Exploring the antiviral inhibitory activity of Niloticin against the NS2B/NS3 protease of Dengue virus (DENV2).

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of 'Niloticin' against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC50 value of niloticin was 0.14 µM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 µM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses.

An Update on the Entomology, Virology, Pathogenesis, and Epidemiology Status of West Nile and Dengue Viruses in Europe (2018-2023).

In recent decades, increases in temperature and tropical rainfall have facilitated the spread of mosquito species into temperate zones. Mosquitoes are vectors for many viruses, including West Nile virus (WNV) and dengue virus (DENV), and pose a serious threat to public health. This review covers most of the current knowledge on the mosquito species associated with the transmission of WNV and DENV and their geographical distribution and discusses the main vertebrate hosts involved in the cycles of WNV or DENV. It also describes virological and pathogenic aspects of WNV or DENV infection, including emerging concepts linking WNV and DENV to the reproductive system. Furthermore, it provides an epidemiological analysis of the human cases of WNV and DENV reported in Europe, from 1 January 2018 to 31 December 2023, with a particular focus on Italy. The first autochthonous cases of DENV infection, with the most likely vector being Aedes albopictus, have been observed in several European countries in recent years, with a high incidence in Italy in 2023. The lack of treatments and effective vaccines is a serious challenge. Currently, the primary strategy to prevent the spread of WNV and DENV infections in humans remains to limit the spread of mosquitoes.

Pro- and anti-inflammatory cytokines signatures at different severity of dengue infection.

Context: Dengue disease severity and progression are determined by the host immune response, with both pro- and anti-inflammatory cytokines are key mediators. Aims: To study pro- and anti-inflammatory cytokines across dengue severity and as a biomarker for predicting severe dengue infection. Settings and Design: Hospital-based cross-sectional study was conducted on 125 dengue-positive subjects across the 5-60 years age group of either gender in 2022. Methods and Materials: Haematological parameters and blood samples were drawn to measure cytokines IL6, IL-10 and TNF alpha using the ELISA technique. Statistical Analysis: One-way ANOVA and the Kruskal - Wallis test were used to compare the dependent variables across categories of the dengue spectrum. Receiver operating characteristic curve was drawn to calculate the predictability of the cytokines as a predictor of severe dengue. A P < 0.05 was considered significant. Results: 34.4% of cases had severe dengue infection with 53.2% of severe cases reported in >40 years of age. Only IL-6 levels significantly increased (P < 0.01) across the spectrum of dengue infection across age groups >20 years with a consistent and significant fall in platelet levels (P < 0.01). The accuracy of IL-6 to predict severe dengue was 74.4% and platelet count was 16.2%. Conclusions: Only IL-6 cytokine levels were significantly increased across the spectrum of dengue infection observed in age >20 years and can significantly predict the probability of severe dengue by 74% (sensitivity 81.4%). A significant decrease in platelet values is consistent with the severity but is not a good predictor for severe dengue infection.

The microbiota's role in flavivirus control: Rosenbergiella_YN46.

The transmission of flaviviruses, such as dengue virus (DENV) and Zika virus (ZIKV), poses a significant threat to global public health. Zhang et al. recently showed that Rosenbergiella sp. YN46 (Rosenbergiella_YN46), a bacterium from the mosquito gut, inhibits flavivirus transmission and thus offers a potential biocontrol strategy with broad public health implications.

The Development of New Primer Sets for the Amplification and Sequencing of the Envelope Gene of All Dengue Virus Serotypes.

Dengue virus (DENV) poses a significant threat to global health, infecting approximately 390 million people annually. This virus comprises four serotypes capable of causing severe disease. Genetic analyses are crucial for understanding the epidemiology, evolution, and spread of DENV. Although previous studies have focused on the envelope protein-coding (E) gene, only a few primers can efficiently detect and amplify the viral genes from multiple endemic countries simultaneously. In this study, we designed degenerate primer pairs for each DENV serotype to amplify and sequence the entire E gene, using globally representative sequences for each serotype. These primers were validated using DENV isolates from various Asian countries and demonstrated broad-spectrum detection capabilities and high-quality sequences. The primers provide effective tools for genetic analysis in the regions affected by dengue, aiding strain identification and epidemiological studies during outbreaks.

Serotype and genomic diversity of dengue virus during the 2023 outbreak in Pakistan reveals the circulation of genotype III of DENV-1 and cosmopolitan genotype of DENV-2.

Dengue, a mosquito-borne viral disease, poses a significant public health challenge in Pakistan, with a significant outbreak in 2023, prompting our investigation into the serotype and genomic diversity of the dengue virus (DENV). NS-1 positive blood samples from 153 patients were referred to the National Institute of Health, Pakistan, between July and October 2023. Among these, 98 (64.1%) tested positive using multiplex real-time PCR, with higher prevalence among males (65.8%) and individuals aged 31-40. Serotyping revealed DENV-1 as the predominant serotype (84.7%), followed by DENV-2 (15.3%). Whole-genome sequencing of 18 samples (DENV-1 = 17, DENV-2 = 01) showed that DENV-1 (genotype III) samples were closely related (>99%) to Pakistan outbreak samples (2022), and approx. > 98% with USA (2022), Singapore and China (2016), Bangladesh (2017), and Pakistan (2019). The DENV-2 sequence (cosmopolitan genotype; clade IVA) shared genetic similarity with Pakistan outbreak sequences (2022), approx. > 99% with China and Singapore (2018-2019) and showed divergence from Pakistan sequences (2008-2013). No coinfection with dengue serotypes or other viruses were observed. Comparisons with previous DENV-1 sequences highlighted genetic variations affecting viral replication efficiency (NS2B:K55R) and infectivity (E:M272T). These findings contribute to dengue epidemiology understanding and underscore the importance of ongoing genomic surveillance for future outbreak responses in Pakistan.

DENV-2 Outbreak Associated With Cosmopolitan Genotype Emergence in Western Brazilian Amazon.

Background: Dengue virus (DENV) causes an important disease and directly affects public health, being the arbovirus that presents the highest number of infections and deaths in the Western Brazilian Amazon. This virus is divided into 4 serotypes that have already circulated in the region. Methodology: Molecular characterization of a cohort containing 841 samples collected from febrile patients between 2021 and 2023 was analyzed using a commercial kit to detect the main arboviruses circulating in Brazil: Zika, DENV-1, DENV-2, DENV-3, DENV-4 and, Chikungunya. Subsequently, Sanger sequencing was performed for positive samples. Results: The cohort detected 162 positive samples, 12 for DENV-1 and 150 identified as DENV-2, indicating co-circulation of serotypes. The samples were subjected to sequencing and the analysis of the sequences that obtained good quality revealed that 5 samples belonged to the V genotype of DENV-1 and 46 were characterized as DENV-2 Cosmopolitan genotype-lineage 5. Conclusion: The results allowed us to identify for the first time the Cosmopolitan genotype in Rondônia, Brazilian Western Amazon, and its fast spread dispersion.

Spatiotemporal models of dengue epidemiology in the Philippines: Integrating remote sensing and interpretable machine learning.

Previous dengue epidemiological analyses have been limited in spatiotemporal extent or covariate dimensions, the latter neglecting the multifactorial nature of dengue. These constraints, caused by rigid and traditional statistical tools which collapse amidst 'Big Data', prompt interpretable machine-learning (iML) approaches. Predicting dengue incidence and mortality in the Philippines, a data-limited yet high-burden country, the mlr3 universe of R packages was used to build and optimize ML models based on remotely sensed provincial and dekadal 3 NDVI and 9 rainfall features from 2016 to 2020. Between two tasks, models differ across four random forest-based learners and two clustering strategies. Among 16 candidates, rfsrc-year-case and ranger-year-death significantly perform best for predicting dengue incidence and mortality, respectively. Therefore, temporal clustering yields the best models, reflective of dengue seasonality. The two best models were subjected to tripartite global exploratory model analyses, which encompass model-agnostic post-hoc methods such as Permutation Feature Importance (PFI) and Accumulated Local Effects (ALE). PFI reveals that the models differ in their important explanatory aspect, rainfall for rfsrc-year-case and NDVI for ranger-year-death, among which long-term average (lta) features are most relevant. Trend-wise, ALE reveals that average incidence predictions are positively associated with 'Rain.lta', reflective of dengue cases peaking during the wet season. In contrast, those for mortality are negatively associated with 'NDVI.lta', reflective of urban spaces driving dengue-related deaths. By technologically addressing the challenges of the human-animal-ecosystem interface, this study adheres to the One Digital Health paradigm operationalized under Sustainable Development Goals (SDGs). Leveraging data digitization and predictive modeling for epidemiological research paves SDG 3, which prioritizes holistic health and well-being.

Toward a Categorization of Virus-ncRNA Interactions in the World of RNA to Disentangle the Tiny Secrets of Dengue Virus.

In recent years, the function of noncoding RNAs (ncRNAs) as regulatory molecules of cell physiology has begun to be better understood. Advances in viral molecular biology have shown that host ncRNAs, cellular factors, and virus-derived ncRNAs and their interplay are strongly disturbed during viral infections. Nevertheless, the folding of RNA virus genomes has also been identified as a critical factor in regulating canonical and non-canonical functions. Due to the influence of host ncRNAs and the structure of RNA viral genomes, complex molecular and cellular processes in infections are modulated. We propose three main categories to organize the current information about RNA-RNA interactions in some well-known human viruses. The first category shows examples of host ncRNAs associated with the immune response triggered in viral infections. Even though miRNAs introduce a standpoint, they are briefly presented to keep researchers moving forward in uncovering other RNAs. The second category outlines interactions between virus-host ncRNAs, while the third describes how the structure of the RNA viral genome serves as a scaffold for processing virus-derived RNAs. Our grouping may provide a comprehensive framework to classify ncRNA-host-cell interactions for emerging viruses and diseases. In this sense, we introduced them to organize DENV-host-cell interactions.

Tanreqing injection demonstrates anti-dengue activity through the regulation of the NF-κB-ICAM-1/VCAM-1 axis.

BACKGROUND: Tanreqing injection (TRQ) has been employed in clinical practice as a treatment for dengue fever (DF). Nevertheless, the precise pharmacological mechanism underlying its efficacy remains elusive. METHOD: Network pharmacology, molecular docking, transcriptome sequencing, and experimental evaluation were employed to analyze and study the inhibitory potential of TRQ against dengue virus (DENV). RESULT: We found that TRQ inhibited the replication of DENV in human umbilical vein endothelial cells, Huh-7 cells, and Hep3B cells. In addition, TRQ prolonged the survival duration of AG129 mice infected with DF, decreased the viral load in serum and organs, and alleviated organ damage. Subsequently, ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of TRQ was performed to identify 314 targets associated with 36 active compounds present in TRQ. Integration of multiple databases yielded 47 DF-related genes. Then, 15 hub targets of TRQ in DF were determined by calculating the network topology parameters (Degree). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these pathways were primarily enriched in the processes of cytokine activation and leukocyte cross-endothelial migration, with significant enrichment of cell adhesion molecules. Molecular docking revealed favorable binding affinity between TRQ's key active compounds and the predicted hub targets. Transcriptome sequencing results showed TRQ's ability to restore the expression of vascular cell adhesion molecule-1 (VCAM-1) post-DENV infection. Finally, TRQ was found to modulate the immune status by regulating the nuclear factor kappa-B (NF-κB)- intercellular cell adhesion molecule-1 (ICAM-1)/VCAM-1 axis, as well as reduce immune cell alterations, inflammatory factor secretion, vascular permeability, and bleeding tendencies induced by DENV infection. CONCLUSION: Our research suggests that TRQ exerts therapeutic effects on DF by regulating the NF-κB-ICAM-1/VCAM-1 axis.

Antiviral drug discovery: Pyrimidine entry inhibitors for Zika and dengue viruses.

Vector-borne diseases, constituting over 17 % of infectious diseases, are caused by parasites, viruses, and bacteria, and their prevalence is shaped by environmental and social factors. Dengue virus (DENV) and Zika virus (ZIKV), some of the most prevalent infectious agents of this type of diseases, are transmitted by mosquitoes belonging to the genus Aedes. The highest prevalence is observed in tropical regions, inhabited by around 3 billion people. DENV infects millions of people annually and constitutes an additional sanitary challenge due to the circulation of four serotypes, which has complicated vaccine development. ZIKV causes large outbreaks globally and its infection is known to lead to severe neurological diseases, including microcephaly in newborns. Besides, not only mosquito control programs have proved to be not totally effective, but also, no antiviral drugs have been developed so far. The envelope protein (E) is a major component of DENV and ZIKV virion surface. This protein plays a key role during the virus cell entry, constituting an attractive target for the development of antiviral drugs. Our previous studies have identified two pyrimidine analogs (3e and 3h) as inhibitors; however, their activity was found to be hindered by their low water solubility. In this study, we performed a low-throughput antiviral screening, revealing compound 16a as a potent DENV-2 and ZIKV inhibitor (EC50 = 1.4 µM and 2.4 µM, respectively). This work was aimed at designing molecules with improved selectivity and pharmacokinetic properties, thus advancing the antiviral efficacy of compounds for potential therapeutic use.

Effects of Arboviral Infections on Transposable Element Transcript Levels in Aedes aegypti.

Transposable elements are mobile repeated sequences found in all genomes. Transposable elements are controlled by RNA interference pathways in most organisms, and this control involves the PIWI-interacting RNA pathway and the small interfering RNA pathway, which is also known to be the first line of antiviral defense in invertebrates. Using Drosophila, we recently showed that viral infections result in the modulation of transposable element transcript levels through modulation of the small RNA repertoire. The Aedes aegypti mosquito is of particular interest because almost half of its genome is made of transposable elements, and it is described as a major vector of viruses (such as the dengue [DENV], Zika [ZIKV], and chikungunya [CHIKV] arboviruses). Moreover, Aedes mosquitoes are unique among insects in that the PIWI-interacting RNA pathway is also involved in the somatic antiviral response, in addition to the transposable element control and PIWI-interacting RNA pathway genes expanded in the mosquito genome. For these reasons, we studied the impacts of viral infections on transposable element transcript levels in A. aegypti samples. We retrieved public datasets corresponding to RNA-seq data obtained from viral infections by DENV, ZIKV, and CHIKV in various tissues. We found that transposable element transcripts are moderately modulated following viral infection and that the direction of the modulation varies greatly across tissues and viruses. These results highlight the need for an in-depth investigation of the tightly intertwined interactions between transposable elements and viruses.

Identifying citrus limonoids as a potential fusion inhibitor of DENV-2 virus through its in silico study and FTIR analysis.

Dengue virus type 2 (DENV-2) is an arthropod-borne deadly RNA human pathogen transmitted through the mosquito Aedes. The DENV-2 roots viral infection by facilitating entry with its envelope glycoprotein to the receptor protein Dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN) through membrane fusion. Here, an organizational path is reported for inhibiting the transition due to fusion activation and by blocking the residues of the DC-SIGN-E-Glyco protein complex through citrus limonoids with its antiviral effect. Based on lower binding affinity obtained with E-glycoprotein, and based on ADMET and drug-likeness study, limonin was selected as having effective interaction with DC-SIGN-E-glycoprotein complex in comparison to other citrus limonoids. The FTIR spectra performed with the limonin-E-glycoprotein sample provide evidence of hydrogen bond formation that indicates the formation of a strong limonin-E-glycoprotein conjugate. Further, the strong physical interaction between DC-SIGN and small limonin molecules in comparison to that of E-glyco with DC-SIGN assures the development of immunity against DENV-2. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00207-2.

Mosquito E-20-Monooxygenase Gene Knockout Increases Dengue Virus Replication in Aedes aegypti Cells.

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.

Anti-dengue therapeutic potential of Tinospora cordifolia and its bioactives.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue is one of the most prevalent mosquito-borne viral infections. Moreover, due to the absence of appropriate curative and preventive measures against it, the mortality rate is increasing alarmingly. However, remarkable docking and clinical advances have been achieved with plant-based natural and conventional therapeutics. Tinospora cordifolia is one of the highly explored panaceas at the local level for its effective anti-dengue formulations. AIM OF THE STUDY: The present article aims for critical assessment of the data available on the anti-dengue therapeutic use of T. cordifolia. Efforts have also been made on the clinical and in-silico anti-dengue efficacy of this plant. The phytochemistry and the antiviral machinery of the plant are also emphasized. MATERIALS AND METHODS: The present article is the outcome of the literature survey on the anti-dengue effect of T. cordifolia. A literature survey was conducted from 2011 to 2024 using different databases with appropriate keywords. RESULTS: The present study confirms the anti-dengue potential of T. cordifolia. The plant can suppress the initiation of 'cytokine storm', vascular leakage and inhibition of various structural and NS proteins to exert its anti-dengue potential. Berberine and magnoflorine phytocompounds were highly explored for their anti-dengue potential. CONCLUSIONS: The present study concluded that T. cordifolia serves as an effective therapeutic agent for treating dengue. Further in-silico and clinical studies are needed so that stable, safe and efficacious anti-dengue drug can be developed. Besides, a precise antiviral mechanism of T. cordifolia against DENV infection is still needed.

Prevalence of arboviruses in sickle cell disease patients from two different regions of Brazil, the North and Northeast.

Sickle Cell Disease (SCD) is a hereditary disease characterized by extravascular and intravascular hemolysis and clinical variability, from mild pain to potentially life-threatening. Arboviruses include mainly Zika (ZIKV), Chikungunya (CHKV), and Dengue (DENV) virus, and are considered a public and social health problem. The present cross-sectional observational study aimed to investigate the prevalence of arbovirus infection in SCD patients from two Brazilian cities, Salvador and Manaus located in Bahia and Amazonas states respectively. A total of 409 individuals with SCD were included in the study, and 307 (75.06 %) patients tested positive for DENV-IgG, 161 (39.36 %) for ZIKV-IgG, and 60 (14.67 %) for CHIKV-IgG. Only one individual was positive for DENV-NS1 and another for DENV-IgM, both from Salvador. No individuals had positive serology for ZIKV-IgM or CHIKV-IgM. Arbovirus positivity by IgG testing revealed that the SCD group presented high frequencies in both cities. Interestingly, these differences were only statistically significant for ZIKV-IgG (p = 0.023) and CHIKV-IgG (p = 0.005) among SCD patients from Manaus. The reshaping of arbovirus from its natural habitat by humans due to disorderly urban expansion and the ease of international Mobility has been responsible for facilitating the spread of vector-borne infectious diseases in humans. We found the need for further studies on arboviruses in this population to elucidate the real association and impact, especially in acute infection. We hope that this study will contribute to improvements in the personalized clinical follow-up of SCD patients, identifying the influence of arbovirus infection in severe disease manifestations.

Case report: dengue fever associated acute macular neuroretinopathy.

Dengue fever (DF), which is caused by the dengue virus (DENV) and transmitted through Aedes mosquitoes, is well recognized for its systemic manifestations, with its ocular involvement gaining recent attention. We present a case of a 41-year-old Taiwanese female who developed acute macular neuroretinopathy (AMN) following a DF diagnosis related to DENV-1, emphasizing the need for awareness of this complication. The patient, with a history of completely resolved optic neuritis (ON) and comorbidities, experienced blurred vision on day 10 after the onset of DF. The ophthalmic examination revealed macular edema, ellipsoid zone (EZ) infiltration, and choriocapillaris involvement. Despite pulse therapy with corticosteroids, visual disturbances persisted, highlighting the challenge of managing ocular complications. Ocular manifestations in DF include hemorrhages, inflammation, and vascular complications. DF-associated AMN, a rare presentation, poses challenges in diagnosis and treatment response evaluation. While most patients recover spontaneously, some face persistent visual impairment, especially with AMN. Our case emphasizes the importance of recognizing ocular complications in DF, necessitating a multidisciplinary approach for optimal management and further research to delineate treatment strategies and outcomes.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

Concurrent infection of dengue virus with malaria parasites among outpatients attending healthcare facilities in Benin city, Nigeria.

Background: Dengue virus (DENV) and malaria parasites (MP) are among the common febrile diseases affecting the tropics and subtropics of the world. Both are mosquito-borne pathogens affecting humans and other animals. Methods: Blood samples were collected from 280 consented out-patients attending the selected hospitals and were analyzed. Malaria parasites were detected using microscopy and Malaria Ag Pf/Pan Rapid Test Device. Dengue virus was detected by serology and heminested reverse transcriptase PCR (hnRT-PCR) to target the flavivirus polymerase (NS5) gene. Results: Malaria parasites recorded a total positivity of 151 patients (53.9%) using microscopy, while DENV antibodies (DENV IgM and DENV IgG) were positive in 16 (5.7%) and 39 (13.9%) patients, respectively. There was a concurrent infection between MP/DENV IgM in 13 (4.6%) patients and MP/DENV IgG in 27 (9.6%) patients. Molecular identification revealed DENV serotype 2 in circulation. Conclusion: This study documents molecular evidence of dengue virus coexisting with malaria parasites in the study population, hence the need for efficient surveillance and control system.

NOD2 Responds to Dengue Virus Type 2 Infection in Macrophage-like Cells Interacting with MAVS Adaptor and Affecting IFN-α Production and Virus Titers.

In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points.