Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations.

Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Antigenic distance between primary and secondary dengue infections correlates with disease risk.

Many pathogens continuously change their protein structure in response to immune-driven selection, resulting in weakened protection even in previously exposed individuals. In addition, for some pathogens, such as dengue virus, poorly targeted immunity is associated with increased risk of severe disease through a mechanism known as antibody-dependent enhancement. However, it remains unclear whether the antigenic distances between an individual's first infection and subsequent exposures dictate disease risk, explaining the observed large-scale differences in dengue hospitalizations across years. Here, we develop a framework that combines detailed antigenic and genetic characterization of viruses with details on hospitalized cases from 21 years of dengue surveillance in Bangkok, Thailand, to identify the role of the antigenic profile of circulating viruses in determining disease risk. We found that the risk of hospitalization depended on both the specific order of infecting serotypes and the antigenic distance between an individual's primary and secondary infections, with risk maximized at intermediate antigenic distances. These findings suggest that immune imprinting helps determine dengue disease risk and provide a pathway to monitor the changing risk profile of populations and to quantifying risk profiles of candidate vaccines.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Circulating serotypes and genotypes of dengue virus in North India: An observational study.

BACKGROUND OBJECTIVES: This study reports observation on circulating serotypes and genotypes of Dengue Virus in North India. METHODS: Serum samples were obtained from suspected cases of dengue referred to the virus diagnostic laboratory during 2014 to 2022. All samples were tested for anti-dengue virus IgM antibodies and NS1Ag by ELISA. NS1Ag positive samples were processed for serotyping and genotyping. RESULTS: Total 41,476 dengue suspected cases were referred to the laboratory of which 12,292 (29.6%) tested positive. Anti-Dengue Virus IgM antibodies, NS1Ag, both IgM and NS1Ag, were positive in 7007 (57.4%); 3200 (26.0%) and 2085 (16.0%) cases respectively. Total 762 strains were serotyped during 9-year period. DENV-1, DENV-2, DENV-3 and DENV-4 serotypes were found in 79 (10.37%), 506 (66.40%), 151 (19.82%) and 26 (3.41%) cases respectively. DENV-1, DENV-2 and DENV-3 were in circulation throughout. Total 105 strains were genotyped. Genotype IV of DENV-1 serotype was circulating till 2014 which was later replaced by genotype V. A distinct seasonality with increase in number of cases in post-monsoon period was seen. INTERPRETATION CONCLUSION: DENV-1, DENV-2 and DENV-3 were found to be in circulation in North India. Predominant serotype/genotype changed at times, but not at regular intervals.

Dengue virus pathogenesis and host molecular machineries.

Dengue viruses (DENV) are positive-stranded RNA viruses belonging to the Flaviviridae family. DENV is the causative agent of dengue, the most rapidly spreading viral disease transmitted by mosquitoes. Each year, millions of people contract the virus through bites from infected female mosquitoes of the Aedes species. In the majority of individuals, the infection is asymptomatic, and the immune system successfully manages to control virus replication within a few days. Symptomatic individuals may present with a mild fever (Dengue fever or DF) that may or may not progress to a more critical disease termed Dengue hemorrhagic fever (DHF) or the fatal Dengue shock syndrome (DSS). In the absence of a universally accepted prophylactic vaccine or therapeutic drug, treatment is mostly restricted to supportive measures. Similar to many other viruses that induce acute illness, DENV has developed several ways to modulate host metabolism to create an environment conducive to genome replication and the dissemination of viral progeny. To search for new therapeutic options, understanding the underlying host-virus regulatory system involved in various biological processes of the viral life cycle is essential. This review aims to summarize the complex interaction between DENV and the host cellular machinery, comprising regulatory mechanisms at various molecular levels such as epigenetic modulation of the host genome, transcription of host genes, translation of viral and host mRNAs, post-transcriptional regulation of the host transcriptome, post-translational regulation of viral proteins, and pathways involved in protein degradation.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Genotype of dengue virus serotype 1 in relation to severe dengue in Guangzhou, China.

Guangzhou has been the city most affected by the dengue virus (DENV) in China, with a predominance of DENV serotype 1 (DENV-1). Viral factors such as dengue serotype and genotype are associated with severe dengue (SD). However, none of the studies have investigated the relationship between DENV-1 genotypes and SD. To understand the association between DENV-1 genotypes and SD, the clinical manifestations of patients infected with different genotypes were investigated. A total of 122 patients with confirmed DENV-1 genotype infection were recruited for this study. The clinical manifestations, laboratory tests, and levels of inflammatory mediator factors were statistically analyzed to investigate the characteristics of clinical manifestations and immune response on the DENV-1 genotype. In the case of DENV-1 infection, the incidence of SD with genotype V infection was significantly higher than that with genotype I infection. Meanwhile, patients infected with genotype V were more common in ostealgia and bleeding significantly. In addition, levels of inflammatory mediator factors including IFN-γ, TNF-α, IL-10, and soluble vascular cell adhesion molecule 1 were higher in patients with SD infected with genotype V. Meanwhile, the concentrations of regulated upon activation normal T-cell expressed and secreted and growth-related gene alpha were lower in patients with SD infected with genotype V. The higher incidence of SD in patients infected with DENV-1 genotype V may be attributed to elevated cytokines and adhesion molecules, along with decreased chemokines.

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.

Molecular and Entomological Characterization of 2023 Dengue Outbreak in Dhading District, Central Nepal.

In 2023, Nepal faced its second largest dengue outbreak ever, following a record-breaking number of dengue cases in 2022, characterized by the expansion of infections into areas of higher altitudes. However, the characteristics of the 2023 circulating dengue virus (DENV) and the vector density remain poorly understood. Therefore, we performed DENV serotyping, clinical and laboratory assessment, and entomological analysis of the 2023 outbreak in central Nepal. A total of 396 fever cases in Dhading hospital suspected of being DENV positive were enrolled, and blood samples were collected and tested by different techniques including PCR. Of these, 278 (70.2%) had confirmed DENV infection. Multiple serotypes (DENV-1, -2, and -3) were detected. DENV-2 (97.5%) re-emerged after six years in Dhading while DENV-3 was identified for the first time. Dengue inpatients had significantly higher frequency of anorexia, myalgia, rash, diarrhea, nausea, vomiting, abdominal pain, and thrombocytopenia (p < 0.05). In this area, Aedes mosquitoes largely predominated (90.7%) with the majority being A. aegypti (60.7%). We also found high levels of Aedes index (20.0%) and container index (16.7%). We confirmed multiple DENV serotype circulation with serotype re-emergence and new serotype introduction, and high vector density in 2023. These findings call for the urgent initiation and scaling up of DENV molecular surveillance in human and mosquito populations for dengue control and prevention in Nepal.

Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

Isolation and molecular detection of dengue and chikungunya virus from field-collected adult mosquitoes in Kelantan, Malaysia.

BACKGROUND OBJECTIVES: Dengue and chikungunya infections are one of the major health problems that have plagued the human population globally. All dengue virus (DENV) serotypes circulate within Malaysia with particular serotypes dominating in different years/outbreaks. In the state of Kelantan, an increasing number of DENV and chikungunya virus (CHIKV) new cases have been reported, including several deaths. This study aimed to isolate and detect these arboviruses from adult mosquitoes in Kelantan. METHODS: Adult mo squito samples were collected from January to August 2019 and were identified according to gender, species and locality. The isolation of the virus was done in C6/36 cells. Dengue NS1 antigen was carried out using direct mosquito lysate and mosquito culture supernatant. Detection and serotyping of the DENV was performed using multiplex RT-PCR and CHIKV detection using a one-step RT-PCR assay. RESULTS: Of 91 mosquito pools, four were positive for NS1 antigen comprising two pools (2.2%) of male Ae. albopictus (Pulau Melaka and Kubang Siput) and two pools (2.2%) of Ae. aegypti (Kampung Demit Sungai). DENV 1 was detected in one pool (0.9%) of female Ae. albopictus among 114 tested Aedes pools. Two pools of 114 pools (1.7%) from both male Aedes species were positive with double serotypes, DENV 1 and DENV 2 (Pulau Melaka). However, no pool was positive for CHIKV. INTERPRETATION CONCLUSION: The presence of DENV and the main vectors of arboviruses in Kelantan are pertinent indicators of the need to improve vector controls to reduce arbovirus infections among people in the localities.

Dual action effects of ethyl-p-methoxycinnamate against dengue virus infection and inflammation via NF-κB pathway suppression.

Dengue virus (DENV) infection can lead to severe outcomes through a virus-induced cytokine storm, resulting in vascular leakage and inflammation. An effective treatment strategy should target both virus replication and cytokine storm. This study identified Kaempferia galanga L. (KG) extract as exhibiting anti-DENV activity. The major bioactive compound, ethyl-p-methoxycinnamate (EPMC), significantly reduced DENV-2 infection, virion production, and viral protein synthesis in HepG2 and A549 cells, with half-maximal effective concentration (EC50) values of 22.58 µM and 6.17 µM, and impressive selectivity indexes (SIs) of 32.40 and 173.44, respectively. EPMC demonstrated efficacy against all four DENV serotypes, targeting the replication phase of the virus life cycle. Importantly, EPMC reduced DENV-2-induced cytokines (IL-6 and TNF-α) and chemokines (RANTES and IP-10), as confirmed by immunofluorescence and immunoblot analyses, indicating inhibition of NF-κB activation. EPMC's role in preventing excessive inflammatory responses suggests it as a potential candidate for dengue treatment. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness for EPMC were predicted using SwissADME and ProTox II servers, showing good drug-like properties without toxicity. These findings highlight KG extract and EPMC as promising candidates for future anti-dengue therapeutics, offering a dual-action approach by inhibiting virus replication and mitigating inflammatory reactions.

Low-dose dengue virus 3 human challenge model: a phase 1 open-label study.

Dengue human infection models present an opportunity to explore the potential of a vaccine, anti-viral or immuno-compound for clinical benefit in a controlled setting. Here we report the outcome of a phase 1 open-label assessment of a low-dose dengue virus 3 (DENV-3) challenge model (NCT04298138), in which nine participants received a subcutaneous inoculation with 0.5 ml of a 1.4 × 103 plaque-forming unit per ml suspension of the attenuated DENV-3 strain CH53489. The primary and secondary endpoints of the study were to assess the safety of this DENV-3 strain in healthy flavivirus-seronegative individuals. All participants developed RNAaemia within 7 days after inoculation with peak titre ranging from 3.13 × 104 to 7.02 × 108 genome equivalents per ml. Solicited symptoms such as fever and rash, clinical laboratory abnormalities such as lymphopenia and thrombocytopenia, and self-reported symptoms such as myalgia were consistent with mild-to-moderate dengue in all volunteers. DENV-3-specific seroconversion and memory T cell responses were observed within 14 days after inoculation as assessed by enzyme-linked immunosorbent assay and interferon-gamma-based enzyme-linked immunospot. RNA sequencing and serum cytokine analysis revealed anti-viral responses that overlapped with the period of viraemia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual's peak viral titre.

Molecular mechanisms in the pathogenesis of dengue infections.

Dengue is the most rapidly emerging climate-sensitive infection, and morbidity/mortality and disease incidence are rising markedly, leading to healthcare systems being overwhelmed. There are currently no specific treatments for dengue or prognostic markers to identify those who will progress to severe disease. Owing to an increase in the burden of illness and a change in epidemiology, many patients experience severe disease. Our limited understanding of the complex mechanisms of disease pathogenesis has significantly hampered the development of safe and effective treatments, vaccines, and biomarkers. We discuss the molecular mechanisms of dengue pathogenesis, the gaps in our knowledge, and recent advances, as well as the most crucial questions to be answered to enable the development of therapeutics, biomarkers, and vaccines.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1ß in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1ß. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Travel surveillance uncovers dengue virus dynamics and introductions in the Caribbean.

Dengue is the most prevalent mosquito-borne viral disease in humans, and cases are continuing to rise globally. In particular, islands in the Caribbean have experienced more frequent outbreaks, and all four dengue virus (DENV) serotypes have been reported in the region, leading to hyperendemicity and increased rates of severe disease. However, there is significant variability regarding virus surveillance and reporting between islands, making it difficult to obtain an accurate understanding of the epidemiological patterns in the Caribbean. To investigate this, we used travel surveillance and genomic epidemiology to reconstruct outbreak dynamics, DENV serotype turnover, and patterns of spread within the region from 2009-2022. We uncovered two recent DENV-3 introductions from Asia, one of which resulted in a large outbreak in Cuba, which was previously under-reported. We also show that while outbreaks can be synchronized between islands, they are often caused by different serotypes. Our study highlights the importance of surveillance of infected travelers to provide a snapshot of local introductions and transmission in areas with limited local surveillance and suggests that the recent DENV-3 introductions may pose a major public health threat in the region.

Cross Talk between MicroRNAs and Dengue Virus.

Dengue fever (DF) is an endemic infectious tropical disease and is rapidly becoming a global problem. Dengue fever is caused by one of the four dengue virus (DENV) serotypes and is spread by the female Aedes mosquito. Clinical manifestations of DF may range from asymptomatic to life-threatening severe illness with conditions of hemorrhagic fever and shock. Early and precise diagnosis is vital to avoid mortality from DF. A different approach is required to combat DF because of the challenges with the vaccines currently available, which are nonspecific; each is capable of causing cross-reaction and disease-enhancing antibody responses against the residual serotypes. MicroRNAs (miRNAs) are known to be implicated in DENV infection and are postulated to be involved in most of the host responses. Thus, they might be a suitable target for new strategies against the disease. The involvement of miRNAs in cellular activities and pathways during viral infections has been explored under numerous conditions. Interestingly, miRNAs have also been shown to be involved in viral replication. In this review, we summarize the role of known miRNAs, specifically the role of miRNA Let-7c (miR-Let-7c), miR-133a, miR-30e, and miR-146a, in the regulation of DENV replication and their possible effects on the initial immune reaction.

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection.

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.