Genetic Analysis of Dengue Virus in Severe and Non-Severe Cases in Dhaka, Bangladesh, in 2018-2022.

Dengue virus (DENV) infections have unpredictable clinical outcomes, ranging from asymptomatic or minor febrile illness to severe and fatal disease. The severity of dengue infection is at least partly related to the replacement of circulating DENV serotypes and/or genotypes. To describe clinical profiles of patients and the viral sequence diversity corresponding to non-severe and severe cases, we collected patient samples from 2018 to 2022 at Evercare Hospital Dhaka, Bangladesh. Serotyping of 495 cases and sequencing of 179 cases showed that the dominant serotype of DENV shifted from DENV2 in 2017 and 2018 to DENV3 in 2019. DENV3 persisted as the only representative serotype until 2022. Co-circulation of clades B and C of the DENV2 cosmopolitan genotype in 2017 was replaced by circulation of clade C alone in 2018 with all clones disappearing thereafter. DENV3 genotype I was first detected in 2017 and was the only genotype in circulation until 2022. We observed a high incidence of severe cases in 2019 when the DENV3 genotype I became the only virus in circulation. Phylogenetic analysis revealed clusters of severe cases in several different subclades of DENV3 genotype I. Thus, these serotype and genotype changes in DENV may explain the large dengue outbreaks and increased severity of the disease in 2019.

Performance of VIDAS® Diagnostic Tests for the Automated Detection of Dengue Virus NS1 Antigen and of Anti-Dengue Virus IgM and IgG Antibodies: A Multicentre, International Study.

Dengue is a serious mosquito-transmitted disease caused by the dengue virus (DENV). Rapid and reliable diagnosis of DENV infection is urgently needed in dengue-endemic regions. We describe here the performance evaluation of the CE-marked VIDAS® dengue immunoassays developed for the automated detection of DENV NS1 antigen and anti-DENV IgM and IgG antibodies. A multicenter concordance study was conducted in 1296 patients from dengue-endemic regions in Asia, Latin America, and Africa. VIDAS® dengue results were compared to those of competitor enzyme-linked immunosorbent assays (ELISA). The VIDAS® dengue assays showed high precision (CV ≤ 10.7%) and limited cross-reactivity (≤15.4%) with other infections. VIDAS® DENGUE NS1 Ag showed high positive and negative percent agreement (92.8% PPA and 91.7% NPA) in acute patients within 0-5 days of symptom onset. VIDAS® Anti-DENGUE IgM and IgG showed a moderate-to-high concordance with ELISA (74.8% to 90.6%) in post-acute and recovery patients. PPA was further improved in combined VIDAS® NS1/IgM (96.4% in 0-5 days acute patients) and IgM/IgG (91.9% in post-acute patients) tests. Altogether, the VIDAS® dengue NS1, IgM, and IgG assays performed well, either alone or in combination, and should be suitable for the accurate diagnosis of DENV infection in dengue-endemic regions.

N-Butanol Extract of Glycyrrhizae Radix et Rhizoma Inhibits Dengue Virus through Targeting Envelope Protein.

BACKGROUND: At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). METHODS: Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. RESULTS: Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. CONCLUSIONS: GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.

Identification of Polyphenol Derivatives as Novel SARS-CoV-2 and DENV Non-Nucleoside RdRp Inhibitors.

The Coronavirus Disease 2019 (COVID-19) and dengue fever (DF) pandemics both remain to be significant public health concerns in the foreseeable future. Anti-SARS-CoV-2 drugs and vaccines are both indispensable to eliminate the epidemic situation. Here, two piperazine-based polyphenol derivatives DF-47 and DF-51 were identified as potential inhibitors directly blocking the active site of SARS-CoV-2 and DENV RdRp. Data through RdRp inhibition screening of an in-house library and in vitro antiviral study selected DF-47 and DF-51 as effective inhibitors of SARS-CoV-2/DENV polymerase. Moreover, in silico simulation revealed stable binding modes between the DF-47/DF-51 and SARS-CoV-2/DENV RdRp, respectively, including chelating with Mg2+ near polymerase active site. This work discovered the inhibitory effect of two polyphenols on distinct viral RdRp, which are expected to be developed into broad-spectrum, non-nucleoside RdRp inhibitors with new scaffold.

Synthesis and biological evaluation of new ß-D-N4-hydroxycytidine analogs against SARS-CoV-2, influenza viruses and DENV-2.

Drug repurposing approach was applied to find a potent antiviral agent against RNA viruses such as SARS-CoV-2, influenza viruses and dengue virus with a concise strategy of small change in parent molecular structure. For this purpose, ß-D-N4-hydroxycytidine (NHC, 1) with a broad spectrum of antiviral activity was chosen as the parent molecule. Among the prepared NHC analogs (8a-g, and 9) from uridine, ß-D-N4-O-isobutyrylcytidine (8a) showed potent activity against SARS-CoV-2 (EC50 3.50 µM), Flu A (H1N1) (EC50 5.80 µM), Flu A (H3N2) (EC50 7.30 µM), Flu B (EC50 3.40 µM) and DENV-2 (EC50 3.95 µM) in vitro. Furthermore, its potency against SARS-CoV-2 was >5-fold, 3.4-fold, and 3-fold compared to that of NHC (1), MK-4482 (2), and remdesivir (RDV) in vitro, respectively. Ultimately, compound 8a was expected to be a potent inhibitor toward RNA viruses as a viral mutagenic agent like MK-4482.

Endothelial Dysfunction, HMGB1, and Dengue: An Enigma to Solve.

Dengue is a viral infection caused by dengue virus (DENV), which has a significant impact on public health worldwide. Although most infections are asymptomatic, a series of severe clinical manifestations such as hemorrhage and plasma leakage can occur during the severe presentation of the disease. This suggests that the virus or host immune response may affect the protective function of endothelial barriers, ultimately being considered the most relevant event in severe and fatal dengue pathogenesis. The mechanisms that induce these alterations are diverse. It has been suggested that the high mobility group box 1 protein (HMGB1) may be involved in endothelial dysfunction. This non-histone nuclear protein has different immunomodulatory activities and belongs to the alarmin group. High concentrations of HMGB1 have been detected in patients with several infectious diseases, including dengue, and it could be considered as a biomarker for the early diagnosis of dengue and a predictor of complications of the disease. This review summarizes the main features of dengue infection and describes the known causes associated with endothelial dysfunction, highlighting the involvement and possible relationship between HMGB1 and DENV.

Case report: A fatal outcome from co-infection of COVID-19 and dengue in the western region of Jeddah, Saudi Arabia.

Introduction: Co-infection of coronavirus disease 2019 (COVID-19) and dengue may coexist, as both viruses share similar laboratory and clinical features, making diagnosis and treatment challenging for health care professionals to prescribe, negatively impacting patient prognosis, and outcomes. Results and discussions: Both cases were positive for PCR and X-ray laboratory investigation at clinical examination, confirming COVID-19 and dengue co-infection, admission, and better management in referral hospitals are presented and discussed. The timeline provides detailed cases of situational analysis and the medical actions taken, as well as the outcomes. Conclusion: Both co-infection cases' (patients) health conditions had a poor prognosis and diagnosis and ended with undesired outcomes. Scaling up dual mosquito-vector linked viral diseases surveillance in understanding the transmission dynamics, early diagnosis, and the timely and safe monitoring of case management in clinical and hospital settings nationwide is paramount in curbing preventable co-infections and mortality.

Dengue Infection - Recent Advances in Disease Pathogenesis in the Era of COVID-19.

The dynamics of host-virus interactions, and impairment of the host's immune surveillance by dengue virus (DENV) serotypes largely remain ambiguous. Several experimental and preclinical studies have demonstrated how the virus brings about severe disease by activating immune cells and other key elements of the inflammatory cascade. Plasmablasts are activated during primary and secondary infections, and play a determinative role in severe dengue. The cross-reactivity of DENV immune responses with other flaviviruses can have implications both for cross-protection and severity of disease. The consequences of a cross-reactivity between DENV and anti-SARS-CoV-2 responses are highly relevant in endemic areas. Here, we review the latest progress in the understanding of dengue immunopathogenesis and provide suggestions to the development of target strategies against dengue.

Bomidin: An Optimized Antimicrobial Peptide With Broad Antiviral Activity Against Enveloped Viruses.

The rapid evolution of highly infectious pathogens is a major threat to global public health. In the front line of defense against bacteria, fungi, and viruses, antimicrobial peptides (AMPs) are naturally produced by all living organisms and offer new possibilities for next-generation antibiotic development. However, the low yields and difficulties in the extraction and purification of AMPs have hindered their industry and scientific research applications. To overcome these barriers, we enabled high expression of bomidin, a commercial recombinant AMP based upon bovine myeloid antimicrobial peptide-27. This novel AMP, which can be expressed in Escherichia coli by adding methionine to the bomidin sequence, can be produced in bulk and is more biologically active than chemically synthesized AMPs. We verified the function of bomidin against a variety of bacteria and enveloped viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), herpes simplex virus (HSV), dengue virus (DENV), and chikungunya virus (CHIKV). Furthermore, based on the molecular modeling of bomidin and membrane lipids, we elucidated the possible mechanism by which bomidin disrupts bacterial and viral membranes. Thus, we obtained a novel AMP with an optimized, efficient heterologous expression system for potential therapeutic application against a wide range of life-threatening pathogens.

The endothelial glycocalyx in critical illness: A pediatric perspective.

The vascular endothelium is the interface between circulating blood and end organs and thus has a critical role in preserving organ function. The endothelium is lined by a glycan-rich glycocalyx that uniquely contributes to endothelial function through its regulation of leukocyte and platelet interactions with the vessel wall, vascular permeability, coagulation, and vasoreactivity. Degradation of the endothelial glycocalyx can thus promote vascular dysfunction, inflammation propagation, and organ injury. The endothelial glycocalyx and its role in vascular pathophysiology has gained increasing attention over the last decade. While studies characterizing vascular glycocalyx injury and its downstream consequences in a host of adult human diseases and in animal models has burgeoned, studies evaluating glycocalyx damage in pediatric diseases are relatively few. As children have unique physiology that differs from adults, significant knowledge gaps remain in our understanding of the causes and effects of endothelial glycocalyx disintegrity in pediatric critical illness. In this narrative literature overview, we offer a unique perspective on the role of the endothelial glycocalyx in pediatric critical illness, drawing from adult and preclinical data in addition to pediatric clinical experience to elucidate how marked derangement of the endothelial surface layer may contribute to aberrant vascular biology in children. By calling attention to this nascent field, we hope to increase research efforts to address important knowledge gaps in pediatric vascular biology that may inform the development of novel therapeutic strategies.

Human Brain Organoids as Models for Central Nervous System Viral Infection.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.

Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

A case of coinfection of a pediatric patient with acute SARS-COV-2 with MIS-C and severe DENV-2 in Mexico: a case report.

BACKGROUND: COVID-19 cases have been increasing since the epidemic started. One of the major concerns is how clinical symptomatology would behave after coinfection with another virus. CASE PRESENTATION: In this case report, a pediatric native patient from Estado de Mexico (EDOMEX), MEX had severe DENV-2 and acute SARS-CoV-2 at the same time. The clinical features were severe thrombocytopenia, secondary septic shock, cerebral edema, pericardial effusion, fluid overload that exhibited bipalpebral edema in all four extremities, hemophagocytic lymphohistiocytosis (HLH), coronary artery ectasia (CAE), multisystemic inflammatory syndrome in children (MIS-C), and probable COVID-19 pneumonia or acute respiratory distress syndrome (ARDS) that triggered patient intubation. The patient presented unusual symptomatology according to the literature. After 15 days of intubation and 15 more days under surveillance, he was released without respiratory sequelae and without treatment after major clinical improvement. CONCLUSION: The aim of this manuscript is to present clinical challenges that coinfection may cause in pediatric patients, even though COVID-19 in children does not tend to be as severe as in other sectors of the population.

SARS-CoV-2/DENV co-infection: a series of cases from the Federal District, Midwestern Brazil.

BACKGROUND: Since the novel coronavirus disease outbreak, over 179.7 million people have been infected by SARS-CoV-2 worldwide, including the population living in dengue-endemic regions, particularly Latin America and Southeast Asia, raising concern about the impact of possible co-infections. METHODS: Thirteen SARS-CoV-2/DENV co-infection cases reported in Midwestern Brazil between April and September of 2020 are described. Information was gathered from hospital medical records regarding the most relevant clinical and laboratory findings, diagnostic process, therapeutic interventions, together with clinician-assessed outcomes and follow-up. RESULTS: Of the 13 cases, seven patients presented Acute Undifferentiated Febrile Syndrome and six had pre-existing co-morbidities, such as diabetes, hypertension and hypopituitarism. Two patients were pregnant. The most common symptoms and clinical signs reported at first evaluation were myalgia, fever and dyspnea. In six cases, the initial diagnosis was dengue fever, which delayed the diagnosis of concomitant infections. The most frequently applied therapeutic interventions were antibiotics and analgesics. In total, four patients were hospitalized. None of them were transferred to the intensive care unit or died. Clinical improvement was verified in all patients after a maximum of 21 days. CONCLUSIONS: The cases reported here highlight the challenges in differential diagnosis and the importance of considering concomitant infections, especially to improve clinical management and possible prevention measures. Failure to consider a SARS-CoV-2/DENV co-infection may impact both individual and community levels, especially in endemic areas.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

Cytokines storm in COVID-19 with dengue co-infection in pregnancy: Fatal maternal and fetal outcome.

We report a 23-year-old pregnant woman who presented with acute high-grade fever, vomiting, and diarrhea for 5 days. She was first hospitalized in RSUD Tangerang-a secondary hospital based in Tangerang and were referred to Cipto Mangunkusumo General Hospital as a tertiary hospital. Initial laboratory results from previous hospital revealed leukopenia, low platelet, elevated aspartate transaminase, and alanine transaminase. Chest radiograph showed no pulmonary infiltration. Reverse transcriptase-PCR (RT-PCR) of the nasopharyngeal swab detected SARS-CoV-2, and NS1 antigen or IgM dengue-specific antibodies were positive. COVID-19 with dengue fever co-infection was diagnosed. Hemorrhagic manifestations were seen in both the mother (gum and gastrointestinal bleeding) and pregnancy (placental abruption). Patient was put on ventilator and was unfortunately lead to her death that were caused by multiorgan dysfunction failure due to co-infection of dengue and COVID-19. Both dengue and COVID-19 had similar manifestation, as it is a warning sign in pregnant patient experienced both that can lead to fatal result in mother and baby. Early diagnosis and management of co-infection is high clinical importance, especially in endemic area of dengue like Indonesia.

Autochthonous dengue outbreak in Italy 2020: clinical, virological and entomological findings.

BACKGROUND: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy. METHODS: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood. RESULTS: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site. CONCLUSIONS: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.

Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection.

BACKGROUND: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh. METHODS: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA. RESULTS: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9-23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27-30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations. CONCLUSIONS: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels.