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Experimental Biology and Medicine 233:401-408 (2008)
doi: 10.3181/0707-MR-198
© 2008 Society for Experimental Biology and Medicine

MINIREVIEW

Alternate Hypothesis on the Pathogenesis of Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) in Dengue Virus Infection

Sansanee Noisakran*,{dagger} and Guey Chuen Perng{ddagger},1

* Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, 12120, Thailand; {dagger} Medical Molecular Biology Unit, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; and {ddagger} Department of Pathology and Laboratory Medicine, Emory Vaccine Center, School of Medicine, Emory University, Atlanta, Georgia 30322

1 To whom requests for reprints should be addressed at Department of Pathology and Laboratory Medicine, Emory Vaccine Center, School of Medicine, Emory University, Dental Building Room 429, 1462 Clifton Road, Atlanta, GA 30322. E-mail: gperng{at}emory.edu


   Abstract
Top
 Abstract
Introduction
 Conclusions
 References
 
Dengue fever, caused by infection with dengue virus, is not a new disease, but recently because of its serious emerging health threats, coupled with possible dire consequences including death, it has aroused considerable medical and public health concerns worldwide. Today, dengue is considered one of the most important arthropod-borne viral diseases in humans in terms of morbidity and mortality. Globally, it is estimated that approximate 50 to 100 million new dengue virus infections occur annually. Among these, there are 200,000 to 500,000 cases of potential life-threatening dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), characterized by thrombocytopenia and increased vascular permeability. The death rate associated with the more severe form DHF/DSS is approximately 5%, predominantly in children under the age of 15. Although intensive efforts have been made to study the early clinical pathophysiology of dengue infection with the objective to identify the potential cause of DHF, results or data that have accumulated from different regions of the world involving studies of different ethnicity groups are inconsistent at present in terms of identifying a unified hypothesis for the pathogenesis of DHF/DSS. Thus, the potential mechanisms involved in the pathogenesis of DHF and DSS remain elusive. The purpose of this review is to identify alternate factors, such as innate immune parameters, hyper-thermal factors, conditioning of neutralizing antibody, concept of vector transmission, and physical status of virus in viremic patients that may play a role in the induction of DHF and DSS, which might have directly or indirectly contributed to the discrepancies that are noted in the literature reported to date. It is the hope that identification of an alternative explanation for the pathogenesis of DHF/DSS will pave the way for the institution of new strategies for the prevention of this complicated disease.

Key Words: Flavivirus • DHF • DSS • emerging infectious disease


   Introduction
Top
 Abstract
 Introduction
Conclusions
 References
 
Dengue Virus.
The dengue viruses, the cause of dengue illness, are members of the Flaviviridae family. Four genetically related but distinct serotypes, designated DENV-1, DENV-2, DENV-3, and DENV-4, are circulating worldwide (1). The main vector for dengue virus transmission is the Aedes aegypti species of mosquitoes. Dengue viruses, similar to other flaviviruses, have a positive single-stranded RNA genome packaged inside a core protein, which is surrounded by an icosahedral scaffold and covered by a lipid envelope (2). The genome of the dengue virus contains an 11-kb plus-sensed RNA encoding 3 structural proteins and 7 nonstructural proteins (Fig. 1Go). Viral protein and RNA synthesis occur predominantly in the cytoplasm of host cells (3). Replication is slow and begins within 15 hrs after infection. Low amounts of dengue virus are released into the supernatant fluid. Dengue virus replication does not significantly affect the metabolic function of the host cell as exemplified by normal levels of protein synthesis by the infected host cells (3).


Figure 1
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  		Figure 1. The dengue viral genome. Noncoding regions with their terminal structures are indicated by black lines. The single open reading frame encodes a poly-protein that is processed by the viral NS2B-NS3 protease and cell proteases to the mature viral proteins. Structural proteins are C, prM, and E. Nonstructural proteins are 1, 2A, 2B, 3, 4A, 4B, and 5. The genome is not drawn to scale. A color figure is available in the online version.

 
Epidemiology of Dengue Infection.
Dengue infections are at present one of the most common mosquito-borne viral diseases of humans worldwide. Initially, dengue infections were primarily recorded when they occurred as epidemics in tropical and subtropical countries. But over time, increasing globalization and human movement coupled by the increase in the geographic area where the Aedes aegypti mosquito vector inhabit, has promoted dengue virus infection to nearly every corner of the world (4, 5). The National Institute of Allergy and Infectious Diseases (NIAID) has listed dengue virus as a category A priority biothreat pathogen (6). Approximately 50 to 100 million people contract dengue fever annually, and about 200,000 to 500,000 contract dengue hemorrhagic fever (DHF), and the mortality rate is about 5%, predominantly in children under 15 years of age (7). The pathogenesis of DHF and dengue shock syndrome (DSS) is poorly understood. Epidemiologically, secondary infections, which occur commonly in dengue-endemic areas, have correlated well with the occurrence of severe dengue viral illness and therefore are considered as one of the major risk factor for severe dengue disease (8–10). This view led to the advancement of the antibody-dependent enhancement theory three decades ago (11). Other notable risk factors for DHF include the strain/serotype of the infecting virus, age of the patient, and the genetic background of the patient (2, 12). Recently, evidence has been presented that suggests that not only dengue virus serotypes, but flaviviruses in general may share peptide sequences, which serve as immunodominant determinants. T cells that recognize such determinants once primed during primary infection with one of the dengue virus serotype or a member of the Flavivirus family respond vigorously (memory T-cell responses) upon exposure to a second infection releasing massive amounts of proinflammatory cytokines, which induce vascular endothelial cell activation and are the likely cause of the capillary leak syndrome and the basis of DHF pathogenesis (13–15). While this concept emerged specially from studies of mice immunized with recombinant proteins of the dengue or Flavivirus, evidence for its occurrence in the clinical setting continues to be a subject of intense debate.

Clinical Manifestations of Dengue Disease.
Each of the four dengue virus serotypes is capable of causing a spectrum of diseases ranging from mild infection to a potentially deadly disease. Infection with one serotype leads to life-long immunity to that serotype but only partial and temporary immunity to the others (16). As outlined above, circulation of more than one serotype upon infection can increase the risk of serious and complicated infections, i.e., DHF and DSS (10). Infection with any serotype can be asymptomatic or lead to one of the four clinical scenarios of increasing severity: undifferentiated fever, dengue fever, DHF, and DSS (1, 17).

Dengue Fever.
Dengue fever (DF) follows the bite of a mosquito carrying infectious dengue virus. DF is an acute and a self-limited dengue disease, and is a syndrome that is associated with the occurrence of fever that lasts from 2 to 7 days, headache, myalgia, bone/joint pain and rash, often accompanied by leucopenia. Occasionally variable degrees of thrombocytopenia and cutaneous hemorrhage are observed. More severe cases with incapacitating bone/joint pain ("break-bone-fever") are common among adults. Infrequently, DF may be accompanied by unusual bleeding complications that may cause death (18).

Dengue Hemorrhagic Fever.
The clinical features of DHF in many aspects are very similar to that of DF during the early febrile phase. The prominent feature of DHF is its potential to develop into fatal DSS. The major pathophysiologic hallmarks that determine disease severity and distinguish DHF from DF are plasma leakage as a result of increased vascular permeability and abnormal hemostasis, occurring in a select group of patients during the course of dengue infection (18). The underlying mechanisms causing DHF/DSS are a subject of intense debate. Current evidence strongly suggests that the intensity of the immune response to dengue virus plays a key role in the pathophysiologic cascade leading to plasma leakage (19–21). In addition, DHF and DSS have been associated with high levels of proinflammatory cytokines in the serum of patients (22–26). In addition, other mediators produced by phagocytic cells and a role for antibody mimicry have been suggested (27–29).

Hypotheses on the Pathogenesis of DHF/DSS.
There are numerous theories on how DHF/DSS develops in infected dengue individuals. These hypotheses are predominantly derived from data obtained on studies conducted within the regions of countries where the disease occurs in an epidemic form, and/or to some extent, from in vitro experiments. These include antibody-mediated pathogenesis or so-called antibody-dependent enhancement, cell-mediated pathogenesis (10), cytokine storm phenomenon (30), individual’s genetic background (31), virus strain differences (32, 33), levels of virus circulating in individuals during the acute phase (34, 35), and the nutritional status of the infected individual (36). The major limitations in our ability to decipher or dissect the mechanisms of pathogenesis of dengue virus infection are a lack of suitable small animal models. Consequently, the precise mechanisms that lead to the development of DHF/DSS remain an enigma.

In addition to the aforementioned hypotheses, the authors reason that other factors, which are closely associated with dengue virus infection, have not been studied in sufficient detail. These include (i) hyper-thermal factors, (ii) physical status of virus in viremic individuals, (iii) conditioning of neutralizing antibody assay in dengue virus infection, (iv) concept of vector transmission, and (v) innate immune system. We, therefore, offer that detailed studies of these events that occur during the acute disease stage are critical in shedding light on the pathogenesis of DHF/DSS. A detailed understanding of these events may provide an alternate scenario in which these factors may contribute directly or indirectly to DHF/DSS and are the subject of this review.

(i) Hyper-thermal Factors.
As the name of the disease implies, dengue fever is one of the hallmarks of dengue virus infection. However, there has been limited attention given to the distinguishing characteristics of this fever that occur in patients who simply develop DF as compared with those that develop DHF or DSS.

The period of fever following dengue virus infection lasts from 2 to 7 days. Fever is the normal physiologic response of the body to mediators that are generated during the acute phase of dengue virus infection. In fact, fever is a condition induced by the host to subdue and contain the adverse effects of the infection, making it unfavorable for the infecting agent, and is designed to promote body health. Heat-inducible factors from host cells are expressed and released into the circulation. Most prominent amongst these heat inducible factors are cytokines that trigger fever (fever inducers), such as TNF-alpha, IL-1 and IL-6, and cytokines to calm down fever (fever inhibitors), such as TGF-beta and IL-10 (37). These cytokines are promiscuous effectors and can be found in febrile patients as a response to a variety of infectious agents, including dengue virus infections. Differences in the quality and/or quantity of these array of fever-related factors circulating in the blood may alter or enhance parameters observed in dengue virus–infected patients and may be critical in defining the outcome of such an infection. In this regard it is of interest to note that hyper-thermal conditions have in fact been reported to increase cell susceptibility to virus infection (38–40). Thus, a more careful analysis of the spectrum and quantitation of cytokines that either induce or are secondary to the thermal response following dengue virus infection may be critical in identifying the possible factors or mechanisms that may distinguish DF from DHF or DSS and shed light on the pathogenesis of DHF/DSS.

(ii) Physical Status of Virus in Viremic Individuals.
Viremia is the major unique feature in dengue virus infection among the Flavivirus family. Although this feature is accepted and well-known, the precise nature of this viremia remains to be established. Thus, physical evidence on whether the virus exists as a free viral particle, or is primarily cell associated and/or is encapsulated by host cell membrane, remains unknown.

Viremia can be caused by the presence of the virus in plasma (free viral particle) or be cell associated, such as within platelets, lymphocytes, monocytes, but not likely within red blood cells. In the case of plasma viremia, the amount of circulating virus is a critical factor for its clearance. Results of one study suggest that most of the virus circulating in the blood is cell-associated (41). These cell-associated viruses are poorly cleared when injected into a normal host, as compared with the clearance of plasma associated virus that is 95% cleared in less than 2 mins (41). Of importance is the finding that the cell-associated virus by its nature helps spread the virus throughout the body.

In dengue viremia, although virus has also been shown to circulate in the form of immune-complexes, a detailed study of the kinetics by which this occurs is still lacking. Thus, in general, a complete picture as to the nature of dengue viremia has not emerged primarily because it has been difficult to study this issue during the acute viremia period in human patients, since such patients are only seen following a febrile course at clinics where this issue is being studied. It is clearly possible that following the entry of the virus into the blood stream, the virus enters a permissive cell where it replicates in sufficient quantity to induce the febrile response, after which it can exist in several forms based on the level of viremia and host response to the viremia. It is therefore critical to establish a detailed analysis of these early events during the course of dengue virus infection, such as kinetics of viremia associated with each specific stage and the form the virus exists at during these specific stage, which may provide unique insights as to the physical properties of circulating dengue viral particles and may lead to the identification of unique targets for preventive vaccine development and the pathogenesis of DHF/DSS.

(iii) Conditioning of Neutralizing Antibody Assay in Dengue Virus Infection.
In dengue endemic regions, a majority of the individuals are sera-positive for all four dengue serotypes by the time they reach adulthood. The intriguing phenomenon is that dengue viremia occurs even in the presence of significant levels of circulating antidengue virus antibody in these individuals, providing suggestive evidence that such antibodies are clearly nonneutralizing antibodies. However, it should be kept in mind that the measurement of neutralizing antibody levels to dengue virus has been predominantly derived on in vitro cultured dengue virus preparations. Thus the sources of viral stocks used for these studies may not be representative of wild-type virus and thus may influence the data obtained by the neutralization assays performed.

In reviewing the literature on this issue, three major sources of dengue virus stocks have been used for the virus neutralization studies. These include viral stocks cultured from C6/36 mosquito cells, Vero cells, and gradient-purified virus preparations. The interpretation of the neutralizing antibody titer data on these stocks needs to be carefully evaluated. Thus as noted in the literature, differences in neutralization titers sometimes by twofold of magnitude have been reported even when using the same sera for the neutralization assay. In the era of evidence-based medicine, it is our opinion that the meaningful clinical relevance of the neutralizing assays can only be derived using primary virus isolates directly from dengue-infected patients, which to the best of our knowledge has not been investigated.

(iv) Concept of Vector Transmission.
Dengue is transmitted by the bite of mosquito carrying the infectious dengue virus. The vector can serve as a biologic host also referred to as biologic transmission, which means that the virus is required to replicate within the vector before the virus can be transmitted to a new target host. In addition, the vector can serve as a transmitting vehicle, or so-called mechanical transmission, which means that the virus does not require replicating within the vector before it can be transmitted to a new target. While there are several reports on the biologic transmission of dengue virus in the mosquito vector, there is a paucity of information on the mechanical transmission of this virus. In endemic areas especially during an epidemic, mechanical transmission may play an important role in helping the spread of dengue virus. In both cases, the vector appears to directly inject the virus into capillary blood vessel (42).

Since dengue virus infection is initiated by injecting virus directly into the blood stream from the mosquito vector, it seems logical that initially at this stage, the virus predominantly encounters the host’s innate immune system (Fig. 2Go). It thus seems reasonable to carry out more detailed studies of these initial events in efforts to identify at least some therapeutic strategies aimed at preventing these initial events of virus infection.


Figure 2
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  		Figure 2. Innate immune encounter upon virus injection in the capillary vessel. There are several immune-related components circulated in the capillary, which is surrounded by multiple cell layers. The enlarged circle indicates the mosquito’s probing site. Prior to the feeding process, mosquito saliva-containing factors (e.g., apyrases) and virus are released into the blood stream. A color figure is available in the online version.

 
(v) Innate Immune System.
The innate immune system is inborn in the host and is a universal and ancient form of host defense against infection. Thus, as the saying goes "phylogeny recapitulates ontogeny." It has been documented that the innate immune system exists without detectable forms of the adaptive immune system in more primitive phylogenetic species, which suggests that the innate immune system has had a longer evolutionary time to evolve. The innate immune system has been shown to consist of several components broadly ranging from skin barriers to cellular effector mechanisms and genome-encoded molecules, which act in concert to provide the host with an initial barrier against foreign organisms. The innate immune system thus includes natural antibody and factors involved in the maintenance of homeostasis (43–45). Key features of innate immunity are that it is not antigen-specific, and that it can respond immediately or within a few hours after encountering a novel intruder (43). The initial defense response by the body is to eliminate or temporarily contain the intruders and thereby to prevent or reduce the infection. The innate immune response to dengue infection, in particular the period between infection and appearance of clinical signs (preillness period, Fig. 3Go), is basically a subject that is to a large extent underexplored. We propose that two important components of the innate immune system, natural IgM antibody and platelets, are likely to play a critical role in the preillness period of dengue virus infection.


Figure 3
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  		Figure 3. Preillness gaps. The innate immune factors between mosquito bites and the appearance of clinical signs are mysterious in dengue disease, either in primary infection or secondary infection. Viremia, IgM, and IgG are all readily detectable during the apparent clinical signs. The oval dotted lines indicate the parameters that are unknown between infection (mosquito bite) and the appearance of illness (day x, normally 2 to 5 days). The solid circles indicate the knowledge gaps in dengue virus infections, primary or secondary. Day y indicates viremia periods, which last from 5 to 7 days. Defervescence is the period that fever and viremia are recessed. A color figure is available in the online version.

 
Natural IgM Antibody.
Humoral immunity is mainly mediated by B cells, which produce different classes of antibodies, both natural and pathogen induced. Natural IgM antibodies are produced mainly by CD5+ B cells (B1 cells) and are a component of innate immunity (45–48). These nonspecific circulating natural antibodies can bind to pathogens, providing early host protection (49). Natural antibodies may facilitate antigen uptake, processing, and presentation by B lymphocytes via complement and Fc-receptors (50, 51). Interestingly, it has been suggested that B cells are the principal circulating mononuclear cells infected by dengue virus (52). Interactions between innate immune mechanisms and adaptive immune responses are now widely viewed as essential for a normal immune response (53–55).

Although in humans about 30% of the circulating antibodies are pentameric IgM molecules, a small amount of hexameric IgM, IgG, and IgA natural antibodies circulate as well (56). The physiologic roles of the natural IgM antibodies are not known. However, natural IgM antibodies have been shown to be involved in early recognition of external invaders and elimination of pathogens like bacteria and viruses (43, 57–65).

The multimeric structure makes IgM a strong complement activator; a single-bound IgM pentamer can trigger the classical pathway of complement activation and can lyse a red blood cell, while approximately a thousand IgG molecules are required to accomplish the same (66). Furthermore, the hexamer IgM, even though it circulates in smaller amounts, is 15 to 20 times more efficient in activating complement than the pentameric form of IgM (67–69). Circulating immune complexes (CIC) in sera of patients with DHF/DSS was first reported by Theofilopoulos et al. in 1976 (70). Dengue antigen can be detected in more than 50% of the CIC, and increased levels of platelet-associated IgM has been observed in DHF cases (71). Interestingly, IgM immune complex have been consistently found in the blood vessel walls of dermal papillae or cutaneous rashes of dengue patients (72–77). However, the origin and the structure of IgM in these immune complexes has never been characterized and described. Furthermore, the role of circulating IgM-immune complex in DHF/DSS is unknown. Perhaps, immune complexes that attach to the surface of platelets may enhance platelet destruction by the reticulo-endothelial system in the liver and the spleen, resulting in thrombocytopenia during the shock phase of disease. Therefore, the levels of IgM, notably natural IgM that has specificity for dengue viruses, in an individual may have an impact on the outcome of dengue infection.

Platelets.
Platelets are an essential element in hemostasis (78). In recent studies, platelets have been viewed as an integral part of the innate immune system and can be potent effectors of the innate immune response (79, 80). In addition, a functional CD154 (CD40L), a molecule shown to be critical for the enhancement of antigen presentation and augmentation of the adaptive immune response, has recently been shown to be expressed on the surface of platelets (81, 82). This further supports a role of platelets in modulating the immune response and inflammation (80, 83).

Platelets are anucleated, composed of a concentric megakaryocyte membrane, cytoplasm, granules, and organelles. Platelets are produced within the bone marrow and the cells from which platelets originate are called megakaryocytes (84). Platelets circulate throughout blood vessels and function to monitor the integrity of the vascular system. There are two forms of platelets, a resting form circulating as discs and an active form circulating as filopodia. Structurally, these forms have different arrangements of internal microtubules and actin cytoskeleton (84).

When the lining of a blood vessel is traumatized, platelets are stimulated to go to the site of the injury, where they form a plug to reduce the loss of blood. All platelet functional responses must be tightly regulated to ensure that the formation of a blood clot is of sufficient size to seal off the damaged area, while not disrupting blood flow to vital organs by causing vessel occlusion (85, 86). Thus, impairment of platelet function can increase risk of vascular fragility leading to hemorrhage. This may be an important mechanism of plasma leakage in severe dengue disease (DHF/DSS) (18).

One of the key clinical manifestations in dengue disease is thrombocytopenia, otherwise known as low platelet counts (17). This arises from both decreased production (87, 88) and increased destruction of platelets (77, 89–92). The degree of thrombocytopenia appears well-correlated not only with the clinical severity of DHF, but also with the activation of the complement system (93, 94).

During dengue virus infection, platelets may provide a wonderful shield for the virus from exposure and binding to neutralizing preexisting antibody. Interestingly, there are a few reports suggesting that dengue virus may associate with platelets, directly or indirectly through antibody (75, 77, 91, 92, 95, 96). Assuming this is the case, we can envision that the virus-platelets-antibody complexes may enhance the phagocytosis or be engulfed by macrophages or monocytes via Fc{gamma} receptor as suggested by others (10). In addition, relatively recently dengue virus RNA has been detected using reverse transcription polymerase chain reaction techniques in RNA purified from platelets from dengue infected patients (71). The interesting point in these observations or reports is that the thrombocytopenia seen in DHF/DSS patients may not only be caused by the destruction of platelets by the virus itself (direct cytotoxicity), but may also be caused by the destruction of platelets following the binding of dengue-specific antibodies to the virus-infected platelets (immune-mediated toxicity) (97. It is also possible that platelets can serve as a reservoir for dengue virus replication; however, this issue is a subject of further investigation.

Alternate Explanation on DHF/DSS.
Our take-home message in this short review on alternate hypothesis is summarized in Figure 4Go. Under this scheme, there is a potentially important role of naturally occurring IgM antibodies that may play an important role in the early acute dengue viral infection, which may set the stage for the clinical course of the disease. In addition, a role for platelets as the source of primary infection and/or as carriers of the virus and the subsequent innate immune response against these virus infected platelets may play a pivotal role in the induction of DHF/DSS. Our laboratory is currently focused on addressing this issue in a formal study.


Figure 4
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  		Figure 4. Role of innate immune parameters in DHF/DSS. The proposed alternate hypothesis on DHF/DSS can potentially divide into two stages, disease induction or defined stage and protective stage. Innate immune-related parameters, such as other factors (cytokines, etc.), natural IgM, or platelets, may directly or indirectly contribute to define the development of DHF/DSS during the course of early dengue virus infection (purple circle, preillness). While in the protective stage, the periods in which fever and viral load are recessed, DHF/DSS may attribute to antigen-induced factors, such as IgG and adaptive immune response (blue circle).The purple arrow indicates the time in which, at or around the peak viremia stage, patients reported to the clinics or hospitals, normally after 2 or 3 days of fever. The orange curve and arrow indicate the typical kinetic of viremia. x and y are defined as in Figure 3Go. A color figure is available in the online version.

 
The dynamic and complexity of dengue disease immediately prompts us to realize that the alternative hypothesis in this short review will not be sufficient to cover all the various other hypothesis that have been forwarded in the extensive amount of literature. Readers, therefore, should refer to recent comprehensive reviews on dengue pathogenesis (44, 98–105).


   Conclusions
Top
 Abstract
 Introduction
 Conclusions
References
 
An alternate hypothesis on the pathogenesis of DHF/DSS is presented in this short review. It is the objective of the authors to draw scientific attention to these new concepts, which we hope contribute to providing a much more complete picture on dengue pathogenesis. Our aim is to encourage more detailed studies of the acute viremia period, which we believe will not only provide useful information of the mechanisms associated with DHF/DSS, but will also assist in the formulation of effective candidate vaccines or drug development.


   Acknowledgments
 
We thank Professor Aftab A. Ansari, Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, for his helpful discussions and knowledgeable suggestions in the writing of this manuscript.

Received for publication July 24, 2007. Accepted for publication October 14, 2007.


   References
Top
 Abstract
 Introduction
 Conclusions
 References
 

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H.-J. Cheng, C.-F. Lin, H.-Y. Lei, H.-S. Liu, T.-M. Yeh, Y.-H. Luo, and Y.-S. Lin
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