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• Out of the seven compounds and phytochemicals, withanolide A demonstrated high binding affinities towards ten targets consisting of DENV structural, NS proteins, and CLEC5A receptor. • It showed high binding energies of −10.8, −10.6 and −10.4 kcal/mol for NS5 MTase of DENV1, DENV2 and DENV3, respectively. • The molecular dynamics simulation revealed that it showed a fair stability with DENV1, DENV2, and DENV3 over a time span of 100 ns. • Withanolide A was predicted to possess good drug-likeness properties, moderate bioavailability, and high gastrointestinal absorptivity. The prevalence of dengue remains a significant global threat to public health with millions of infections reported annually due to the lack of effective treatment options. Natural resources such as plants contain phytochemicals with desirable therapeutic effects. For instance, the Indian ginseng, Withania somnifera L. (Solanaceae), contains steroidal lactones, saponins, glycosides, and alkaloids which possessed antiviral activities against various viruses. In this study, docking analysis of seven compounds and phytochemicals were performed against the structural and non-structural (NS) proteins of all four dengue virus (DENV) serotypes as well as host cell receptors such as DC-SIGN and CLEC5A. Withanolide A (L5) was selected as the best inhibitor with high binding affinities towards ten targets consisting of DENV structural and NS proteins as well as the CLEC5A receptor. L5 also showed a fair stability with DENV1, DENV2, and DENV3 over a time span of 100 ns via molecular dynamics simulation. L5 demonstrated good drug-likeness properties, a moderate bioavailability score of 0.55, and is predicted to have high gastrointestinal absorptivity. The data from this in silico study revealed the potential of withanolide A to be further developed as an antiviral against dengue. This is the first study to report the in silico analyses of phytochemicals derived from W. somnifera L. (Solanaceae) against the viral proteins of DENV1 to 4 and their respective host cell receptors. [ABSTRACT FROM AUTHOR]

Dengue hemorrhagic fever (DHF) is a severe condition resulting from the dengue virus, with four serotypes known as DEN-1, DEN-2, DEN-3, and DEN-4. Genetic variations play a crucial role in influencing susceptibility to DHF. Therefore, this investigation conducted a meta-analysis to uncover genetic changes that might have remained undetected in individual studies due to small sample sizes or methodological differences. Among 2212 initially identified studies, 23 were deemed suitable for analysis based on PRISMA guidelines. Toll-like receptors (TLR) and CD209 showed significant association with DHF (odds ratios: TLR=0.56, CD209 =0.55), indicating protective effects. However, tumor necrosis factor (TNF) and human leukocyte antigen (HLA) did not exhibit a statistically significant relationship with DHF. This study emphasizes the relevance of TLR and CD209 in DHF susceptibility and resistance across diverse geographical locations. [ABSTRACT FROM AUTHOR]

Due to dengue fever's swift global expansion and the lack of effective antiviral remedies, it is crucial to discover and develop new antiviral drugs. The study aimed to assess the antiviral potential of the extract and fractions from the leaves of Goniothalamus lanceolatus Miq. (Annonaceae). New Guinea C strain DENV-2, at a multiplicity of infection of 0.4, was used to infect Vero cells. The assessment of antiviral effectiveness was conducted through the plaque reduction assay. Deep metabolome analysis of the active fraction identified nine styryllactones based on reference standards and eighteen styryllactones were further annotated through a molecular database search. One new 2 H -tetrahydropyran derivative, 3- epi -goniothalesdiol A (19), together with seven known styryllactones (8 , 11 , 14 , 16 , 17 , 18 , and 22) were isolated from the leaves of G. lanceolatus. The active bis-styryllactone goniolanceolatin A (22) with the highest selectivity index (SI) underwent further evaluation using quantitative reverse transcription qRT-PCR to determine the viral RNA level. The qRT-PCR data showed that the IC 50 value for the active compound was 5.07 µg/mL, and its corresponding SI value was 5.30. Following comprehensive docking studies, the bis-styryllactone derivative 22 showed potential binding interactions with crucial amino acids of the Envelope (E) of DENV proteins comparable to those of ribavirin. [Display omitted] • In vitro evaluation of Goniothalamus lanceolatus leaf dichloromethane (GLLD) extract showed the strongest anti-dengue activity. • One new 2H-tetrahydropyran derivative was successfully isolated from the leaves of G. lanceolatus during a bioactivity-directed. • In vitro and in silico evaluation of bis-styryl lactone isolated from the most active fraction of leaves of G. lanceolatus showed the most potential anti-dengue activity. [ABSTRACT FROM AUTHOR]

• TAK-003 had an acceptable safety profile in healthy US adults. • TAK-003 induced robust neutralizing antibody (Ab) responses vs. all DENV serotypes. • Eight of the 12 lot-to-lot immunogenicity consistency comparisons were met. • Elicited Ab titers were similar to those in the efficacy trial at same timepoints. • These data support acceptable consistency of TAK-003 manufacturing process. We conducted a trial to demonstrate immunogenic equivalence of three consecutive manufacturing lots of Takeda's tetravalent dengue vaccine candidate, TAK-003, and further assessed its safety and reactogenicity. Healthy US adults (n = 923) randomized 2:2:2:1 to four groups received two doses of one of three TAK-003 lots or placebo on Days 0 and 90, with follow-up to Day 270. Primary endpoint evaluated lot-to-lot equivalence of geometric mean neutralizing titers at Day 120 against each of 4 dengue serotypes in baseline seronegative participants. Solicited local and systemic, and unsolicited adverse events (AEs) were assessed for 7, 14 and 28 days after each dose, respectively. Serious AEs (SAE) were monitored throughout the study. Eight of 12 prespecified equivalence comparisons were met in the per-protocol set but failed marginally in the other 4 mainly due to loss of statistical power following higher than anticipated baseline seropositivity and drop-out rates. All three TAK-003 lots elicited high rates of tetravalent dengue seropositivity (96.7 %, 93.0 % and 97.5 % at Day 120; 91.0 %, 80.5 % and 85.7 % at Day 270) and had similar reactogenicity profiles with no vaccine-related SAEs. The three lots of TAK-003 were immunogenic for all four dengue serotypes and well tolerated in healthy adults. Despite not meeting all equivalence comparisons, no major differences were observed between lots and the data support acceptable consistency of the manufacturing process. Trial registration ClinicalTrials.gov identifier: NCT03423173. [ABSTRACT FROM AUTHOR]

Dengue is caused by the dengue virus (DENVs) infection and clinical manifestations include dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). Due to a lack of antiviral drugs and effective vaccines, several therapeutic and control strategies have been proposed. A systemic literature review was conducted according to PRISMA guidelines to select proper references to give an overview of DENV infection. Results indicate that understanding the virus characteristics and epidemiology are essential to gain the basic and clinical knowledge as well as dengue disseminated pattern and status. Different factors and mechanisms are thought to be involved in the presentation of DHF and DSS, including antibody-dependent enhancement, immune dysregulation, viral virulence, host genetic susceptibility, and preexisting dengue antibodies. This study suggests that dissecting pathogenesis and risk factors as well as developing different types of therapeutic and control strategies against DENV infection are urgently needed. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral disease. Stroke is a severe manifestation of dengue. However, few large-scale studies have investigated post-dengue risk of stroke. This population-based cohort study included 57,934 newly diagnosed, laboratory-confirmed dengue patients in Taiwan from 2002 to 2015; patients were matched to nondengue individuals by age, sex, and area of residence at a ratio of 1:4 (n = 231,736). We used subdistribution hazard regression to evaluate short-term (≤ 30 days), medium-term (31–365 days), and long-term (1–3 years) risk of stroke after DENV infection. The robustness of the results to unmeasured confounding was assessed with E-values. DENV infection was associated with a significantly increased risk of overall stroke (aSHR 4.51; 95% CI: 3.23–6.32; P < 0.0001; E-value = 8.49), hemorrhagic stroke (aSHR 4.13; 95% CI: 2.20–7.76; P < 0.0001; E-value =7.73), and ischemic stroke (aSHR 3.80; 95% CI: 2.37–6.11; P < 0.0001; E-value = 7.06) within 30 days. Stratified analysis by age showed that the aSHRs for overall stroke, hemorrhagic stroke, and ischemic stroke were larger among dengue patients aged ≥ 65 during the first 30 days. The 30-day risks of overall stroke, hemorrhagic stroke, and ischemic stroke among elderly dengue patients were 6.71, 1.29, and 3.49 per 1000, respectively. No increased risk was observed after 30 days. DENV infection was associated with a significant short-term increased risk of stroke. Clinical practitioners should remain alert to patients with stroke-associated symptoms during epidemic seasons, especially elderly patients. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) has caused about 12 large outbreaks in Pakistan, resulting in 286,262 morbidities and 1108 deaths. The most affected province is Khyber Pakhtunkhwa (KP). This study was conducted to determine the average DENV prevalence in different areas of the Haripur endemic district of KP and the causing factors of DENV. This work was a cross-sectional study that was performed in the DENV endemic district Haripur. A total of 761 individuals were included in this study. The data were categorized according to sex, age and symptoms (like fever, body aches, bleeding, and skin rash). For data analysis, SPSS 23 version was applied. ArcGIS version 10.8 was used to map the study area. In this study, there were 716 confirmed cases of DENV fever, including 421 males (58.8%) and 295 females (41.2%). The most affected age range, 16–30 years, reported by 301 (42.0%), was followed by 31–45 years, 184 (25.7%), above 46 years, 132 (18.4%), and 0–15 years, 99 (13.8%). The positive IgG cases were 581(81.0%). Those whose age ranges from 1 to 15 years 82 (8.7%) cases, 16–30 years 244 (34.1%), 31–45 years 156 (21.8%), above 46-year age 99 (13.8%) cases. In addition, this suggests that those between the ages of 16 and 30 are at the highest risk for DENV infection. However, this might be the fact that individuals in this age range are more likely to be out in the environment, making them more vulnerable to the virus. Over the past ten years, DENV fever has become increasingly prevalent in Pakistan. The risk is substantially higher for males. Dengue outbreaks hit those between the ages of 16 and 30 the hardest. The proper monitoring and assessment of DENV are necessary for prevention and controlling the disease. Disease surveillance includes identification and molecular characterization of infected persons and monitoring mosquito populations in high-risk locations for the purpose of vector surveillance. In order to assess the community's willingness to participate in DENV preventive efforts, behavioral impact surveillance is also necessary. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) exploits various cellular pathways including autophagy to assure enhanced virus propagation. The mechanisms of DENV mediated control of autophagy pathway are largely unknown. Our investigations have revealed a novel role for high-mobility group box1 protein (HMGB1) in regulation of cellular autophagy process in DENV-2 infected A549 cell line. While induction of autophagy by rapamycin treatment resulted in enhanced DENV-2 propagation, the blockade of autophagy flux with bafilomycin A1 suppressed viral replication. Furthermore, siRNA-mediated silencing of HMGB1 significantly abrogated dengue induced autophagy, while LPS induced HMGB1 expression counteracted these effects. Interestingly, silencing of HMGB1 showed reduction of BECN1 and stabilization of BCL-2 protein. On the contrary, LPS induction of HMGB1 resulted in enhanced BECN1 and reduction in BCL-2 levels. This study shows that the modulation of autophagy by DENV-2 is HMGB1/BECN1 dependent. In addition, glycyrrhizic acid (GA), a potent HMGB1 inhibitor suppressed autophagy as well as DENV-2 replication. Altogether, our data suggests that HMGB1 induces BECN1 dependent autophagy to promote DENV-2 replication. • DENV infection in A549 cell line induces autophagy for successful viral replication. • DENV induced translocation of HMGB1 is the critical host factor for autophagy induction. • Glycyrrhizic acid can inhibit HMGB1 mediated autophagy during DENV infection. • HMGB1 induced autophagy during DENV infection is mediated by BECN1/BCL2 pathway. [ABSTRACT FROM AUTHOR]

Lethal Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) caused by Dengue virus (DENV) infection necessitate the development of effective treatments. Peptides derived from the N-terminal amphipathic α-helix of hepatitis C virus (HCV) NS5A exhibit antiviral activity by disrupting liposomes with high curvatures, such as virus envelopes. This study engineered five peptides from HCV genotype 3a NS5A N-terminal α-helix and screened them for neutralizing efficacy against three DENV serotypes. Two peptides, 3a 3/20 and DS-05, showed superior therapeutic efficacy against DENV and were further evaluated in treating DHF/DSS induced by mouse-adapted DENV infection. Administration of 3a 3/20 and DS-05 post-infection significantly improved mortality and weight loss associated with DHF/DSS in AG6 mice. These peptides reduced viral load in internal organs and viremia to levels comparable with the positive control drug, JNJ-A07, a DENV NS3-NS4B inhibitor. Additionally, they attenuated the cytokine storm in the blood and expression of inflammatory cytokines in internal organ tissues, ameliorating liver and kidney dysfunction after DENV infection. Histopathological analysis revealed significant suppression of damages in internal organs. These findings suggest that the 3a 3/20 and DS-05 peptides improve clinical symptoms of DHF/DSS induced by DENV infection, indicating their potential for clinical application. • N-terminal α-helix peptides from HCV NS5A disrupt high-curvature liposome, exhibiting antiviral activity. • Peptides 3a 3/20 and DS-05 display superior antiviral activity against three dengue virus serotypes. • Peptides 3a 3/20 and DS-05 exhibit therapeutic potential in treating DHF/DSS caused by DenV infection. • Peptides 3a 3/20 and DS-05 are proposed as clinically promising candidates for DHF/DSS. [ABSTRACT FROM AUTHOR]

• Pooled DENV, ZIKV, CHIKV prevalence in Asian mosquitoes were 5.85%, 2.15%, 1.26%. • Southeast Asia had the highest prevalence of DENV in mosquitoes at 6.92%. • Indoor mosquito capture was less likely to detect DENV than outdoor or both sites. • East Asia showed higher ZIKV detection in mosquitoes than South and Southeast Asia. Dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) continue to pose significant public health risks. This study aims to assess the prevalence of these arbovirus infections in field-caught mosquitoes across Asia. Studies published after the year 2000 on DENV, ZIKV, and/or CHIKV infections in Asian mosquitoes were identified from Embase, Scopus, PubMed, and Ovid. A random-effects model estimated the pooled prevalence, defined as the overall prevalence from included studies, adjusted for variability among the studies. Meta-regression models were used to evaluate the association between predictors and their prevalence. A total of 2529 articles were retrieved; 57 met the inclusion criteria. Pooled prevalence of DENV, ZIKV, and CHIKV infections in Asian mosquitoes were 5.85%, 2.15%, and 1.26%, respectively. Subgroup analysis revealed varying DENV prevalence across regions: East Asia (3.32%), South Asia (5.26%), and Southeast Asia (6.92%). Univariate regression analysis demonstrated significant associations between mosquito capture site and DENV prevalence (P < 0.001), and between study region and ZIKV prevalence (P = 0.005). However, no significant predictors were identified for CHIKV prevalence. Our findings provide reference pooled summary estimates of arbovirus infections in mosquitoes, offering crucial insight into the regional disease burden and - guidance in the development and implementation of arbovirus surveillance in mosquitoes. [ABSTRACT FROM AUTHOR]

Dengue, caused by the dengue virus (Orthoflavivirus dengue , encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. This study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. To assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. The DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. The results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. This study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection. [Display omitted] [ABSTRACT FROM AUTHOR]

The first dengue "endgame" summit was held in Syracuse, NY over August 9 and 10, 2023. Organized and hosted by the Institute for Global Health and Translational Sciences at SUNY Upstate Medical University, the gathering brought together researchers, clinicians, drug and vaccine developers, government officials, and other key stakeholders in the dengue field for a highly collaborative and discussion-oriented event. The objective of the gathering was to discuss the current state of dengue around the world, what dengue "control" might look like, and what a potential roadmap might look like to achieve functional dengue control. Over the course of 7 sessions, speakers with a diverse array of expertise highlighted both current and historic challenges associated with dengue control, the state of dengue countermeasure development and deployment, as well as fundamental virologic, immunologic, and medical barriers to achieving dengue control. While sustained eradication of dengue was considered challenging, attendees were optimistic that significant reduction in the burden of dengue can be achieved by integration of vector control with effective application of therapeutics and vaccines. [ABSTRACT FROM AUTHOR]

This manuscript introduces highly sensitive surface plasmon resonance (SPR) sensor for early detection of the dengue virus. Through extensive optimization using MATLAB, the sensor architecture is meticulously constructed with a BK7 prism, Copper (Cu) layer, Gallium selenide (GaSe), Tungsten disulphide (WS 2), and a sensing medium (SM) containing blood components (infected platelets and normal platelets) to ensure optimal performance. Use of WS 2 emerges as a highly effective biomolecular recognition element (BRE) layer, leading to exceptional sensor capabilities. The proposed sensor achieves a peak sensitivity (S) of 303.28 (˚/RIU) and a resonance angle shift (∆θ res.) of 10˚ specifically during the detection of infected platelets. Computational modeling using COMSOL Multiphysics validates the ability of the sensor to generate an intense electric field with a magnitude of 1.68×105 (V/m) and a penetration depth (PD) extending to 153.24 nm in the SM. This unique combination of PD and enhanced performance parameters positions the proposed SPR biosensor as a promising tool for the early-stage detection of the dengue virus. [ABSTRACT FROM AUTHOR]

The antiviral defense directed by the RNAi pathway employs distinct specificity and effector mechanisms compared with other immune responses. The specificity of antiviral RNAi is programmed by siRNAs processed from virus-derived double-stranded RNA by Dicer endonuclease. Argonaute-containing RNA-induced silencing complex loaded with the viral siRNAs acts as the effector to mediate specific virus clearance by RNAi. Recent studies have provided evidence for the production and antiviral function of virus-derived siRNAs in both undifferentiated and differentiated mammalian cells infected with a range of RNA viruses when the cognate virus-encoded suppressor of RNAi (VSR) is rendered nonfunctional. In this review, we discuss the function, mechanism, and evolutionary origin of the validated mammalian VSRs and cell culture assays for their identification. RNA viruses from the Flaviviridae , Nodaviridae , Orthomyxoviridae , and Picornaviridae encode functionally validated viral suppressors of RNAi (VSRs) to promote mammalian cell infection in the presence or absence of the interferon signaling. Therapeutic targeting of closely related human enteroviral VSRs confers protection in mice against lethal challenges, revealing VSRs as a novel class of antiviral drug targets. Validated VSRs from different virus families show no sequence similarity, but all suppress Dicer-dependent production of virus-derived siRNAs by sequestering long dsRNA likely as a homodimer with a similar fold of two antiparallel α-helices, suggesting convergent evolution. Additional families of human RNA viruses, including Ebola virus and SARS-CoV-1 and 2, encode unrelated VSRs awaiting further functional validation. [ABSTRACT FROM AUTHOR]

Dengue virus is transmitted by Aedes mosquitoes and dengue is endemic in many regions of the world. Severe dengue results in complications that may lead to death. Although some vaccine candidates are in clinical trials and one vaccine Dengvaxia, with restricted efficacy, is available, there are currently no specific therapies to completely prevent or treat dengue. The dengue virus structural protein E (envelope) exists as a head-to-tail dimer on mature virus, is targeted by broadly neutralizing antibodies and is suitable for developing vaccine immunogens. Here, we have used a redesigned dengue prME expression construct and immunoaffinity chromatography with conformational/quaternary antibody A11 to purify soluble DENV4 sE(A259C) (E ectodomain) dimers from mammalian expression system to ~99 % purity. These dimers retain glycosylation reported for native DENV E, display the three major broadly neutralizing antibody epitopes, and form well-ordered structure. This strategy can be used for developing subunit vaccine candidates against dengue and other flaviviruses. • The dengue virus E (envelope) protein is the major target of neutralizing antibodies. • The DENV E protein exists as a head-to-tail dimer on virus. • We have purified to ~99 % homogeneity DENV4 sE(A259C) dimers. • These dimers are cleaved, well-ordered and native-like. • These dimers are suitable for developing subunit vaccine immunogens. [ABSTRACT FROM AUTHOR]

• Thirty-six new 5-arylmethylene-1,3-thiazolidin-4-one derivatives were synthesized. • The structures of target compounds were confirmed by spectral techniques. • Antiviral activities of the synthesized compounds were screened against HCV and DENV. • Active compounds suppressed the HCV NS5B protein by downregulating the COX-2. • In silico and in vitro study of selected compounds against COX-2 enzyme were performed. Hepatitis C (HCV) is a viral infection that leads to forms of acute and chronic liver disease, including cirrhosis (scarring of the liver) and liver cancer. The World Health Organization (WHO) estimated in 2019 that approximately 290,000 people died from hepatitis C (mostly from cirrhosis and hepatocellular carcinoma). Direct-acting antiviral drugs (DAAs) can cure more than 95% of individuals with hepatitis C infection, while research on the discovery of new antiviral agents is still ongoing. The Hepatitis C virus (HCV) can cause various biochemical changes in liver cells, and some of these changes are associated with the COX-2 enzyme. The identification of its role in promoting growth in liver cells as well as its involvement in various cancer types, including hepatocellular carcinoma, has made COX-2 an important target in the development of new agents effective against HCV. In this study, thirty-six new 5-arylmethylene-2-imino-1,3-thiazolidin-4-one derivatives (5a - s, 6a - s) were synthesized through Knoevenagel condensation of 2-[(4-substitutedpyridin-2-yl)imino]-1,3-thiazolidin-4-one derivatives with various aldehydes. Structures of the synthesized compounds were elucidated by the use of spectral and chromatographic techniques, besides elemental analyses. Four compounds were selected for further studies as they were found to suppress the NS5B protein with anti-HCV activity using the Western Blotting method. The selected compounds 5o, 6m, 6r , and 6s inhibited HCV with EC 50 values of 8.0 ± 0.2 µM, 13.9 ± 0.45, 9.2 ± 0.2 µM, and 12.1 ± 0.1 µM, respectively. It was determined that these compounds reduced HCV-induced COX-2 promoter activity in Ava5 cells compared to Huh7 cells. The antiviral effects of the compounds were also investigated on DENV, closely related to HCV due to sharing certain biological, structural, and mechanical properties throughout their life cycles. However, no significant effect was observed in the preliminary screening study, indicating the compounds' specificity for HCV. Considering the relationship between HCV, DENV, and COX-2, the compounds' COX-1 and COX-2 enzyme inhibition potentials were investigated both in vitro and in silico. Compounds 6d, 6e, 6f , and 6m , which exhibited high selective COX-2 inhibition, were discussed for their interactions with the active site. Our study revealed that our target compounds suppressed COX-2 both at the protein level and through enzyme inhibition, thus providing promising findings for the discovery of new anti-HCV effective COX-2 inhibitors. [Display omitted] [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection poses a global health threat, leading to severe conditions with the potential for critical outcomes. Currently, there are no specific drugs available whereas the vaccine does not offer comprehensive protection across all DENV serotypes. Therefore, the development of potential antiviral agents is necessary to reduce the severity risk and interrupt the transmission circuit. The search for effective antiviral agents against DENV has predominantly focused on natural resources, particularly those demonstrating diverse biological activities and high safety profiles. Cyanobacteria and algae including Leptolyngbya sp., Spirulina sp., Chlorella sp., and Sargassum spp., which are prevalent species in Thailand, have been reported for their diverse biological activities and high safety profiles. However, their anti-DENV activity has not been documented. In this study, the screening assay was performed to compare the antiviral activity against DENV of crude polysaccharide and ethanolic extracts derived from 4 species of cyanobacteria and algae in Vero cells. Polysaccharide extracts from Sargassum spp. were the most effective in inhibiting DENV-2 infection under co-infection conditions, where the virus was exposed to the extract at the time of infection. Treatment of the extract significantly reduced the ability of DENV to bind to the host cells to 47.87 ± 3.88 % while treatment upon virus binding step had no antiviral effect suggesting the underlaying mechanism of the extract on interfering virus binding step. Fucoidan, a key bioactive substance in Sargassum polysaccharide, showed to reduce DENV-2 infection to 26.59 ± 5.01 %, 20.46 ± 6.58 % under the co-infection condition in Vero and A549 cells, respectively. In accompanied with Sargassum polysaccharide, fucoidan disturbed the virus binding to the host cells. These findings warrant further development and exploration of the Sargassum -derived polysaccharide, fucoidan, as a promising candidate for combating DENV infections. [Display omitted] [ABSTRACT FROM AUTHOR]

• Land use affects mosquito species richness in tropical deciduous forest. • Zika and dengue virus were detected in 85 % of captured mosquito species. • The first reports of Zika and dengue virus for multiple Aedes and Culex species. • Entomovirological surveillance needs to advocate for a deeper understanding of vector ecology. Fragmented landscapes in Mexico, characterized by a mix of agricultural, urban, and native vegetation cover, presents unique ecological characteristics that shape the mosquito community composition and mosquito-borne diseases. The extent to which landscape influences mosquito populations and mosquito-borne diseases is still poorly understood. This work assessed the effect of landscape metrics -agriculture, urban, and native vegetation cover- on mosquito diversity and arbovirus presence in fragmented tropical deciduous forests in Central Mexico during 2021. Among the 21 mosquito species across six genera we identified, Culex quinquefasciatus was the most prevalent species, followed by Aedes aegypti, Ae. albopictus, and Ae. epactius. Notably, areas with denser native vegetation cover displayed higher mosquito species richness, which could have an impact on phenomena such as the dilution effect. Zika and dengue virus were detected in 85% of captured species, with first reports of DENV in several Aedes species and ZIKV in multiple Aedes and Culex species. These findings underscore the necessity of expanding arbovirus surveillance beyond Ae. aegypti and advocate for a deeper understanding of vector ecology in fragmented landscapes to adequately address public health strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5′ and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus. [Display omitted] • A Field-deployable viral diagnostic tool was developed for rapid and sensitive detection of dengue virus. • We developed a Paper-based strip strategy for visual diagnosis of dengue virus. • Owing to the cascade enzymatic amplification of Cas13a and Cas12a, the detection limit of the developed method was down to 190 fM. [ABSTRACT FROM AUTHOR]

• The NKp30 receptor's expression on activated NK cells is more pronounced in DF patients than in DHF patients, suggesting a potential role of this receptor in the milder disease manifestation observed in pediatric dengue patients. • Natural killer cells are activated upon engagement with dengue infected dendritic cells and the outcome of cellular activation depends on the type of receptor expressing on NK cells and the intracellular signaling pathway. • The NK cells response to the dengue virus operates through distinct mechanisms. Degranulation is initiated by direct interaction of NK cells and dengue infected cells and signaling from type I IFNs. The production of IFN-γ is first initiated by direct cell-cell contact and type i IFNs followed by several cytokines including type I IFNs, IL-12, IL-15, and IL-18. Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease. [ABSTRACT FROM AUTHOR]

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1–4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1–4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks. • A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus. • Mapping rate, base coverage, viral genome coverage, and genome coverage uniformity are tested. • The assay shows its ability to recover the full-length dengue genome across various viral abundances in clinical specimens. • It is suitable for precise examination of intra-host diversity. [ABSTRACT FROM AUTHOR]

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B−NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure−activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B−NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo. Erythrosin B (EB), an FDA-approved food dye, can protect 3D mini-brain organoid from infection of Zika virus. This figure shows the immunofluorescence imaging of slices of organoid infected with mock solution or ZIKV PRVABC59 (green) treated with EB or DMSO control. Blue, DAPI. [Display omitted] [ABSTRACT FROM AUTHOR]

An emergent positive-stranded RNA virus, transmitted by mosquitoes with its first case of vertical transmission confirmed in 2015 in Brazil. The Zika virus (ZIKV) fever has received particular attention, mainly related to neurological diseases such as microcephaly in newborns. However, the laboratory diagnosis for ZIKV still faces some challenges due to its cross-reactivity with other flaviviruses, requiring a correct and differential diagnosis, contributing to the good prognosis of patients, especially in pregnant women. Among these, for early diagnosis, the CDC considers the RT-PCR the gold standard, more sensitive and specific, but expensive. Serological tests for the diagnosis of ZIKV can also be found beyond the period when the viral components are detectable in the serum. Inputs to produce more sensitive and specific diagnostic kits and the possibility of viral detection in less invasive samples are among the objectives of recent research on ZIKV. This review outlines recent advances in developing recombinant antigen and antibody-based diagnostic tools for the main flaviviruses in Northeast Brazil, such as ZIKV and Dengue virus (DENV). [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is a positive-sense single-stranded RNA virus and that the detection of viral RNA itself is highly desirable, which can be achieved by using RNA biosensor diagnostic method. Herein, acrylic micropolymer-based optical RNA biosensor was developed by binding anionic copper(II) phthalocyanine (CPC) planar aromatic ligand to the G-quadruplex DNA probe via end-stacking with π-system of the guanine (G) quartet, and a blue coloration was developed on the G-quadruplex microspheres. Hybridization of G-quadruplex DNA probe with target DENV serotype 2 (DENV2) RNA unfolded the G-quadruplex, and rendering release of the CPC planar optical label, causing discoloration of the G-quadruplex microbiosensor. Optical characterization of the RNA biosensor was performed by means of fiber optic reflectance spectrophotometer at maximum reflectance wavelength of 774 nm. The reflectance response enhancement of the RNA-responsive G-quadruplex-based reflectometric biosensor was linearly proportional to the target oligo DENV2 RNA concentration in the range of 2 zM–2 μM, with a 0.447 zM limit of detection and a rapid response time of 30 min. Heightening in the reflectance signal based on structural transition of G-quadruplex in response to target RNA was successfully implemented in real-time DENV2 detection in non-invasive human fluid samples (i.e. saliva and urine) under informed consent. • RNA-responsive G-quadruplex microspheres-based reflectometric biosensor. • Structural transition of G-quadruplex in response to RNA releasing CPC optical label. • Hybridization of G-quadruplex with RNA rendered discoloration of the microbiosensor. • Heightening of reflectance observed in real-time DENV assay in non-invasive samples. [ABSTRACT FROM AUTHOR]

Urbanization impacts the community composition, abundance, and richness of mosquitoes. As urbanization processes increase globally, it is important to better understand the biodiversity loss caused by anthropogenic changes and associated impacts on vector-borne diseases. Mosquito surveillance and control are key for reducing the risk of mosquito-borne pathogen transmission. [ABSTRACT FROM AUTHOR]

• The LDL receptor and serum LDL-C levels play a critical role in various pathogens. • Decreasing LDLR expression may improve outcomes with VSV, T. Cruzi, T. gondii. • Increasing LDLR expression may improve outcomes with HCV, PL's, and DENV. • Lower serum LDL-C may improve outcomes with T. Cruzi , and T. gondii. • Greater serum LDL-C may improve outcomes with HCV, and DENV. Although the role of the LDL receptor concerning lipids is well known, its role in various viral and parasitic infections, and in regulating the inflammatory response is poorly understood. Several infectious agents use the LDL receptor as a port of entry, and others depend on it for their cycle of infection. In this review, we focus on the discovery, structure, and normal function of the LDL receptor, as well as its role in a selection of infections. The LDL receptor plays an important role in certain infections and is a potential target for treatment deserving further research. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel variable-order nonlinear model of dengue virus is analyzed as an optimal control problem. • The bang-bang control is suggested to minimize the dose and duration of the intervention for model. • Necessary conditions for the control problem are derived. • Two numerical methods are constructed to solve the proposed optimal control problem. • Comparative studies and numerical simulations are implemented. Dengue and Malaria are the most important mosquito-borne viral diseases affecting humans. Fever is transmitted between human hosts by infected female aedes mosquitoes. The modeling study of viral infections is very useful to show how the virus replicates in an infected individual and how the human antibody response acts to control that replication, which antibody playing a key role in controlling infection. Optimal control of a novel variable-order nonlinear model of dengue virus is studied in the present work. Bang-bang control is suggested to minimize the viral infection as well as quick clearance of the virus from the host. Necessary conditions for the control problem are given. The variable-order derivatives are given in the sense of Caputo. Moreover, the parameters of the proposed model are dependent on the same variable-order fractional power. Two numerical schemes are constructed for solving the optimality systems. Comparative studies and numerical simulations are implemented. The variable-order fractional derivative can be describe the effects of long variable memory of time dependent systems than the integer order and fractional order derivatives. Both the nonstandard generalized fourth order Runge-Kutta and the nonstandard generalized Euler methods are presented. We have successfully applied a kind of Pontryagin's maximum principle with bang-bang control and were able to reduce the viraemia level by adding the dose of DI particles. The nonstandard generalized fourth order Runge-Kutta method has the best results than nonstandard generalized Euler method. The combination of the variable-order fractional derivative and bang-bang control in the Dengue mathematical model improves the dynamics of the model. The nonstandard generalized Euler method and the nonstandard generalized fourth order Runge-Kutta method can be used to study the variable order fractional optimal control problem simply. [ABSTRACT FROM AUTHOR]

Dengue is one of the highest and rapidly spreading vector-borne viral diseases with high mortality rates. The infection causes acute febrile illness, a major public health concern in the tropics and subtropics globally. The disease is caused by an RNA virus that belongs to the Flaviviridae family. The virus is transferred to humans by the mosquito vector called Aedvrves aegypti, which is the cause of new prevalent sicknesses worldwide. These vector-borne viral diseases spread very fast and pose public health and economic challenges that deemed various prevention and control techniques. The Flavivirus genus consists of five different types of viruses starting from DENV-1 to DENV-5. Thus, the present review focuses on the origin of the virus, how the Dengue virus can be detected, infection, the morphology of the virus, its classifications as proposed by ICTV, the replication and genome of the dengue virus, translation, receptor binding, and some vaccine trial volunteers. In addition, it highlights the current challenges and limitations of effective dengue treatment. [ABSTRACT FROM AUTHOR]

• Dengue virus induces RIG-I (at 24hr post infection) and MDA-5 (at 48hr and 72hr post infection) in MEG-01 cells. • MDA-5 induces type-I IFN-β and ISG-15 in MEG-01 cells during dengue virus infection. • Dengue virus reduce CD61 expression, an activation marker of megakaryocytes which may impaired megakaryocyte development. Activation of innate receptors in megakaryocytes (MKs) may affect the ability to produce functional platelets. Low platelet count is one of the clinical manifestations of dengue virus (DENV) infection. In MKs, the effect of innate receptors during DENV-infection is not well studied. Here we used MEG-01 cells to investigate DENV serotype 2 induced innate receptors in these cells. DENV RNA was estimated by qRT-PCR in the culture supernatant. The expression of innate receptors was determined by western blot and qPCR. DENV infection led to increased expression of RIG-I at 24 hrs post-infection (hpi) and MDA-5 at 48 and 72 hpi (p<0.05). However, no change in the expression of TLR3 at protein level was observed. Activation of MDA-5 resulted in increased expression of IFN-β and ISG-15 in DENV infected MEG-01 cells, which was further confirmed by MDA-5 siRNA treatment. Apart from inducing innate receptors, DENV significantly decreases the expression of CD61, an activation marker of megakaryocyteson MEG-01 cells as observed by flow cytometry analysis (p<0.01). Results from this study confirm that DENV infection activates the type-I interferon in megakaryocytes and may play a significant role in maturation and development. [ABSTRACT FROM AUTHOR]

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (−57.3 ± 2.8 and − 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection. [ABSTRACT FROM AUTHOR]

Brucea javanica , a valued traditional medicinal plant in Malaysia, known for its fever-treating properties yet remains underexplored for its potential antiviral properties against dengue. This study aims to simultaneously identify chemical classes and metabolites within B. javanica using molecular networking (MN), by Global Natural Product Social (GNPS), and SIRIUS in silico annotation. Liquid chromatography-mass spectrometry (LC-MS2)-based MN explores chemical diversity across four plant parts (leaves, roots, fruits, and stem bark), revealing diverse metabolites such as tryptophan-derived alkaloids, terpenoids, and octadecadenoids. Simultaneous LC-MS2 and MN analyses reveal a discriminative capacity for individual plant components, with roots accumulating tryptophan alkaloids, fruits concentrating quassinoids, leaves containing fusidanes, and stem bark primarily characterised by simple indoles. Subsequently, extracts were evaluated for dengue antiviral activity using adenosine triphosphate (ATP) and plaque assays, indicates potent efficacy in the dichloromethane (DCM) extract from roots (EC 50 = 0.3 μg/mL, SI = 10). Molecular docking analysis of two major compounds; canthin-6-one (264) and 1-hydroxy-11-methoxycanthin-6-one (275) showed potential binding interactions with active sites of NS5 RNA-dependent RNA polymerase (RdRp) of dengue virus (DENV) protein. Subsequent in vitro evaluation revealed compounds 264 and 275 had a promising dengue antiviral activity with SI value of 63 and 1.85. These identified metabolites emerge as potential candidates for further evaluation in dengue antiviral activities. [Display omitted] • Brucea javanica highlights prominent alkaloids in root, quassinoids in fruit, indoles in stem bark, and fusidanes in leaves. • Molecular docking analysis annotated canthin-6-one derivatives as candidates for further evaluation in dengue assays. • Canthin-6-one showed potent EC 50 of 0.86 μM (SI = 63) and 1-hydroxy-11-methoxycanthin-6-one an EC 50 of 6.1 μM (SI = 1.85) [ABSTRACT FROM AUTHOR]

Dengue and chikungunya, caused by dengue virus (DENV) and chikungunya virus (CHIKV) respectively, are the most common arthropod-borne viral diseases worldwide, for which there are no FDA-approved antivirals or effective vaccines. Arctigenin, a phenylpropanoid lignan from the seeds of Arctium lappa L. is known for its anti-inflammatory, anti-cancer, antibacterial, and immunomodulatory properties. Arctigenin's antimicrobial and immunomodulatory capabilities make it a promising candidate for investigating its potential as an anti-DENV and anti-CHIKV agent. The aim of the study was to explore the anti-DENV and anti-CHIKV effects of arctigenin and identify the possible mechanisms of action. The anti-DENV or anti-CHIKV effects of arctigenin was assessed using various in vitro and in silico approaches. Vero CCL-81 cells were infected with DENV or CHIKV and treated with arctigenin at different concentrations, temperature, and time points to ascertain the effect of the compound on virus entry or replication. In silico molecular docking was performed to identify the interactions of the compound with viral proteins. Arctigenin had no effects on DENV. Various time- and temperature-dependent assays revealed that arctigenin significantly reduced CHIKV RNA copy number and infectious virus particles and affected viral entry. Entry bypass assay revealed that arctigenin inhibited the initial steps of viral replication. In silico docking results revealed the high binding affinity of the compound with the E1 protein and the nsp3 macrodomain of CHIKV. This study demonstrates the in-vitro anti-CHIKV potential of arctigenin and suggests that the compound might affect CHIKV entry and replication. Further preclinical and clinical studies are needed to identify its safety and efficacy as an anti-CHIKV drug. [Display omitted] [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection is mostly prevalent in tropical and sub-tropical regions of the world. Though most DENV infections are self-limiting febrile like-illness, a small proportion of secondary infection is fatal, if untreated symptomatically. Among various factors involved in severe dengue, immune enhancement by cytokine is the major one. The objective of the study is to elucidate serum cytokine expression among primary and secondary infection and determine if any signature cytokine is correlated with disease severity. Seventy-six serum samples at acute time points were collected during the 2017 DENV outbreak in Madurai, Tamil Nadu. Among the 76 serum samples, 49 belong to primary and 27 to secondary DENV infection. Interestingly, a large number of primary infection presented with DHF/DSS symptoms and, children were found prone to DHF and DSS in secondary infection. The serum samples were analysed for inflammatory cytokines, namely IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFN-γ, TNF-α, IP-10 and GM-CSF using ELISA assay as well as mRNA analysis using qPCR. Among the 12 inflammatory cytokines analysed IP-10 and GMCSF mRNA and protein shows significant upregulation in secondary infection. Similarly, a strong correlation was observed between GM-CSF and IP-10 with thrombocytopenia, ascites, serous effusion and spontaneous bleeding. Based on the observations, GM-CSF and IP-10 could be a potential prognostic biomarkers for secondary DENV infection. [ABSTRACT FROM AUTHOR]

Dengue virus causes the dengue fever as well as hemorrhagic fever in tropical and sub-tropical countries. It is now endemic in most parts of the South East Asia. Full-genome information of dengue virus 3 is not available from Yemen. In this study, the dengue virus 3 was detected by diagnostic tools like serology and RT-PCR in the samples isolated from a patient in Yemen. The full-genome was sequenced, and the identity, phylogenetic relationship and recombination analysis was performed by using BioEdit, MEGA X and RDP4 softwares. The full-genome of the Yemen isolate was found to be 10,643 nt long with 3390 amino acids. The Yemen dengue virus 3 isolate showed the sequence similarity (98.5–92.4%) with dengue virus 3 isolates from China, Pakistan, India and Bangladesh respectively. The significant non-synonymous substitutions of amino acid in Yemen isolate were observed with selected isolates. The phylogenetic tree of Yemen isolate formed a unique clade within genotype III and sub-clade into lineage III. The Dengue virus isolate from Jeddah formed separated cluster with lineage IV. This reveals the unique genetic variability among DENV-3 serotypes from Jeddah and earlier reported isolates from other regions. [ABSTRACT FROM AUTHOR]

Virion assembly is an important step in the life cycle of all viruses. For viruses of the Flavivirus genus, a group of enveloped positive-sense RNA viruses, the assembly step represents one of the least understood processes in the viral life cycle. While assembly is primarily driven by the viral structural proteins, recent studies suggest that several nonstructural proteins also play key roles in coordinating the assembly and packaging of the viral genome. This review focuses on describing recent advances in our understanding of flavivirus virion assembly, including the intermolecular interactions between the viral structural (capsid) and nonstructural proteins (NS2A and NS2B-NS3), host factors, as well as features of the viral genomic RNA required for efficient flavivirus virion assembly. Flaviviruses, including Zika, dengue, and yellow fever viruses, annually impact millions of people. For infection to spread, virions must be packaged and assembled at the host endoplasmic reticulum (ER) membrane. Recent research provides insight into how flavivirus virions are assembled and how both viral structural and nonstructural (NS) proteins participate in this process. The α5 helix of the capsid protein, previously thought to be removed, may be partially retained on some capsid molecules and aids in capsid oligomerization for assembly. The viral NS2A protein appears to be a central hub in the packaging process, while the NS3 protein contributes to assembly through protease-mediated as well as nonenzymatic functions. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is the most common mosquito-borne flavivirus, and it affects millions of people globally every year. Currently, there are no approved drugs for the treatment of dengue infection. By screening a natural product library, we identified a novel compound, cyclovirobuxine D (Cvb D), that displays anti-DENV activity. Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection. Mechanistically, Cvb D regulates the expression of genes related to the cellular cholesterol pathway. As a result, Cvb D increases cellular cholesterol synthesis and accumulation, activates mTOR, and inhibits viral-dependent autophagy. Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB. In addition, Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3. We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication. [ABSTRACT FROM AUTHOR]

• Pathogenic and protective effects of complement in flavivirus infections. • Protective role of classical pathway activation on ADE of flavivirus infections. • Binding of human IgG isotypes to C1q depends on Fc domain flexibility and conformation. • C1q modulation of ADE of flavivirus infections by different potential mechanisms. Flavivirus infections have increased dramatically in the last decades in tropical and subtropical regions of the world. Antibody-dependent enhancement of dengue virus infections has been one of the main hypotheses to explain severity of disease and one of the major challenges to safe and effective vaccine development. In the presence of cross-reactive sub-neutralizing concentrations of anti-dengue antibodies, immune complexes can amplify viral infection in mononuclear phagocytic cells, triggering a cytokine cascade and activating the complement system that leads to severe disease. The complement system comprises a family of plasma and cellular surface proteins that recognize pathogen associated molecular patterns, modified ligands and immune complexes, interacting in a regulated manner and forming an enzymatic cascade. Pathogenic as well as protective effects of complement have been reported in flavivirus infections. This review provides updated knowledge on complement activation during flavivirus infection, including antiviral effects of complement and its regulation, as well as mechanisms of complement evasion and dysregulation of complement activity during viral infection leading to pathogenesis. Particularly, insights into classical pathway activation and its protective role on antibody-dependent enhancement of flavivirus infections are highlighted. [ABSTRACT FROM AUTHOR]

Dengue fever, Chikungunya fever, and Zika virus infection have similar symptoms and overlapping areas of endemicity and so cannot be distinguished clinically. Rapid diagnosis tests are available for each infection but each test covers only single target. There are PCR-based methods available that test for all three infections simultaneously, not applicable for in-vitro diagnostic use. The aim of this study was to evaluate the diagnostic accuracy of a new point-of-care testing (LAMP POCT) based on loop-mediated isothermal amplification method that tests for all three viruses simultaneously, using serum or urine samples, and takes about 45 min. LAMP POCT was evaluated as the comparator on 67 individuals, of whom 56 had dengue, three had Chikungunya, two had Zika virus infection and six did not have any kind of these infections confirmed by RT-PCR. Among individuals with dengue, the sensitivity of LAMP POCT was 100% in samples collected in the first 3 days after illness onset, all three patients with Chikungunya were LAMP POCT positive, and both patients with Zika virus infection were LAMP POCT negative. There were no false-positive test results. LAMP POCT has potential utility as a point-of-care combination test for dengue and Chikungunya viruses, but further prospective studies are needed among individuals with Chikungunya virus and Zika virus infection, using serum and urine samples. [ABSTRACT FROM AUTHOR]

• The four serotypes of the dengue virus circulate in the department of Cordoba, Colombia. • The predominant serotype of the dengue virus was DENV-2. • Was detected DENV and ZIKV coinfections. To determine simultaneous circulation of DENV serotypes and ZIKV in Córdoba, Colombia, during 2015 and 2016. A total of 294 samples from patients with clinical diagnosis of febrile syndrome compatible with dengue were collected between June 2015 and December 2016. All samples were tested for DENV and ZIKV by RT-PCR using C6/36 cells culture supernatant. Thirty-three percent of the samples were positive (97/294); from these, 61.8% were positive for DENV and 31% were positive for Zika. The predominant serotype was DENV-2 (70.1%), followed by DENV-3 (8.9%), DENV-4 (6%), and DENV-1 (3%). DENV/ZIKV coinfection was identified in 7.2% of the cases associated with DENV-1 and DENV-3 serotypes. The confirmed cases of dengue, Zika, and DENV/ZIKV coinfections were clinically mild and self-limited. We reported the co-circulation of all four DENV serotypes, with a higher frequency of DENV-2, and ZIKV introduction in Córdoba department-Colombia in August 2015. This scenario favored the appearance of DENV/ZIKV coinfections. [ABSTRACT FROM AUTHOR]

Five years of laboratory records were reviewed to establish the performance of two dengue rapid assay (RDT) for acute dengue diagnosis in a non-endemic country: • The two rapid assays showed high specificity and sensitivity. • NS1 antigen was the most reliable marker of dengue acute infection. • IgM positivity in the absence of NS1 and IgG often results from non-specific reactivity of the RDTs. • The sole detection of IgG was never indicative of acute dengue infection. • No secondary infections were observed in the studied population. • The combined use of NS1 and IgM/IgG detecting RDTs increases diagnostic sensitivity. In Italy, dengue virus is the most frequent agent of imported viral infections. The use of rapid diagnostic tests (RDTs) may be of help as a preliminary user-friendly quick assay to facilitate dengue diagnosis, as ordinary laboratory diagnosis of dengue fever may require special efforts in terms of tools availability, interpretation of results, and skilled personnel. The performance of RDTs, however, may vary according to different epidemiological and laboratory background. We reviewed five years of laboratory records of two dengue RDT results (Colorimetric SD-Bioline Dengue-Duo-RDT and Fluorimetric SD-Biosensor-STANDARD-F-Dengue-RDT), able to detect viral NS1 antigen and specific IgM and IgG. Diagnostic parameters were calculated using as reference the results of molecular (RT-PCR) and serological (immunofluorescence, IFA) tests. Overall performance, calculated considering the final case definition, was included in the accuracy assessment of RDTs. The combined use of NS1 and IgM/IgG RDT for the detection of acute dengue cases resulted in an overall sensitivity and specificity of 87.2% and 97.9% for Colorimetric RDT, 96.2% and 96.2% for Fluorimetric RDT. NS1 was the most reliable marker of acute infection, while IgM resulted falsely positive in nine samples, including sera derived from 2 Zika and 4 non-arbovirus infected patients. The inclusion of RDT in the diagnostic algorithm is of undeniable help in the prompt management and surveillance of dengue infection in non-endemic areas. Confirmatory tests are, however, necessary to rule in or rule out dengue fever diagnosis. [ABSTRACT FROM AUTHOR]

• A novel point-of-care test for the detection of dengue virus (DENV) RNA was developed by combining reverse transcription recombinase-aided amplification (RT-RAA) with lateral-flow dipstick assay (LFD). • The developed RT-RAA-LFD assay can be performed in the field or low-resource settings to rapidly detect DENV RNA at 37 °C within 30 minutes. • The detection limit of the RT-RAA-LFD assay was 10 copies/μL and was specific, showing no cross-detection with other viruses. • The RT-RAA-LFD assay offers excellent performance for the detecting dengue virus the same as RT-qPCR in a clinical setting. Dengue caused by infection with the dengue virus (DENV) is endemic in the tropical and subtropical regions of the world and of greatest public health concern. With more large outbreaks in rural areas, the purpose of this study was to develop a point-of-care test using recombinase-aided amplification and lateral-flow dipsticks for rapidly detecting DENV in low-resource settings. The primers for the recombinase-aided amplification (RAA) assay were designed based on 3' UTR of the DENV genome and screened. The RAA temperature, time and the concentration of primers were then optimized, as well as the lateral-flow dipstick assay (LFD) time. Finally, the diagnostic performance of the reverse transcription (RT)-RAA-LFD assay was evaluated using blood samples from 247 patients who were clinically suspected to be infected with DENV. The RAA primer pair F1/R2 was the optimal combination for detecting DENV. The RT-RAA was performed in an incubator block at 37 °C for 20 minutes, and the amplicons were visible in the flow dipsticks from a naked eye within 3 minutes. The detection limit of the developed RT-RAA-LFD assay was 10 copies/μL with high specificity for DENV. Compared with commercial reverse transcription quantitative PCR assay, the kappa value of RT-RAA-LFD in the 247 clinical samples was 0.957. In this study, a rapid and visual point-of-care test based on RT-RAA and LFD assay was developed. It was found to be suitable for reliable detection of DENV in low-resource settings with limited laboratory capabilities and optimal storage conditions. [ABSTRACT FROM AUTHOR]

A conference on «ARBOVIRUSES, A GLOBAL PUBLIC HEALTH THREAT» was organized on June 20–22, 2018 at the Merieux Foundation Conference Center in Veyrier du Lac, France, to review and raise awareness to the global public health threat of epidemic arboviruses, and to advance the discussion on the control and prevention of arboviral diseases. The presentations by scientists and public health officials from Asia, the Americas, Europe and Africa strengthened the notion that arboviral diseases of both humans and domestic animals are progressively becoming dominant public health problems in the world. The repeated occurrence of recent deadly epidemics strongly reinforces the call for action against these viral diseases, and the need for developing effective vaccines, drugs, vector control tools and strong prevention programs. [ABSTRACT FROM AUTHOR]

• Two-step purification of Dengue virus using Chromatographic methods. • Psoralen-inactivation of Dengue virus. • Evaluation of psoralen-inactivated Dengue virus vaccines in mice. • Evaluation of psoralen-inactivated Dengue virus vaccines in nonhuman primates. Dengue fever, caused by dengue viruses (DENV 1–4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1–4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV. [ABSTRACT FROM AUTHOR]

• Dengue virus serotype 3 (DENV-3) was detected in febrile patients in Gabon. • Most of the DENV-3-positive patients were also positive for anti-DENV IgG. • The phylogenetic analysis inferred that DENV-3 has been stably maintained in Gabon. • Whole-genome sequencing of DENV-3 revealed a number of amino acid substitutions. Dengue outbreaks, mainly caused by dengue virus serotype 2 (DENV-2), occurred in 2007 and in 2010 in Gabon, Central Africa. However, information on DENV infections has been insufficient since 2010. The aim of this study was to investigate the current DENV infection scenario and the risk of repeated infections in Gabon. During 2015–2017, serum samples were collected from enrolled febrile participants and were tested for DENV infection using RT-qPCR. DENV-positive samples were analyzed for a history of previous DENV infection(s) using ELISA. The complete DENV genome was sequenced to analyze the phylogeny of Gabonese DENV strains. DENV-3 was exclusively detected, with a high rate of anti-DENV IgG seropositivity among DENV-3-positive participants. DENV-3 showed higher infection rates in adults and the infection was seasonal with peaks in the rainy seasons. Phylogenetic analysis revealed that Gabonese DENV-3 originated from West African strains and has been circulating continuously in Gabon since at least 2010, when the first DENV-3 case was reported. These findings indicate stable DENV-3 circulation and the risk of repeated DENV infections in Gabon, highlighting the need for continuous monitoring to control DENV infections. [ABSTRACT FROM AUTHOR]

The aim of this study was to update the epidemic situation of dengue fever (DF) and provide new insights for the consideration of disease control in Fujian province, China. Details about DF cases in Fujian reported during 2004–2017 were collected and analyzed. The envelope (E) genes of isolates of dengue virus (DENV) were sequenced for phylogenetic analysis. The number of imported DF cases had increased dramatically since 2013, and the source regions expanded from Southeast Asia to South Asia, America, Oceania, and Africa, as well as the surrounding provinces. This resulted in local outbreaks and indigenous cases of DF that occurred more frequently, with 10 of 13 local outbreaks and 85.9% (1,252/1,458) of indigenous cases reported in 2013–2017. Compared with only two coastal cities before 2013, four coastal and one inland city in 2013–2017 experienced the local DF outbreaks. The phylogenetic analysis of E genes confirmed that the import of DENV, not only from abroad but also from the surrounding provinces, played an important role in dissemination and local outbreaks of DF in Fujian. The frequent import of DF cases from not only abroad but also the surrounding provinces resulted in increased incidence, frequent local outbreaks, and expansion of distribution in Fujian in recent years. There is a need for urgent measures to improve disease control in this province. [ABSTRACT FROM AUTHOR]

A recent article by Sung et al. identified the CLEC2 platelet receptor as an important factor of lethal dengue virus infection. Formation of neutrophil extracellular traps via crosstalk with CLEC5A and TLR2 neutrophils were ascribed a causative role in DENV infection. This provides new insights for the development of candidate broad-spectrum therapies against hemorrhagic virus infections. [ABSTRACT FROM AUTHOR]

• Zika virus (ZIKV) infection was detected in an important fraction of pediatric encephalitis cases. • ZIKV encephalitis patients displayed a higher frequency of exanthema and shorter duration of hospitalization compared to patients with encephalitis caused by other etiological agents. • Cerebrospinal fluid analysis shown a remarkable lymphocytic pleocytosis in children with ZIKV encephalitis. • ZIKV encephalitis induced a selective encephalic but not systemic expression of cytokines. To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae , group B Streptococcus , Staphylococcus epidermidis , and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n = 3), enterovirus (n = 2), and dengue virus type 2 (DENV-2; n = 1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines. [ABSTRACT FROM AUTHOR]

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. • Mutation of H144, H244, H261, and H282 of envelop protein impaired Dengue viral like particle secretion • Neutralisation of secretory vesicles rescued VLP defective mutants • Furin over expression increased mutants VLP secretion but did not change furin processing • Histidine mutants-H244A, H261A and H282A lost the ability to interact with prM at acidic pH [ABSTRACT FROM AUTHOR]