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Liver involvement is an unusual yet frequently overlooked dengue complication. Pivotal for an efficient clinical management, the early diagnosis of dengue-associated liver involvement relies on an accurate description of its clinical and biological characteristics, its prognosis factors, its association with severe dengue and its clinical management. We conducted a systematic review by searching PubMed and Web of Science databases for original case reports, cohort and cross-sectional studies reporting the clinical and/or biological features of dengue-associated liver involvement. The study was registered in PROSPERO (CRD42021262657). Of the 2552 articles identified, 167 were included. Dengue-associated liver involvement was characterised by clinical features including abdominal pain, hepatomegaly, jaundice, nausea/vomiting, and an echogenic liver exhibiting hepatocellular necrosis and minimal inflammation. Elevated Aspartate Aminotransferase and Alanine Aminotransferase but also elevated bilirubin, Alkaline Phosphatase, gamma-glutamyl transferase, increased International Normalised Ratio, creatinine and creatine kinase, lower albumin and prolonged prothrombin and activated partial thromboplastin time were prevalent in dengue-associated liver involvement. Cardiovascular and haematological systems were frequently affected, translating in a strong association with severe dengue. Liver involvement was more common in males and older adults. It was associated with dengue virus serotype-2 and secondary infections. Early paracetamol intake increased the risk of liver involvement, which clinical management was mostly conservative. In conclusion, this systematic review demonstrates that early monitoring of transaminases, clinical assessment, and ultrasound examination allow an efficient diagnosis of dengue-associated liver involvement, enabling the early identification and management of severe dengue., (© 2024 The Author(s). Reviews in Medical Virology published by John Wiley & Sons Ltd.)

Tumefactive demyelinating lesions (TDL) are a rare occurrence among inflammatory demyelinating diseases of the central nervous system, distinguished by tumor-like lesions exceeding 2 cm in diameter. While various etiologies have been associated with TDL, only a limited number of case reports document the coexistence of acute disseminated encephalomyelitis (ADEM) and TDL. Here, we present the case of a female diagnosed with dengue fever two weeks prior, who subsequently developed left hemiparesis and encephalopathy. Both her brain magnetic resonance imaging (MRI) and clinical course align with the characteristics of tumefactive ADEM., (© 2024. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Importance: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR + CD38 + phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR + CD38 + CD8 T cells were also CD69 high when examined ex vivo , only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR + CD38 + CD8 T cells and the transcriptional profiles that distinguish the CD69 + IFNγ + , CD69 + IFNγ - , and CD69 - IFNγ - subsets., Competing Interests: The authors declare no conflict of interest.

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the β-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) infection causes ocular and neurological pathologies with ZIKV-induction of developmental abnormalities following in utero infection a major concern. The study here has compared ZIKV and the related dengue virus (DENV) infection in the eye and brain. In vitro, both ZIKV and DENV could infect cell lines representing the retinal pigmented epithelium, endothelial cells, and Mueller cells, with distinct innate responses in each cell type. In a 1-day old mouse challenge model, both ZIKV and DENV infected the brain and eye by day 6 post-infection (pi). ZIKV was present at comparable levels in both tissues, with RNA increasing with time post-infection. DENV infected the brain, but RNA was detected in the eye of less than half of the mice challenged. NanoString analysis demonstrated comparable host responses in the brain for both viruses, including induction of mRNA for myosin light chain-2 (Mly2), and numerous antiviral and inflammatory genes. Notably, mRNA for multiple complement proteins were induced, but C2 and C4a were uniquely induced by ZIKV but not DENV. Consistent with the viral infection in the eye, DENV induced few responses while ZIKV induced substantial inflammatory and antiviral responses. Compared to the brain, ZIKV in the eye did not induce mRNAs such as C3, downregulated Retnla, and upregulated CSF-1. Morphologically, the ZIKV-infected retina demonstrated reduced formation of specific retinal layers. Thus, although ZIKV and DENV can both infect the eye and brain, there are distinct differences in host cell and tissue inflammatory responses that may be relevant to ZIKV replication and disease., (© 2023. The Author(s).)

Dengue virus is reported to activate endothelial cells (EC), but the precise cause for severe dengue (SD) is not known. Guanylate binding proteins (GBPs) are IFN-inducible proteins secreted by ECs and are involved in the anti-oxidant and anti-viral response. The involvement of GBPs in the pathogenesis of dengue remains under explored. In the present study, we quantified the mRNA and protein levels of GBP1 and 2 during acute, defervescence and convalescent phase in SD-10, dengue without warning sign-15 and dengue with warning sign-25 compared to other febrile illnesses-10 and healthy controls-8 using RT-PCR and ELISA respectively. Lipid peroxidation in plasma samples were measured using the Kei Satoh method. Protein and DNA oxidation were determined by ELISA. The efficacy of the proteins in predicting disease severity was done by Support Vector Machine (SVM) model. A significant (P ≤ 0.01) decrease in the levels of mRNA and protein of both GBP1 and GBP2 was observed during defervescence in both SD and DWW cases. The levels were significantly (P ≤ 0.05) tapered off in SD cases from acute till critical phases compared to other study groups. DNA, protein and lipid oxidation markers showed an increasing trend in SD (P ≤ 0.01). Both GBP1 & 2 were found to be negatively associated plasma leakage and oxidative stress markers. EC's activated with SD serum showed a reduced expression of GBP 1 and 2. Nevertheless, the SVM model revealed that plasma levels of proteins along with clinical symptoms could predict the disease outcomes with higher precision. This is the first study reporting a downregulated expression of GBP1 & 2 and their association with oxidative stress and plasma leakage in dengue cases. This suggests the importance of GBPs in regulating disease manifestation. However, further investigations are required to ascertain its role as a biomarker or therapeutic target in dengue infection., Competing Interests: Declaration of competing interest The author declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mitochondrial fitness is governed by mitochondrial quality control pathways comprising mitochondrial dynamics and mitochondrial-selective autophagy (mitophagy). Disruption of these processes has been implicated in many human diseases, including viral infections. Here, we report a comprehensive analysis of the effect of dengue infection on host mitochondrial homeostasis and its significance in dengue disease pathogenesis. Despite severe mitochondrial stress and injury, we observed that the pathways of mitochondrial quality control and mitochondrial biogenesis are paradoxically downregulated in dengue-infected human liver cells. This leads to the disruption of mitochondrial homeostasis and the onset of cellular injury and necrotic death in the infected cells. Interestingly, dengue promotes global autophagy but selectively disrupts mitochondrial-selective autophagy (mitophagy). Dengue downregulates the expression of PINK1 and Parkin, the two major proteins involved in tagging the damaged mitochondria for elimination through mitophagy. Mitophagy flux assays also suggest that Parkin-independent pathways of mitophagy are also inactive during dengue infection. Dengue infection also disrupts mitochondrial biogenesis by downregulating the master regulators PPARγ and PGC1α. Dengue-infected cells release mitochondrial damage-associated molecular patterns (mtDAMPs) such as mitochondrial DNA into the cytosol and extracellular milieu. Furthermore, the challenge of naive immune cells with culture supernatants from dengue-infected liver cells was sufficient to trigger proinflammatory signaling. In correlation with our in vitro observations, dengue patients have high levels of cell-free mitochondrial DNA in their blood in proportion to the degree of thrombocytopenia. Overall, our study shows how defective mitochondrial homeostasis in dengue-infected liver cells can drive dengue disease pathogenesis. IMPORTANCE Many viruses target host cell mitochondria to create a microenvironment conducive to viral dissemination. Dengue virus also exploits host cell mitochondria to facilitate its viral life cycle. Dengue infection of liver cells leads to severe mitochondrial injury and inhibition of proteins that regulate mitochondrial quality control and biogenesis, thereby disrupting mitochondrial homeostasis. A defect in mitochondrial quality control leads to the accumulation of damaged mitochondria and promotes cellular injury. This leads to the release of mitochondrial damage-associated molecular patterns (mt-DAMPs) into the cell cytoplasm and extracellular milieu. These mt-DAMPs activate the naive immune cells and trigger proinflammatory signaling, leading to the release of cytokines and chemokines, which may trigger systemic inflammation and contribute to dengue disease pathogenesis. In correlation with this, we observed high levels of cell-free mitochondrial DNA in dengue patient blood. This study provides insight into how the disruption of mitochondrial quality control in dengue-infected cells can trigger inflammation and drive dengue disease pathogenesis.

Purpose of Review: To discuss the neurological complications of dengue virus (DENV) infection and their pathogenesis., Recent Findings: Include recognition of the four different serotypes of DENV and their epidemiology as well as recognition of the expanded dengue syndrome encompassing multisystem involvement in the severe form of the disease including involvement of the central nervous system (CNS). DENV is a neurotropic virus with the ability to infect the supporting cells of the CNS. Neural injury during the acute stage of the infection results from direct neuro-invasion and/or the phenomenon of antibody-dependent enhancement, resulting in plasma leakage and coagulopathy. Immune mechanisms have been implicated in the development of the delayed neurological sequelae through molecular mimicry. A myriad of neurological syndromes has been described as a result of the involvement of the CNS, the peripheral nervous system (PNS), or both. Neurological manifestations in DENV infection are increasingly being recognized, some of which are potentially fatal if not treated promptly. DENV encephalopathy and encephalitis should be considered in the differential diagnosis of other acute febrile encephalopathies, autoimmune encephalitides, and in cases of encephalopathy/encephalitis related to SARS-CoV2 infection, especially in dengue-endemic areas. Acute disseminated encephalomyelitis (ADEM) may be occasionally encountered. Clinicians should be knowledgeable of the expanded dengue syndrome characterized by the concurrent compromise of cardiac, neurological, gastrointestinal, renal, and hematopopoietic systems. Isolated cranial nerve palsies occur rather uncommonly and are often steroid responsive. These neuropathies may result from the direct involvement of cranial nerve nuclei or nerve involvement or may be immune-mediated. Even if the diagnosis of dengue is confirmed, it is absolutely imperative to exclude other well-known causes of isolated cranial nerve palsies. Ischemic and hemorrhagic strokes may occur following dengue fever. The pathogenesis may be beyond the commonly observed thrombocytopenia and include cerebral vasculitis. Involvement of ocular blood vessels may cause maculopathy or retinal hemorrhages. Posterior reversible encephalopathy syndrome (PRES) is uncommon and possibly related to dysregulated cytokine release phenomena. Lastly, any patient developing acute neuromuscular weakness during the course or within a fortnight of remission from dengue fever must be screened for acute inflammatory demyelinating polyneuropathy (AIDP), hypokalemic paralysis, or acute myositis. Rarely, a Miller-Fisher-like syndrome with negative anti-GQ1b antibody may develop., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Dengue fever is one of the most important arboviral diseases in the world, and its severe forms are characterised by a broad spectrum of systemic and cardiovascular hallmarks. However, much remains to be elucidated regarding the pathogenesis triggered by Dengue virus (DENV) in the heart. Herein, we evaluated the cardiac outcomes unleashed by DENV infection and the possible mechanisms associated with these effects., Methods: A model of an adapted DENV-3 strain was used to infect male BALB/c mice to assess haemodynamic measurements and the functional, electrophysiological, inflammatory and oxidative parameters in the heart., Results: DENV-3 infection resulted in increased systemic inflammation and vascular permeability with consequent reduction of systolic blood pressure and increase in heart rate. These changes were accompanied by a decrease in the cardiac output and stroke volume, with a reduction trend in the left ventricular end-systolic and end-diastolic diameters and volumes. Also, there was a reduction trend in the calcium current density in the ventricular cardiomyocytes of DENV-3 infected mice. Indeed, DENV-3 infection led to leucocyte infiltration and production of inflammatory mediators in the heart, causing pericarditis and myocarditis. Moreover, increased reactive oxygen species generation and lipoperoxidation were also verified in the cardiac tissue of DENV-3 infected mice., Conclusions: DENV-3 infection induced a marked cardiac dysfunction, which may be associated with inflammation, oxidative stress and electrophysiological changes in the heart. These findings provide new cardiac insights into the mechanisms involved in the pathogenesis triggered by DENV, contributing to the research of new therapeutic targets for clinical practice., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Our objective was to analyze if there was a significant relationship between platelet parameters (PLT, MPV, PDW, P-LCR, PCT) among dengue, its serological subgroups and controls. Serologically proven adult patients with dengue {(n = 238) (NS1 positive = 218, IgM positive = 14, NS1 & IgM positive = 6)} and age- and gender-matched controls (n = 254) were included. The MPV, PDW and P-LCR were significantly higher, and PLT and PCT were significantly lower in cases compared with controls. Cases as well as controls showed a positive correlation between PLT and PCT, both parameters individually showed negative correlation with MPV, PDW, P-LCR. MPV, PDW and P-LCR showed positive correlation with each other. The results were similar in the serological subgroups. Comparison of our results with other studies points toward an overall hyperdestructive etiology for thrombocytopenia in dengue. There were two subgroups of cases based on the severity of thrombocytopenia. The mean/median value of all the platelet parameters was lesser in the ≤20k group than the >20k group, except for PDW, which was high although not statistically significant. Suppression of megakaryopoiesis with concomitant immune destruction of platelets in severe dengue could explain low MPV and P-LCR with a high PDW in view of the presence of microthrombocytes as a result of immune destruction. Although an overall hyperdestructive mechanism contributes to thrombocytopenia in dengue, regular monitoring of the platelet indices could reflect the status of megakaryopoiesis and thrombokinetic axis, thus aiding easy determination of pathophysiology and treatment.

The purpose of this study is to examine the awareness and perception of malaria and dengue fever in Multan Punjab, Pakistan while taking into account the important role of government policies and other variables. The goal of this study is to examine the awareness of students in Multan, Pakistan on malaria and dengue. This study is based on a quantitative approach of secondary evidence from scientific journals and questionnaire surveys. It is also based on observational evidence gathered in Multan Punjab Pakistan, in a field study. The survey with school children, teachers and healthcare professionals were both formal and semi-structuralize. Studies have found that malaria and dengue mainly affect children's schooling through their absence, but can also induce brain loss and cognitive disability. In questionnaires, students were seen to have different understanding of the illness, but also to be able to serve as agents of health reform only through teachers. A sample size of 500 respondents has been selected from different colleges of district Multan Punjab, Pakistan. Correlation technique is used for the data analysis. According to our results it is concluded that the students at college level are aware of malaria and dengue diseases, but they are not capable of engaging and serving as agents for health reform. On the basis of results it is recommended that students must teach about epidemics diseases regarding how to handle these diseases., Competing Interests: The authors have declared that no competing interests exist.

The spread of the Dengue virus over the world, as well as multiple outbreaks of different serotypes, has resulted in a large number of deaths and a medical emergency, as no viable medications to treat Dengue virus patients have yet been found. In this paper, we provide an in silico virtual screening and molecular dynamics-based analysis to uncover efficient Dengue infection inhibitors. Based on a Google search and literature mining, a large phytochemical library was generated and employed as ligand molecules. In this investigation, the protein target NS2B/NS3 from Dengue was employed, and around 27 compounds were evaluated in a docking study. Phellodendroside (-63 kcal/mole), quercimeritrin (-59.5 kcal/mole), and quercetin-7- O -rutinoside (-54.1 kcal/mole) were chosen based on their binding free energy in MM-GBSA. The tested compounds generated numerous interactions at Lys74, Asn152, and Gln167 residues in the active regions of NS2B/NS3, which is needed for the protein's inhibition. As a result, the stable mode of docked complexes is defined by various descriptors from molecular dynamics simulations, such as RMSD, SASA, Rg, RMSF, and hydrogen bond. The pharmacological properties of the compounds were also investigated, and no toxicity was found in computational ADMET properties calculations. As a result, this computational analysis may aid fellow researchers in developing innovative Dengue virus inhibitors.

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

A synergistic impact of virulence capacity of dengue viruses and host immune response contributes to the pathogenesis of dengue virus infection (DENV). Elevation of hepatic enzyme and cytokine storm is the major contributory factor producing severity. IL-2 and IL-18 both play a critical role in generating immunogenicity during viral infection. The goal of the study was to evaluate the correlation between alterations in IL-2 and IL-18 levels with alterations in hepatic enzymes in dengue-infected patients. Accordingly, a total of 91 NS1/IgM confirmed DENV infected patients were selected for the IL-2 and IL-18 evaluation using a standard ELISA assay. Biochemical, haematological parameters were recorded at the time of admission. Interestingly, raised levels of IL-2 (p < 0.0001) and IL-18 (p < 0.0001) was obtained in severe dengue as compared to non-severe dengue patients. A significant positive correlation was observed between IL and 2 levels and SGOT (r = 0.3168 with p = 0.002), SGPT (r = 0.569 with p < 0.0001). Similarly IL-18 was also highly associated with SGOT (r = 0.387 with p = 0.0002) and SGPT (r = 0.407 with p < 0.0001) in dengue infected patient.Our data reveal that a rising amount of IL-2 and IL-18 in initial stage of the disease could be predictors for developing severe form of dengue infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it may also be linked to viral pathogenesis. Intending to shed light on the viral determinants for severe dengue pathogenesis, we previously analyzed the DENV-2 intrahost genetic diversity in 68 patients clinically classified as dengue fever ( n  = 31), dengue with warning signs ( n  = 19), and severe dengue ( n  = 18), performing viral whole-genome deep sequencing from clinical samples with an amplicon-free approach. From it, we identified a set of 141 relevant mutations distributed throughout the viral genome that deserved further attention. Therefore, we employed molecular modeling to recreate three-dimensional models of the viral proteins and secondary RNA structures to map the mutations and assess their potential effects. Results showed that, in general lines, disruptive variants were identified primarily among dengue fever cases. In contrast, potential immune-escape variants were associated mainly with warning signs and severe cases, in line with the latter's longer intrahost evolution times. Furthermore, several mutations were located on protein-surface regions, with no associated function. They could represent sites of further investigation, as the interaction of viral and host proteins is critical for both host immunomodulation and virus hijacking of the cellular machinery. The present analysis provides new information about the implications of the intrahost genetic diversity of DENV-2, contributing to the knowledge about the viral factors possibly involved in its pathogenesis within the human host. Strengthening our results with functional studies could allow many of these variants to be considered in the design of therapeutic or prophylactic compounds and the improvement of diagnostic assays. IMPORTANCE Previous evidence showed that intrahost genetic diversity in arboviruses may be linked to viral pathogenesis and that one or a few amino acid replacements within a single protein are enough to modify a biological feature of an RNA virus. To assess dengue virus serotype 2 determinants potentially involved in pathogenesis, we previously analyzed the intrahost genetic diversity of the virus in patients with different clinical outcomes and identified a set of 141 mutations that deserved further study. Thus, through a molecular modeling approach, we showed that disruptive variants were identified primarily among cases with mild dengue fever, while potential immune-escape variants were mainly associated with cases of greater severity. We believe that some of the variants pointed out in this study were attractive enough to be potentially considered in future intelligent designs of therapeutic or prophylactic compounds or the improvement of diagnostic tools. The present analysis provides new information about DENV-2 viral factors possibly involved in its pathogenesis within the human host.

Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG <  - 7.5) with ERα at the same site as estrogen. The expression signatures in the connectivity map ( https://clue.io/; ) revealed high positive scores with translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is a reported ERα coactivator. Further, gene knockdown experiments revealed that Cipa exhibits antiviral activity in dengue infected MCF7 cells potentially modulated through estrogen receptor 1. This approach reveals a novel pathway involving the ESR1-RPL7 axis which could be a potential target in dengue viral infection., (© 2021. The Author(s).)

The novel coronavirus disease 2019 (COVID-19) presents with non-specific clinical features. This may result in misdiagnosis or delayed diagnosis, and lead to further transmission in the community. We aimed to derive early predictors to differentiate COVID-19 from influenza and dengue. The study comprised 126 patients with COVID-19, 171 with influenza and 180 with dengue, who presented within 5 days of symptom onset. All cases were confirmed by reverse transcriptase polymerase chain reaction tests. We used logistic regression models to identify demographics, clinical characteristics and laboratory markers in classifying COVID-19 versus influenza, and COVID-19 versus dengue. The performance of each model was evaluated using receiver operating characteristic (ROC) curves. Shortness of breath was the strongest predictor in the models for differentiating between COVID-19 and influenza, followed by diarrhoea. Higher lymphocyte count was predictive of COVID-19 versus influenza and versus dengue. In the model for differentiating between COVID-19 and dengue, patients with cough and higher platelet count were at increased odds of COVID-19, while headache, joint pain, skin rash and vomiting/nausea were indicative of dengue. The cross-validated area under the ROC curve for all four models was above 0.85. Clinical features and simple laboratory markers for differentiating COVID-19 from influenza and dengue are identified in this study which can be used by primary care physicians in resource limited settings to determine if further investigations or referrals would be required., (© 2021. The Author(s).)

Background: Intracranial hemorrhage (ICH) or infarction in dengue cases is rare but very challenging for clinicians. We report these uncommon complications of dengue patients and focused on the significant factors associated with ICH or infarction in dengue patients., Methods: This investigation was a retrospective study of 182 adult dengue patients who received brain computed tomography at three Taiwan hospitals during the 2014 and 2015 dengue outbreaks. This included 13 hemorrhage cases, 26 infarction cases and 143 cases without brain infarction or hemorrhage., Results: Among them, 13 (7.14%) suffered from ICH (6 had subdural hemorrhage, 3 had subarachnoid hemorrhage, 1 had subdural and subarachnoid hemorrhage, and 3 had intracerebral hemorrhage) and 26 (14.3%) had brain infarction. The overall mortality rate was 4/13 (30.8%) in the ICH group and 3/26 (11.5%) in the infarction group. The significant variables from the univariate analysis, including difference between 2014 and 2015, age, history of cerebrovascular accident, bone pain, arthralgia, dizziness, altered consciousness, and a higher Charlson comorbidity score. Multivariate analysis revealed that significant risk factors for ICH/infarction in dengue cases were the year of occurrence, 2014 vs. 2015 (p < 0.0001, OR = 25.027, 95% CI = 8.205-76.336), Charlson score >4 (p = 0.01, OR = 3.764, 95% CI = 1.364-10.386) and altered consciousness (p < 0.0001, OR = 6.3, 95% CI = 2.242-17.7). The factors physicians should notice in dengue endemic regions for brain infarction or ICH include altered consciousness and a Charlson score >4, especially in the year that a higher frequency of infarction/ICH was observed., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Purpose: The present study was intended to investigate whether monocyte immune activation shapes plasma positive to negative acute phase reactants (APRs) ratio and predicts disease severity in dengue infection., Methods: Serum level of ferritin, ceruloplasmin and transferrin was measured by means of electrochemiluminescence and immunoturbidimetry, respectively. Gene expression and plasma level for TNF-α, IL-6 and IL1-β was measured by means of RT-qPCR and ELISA., Results: A significant increased serum ferritin to transferrin [6.6 (3-11.7) vs 3.4 (1.9-6.1)] and ceruloplasmin to transferrin ratio [0.48 (0.21-0.87) vs 0.22 (0.13-0.43)] has been detected among the subjects with secondary dengue infection (SDENI) compared to primarily infected (PDENI) subjects (P < 0.001). Significant increased expression for CD14 + monocyte TNF-α, IL-6 and IL-1β has been detected in SDENI patients (vs PDENI and control, P < 0.001). Plasma ferritin to transferrin ratio was found in a significant association with high level of plasma TNF-α [ρ = 0.6522, 95% CI (0.4714-0.7805)], IL-6 [ρ = 0.6181, 95% CI (0.4257-0.7571)] and IL- 1β [ρ = 0.4119, 95% CI (0.1689-0.6077)] level among SDENI patients at 5th day time point after progression of the disease, with significantly low platelet [P < 0.001] and prolonging prothrombin time [P < 0.001] compared to control and PDENI subjects, respectively., Conclusion: Acute proinflammatory cytokine response is significantly associated with increased positive to negative APRs ratio in SDENI patients, which predicts intense immune activation, and renders SDENI patients extremely susceptible to hemostatic derangement., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future., (© 2021. The Author(s).)

Introduction/background: Dengue fever remains a public health threat despite being preventable. A solution to the constant problem of dengue infection will require active intervention and a paradigm shift. Assessing perceived risk and correlating it with the attitude and practice of the community will help in designing appropriate measures. However, possible instruments for these assessments come with limitations., Objective: The aim is to develop and validate a new scoring-based questionnaire, using dual statistical approaches to measure risk perception, attitude, and practices (RPAP) related to dengue in the community., Methods: The RPAP questionnaire was developed bilingually using the International Society for Pharmacoeconomics and Outcome Research (ISPOR) guidelines. Content analysis was reviewed scrupulously by four expert panels. The initial 35-item scale was tested among 253 Malaysian respondents recruited non-probabilistically via multiple online platforms. Two statistical methods were employed to measure the construct validity: Exploratory Factor Analysis (EFA) as part of the Classical Test Theory (CTT) measurement, while Rasch Measurement Analysis (Rasch) was performed for the Item Response Theory (IRT) measurement. All results were cross-validated with their counterpart to ensure stability. Confirmatory Factor Analysis (CFA) was used to obtain a model fit index., Results: 29 questions were retained after the final analysis. Both EFA and Rasch analysis detect multidimensionality. Nine latent factors were extracted from EFA, while only eight factors remained in the final model following CFA: 1) perceived susceptibility; 2) perceived severity; 3) perceived barrier; 4) perceived benefit; 5) cues to action; 6) self-efficacy; 7) attitude; and 8) practice. All items had adequate factor loadings and showed good internal consistency. The final model after CFA achieved a good fit with an RMSEA value of 0.061, SRMR of 0.068, PNFI of 0.649, and GFI of 0.996., Conclusion: The RPAP questionnaire contains 29 items and is a reliable and accurate psychometric instrument for measuring the risk perception of dengue fever, attitude, and practice of the community in dengue prevention. The Rasch measurement provides additional rigour to complement the CTT analysis. This RPAP questionnaire is suitable for use in studies related to dengue prevention in the community., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Viral infection is a global public health threat causing millions of deaths. A suitable small animal model is essential for viral pathogenesis and host response studies that could be used in antiviral and vaccine development. The tree shrew ( Tupaia belangeri or Tupaia belangeri chinenesis ), a squirrel-like non-primate small mammal in the Tupaiidae family, has been reported to be susceptible to important human viral pathogens, including hepatitis viruses (e.g., HBV, HCV), respiratory viruses (influenza viruses, SARS-CoV-2, human adenovirus B), arboviruses (Zika virus and dengue virus), and other viruses (e.g., herpes simplex virus, etc.). The pathogenesis of these viruses is not fully understood due to the lack of an economically feasible suitable small animal model mimicking natural infection of human diseases. The tree shrew model significantly contributes towards a better understanding of the infection and pathogenesis of these important human pathogens, highlighting its potential to be used as a viable viral infection model of human viruses. Therefore, in this review, we summarize updates regarding human viral infection in the tree shrew model, which highlights the potential of the tree shrew to be utilized for human viral infection and pathogenesis studies.

The American Zika virus (ZIKV) epidemic has highlighted the need to gain a better understanding of this emerging virus. The goal of this study was to describe the clinical symptoms, laboratory findings, and risk factors for symptomatic ZIKV infection in an area with ongoing transmission of other arboviral infections. We recruited patients at least 2 years of age seeking care at public health centers in León, Nicaragua, between January 2016 and August 2017, for fever, maculopapular rash, and/or nonsuppurative conjunctivitis with a duration of less than 1 week. A laboratory diagnosis of ZIKV was established using a combination of molecular and serological tests. Clinical and laboratory findings and potential risk factors were compared between participants with and without acute ZIKV infection. Fifty-eight (26%) of the 225 participants included in the analysis were found to have acute ZIKV infection. Pregnancy and reports of previous arboviral infection were associated with a higher risk of ZIKV infection. Rash, conjunctivitis, sore throat, and lower absolute neutrophil counts were associated with acute ZIKV infection. The clinical characteristics and risk factors identified were consistent with those identified by previous studies; however, we found sore throat to be a feature of ZIKV infection. We also found that neutrophil counts were lower in ZIKV-infected subjects. These clinical symptoms and laboratory data may help clinicians suspect ZIKV infection during future outbreaks.

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses , in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV&#95;BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV&#95;BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo , mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV&#95;BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.

Globally, the burden due to dengue infection is increasing with a recent estimate of 96 million progressing to the disease every year. Dengue pathogenesis and the factors influencing it are not completely known. It is now widely speculated that there is an important role of matrix metalloproteinases (MMPs) in the initiation and progression of dengue pathogenesis; however, their exact roles are not fully understood. Overactivation of matrix metalloproteinases may contribute to the severity of dengue pathogenesis. Cytokines and various other mediators of inflammation interact with the vascular endothelium and matrix metalloproteinases may be one of the components among them. Extensive plasma leakage into tissue spaces may result in a shock. It is evident in the literature that MMP2 and MMP9 increase in dengue patients is correlated with the severity of the disease; however, the underlying mechanism is still unknown. Activation of innate cells and adaptive immune cells which include, B and T cells, macrophages or monocytes and dendritic cells also contribute to the dengue pathology. Newer therapeutic strategies include microRNAs, such as miR-134 (targets MMP3 and MMP1) and MicroRNA-320d, (targets MMP/TIMP proteolytic system). The use of antibodies-based therapeutics like (Andecaliximab; anti-matrix metalloproteinase-9 antibody) is also suggested against MMPs in dengue. In this review, we summarize some recent developments associated with the involvement of immune cells and their mediators associated with the matrix metalloproteinases mediated dengue pathogenesis. We highlight that, there is still very little knowledge about the MMPs in dengue pathogenesis which needs attention and extensive investigations., (© 2021 Wiley Periodicals LLC.)

Background: The outbreak of Coronavirus disease 2019 (COVID-19) has become an international public health crisis, and the number of cases with dengue co-infection has raised concerns. Unfortunately, treatment options are currently limited or even unavailable. Thus, the aim of our study was to explore the underlying mechanisms and identify potential therapeutic targets for co-infection., Methods: To further understand the mechanisms underlying co-infection, we used a series of bioinformatics analyses to build host factor interaction networks and elucidate biological process and molecular function categories, pathway activity, tissue-specific enrichment, and potential therapeutic agents., Results: We explored the pathologic mechanisms of COVID-19 and dengue co-infection, including predisposing genes, significant pathways, biological functions, and possible drugs for intervention. In total, 460 shared host factors were collected; among them, CCL4 and AhR targets were important. To further analyze biological functions, we created a protein-protein interaction (PPI) network and performed Molecular Complex Detection (MCODE) analysis. In addition, common signaling pathways were acquired, and the toll-like receptor and NOD-like receptor signaling pathways exerted a significant effect on the interaction. Upregulated genes were identified based on the activity score of dysregulated genes, such as IL-1, Hippo, and TNF-α. We also conducted tissue-specific enrichment analysis and found ICAM-1 and CCL2 to be highly expressed in the lung. Finally, candidate drugs were screened, including resveratrol, genistein, and dexamethasone., Conclusions: This study probes host factor interaction networks for COVID-19 and dengue and provides potential drugs for clinical practice. Although the findings need to be verified, they contribute to the treatment of co-infection and the management of respiratory disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zheng, Wu, Liu, Yan, Wang, Wu, Liu, Jiang and Zhan.)

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients' sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients., Competing Interests: The authors have declared that no competing interests exist.

Dengue remains a major problem in the tropics. Several Asian countries have reported an increasing trend in the proportion of infants with dengue fever. However, most studies are limited to case reports or small case series from isolated outbreaks. We planned this study to look at clinico-laboratory profile, outcome, and predictors of severity in a large cohort of infants over a decade. Electronic medical records of infants admitted at a tertiary center of South India, with laboratory confirmed dengue infection between 2009 and 2019 were reviewed. Diagnosis was based on detection of NS-1 antigen and/or immunoglobulin M antibody against DENV(dengue virus) or positive DENV RNA polymerase chain reaction in infants presenting with acute febrile illness and clinical features consistent with dengue. Of 395 children with dengue admitted during study period, 99 (25%) were infants. A cyclical incidence pattern was noted, with higher cases in alternate years. Fever (99%) was most common, followed by gastrointestinal symptoms (vomiting, diarrhea-28%) and upper respiratory symptoms (cough, coryza-22%). Fifty-three infants had severe dengue, and 39 had shock. Fourteen children had multiorgan dysfunction syndrome, and 13 died. Infants with severe dengue were older than those with nonsevere disease, had lower serum albumin and greater frequency of severe thrombocytopenia, and had coagulopathy. On multivariable analysis, low serum albumin predicted development of severe dengue [P = 0.003, odds ratio 12.4 (95% confidence interval: 2.42-63.7)]. Dengue in infants may be challenging to recognize because of its undifferentiated presentation, with gastrointestinal and upper respiratory symptoms that are similar to other viral illness. Severe dengue is common in this sample, and lower serum albumin at presentation was predictive of severe disease.

Background: Sanofi-Pasteur's CYD-TDV is the only licensed dengue vaccine. Two phase three trials showed higher efficacy in seropositive than seronegative recipients. Hospital follow-up revealed increased hospitalisation in 2-5- year-old vaccinees, where serostatus and age effects were unresolved., Methods: We fit a survival model to individual-level data from both trials, including year 1 of hospital follow-up. We determine efficacy by age, serostatus, serotype and severity, and examine efficacy duration and vaccine action mechanism., Results: Our modelling indicates that vaccine-induced immunity is long-lived in seropositive recipients, and therefore that vaccinating seropositives gives higher protection than two natural infections. Long-term increased hospitalisation risk outweighs short-lived immunity in seronegatives. Independently of serostatus, transient immunity increases with age, and is highest against serotype 4. Benefit is higher in seropositives, and risk enhancement is greater in seronegatives, against hospitalised disease than against febrile disease., Conclusions: Our results support vaccinating seropositives only. Rapid diagnostic tests would enable viable 'screen-then-vaccinate' programs. Since CYD-TDV acts as a silent infection, long-term safety of other vaccine candidates must be closely monitored., Funding: Bill & Melinda Gates Foundation, National Institute for Health Research, UK Medical Research Council, Wellcome Trust, Royal Society., Clinical Trial Number: NCT01373281 and NCT01374516., Competing Interests: DL, ID, WH, GN, NF No competing interests declared, LC Laurent Coudeville is employed by Sanofi-Pasteur, (© 2021, Laydon et al.)

Background: Early identification of severe dengue patients is important regarding patient management and resource allocation. We investigated the association of 10 biomarkers (VCAM-1, SDC-1, Ang-2, IL-8, IP-10, IL-1RA, sCD163, sTREM-1, ferritin, CRP) with the development of severe/moderate dengue (S/MD)., Methods: We performed a nested case-control study from a multi-country study. A total of 281 S/MD and 556 uncomplicated dengue cases were included., Results: On days 1-3 from symptom onset, higher levels of any biomarker increased the risk of developing S/MD. When assessing together, SDC-1 and IL-1RA were stable, while IP-10 changed the association from positive to negative; others showed weaker associations. The best combinations associated with S/MD comprised IL-1RA, Ang-2, IL-8, ferritin, IP-10, and SDC-1 for children, and SDC-1, IL-8, ferritin, sTREM-1, IL-1RA, IP-10, and sCD163 for adults., Conclusions: Our findings assist the development of biomarker panels for clinical use and could improve triage and risk prediction in dengue patients., Funding: This study was supported by the EU's Seventh Framework Programme (FP7-281803 IDAMS), the WHO, and the Bill and Melinda Gates Foundation., Competing Interests: NV, PL, HD, NN, DT, KD, NC, NC, EP, CS, KR, DB, NA, CH, PO No competing interests declared, DM reports receiving personal fees from the Wellcome Trust (grant number 215010/Z/18/Z), during the conduct of the study. LL reports receiving personal fees from ROCHE Advisory Board on Severe Dengue. TJ reports receiving personal fees as members of the ROCHE Advisory Board on Severe Dengue, outside the submitted work. BW reports personal fees as a member of the Data Monitoring and Adjudication Committees for the Takeda dengue vaccine trials and as a member of the ROCHE Advisory Board on Severe Dengue, outside the submitted work. RG reports receiving personal fees from the Wellcome Trust (grant number 106680/Z/14/Z), during the conduct of the study. SY reports receiving personal fees as a member of the ROCHE Advisory Board on Severe Dengue, for work on Janssen Pharmaceuticals Advisory Board for Dengue Antiviral Development, and from the Wellcome Trust (grant number 106680/Z/14/Z)., (© 2021, Vuong et al.)

Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo . IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia -resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia . While there are hurdles to dengue viruses developing resistance to Wolbachia , we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia -introgression biocontrol programs.

Dengue virus (DENV), a Flavivirus, causes a broad spectrum of disease in humans with key clinical signs including thrombocytopenia, vascular leakage and hemorrhaging. A major obstacle to understanding DENV immunity has been the lack of a validated immune-competent mouse model. Here, we report the infection profiles of human clinical isolates of DENV serotypes 1-4 in an immune-competent mouse model. We detected replicating DENV in the peritoneal cells, liver and the spleen that was generally resolved within 2 weeks. The DENV target cell types for infection were monocytes/macrophages, dendritic cells, endothelial cells, and we identified a novel DENV cellular target, fibroblast reticular cells of the spleen. We observed gross pathologies in the spleen and liver that are consistent with dengue disease, including hemorrhaging as well as transcriptional patterns suggesting that antiviral responses and tissue damage were induced. Key clinical blood parameters that define human DENV disease such as hemoconcentration, leukopenia and reduced number of platelets were also observed. Thus, immune-competent mice sustain replicating infection and experience signs, such as hemorrhaging, that define DENV disease in humans. This study thoroughly characterizes DENV1-4 infection in immune-competent mice and confirms the wild-type mouse model as a valid and reproducible system for investigating the mechanisms of DENV pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rathore, Mantri, Tan, Shirazi, Nishida, Aman, Morrison and St. John.)

Background: In 2017, New Caledonia experienced an outbreak of severe dengue causing high hospital burden (4379 cases, 416 hospital admissions, 15 deaths). We decided to build a local operational model predictive of dengue severity, which was needed to ease the healthcare circuit., Methods: We retrospectively analyzed clinical and biological parameters associated with severe dengue in the cohort of patients hospitalized at the Territorial Hospital between January and July 2017 with confirmed dengue, in order to elaborate a comprehensive patient's score. Patients were compared in univariate and multivariate analyses. Predictive models for severity were built using a descending step-wise method., Results: Out of 383 included patients, 130 (34%) developed severe dengue and 13 (3.4%) died. Major risk factors identified in univariate analysis were: age, comorbidities, presence of at least one alert sign, platelets count < 30 × 10 9 /L, prothrombin time < 60%, AST and/or ALT > 10 N, and previous dengue infection. Severity was not influenced by the infecting dengue serotype nor by previous Zika infection. Two models to predict dengue severity were built according to sex. Best models for females and males had respectively a median Area Under the Curve = 0.80 and 0.88, a sensitivity = 84.5 and 84.5%, a specificity = 78.6 and 95.5%, a positive predictive value = 63.3 and 92.9%, a negative predictive value = 92.8 and 91.3%. Models were secondarily validated on 130 patients hospitalized for dengue in 2018., Conclusion: We built robust and efficient models to calculate a bedside score able to predict dengue severity in our setting. We propose the spreadsheet for dengue severity score calculations to health practitioners facing dengue outbreaks of enhanced severity in order to improve patients' medical management and hospitalization flow.

Background: Dengue virus (DENV) infection has a global impact on public health. The clinical outcomes (of DENV) can vary from a flu-like illness called dengue fever (DF), to a more severe form, known as dengue hemorrhagic fever (DHF). The underlying innate immune mechanisms leading to protective or detrimental outcomes have not been fully elucidated. Helper innate lymphoid cells (hILCs), an innate lymphocyte recently discovered, functionally resemble T-helper cells and are important in inflammation and homeostasis. However, the role of hILCs in DENV infection had been unexplored., Methods: We performed flow cytometry to investigate the frequency and phenotype of hILCs in peripheral blood mononuclear cells from DENV-infected patients of different disease severities (DF and DHF), and at different phases (febrile and convalescence) of infection. Intracellular cytokine staining of hILCs from DF and DHF were also evaluated by flow cytometry after ex vivo stimulation. Further, the hILCs were sorted and subjected to transcriptome analysis using RNA sequencing. Differential gene expression analysis was performed to compare the febrile and convalescent phase samples in DF and DHF. Selected differentially expressed genes were then validated by quantitative PCR., Results: Phenotypic analysis showed marked activation of all three hILC subsets during the febrile phase as shown by higher CD69 expression when compared to paired convalescent samples, although the frequency of hILCs remained unchanged. Upon ex vivo stimulation, hILCs from febrile phase DHF produced significantly higher IFN-γ and IL-4 when compared to those of DF. Transcriptomic analysis showed unique hILCs gene expression in DF and DHF, suggesting that divergent functions of hILCs may be associated with different disease severities. Differential gene expression analysis indicated that hILCs function both in cytokine secretion and cytotoxicity during the febrile phase of DENV infection., Conclusions: Helper ILCs are activated in the febrile phase of DENV infection and display unique transcriptomic changes as well as cytokine production that correlate with severity. Targeting hILCs during early innate response to DENV might help shape subsequent immune responses and potentially lessen the disease severity in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Poonpanichakul, Chan-In, Opasawatchai, Loison, Matangkasombut, Charoensawan, Matangkasombut and DENFREE Thailand.)

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG + PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM + PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67 + ) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Raúl A. Maqueda-Alfaro et al.)

Clinical presentations of dengue fever (DF) are diverse and non-specific, causing unpredictable progression and outcomes. Its progression and severity have been associated with cytokine levels alteration. In this study, dengue patients were classified into groups following the 2009 WHO dengue classification scheme to investigate the cytokine signature at different severity of the disease: dengue without warning sign symptoms (A); dengue with warning signs (B); severe dengue (C); other fever (OF) and healthy (Healthy). We analyzed 23 different cytokines simultaneously, namely IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-33, CD14, CD54, CD62E, CD62L, CD62p, CD106, CD121b, CD154, CD178, GM-CSF, IFN-g, MIF, ST2 and TNF from patients admitted to National Cheng Kung University Hospital during the 2015 Taiwan dengue outbreak. Cytokines TNF, CD54, CD62E, CD62L, CD62P, GM-CSF, IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, INF-g and MIF were elevated while CD106, CD154, IL-4 and L-33 were decreased when compared to the control. IL-10 demonstrated to be a potential diagnostic marker for DF (H and A group; AUC = 0.944, H and OF group; AUC = 0.969). CD121b demonstrated to be predictive of the SD (A and B group; AUC = 0.744, B and C group; AUC = 0.775). Our results demonstrate the cytokine profile changes during the progression of dengue and highlight possible biomarkers for optimizing effective intervention strategies.

Exposure to environmental hormones such as di(2-ethylhexyl) phthalate (DEHP) has become a critical human health issue globally. This study aimed to investigate the correlations between DEHP/mono-(2-ethylhexyl) phthalate (MEHP) levels and macrophage-associated immune responses and clinical manifestations in dengue virus (DV)-infected patients. Among 89 DV-infected patients, those with DV infection-related gastrointestinal (GI) bleeding (n = 13, 15% of patients) had significantly higher DEHP exposure than those without GI bleeding (n = 76, 85% of patients), which were 114.2 ng/ml versus 52.5 ng/ml ΣDEHP in urine; p = 0.023). In an in vitro study using cultured human monocyte-derived macrophages (MDMs) to investigate the effects of MEHP, treatment increased IL-1β and TNF-α release but decreased IL-23 release, with negative correlations observed between urine ΣDEHP and serum IL-23 levels in patients. MEHP-treated MDMs had lower antiviral Th17 response induction activity in mixed T-cell response tests. The in vitro data showed that MEHP increased DV viral load and decreased IL-23 release dose-dependently, and adding IL-23 to MEHP-exposed MDMs significantly reduced the DV viral load. MEHP also suppressed IL-23 expression via the peroxisome proliferator-activated receptor-gamma (PPAR-γ) pathway. Further, the PPAR-γ antagonist GW9662 significantly reversed MEHP-induced IL-23 suppression and reduced the DV viral load. These study findings help to explain the associations between high MEHP levels and the high global burden of dengue disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Huang, Wang, Tenhunen, Hung, Lin, Chen, Chen and Liao.)

Dengue is an acute viral disease caused by dengue virus (DENV), which is transmitted by Aedes mosquitoes. Symptoms of DENV infection range from inapparent to severe and can be life-threatening. DENV replicates in primary immune cells such as dendritic cells and macrophages, which contribute to the dissemination of the virus. Susceptibility of other immune cells such as B cells to direct infection by DENV and their subsequent response to infection is not well defined. In a cohort of 60 Cambodian children, we showed that B cells are susceptible to DENV infection. Moreover, we show that B cells can support viral replication of laboratory adapted and patient-derived DENV strains. B cells were permissive to DENV infection albeit low titers of infectious virions were released in cell supernatants CD300a, a phosphatidylserine receptor, was identified as a potential attachment factor or receptor for entry of DENV into B cells. In spite of expressing Fc γ -receptors, antibody-mediated enhancement of DENV infection was not observed in B cells in an in vitro model. Direct infection by DENV induced proliferation of B cells in dengue patients in vivo and plasmablast/plasma cell formation in vitro . To summarize, our results show that B cells are susceptible to direct infection by DENV via CD300a and the subsequent B cell responses could contribute to dengue pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Upasani, Vo, Auerswald, Laurent, Heng, Duong, Rodenhuis-Zybert, Dussart and Cantaert.)

Background: In Africa, information on dengue is limited to outbreak reports and focused on some countries with continuing transmission in West and East Africa. To estimate the proportion of dengue-positive cases among febrile patients and identify clinical indicators of dengue cases, we conducted passive facility-based fever surveillance in a catchment area population of 70,000 residents of Lambaréné and its surroundings in Gabon., Methods: Non-malarial febrile patients with current fever or history of fever (≤7 days) between 1 and 55 years of age, were enrolled at Albert Schweitzer Hospital (ASH). Acute (visit 1, day of enrollment) and convalescent blood samples were collected between 10 and 21 days after enrollment. Acute/convalescent samples were tested with IgM/IgG ELISA, and a selected subset of acute samples with RT-PCR., Results: Among 682 non-malarial febrile patients enrolled, 119 (17.4%) were identified as dengue-positive (94 dengue-confirmed and 25 dengue-probable cases). Of these dengue-positive cases, 14 were confirmed with PCR, and based on serotyping, two infections were identified to be DENV-2 and two were DENV-3. The majority of our enrolled patients were <25 years of age and close to 80% of our dengue-positive cases were <15 years of age. In adjusted analyses, retro-orbital pain and abdominal pain were 2.7 and 1.6 times more frequently found among dengue-positive cases, compared to non-dengue cases., Conclusion: Lambaréné is not considered dengue-endemic. However, one in six non-malarial febrile episodes was found to be dengue-positive in the study period. Dengue should be considered more frequently in clinicians' diagnosis among non-malarial febrile patients in Lambaréné. Given the lack of data on dengue in Gabon, additional prospective and longitudinal studies would help to further define the burden and patterns of dengue for improved case detection., Competing Interests: The authors have declared that no competing interests exist.

Formalin-fixed paraffin-embedded (FFPE) tissues are an important source for investigation of dengue virus (DENV) infection, particularly when blood or fresh frozen (FF) samples are unavailable. Histopathologic features and immunohistochemistry may have poor sensitivity and serotype determination is not always possible. Viral RNA genome detection tests are faster and considered the most sensitive technique for this kind of analysis, however, the use of molecular methods applied to FFPE tissues is still limited. The authors applied a single-step multiplex reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for the investigation of DENV infection and typing to FFPE samples of 32 fatal cases received during the 2019 outbreak that occurred in São Paulo state, Brazil. The authors compared the results with those obtained using FF tissues. Of the 24 cases with both FF and FFPE samples, 22 (91.67%) of the FF and 19 (76.20%) of the FFPE specimens were positive. Two cases (8.33%) tested negative in both types of samples. All 8 cases with only FFPE samples available were positive. The accuracy (87.5%) of the RT-qPCR for DENV in FFPE samples were satisfactory. Although the cycle quantification (Cq) values were significantly higher in these materials (P<0.0001, Wilcoxon signed-rank test) when compared with FF tissues, Spearman's rank coefficient indicated a good correlation between the Cq values from both sample types (P=0.0063; rho=0.576). RT-qPCR applied to FFPE samples improved detection of DENV in fatal cases and represents a useful tool for diagnosis and epidemiologic studies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses., (Copyright © 2021 American Society for Microbiology.)

Dengue infections are increasing globally and account for significant morbidity and mortality. Severe dengue results in microvascular changes and coagulopathy that may make surgical intervention risky and the overall surgical management challenging. We outline the potential surgical manifestations and complications following dengue infections and describe the clinical, pathogenetic, diagnostic, and treatment aspects of dengue and surgical patients. The main surgical presentations were acute cholecystitis, acute pancreatitis, acute appendicitis, splenic rupture, bowel perforation, gastrointestinal bleeding, and hematomas. Dengue may also mimic an acute abdomen without any true surgical complications. A majority were treated nonoperatively. Misdiagnosis and unnecessary surgical intervention resulted in poor outcomes. Better knowledge of the potential surgical complications would help in early diagnosis, treatment, and referral to specialized centers and thus improve outcomes. A high degree of suspicion of dengue fever is necessary when patients in a dengue-epidemic area present with acute abdomen or bleeding manifestations. In endemic areas, early dengue antigen testing and abdominal imaging before surgical intervention may help in the diagnoses. Multidisciplinary team involvement with case-by-case decision-making is needed for optimal care.

Dengue virus (DENV) infection is increasing with rapid urbanization in India. Treatment of DENV infection is mainly supportive with no specific antiviral therapy. Although most patients show mild illness, some have a severe disease course such as dengue hemorrhagic syndrome, dengue shock, multi-organ failure, and death. The cause for severity is not fully understood. Currently, there are no methods available to predict the course of the illness. Hence, it is crucial to develop an early biomarker to predict the course of dengue illness which can aid in vigorous monitoring and early intervention. Here, we tried to establish a correlation between serum ferritin and severity of dengue illness. We measured ferritin levels in 100 dengue-positive cases on day 1 (D1) (febrile phase) and day 4 (D4) (defervescence or convalescent) of admissions to compare the levels with the severity of the disease. On D1, the serum ferritin level was a "good" predictor of severe dengue, with an area under the curve (AUC) of 0.863 with standard error (SE) = 0.043 and a 95% CI from 0.778 to 0.947 (P < 0.05). On D4, serum ferritin was an "excellent" predictor of severe dengue, with an AUC of 0.947 with SE = 0.021 and a 95% CI from 0.907 to 0.988 (P < 0.05). Serum ferritin is an inexpensive and easily accessible biomarker that can assist in monitoring and prognosticating the dengue-positive patients. This biomarker also directs us to explore the underlying pathogenetic mechanism in severe dengue, which can lay a foundation for future targeted therapeutic options to combat severe illness.

Phenolic compounds have been related to multiple biological activities, and the antiviral effect of these compounds has been demonstrated in several viral models of public health concern. In this review, we show the antiviral role of phenolic compounds against dengue virus (DENV), the most widespread arbovirus globally that, after its re-emergence, has caused multiple epidemic outbreaks, especially in the last two years. Twenty phenolic compounds with anti-DENV activity are discussed, including the multiple mechanisms of action, such as those directed against viral particles or viral proteins, host proteins or pathways related to the productive replication viral cycle and the spread of the infection.

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.