In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.

Boletim Arboviroses: semana 36/2024

Coordenadoria de Vigilância em Saúde da Secretaria Municipal da Saúde de São Paulo (COVISA/SMS-SP) disponibiliza semanalmente o boletim epidemiológico de Arboviroses: Dengue, Zika e Chikungunya.

Boletim Arboviroses: semana 35/2024

Coordenadoria de Vigilância em Saúde da Secretaria Municipal da Saúde de São Paulo (COVISA/SMS-SP) disponibiliza semanalmente o boletim epidemiológico de Arboviroses: Dengue, Zika e Chikungunya.

Boletim Arboviroses: semana 34/2024

Coordenadoria de Vigilância em Saúde da Secretaria Municipal da Saúde de São Paulo (COVISA/SMS-SP) disponibiliza semanalmente o boletim epidemiológico de Arboviroses: Dengue, Zika e Chikungunya.

Effectiveness of Wolbachia-mediated sterility coupled with sterile insect technique to suppress adult Aedes aegypti populations in Singapore: a synthetic control study.

BACKGROUND: Incompatible insect technique (IIT) coupled with sterile insect technique (SIT) via the release of sterile male Wolbachia-infected mosquitoes is a promising tool for Aedes-borne disease control. Yet, real-world evidence on the suppressive effectiveness of IIT-SIT on mosquito abundance remains mostly limited to small semi-rural village and suburban localities over short trial durations. However, a large proportion of Aedes-borne diseases occur in dense, urban, and high-rise locations, limiting the applicability of previous studies for these settings with high disease burden. The sustainability and use of this technology over multiple years is also unknown. METHODS: In this synthetic control study, we conducted a large-scale, field trial of IIT-SIT targeting Aedes aegypti among high-rise public housing estates in Singapore, an equatorial city state. Routinely collected data from a large, nationwide surveillance system of 57 990 unique mosquito traps, combined with a high-dimensional set of anthropogenic and environmental confounders were collected to ascertain mosquito abundance and its key drivers. Four townships were selected as the intervention groups (approximate population size of 607 872 residents as of 2022), wherein interventions that combined ITT with SIT over the course of the study period were conducted. Townships were subject to releases of wAlbB-SG male A aegypti mosquitoes twice a week. Data were assessed over the course of epidemiological weeks (EWs), which provide the finest temporal resolution of recorded Wolbachia release schedule and mosquito abundance data. A novel synthetic control framework was then developed to account for the non-randomised and staggered adoption setting of the intervention across trial sectors to identify the direct suppressive effectiveness of IIT-SIT on female A aegypti populations, the spillover effects in non-release areas, and the effect of the intervention on other mosquito populations such as Aedes albopictus. Furthermore, we recalculated effectiveness in terms of calendar time, time since intervention, and over multiple sites to examine heterogeneities in IIT-SIT effectiveness. FINDINGS: Between EW27 2018 and EW26 2022, Wolbachia releases were conducted across 117 sectors, of which 97 had sufficient trap data, which were collected between EW8 2019 and EW26 2022. We found that Wolbachia-based IIT-SIT reduced wild-type female A aegypti populations by a mean of 62·01% (95% CI 60·68 to 63·26) by 3 months, 78·40% (77·56 to 79·18) by 6 months, and 91·32% (90·95 to 91·66) by at least 18 months of releases. We also found a smaller but non-negligible spillover suppression effect that gradually increased over time (mean spillover intervention effectiveness 61·02% [95% CI 57·89 to 63·72] in adjacent, non-intervention sectors). Although no consistent change in A albopictus populations was seen across the four intervention townships after Wolbachia releases, the average intervention effectiveness on the A albopictus population across all release sectors was -25·80% (95% CI -30·93 to -21·05), which was driven by increases in two towns. INTERPRETATION: Our results demonstrate the potential of IIT-SIT for strengthening long-term, large-scale vector control in tropical cities, where dengue burden is the greatest. The effect of these interventions in different geographical settings should be assessed in future work. FUNDING: Singapore's Ministry of Finance, Ministry of Sustainability and the Environment, National Environment Agency, and National Robotics Program.

Risks of infectious disease hospitalisations in the aftermath of tropical cyclones: a multi-country time-series study.

BACKGROUND: The proportion of intense tropical cyclones is expected to increase in a changing climate. However, there is currently no consistent and comprehensive assessment of infectious disease risk following tropical cyclone exposure across countries and over decades. We aimed to explore the tropical cyclone-associated hospitalisation risks and burden for cause-specific infectious diseases on a multi-country scale. METHODS: Hospitalisation records for infectious diseases were collected from six countries and territories (Canada, South Korea, New Zealand, Taiwan, Thailand, and Viet Nam) during various periods between 2000 and 2019. The days with tropical cyclone-associated maximum sustained windspeeds of 34 knots or higher derived from a parametric wind field model were considered as tropical cyclone exposure days. The association of monthly infectious diseases hospitalisations and tropical cyclone exposure days was first examined at location level using a distributed lag non-linear quasi-Poisson regression model, and then pooled using a random-effects meta-analysis. The tropical cyclone-attributable number and fraction of infectious disease hospitalisations were also calculated. FINDINGS: Overall, 2·2 million people who were hospitalised for infectious diseases in 179 locations that had at least one tropical cyclone exposure day in the six countries and territories were included in the analysis. The elevated hospitalisation risks for infectious diseases associated with tropical cyclones tended to dissipate 2 months after the tropical cyclone exposure. Overall, each additional tropical cyclone day was associated with a 9% (cumulative relative risk 1·09 [95% CI 1·05-1·14]) increase in hospitalisations for all-cause infectious diseases, 13% (1·13 [1·05-1·21]) for intestinal infectious diseases, 14% (1·14 [1·05-1·23]) for sepsis, and 22% (1·22 [1·03-1·46]) for dengue during the 2 months after a tropical cyclone. Associations of tropical cyclones with hospitalisations for tuberculosis and malaria were not significant. In total, 0·72% (95% CI 0·40-1·01) of the hospitalisations for all-cause infectious diseases, 0·33% (0·15-0·49) for intestinal infectious diseases, 1·31% (0·57-1·95) for sepsis, and 0·63% (0·10-1·04) for dengue were attributable to tropical cyclone exposures. The attributable burdens were higher among young populations (aged ≤19 years) and male individuals compared with their counterparts, especially for intestinal infectious diseases. The heterogeneous spatiotemporal pattern was further revealed at the country and territory level-tropical cyclone-attributable fractions showed a decreasing trend in South Korea during the study period but an increasing trend in Viet Nam, Taiwan, and New Zealand. INTERPRETATION: Tropical cyclones were associated with persistent elevated hospitalisation risks of infectious diseases (particularly sepsis and intestinal infectious diseases). Targeted interventions should be formulated for different populations, regions, and causes of infectious diseases based on evidence on tropical cyclone epidemiology to respond to the increasing risk and burden. FUNDING: Australian Research Council, Australian National Health, and Medical Research Council.

Bis-benzylisoquinoline alkaloids inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy.

Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), represent a substantial public health challenge as there are currently no approved treatments available. Here, we investigated the antiviral effects of bis-benzylisoquinoline alkaloids (BBAs) on flavivirus infections. We evaluated five specific BBAs-berbamine, tetrandrine, iso-tetrandrine, fangchinoline, and cepharanthine-and found that they effectively inhibited infections by ZIKV, DENV, or JEV by blocking virus entry and genome replication stages in the flavivirus life cycle. Furthermore, we synthesized a fluorophore-conjugated BBA and showed that BBAs targeted endolysosomes, causing lysosomal pH alkalization. Mechanistic studies on inhibiting ZIKV infection by BBAs revealed that these compounds blocked TRPML channels, leading to lysosomal dysfunction and reducing the expression of NCAM1, a key receptor for the entry of ZIKV into cells, thereby decreasing cells susceptibility to ZIKV infection. Additionally, BBAs inhibited the fusion of autophagosomes and lysosomes, significantly reducing viral RNA replication. Collectively, our results suggest that BBAs inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy, respectively, underscoring the potential of BBAs as therapeutic agents against flavivirus infections.

Harnessing Natural Resources for Advancements in Dengue Virus Treatment.

Dengue fever, caused by the Dengue virus (DENV) and transmitted by Aedes aegypti mosquitoes, has become endemic in over 100 countries. Despite considerable research, there is a lack of specific drugs for clinical use against dengue. Hence, further exploration to identify anti-- dengue compounds is essential. In recent years, natural products have gained attention for their antiviral properties. Plant-based medicines are particularly appealing due to their safety and low toxicity. This review summarizes natural compounds with potential antiviral activity against DENV, highlighting their mechanisms of action. Various compounds, from traditional herbal remedies to novel plant isolates, show promise against dengue, targeting crucial viral proteins like the envelope protein, proteases, and RNA polymerase. Exploring natural sources of antiviral agents against dengue is crucial. These compounds offer hope for effective treatments and mitigating dengue's global impact.

Olfactory receptor coexpression and co-option in the dengue mosquito.

The olfactory sensory neurons of vinegar flies and mice tend to express a single ligand-specific receptor. While this 'one neuron-one receptor' motif has long been expected to apply broadly across insects, recent evidence suggests it may not extend to mosquitoes. We sequenced and analyzed the transcriptomes of 46,000 neurons from antennae of the dengue mosquito Aedes aegypti to resolve all olfactory, thermosensory, and hygrosensory neuron subtypes and identify the receptors expressed therein. We find that half of all olfactory subtypes coexpress multiple receptors. However, coexpression occurs almost exclusively among genes from the same family-among odorant receptors (ORs) or among ionotropic receptors (IRs). Coexpression of ORs with IRs is exceedingly rare. Many coexpressed receptors are recent duplicates. In other cases, the recruitment or co-option of single receptors by multiple neuron subtypes has placed these genes together in the same cells with distant paralogs. Close examination of data from Drosophila reveal rare cases of both phenomena, indicating that the olfactory systems of these two species are not fundamentally different, but instead fall at different locations along a continuum likely to encompass diverse insects.

Chronic innate immune impairment and ZIKV persistence in the gastrointestinal tract during SIV infection in pigtail macaques.

Mosquito borne flaviviruses, including dengue (DENV) and Zika (ZIKV) viruses, have caused global epidemics in areas with high HIV prevalence due to the expanded geographic range of arthropod vectors. Despite the occurrence of large flavivirus outbreaks in countries with high HIV prevalence, there is little knowledge regarding the effects of flavivirus infection in people living with HIV (PLWH). Here, we use a pigtail macaque model of HIV/AIDS to investigate the impact of simian immunodeficiency virus (SIV)-induced immunosuppression on ZIKV replication and pathogenesis. Early acute SIV infection induced expansion of peripheral ZIKV cellular targets and increased innate immune activation and peripheral blood mononuclear cells (PBMC) from SIV infected macaques were less permissive to ZIKV infection in vitro. In SIV-ZIKV co-infected animals, we found increased persistence of ZIKV in the periphery and tissues corresponding to alterations in innate cellular (monocytes, neutrophils) recruitment to the blood and tissues, decreased anti-ZIKV immunity, and chronic peripheral inflammatory and innate immune gene expression. Collectively, these findings suggest that untreated SIV infection may impair cellular innate responses and create an environment of chronic immune activation that promotes prolonged ZIKV viremia and persistence in the gastrointestinal tract. These results suggest that PLWH or other immunocompromised individuals could be at a higher risk for chronic ZIKV replication, which in turn could increase the timeframe of ZIKV transmission. Thus, PLWH are important populations to target during the deployment of vaccine and treatment strategies against ZIKV.

Dengue Infection During Pregnancy and Adverse Birth Outcomes: A Systematic Review and Meta-Analysis.

Dengue is a rapidly spreading mosquito-borne viral disease, posing significant public health challenges in tropical and subtropical regions. This systematic review and meta-analysis aimed to evaluate the relationship between maternal dengue virus infection and adverse birth outcomes. A literature search was conducted in PubMed, Embase, and web of science databases until April 2024. Observational studies examining the association between laboratory-confirmed maternal dengue infection and adverse birth outcomes such as preterm birth, low birth weight (LBW), small for gestational age (SGA), stillbirth, and postpartum haemorrhage were included. Data were extracted, and risk of bias was assessed using the Newcastle-Ottawa Scale. Random-effects meta-analysis models were used to pool data in R software (V 4.3). Twenty studies met the inclusion criteria. The pooled prevalence of preterm birth among dengue-affected pregnancies was 18.3% (95% CI: 12.6%-25.8%), with an OR of 1.21 (95% CI: 0.78-1.89). For LBW, the pooled prevalence was 17.1% (95% CI: 10.4%-26.6%), with an OR of 1.00 (95% CI: 0.69-1.41). SGA had a pooled prevalence of 11.2% (95% CI: 2.7%-36.9%) and an OR of 0.93 (95% CI: 0.41-2.14). The prevalence of stillbirth was 3.3% (95% CI: 1.6%-6.8%), with significant associations found in some studies (RR: 2.67; 95% CI: 1.09-6.57). Postpartum haemorrhage had an OR of 1.97 (95% CI: 0.53-2.69). While maternal dengue infection was associated with a higher prevalence of preterm birth and LBW, the associations were not statistically significant. Significant associations were observed for stillbirth in specific studies. Further research with standardized methodologies is needed to clarify these relationships and identify potential mechanisms.

Evaluation of developmental milestones and of brain measurements in rats exposed to the pesticide pyriproxyfen in prenatal period.

Pyriproxyfen is a pesticide used in Brazil to control the Aedes aegypti mosquito, vector of arboviruses like Zika and dengue. However, this pesticide is structurally similar to retinoic acid, a metabolite of vitamin A that regulates neuronal differentiation and hindbrain development during the embryonic period. Due to the similarity between pyriproxyfen and retinoic acid, studies indicate that this pesticide may have cross-reactivity with retinoid receptors. Thus, pregnant exposure to pyriproxyfen could interfere in the nervous system development of the fetal. In this context, the present study evaluated whether prenatal exposure to pyriproxyfen affects neonatal development and brain structure in rats. Wistar rat pups were divided in three experimental groups: (1) negative control (CT-)-offspring of rats that drink potable water during pregnancy; (2) pyriproxyfen (PIR)-offspring of rats exposed to Sumilarv® prenatally, a pesticide that has pyriproxyfen as active ingredient; and (3) positive control (CT+)-offspring of rats exposed to an excess of vitamin A prenatally. Only vitamin A treated-pregnant showed lower weight gain, but gestation length was similar among pregnant that received potable water, water containing vitamin A and water containing Sumilarv. In relation to the offspring, PIR group exhibits a delayed front-limb suspension response but performed early the negative geotaxis reflex. On the other hand, CT+ group exhibited lower body weight in the 1st postnatal day, delayed audio startle response, but performed early the eyelids opening and hindlimb placing response. A reduction in the maximum brain width was observed both in PIR and CT+ groups, but a reduction in the number of neurons in the M1 cortex was showed only in CT+ group. The number of glial cells in this brain area was similar between the three experimental groups studied. Although prenatal exposure to pyriproxyfen did not alter neonatal milestones in the same way as vitamin A in excess, both substances caused a reduction in the maximum width of the brain, suggesting that this pesticide can produce neurotoxic effects during the embryonic period.

TIME OF HOST-SEEKING OF MOSQUITO VECTOR SPECIES ON THE TEMPE CAMPUS OF ARIZONA STATE UNIVERSITY.

The Arizona State University (ASU) Tempe campus is inhabited by some 55,000 enrolled students as well as several mosquito species that can transmit West Nile virus, dengue, Zika, chikungunya, and yellow fever. The time of host-seeking of these vectors has not been quantified on the ASU Tempe campus, but this information is important to inform ground and/or truck-mount fogging operations targeting mosquitoes to prevent or control disease outbreaks. We quantified the time of host-seeking of the predominant mosquito vector species at the ASU Tempe campus during the post-monsoon season in 2021, using collection bottle rotators with encephalitis vector survey traps that were baited with CO2, at 3 h intervals during a full day. Culex quinquefasciatus, Aedes aegypti, and Culex tarsalis were the most abundant species captured. Pre-midnight host-seeking (18:00-00:00) accounted for 52% of all captures, whereas post-midnight host-seeking (00:00-06:00) accounted for 35% of all captures. Peak activity times were between 21:00 and 00:00 for Cx. quinquefasciatus and Cx. tarsalis, and between 15:00 and 18:00 for Ae. aegypti. Data can be used to inform local mosquito surveillance and control programs.

Study of Clinicoetiological Spectrum of Bicytopenia and Pancytopenia in Hospitalized Children.

Background The etiological profile of children with bicytopenia and pancytopenia has a very wide spectrum, ranging from transient causes like infections or nutritional deficiencies to bone marrow failure syndromes. Timely diagnosis and treatment impart favorable prognosis to this entity. There is a paucity of data regarding the etiology of cytopenia in hospitalized children at a tertiary center in India. Additionally, only a few studies have discussed the possible association between the severity of cytopenia at presentation and the possible etiology. Methods This is a cross-sectional observational study analyzing bicytopenia and pancytopenia in hospitalized children. Patient details, along with clinical findings and relevant investigations, were recorded on predesigned pro forma and analyzed statistically. Results Out of 202 children, 174 (86.13%) had bicytopenia, and 28 (13.86%) had pancytopenia, with a male predominance resulting in a male-to-female ratio of 1.65:1. The commonest age group affected was pre-adolescent age group (6-12 years). The causes of bicytopenia and pancytopenia in hospitalized children in the decreasing order of frequency were infections (65.84%), benign hematological disorders (18.81%), systemic illness (10.39%), and malignancies (4.95%). The cytopenia was more severe in children with pancytopenia than bicytopenia. Conclusions Infections outweigh the other causes of bicytopenia and pancytopenia. The severity of the cell line affected can help narrow down a diagnosis of cytopenia etiologies. Most of the children with bicytopenia and pancytopenia had treatable etiology and favorable outcomes.

Rare occurrence of sagittal sinus thrombosis and haemorrhagic infarction with dengue fever: jumping from traditional symptoms to lethal neurological consequences.

Introduction and importance: Dengue virus (DENV) is an RNA virus transmitted by Aides mosquito causing dengue fever. There is growing recognition of neurological symptoms associated with DENV infection, some of which might be lethal if left untreated. Case reports describing sagittal sinus thrombosis, as a serious neurologic consequence of dengue infection, are rare. It is still unknown how often sagittal sinus thrombosis occurs and what variables increase the risk in dengue patients. Case presentation: Herein the authors presented an elderly Sudanese patient diagnosed with dengue fever. He was admitted, then 2 days after admission, the condition was complicated by atrial fibrillation, sagittal sinus thrombosis complicated by massive left temporal lobe infarction with haemorrhagic transformation and recurrent episodes of status epilepticus. After receiving the necessary care, his condition remained the same and no progress or deterioration was seen. Clinical discussion: Sagittal sinus thrombosis can happen due to several underlying causes. DENV can very rarely lead to such condition. The authors' patient developed this condition, which was later complicated by ischaemic stroke with haemorrhagic transformation and status epilepticus. In addition to a familial history of DVT and a history of myocardial infarction, our patient also acquired cardiac mural thrombus and DVT throughout his illness, which increased the suspicion of a protein C, protein S, or antithrombin 3 deficiency. Conclusion: Sagittal sinus thrombosis with haemorrhagic infarction associated with thrombocytopenia is a very rare kind of stroke that occurs in dengue. Dengue as a pathogenic mechanism of ischaemic stroke requires validation with further data.

Peri-domestic entomological surveillance using private traps allows detection of dengue virus in Aedes albopictus during an autochthonous transmission event in mainland France, late summer 2023.

While locally-acquired dengue virus (DENV) human infections occur in mainland France since 2010, data to identify the mosquito species involved and to trace the virus are frequently lacking. Supported by a local network gathering public health agencies and research laboratories, we analysed, in late summer 2023, mosquitoes from privately-owned traps within a French urban neighbourhood affected by a dengue cluster. The cluster, in Auvergne-Rhône-Alpes, comprised three cases, including two autochthonous ones. Upon return from a recent visit to the French Caribbean Islands, the third case had consulted healthcare because of dengue-compatible symptoms, but dengue had not been recognised. For the two autochthonous cases, DENV-specific antibodies in serum or a positive quantitative PCR for DENV confirmed DENV infection. The third case had anti-flavivirus IgMs. No DENV genetic sequences were obtained from affected individuals but Aedes albopictus mosquitoes trapped less than 200 m from the autochthonous cases' residence contained DENV. Genetic data from the mosquito-derived DENV linked the cluster to the 2023-2024 dengue outbreak in the French Caribbean Islands. This study highlights the importance of raising mosquito-borne disease awareness among healthcare professionals. It demonstrates Ae. albopictus as a DENV vector in mainland France and the value of private mosquito traps for entomo-virological surveillance.

Sulfoglycodendron Antivirals with Scalable Architectures and Activities.

Many viruses initiate their cell-entry by binding their multisubunit receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multiprotein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed the detailed nature of binding between these antivirals and viral protein receptors and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.

Circulating serotypes and genotypes of dengue virus during the 2023 outbreak in Eastern Nepal.

Dengue virus (DENV) is one of the most significant mosquito-borne diseases in Nepal. In 2023, DENV outbreaks began in Eastern Nepal, near the border with India, and rapidly spread nationwide. The study aims to describe the outbreak's epidemiological pattern, laboratory characteristics, DENV serotypes, and genotypes. A hospital-based cross-sectional study was conducted in four hospitals in Jhapa, Eastern Nepal, in 2023. Acute serum samples were obtained from dengue suspected patients within 7 days of illness and subjected to virus isolation, conventional and real-time polymerase chain reaction (RT-PCR), and phylogenetic analysis. Out of 60 samples, 42 (70 %), 11 (18.3 %) and 7 (11.7 %) were primary, secondary and non-dengue infection, respectively. Among 53 dengue confirmed patients, 46 (86.7 %) were positive for NS1 and 12 (22.6 %) were positive for both NS1 and IgM. Out of 42 dengue isolates, a new clade of the cosmopolitan genotype of DENV-2 was the most prevalent (28, 66.7 %), followed by genotype III of DENV-3 (11, 26.2 %) and genotype V of DENV-1 (3, 7.1 %). Genotype III of DENV-3 was first introduced in 2022-2023 in Nepal. Phylogenetic analysis of the E gene revealed the DENV-2 isolates from Nepal had 98 % homologous nucleotide similarity with the strains from India and Bangladesh. To our knowledge, this is the first report of circulating serotypes and genotypes of DENV in Jhapa. Integrating molecular findings into the dengue control plan can enhance surveillance efforts, monitor disease trends, and implement proactive measures to reduce the burden of dengue and prevent fatalities in future outbreaks.

Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

BACKGROUND: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there has been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. METHODS: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF = 8) or dengue haemorrhagic fever (DHF = 8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. RESULTS: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p = 0.01, median = 45.69 pg/ml), but lower levels when NS1 was co-cultured with HDL (p = 0.05, median = 4.617 pg/ml). MDMs of those with past DHF produced higher levels of IL-1ß when NS1 was co-cultured with PAF (p = 0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. CONCLUSIONS: As RPLP2 is important in DENV replication, regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Larvicidal potential of Trachyspermum ammi essential oil and Delphinium speciosum extract against malaria, dengue, and filariasis mosquito vectors.

Mosquito-borne diseases, such as malaria, dengue, and Zika, pose major public health challenges globally, affecting millions of people. The growing resistance of mosquito populations to synthetic insecticides underscores the critical need for effective and environmentally friendly larvicides. Although chemical pesticides can initially be effective, they often lead to negative environmental consequences and health hazards for non-target species, including humans. This study aimed to evaluate the larvicidal effects of Trachyspermum ammi essential oil and Delphinium speciosum extract on the larvae of three major mosquito species: Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Mosquito larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were reared under controlled laboratory conditions. The larvicidal activity of T. ammi essential oil and D. speciosum extract was evaluated through standard bioassays, using various concentrations of essential oils (10, 20, 40, 80, and 160 ppm) and extracts (160, 320, 640, 1280, and 2560 ppm) to determine the lethal concentration (LC50) values after 24 h of exposure. Fresh plant materials were collected, with the essential oil extracted via hydro-distillation, and the extract prepared using methanol solvent extraction. The chemical composition of T. ammi essential oil was examined using gas chromatography-mass spectrometry (GC-MS). Additionally, the preliminary analysis of the chemical compounds in D. speciosum extract was carried out using thin layer chromatography (TLC) and nuclear magnetic resonance spectroscopy (NMR) techniques. The results indicated that the essential oil of T. ammi exhibited more effective larvicidal activity compared to the D. speciosum extract. Specifically, the essential oil demonstrated LC50 values of 18 ppm for Cx. quinquefasciatus and 19 ppm for Ae. aegypti. In contrast, the D. speciosum extract showed the strongest larvicidal effect against An. stephensi, with an LC50 of 517 ppm. Concentrations of 40 ppm of the essential oil and 1280 ppm of the extract resulted in 100% mortality across all three species. Both the essential oil of T. ammi and the D. speciosum extract exhibited concentration-dependent larvicidal activity, and these results were statistically significant (p < 0.001) compared to the no-treatment group. GC-MS analysis revealed thymol (88.95%), o-cymen-5-ol (4.11%), and γ-terpinene (2.10%) as the major constituents of the T. ammi essential oil. Additionally, TLC verified the presence of alkaloids in both chloroform and methanolic extracts. Proton NMR identified a diterpene structure for these alkaloids. These findings suggest that T. ammi essential oil is a promising candidate for natural mosquito control strategies. Given its efficacy, further research is warranted to explore its potential in integrated vector management programs.