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Dengue is a significant public health concern across tropical and subtropical regions worldwide, principally causing disease in children. Very young children are at increased risk of severe manifestations of dengue infection. The mechanism of dengue disease in this population is not fully understood. In this study, we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis. Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice, showing increased liver enzymes, extended viremia, dissemination to organs and histological alterations in liver and small intestine. Furthermore, the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice. These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study age-related mechanisms of dengue pathogenesis., Competing Interests: None., (© 2020 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Dengue virus (DENV) infection is a significant public health threat in tropical and subtropical regions; however, there is no specific antiviral drug. Accumulated studies have revealed that DENV infection induces several cellular responses, including autophagy and apoptosis. The crosstalk between autophagy and apoptosis is associated with the interactions among components of these two pathways, such as apoptotic caspase-mediated cleavage of autophagy-related proteins. Here, we show that DENV-induced autophagy inhibits early cell apoptosis and hence enhances DENV replication. Later, the apoptotic activities are elevated to suppress autophagy through cleavage of Beclin-1, an essential autophagy-related protein. Inhibition of cleavage of Beclin-1 by a pan-caspase inhibitor, Z-VAD, increases both autophagy and viral replication. Regarding the mechanism, we further found that DENV nonstructural protein 1 (NS1) is able to interact with Beclin-1 during DENV infection. The interaction between Beclin-1 and NS1 attenuates Beclin-1 cleavage and facilitates autophagy to prevent cell apoptosis. Our study suggests a novel mechanism whereby NS1 preserves Beclin-1 for maintaining autophagy to antagonize early cell apoptosis; however, elevated caspases trigger apoptosis by degrading Beclin-1 in the late stage of infection. These findings suggest implications for anti-DENV drug design.

In this paper, we generalize a previous model of within-host dengue infection with a nonconstant monocyte production rate. We establish the existence of three equilibria and give some local stability results. We then estimate three parameters in the model from clinical data for dengue virus serotype 2. It is then shown that the model can exhibit behaviours that are not possible under the assumption of constant monocyte production. Lastly, we perform a sensitivity analysis of the model in two contexts, antiviral treatment and immunostimulatory treatment. The results predict that antiviral treatments that reduce the viral replication rate in infected monocytes are the most effective, while immunostimulatory treatments that increase the rate at which infected monocytes are removed are best.

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects., Competing Interests: The authors have declared that no competing interests exist.

Management of severe malaria remains a critical global challenge. In this study, using a multiplexed quantitative proteomics pipeline we systematically investigated the plasma proteome alterations in non-severe and severe malaria patients. We identified a few parasite proteins in severe malaria patients, which could be promising from a diagnostic perspective. Further, from host proteome analysis we observed substantial modulations in many crucial physiological pathways, including lipid metabolism, cytokine signaling, complement, and coagulation cascades in severe malaria. We propose that severe manifestations of malaria are possibly underpinned by modulations of the host physiology and defense machinery, which is evidently reflected in the plasma proteome alterations. Importantly, we identified multiple blood markers that can effectively define different complications of severe falciparum malaria, including cerebral syndromes and severe anemia. The ability of our identified blood markers to distinguish different severe complications of malaria may aid in developing new clinical tests for monitoring malaria severity.

Severe pneumonia and multiorgan dysfunction in COVID-19 and dengue haemorrhagic fever (DHF) are two diseases that can associate with an altered immune response to the infecting virus. To determine the similarities and differences in the cytokine and chemokine responses in these two infections, we compared responses in patients with varying severity of COVID-19 and acute dengue at different time points of illness. During early disease, patients who proceeded to develop COVID-19 severe pneumonia (SP) and DHF had significantly higher levels of IL-6, IL-10 and MIP3α than those who developed mild illness. The lowest levels of IFNγ in early illness were seen in those who succumbed to their illness due to COVID-19. Levels of serum IL-10 (p = 0.0001), IL-6 (p = 0.002), MIP-3α (p = 0.02) and CD40-L levels (p = 0.002) significantly increased from 5 to 9 day of illness to 10-21 day of illness in patients with moderate-to-severe COVID-19, but not in those with mild illness. In contrast, these cytokine/chemokine levels remained unchanged in those with DHF or dengue fever (DF) during febrile and critical phases. Although IL-10 levels were significantly higher in COVID-19 patients with SP, patients with DHF had 25-fold higher levels, whereas IL-6 levels were 11-fold higher in those with COVID-19 SP. IL-10 and other cytokines were evaluated in a larger cohort of patients during early illness (≤ 4 days) who proceeded to develop DF (n = 71) or DHF (n = 64). Of the cytokines evaluated, IL-10 was significantly higher (p < 0.0001) in those who went on to develop DHF compared to DF. Low IFNγ response to the SARS-CoV2 and high levels of immunosuppressive IL-10 in both COVID-19 and dengue during early illness are indicators of an altered antiviral response potentially contributing to disease severity.

Ocular complications are rare in patients with dengue fever, but may cause permanent loss of vision. We present the case of a 29-year-old German woman who developed severe acute vision loss because of dengue-associated maculopathy after traveling to Vietnam and Cambodia. Initially, the optical coherence tomography showed detachment of the retinal pigment epithelium, a central shift in the retinal pigmentation and intraretinal cysts. The patient was hospitalized and treated with a short course of intravenous prednisolone. Vision improved, and the patient showed full recovery at 9 months after the onset. This case highlights the importance of awareness and adequate management for ocular involvement in patients with dengue fever, including travelers.

We previously reported that dengue virus (DENV)-induced autophagy plays a promoting role in viral replication and pathogenesis both in vitro and in vivo. Although it is known that DENV infection increases glycolysis, which promotes viral replication, the role of glucose metabolism together with autophagic activity in DENV replication remains unclear. In this study, we reveal that DENV2 infection increased autophagic activity, glucose uptake, protein levels of glucose transporter-1 (GLUT1), and glycolysis rate-limiting enzyme hexokinase-2 (HK2) in cells. Furthermore, the protein levels of LC3-II and HK2 were increased in the brain tissues of the DENV2-infected suckling mice. However, DENV2 infection decreased ATP level and showed no effect on mRNA expression of HK2 and phosphofructokinase, as well as lactate production, indicating that DENV2-regulated glycolytic flux occurs at the post-transcriptional level and is lactate pathway-independent. Moreover, amiodarone-induced autophagic activity, glucose uptake, HK2 level, and viral titer were reversed by the autophagy inhibitor spautin-1 or silencing of Atg5 gene expression. Intriguingly, blocking of glycolysis, HK2 protein level, and viral titer were accordingly decreased, but autophagic activity was increased, suggesting the existence of another regulation mechanism that influences the relationship between glycolysis and autophagy. This is the first report to reveal that DENV2-induced autophagy positively regulates glycolysis and viral replication in vitro and in vivo. Our findings open a new avenue wherein metabolic modulation could be used as a target for the treatment of DENV infection., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14 + monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin., (Copyright © 2020 Montes-Gómez, García-Cordero, Marcial-Juárez, Shrivastava, Visoso-Carvajal, Juárez-Delgado, Flores-Romo, Sanchez-Torres, Santos-Argumedo, Bustos-Arriaga and Cedillo-Barrón.)

Backgrounds: Approximately, half of the population in the world including tropical and sub-tropical climates region is at risk of dengue. Being an endemic country, Bangladesh has experienced the largest dengue epidemic in 2019. The present study aimed at evaluating the clinical and laboratory profile of dengue patients in northern Bangladesh during the epidemic., Methods: This cross-sectional study included 319 serologically confirmed dengue patients admitted in Shaheed Ziaur Rahman Medical College Hospital in Bogra district. It is one of the main tertiary care hospitals in northern Bangladesh. Data were collected from July to September 2019. Patients' clinical and laboratory data were extracted from clinical records. Patients were classified into two classes according to the WHO 2009 dengue classification such as (i) non-severe dengue and (ii) severe dengue. Chi-square test and independent t-test were used in this study., Results: Of the 319 patients, 94.1% had non-severe dengue and the remaining 5.9% had severe dengue (severe plasma leakage 68.4%, severe organ involvement 68.4%, and severe clinical bleeding 10.5%). Most of the patients were suffering from primary dengue infection. The most common clinical presentation was fever followed by headache and myalgia. Vomiting and abdominal pain were the most prevalent warning signs. The common hematological findings on admission were leukopenia (63.3%), thrombocytopenia (30.4%) and increased hematocrit (26.6%). Raised serum ALT or AST was observed in 14.1% cases whereas raised serum creatinine was observed in 6.6% cases. Signs of plasma leakage (pleural effusion, respiratory distress, and ascites, rise of hematocrit >20% during hospital stay) and hepatic or renal involvement (serum ALT >42UI/L or serum creatinine >1.2 mg/dL) on admission were mostly associated with severe dengue., Conclusion: The study provides clinical evidence on presentation as well as hematological and biochemical profile of dengue patients in northern Bangladesh that should be implicated in effective patient management., Competing Interests: The authors have declared that no competing interests exist.

Dengue is characterized as one of the most important arthropod-borne human viral diseases, representing a public health problem. Increased activation of immune cells is involved in the progression of infection to severe forms. Recently, our group demonstrated the contribution of platelet-monocyte interaction to inflammatory responses in dengue, adding to evolving evidence that platelets have inflammatory functions and can regulate different aspects of innate immune responses. Furthermore, stimuli-specific-activated platelets can promote phenotypic changes and metabolic reprogramming in monocytes. Thus, this study aimed to evaluate the roles of dengue virus (DENV)-activated platelets on immunometabolic reprogramming of monocytes in vitro, focusing on lipid droplet (LD) biogenesis. We demonstrated that platelets exposed to DENV in vitro form aggregates with monocytes and signal to LD formation and CXCL8/IL-8, IL-10, CCL2, and PGE 2 secretion. Pharmacologic inhibition of LD biogenesis prevents PGE 2 secretion, but not CXCL8/IL-8 release, by platelet-monocyte complexes. In exploring the mechanisms involved, we demonstrated that LD formation in monocytes exposed to DENV-activated platelets is partially dependent on platelet-produced MIF. Additionally, LD formation is higher in monocytes, which have platelets adhered on their surface, suggesting that beyond paracrine signaling, platelet adhesion is an important event in platelet-mediated modulation of lipid metabolism in monocytes. Together, our results demonstrate that activated platelets aggregate with monocytes during DENV infection and signal to LD biogenesis and the secretion of inflammatory mediators, which may contribute to dengue immunopathogenesis., (©2020 Society for Leukocyte Biology.)

Immunological adaptations in pregnancy allow maternal tolerance of the semi-allogeneic fetus but also increase maternal susceptibility to infection. At implantation, the endometrial stroma, glands, arteries and immune cells undergo anatomical and functional transformation to create the decidua, the specialized secretory endometrium of pregnancy. The maternal decidua and the invading fetal trophoblast constitute a dynamic junction that facilitates a complex immunological dialogue between the two. The decidual and peripheral immune systems together assume a pivotal role in regulating the critical balance between tolerance and defense against infection. Throughout pregnancy, this equilibrium is repeatedly subjected to microbial challenge. Acute viral infection in pregnancy is associated with a wide spectrum of adverse consequences for both mother and fetus. Vertical transmission from mother to fetus can cause developmental anomalies, growth restriction, preterm birth and stillbirth, while the mother is predisposed to heightened morbidity and maternal death. A rapid, effective response to invasive pathogens is therefore essential in order to avoid overwhelming maternal infection and consequent fetal compromise. This sentinel response is mediated by the innate immune system: a heritable, highly evolutionarily conserved system comprising physical barriers, antimicrobial peptides (AMP) and a variety of immune cells-principally neutrophils, macrophages, dendritic cells, and natural killer cells-which express pattern-receptors that detect invariant molecular signatures unique to pathogenic micro-organisms. Recognition of these signatures during acute infection triggers signaling cascades that enhance antimicrobial properties such as phagocytosis, secretion of pro-inflammatory cytokines and activation of the complement system. As well as coordinating the initial immune response, macrophages and dendritic cells present microbial antigens to lymphocytes, initiating and influencing the development of specific, long-lasting adaptive immunity. Despite extensive progress in unraveling the immunological adaptations of pregnancy, pregnant women remain particularly susceptible to certain acute viral infections and continue to experience mortality rates equivalent to those observed in pandemics several decades ago. Here, we focus specifically on the pregnancy-induced vulnerabilities in innate immunity that contribute to the disproportionately high maternal mortality observed in the following acute viral infections: Lassa fever, Ebola virus disease (EVD), dengue fever, hepatitis E, influenza, and novel coronavirus infections., (Copyright © 2020 Cornish, Filipovic, Åsenius, Williams and McDonnell.)

Background: Dengue is a systemic and dynamic disease with symptoms ranging from undifferentiated fever to dengue shock syndrome. Assessment of patients' severity of dehydration is integral to appropriate care and management. Urine colour has been shown to have a high correlation with overall assessment of hydration status. This study tests the feasibility of measuring dehydration severity in dengue fever patients by comparing urine colour captured by mobile phone cameras to established laboratory parameters., Methodology/principal Findings: Photos of urine samples were taken in a customized photo booth, then processed using Adobe Photoshop to index urine colour into the red, green, and blue (RGB) colour space and assigned a unique RGB value. The RGB values were then correlated with patients' clinical and laboratory hydration indices using Pearson's correlation and multiple linear regression. There were strong correlations between urine osmolality and the RGB of urine colour, with r = -0.701 (red), r = -0.741 (green), and r = -0.761 (blue) (all p-value <0.05). There were strong correlations between urine specific gravity and the RGB of urine colour, with r = -0.759 (red), r = -0.785 (green), and r = -0.820 (blue) (all p-value <0.05). The blue component had the highest correlations with urine specific gravity and urine osmolality. There were moderate correlations between RGB components and serum urea, at r = -0.338 (red), -0.329 (green), -0.360 (blue). In terms of urine biochemical parameters linked to dehydration, multiple linear regression studies showed that the green colourimetry code was predictive of urine osmolality (β coefficient -0.082, p-value <0.001) while the blue colourimetry code was predictive of urine specific gravity (β coefficient -2,946.255, p-value 0.007)., Conclusions/significance: Urine colourimetry using mobile phones was highly correlated with the hydration status of dengue patients, making it a potentially useful hydration status tool., Competing Interests: The authors have declared that no competing interests exist.

Nine criteria regarding the infectious agent, mode of transmission, portal of entry, route of spread, target organs, target cells, pathologic lesions, incubation period, and modifiable spectrum of disease and outcomes appropriate to the intended experimental purpose are described. To provide context for each criterion, mouse models of two vector-borne zoonotic infectious diseases, scrub typhus and dengue, are summarized. Application of the criteria indicates that intravenous inoculation of Orientia tsutsugamushi into inbred mice is the best current model for life-threatening scrub typhus, and intradermal inoculation accurately models sublethal human scrub typhus, whereas the immunocompromised mouse models of dengue provide disease outcomes most closely associated with human dengue. In addition to addressing basic questions of immune and pathogenic mechanisms, mouse models are useful for preclinical testing of experimental vaccines and therapeutics. The nine criteria serve as guidelines to evaluate and compare models of vector-borne infectious diseases.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers. IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines., (Copyright © 2020 American Society for Microbiology.)

Competing Interests: The authors have declared that no competing interests exist.

Dengue disease is an inflammatory-driven pathology, and complement overactivation is linked to disease severity and vascular leakage. Additionally, dysregulation of complement alternative pathway (AP) components has been described, such as upregulation of complement factor D and downregulation of complement factor H (FH), which activate and inhibit the AP, respectively. Thus, the pathology of severe dengue could in part result from AP dysfunction, even though complement and AP activation usually provide protection against viral infections. In dengue virus-infected macrophages and endothelial cells (ECs), the site of replication and target for vascular pathology, respectively, the AP is activated. The AP activation, reduced FH and vascular leakage seen in dengue disease in part parallels other complement AP pathologies associated with FH deficiency, such as atypical haemolytic uraemic syndrome (aHUS). aHUS can be therapeutically targeted with inhibitors of complement terminal activity, raising the idea that strategies such as inhibition of complement or delivery of FH or other complement regulatory components to EC may be beneficial to combat the vascular leakage seen in severe dengue., (© 2020 Federation of European Biochemical Societies.)

Single nucleotide polymorphisms (SNPs) in immune-related genes have been shown to play a role in driving the development of the severe phenotypes of dengue virus (DENV) infection. We assessed the association between IFNL3 gene SNP (rs12979860) and dengue clinical outcomes in children. Patients with dengue-related symptoms (aged 1-15 years) admitted at a public hospital in Northeast Brazil were invited to participate. The association between rs12979860 polymorphism and dengue classification and clinical signs and symptoms were analysed. A total of 206 DENV-infected children were included: 53.4% of the infections were classified as severe dengue. The T allele carriers had higher risk of developing severe dengue when compared to CC genotype carriers (OR: 1.81; 95% CI: 0.98-3.32 p = .054). The T allele carriers also showed longer fever episodes when compared to patients with the CC genotype (OR: 1.90; 95%CI: 1.07-3.38; p = .027). On the other hand, the ones carrying the CT/TT genotype had 70% lower chance of developing thrombocytopenia when compared to those with the CC genotype (OR: 0.30; 95%CI: 0.08-0.88; p = .042). Our findings demonstrated that the T allele carriers of the IFNL3 gene had higher risk of developing severe dengue, suggesting a link between IFN-λ expression and DENV immunopathogenesis., (© 2020 John Wiley & Sons Ltd.)

Dengue virus (DENV) causes the most prevalent arbovirus illness worldwide and is responsible for many debilitating epidemics. The four circulating DENV serotypes infect humans and can cause asymptomatic, mild, moderate, or severe Dengue. Because of the global morbidity and mortality due to Dengue, deployment of a safe and effective tetravalent vaccine has been a high priority, and to date, a partially realized goal. The study of pathogenesis and development of DENV therapeutics and vaccines has been limited by few animal models that recapitulate key features of human disease. Over the past two decades, mouse models of DENV infection have evolved with increasing success. Here, we review the utilization and limitations of mice for studying DENV pathogenesis and evaluating countermeasures., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The first documented outbreak of dengue which included cases with haemorrhage occurred in Papua New Guinea in 2016., Aim: To document the presentation and outcome of children with dengue in Port Moresby., Methods: This prospective cross-sectional descriptive study was conducted in Port Moresby General Hospital during a 6-month period from 6 January to 6 July 2016. Altogether, 165 children aged 1-14 years who met the WHO criteria for probable dengue were assessed and treated. Clinical features, presence of warning signs and signs of severe dengue, date of onset, management and outcome were recorded. Blood specimens were collected for serological testing and full blood count., Results: The median age was 6 years (interquartile range 3-8). Eighty-eight (53%) children had no warning signs and were managed as outpatients. Of the 165 patients, 42 (25%) had abdominal pain, 28 (17%) had bleeding and 3 (2%) had clinical evidence of fluid accumulation. The median (IQR) lowest platelet count in those tested was 34 × 10 9 /L (22-54). Two children were transfused with packed red blood cells and one received a platelet transfusion. No child developed dengue shock and none died. Non-structural protein 1 (NS1) and dengue IgM were positive in 122/144 (85%) and 36/111 (32%) of blood samples, respectively. 150/151 blood samples tested for dengue were positive on one or more tests., Conclusion: There is the potential for future outbreaks of increased severity in Papua New Guinea. Surveillance, mosquito reduction initiatives and health education programmes are needed to reduce the impact of future outbreaks.

Introduction: Due to the shortcomings in the 1997-World Health Organisation (WHO) dengue case classification (DCC), a revised classification was proposed in 2009. This study was aimed to assess the clinical usefulness of the two classifications during a large dengue epidemic., Methodology: Clinical data of dengue patients admitted to selected units at National Hospital of Sri Lanka, Panadura Base Hospital and Nawaloka Hospital Colombo between June and August 2017 were collected prospectively. Cases were classified using the 1997 and 2009 WHO DCCs., Results: 1,878 patients [adult = 1,573 (83.8%)] were studied. Based on 1997-WHO-DCC-DF (Dengue Fever): 1,316 (70.1%), DHF (Dengue Haemorrhagic Fever) -1: 468 (24.9%), DHF-2: 86 (4.6%) and DHF-3: 8 (0.4%). Based on 2009-WHO-DCC-Dengue with warning signs (WS): 1647 (87.7%), Dengue without WS: 231 (12.3%) and severe dengue (SD): 41 (2.18%). A total of 1,088 (82.7%) DF and 559 (99.5%) DHF patients developed WS. Of those without WS, 228 (17.3%) were DF patients and 3 (0.5%) were DHF patients. Three (0.23%) DF and 38 (6.76%) DHF patients had SD. All SD patients had WS. The level of agreement between the two systems of classification was poor (Kappa = - 0.035, p < 0.001)., Conclusions: The 2009-WHO-DCC was more useful than 1997-WHO-DCC in predicting dengue disease severity as few DF patients also had SD. Furthermore, the presence of WS identified patients with SD. However, the 2009-WHO-DCC may not suit the resource limited countries as WS are non-specific, and lack of diagnostic tests can result in case overload., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Umesh Jayarajah, Visula Abeysuriya, Pradeep K de Silva, Priyankara Jayawardena, Aruna Kulatunga, Harshini Fernando, Manohari Madarasinghe, Damayanthi Hapugoda, Lakshika Perera, Vibhavee Kannangara, Champika Udayangani, Ranga Peiris, Shuaib Faizer, Pamodh Yasawardene, Sanjay de Mel, Ishan de Zoysa, Suranjith L Seneviratne.)

Previous studies measuring the health-related quality of life (HRQoL) of individuals with dengue focused on treatment seeking populations. However, the vast majority of global dengue cases are unlikely to be detected by health systems. Representative measurements of HRQoL should therefore include patients with disease not likely to trigger treatment-seeking behavior. This study based in Iquitos, Peru used the Quality of Wellbeing Scale-Self Administered, a survey that enquires about not only physical health, but also psychological health, self-care, mobility, and usual social activities, and rates HRQoL between 0 (death) and 1 (optimum function), to evaluate the impact of dengue on HRQoL. In order to enroll treatment and non treatment-seeking participants, three modalities of participant recruitment were used. In addition to clinic and community-based febrile surveillance, a contact-cluster methodology was also employed to identify infected individuals less likely to seek treatment. We measured changes in HRQoL and identified common areas of health impairment in 73 virologically confirmed dengue cases at 3 time points during the participant's illness; the early-acute (days 0-6 post symptom onset), late-acute (days 7-20), and convalescent illness phases (days 21 +). Participants reported HRQoL related impairments at significantly higher frequency during the early-acute versus convalescent illness phase (Fisher's exact: P<0.01). There was substantial heterogeneity in scores during each illness phase with median scores in the early-acute, late-acute and convalescent phases of 0.56 (IQR: 0.41-0.64), 0.70 (IQR: 0.57-0.94), and 1 (IQR: 0.80-1.00), respectively. In all illness phases participants recruited in clinics had on average the lowest HRQoL scores where as those recruited in the contact clusters had the highest. Only 1 individual who was recruited in the contact-clusters had no reduction in HRQoL score during their illness. These data illustrate that dengue should be considered as a disease that may have significant implications for not only physical health but also psychological health and social functioning. The impact of dengue on the HRQoL of non-treatment-seeking individuals, although lower than the impact among treatment-seeking individuals, is not necessarily trivial., Competing Interests: The authors have declared that no competing interests exist.

Dengue infection causes dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). CD4 + Foxp3 + Tregs are expanded in patients during dengue infection, and appear to be associated with clinical severity. However, molecular pathways involved in Treg proliferation and the reason for their insufficient control of severe diseases are poorly understood. Here, dengue infection induced the proliferation of functional CD4 + Foxp3 + Tregs via TLR2/MyD88 pathway. Surface TLR2 on Tregs was responsible for their proliferation, and dengue-expanded Tregs subverted in vivo differentiation of effector CD8 + T cells. An additional interesting finding was that dengue-infected hosts displayed changed levels of susceptibility to other diseases in TLR2-dependent manner. This change included enhanced susceptibility to tumors and bacterial infection, but highly enhanced resistance to viral infection. Further, the transfer of dengue-proliferated Tregs protected the recipients from dengue-induced DHF/DSS and LPS-induced sepsis. In contrast, dengue-infected hosts were more susceptible to sepsis, an effect attributable to early TLR2-dependent production of proinflammatory cytokines. These facts may explain the reason why in some patients, dengue-proliferated Tregs is insufficient to control DF and DHF/DSS. Also, our observations lead to new insights into Treg responses activated by dengue infection in a TLR2-dependent manner, which could differentially act on subsequent exposure to other disease-producing situations., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The usefulness of laboratory tests in the decision-making process with regard to early identification of dengue virus infection has not been widely reported, particularly the aspartate aminotransferase (AST)/platelet count ratio index during a patient's days of illness. The aim of this study was to examine the pattern of the ratio index over the course of illness and identify whether it is a marker of dengue virus infection in dengue patients, as well as to assess the role of other laboratory tests., Methods: A chart review of 205 dengue patients was analyzed using available records of 845 laboratory results within different time intervals or exam dates during the course of illness. We used repeated measures mixed binary logistic regression analyses to model the dengue virus infection, defined as giving at least one positive antibody test (yes/no)., Results: The high risk of dengue virus infection in dengue patients was found in the male gender (adjusted OR=4.316, 95% CI: 1.285-14.498, P=0.018), in patients with a high AST/platelet count ratio index (adjusted OR=1.438, 95% CI: 1.057-1.957, P=0.021), in patients with a low MCV level (adjusted OR=0.815, 95% CI: 0.679-0.978, P=0.028), and in patients with a low ALT level (adjusted OR=0.996, 95% CI: 0.993-0.999, P=0.010)., Conclusion: Laboratory markers, in particular the AST/platelet count ratio index, can be useful for clinicians to strengthen the decision-making process in primary care settings. Furthermore, our model revealed that low MCV and low ALT are predictors of the dengue virus infection, while being a male increases the risk of dengue virus infection. More studies are needed to evaluate the impact of the AST/platelet count ratio index on the severity of dengue fever infection during the onset of symptoms and course of treatment., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

SARS-CoV-2 pandemic and recurrent dengue epidemics in tropical countries have turned into a global health threat. While both virus-caused infections may only reveal light symptoms, they can also cause severe diseases. Here, we review the possible antibody-dependent enhancement (ADE) occurrence, known for dengue infections, when there is a second infection with a different virus strain. Consequently, preexisting antibodies do not neutralize infection, but enhance it, possibly by triggering Fcγ receptor-mediated virus uptake. No clinical data exist indicating such mechanism for SARS-CoV-2, but previous coronavirus infections or infection of SARS-CoV-2 convalescent with different SARS-CoV-2 strains could promote ADE, as experimentally shown for antibodies against the MERS-CoV or SARS-CoV spike S protein. © 2020 International Society for Advancement of Cytometry., (© 2020 International Society for Advancement of Cytometry.)

Arthropod-borne viruses (arboviruses) of the genus Flavivirus are distributed globally and cause significant human disease and mortality annually. Flavivirus infections present a spectrum of clinical manifestations, ranging from asymptomatic to severe manifestations, including hemorrhage, encephalitis and death. Herein, we describe 3 case reports of cerebrovascular involvement in patients infected by dengue and Zika viruses in Sao Jose do Rio Preto, São Paulo State, Brazil, a hyperendemic area for arbovirus circulation, including dengue, yellow fever, chikungunya and Saint Louis encephalitis viruses. Our findings highlight the potential threat that unusual clinical manifestations may pose to arbovirus disease management and recovery.

Dengue virus infection is associated with the upregulation of metabolic pathways within infected cells. This effect is common to infection by a broad array of viruses. These metabolic changes, including increased glucose metabolism, oxidative phosphorylation and autophagy, support the demands of viral genome replication and infectious particle formation. The mechanisms by which these changes occur are known to be, in part, directed by viral nonstructural proteins that contact and control cellular structures and metabolic enzymes. We investigated the roles of host proteins with overarching control of metabolic processes, the transcriptional regulators, cyclin-dependent kinase 8 (CDK8) and its paralog, CDK19, as mediators of virally induced metabolic changes. Here, we show that expression of CDK8, but not CDK19, is increased during dengue virus infection in Huh7 human hepatocellular carcinoma cells, although both are required for efficient viral replication. Chemical inhibition of CDK8 and CDK19 with Senexin A during infection blocks virus-induced expression of select metabolic and autophagic genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3), and reduces viral genome replication and infectious particle production. The results further define the dependence of virus replication on increased metabolic capacity in target cells and identify CDK8 and CDK19 as master regulators of key metabolic genes. The common inhibition of CDK8 and CDK19 offers a host-directed therapeutic intervention that is unlikely to be overcome by viral evolution.

Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a challenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001-2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-antithrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lymphocytes (%) and thrombin time were increased. In dengue patients, the absolute lymphocytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamination events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies., Competing Interests: IB and BBH are co-founders of E25Bio Inc. (www.e25bio.com), a company that develops point-of-care diagnostics for fever-causing infectious agents.

Background: Dengue is a mosquito-borne viral infection which has been estimated to cause a global economic burden of US$8.9 billion per year. 40% of this estimate was due to what are known as productivity costs (the costs associated with productivity loss from both paid and unpaid work that results from illness, treatment or premature death). Although productivity costs account for a significant proportion of the estimated economic burden of dengue, the methods used to calculate them are often very variable within health economic studies. The aim of this review was to systematically examine the current estimates of the productivity costs associated with dengue episodes in Asia and to increase awareness surrounding how productivity costs are estimated., Method: We searched PubMed and Web of Knowledge without date and language restrictions using terms related to dengue and cost and economics burden. The titles and abstracts of publications related to Asia were screened to identify relevant studies. The reported productivity losses and costs of non-fatal and fatal dengue episodes were then described and compared. Costs were adjusted for inflation to 2017 prices., Results: We reviewed 33 relevant articles, of which 20 studies reported the productivity losses, and 31 studies reported productivity costs. The productivity costs varied between US$6.7-1445.9 and US$3.8-1332 for hospitalized and outpatient non-fatal episodes, respectively. The productivity cost associated with fatal dengue episodes varied between US$12,035-1,453,237. A large degree of this variation was due to the range of different countries being investigated and their corresponding economic status. However, estimates for a given country still showed notable variation., Conclusion: We found that the estimated productivity costs associated with dengue episodes in Asia are notable. However, owing to the significant variation in methodology and approaches applied, the reported productivity costs of dengue episodes were often not directly comparable across studies. More consistent and transparent methodology regarding the estimation of productivity costs would help the estimates of the economic burden of dengue be more accurate and comparable across studies.

Dengue virus (DENV) infection is a major cause of morbidity and mortality in Vietnam, and the incidence is higher and more consistent in the southern part of the country. This study investigated the circulation of DENV serotypes, viremia levels, immunological status, and cytokine levels, with disease severities among children infected in 2017 in Ho Chi Minh City, Southern Vietnam. Acute and convalescent serum samples were collected from clinically diagnosed dengue children. They were confirmed to have DENV infection by NS1 antigen, IgM and IgG ELISAs, virus isolation, and conventional and real-time RT-PCR. Measurement of 10 cytokine levels was performed in the serum samples. All the children were dengue IgM positive; 28% and 72% of them had primary and secondary DENV infections, respectively, whereas 54% of those with secondary infection were children with dengue with warning signs and with severe dengue. Any or mixed infection of the four serotypes of DENV RNA was detected in 58 children. Twenty DENV strains (DENV-1 = 16 and DENV-4 = 4) were isolated. Levels of IFN-γ, TNF-α, MCP-1, IL-10, and IL-6 were significantly higher in severe dengue cases. We report the predominance of DENV-1 over other serotypes in the 2017 dengue outbreak in Southern Vietnam. Our data showed that cytokine expressions were correlated with dengue pathogenesis and may help in identifying an effective therapeutic strategy.

Dengue is an arboviral disease caused by dengue virus (DENV), which is transmitted to humans by Aedes aegypti mosquitoes. Infection by DENV most commonly results in a mild flu-like illness; however, the disease has been increasingly associated with neurological symptomatology. This association draws attention to further investigations on the impact of DENV infection in the host's central nervous system. Here, we analyzed brain samples of three fatal dengue cases that occurred in 2002 during an outbreak in Rio de Janeiro, Brazil. Brain tissues of these cases were marked by histopathological alterations, such as degenerated neurons, demyelination, hemorrhage, edema, and increased numbers of astrocytes and microglial cells. Samples were also characterized by lymphocytic infiltrates mainly composed of CD8 T cells. DENV replication was evidenced in neurons, microglia and endothelial cells through immunohistochemistry and in situ hybridization techniques. Pro-inflammatory cytokines, such as TNF-α and IFN-γ were detected in microglia, while endothelial cells were marked by the expression of RANTES/CCL5. Cytoplasmic HMGB1 and the production of nitric oxide were also found in neurons and microglial cells. This work highlights the possible participation of several local pro-inflammatory mediators in the establishment of dengue neuropathogenesis., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: Dengue manifestations can range from subclinical to fatal. The study of factors that influence dengue's clinical severity can provide information to potentially limit or predict severe cases. Secondary infection (SI) with a different dengue serotype has been recognized as an important determinant of severity. However, severe dengue (SD) manifestations, including shock, can happen during primary infection (PI) too and the mechanisms involved are less understood. To characterize the severe manifestations associated to PI, we distinguished between primary and secondary dengue cases in hospitalized patients from a region of low and recent dengue incidence in central Mexico. This region can serve as a model for dengue's behavior as it spreads to new areas worldwide. Methods: Dengue-specific immunoglobulin M (IgM) and IgG concentrations were measured in the serum of 78 hospitalized patients with dengue hemorrhagic fever, and their ratios were used to discriminate between PI and SI, as recommended by World Health Organization. Clinical and laboratory manifestations were compared between PI and SI. Results and Conclusions: PI was detected in 23% of hospitalized dengue cases, a proportion similar to that reported in high-incidence regions in Mexico. PI was more frequent in 16- to 40-year-olds, and was absent in patients older than 60 years. Only dengue with warning signs and SD were present in the studied population of hospitalized patients, and case frequency decreased with clinical severity both in PI and SI groups. No significant differences in demographics, laboratory tests, or symptoms were found between PI and SI, which illustrates that cases requiring hospitalization during outbreaks can be severe, even if they are PI. This information can help plan for sanitary contingencies in places where dengue is recently emergent and numerous PI cases are expected. The mechanisms involved in PI clinical severity need to be studied further.

Dengue is the most common mosquito-borne flaviviral infection in the world today. Several factors contribute and act synergistically to cause severe infection. One of these is dysregulated host immunological mediators that cause transient pathophysiology during infection. These mediators act on the endothelium to increase vascular permeability, which leads to plasma leakage compromising hemodynamics and coagulopathy. We conducted a prospective study to explore the expression of pro- and anti-inflammatory cytokines and how they relate to clinical dengue manifestations, by assessing their dynamics through acute dengue infection in adults admitted to the Hospital for Tropical Diseases, Bangkok, Thailand. We performed cytokine analysis at three phases of infection for 96 hospitalized adults together with serotyping of confirmed dengue infection during the outbreaks of 2015 and 2016. The serum concentrations of seven cytokines (interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha, and interferon gamma) were measured in duplicate using a commercial kit (Bio-Plex Human Cytokine Assay). In this study, the cytokine profile was suggestive of a T-helper 2 response. Most patients had secondary infection, and the levels of viremia were higher in patients with plasma leakage than those without plasma leakage. In addition, we observed that bleeding and hepatitis were associated with significantly higher levels of IL-8 during the early phases of infection. Furthermore, IL-6 levels in the early phase of infection were also elevated in bleeding patients with plasma leakage. These results suggest that IL-6 and IL-8 may act in synergy to cause bleeding in patients with plasma leakage.

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14 ++ CD16 - ) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

Aedes aegypti transmitted arboviral diseases are of significant importance in Colombia, particularly since the 2014/2015 introduction of chikungunya and Zika in the Americas and the increasing spread of dengue. In response, the Colombian government initiated the scaling-up of a community-based intervention under inter and multi-sector partnerships in two out of four sectors in Girardot, one of the most hyper-endemic dengue cities in the country. Using a quasi-experimental research design a scaled-up community-led Aedes control intervention was assessed for its capacity to reduce dengue from January 2010 to August 2017 in Girardot, Colombia. Reported dengue cases, and associated factors were analysed from available data sets from the Colombian disease surveillance systems. We estimated the reduction in dengue cases before and after the intervention using, Propensity Score Matching and an Autoregressive Moving Average model for robustness. In addition, the differences in dengue incidence among scaling-up phases (pre-implementation vs sustainability) and between treatment groups (intervention and control areas) were modelled. Evidence was found in favour of the intervention, although to maximise impact the scaling-up of the intervention should continue until it covers the remaining sectors. It is expected that a greater impact of the intervention can be documented in the next outbreak of dengue in Girardot., Competing Interests: The authors have declared that no competing interests exist.

This study was performed to determine the risk factors and predictors of severe dengue fever (SDF) in Saudi population in Jeddah, Western Saudi Arabia. This 7-year retrospective study included children and adults with confirmed dengue from 2010 to 2016. Demographic, clinical, laboratory, serological, and virologic data were collected. Comparative analyses were performed between pediatric and adult SDF cases defined according to the WHO 2009 dengue classification. During the study period, dengue was confirmed in 17,646 cases with predominant infection of adults (6.5 times that of children) and males (3.8 times that of females). May and June were associated with 43.9% of total dengue cases. All 56 pediatric and 187 adult SDF cases were hospitalized. At least one warning sign of severe illness was present in 92.2% of total SDF cases. Mortality rates were 8.9% and 10.7% of pediatric and adult SDF cases, respectively. Multiple logistic regression detected that the most significant risk factors and predictors of SDF in adults versus children were significantly more secondary dengue infection (adjusted odds ratio [AOR]: 2.20, 95% CI: 1.09-4.44, P = 0.02), significantly less clinical fluid accumulation (AOR: 0.17, 95% CI: 0.07-0.44, P < 0.001) and significantly less neutropenia (AOR: 0.41, 95% CI: 0.17-0.97, P = 0.04). This was the first large multicenter study evaluating SDF in Saudi population and considering the WHO 2009 dengue classification, which showed predominant infection of adults and males with dengue, few SDF cases with low mortality and highlighted predictors of SDF in adults versus children. Consideration of warning signs for severe dengue may result in hospital admission, prompting closer monitoring, timely and proper interventions and reduced mortality in SDF cases.

After the mass campaign of Measles and Rubella vaccination in 2003 in Iran, the cases of measles and rubella infection decreased but still, the cases of rash and fever were reported. It is worth noting that some other viral infections show signs similar to measles and rubella such as some arboviruses. Considering the epidemic outbreak of arbovirus infections in countries neighbouring Iran, we performed this study to estimate the possibility of chikungunya and dengue fever among measles and rubella IgM negative patients presenting with rash and fever from December 2016 to November 2017 in the National Measles Laboratory at Tehran University of Medical Sciences. Serum samples were selected at random from patients from eight provinces. The presence of DENV IgM and CHIKV IgM was examined by enzyme-linked immunosorbent assay. Of the 1306 sera tested, 210 were CHIKV seropositive and 82 were dengue seropositive. Statistical analysis demonstrated a significant increase in the CHIKV IgM antibody seropositivity rate in Kerman (OR = 2.07, 95% CI: 1.10-3.92; P = 0.024) and Fars (OR = 1.77, 95% CI: 1.06-2.93; P = 0.027). The DENV and CHIKV seropositivity rate in summer is higher than in other seasons (P < 0.01). Our seropositive samples suggest possible CHIKV and DENV infection in Iran. It is likely that these viruses are circulating in Iran and there is a need to study vector carriage of these two viruses.

In 2013, cases of chikungunya virus (CHIKV) infection were first detected in the Caribbean. Chikungunya virus rapidly spread through Central and South America, causing explosive outbreaks in naive populations. Since its emergence in 2004, the number of case and series reports describing severe, atypical manifestations seen in chikungunya patients has increased substantially, calling into question whether clinicians and health services are failing to diagnose these atypical cases because of not only insufficient knowledge but also limitations in the case classification. Although this classification based on the duration of the musculoskeletal (acute, subacute, and chronic forms) complaints helped guide therapeutic approaches directed to these manifestations, patients presenting severe or complicated forms, which are less frequent but produce most of the fatal outcomes, were not properly addressed. In Brazil and the Caribbean, a clear temporal and spatial association between excess overall mortality and the occurrence of chikungunya epidemics has been shown, supporting the hypothesis that many of these excess deaths were a consequence of CHIKV infections. Thus, accumulated experience has highlighted that the current chikungunya case classification does not encompass the actual needs presented by certain cases with atypical features nor does it contribute to early detection and management of potentially severe cases. With continued CHIKV circulation in three continents and recent reemergence in Asia and Europe, we need a classification that is prospective and informed both by initial clinical presentation and by progression of signs and symptoms.

Background: In 2014, a serious dengue outbreak occurred in Guangzhou, South China. In this study, the clinical and laboratory characteristics of dengue fever (DF) group and other febrile illnesses (OFI) group in Guangzhou were described., Methods: Clinical and laboratory data collected by studying 1,792 patients from Nanfang Hospital, Southern Medical University during 2014 and 2018. Laboratory data was analyzed by SPSS 22.0 statistical software including Full blood counts (SYSMEX XE-5000), Laboratory Biochemical tests (Roche Cobas 8000), Dengue virus RNA (RT-PCR-Fluorescence Probing) and Dengue IgG/IgM Antibody (Colloidal Gold), Dengue Virus NS1 (ELISA)., Results: In the DF group and OFI group, gender ratios were 1.08:1 (male/female, P>0.05) and 1.45:1 (male/female, P<0.05). Adults aged 25-34 years old (29.4%) with the main peak appeared in the DF group, and the same main peak appeared in the OFI group: 25-34 years old (25.13%). Patients were from Medical emergency (41.2% DF group, 29.4% OFI group). The distribution of fever days before treatment was mainly focused within 5 days, with a main peak in the 2 fever days before treatment (24.6%) in the DF group and the main peak in 1 fever day before treatment (46.9%) in OFI group. The major symptoms of the DF group were presented with were fever (100%), myalgia (34.77%), pharyngeal hyperemia (31.33%), headache (25.65%), adenoids (19.62%), and rash (13.25%). In the OFI group, Pharyngeal hyperemia was the most common clinical symptom, accounting for 27.24%, and the next symptom was adenoids (21.26%). The sensitivity and specificity of DV RNA were 61.54%, 100%, respectively, compared to the DF Nonstructural protein 1 (NS1). Dengue virus (DENV) Immunoglobulin M (IgM) IgM in both groups was statistically significant, with DENV-IgM in the DF group were stronger (Z=-7.863, P<0.001), and DENV immunoglobulin G (IgG) were no statistically significant (Z=-1.212, P=0.226). In DF group, 37.14% of serum samples had elevated Alanine transaminase (ALT) levels, 76.85% of them had elevated aspartate aminotransferase (AST) levels, 32.08% of them had elevated creatine kinase (CK) levels, and 2.67% of them had elevated C-reaction protein (CRP) levels, compared with 13.51% of them had elevated ALT levels, 30.65% of them had elevated AST levels, 6.06% of them had elevated CK levels and 69.35% of them had elevated CRP levels of the OFI patients. The prominent manifestations were thrombocytopenia (occurring in 28.07% of the DF group, compared to 5.18% of OFI group) and leucopenia (occurring in 43.27% of DF group and 3.63% of OFI group). The DF incidence of all fever cases was 49.0% within three months in 2014, compared with 1.4% in 2015, 0% in 2016, 0.9% in 2017 and 6.4% in 2018 (P<0.001). DF and OFI can occur in any age and sex. DF occurred in the young and the old, OFI occurred in children and youth. The clinical symptoms of myalgia, headache, rash, weak, Chills, follicular hyperplasia in both groups were statistically significant (P<0.001)., Conclusions: IgM can be easily recognized for early diagnoses, DENV-RNA had lower sensitivity and higher specificity, and DF NS1 enzyme-linked immunosorbent assay (ELISA) has a higher sensitive and specificity. DF is a serious public health problem and an emerging continuous threat in Guangzhou. In high-prevalence areas, effective epidemic monitoring and prevention measures need to be undertaken. After the unprecedented outbreak in 2014, on account of the government and citizen paying more attention to the DF epidemic, the cases of DF were decreased significantly from 2015 to 2018.

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 10 days. 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 4000 IU/day of vitD were less susceptible to DENV-2 infection than MDDCs from donors who received 1000 IU/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 4000 IU/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.

Tonate virus (TONV) is an arbovirus discovered in 1973 in French Guiana (FG) belonging to the Venezuelan equine encephalitis virus complex, Alphavirus genus. Only few publications and cases have been reported in FG. The objectives of the present study were to describe the clinical picture of TONV and to compare its presentation with that of dengue virus (DENV). A retrospective study was performed in Cayenne hospital from 2003 to 2016 including all patients exclusively positive for TONV IgM and not for other alphaviruses. They were classified as high probability: typical clinical picture of arbovirus infection (i.e., fever, chills, headaches, muscle, and joint pains) and IgM seroconversion; medium probability: typical clinical picture + single positive IgM on a unique serum sample without control; and low probability: atypical clinical picture of infection and single positive IgM. Only patients with high and medium probability were included in the analysis and compared with a gender- and age-matched control group of DENV diagnosed by NS1 antigen (two controls per case). During the study period, 45 cases of TONV were included and compared with 90 cases of DENV. Twenty-eight (62.2%) were men; the median age was 34 years (IQ [22-49]). In the bivariate analysis, variables significantly associated with TONV versus DENV were the presence of cough (33.3% versus 10.3%) and anemia (32.5% versus 11.1%) and the absence of nausea (4.4% versus 32.2%), rash (2.2% versus 27.4%), fatigue (17.8% versus 41.0%), anorexia (6.7% versus 30.1%), muscle pain (42.2% versus 61.4%), headache (53.3% versus 70.8%), leukopenia (9.8% versus 44.4), and lymphopenia (42.5% versus 89.9%). There were no cases with severe neurological involvement, and there were no deaths. Tonate virus may be evoked as a cause of fever in patients living or returning from the Amazonian area. Positive TONV IgM does not prove the diagnosis and should not preclude from searching for alternative infectious diagnoses.

Background: In Africa, the magnitude of dengue virus (DENV) transmission is largely unknown. In Burkina Faso, several outbreaks have been reported and data are often based on findings from outbreak investigations., Methods: To better understand dengue epidemiology and clinical characteristics in Burkina Faso, a fever surveillance study was conducted among patients aged 1-55 years, who presented with non-malarial febrile illness at five primary healthcare facilities in Ouagadougou, Burkina Faso from December 2014 to February 2017, encompassing a 3-month dengue outbreak in September-November 2016. Acute and convalescent blood samples were collected within an interval of 10-21 days between visits. Acute samples were tested with dengue rapid diagnostic tests (RDT) and a selected subset with RT-PCR, and all acute/convalescent samples with IgM/IgG ELISA., Results: Among 2929 non-malarial febrile patients, 740 (25%) were dengue-positive based on RT-PCR and/or IgM/IgG ELISA; 428 out of 777 patients (55%) and 312 out of 2152 (14%) were dengue-positive during outbreak and non-outbreak periods, respectively. There were 11% (316/2929) and 4% (129/2929) patients showing positive for NS1 and IgM, on the RDT, respectively. DENV 2 predominated during the outbreak, whereas DENV 3 predominated before the outbreak. Only 25% of dengue-positive cases were clinically diagnosed with suspected dengue. The odds of requiring observation for ≤3 days (versus routine outpatient care) were 11 times higher among dengue-positive cases than non-dengue cases. In adjusted analyses, dengue-positivity was associated with rash and retro-orbital pain (OR = 2.6 and 7.4, respectively) during the outbreak and with rash and nausea/vomiting (OR = 1.5 and 1.4, respectively) during the non-outbreak period., Conclusion: Dengue virus is an important pathogen in Burkina Faso, accounting for a substantial proportion of non-malarial fevers both during and outside outbreak, but is only infrequently suspected by clinicians. Additional longitudinal data would help to further define characteristics of dengue for improved case detection and surveillance., Competing Interests: I certify that the authors do not have any relevant financial relationships or potential conflicts of interest to disclose regarding the material discussed in this manuscript.

The protective roles of endosomal toll-like receptors (TLRs) and cytosolic nucleic acid sensors are well elucidated, but the pathogenic host factors during viral infections remain unclear. Spleen tyrosine kinase (Syk)-coupled C-type lectins (CLECs) CLEC2 and CLEC5A are highly expressed on platelets and myeloid cells, respectively. CLEC2 has been shown to recognize snake venom aggretin and the endogenous ligand podoplanin and acts as a critical regulator in the development and immunothrombosis. Although CLEC2 has been reported to interact with type I immunodeficiency virus (HIV-1), its role in viral infections is still unclear. CLEC5A binds to fucose and mannose moieties of dengue virus membrane glycans, as well as to N-acetylglucosamine (GlcNAc)/N-acetylmuramic acid (MurNAc) disaccharides that form the backbone of L. monocytogenes peptidoglycans. Recently, we demonstrated that both CLEC2 and CLEC5A are critical in microbe-induced "neutrophil extracellular trap" (NET) formation and proinflammatory cytokine production. Moreover, activation of CLEC2 by dengue virus (DV) and H5N1 influenza virus (IAV) induces the release of extracellular vesicles (EVs), which further enhance NETosis and proinflammatory cytokine production via CLEC5A and Toll-like receptor 2 (TLR2). These findings not only illustrate the immunomodulatory effects of EVs during platelet-leukocyte interactions, but also demonstrate the critical roles of CLEC2 and CLEC5A in acute viral infections., (Copyright © 2019 Sung and Hsieh.)

Background: Hepatic dysfunction and coagulopathy are common in acute dengue illness. We analyzed the trajectories of the above parameters in the survivors and fatal patients in the outbreak in Tainan, 2015., Methods: A retrospective study was conducted using data from a tertiary hospital between January and December 2015. Multilevel modeling (MLM) was used to identify the changes in aminotransferase (AST), alanine aminotransferase (ALT), activated partial thromboplastin time (aPTT), and platelet counts from Day 0 to Day 7 of the onset of dengue infection. The machine-learning algorithm was used by purity measure assumption to calculate the accuracy of serum transaminases and coagulation variables to discriminate between the fatal and survival groups., Results: There were 4,069 dengue patients, of which 0.9% died in one week after illness onset (i.e., early mortality). Case fatality rate was the highest for those aged ≥70 years. Both AST and ALT values of the fatal group were significantly higher than those of the survivor group from Day 3 (AST median, 624 U/L vs. 60 U/L, p < 0.001; ALT median, 116 U/L vs. 29 U/L, p = 0.01) of illness onset and peaked on Day 6 (AST median, 9805 U/L vs. 90 U/L, p < 0.001; ALT median, 1504 U/L vs. 49 U/L, p < 0.001). AST ≥ 203 U/L, ALT ≥ 55 U/L, AST2/ALT criteria ≥337.35, or AST/platelet count ratio index (APRI) ≥ 19.18 on Day 3 of dengue infection had a high true positive rate, 90%, 78%, 100%, or 100%, respectively, of early mortality. The platelet counts of the fatal group declined significantly than those of the survivor group since Day 3 of illness onset (median, 19 x103/μl vs. 91 x103/μl, p < 0.01), and aPTT values of the fatal group significantly prolonged longer since Day 5 (median, 68.7 seconds vs. 40.1 seconds, p < 0.001)., Conclusions: AST, ALT, and platelet counts should be monitored closely from Day 0 to Day 3 of dengue infection, and aPTT be followed up on Day 5 of infection to identify the individuals at risk for early mortality., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Although the role of dengue virus (DENV)-specific T cells in the pathogenesis of acute dengue infection is emerging, the functionality of virus-specific T cells associated with milder clinical disease has not been well studied. We sought to investigate how the functionality of DENV-NS3 and DENV-NS5 protein-specific T cells differ in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF)., Methods: Using intracellular cytokine assays, we assessed the production of interferon γ (IFNγ), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-1β (MIP-1β), and CD107a expression in adult patients with acute DF (n = 21) and DHF (n = 22)., Results: Quadruple cytokine-producing, polyfunctional DENV-NS3- and DENV-NS5-specific T cells were more frequent in those with DF when compared to those with DHF. While DENV-NS3- and DENV-NS5-specific T cells in patients with DF expressed IFNγ > TNF-α > MIP-β > CD107a, T cells of those with DHF predominantly expressed CD107a > MIP-1β > IFNγ > TNF-α. Overall production of IFNγ or TNF-α by DENV-NS3- and DENV-NS5-specific T cells was significantly higher in patients with DF. The majority of NS3-specific T cells in patients with DF (78.6%) and DHF (68.9%) were single-cytokine producers; 76.6% of DENV-NS5-specific T cells in those with DF and 77.1% of those with DHF, produced only a single cytokine. However, no significant association was found with polyfunctional T-cell responses and the degree of viraemia., Conclusions: Our results suggest that the functional phenotype of DENV-specific T cells are likely to associate with clinical disease severity., (© 2019 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7 th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.