Severe Dengue Virus Infection Research Articles

Role of auto-antibodies in the mechanisms of dengue pathogenesis and its progression: a comprehensive review.

A complex interaction among virulence factors, host-genes and host immune system is considered to be responsible for dengue virus (DENV) infection and disease progression. Generation of auto-antibodies during DENV infection is a major phenomenon that plays a role in the pathophysiology of dengue hemorrhagic fever and dengue shock syndrome. Hemostasis, thrombocytopenia, hepatic endothelial dysfunction, and autoimmune blistering skin disease (pemphigus) are different clinical manifestations of dengue pathogenesis; produced due to the molecular mimicry of DENV proteins with self-antigens like coagulation factors, platelets and endothelial cell proteins. This review elaborately describes the current advancements in auto-antibody-mediated immunopathogenesis which inhibits coagulation cascade and promotes hyperfibrinolysis. Auto-antibodies like anti-endothelial cell antibodies-mediated hepatic inflammation during severe DENV infection have also been discussed. Overall, this comprehensive review provides insight to target auto-antibodies that may act as potential biomarkers for disease severity, and a ground for the development of therapeutic strategy against DENV.

HEMATOLOGY PROFILES AND DISEASE SEVERITY OF PEDIATRIC DENGUE VIRUS INFECTION AT A TERTIARY HOSPITAL IN SURABAYA, INDONESIA

Highlights Dengue virus infections exhibit a spectrum of clinical manifestations, ranging from asymptomatic cases to severe disease, with the potential for fatalities if not managed effectively. Hematology factors significantly contribute to the severity of dengue virus infection. Abstract Background: The escalating incidence of dengue cases in Surabaya, Indonesia, underscores the imperative to comprehend the hematology profiles and disease severity in pediatric patients affected by dengue virus infections (DVI). As the prevalence of DVI continues to surge, understanding the nuanced clinical manifestations becomes paramount for effective management and mitigation of the disease burden. Objective: This study aimed to characterize the hematology profiles and the disease severity of dengue virus infections (DVI) among pediatric patients hospitalized at Dr. Soetomo General Academic Hospital, Surabaya, Indonesia throughout 2019. Material and Method: A retrospective descriptive cross-sectional study was conducted using secondary data from medical records. Pediatric patients aged six months to 18 years were enrolled. A total sampling method comprised 67 patients meeting the inclusion criteria. Result: Severe thrombocytopenia was most commonly observed in dengue hemorrhagic fever (DHF) III cases (36.4%), while leukopenia was predominant in DF cases (42.2%). High hematocrit levels were more prevalent in DHF III cases (27.3%), and high hemoglobin levels were most frequently identified in DHF II and DHF III cases (33% in each case). Significant differences in DVI severity were observed in platelets and hemoglobin levels (p=0.0002 and p=0.0066, respectively) but not in leukocyte and hematocrit levels. Conclusion: Mild thrombocytopenia was prevalent in Dengue Fever (DF), while severe thrombocytopenia was most prevalent in Dengue Hemorrhagic Fever (DHF) grade III. Leukopenia was prominent in DF patients, and platelets and hemoglobin levels varied across severity of DVI. These findings provide insights for improved clinical management and diagnostic criteria refinement.

Kinetics of severe dengue virus infection and development of gut pathology in mice.

Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.

Increased In Vivo Exposure of N-(4-Hydroxyphenyl) Retinamide (4-HPR) to Achieve Plasma Concentrations Effective against Dengue Virus.

N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretinide) has promising in vitro and in vivo antiviral activity against a range of flaviviruses and an established safety record, but there are challenges to its clinical use. This study evaluated the in vivo exposure profile of a 4-HPR dosage regime previously shown to be effective in a mouse model of severe dengue virus (DENV) infection, comparing it to an existing formulation for human clinical use for other indications and developed/characterised self-emulsifying lipid-based formulations of 4-HPR to enhance 4-HPR in vivo exposure. Pharmacokinetic (PK) analysis comprising single-dose oral and IV plasma concentration-time profiles was performed in mice; equilibrium solubility testing of 4-HPR in a range of lipids, surfactants and cosolvents was used to inform formulation approaches, with lead formulation candidates digested in vitro to analyse solubilisation/precipitation prior to in vivo testing. PK analysis suggested that effective plasma concentrations could be achieved with the clinical formulation, while novel lipid-based formulations achieved > 3-fold improvement. Additionally, 4-HPR exposure was found to be limited by both solubility and first-pass intestinal elimination but could be improved through inhibition of cytochrome P450 (CYP) metabolism. Simulated exposure profiles suggest that a b.i.d dosage regime is likely to maintain 4-HPR above the minimum effective plasma concentration for anti-DENV activity using the clinical formulation, with new formulations/CYP inhibition viable options to increase exposure in the future.

Cepharanthine inhibits dengue virus production and cytokine secretion

Dengue virus (DENV) infection is a public health problem in tropical and subtropical regions. It can cause a spectrum of clinical manifestations ranging from mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF) and potentially life-threatening disease including dengue shock syndrome (DSS). Severe DENV infection is caused by high viral load and cytokine storm in dengue-infected patients. Currently, there is no specific antiviral drug for DENV infection. An anti-DENV agent that demonstrates inhibitory effects on both DENV replication and cytokine secretion is urgently needed. In this study, cepharanthine (CEP), which is an anti-inflammatory, anti-HIV, and anti-tumor compound isolated from Stephania cepharantha Hayata, was tested for inhibition of DENV infection. We investigated the efficacy of CEP to inhibit DENV infection, replication, and cytokine production. The inhibitory effect of CEP treatment was studied in DENV-infected human chronic myeloid leukemia (K562) cells. The levels of DENV E protein and DENV production were determined by flow cytometry and FFU assay, respectively. CEP treatment significantly reduced viral E protein and viral production in all DENV-1, 2, 3, 4 serotypes. In addition, CEP treatment reduced the IL-6 proinflammatory cytokine production in DENV-infected A549 cells. Taken together, CEP has inhibitory effects on DENV infection specifically at the initial viral replication states and proinflammatory cytokine secretion, and is a promising candidate for further development as an anti-DENV treatment.

TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to non-infectious immature dengue virus particles.

Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF-α and IL-1β by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF-α production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection.

Alpha-mangostin inhibits viral replication and suppresses nuclear factor kappa B (NF-κB)-mediated inflammation in dengue virus infection

Severe dengue virus (DENV) infection results from viral replication and dysregulated host immune response, which trigger massive cytokine production/cytokine storm. The result is severe vascular leakage, hemorrhagic diathesis, and organ dysfunction. Subsequent to previously proposing that an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine storm, we discovered that α-mangostin (α-MG) from the pericarp of the mangosteen fruit could inhibit both DENV infection and cytokine/chemokine production. In this study, we investigated the molecular mechanisms underlying the antiviral and anti-inflammatory effects of α-MG. Time-of-drug-addition and time-of-drug-elimination studies suggested that α-MG inhibits the replication step of the DENV life cycle. α-MG inhibited polymerization activity of RNA-dependent RNA polymerase (RdRp) with IC50 values of 16.50 μM and significantly reduced viral RNA and protein syntheses, and virion production. Antiviral and cytokine/chemokine gene expression profiles of α-MG-treated DENV-2-infected cells were investigated by polymerase chain reaction array. α-MG suppressed the expression of 37 antiviral and cytokine/chemokine genes that relate to the NF-κB signaling pathway. Immunofluorescence and immunoblot analyses revealed that α-MG inhibits NF-κB nuclear translocation in DENV-2-infected cells in association with reduced RANTES, IP-10, TNF-α, and IL-6 production. These results suggest α-MG as a potential treatment for DENV infection.

Secondary cross infection with dengue virus serotype 2/3 aggravates vascular leakage in BALB/c mice.

Dengue virus (DV) has occasionally emerged at epidemic levels in Yunnan, China. Vaccine development is limited by antibody-dependent enhancement and a lack of good animal models. Thus, the study investigated cross infection based on maternal immunity in BALB/c mice and assessed the risk of cross infection by DV2-D13113 and DV3-YNWS2 epidemic virus strains. DV replicated within the organs of the BALB/c infant mice, even causing death. Particularly, DV3-infected infant mice were at higher risk of severe disease if their mothers were infected with DV2. Although BALB/c adults and pups survived DV2/DV3 infection and produced anti-DV antibodies after 5-8 days, extensive subcutaneous vascular leakage was observed after secondary DV infection. Furthermore, vascular permeability in the lung and kidney significantly increased in offspring born to heterotypic virus-infected mothers. Thus, vascular leakage indicates severe DV infection. The results indicate that maternal immunity increases the severity of subsequent heterotypic infection. Additionally, secondary cross infection by D13113 and YNWS2 represents a risk of serious disease. This study has implications for studies of DV cross infection and vaccine development.

Dengue Virus and Platelets: From the Biology to the Clinic.

Dengue is one of the most important vector-borne viral illnesses found in tropical and subtropical regions. Colombia has one of the highest rates of dengue cases in the Americas. Severe dengue virus (DENV) infection presents with capillary leakage, hemorrhage, and organ compromise, eventually leading to death. Over the years, there have been many efforts to develop a vaccine that guarantees protective immunity, but they have been partially successful, as such immunity would need to guarantee protection against four distinct viral serotypes. Absolute platelet count is a laboratory parameter used to monitor the clinical progression of DENV, as infection is often accompanied by thrombocytopenia. Although this finding is well described with respect to the natural history of the disease, there are various hypotheses as to the cause of this rapid decrease, and several in vivo and ex vivo models have been used to explain the effect of DENV infection on platelets and their precursors. DENV infects and activates platelets, facilitating their elimination through recognition by phagocytic cells and peripheral margination. However, infection also affects the precursors in the bone marrow by modulating megakaryopoiesis. The objective of this article is to explore various proposed mechanisms of DENV-induced thrombocytopenia to better understand the pathophysiology and clinical presentations of this highly relevant viral infection.

Increased Plasma Heparanase Activity and Endothelial Glycocalyx Degradation in Dengue Patients Is Associated With Plasma Leakage

BackgroundEndothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection.MethodsHeparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo.ResultsHeparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets.ConclusionTaken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase.

Detection of Proteinuria by Colorimetric Method and Correlation with Disease Severity in Patients Dengue

Severe dengue virus infection often causes proteinuria through several mechanisms, such as leakage of plasma proteins caused by higher protein filtering in the kidneys. The condition of proteinuria in patients with dengue shock syndrome will affect the management, prognosis, and mortality of patients with dengue virus infection. The objectives of this study were to determine the presence of protein in urine samples in patients in which IgG/IgM dengue was detected, to determine the presence of protein in urine samples in patients without dengue infection, and to determine the relationship between proteinuria in dengue patients and the degree of disease severity. The study was conducted in March-June 2020 at the Clinical Laboratory and Inpatient Clinic of Vita Medika Kepung, Kediri Regency. The method used was the consecutive sampling technique. The minimum sample size was 100 respondents with criteria for suspected dengue infection, onset fever > 4 days, age > 2 years, and no indication of other infectious diseases (blood and urine), divided into two groups, group with positive dengue infection and non-dengue group. The results showed that proteinuria was detected in 26% of respondents, secondary dengue infection and positive serological test of IgG, IgM only IgG were detected in 24 (92.3%) respondents and positive IgG and IgM were found in 2 (7.7%) respondents, this was shown by the p-value = 0.000 in the Chi-Square test with a Prevalence Ratio (PR) value of 11,987. In addition, Chi-Square test results indicated that a higher urine protein/albumin led to more severe disease, with p-value = 0.012 and 0.025 (p <0.05) and PR values of 3.333 and 2.800. The results of this study concluded that there was a close relationship between high levels of protein/albumin in the urine with the degree of disease severity in patients with dengue infection. In addition, an increase in urine protein/albumin levels is always followed by a decrease in serum protein/albumin levels.

Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infection.

Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.

Crocetin Improves Dengue Virus-Induced Liver Injury

Dengue virus (DENV) infection is one of the most widespread mosquito-borne viral infections. Liver injury is commonly observed in severe DENV infection, and the present study aimed to examine the efficacy of crocetin treatment in an immunocompetent mouse model of DENV infection exhibiting liver injury. The efficacy of crocetin treatment in DENV-induced liver injury was assessed via both transaminase levels and histopathology analysis. A real-time polymerase chain reaction array was then used to describe the expression of 84 apoptosis-related genes. Using real-time RT-PCR and Western blot analysis, the gene expressions of host factors were investigated. Additionally, the effect of crocetin in NF-kB signaling during DENV infection was studied. We did not observe any significant reduction in virus production when DENV-infected mice were treated with crocetin. However, DENV-infected mice treated with crocetin showed reduced DENV-induced apoptosis. The real-time polymerase chain reaction array revealed pro-inflammatory cytokine expressions to be significantly reduced in the crocetin-treated DENV-infected mice. We also found that crocetin could effectively modulate antioxidant status in DENV-infected mice. Moreover, crocetin demonstrated the ability to reduce the nuclear translocation of NF-kB in DENV-infected mice. Our results suggest that crocetin treatment does not inhibit DENV replication in the liver of DENV-infected mice; however, we did find that crocetin improves host responses that reduce liver injury.

TLR2 on blood monocytes senses dengue virus infection and its expression correlates with disease pathogenesis

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.

A Study of Serum Lipid Profile Changes in Children with Dengue Haemorrhagic Fever and its Correlation with Severity in a Tertiary Care Hospital

Introduction: Dengue Virus (DENV) infection now remains as the most rapidly spreading viral disease worldwide. Lower lipid level occurs in critically ill patients with DENV infection and is postulated as an independent predictor of the clinical outcome. Aim: To find the correlation between serum lipid levels and severity in children admitted with various grades of Dengue Haemorrhagic Fever (DHF) and secondarily to correlate serum lipid levels with factors like platelet count and duration of hospital stay. Materials and Methods: This was a prospective descriptive study done in the Paediatric ward from October 2019 to December 2019. Febrile Children with a platelet count of less than 1 lakh/cu.mm and positive for DENV IgM antibodies by Enzyme Linked Immuno Sorbent Assay (ELISA) formed the study group. Demographic details, number of days of fever on admission, duration of stay in hospital and severity grading of DENV infection as per the National guidelines were collected. These children were evaluated with Complete Blood Counts (CBC), fasting lipid profile, X-ray chest and ultrasound abdomen. Mean and standard deviation was used for categorical variables. Chi-square test was used to assess the strength of association between lipids and severity of DENV infections. Pearson correlation coefficient was used to assess R value between length of stay and serum lipid levels. Results: The Total Cholesterol, Low Density Lipoprotein (LDL), Triglyceride and Very Low Density Lipoprotein (VLDL) showed a simple linear decline as the severity grade advanced from grade I to grade IV. However, statistical significance was found only for total cholesterol and LDL with p-values of 0.046 and 0.041 respectively. The distribution of High Density Lipoprotein (HDL) across the grades of severity did not follow any pattern and was randomly distributed. On analysis of Pearson correlation between number of days of hospital stay and lipid profile, statistical significance was observed for LDL alone (p=0.023). Conclusion: The serum levels of total cholesterol and LDL were significantly reduced in severe grades of DHF. Serum levels of total cholesterol and LDL can be used as an economical surrogate marker to predict the severity of DHF at the time of admission.

Dynamic changes of soluble ST2 levels predicted fatality and were involved in coagulopathy in dengue fever in the elderly.

BackgroundSevere dengue virus (DENV) infection involves plasma leakage and vascular collapse, and leads to significant morbidity and death. Serum soluble ST2 (sST2 [interleukin (IL)-1 receptor like-1 protein: IL-1-RL-1]) levels are high in pediatric cases of DENV infection, and the disease progresses. However, the correlation between serum sST2 levels and the outcomes of DENV infection in the elderly (≥65 years) is unclear. We thus explored the mechanisms of serial sST2 level changes involved in the coagulopathy and bloodstream infections of elderly patients in Taiwan’s 2015 DENV outbreak.MethodsThis retrospective study was done in a tertiary medical center in southern Taiwan during the outbreak. All DENV-infected patients who, between July 1, 2015, and December 31, 2015, provided a written informed consent for at least two blood sample analyses were enrolled and reviewed. The serum levels of sST2 were quantified. ΔsST2 is defined as the “changes of sST2 levels in serially paired samples”. Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) analyses were used to evaluate the prognostic ability of ΔsST2.ResultsForty-three patients with DENV infection were enrolled. Mean patient age was 75.0 ± 12.2 years and the case fatality rate was 44.2% (19/43). Significantly more non-survivors than survivors had increased ST2 level (78.9% vs. 12.5%, p < 0.001). The AUC value for serum ΔsST2 level was 0.857 for predicting DENV fatality. Moreover, patients given frozen fresh plasma (FFP) transfusions were significantly (p = 0.025) more likely to have higher serum ST2 level changes than were those who had not. DENV-infected patients with early bloodstream infections (BSIs) seemed to have higher ST2 levels than those who did not have BSIs.ConclusionsSerum ST2 levels increased in the elderly (≥ 65 years of age) with DENV infection. The changes in serum sST2 levels might be a critical indicator of DENV infection severity for the elderly; sST2 is an important modulator of coagulopathy in severe DENV infections.

Effect of high doses of vitamin D supplementation on dengue virus replication, Toll-like receptor expression, and cytokine profiles on dendritic cells.

Dengue, caused by dengue virus (DENV) infection, is a public health problem worldwide. Although DENV pathogenesis has not yet been fully elucidated, the inflammatory response is a hallmark feature in severe DENV infection. Although vitamin D (vitD) can promote the innate immune response against virus infection, no studies have evaluated the effects of vitD on DENV infection, dendritic cells (DCs), and inflammatory response regulation. This study aimed to assess the impact of oral vitD supplementation on DENV-2 infection, Toll-like receptor (TLR) expression, and both pro- and anti-inflammatory cytokine production in monocyte-derived DCs (MDDCs). To accomplish this, 20 healthy donors were randomly divided into two groups and received either 1000 or 4000 international units (IU)/day of vitD for 10days. During pre- and post-vitD supplementation, peripheral blood samples were taken to obtain MDDCs, which were challenged with DENV-2. We found that MDDCs from donors who received 4000IU/day of vitD were less susceptible to DENV-2 infection than MDDCs from donors who received 1000IU/day of vitD. Moreover, these cells showed decreased mRNA expression of TLR3, 7, and 9; downregulation of IL-12/IL-8 production; and increased IL-10 secretion in response to DENV-2 infection. In conclusion, the administration of 4000IU/day of vitD decreased DENV-2 infection. Our findings support a possible role of vitD in improving the innate immune response against DENV. However, further studies are necessary to determine the role of vitD on DENV replication and its innate immune response modulation in MDDCs.

High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity.

BackgroundA main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known.MethodsA cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Serum hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability.FindingsElevated serum hyaluronan levels (≥70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity.InterpretationPharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection.FundThis work was supported in part by grants from the Swedish Cancer Society (2018/337; 2016/445), the Swedish Research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106–2314-B-037-088- and 106–2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3 T05) and Academy of Finland. The funders played no role in the design, interpretation or writing of the manuscript.

High Levels of Serum Hyaluronan Predicts Severe Dengue Infection and Perturbs Vascular Integrity

A main pathological feature of severe dengue virus infection is endothelial hyperpermeability. We demonstrate that elevated serum hyaluronan levels (≥70 ng/ml)during early infection is an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlate with high concentration of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells recruited CD44-expressing macrophages, thus perpetuating inflammation. Perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VEcadherin and cytoskeleton re-organization, and exacerbated the NS1 induced disruption of endothelial integrity. Thus, pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during severe dengue virus infection. Funding Statement: This work was supported in part by grants from the Swedish Cancer Society (2015- 0778), the Swedish research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106- 2314-B-037-088- and 106-2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3T05) and Academy of Finland. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: The human study protocol was approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (IRB Number: KMUH-IRB-20110451). Informed consent was obtained from all subjects.

Drug repurposing of N-acetyl cysteine as antiviral against dengue virus infection

Liver injury is one of the hallmark features of severe dengue virus (DENV) infection since DENV can replicate in the liver and induce hepatocytes to undergo apoptosis. N-acetyl cysteine (NAC), which is a clinically-used drug for treating acetaminophen toxicity, was found to benefit patients with DENV-induced liver injury; however, its mechanism of action remains unclear. Accordingly, our aim was to repurpose NAC in the preclinical studies to investigate its mechanism of action. Time of addition experiments in HepG2 cells elucidated effectiveness of NAC to reduce infectious virion at pre-, during- and post infection. In DENV-infected mice, NAC improved DENV-associated clinical manifestations, including leucopenia and thrombocytopenia, and reduced liver injury and hepatocyte apoptosis. Interestingly, we discovered that NAC significantly reduced DENV production in HepG2 cells and in liver of DENV-infected mice by induction of antiviral responses via interferon signaling. NAC treatment in DENV-infected mice helped to maintain antioxidant enzymes and redox balance in the liver. Therefore, NAC reduces DENV production and oxidative damage to ameliorate DENV-induced liver injury. Taken together, these findings suggest the novel therapeutic potential of NAC in DENV-induced liver injury and recommend evaluating its efficacy and safety in humans with DENV-induced liver injury.