Dengue virus type 3 infection in a traveler returning from Costa Rica to Japan in 2023.

The number of dengue cases has increased dramatically in recent years. In Latin America, the number of cases and deaths in 2023 was the highest ever recorded. We report on a patient who had been infected with dengue virus during his stay in Costa Rica in September 2023, and developed the disease after returning to Japan. Plasma obtained from the patient was used for diagnosis and dengue virus serotyping by real-time PCR. The nucleotide sequence of the envelope region of dengue virus was then determined by the direct sequencing method, and this sequence was used for phylogenetic analyses. The patient was found to be infected with dengue virus type 3 genotype III. The sequence from the present case was more homologous with sequences registered in Florida, USA, associated with travel to Cuba in 2022 than with sequences registered in Costa Rica 10 years ago. The Pan American Health Organization reported that only dengue virus type 1 and 2 cases were reported in Costa Rica in 2019-2021, whereas dengue virus type 3 and 4 cases started being reported in 2022. In 2023, the reported numbers of cases with dengue virus types 3 and 4 exceeded those of dengue virus types 1 and 2. In addition, regional differences in endemic strains have been observed in Costa Rica. Our findings suggest that the dengue virus type 3 that infected the patient was more likely an influx of a strain that had been circulating in Caribbean countries such as Cuba in recent years, rather than a re-emergence of an indigenous virus in Costa Rica. The serotypes of dengue virus prevalent in Costa Rica have been changing since 2022. All four serotypes were prevalent in 2023, with a particularly sharp increase in the number of cases of dengue virus types 3 and 4. Future monitoring and surveillance are essential because changes in endemic serotypes can cause antibody-dependent enhancement, which can lead to severe dengue disease presentations.

[Baicalin suppresses type 2 dengue virus-induced autophagy of human umbilical vein endothelial cells by inhibiting the PI3K/AKT pathway].

OBJECTIVE: To investigate the effect of type 2 dengue virus (DENV-2) infection on autophagy in human umbilical vein endothelial cells (HUVECs) and the mechanism mediating the inhibitory effect of baicalin against DENV-2 infection. METHODS: Cultured HUVECs with DENV-2 infection were treated with different concentrations of baicalin, and the changes in autophagy of the cells were detected using transmission electron microscopy. Lyso Tracker Red staining was used to examine pH changes in the lysosomes of the cells, and the expressions of ATG5, beclin-1, LC3, P62, STX17, SNAP29, VAMP8, and PI3K/AKT signaling pathway-related proteins were detected by Western blotting. DENV-2 replication in the cells were evaluated using RT-qPCR. The differentially expressed proteins in DENV-2-infected HUVECs were identified by proteomics screening. RESULTS: Treatment with baicalin did not significantly affect the viability of cultured HUVECs. Proteomic studies suggested that the PI3K-AKT pathway played an important role in mediating cell injury induced by DENV-2 infection. The results of RT-qPCR demonstrated that baicalin dose-dependently inhibited DENV-2 replication in HUVECs and produced the strongest inhibitory effect at the concentration of 50 µg/mL. Transmission electron microscopy, Lyso Tracker Red staining, RT-qPCR, and Western blotting all showed significant inhibitory effect of baicalin on DENV-2-induced autophagy in HUVECs. DENV-2 infection of HUVECs caused increased cellular expressions of LC3 and P62 proteins, which were significantly lowered by treatment with LY294002 (a PI3K inhibitor). CONCLUSION: Baicalin inhibits DENV-2 replication in HUVECs and suppresses DENV-2-induced cell autophagy by inhibiting the PI3K/AKT signaling pathway.

Using Multiscale Molecular Modeling to Analyze Possible NS2b-NS3 Protease Inhibitors from Philippine Medicinal Plants.

Within the field of Philippine folkloric medicine, the utilization of indigenous plants like Euphorbia hirta (tawa-tawa), Carica papaya (papaya), and Psidium guajava (guava) as potential dengue remedies has gained attention. Yet, limited research exists on their comprehensive effects, particularly their anti-dengue activity. This study screened 2944 phytochemicals from various Philippine plants for anti-dengue activity. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling provided 1265 compounds demonstrating pharmacokinetic profiles suitable for human use. Molecular docking targeting the dengue virus NS2b-NS3 protease's catalytic triad (Asp 75, Ser 135, and His 51) identified ten ligands with higher docking scores than reference compounds idelalisib and nintedanib. Molecular dynamics simulations confirmed the stability of eight of these ligand-protease complexes. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) analysis highlighted six ligands, including veramiline (-80.682 kJ/mol), cyclobranol (-70.943 kJ/mol), chlorogenin (-63.279 kJ/mol), 25beta-Hydroxyverazine (-61.951 kJ/mol), etiolin (-59.923 kJ/mol), and ecliptalbine (-56.932 kJ/mol) with favorable binding energies, high oral bioavailability, and drug-like properties. This integration of traditional medical knowledge with advanced computational drug discovery methods paves new pathways for the development of treatments for dengue.

Inhibition of dengue virus infection in vitro by fucoidan and polysaccharide extract from marine alga Sargassum spp.

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.

Concurrently Affected by Dengue and Hepatitis A: Exploring the Intricacies of Co-infection in a Comprehensive Case Series.

Based on the examination of four distinct cases, this case series offers a thorough investigation of the intricate relationship between dengue fever and hepatitis A infection. Despite their distinct origins, both illnesses manifest overlapping clinical features, posing considerable diagnostic hurdles, particularly in endemic regions. The cases reveal consistent symptoms such as elevated fever, abdominal discomfort, jaundice, and irregular liver function test results, underscoring the intricate nature of an accurate diagnosis. Variations in age distribution and the severity of symptoms underscore the necessity for tailored treatment approaches. Diagnostic challenges stem from the similarity in clinical presentations and shared laboratory abnormalities, necessitating comprehensive serological assessments. Therapeutic strategies entail a multidisciplinary approach addressing both hepatic and systemic manifestations, with supportive measures ensuring favorable clinical outcomes. Despite the complexities involved, timely interventions facilitate gradual symptom amelioration and successful patient recovery. Informing clinical practice and directing public health actions, this case series provides insightful information about the diagnostic and treatment complications associated with co-occurring dengue fever and hepatitis A infection.

Repurposing of FDA-approved drugs against oligomerization domain of dengue virus NS1 protein: a computational approach.

Dengue fever is a serious health hazard on a global scale and its primary causative agent is the dengue virus (DENV). The non-structural protein 1 (NS1) of DENV plays a pivotal role in pathogenesis. It is associated with several autoimmune events, endothelial cell apoptosis, and vascular leakage, which increase mainly during the critical phase of infection. In this study, important residues of the oligomerization domain of NS1 protein were identified by literature searches. Virtual screening has been conducted using the entire dataset of the DrugBank database and the potential small-molecule inhibitors against the NS1 protein have been chosen on the basis of binding energy values. This is succeeded by molecular dynamics (MD) simulations of the shortlisted compounds, ultimately giving rise to five compounds. These five compounds were further subjected to RAMD simulations by applying a random direction force of specific magnitude on the ligand center of mass in order to push the ligand out of the protein-binding pocket, for the quantitative estimation of their binding energy values to determine the interaction strength between protein and ligand which prevents ligand unbinding from its binding site, ultimately leading to the selection of three major compounds, DB00826 (Natamycin), DB11274 (Dihydro-alphaergocryptine), and DB11275 (Epicriptine), with the DB11274 having a role against idiopathic Parkinson's disease, and thus may have possible important roles in the prevention of dengue-associated Parkinsonism. These compounds may act as prospective drugs against dengue, by preventing the oligomerization of the NS1 protein, thereby preventing disease progression and pathogenesis.

Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Neuro-Ophthalmic Dengue Infection: A Case Report with a Multiple Body Site Sampling Strategy and Review of Laboratory Data.

Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease.

Unraveling Dengue Virus Diversity in Asia: An Epidemiological Study through Genetic Sequences and Phylogenetic Analysis.

Dengue virus (DENV) is the causative agent of dengue. Although most infected individuals are asymptomatic or present with only mild symptoms, severe manifestations could potentially devastate human populations in tropical and subtropical regions. In hyperendemic regions such as South Asia and Southeast Asia (SEA), all four DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) have been prevalent for several decades. Each DENV serotype is further divided into multiple genotypes, reflecting the extensive diversity of DENV. Historically, specific DENV genotypes were associated with particular geographical distributions within endemic regions. However, this epidemiological pattern has changed due to urbanization, globalization, and climate change. This review comprehensively traces the historical and recent genetic epidemiology of DENV in Asia from the first time DENV was identified in the 1950s to the present. We analyzed envelope sequences from a database covering 16 endemic countries across three distinct geographic regions in Asia. These countries included Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan, and Sri Lanka from South Asia; Cambodia, Laos, Myanmar, Thailand, and Vietnam from Mainland SEA; and Indonesia, the Philippines, Malaysia, and Singapore from Maritime SEA. Additionally, we describe the phylogenetic relationships among DENV genotypes within each serotype, along with their geographic distribution, to enhance the understanding of DENV dynamics.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

Cross-Neutralizing Anti-Chikungunya and Anti-Dengue 2 IgG Antibodies from Patients and BALB/c Mice against Dengue and Chikungunya Viruses.

Dengue (DENV) and Chikungunya (CHIKV) viruses can be transmitted simultaneously by Aedes mosquitoes, and there may be co-infections in humans. However, how the adaptive immune response is modified in the host has yet to be known entirely. In this study, we analyzed the cross-reactivity and neutralizing activity of IgG antibodies against DENV and CHIKV in sera of patients from the Mexican Institute of Social Security in Veracruz, Mexico, collected in 2013 and 2015 and using IgG antibodies of BALB/c mice inoculated with DENV and/or CHIKV. Mice first inoculated with DENV and then with CHIKV produced IgG antibodies that neutralized both viruses. Mice were inoculated with CHIKV, and then with DENV; they had IgG antibodies with more significant anti-CHIKV IgG antibody neutralizing activity. However, the inoculation only with CHIKV resulted in better neutralization of DENV2. In sera obtained from patients in 2013, significant cross-reactivity and low anti-CHIKV IgG antibody neutralizing activity were observed. In CHIKV-positive 2015 sera, the anti-DENV IgG antibody neutralizing activity was high. These results suggest that CHIKV stimulates DENV2-induced memory responses and vice versa. Furthermore, cross-reactivity between the two viruses generated neutralizing antibodies, but exchanging CHIKV for DENV2 generated a better anti-CHIKV neutralizing response.

Unraveling the Genomic Evolution of Dengue Virus Serotype 1: A Case Study from Yantai, China.

In August 2023, we identified a case of dengue fever in Yantai City, which was imported from Xishuangbanna, China. To investigate its evolutionary history and population dynamics, we utilized the metatranscriptomic method to obtain the virus' whole genome sequence. Together with 367 selected dengue virus whole genome sequences from the NCBI database, we constructed a time-scaled Maximum Clade Credibility (MCC) tree. We found that our sequence exhibited a high homology with a sequence of DENV1 (OR418422.1) uploaded by the Guangzhou Center for Disease Control and Prevention in 2023, with an estimated divergence time around 2019 (95% HPD: 2017-2023), coinciding with the emergence of SARS-CoV-2. The DENV strain obtained in this study belongs to genotype I of DENV1. Its ancestors experienced a global epidemic around 2005 (95% HPD: 2002-2010), and its progeny strains have spread extensively in Southeast Asia and China since around 2007 (95% HPD: 2006-2011). The Bayesian skyline plot indicates that the current population of DENV1 has not been affected by SARS-CoV-2 and is expected to maintain stable transmission. Hence, it is imperative to track and monitor its epidemiological trends and genomic variations to prevent potential large-scale outbreaks in the post-SARS-CoV-2 era.

Epidemiology of Travel-Associated Dengue from 2007 to 2022: A GeoSentinel Analysis.

BACKGROUND: Dengue is a leading cause of febrile illness among international travellers. We aimed to describe the epidemiology and clinical characteristics of imported dengue in returning travellers evaluated at GeoSentinel sites from 2007-2022. METHODS: We retrieved GeoSentinel records of dengue among travellers residing in non-endemic countries. We considered dengue confirmed when diagnosed by a positive DENV-specific RT-PCR, positive NS-1 antigen, and/or anti-DENV IgG seroconversion, and probable when diagnosed by single anti-DENV IgM or high titre anti-DENV IgG detection. Severe dengue was defined as evidence of clinically significant plasma leakage or bleeding, organ failure, or shock, according to the 2009 WHO guidance. Complicated dengue was defined as either severe dengue or dengue with presence of any warning sign. Analyses were descriptive. RESULTS: This analysis included 5958 travellers with confirmed (n = 4859; 81.6%) or probable (n = 1099; 18.4%) dengue. The median age was 33 years (range: < 1-91); 3007 (50.5%) travellers were female. The median travel duration was 21 days (interquartile range [IQR]: 15-32). The median time between illness onset and GeoSentinel site visit was 7 days (IQR: 4-15). The most frequent reasons for travel were tourism (67.3%), visiting friends or relatives (12.2%), and business (11.0%). The most frequent regions of acquisition were Southeast Asia (50.4%), South-Central Asia (14.9%), the Caribbean (10.9%), and South America (9.2%). Ninety-five (1.6%) travellers had complicated dengue, of whom 27 (0.5%) had severe dengue, and one died. Of 2710 travellers with data available, 724 (26.7%) were hospitalized. The largest number of cases (n = 835) was reported in 2019. CONCLUSIONS: A broad range of international travellers should be aware of the risk of acquiring dengue and receive appropriate pretravel counselling regarding preventive measures. Prospective cohort studies are needed to further elucidate dengue risk by destination and over time, as well as severe outcomes and prolonged morbidity (long-dengue) due to travel-related dengue.

In silico homology modeling of dengue virus non-structural 4B (NS4B) protein and its molecular docking studies using triterpenoids.

BACKGROUND: Dengue fever has become a significant worldwide health concern, because of its high morbidity rate and the potential for an increase in mortality rates due to lack of adequate treatment. There is an immediate need for the development of effective medication for dengue fever. METHODS: Homology modeling of dengue virus (DENV) non-structural 4B (NS4B) protein was performed by SWISS-MODEL to predict the 3D structure of the protein. Structure validation was conducted using PROSA, PROCHECK, Ramachandran plot, and VERIFY-3D. MOE software was used to find out the in-Silico inhibitory potential of the five triterpenoids against the DENV-NS4B protein. RESULTS: The SWISS-MODEL was employed to predict the three-dimensional protein structure of the NS4B protein. Through molecular docking, it was found that the chosen triterpenoid NS4B protein had a high binding affinity interaction. It was observed that the NS4B protein binding energy for 15-oxoursolic acid, betulinic acid, ursolic acid, lupeol, and 3-o-acetylursolic acid were - 7.18, - 7.02, - 5.71, - 6.67 and - 8.00 kcal/mol, respectively. CONCLUSIONS: NS4B protein could be a promising target which showed good interaction with tested triterpenoids which can be developed as a potential antiviral drug for controlling dengue virus pathogenesis by inhibiting viral replication. However, further investigations are necessary to validate and confirm their efficacy.

North-south pathways, emerging variants, and high climate suitability characterize the recent spread of dengue virus serotypes 2 and 3 in the Dominican Republic.

BACKGROUND: Dengue fever remains a significant public health challenge in tropical and subtropical regions, with its transmission dynamics being influenced by both environmental factors and human mobility. The Dominican Republic, a biodiversity hotspot in the Caribbean, has experienced recurrent dengue outbreaks, yet detailed understanding of the virus's transmission pathways and the impact of climatic factors remains limited. This study aims to elucidate the recent transmission dynamics of the dengue virus (DENV) in the Dominican Republic, utilizing a combination of genomic sequencing and epidemiological data analysis, alongside an examination of historical climate patterns. METHODS: We conducted a comprehensive study involving the genomic sequencing of DENV samples collected from patients across different regions of the Dominican Republic over a two-year period. Phylogenetic analyses were performed to identify the circulation of DENV lineages and to trace transmission pathways. Epidemiological data were integrated to analyze trends in dengue incidence and distribution. Additionally, we integrated historical climate data spanning several decades to assess trends in temperature and their potential impact on DENV transmission potential. RESULTS: Our results highlight a previously unknown north-south transmission pathway within the country, with the co-circulation of multiple virus lineages. Additionally, we examine the historical climate data, revealing long-term trends towards higher theoretical potential for dengue transmission due to rising temperatures. CONCLUSION: This multidisciplinary study reveals intricate patterns of dengue virus transmission in the Dominican Republic, characterized by the co-circulation of multiple DENV lineages and a novel transmission pathway. The observed correlation between rising temperatures and increased dengue transmission potential emphasizes the need for integrated climate-informed strategies in dengue control efforts. Our findings offer critical insights for public health authorities in the Dominican Republic and similar settings, guiding resource allocation and the development of preparedness strategies to mitigate the impacts of climate change on dengue transmission.

Dengue virus non-structural protein 3 inhibits mitochondrial respiration by impairing complex I function.

Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

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.

Mosquito (Diptera: Culicidae) diversity and arbovirus detection across an urban and agricultural landscape.

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.