Vascular endothelium: the battlefield of dengue viruses

Increased vascular permeability without morphological damage to the capillary endothelium is the cardinal feature of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). Extensive plasma leakage in various tissue spaces and serous cavities of the body, including the pleural, pericardial and peritoneal cavities in patients with DHF, may result in profound shock. Among various mechanisms that have been considered include immune complex disease, T-cell-mediated, antibodies cross-reacting with vascular endothelium, enhancing antibodies, complement and its products, various soluble mediators including cytokines, selection of virulent strains and virus virulence, but the most favoured are enhancing antibodies and memory T cells in a secondary infection resulting in cytokine tsunami. Whatever the mechanism, it ultimately targets vascular endothelium (making it a battlefield) leading to severe dengue disease. Extensive recent work has been done in vitro on endothelial cell monolayer models to understand the pathophysiology of vascular endothelium during dengue virus (DV) infection that may be translated to help understand the pathogenesis of DHF/DSS. The present review provides a broad overview of the effects of DV infection and the associated host responses contributing towards alterations in vascular endothelial cell physiology and damage that may be responsible for the DHF/DSS.     

Tracking the Evolution of Dengue Virus Strains D2S10 and D2S20 by 454 Pyrosequencing

Dengue virus is the most prevalent mosquito-borne virus worldwide. In this study, we used pyrosequencing to analyze the whole viral genome of two mouse-adapted strains, D2S10 and D2S20, that induce a dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in mice lacking the type I and/or type II interferon receptors. Previous experiments with D2S10 indicated that N124D and K128E mutations in the envelope protein were responsible for the severe disease induced in mice compared to its parental strain PL046. Here we demonstrate that D2S20 is more virulent than D2S10 and captured the presence of five key amino acid mutations – T70I, N83D, and K122I in envelope (E), and A62T in nonstructural protein 2A (NS2A) and G605V in nonstructural protein 5 (NS5) – that may account for this. These findings set the foundation for further dissection of the viral determinants responsible for dengue disease manifestations in mouse models.     

Endothelial cells elicit immune-enhancing responses to dengue virus infection

Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.     

Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability

Dengue virus (DENV) infection can cause mild dengue fever or severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Serum levels of the macrophage migration inhibitory factor (MIF) have been shown to be correlated with severity and mortality in DENV patients, but the pathogenic roles of MIF in DHF/DSS are still unclear. Increase in vascular permeability is an important hallmark of DHF/DSS. In this study, we found that DENV infection of the human hepatoma cell line (Huh 7) induced MIF production. Conditioned medium collected from DENV-infected Huh 7 cells enhanced the permeability of the human endothelial cell line (HMEC-1) which was reduced in the presence of a MIF inhibitor, ISO-1 or medium from DENV-infected MIF knockdown Huh 7 cells. To further identify whether MIF can alter vascular permeability, we cloned and expressed both human and murine recombinant MIF (rMIF) and tested their effects on vascular permeability both in vitro and in vivo. Indirect immunofluorescent staining showed that the tight junction protein ZO-1 of HMEC-1 was disarrayed in the presence of rMIF and partially recovered when cells were treated with ISO-1 or PI3K/MEK-ERK/JNK signaling pathway inhibitors such as Ly294002, U0126, and SP600215. In addition, ZO-1 disarray induced by MIF was also recovered when CD74 or CXCR2/4 expression of HMEC-1 were inhibited. Last but not least, the vascular permeabilities of the peritoneal cavity and dorsal cutaneous capillary were also increased in mice treated with rMIF. Taken together; these results suggest that MIF induced by DENV infection may contribute to the increase of vascular permeability during DHF/DSS. Therapeutic intervention of MIF by its inhibitor or neutralizing antibodies may prevent DENV-induced lethality.     

Alternate hypothesis on the pathogenesis of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) in dengue virus infection

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.     

A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.     

Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS)

The past four decades has witnessed a consolidation of the original observations made in the 1970s that dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) have an immunological basis. Following reinfection with a dengue virus of different serotype, severe disease is linked to high levels of antibody-enhanced viral replication early in illness which is followed by a cascade of memory T-cell activation and a 'storm' of inflammatory cytokines and other chemical mediators. These compounds are released mainly from T cells, monocytes/macrophages and endothelial cells, and ultimately cause an increase in vascular permeability. The consolidation of the evidence has been largely due to several important prospective sero-epidemiological studies in areas endemic for DHF/DSS, which have shown that risk of severe disease is significantly higher in secondary dengue infections. These advances have underscored the fact that DHF/DSS pathogenesis is a complex, multifactorial process involving cocirculation of various dengue virus serotypes and the interplay of host and viral factors that influence disease severity. The continued search to define risk factors in susceptible populations must be combined with the new techniques of molecular virology and innovative approaches in vaccine design to achieve the ultimate objective of developing a safe and effective vaccine.     

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients’ sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.     

First Experimental In Vivo Model of Enhanced Dengue Disease Severity through Maternally Acquired Heterotypic Dengue Antibodies

Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations.Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.     

Bispecific monoclonal antibodies mediate binding of dengue virus to erythrocytes in a monkey model of passive viremia

Dengue viruses (DEN), causative agents of dengue fever (DF) and more severe dengue hemorrhagic fever (DHF)/dengue shock syndrome, infect over 100 million people every year. Among those infected, up to one-half million people develop DHF, which requires an extensive hospital stay. Recent reports indicate that there is a significant correlation between virus titer in the bloodstream of infected individuals and the severity of the disease, especially the development of DHF. This suggests that if there is a procedure to reduce viremia in infected subjects, then the severity of the disease may be controlled during the critical early stages of the disease before it progresses to DHF. We have generated bispecific mAb complexes (heteropolymer(s), HP), which contain a mAb specific for the DEN envelope glycoprotein cross-linked with a second mAb specific for the primate E complement receptor 1. These HP facilitate rapid binding of DEN to human and monkey E in vitro, with approximately 90% bound within 5 min. Furthermore, in a passive viremia monkey model established by continuous steady state infusion of DEN, injection of HP during the steady state promoted rapid binding of DEN to the E, followed by subsequent clearance from the vascular system. Moreover, HP previously infused into the circulation is capable of efficiently capturing a subsequent challenge dose of DEN and binding it to E. These data suggest that HP potentially can be useful for alleviating DEN infection-associated symptoms by reducing titers of free virus in the vascular system.     

Immunopathogenesis of dengue virus infection

Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogeneses are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNFalpha-mediated immunopathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration. Dengue virus infection induces transient immune aberrant activation of CD4/CD8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. The coagulation and fibrinolysis systems are also activated after dengue virus infection. We propose a new hypothesis for the immunopathogenesis for dengue virus infection. The aberrant immune responses not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. Platelets are destroyed by crossreactive anti-platelet autoantibodies. Dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the dysregulation of coagulation and fibrinolysis persists. The overproduced IL-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Capillary leakage is triggered by the dengue virus itself or by antibodies to its antigens. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.     

Dengue type 4 live-attenuated vaccine viruses passaged in vero cells affect genetic stability and dengue-induced hemorrhaging in mice

Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3' NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q(438)H, E-V(463)L, NS2B-Q(78)H, and NS2B-A(113)T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.     

Re-evaluation of the pathogenic roles of nonstructural protein 1 and its antibodies during dengue virus infection

Dengue virus (DENV) infection can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and abnormal hemorrhage are the two major pathogenic changes found in these patients. From previous studies, it is known that both antibodies and cytokines induced in response to DENV infection are involved in the immunopathogenesis of DHF/DSS. However, the role of viral factors during DENV infection remains unclear. Nonstructural protein 1 (NS1), which is secreted in the sera of patients, is a useful diagnostic marker for acute DENV infection. Nevertheless, the roles of NS1 and its antibodies in the pathogenesis of DHF/DSS are unclear. The focus of this review is to evaluate the possible contributions of NS1 and the antibodies it induces to vascular leakage and abnormal hemorrhage during DENV infection, which may provide clues to better understanding the pathogenesis of DHF/DSS.     

Apoptosis in the Mouse Central Nervous System in Response to Infection with Mouse-Neurovirulent Dengue Viruses

Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090–4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.     

Severity-related molecular differences among nineteen strains of dengue type 2 viruses

Comparative nucleotide sequencing was carried out on dengue type 2 virus (DEN-2) strains isolated from patients in Northeast Thailand during the epidemic season in 1993. The patients exhibited different clinical manifestations ranging from dengue fever (DF) to dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). The results classified 19 DEN-2 strains into 3 subtypes according to nonsynonymous amino acid replacements. The strain isolated from a DSS patient eliciting secondary serological response belonged to subtype I, whereas 13 strains isolated from DHF patients with secondary response and 2 strains from DF patients with primary response belonged to subtype II. On the other hand, 3 strains isolated from DF cases evoking either primary or secondary response belonged to subtype III. These results suggest that subtype III virus infection could result in clinically milder manifestation irrespective of the serological response compared with subtype I or II viruses. The RNA secondary structure predicted for the 3' noncoding region showed 4 different structures (A, B, C, and D). The result also indicates that different subtypes of DEN-2 serotypes are circulating in a single epidemic in Thailand.     

Role of mitogen-activated protein kinase signaling in the pathogenesis of dengue virus infection

Dengue virus (DENV) infection is a disease that is endemic to many parts of the world, and its increasing prevalence ranks it among the diseases considered to be a significant threat to public health. The clinical manifestations of DENV infection range from mild dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased proinflammatory cytokines and vascular permeability, both of which cause organ injury, are the hallmarks of severe dengue disease. Signs of liver injury were observed in studies using hepatic cell lines, mouse models, and autopsy specimens from DENV-infected patients, and these signs substantiated the effects of inflammatory responses and hepatic cell apoptosis. Mitogen-activated protein kinases (MAPK) are involved in inflammatory responses and cellular stress during viral infections. The roles of MAPK signaling in DENV infection were reviewed, and published data indicate MAPK signaling to be involved in inflammatory responses and hepatic cell apoptosis in both in vitro cultures and in vivo models. Modulation of MAPK signaling ameliorates the inflammatory responses and hepatic cell apoptosis in DENV infection. This accumulation of published data relative to the role of MAPK signaling in inflammatory responses and cell apoptosis in DENV infection is elucidatory, and may help to accelerate the development of novel or repositioned therapies to treat this unpredictable and often debilitating disease.     

Hypertonic Saline Reduces Vascular Leakage in a Mouse Model of Severe Dengue

Dengue (DEN) is a mosquito-borne viral disease and represents a serious public health threat and an economical burden throughout the tropics. Dengue clinical manifestations range from mild acute febrile illness to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). Currently, resuscitation with large volumes of isotonic fluid remains the gold standard of care for DEN patients who develop vascular leakage and shock. Here, we investigated the ability of small volume of hypertonic saline (HTS) suspensions to control vascular permeability in a mouse model of severe DEN associated with vascular leakage. Several HTS treatment regimens were considered and our results indicated that a single bolus of 7.5% NaCl at 4 mL per kg of body weight administered at the onset of detectable vascular leakage rapidly and significantly reduced vascular leak for several days after injection. This transient reduction of vascular leakage correlated with reduced intestine and liver damage with restoration of the hepatic functions, and resulted in delayed death of the infected animals. Mechanistically, we showed that HTS did not directly impact on the viral titers but resulted in lower immune cells counts and decreased systemic levels of soluble mediators involved in vascular permeability. In addition, we demonstrated that neutrophils do not play a critical role in DEN-associated vascular leakage and that the therapeutic effect of HTS is not mediated by its impact on the neutrophil counts. Together our data indicate that HTS treatment can transiently but rapidly reduce dengue-associated vascular leakage, and support the findings of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children.     

Dengue 2 virus enhancement in asthmatic and non asthmatic individual.

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorrhagic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X2 p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data.     

A single amino acid in nonstructural protein NS4B confers virulence to dengue virus in AG129 mice through enhancement of viral RNA synthesis

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.     

Virus-induced decline in soluble vascular endothelial growth receptor 2 is associated with plasma leakage in dengue hemorrhagic Fever

Some individuals infected with dengue virus develop dengue hemorrhagic fever (DHF), a viral hemorrhagic disease characterized by a transient period of localized plasma leakage. To determine the importance of vascular endothelial growth factor A (VEGF-A) in this syndrome, we compared plasma levels of VEGF-A and the soluble forms of its receptors in patients with DHF to patients with dengue fever (DF), a milder form of dengue virus infection without plasma leakage. We observed a rise in the plasma levels of free, but not total VEGF-A in DHF patients at the time of plasma leakage. This was associated with a decline in the soluble form of VEGF receptor 2 (VEGFR2) and VEGF-soluble VEGFR2 complexes, but not the soluble form of VEGFR1. The severity of plasma leakage in patients inversely correlated with plasma levels of soluble VEGFR2. In vitro, dengue virus suppressed soluble VEGFR2 production by endothelial cells but up-regulated surface VEGFR2 expression and promoted response to VEGF stimulation. In vivo, plasma viral load correlated with the degree of decline in plasma soluble VEGFR2. These results suggest that VEGF regulates vascular permeability and its activity is controlled by binding to soluble VEGFR2. Dengue virus-induced changes in surface and soluble VEGFR2 expression may be an important mechanism of plasma leakage in DHF.