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IMMUNOLOGY AND PATHOGENESIS OF VIRAL HEMORRHAGIC FEVERS

A Re-evaluation of the Mechanisms Leading to Dengue Hemorrhagic Fever


Sansanee Noisakran,a,b,c Kulkanya Chokephaibulkit,d Pucharee Songprakhon,c Nattawat Onlamoon,a Hui-Mien Hsiao,a Francois Villinger,a Aftab Ansari,a and Guey Chuen Pernga
Department of Pathology and Laboratory Medicine, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georg a, USA
b a

Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
c

Medical Molecular Biology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
d

Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

Viremia is one of the features of dengue virus infection among the aviviruses. Dengue virus infection results in a spectrum of clinical symptoms, ranging from undifferentiated u-like illness, mild dengue fever, to dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a life-threatening illness. Several mechanisms have been hypothesized based primarily on data collected from post-acute clinical phase to account for DHF/DSS. Lack of a suitable animal model for DHF/DSS has hindered progress in dening the etiology of DHF/DSS. Levels of circulating dengue virus have been wellcorrelated to severe dengue disease. However, the cell lineage(s) serving as a primary target for the source of viremia are largely unknown. Results from in vivo and in vitro pilot studies using molecular and more advanced technologies reveal that dengue virus appears to be associated with platelets and the megakaryocytic lineage. The observation may partially explain the dysfunction of platelets observed in dengue affected patients.

Key words: avivirus; dengue virus; DHF; DSS

Dengue virus infection and dengue disease are recognized as a major public health issue internationally.1 Dengue virus infection is the most important mosquito borne human disease in terms of morbidity and mortality in urban tropical area. Climate changes, such as global warming and El Nino, poorly urbanization planning, an increase in international travel, and lack of effective vector-control programs, are the predominant cause of the expan-

Address for correspondence: Guey Chuen Perng, Ph.D., Dental School Building, Room 429, 1462 Clifton Road, Emory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322. Voice: 404-727-5490. gperng@emory.edu

sion of dengue threat.2 The National Institute of Allergy and Infectious Diseases (NIAID) has listed dengue virus as a Category A priority biothreat pathogen.3 Over 100 million people living in tropical and subtropical areas are at risk of infection with the dengue virus. Estimated 50 million cases of dengue illness are documented per year globally. The death rate from dengue illness ranges from 2 to 5%, predominantly in children under 15 years of age.4 The recent outbreak in Brazil highlights the potential of dengue virus spread to the Americas and as outlined by Dr. David M. Morens and Dr. Anthony S. Fauci, NIAID,5 dengue disease is a potential threat to public health in the United

Immunology and Pathogenesis of Viral Hemorrhagic Fevers: Ann. N.Y. Acad. Sci. 1171: E24E35 (2009). doi: 10.1111/j.1749-6632.2009.05050.x c 2009 New York Academy of Sciences.

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Figure 1. Seven key questions concerning DHF. We hypothesize that there are at least seven distinct issues with regards to dengue virus infection that require address. These issues are based on our current knowledge and the available data from post-clinical specimens. Besides the lack of a suitable animal model, several critical parameters playing important roles in the pathogenesis of DHF/DSS which require further study are outlined. These include (I) the detailed anatomy of the mosquito bite, (II) the role of innate immune parameters contributing to DHF/DSS development, (III) the cell lineage that serves as a source of dengue virus, (IV) the precise nature of the platelet-associated antibodies, (V) the exact role of monocytes in DHF/DSS development, (VI) mechanisms leading to thrombocytopenia and neutropenia, and (VII) what are the causes and consequences of these factors in DHF/DSS.

States. Currently, no effective vaccine and/ or effective chemotherapeutic drug treatment exist. Dengue virus is a single-stranded, positive sense RNA virus whose genome is 11 kb in length. The dengue viral particles prepared from infected tissue culture are a spherical shape 4050 nm in diameter though in vivo the actual physical status of the viral particles remains to be further dened. A single large protein is translated from the positive RNA and is digested into three major structural proteins, C, PrM/M, and E, and seven nonstructural proteins (NS), NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, by viral or host proteases. There are four distinct but genetically related dengue virus serotypes, namely serotypes 1, 2, 3, and 4. Aedes aegypti mosquito is the major vector responsible for dengue virus transmission.4

The spectrum of dengue disease ranges from asymptomatic to dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS).4 Thus, dengue fever infections can be life threatening. The more severe forms are usually a result of secondary infection with heterologous virus serotype.4 The incubation period of dengue fever infection varies from 5 to 10 days. Viremia occurs roughly 2 days prior to clinical manifestations and lasts for 56 days.6 The cell lineage(s) that serve as an initial target and subsequently serve as a reservoir of the virus during and following acute infection remains to be dened. The kinetics and spectrum of disorders associated with dengue virus infection have led to the denition and identication of a number of unresolved mechanisms and questions, respectively, some of which are outlined in Figure 1. The rationale for the categorization

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is based upon the nature of the infection, the early events of the clinical symptoms, and the observations of the clinical outcomes in the affected patients. There are several other potential contributing mechanisms within the disease process, such as the role of T and B cells, cytokines storms, complement factors, and antibody factors, which are not within the scope of the current review. The focus of the studies reported herein is on the relationship between platelets and dengue virus. The readers may refer a series of comprehensive reports for the other issues identied as playing a potential role leading to disease development.713 Thus, as stated above, it is the role of a few of the early events observed during dengue infection which will be the focus of this report and therefore elaborated in more detail below. The Concepts Relevant to a Mosquito Bite and Route of Transmission Is it subcutaneous or intravenous injection? It has been well-established that Aedes aegypti takes up blood either directly from a capillary (capillary feeding) or from a pool formed in the tissue by the leakage of blood (pool feeding) from a capillary previously lacerated by the mosquitos proboscis and the former appears to take far shorter time for repletion than the latter.14,15 However, the current general assumption is that the mosquito bite is similar to that of a subcutaneous injection even though the process does not appear similar. According to the concept that the virus is introduced into the host via the subcutaneous route, during the probing process, the mosquito is thought to deposit dengue virus within the epidermis or the dermis, and the rst cell lineages encountering the virus upon deposition are reasoned to be the epidermal Langerhans cells or the dermal dendritic cells. These concepts have led to numerous studies and a subject of intense discussion1620 with a failure to arrive at a consensus accompanied by conicting reports.2124 In addition, the feeding behavior of the vector in question, such as multiple feeding and mul-

tiple host contacts during each gonotrophic cycle, does not change the virus transmission capability of the dengue virusinfected Aedes aegypti. Thus, it remains infective regardless of how many times it has bitten, probed, or its engorging history.25 It is important to note that dengue virusinfected Aedes aegypti can interrupt feeding and y away following even slight movements by the human host.25 Therefore, every mosquito probe, including probing which results in failing to obtain blood, has the potential to deposit dengue virus within the epidermis or dermis cells. Results of one study have shown that an Aedes aegypti mosquito can feed up to a few times per day, and each feed takes about 1020 probings before exposure to the capillary blood.25 Thus, if the subcutaneous injection concept truly reects what happens in nature, and Langerhans or dendritic cells are the primary cell lineage that is responsible for amplication of the dengue virus, we would see a higher incidence of DHF/DSS. However, the incidence of DHF/DSS in endemic regions is less than or about 1% of the total population.26 These lines of evidence suggest that the role of these antigen presenting cells may be more important for the induction of hosts defense rather than serve as a primary amplication site or vehicle for the dengue virus as suggested by other.27 Importantly, it is of benet to the host that the virus can be engulfed and processed to generate an adequate immune response against the virus to protect the host from further infection. Since such phagocytic cell lineages are the rst line of defense in our body, this may perhaps explain why a majority of dengue cases are asymptomatic, based upon serological results.26,28 Interestingly, previous studies with Aedes aegypti feeding on human volunteers reveal that capillary feeding occurs more frequently than pool feeding, which was attributed to the exibility of the fascicles of the Aedes aegypti , which are designed to seek capillary penetration after the fascicles break through the skin barrier.14,15 This natural feeding of the mosquito is more akin to intravenous injection. Following such

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intravenous inoculation, although the majority of the virus may be cleared by immune components in the circulation, a small amount of virus may encounter a potential permissive target cell lineage, infect this cell lineage, and potentially replicate within the target cell and expand accordingly. The circulating levels of the permissive target cell during the time of mosquito bite therefore may have a critical role for dengue virus dissemination and the severity of dengue disease development. Nevertheless, it is clear that the subcutaneous and intravenous routes of transmission are unlikely to be mutually exclusive. However, the frequency of the virus infection, susceptible cell lineage in the circulation and the micro-anatomy of mosquito probing, and successful penetration rate need to be documented before we can develop a better understanding of the route of infection and its role in the development of DHF/DSS. Innate Immune Response The lack of a suitable animal model to study dengue virus infection precludes a thorough study of the role of the innate immune system and dengue virus infection and hence the paucity in our knowledge in this subject area. It is thus clear that developing a suitable animal model for dengue virus infections is urgently needed. Dengue Viremia in Dengue Patients Viremia is the major unique feature in dengue virus infection among the avivirus family. Although this feature is accepted and well known, the precise nature of this viremia remains to be established. Viremia can be due to the presence of the virus in plasma (as a free mature viral particle) or be cell associated, such as within platelets, lymphocytes, and monocytes, but not likely within red blood cells. Although virus has been shown to circulate in the form of immune complexes, a detailed study of the kinetics by which this occurs is still lacking. Thus, physical evidence

on whether the virus exists as a cell free fully mature replication competent viral particle, or is primarily cell associated and/or is encapsulated by host cell membrane, remains currently unknown. For details of this subject area of dengue infection, readers are referred to a series of recent reviews by others.6,29 The ndings that thrombocytopenia is one of the key clinical features during the acute infection period of dengue infected patients prompted us to carry out studies aimed at attempts to understand the potential role of platelets during acute phase of dengue virus infection and its relationship with dengue virus. Consequently, we focused our studies on the understanding of the nature of dengue viremia during the acute phase of dengue virus infection in patients and used the knowledge gained to carry out in vitro studies aimed at providing proof of concept to document the fact that there exists intimate interactions between dengue virus and platelets and that the level of such an interaction which we submit has not been previously appreciated. Platelets Platelets are anuclear cells and derive from the demarcation of megakaryocytes. Although the size of the platelet is small, platelets perform biological functions, including protein synthesis and protein modications, and possess receptors on the surface for signal transduction. Platelets circulate throughout blood vessels during which they monitor the integrity of the vascular system. All functional platelet responses must be tightly regulated to ensure that the formation of a blood clot is of sufcient size to seal off the damaged area, whilst not disrupting blood ow to vital organs by causing vessel occlusion.30 Importantly, several receptors linked to the entry of the dengue virus, such as dendritic cell-specic intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the putative enhancement receptor (FcII), can be found on the surface of platelets.31 With

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the era of microarray and proteomic technologies, the understanding of the functions of platelets has markedly expanded in recent years. These include ndings which suggest that platelets can function as immune cells,32 crosstalk with lymphocytes,33 and become actively involved in shaping the immune response upon encountering infectious agents.34 One of the key clinical manifestations in dengue disease is thrombocytopenia.35 This arises from both decreased production36,37 and increased destruction of platelets.38,39 Interestingly, the degree of thrombocytopenia appears well correlated not only with the clinical severity of DHF but also with the activation of the complement system.40 Thus, impairment of platelet function can increase the risk of vascular fragility leading to hemorrhage. This may be involved in the mechanism contributing to plasma leakage in severe dengue disease (DHF/DSS).4 In dengue virus infection, the detection of viral antigens on the surface of platelets, immunecomplex containing platelets in skin biopsy specimens, and the association of dengue virus with platelets in vitro have been well documented.39,41,42 However, to date no evidence for replication competent dengue virus has been demonstrated in enriched preparations of platelets from dengue virusinfected patients, and consequently the exact role of platelets in the pathogenesis of dengue virus infection related to DHF or DSS is still unknown. Importantly, the precise mechanisms accounting for the development of thrombocytopenia, whether platelets are directly infected, and their role in dengue viral infections remain elusive.

In Vivo Studies of Dengue Virus and Platelets


In efforts to study the role of platelets in dengue virus infection, we rst attempted to optimize procedures for the isolation of highly enriched preparations of human platelets from

the peripheral blood of healthy human volunteers. As seen in Figure 2A, we isolated the platelet rich band using the commercially available OptiPrep isolation kit (BD Biosciences, San Jose, CA). The purity of the platelet population was assessed by standard ow cytometry utilizing CD41 (a marker specic for human platelets) specic monoclonal antibody. As seen in Figure 2B (typical prole) following appropriate gating strategies we determined the gated population of cells was > 99% CD41+ . In addition, centrifugation of the leftover plasma from the same blood specimen also yielded a platelet population that upon staining and ow cytometric analysis gave a similar prole (data not shown). In a pilot study, we observed that highly enriched preparation of platelets isolated from the peripheral blood of clinically and serologically conrmed dengue patients during the acute phase contained dengue virallike particles within the platelets.43 In efforts to conrm these preliminary data, we utilized the same approach as outlined above for the isolation of platelets from the cellular elements and the plasma and analyzed these for the presence of dengue viral antigens. First of all, we noted that there exist two populations of platelets in both the peripheral blood isolated population and the plasma origin platelets in specimens from dengue infected patients. Figure 3A shows the typical forward and side scatter prole seen in specimens from dengue infected patients. Thus, the platelets were either of normal size (termed G1) or those that display a relatively smaller size (termed G2). There was, however, a considerable amount of variability in the frequency of CD41+ cells in such preparations from dengue infected patients and Figure 3A shows examples of such variability in both the cellular source and the plasma source of the platelets. In general, the cellular source of platelets always contained a G1 population that were highly enriched for CD41+ cells. The relative expression and frequency of CD41 expressing platelets in the G2 specimen from the cellular source or the G1/G2 source of platelets from the plasma was highly variable.

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Figure 2. Isolation of human platelets. Platelets were isolated from Ethlylenediaminetetraacetic acid (EDTA)-treated peripheral blood of healthy individuals using OptiPrep gradient centrifugation according to procedure outlined by the manufacturer (A). The isolated cells were analyzed for the level of purity using standard ow cytometry. Aliquots of the cell preparation were stained either with an isotype control or with anti-CD41 monoclonal antibody. Cells to be analyzed were gated based on forward and side scatter proles (B, left panel) and a single major population of cells was noted. The CD41 prole of the gated population is seen (B, right panel). A total of 5000 events were analyzed and shown to contain > 99% CD41+ cells.

The reasons for such variability are currently unknown and are a subject of further study. The puried population of platelets was smeared onto slides for immunouorescence staining and confocal microscopy to conrm the presence of dengue antigen (Fig. 3B). Formation of micro-aggregates with CD41 clustering of platelets and the co-localization of dengue viral antigen and CD41 expressing platelets were consistently observed in each of the dengue infected patients studied.

Electron microscopic techniques were also utilized to conrm the above ndings using uorescence microscopy. Morphologically the platelets isolated from dengue patients appeared to be activated (Fig. 4A, B, and C). In addition, frequently, a cluster of dengue viral-like particles was observed inside vacuoles or vesicles in platelets isolated from dengue patients (Fig. 4A and B). Occasionally, a single isolated dengue virallike particle inside the platelet was observed (Fig. 4C). The morphological

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Figure 3. Expression of CD41 and dengue viral E antigen in platelets isolated from infected patients. (A) Isolated platelets from the peripheral blood and enriched platelet preparations from the plasma from dengue virusinfected patients were analyzed for the frequency of CD41 expressing cells using phycoerythrin (PE)conjugated anti-CD41 monoclonal antibody and standard ow cytometry. A total of approximately 5000 15,000 gated events were analyzed. Isotype-matched antibody was used as a control. Proles of CD41 expression in platelets isolated from the cellular elements using OptiPrep and the leftover platelets in the plasma from two representative patients (top and bottom rows, respectively) are shown. Results demonstrate the forward and side scatter prole (left panel), the CD41 prole of the G1 gated population (middle panel) and the CD41 prole of the G2 gated population (right panel). (B) Fixed isolated platelets on a glass slide were subjected to double immuouorescence staining and confocal microscopy to determine the presence of dengue viral E antigen (green) and CD41 (red) and the overlay of the two images as well as a bright eld view of the same eld. The results shown are images of stained platelets derived from the two representative patients (top and bottom panels) with a magnication of 63 (objective lens). Scale bar, 10 m.

appearance of dengue viral particles inside a vesicle is very similar to what others have reported in Vero cells infected in vitro with dengue virus.44,45 These data provide suggestive evidence for the presence of dengue virus antigens and/or dengue virallike particles within platelets from dengue infected patients during the acute stage of infection.

In Vitro Platelets and Dengue Virus Studies


To further investigate the role of platelets and dengue virus, in vitro experiments were performed. Highly enriched preparations of platelets isolated from the peripheral blood of healthy individuals were experimentally

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Figure 4. Electron microscopic evidence for the presence of dengue virallike particles in platelets from infected patients. Platelets from dengue-infected patients were isolated and processed for negative staining and subjected to electron microscopy. Images show clustering or isolated dengue virallike particles with an estimated size of about 4050 nm inside platelets isolated from three patients. Inserts with green square box indicate the infected platelets with activated morphology. Red circles indicate the enlargement of the area. Scale bar, for the enlarged image, is 100 nm. Arrow indicates the dengue virallike particles.

infected in vitro with dengue virus serotype 2 at a multiplicity of origin (MOI) of 0.03. An aliquot of the infected platelets was harvested, washed, and stained with dengue NS1 monoclonal antibody after 24 h of infection and subjected to ow cytometry. Expression of NS1 was observed in in vitro dengue virusinfected platelets (Fig. 5). Another aliquot of the infected

platelets was smeared onto slides, and standard immunouorescence was performed using the dengue E monoclonal antibody (clone 3H5) and platelet specic marker, CD41. Dengue E antigen was detected on such platelets, either on the surface, within platelets, or formation of platelet micro-aggregate (Fig. 6). The expression of dengue proteins was further conrmed

Figure 5. Detection of dengue viral NS1 on the surface of infected platelets. Platelets were isolated from healthy individuals and experimentally infected in vitro with dengue virus serotype 2 (DENV-2, 16681) at 0.03 MOI. Following overnight incubation, the platelets were washed three times with media to remove unbound virus and stained with either FITC-conjugated anti-NS1 monoclonal antibody or with an isotype control antibody. Platelets that had not been exposed to DENV-2 served as a control (mock) and were performed in parallel. The stained cells were analyzed by ow cytometry. Histograms demonstrated the expression prole of NS1 antigen on the surface of platelets: left panel (mock), middle panel (DENV-2 infected), and right panel, combinations (mock, black line and DENV-2 infected, red line).

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Figure 6. Expression of CD41 and dengue viral E antigen in in vitro infected platelets. Platelets isolated from healthy individuals were experimentally infected with dengue virus as described under Figure 5. Platelets were washed three times after 2 h of absorption, and further cultured for 18 h. The infected platelets were then subjected to double immunouorescence staining protocol using the platelet specic marker (CD41), and dengue E antibody (3H5). The stained cells were analyzed by confocal microscopy to determine the expression of CD41 (red) and dengue viral E antigen (green). Images were captured at a magnication of 63 (objective lens). Representative results are shown.

by Western blot analysis (Fig. 7) using either human anti-dengue polyclonal antibody (Fig. 7A) or NS1-specic monoclonal antibody (Fig. 7B). Both E and NS1 proteins could be observed with human polyclonal sera (Fig. 7A).

These results suggest that infectious dengue virus can gain access and most likely replicate transiently in platelets. In addition, these results imply that platelets may also help disseminate dengue virus during acute infection.

Figure 7. Dengue viral antigens in in vitro infected platelets. Platelets were isolated from healthy individuals, experimentally infected with dengue virus as described under Figure 5. Platelets that had not been exposed with the virus served as a control (mock). Lysates prepared from mock or dengue infected platelets were subjected to SDS-PAGE and immunoblot analysis. Pooled convalescent serum from dengue patients was used to determine the dengue viral components (A) and anti-NS1 monoclonal antibody was used to verify the identity of NS1 protein in platelet lysates (B). Dengue virusinfected Vero cell lysate was used as a positive control for detection of dengue E and NS1 antigens by immunoblot analysis (labeled DV, A and B).

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Future Perspective Among all the hypothesized mechanisms that have been forwarded to date concerning the etiology of DHF, the understanding of the dynamics of dengue viremia is perhaps the most critical since this can potentially inuence vaccine design to prevent dengue virus infection and dengue disease development. In this communication, we present a novel alternative concept regarding how dengue virus disseminates during the acute viremia period based on its intimate relationship with platelets. We demonstrated that dengue viral particles are present within platelets isolated from dengue patients and that dengue viral products could be readily detected within platelets experimentally infected in vitro. Our results infer that platelets may shield dengue virus from the host immune system. Additionally, our results suggest that there is a high likelihood that active viral replication may occur inside platelets. This may be very critical for the dengue virus survival strategy since platelets do not have the ability to generate interferon related products, which play an important role in controlling dengue virus replication. Importantly, with the observation of dengue virallike particles inside the platelets, we provide an additional piece of information regarding the status of dengue virus in circulation during acute infection. However, whether there is (are) receptor(s) involving in the process of viral entry or the virus gains access via the open canalicular system of platelets remain to be dened. Although numerous animal models have been utilized to investigate certain aspects of DHF/DSS, a suitable animal model for not only dengue virus infection but also one that mimics the clinical manifestations of dengue virus infection is urgently needed in order to delineate the mechanisms which are the basis of the origin of DHF/DSS pathogenesis. Importantly, such a model would allow for studies of some of the currently understudied mechanisms, such as the role of innate immune sys-

tem in DHF/DSS, the likely sources of dengue viremia, and differentiation of the factors that cause DHF/DSS from the factors that are the consequences of DHF/DSS.
Acknowledgments

We thank the clinical staffs in the Department of Pediatric, Siriraj Hospital for technical assistance in patient blood collection, Drs. Chunya Puttikhunt and Watchara Kasinrerk for providing the anti-dengue monoclonal antibodies for immunouorescence staining, and Dr. Bunpote Siridechadilok for technical guidance in sample preparation for electron microscopy. The authors also appreciate the kind and knowledgeable assistance of Dr. Hong Yi from Electron Microscopy Core Facility of the Emory University School of Medicine. The research is partially supported by U19 Pilot Project Funds (RFA-AI-02-042), NIH/SERCEB, and Emory URC grants.
Conicts of Interest

The authors declare no conicts of interest.

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