hospitals with the right equipment; mainly IVs, solution, and blood for transfusions. Research would entail spending a lot of time studying the habits of Aedes aegypti ; when they are most likely to feed, type of areas they live in, if there is a pattern of outbreaks, etc. Outbreaks are a great time for research, unfortunately, as they enable scientists to learn the infectious habits of Aedes aegypti, as was exemplified in the Taiwan outbreak mentioned at the beginning of the talk. Hopefully, with the number of outbreaks the world has experienced in recent years, advances will be made in stopping dengue’s spread and rate of infection.
  • The following slides are to show that the spread of dengue is not a problem that can be solved solely with biotechnology. There are social measures that need to be taken to decrease the incidence and prevalence of this potentially fatal virus, which include providing people not just with vaccines but with mosquito nets or long clothing to protect them from bites. In the U.S., we may be able to eliminate standing water by cleaning out our backyard, but much more drastic measurements are required in other countries where such sewage and/or drainage capabilities do not exist. Children need to know where it is safe to play and where it is dangerous. In America, we have filtration systems so our sewage does not mix with the water we swim in and drink from, but that is a luxury. These are the things that can be done right now to start decreasing dengue’s threat. Vaccines are in the future, but preparation at a grassroots level can start immediately.
  • Mosquito nets are useful for people during the night. For many mosquito-borne illnesses, mosquito nets are ideal. However, Aedes aegypti do not feed during the night. They feed in the morning and afternoon, and are apt to live inside as well as outside; wherever there is standing water. This makes mosquito nets minimally protective. However, they are inexpensive and do offer some protection for the morning hours, so they could definitely help, especially if they are dipped in insecticide so the mosquitoes die upon contact instead of just get temporarily deterred (, 1999).
  • The elimination of standing water is key to preventing mosquitoes from breeding nearby. Besides pails like these, swimming pools, bird baths, even tires are great breeding grounds for mosquitoes, especially if it’s warm out because Aedes aegypti flourish in warm climates.
  • Latin America Some people do not have the luxury of removing the standing water surrounding their homes. In many places, there is no clean-up patrol after floods, there are no knee-high rubber boots, and there is no difference between the places the sewage goes and the places the kids play.
  • Their innocence shields them from fear, but the ramifications of them playing in the sewage go beyond smelling bad. Besides mosquito-borne illnesses, these kids are exposing themselves to numerous other diseases, such as E. Coli , Shigella , and Giardia .
  • Scientists are still trying to determine dengue virus’s inner workings, and are still working on finding out the root of its virulence. Until this is understood more in depth, it is hard to know what to target when creating vaccines. Though it is known that the E protein is where the as of yet discernible differences lie, it is not clear whether or not this is the only place where there are differences that could also affect virulence (Halstead, 1988). Socially, it is very hard to enforce mosquito eradication programs because people commonly have standing water all around their property, whether it is from negligence (leaving things out that may catch rain) or luxury (pools, bird baths). Especially in the areas where dengue is endemic, there is an abundance of breeding grounds for mosquitoes that people do not have the power to empty. Until public health specialists figure out a way to enforce eradication attempts in developing countries, it will be hard for the public health strategies to be very effective in dengue control. Since marketable vaccines are non-existent right now, though, it is important to at least try to enforce the eradication techniques such as insecticide and surveillance, the latter enabling scientists and physicians to know when to prepare for an outbreak and perhaps stop it before it gets out of control as it has in the past (WHO, 2002). With increased air travel, people are capable of bringing a disease to an area where it has not posed a threat in recent years and potentially make it endemic. If someone visits Singapore and contracts dengue-1, then returns to their home in northeastern Australia where dengue-3 is present, and gets bitten by an Aedes aegypti mosquito, that mosquito could then become a transmitter of dengue-1 to another individual and dengue could start spreading and the incidence of hemorrhagic fever could increase having two established serotypes. Demographically, the increase in the world’s population means overcrowding, which results in substandard sanitation. This not only enables mosquitoes to breed, but also helps the disease to spread rapidly (WHO, 2002). Finally, and surprisingly, WHO acknowledged the lack of a centralized public health system being a big problem. This results in people looking to respond to the emergency outbreaks instead of prevent them. It is unclear who has the jurisdiction to implement programs, and who can enforce them. Until there is a more centralized, unified system, dengue, and other infectious diseases, will continue to spread (WHO, 2002).
  • Caitlin Reed- "Dengue Virus: No One is Safe"

    1. 1. DENGUE VIRUS: NO ONE IS SAFE Caitlin Reed Smith College April 29, 2005
    2. 2. OVERVIEW OF THEMES <ul><li>Background Information </li></ul><ul><li>Clinical Presentation & Diagnosis </li></ul><ul><li>Biology </li></ul><ul><li>Vaccination Prospects </li></ul><ul><li>Public Health </li></ul>
    3. 3. WHAT IS DENGUE? <ul><li>Flavivirus (type of arbovirus) </li></ul><ul><li>Transmitted from Aedes aegypti and Aedes albopictus mosquitoes </li></ul><ul><li>Four Serotypes (Dengue 1-4) </li></ul>
    4. 4. DENGUE (cont’d) <ul><li>Three Manifestations: </li></ul><ul><li>1. Dengue Fever </li></ul><ul><li>2. Dengue Hemorrhagic Fever </li></ul><ul><li>3. Dengue Shock Syndrome </li></ul><ul><li>Leads to death in 5% of cases </li></ul><ul><li>More dangerous if infected second time by different serotype </li></ul>
    5. 5. WHY DO WE CARE ABOUT DENGUE? <ul><li>CDC Category A Infectious Disease </li></ul><ul><li>Infects 50-100 million people every year </li></ul><ul><li>About half the world lives in a “hot zone” </li></ul><ul><li>Very hard to create vaccine </li></ul><ul><li>Mosquito evolution = threat to U.S. </li></ul><ul><li>Global warming </li></ul>
    6. 6. WHY NOW? <ul><li>Failed eradication attempt in the Americas in 1970 </li></ul><ul><li>Previously unestablished serotypes are establishing themselves in various countries </li></ul><ul><li>Recent Outbreaks: </li></ul><ul><li>1. India, 2003 </li></ul><ul><li>2. Hawaii, 2001 </li></ul><ul><li>3. Taiwan, 2001 </li></ul><ul><li>4. Puerto Rico, 1994-1995 </li></ul>
    9. 9. <ul><li>Mosquitoes transmit </li></ul><ul><li>dengue to human dendritic </li></ul><ul><li>cells </li></ul><ul><li>2. Dengue targets areas </li></ul><ul><li>with high WBC counts </li></ul><ul><li>(liver, spleen, lymph </li></ul><ul><li>nodes, bone marrow, and </li></ul><ul><li>glands) </li></ul>3. Dengue enters WBCs & lymphatic tissue 4. Dengue enters blood circulation HOW DENGUE SPREADS 3 4 1 2 3
    11. 11. SYMPTOMS OF DHF GRADE I: Fever with other symptoms such as vomiting, headache, muscle and joint pain: positive tourniquet test is the only evidence of hemorrhaging GRADE II: Grade I symptoms + spontaneous bleeding GRADE III*: Failure of circulatory system, clammy skin, rapid & weak pulse, restlessness GRADE IV*: Severe shock, no measurable blood pressure or pulse *Considered Dengue Shock Syndrome (DSS)
    13. 13. P E T E C H I A E
    14. 14. P U R P U R A
    15. 15. E C C H Y M O S I S
    18. 18. BASIC BIOLOGY <ul><li>Single, positive-stranded RNA surrounded by an icosahedral core </li></ul><ul><li>90 glycoprotein E dimers overly M proteins </li></ul><ul><li>Protein E is most important characteristic of dengue </li></ul>Modis, Ogata, Clements, et. al., 2004
    20. 20. FUSION PROTEIN E Modis, Ogata, et. al., 2004 .
    22. 22. FIRST INFECTION <ul><li>Humoral and cellular immune response </li></ul><ul><li>- Ab serum neutralizing levels increase </li></ul><ul><li>- T-lymphocytes activated by dendritic cells </li></ul><ul><li>- Memory cells develop antibodies to fight off future infection of same serotype </li></ul>
    23. 23. SECOND INFECTION <ul><li>Antibody dependent enhancement </li></ul><ul><ul><li>- Enhancing immunoglobulin G (IgG) antibodies </li></ul></ul><ul><ul><li>- Fc Receptors </li></ul></ul>
    25. 25. ENTRY INTO CELL <ul><li>Dengue infection </li></ul><ul><li>Endosome entry & pH change </li></ul><ul><li>E protein conformational change </li></ul><ul><li>Release of viral RNA into cell </li></ul><ul><li>Replication & further infection </li></ul>
    27. 27. PROTEIN E INSERTION INTO PM Modis, Ogata, et. al., 2004.
    30. 30. DENGUE IN THE CELL <ul><li>Dendritic cell infection  T-cell activation </li></ul><ul><li>Previous infection = increase in viral load and decrease in incubation period </li></ul><ul><li>ADE is problem for 20 years after first infection </li></ul>
    31. 31. PATHOGENIC STRATEGIES OF DENGUE <ul><li>Invades circulatory system, causing: </li></ul><ul><li>- vascular permeability </li></ul><ul><li>- Disseminated intravascular coagulation </li></ul><ul><li>- Potentially death </li></ul>
    33. 33. LABORATORY DIAGNOSIS OF DENGUE <ul><li>METHODS: </li></ul><ul><li>1. Viral Isolation & Characterization </li></ul><ul><li>2. Genomic Sequencing </li></ul><ul><li>3. Antibody Detection </li></ul>
    34. 34. VIRAL ISOLATION & CHARACTERIZATION <ul><li>Old “Gold Standard” </li></ul><ul><li>Cell Culture (mammals & mosquitoes) </li></ul><ul><li>-Indirect Immunofluorescence </li></ul><ul><li>Useful to study basic virology, epidemiology, </li></ul><ul><li>and pathogenesis </li></ul><ul><li>Impractical for rapid diagnosis & treatment </li></ul>
    35. 35. GENOMIC SEQUENCING <ul><li>Quicker, more reliable means of diagnosis </li></ul><ul><li>NASBA method (RNA-specific amplification assay) </li></ul><ul><li>RT-PCR method to provide most accuracy, uses 5’-3’ nuclease oligonucleotide probe (which may not be able to distinguish among serotypes) – new “Gold Standard” </li></ul><ul><li>Beware of false-positives due to contamination </li></ul>
    36. 36. ANTIBODY DETECTION <ul><li>Most common methods </li></ul><ul><li>1. Hemagglutinin inhibition test (HI test) </li></ul><ul><li>2. ELISA </li></ul><ul><li>3. Rapid immunochromatography test (commercial kits available) </li></ul>
    38. 38. MOST PROMISING VACCINE <ul><li>ChimeriVax-Dengue </li></ul><ul><li>- Tetravalent </li></ul><ul><li>- Uses yellow fever vaccine as base </li></ul><ul><li>- 92% of monkeys passed “virulent virus challenge” </li></ul>Guirakoo, Pugachev, and Zhang, 2004
    39. 39. WHAT ABOUT HUMANS? <ul><li>Tetravalent vaccine </li></ul><ul><ul><li>ChimeriVax-Dengue? </li></ul></ul><ul><ul><li>20% seroconversion rate </li></ul></ul><ul><li>More research necessary! </li></ul>
    40. 40. PUBLIC HEALTH STRATEGIES <ul><li>Vector Control </li></ul><ul><li>Surveillance </li></ul><ul><li>Preparation for outbreaks </li></ul><ul><li>Research </li></ul>
    42. 42. MOSQUITO NETS images/Bed%20net.jpg
    43. 43. NO MORE MOSQUITOES! images/tincan.jpg
    44. 44. images/unsanitary.jpg ABOUT THAT STANDING WATER…
    45. 45. “ Children play in sewage in Nairobi's sprawling Mukuru Kaiyaba slum.”
    46. 46. IMPEDIMENTS <ul><li>Still lack complete understanding of dengue virus virulence </li></ul><ul><li>Social/socioeconomic </li></ul><ul><li>Travel spreads different serotypes </li></ul><ul><li>Demographic changes </li></ul><ul><li>Decentralized and therefore weak public health systems </li></ul>
    47. 47. REFERENCES <ul><li>“ Arthropod-borne Viruses Infection” </li></ul><ul><ul><li> (accessed on April 2, </li></ul></ul><ul><ul><li>2005). </li></ul></ul><ul><li>“ Bioterrorism Agents/Diseases” (2004). category.asp (accessed on April 12, 2005). </li></ul><ul><li>“ Bleeding Into the Skin.” (2003). (accessed on April 12, 2005). </li></ul><ul><li>“ Capillary Fragility Test.” (1998). (accessed on April 5, 2005). </li></ul><ul><li>CDC Dengue Fever Homepage. (2005). . (accessed on March 3, 2005). </li></ul><ul><li>CDC Slideshow. (1999). “Dengue: Virus, Vector, and Epidemiology.” (accessed on April 1, 2005) </li></ul><ul><li>Chambers, T.J., Y. Liang, D.A. Droll, J.J. Schlesinger, A.D. Davidson, P.J. Wright, X. Jiang (2003). Yellow Fever Virus/Dengue-2 Virus and Yellow Fever Virus/Dengue-4 Virus Chimeris: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model. Journal of Virology. 77 :3655-3668. </li></ul><ul><li>“ Dengue Triad.” (2005). http:// . (accessed on March 31, 2005). </li></ul>
    48. 48. REFERENCES <ul><li>“ Dengue Virus Profile.” (2000). . (accessed on April 4, 2005). </li></ul><ul><li>Guirakhoo, F., K Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath. (2004). Safety and efficacy of chimeric yellow fever-dengue virus tetravalent vaccine formulations in nonhuman primates. Journal of Virology. 78 :4761-4775. </li></ul><ul><li>Halstead, S.B. (1988). Pathogenesis or dengue: challenges to molecular biology. Science. 239: 476-481. </li></ul><ul><li>“ Hemorrhagic Fevers.” 2005. (accessed on April 3, 2005). </li></ul><ul><li>Ho, L., J. Wang, M Shaio, C. Kao, D. Chang, S. Han, and J. Lai. (2001). Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production. The Journal of Immunology. 166: 1499-1506. </li></ul><ul><li>Kalayanarooj, S. (1999). Standardized clinical management: evidence of reduction in dengue hemorrhagic fever child fatality rate in Thailand. Dengue Bulletin. 23 . (accessed on April 2, 2005). </li></ul>
    49. 49. REFERENCES <ul><li>Kao, C., C. King, D. Chao, H. Wu, and G. Chang. (2005). Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health. J. Microbiol. Immunol. Infect. 38 : 5-16. </li></ul><ul><li>Kuhn, R.J., W. Zhang, M.G. Rossmann, S.V. Pletney, J. Corver, E. Lenches, C.T. Jones, S. Mukhopadhyay, P.R. Chipman, E.G. Strauss, T.S. Baker, and J.H. Strauss. (2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell Press. 108 :717-725. </li></ul><ul><li>Lai, P., S. Lee, C. Kao, Y. Chan, C. Huang, W. Lia, S. Wann, H. Lin, M Yen, and Y. Liu. (2004). Characteristics of a dengue hemorrhagic fever oubreak in 2001 in Kaohsiung. J. Microbiol. Immunolo. Infect. 37 : 266-270. </li></ul><ul><li>“ Lymph Nodes.” (accessed on April 1, 2005). </li></ul><ul><li>Mady, B.J., D.V. Erbe, I. Kurane, M.W. Fanger, and F.A. Ennis. (1991). Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. Journal of Immunology. 147 :3139- 3144. </li></ul><ul><li>Modis, Y., S. Ogata., D. Clements, S. Harrison. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature. 427 :313-318. </li></ul><ul><li>Perez, J., A Vorndam, and G. Clark. (2001). The dengue and dengue hemorrhagic fever epidemic in Puerto Rico, 1994-1995. Am. J. Trop. Med. Hyg. 64 : 67-74. </li></ul><ul><li>“ Petechiae.” (2001). http:// / (accessed on April 3, 2005). </li></ul><ul><li>Shepherd, S., P. Hinfrey, and W.H. Shoff. (2002). Dengue Fever. . (accessed April 12, 2005). </li></ul><ul><li>Shrivastava, R. (2004). Dengue haemorrhagic fever: a global challenge. Indian Journal of Medical Microbiology. 22 :5-6. </li></ul>
    50. 50. REFERENCES <ul><li>Shu, P. and J. Huang. (2004). Current advances in dengue diagnosis. Clinical and Diagnostic Laboratory Immunology. 11 :642-650. </li></ul><ul><li>Stephenson, J. (2005). Understanding dengue pathogenesis: implications for vaccine design. Bulletin of the WHO. 83 : 308-314. </li></ul><ul><li>Sun, W., R. Edelman, N. Kanesa-Thasan, K.H. Eckels, J.R. Putnak, A.D. King, H. Houng, D. Tang, J. M. Scherer, C.H. Hoke, and B. Innis. 2003. Vaccination of human volunteers with monovalent and tetravalent live-attenuated dengue vaccine candidates. Am. J. Trop. Med. Hyg. 69 : 24-31. </li></ul><ul><li>“ Thrombocytopenia.” (2005). (accessed on April 2, 2005). </li></ul><ul><li>World Health Organization. (2002). Dengue: Strategic direction for research. . (accessed on March 20, 2005). </li></ul><ul><li>World Health Organization. (1999). Regional guidelines on dengue/DHF Prevention and Control: Clinical manifestations and diagnosis. (accessed on April 1, 2005). </li></ul><ul><li>Wilson, M. and L. Chen. (2002). Dengue in the Americas. Dengue Bulletin. 26 : 44-61. </li></ul>
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