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Background: Yellow fever virus (YFV) and dengue virus (DENV) are among the major re-emerging arboviruses that pose a significant threat to public health. Their associated burden and prevalence can be substantially underestimated due to insufficient surveillance and inadequate diagnosis. This study aimed to determine evidence of dengue, yellow and related flaviviruses circulation among the rural human populations residing in Nguruman (Kajiado County) and Kerio Valley (Baringo County), two dryland ecosystems in the Kenyan Rift Valley. Methods: Serum samples obtained from febrile patients between 5 and 85 years through a hospital-based cross-sectional survey from July 2020 – May 2023, were screened for neutralizing antibodies to YFV, DENV-2 and related flaviviruses, West Nile virus (WNV) and Zika virus (ZIKV) via Plaque reduction neutralization test (PRNT). The study sites and important demographic characteristics were obtained using a structural questionnaire and the data analyzed and seroprevalence compared. A multinomial logistic regression model was done to predict risk for each of the most prevalent viruses with covariates; age, gender, and occupation. Results: Overall, 54.5% (50.1–59.0% 95% confidence interval (CI) of the samples tested positive for at least one of the four Flaviviruses. The percentage was significantly higher in Kerio Valley (64.34%, 184/286) than in Nguruman (40.2%, 78/194) (P<0.0001). YFV had the highest prevalence, followed by WNV (16.25%), ZIKV (5.2%), and DENV-2 (1%). Kerio Valley had a significantly higher YFV seroprevalence (51%) than Nguruman (6%) (P<0.0001), while DENV-2 was observed only in Nguruman with a low seropositivity of 2%. In contrast to Nguruman, where seropositivity rates were higher in males at 47.47% (P=0.049), in Kerio Valley, females showed considerably higher viral seropositivity at 60.82% than males (P<0001). Conclusion: The study suggests that there is significant circulation of Flaviviruses in both regions, posing a public health risk, that could potentially contribute to clinical disease. However, seropositivity rates vary for each specific site. Furthermore, there could be a risk of YFV, WNV, and ZIKV transmission in both sites with DENV transmission specifically noted in Nguruman. The study findings inform direct cost-effective actions (such as YF vaccines) and precise surveillance data of vector populations for improved disease risk prediction. [ABSTRACT FROM AUTHOR]

Flavivirus infection is tightly connected to host lipid metabolism. Here, we performed shotgun lipidomics of cells infected with neurotropic Zika, West Nile, and tick-borne encephalitis virus, as well as dengue and yellow fever virus. Early in infection specific lipids accumulate, e.g., neutral lipids in Zika and some lysophospholipids in all infections. Ceramide levels increase following infection with viruses that cause a cytopathic effect. In addition, fatty acid desaturation as well as glycerophospholipid metabolism are significantly altered. Importantly, depletion of enzymes involved in phosphatidylserine metabolism as well as phosphatidylinositol biosynthesis reduce orthoflavivirus titers and cytopathic effects while inhibition of fatty acid monounsaturation only rescues from virus-induced cell death. Interestingly, interfering with ceramide synthesis has opposing effects on virus replication and cytotoxicity depending on the targeted enzyme. Thus, lipid remodeling by orthoflaviviruses includes distinct changes but also common patterns shared by several viruses that are needed for efficient infection and replication. Viruses need host cell lipids for multiplication. Here, the authors use lipidomics to show that lipid remodeling by orthoflaviviruses includes distinct changes but also shared patterns, some of which are needed for efficient viral replication. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junı́n mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts. [ABSTRACT FROM AUTHOR]

The annual number of reported human cases of flavivirus infections continues to increase. Measures taken by local healthcare systems and international organizations are not fully successful. In this regard, new approaches to treatment and prevention of flavivirus infections are relevant. One promising approach is to use monoclonal antibody preparations. The mouse mAb 10H10 is capable of interacting with viruses belonging to the genus Orthoflavivirus which are pathogenic to humans. ELISA and molecular modeling data can indicate that mAb 10H10 recognizes the fusion loop region of E protein. The KD of interaction between the mAb 10H10 and recombinant analogs of the E protein of the tick-borne encephalitis (TBEV), Zika (ZIKV) and dengue (DENV) viruses range from 1.5 to 4 nM. The aim of this study was to map the epitope of this antibody using phage display technology. After three rounds of biopanning, 60 individual phage clones were chosen. The amino acid sequences of the selected peptides were conveniently divided into five groups. Based on the selected peptides, bacteriophages were obtained carrying peptides on the surfaces of the pIII and pVIII proteins, which were tested for binding to the antibody in ELISA. Thus, the epitope of the mAb 10H10 is the highly conserved region 98-DRGWGNXXGLFGK-110 of the flavivirus E protein. The structures of the complexes of the identified peptides with the antibody paratope are proposed using the molecular docking and dynamics methods. [ABSTRACT FROM AUTHOR]

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Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN–induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3′–5′ exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3′ untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms. Here, the authors identify in the 3′ untranslated region of the genome of Usutu virus, an emerging mosquito-borne flavivirus, a sequence that confers a unique capacity to resist degradation by the ISG20 exonuclease. [ABSTRACT FROM AUTHOR]

Norbelladine derivatives have garnered attention in recent years due to their diverse biological activities and pivotal role in the biosynthetic pathway of Amaryllidaceae alkaloids. This study reports the synthesis and biological evaluation of four O,N-methylated derivatives of norbelladine. These derivatives were synthesized through a three-step process: forming imine intermediates from benzaldehydes with tyramine, hydrogenating them to secondary amines, and N-methylating these amines. The products were purified and characterized by 1H and 13C NMR spectroscopy. Their biological activities were assessed by evaluating their ability to inhibit Alzheimer's disease-related enzymes acetylcholinesterase and butyrylcholinesterase. Additionally, the cytotoxic activity of the novel derivatives was tested against cancer cell lines derived from hepatocarcinoma (Huh7), adenocarcinoma (HCT-8), and acute myeloid leukemia (THP-1) cells, and their antiviral properties against a human coronavirus (HCoV-OC43), a flavivirus (dengue virus), and a lentivirus (pseudotyped HIV-1). Docking analysis was performed to understand the impact of the N-methylation on their pharmacological relevance. The results indicate that while N-methylation does not significantly affect antiviral activity, it enhances butyrylcholinesterase inhibition for N-methylnorbelladine and 4′-O,N-dimethylnorbelladine. Overall, this work enhances our understanding of norbelladine derivatives, provides new tools for Alzheimer's disease research, and lays the groundwork for future pharmaceutical developments. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are molecules that influence messenger RNA (mRNA) expression levels by binding to the 3′ untranslated region (3′ UTR) of target genes. Host miRNAs can influence flavivirus replication, either by inducing changes in the host transcriptome or by directly binding to viral genomes. The 3′ UTR of the flavivirus genome is a conserved region crucial for viral replication. Cells might exploit this well-preserved region by generating miRNAs that interact with it, ultimately impacting viral replication. Despite significant efforts to identify miRNAs capable of arresting viral replication, the potential of all these miRNAs to interact with the flavivirus 3′ UTR is still poorly characterised. In this context, bioinformatic tools have been proposed as a fundamental part of accelerating the discovery of interactions between miRNAs and the 3′ UTR of viral genomes. In this study, we performed a computational analysis to reveal potential miRNAs from human and mosquito species that bind to the 3′ UTR of flaviviruses. In humans, miR-6842 and miR-661 were found, while in mosquitoes, miR-9-C, miR-2945-5p, miR-11924, miR-282-5p, and miR-79 were identified. These findings open new avenues for studying these miRNAs as antivirals against flavivirus infections. [ABSTRACT FROM AUTHOR]

High-throughput experiments often produce ranked gene outputs, with forward genetic screening being a notable example. While there are various tools for analyzing individual datasets, those that perform comparative and meta-analytical examination of such ranked gene lists remain scarce. Here, we introduce Gene Rank Meta Analyzer (GeneRaMeN), an R Shiny tool utilizing rank statistics to facilitate the identification of consensus, unique, and correlated genes across multiple hit lists. We focused on two key topics to showcase GeneRaMeN: virus host factors and cancer dependencies. Using GeneRaMeN 'Rank Aggregation', we integrated 24 published and new flavivirus genetic screening datasets, including dengue, Japanese encephalitis, and Zika viruses. This meta-analysis yielded a consensus list of flavivirus host factors, elucidating the significant influence of cell line selection on screening outcomes. Similar analysis on 13 SARS-CoV-2 CRISPR screening datasets highlighted the pivotal role of meta-analysis in revealing redundant biological pathways exploited by the virus to enter human cells. Such redundancy was further underscored using GeneRaMeN's 'Rank Correlation', where a strong negative correlation was observed for host factors implicated in one entry pathway versus the alternate route. Utilizing GeneRaMeN's 'Rank Uniqueness', we analyzed human coronaviruses 229E, OC43, and SARS-CoV-2 datasets, identifying host factors uniquely associated with a defined subset of the screening datasets. Similar analyses were performed on over 1000 Cancer Dependency Map (DepMap) datasets spanning 19 human cancer types to reveal unique cancer vulnerabilities for each organ/tissue. GeneRaMeN, an efficient tool to integrate and maximize the usability of genetic screening datasets, is freely accessible via https://ysolab.shinyapps.io/GeneRaMeN. [ABSTRACT FROM AUTHOR]

Bagaza virus (BAGV) is a flavivirus that affects avian species. In Europe, it was detected for the first time in Spain in 2010, exhibiting high genetic relatedness to Israel turkey meningoencephalomyelitis virus (ITMV) isolates from Israel. After a period of epidemiological silence, BAGV re-emerged, causing important outbreaks in 2019 and 2021. This study aims to characterize the newly detected strains and to elucidate if these recent outbreaks were caused by single or different virus introductions into the country. Hence, Spanish BAGV isolates from 2019 (n = 3) and 2021 (n = 1) outbreaks, obtained from red-legged partridges in Cádiz, were sequenced and further characterized. The phylogenetic analyses showed that they belong to two different genotypes: BAGV-Genotypes 1 and 2. Isolates from 2019 belong to BAGV-Genotype 1, closely related to isolates from Senegal, where BAGV has been circulating for decades. In turn, the 2021 isolates belong to BAGV-Genotype 2, closely related to those detected in Spain in 2010. Additionally, the comparison of the viral polyproteins of several BAGV isolates from both genotypes supports and confirms the phylogenetic findings. To conclude, BAGV has been introduced into Spain on at least three independent occasions, with alternating genetic clades, thus confirming that BAGV is able to sporadically reach Southern Europe. [ABSTRACT FROM AUTHOR]

Flaviviruses pose significant global health threats, infecting over 300 million people annually. Among their evasion strategies, the production of subgenomic flaviviral RNAs (sfRNAs) from the 3′ UTR of viral genomes is particularly notable. Utilizing a comprehensive in silico approach with the catRAPID algorithm, we analyzed over 300,000 interactions between sfRNAs and human proteins derived from more than 8000 flavivirus genomes, including Dengue, Zika, Yellow Fever, West Nile, and Japanese Encephalitis viruses. By providing the first extensive atlas of sfRNA interactions, we offer new insights into how flaviviruses can manipulate host cellular machinery to facilitate viral survival and persistence. Our study not only validated known interactions but also revealed novel human proteins that could be involved in sfRNA-mediated host defense evasion, including helicases, splicing factors, and chemokines. These findings significantly expand the known interactome of sfRNAs with human proteins, underscoring their role in modulating host cellular pathways. Intriguingly, we predict interaction with stress granules, a critical component of the cellular response to viral infection, suggesting a mechanism by which flaviviruses inhibit their formation to evade host defenses. Moreover, a set of highly-interacting proteins in common among the sfRNAs showed predictive power to identify sfRNA-forming regions, highlighting how protein signatures could be used to annotate viruses. This atlas not only serves as a resource for exploring therapeutic targets but also aids in the identification of sfRNA biomarkers for improved flavivirus diagnostics.

The basis for criteria of the taxonomic classification of DNA and RNA viruses based on data of the genomic sequencing are viewed in this review. The genomic sequences of viruses, which have genome represented by double-stranded DNA (orthopoxviruses as example), positive-sense single-stranded RNA (alphaviruses and flaviviruses as example), non-segmented negative-sense single-stranded RNA (filoviruses as example), segmented negative-sense single-stranded RNA (arenaviruses and phleboviruses as example) are analyzed. The levels of genetic variability that determine the assignment of compared viruses to taxa of various orders are established for each group of viruses.

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-β family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses. Author summary: The Flaviviridae family, which includes Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV), possesses a single-stranded positive-sense RNA and conducts viral RNA replication, translation, and particle formation in the endoplasmic reticulum. The core proteins of flaviviruses, which form viral capsid, have been recognized to localize in not only the cytoplasm but also the nucleus, particularly in the nucleolus. Although this nuclear translocation of the core proteins is conserved within the Flaviviridae family, the underlying molecular mechanisms and their contribution to the viral life cycle remain largely unknown. Our in vitro biochemical assay and mass spectrometry analysis identified IPO7 as a nuclear carrier for Flaviviridae core proteins, such as those in JEV, DENV, ZIKV, and WNV. Moreover, we found that IPO7-mediated nuclear translocation of core proteins is important for the release of infectious viral particles. Our data suggest that the Flaviviridae family evolved to use the IPO7-mediated translocation of core proteins for efficient viral production. Therefore, IPO7 might be a target for the development of antiviral drugs against a broad range of Flaviviruses. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) is a neurotropic member of the genus Orthoflavivirus (former Flavivirus) and is of significant health concern in Europe and Asia. TBEV pathogenesis may occur directly via virus-induced damage to neurons or through immunopathology due to excessive inflammation. While primary cells isolated from the host can be used to study the immune response to TBEV, it is still unclear how well these reflect the immune response elicited in vivo. Here, we compared the transcriptional response to TBEV and the less pathogenic tick-borne flavivirus, Langat virus (LGTV), in primary monocultures of neurons, astrocytes and microglia in vitro, with the transcriptional response in vivo captured by single-nuclei RNA sequencing (snRNA-seq) of a whole mouse cortex. We detected similar transcriptional changes induced by both LGTV and TBEV infection in vitro, with the lower response to LGTV likely resulting from slower viral kinetics. Gene set enrichment analysis showed a stronger transcriptional response in vivo than in vitro for astrocytes and microglia, with a limited overlap mainly dominated by interferon signaling. Together, this adds to our understanding of neurotropic flavivirus pathogenesis and the strengths and limitations of available model systems. [ABSTRACT FROM AUTHOR]

Flaviviruses comprise a large number of arthropod-borne viruses, some of which are associated with life-threatening diseases. Flavivirus infections are rising worldwide, mainly due to the proliferation and geographical expansion of their vectors. The main human pathogens are mosquito-borne flaviviruses, including dengue virus, Zika virus, and West Nile virus, but tick-borne flaviviruses are also emerging. As with any viral infection, the body's first line of defense against flavivirus infections is the innate immune defense, of which type I interferon is the armed wing. This cytokine exerts its antiviral activity by triggering the synthesis of hundreds of interferon-induced genes (ISGs), whose products can prevent infection. Among the ISGs that inhibit flavivirus replication, certain tripartite motif (TRIM) proteins have been identified. Although involved in other biological processes, TRIMs constitute a large family of antiviral proteins active on a wide range of viruses. Furthermore, whereas some TRIM proteins directly block viral replication, others are positive regulators of the IFN response. Therefore, viruses have developed strategies to evade or counteract TRIM proteins, and some even hijack certain TRIM proteins to their advantage. In this review, we summarize the current state of knowledge on the interactions between flaviviruses and TRIM proteins, covering both direct and indirect antiviral mechanisms. [ABSTRACT FROM AUTHOR]

Background and Objectives: West Nile virus (WNV) and Usutu virus (USUV) are mosquito‐borne flaviviruses (Flaviviridae) that originated in Africa, have expanded their geographical range during the last decades and caused documented infections in Europe in the last years. Acute WNV and USUV infections have been detected in asymptomatic blood donors by nucleic acid testing. Thus, inactivation of both viral pathogens before blood transfusion is necessary to ensure blood product safety. This study aimed to investigate the efficacy of the THERAFLEX UV‐Platelets system to inactivate WNV and USUV in platelet concentrates (PCs). Materials and Methods: Plasma‐reduced PCs were spiked with the virus suspension. Spiked PC samples were taken after spiking (load and hold sample) and after UVC illumination on the Macotronic UV illumination machine with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) J/cm2). Virus loads of WNV and USUV before and after illumination were measured by titration. Results: Infectivity assays showed that UVC illumination inactivated WNV and USUV in a dose‐dependent manner. At a UVC dose of 0.2 J/cm2, the WNV titre was reduced by a log10 factor of 3.59 ± 0.43 for NY99 (lineage 1) and 4.40 ± 0.29 for strain ED‐I‐33/18 (lineage 2). USUV titres were reduced at the same UVC dose by a log10 factor of 5.20 ± 0.70. Conclusions: Our results demonstrate that the THERAFLEX UV‐Platelets procedure is an effective technology to inactivate WNV and USUV in contaminated PCs. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that has been associated with congenital neurological defects in fetuses born to infected mothers. At present, no vaccine or antiviral therapy is available to combat this devastating disease. Repurposing drugs that target essential host factors exploited by viruses is an attractive therapeutic approach. Here, we screened a panel of clinically approved small-molecule kinase inhibitors for their antiviral effects against a clinical isolate of ZIKV and thoroughly characterized their mechanisms of action. We found that the Raf kinase inhibitors Dabrafenib and Regorafenib potently impair the replication of ZIKV, but not that of its close relative dengue virus. Time-of-addition experiments showed that both inhibitors target ZIKV infection at post-entry steps. We found that Dabrafenib, but not Regorafenib, interfered with ZIKV genome replication by impairing both negative- and positive-strand RNA synthesis. Regorafenib, on the other hand, altered steady-state viral protein levels, viral egress, and blocked NS1 secretion. We also observed Regorafenibinduced ER fragmentation in ZIKV-infected cells, which might contribute to its antiviral effects. Because these inhibitors target different steps of the ZIKV infection cycle, their use in combination therapy may amplify their antiviral effects which could be further explored for future therapeutic strategies against ZIKV and possibly other flaviviruses. IMPORTANCE There is an urgent need to develop effective therapeutics against re-emerging arboviruses associated with neurological disorders like Zika virus (ZIKV). We identified two FDA-approved kinase inhibitors, Dabrafenib and Regorafenib, as potent inhibitors of contemporary ZIKV strains at distinct stages of infection despite overlapping host targets. Both inhibitors reduced viral titers by ~1 to 2 log10 (~10-fold to 100-fold) with minimal cytotoxicity. Furthermore, we show that Dabrafenib inhibits ZIKV RNA replication whereas Regorafenib inhibits ZIKV translation and egress. Regorafenib has the added benefit of limiting NS1 secretion, which contributes to the pathogenesis and disease progression of several flaviviruses. Because these inhibitors affect distinct post-entry steps of ZIKV infection, their therapeutic potential may be amplified by combination therapy and likely does not require prophylactic administration. This study provides further insight into ZIKV-host interactions and has implications for the development of novel antivirals against ZIKV and possibly other flaviviruses. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain. [ABSTRACT FROM AUTHOR]

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein. [ABSTRACT FROM AUTHOR]

The co-circulation of West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV) in Europe is a public health concern, and the lack of active surveillance programs and high-quality specific serology tests make it difficult to estimate the true disease burden. The potential for introducing other pathogens, such as the related Japanese encephalitis virus (JEV), could further complicate this picture. The standard method for diagnosing flavivirus infections involves RT-(q)PCR to detect the viral RNA during the acute phase of the disease, followed by antibody detection in the convalescence phase. Both methods suffer from several limitations that make flavivirus diagnosis challenging, especially in areas of co-circulation. Non-structural protein 1 (NS1) represents a promising marker for discriminating between co-circulating flaviviruses. However, NS1 antigen-capture tests are lacking on the commercial market for WNV, USUV, TBEV, and JEV. In recent decades, the presence of flaviviruses of concern for human health in Europe has drastically increased,exacerbated by the effects of climate change – which has allowed the vectors of these viruses to expand into new territories. Co-circulation of West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV) represents a threat to the European continent, and this is further complicated by the difficulty of obtaining an early and discriminating diagnosis of infection. Moreover, the possibility of introducing non-endemic pathogens, such as Japanese encephalitis virus (JEV), further complicates accurate diagnosis. Current flavivirus diagnosis is based mainly on RT-PCR and detection of virus-specific antibodies. Yet, both techniques suffer from limitations, and the development of new assays that can provide an early, rapid, low-cost, and discriminating diagnosis of viral infection is warranted. In the pursuit of ideal diagnostic assays, flavivirus non-structural protein 1 (NS1) serves as an excellent target for developing diagnostic assays based on both the antigen itself and the antibodies produced against it. This review describes the potential of such NS1-based diagnostic methods, focusing on the application of flaviviruses that co-circulate in Europe. [ABSTRACT FROM AUTHOR]

Objectives: Tick-borne encephalitis virus Siberian subtype (TBEV-Sib) and Omsk hemorrhagic fever virus (OHFV) are causative agents of natural focal infections in Western Siberia, Russia. The distribution of TBEV phylogenetic lineages and OHFV in the Kemerovo Region of Western Siberia remains poorly investigated. Methods: The phylogenetic analyses of fragment genome sequences 26 flaviviruses identified in 2019 were performed, and the amino acid variation was determined to reveal to which clusteron they belong. The age of Baltic and Asian lineages of the TBEV-Sib was calculated for Kemerovo District and Region, respectively. Results: Twenty-five isolates were members of three TBEV-Sib phylogenetic lineages: Baltic (48%), Asian (36%), and East Siberian (16%). The Baltic lineage's eastern boundary is commonly thought to be in the Novosibirsk Region, but our data suggest that it may reach further east. Analysis of the Baltic lineage clusteron structure showed that the isolates found are unique (6) or belong to clusteron-founder 3D (1) and derived clusteron 3O (5). Based on the age of 3O clusteron, Baltic lineage could have appeared in the Kemerovo Region by the late 1970s. One of the isolated viruses turned out to be the OHFV of the first subtype and not to belong to any known clusteron. This finding is the first known detection of the virus outside the endemic area of Russia. Given the recent discovery of OHFV in Kazakhstan, it can be assumed that the area of this virus distribution is much wider than previously thought. Conclusions: This report provides insights into the population structure of TBEV and OHFV, which may be helpful for epidemiological investigation and surveillance of the viruses. [ABSTRACT FROM AUTHOR]

Live-attenuated flavivirus vaccines confer long-term protection against disease, but the design of attenuated flaviviruses does not follow a general approach. The non-coding, subgenomic flavivirus RNA (sfRNA) is produced by all flaviviruses and is an essential factor in viral pathogenesis and transmission. We argue that modulating sfRNA expression is a promising, universal strategy to finetune flavivirus attenuation for developing effective flavivirus vaccines of the future. [ABSTRACT FROM AUTHOR]

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections. Author summary: Zika virus (ZIKV) belongs to vector-borne flaviviruses, associated with Guillain-Barré syndrome and neonatal microcephaly. Stress granules (SGs) which forms under stress in eukaryotic cells can interfere with viral replication. Although previous studies have indicated that ZIKV can interfere with SG formation, the precise molecular mechanism and function remain unclear. Exploration of the mechanisms by which ZIKV antagonizes SG formation may help to identify new antiviral approaches. Here, we found that ZIKV NS2B bridge PP1α and eIF2α to promote eIF2α dephosphorylation and SG inhibition. Meanwhile, Reciprocal inhibition of K48-linked ubiquitination stabilized NS2B and PP1α to further facilitate the dephosphorylation of eIF2α and the concurrent inhibition of SG formation. Conversely, we demonstrated that a mutant form of NS2B, NS2BV35A, which interacted solely with eIF2α and not PP1α, attenuated virus replication and mitigated neurotoxicity in brain organoid development. The NS2B-PP1α-eIF2α axis reveals a novel biological function of NS2B as a general mechanism for flavivirus evasion of host antiviral effects, providing new therapeutic strategies for antiviral study. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material. [ABSTRACT FROM AUTHOR]

Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences. Zika and Dengue virus non-structural protein 5 can antagonise STAT2 modulating the host response and this interaction is involved in determining tropism. Here the authors show mammals independently evolved resistance to flavivirus NS5 multiple times, involving complex genetic changes in STAT2 which balance viral defence whilst maintaining STAT2's critical functions. [ABSTRACT FROM AUTHOR]

Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals. Developing broad antiviral drugs against dengue is challenging due to genetic diversity. Here, the authors generate PROTACs targeting the dengue virus envelope protein demonstrating improved potency and broadened activity against other mosquito-borne flaviviruses as compared to the parental inhibitors. [ABSTRACT FROM AUTHOR]

Background: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. Methods: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. Results: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. Conclusions: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections. [ABSTRACT FROM AUTHOR]

Mosquito‐borne diseases (MBDs) annually kill nearly half a million people. Due to the lack of effective vaccines and drugs on most MBDs, disease prevention relies primarily on controlling mosquitoes. Despite huge efforts having been put into mosquito control, eco‐friendly and sustainable mosquito‐control strategies are still lacking and urgently demanded. Most mosquito‐transmitted pathogens have lost the capacity of de novo nutrition biosynthesis, and rely on their vertebrate and invertebrate hosts for sustenance during the long‐term obligate parasitism process. Therefore, a better understanding of the metabolic interactions between mosquitoes and pathogens will contribute to the discovery of novel metabolic targets or regulators that lead to reduced mosquito populations or vector competence. This review summarizes the current knowledge about the effects of mosquito metabolism on the transmission of multiple pathogens. We also discuss that research in this area remains to be explored to develop multiple biological prevention and control strategies for MBDs. [ABSTRACT FROM AUTHOR]

Mosquito‐borne viruses (MBVs) are a large class of viruses transmitted mainly through mosquito bites, including dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and chikungunya virus, which pose a major threat to the health of people around the world. With global warming and extended human activities, the incidence of many MBVs has increased significantly. Mosquito saliva contains a variety of bioactive protein components. These not only enable blood feeding but also play a crucial role in regulating local infection at the bite site and the remote dissemination of MBVs as well as in remodeling the innate and adaptive immune responses of host vertebrates. Here, we review the physiological functions of mosquito salivary proteins (MSPs) in detail, the influence and the underlying mechanism of MSPs on the transmission of MBVs, and the current progress and issues that urgently need to be addressed in the research and development of MSP‐based MBV transmission blocking vaccines. [ABSTRACT FROM AUTHOR]

This document is a summary of a special issue of the journal "Viruses" focused on advances in research on alphaviruses and flaviviruses. The special issue includes research articles, review articles, and brief reports covering various aspects of these viruses. The research articles explore topics such as the susceptibility of chickens to Zika virus, serological misdiagnosis of certain viruses in Australia, the evolutionary history of dengue virus in Brazil, and the pathogenesis of Tembusu virus in mammals. The review articles discuss the mechanisms and environmental factors of yellow fever virus and the immune responses to chikungunya virus. The brief reports present a phylogenetic analysis of dengue virus serotypes and the development of a mouse infection model for vaccine testing. Overall, this special issue provides new insights into alphavirus and flavivirus research. [Extracted from the article]

The complete lack of yellow fever virus (YFV) in Asia, and the lack of urban YFV transmission in South America, despite the abundance of the peridomestic mosquito vector Aedes (Stegomyia.) aegypti is an enigma. An immunologically naïve population of over 2 billion resides in Asia, with most regions infested with the urban YF vector. One hypothesis for the lack of Asian YF, and absence of urban YF in the Americas for over 80 years, is that prior immunity to related flaviviruses like dengue (DENV) or Zika virus (ZIKV) modulates YFV infection and transmission dynamics. Here we utilized an interferon α/β receptor knock-out mouse model to determine the role of pre-existing dengue-2 (DENV-2) and Zika virus (ZIKV) immunity in YF virus infection, and to determine mechanisms of cross-protection. We utilized African and Brazilian YF strains and found that DENV-2 and ZIKV immunity significantly suppresses YFV viremia in mice, but may or may not protect relative to disease outcomes. Cross-protection appears to be mediated mainly by humoral immune responses. These studies underscore the importance of re-assessing the risks associated with YF outbreak while accounting for prior immunity from flaviviruses that are endemic. [ABSTRACT FROM AUTHOR]

Author Summary: The Powassan virus causes a nationally notifiable disease which can cause severe neurological disease in humans owing to the lack of approved vaccines or therapeutics. Although two distinct lineages circulate in North America, clinical differentiation does not typically occur because pathology has been assumed to be similar between lineages. In this work, a direct comparison of lineage I (Powassan virus) and lineage II (deer tick virus) demonstrated distinct differences in the clinical presentation, pathology of the central nervous system, and immune response in immunocompetent mice. These differences suggest that the deer tick virus and Powassan virus do not utilize the same mechanisms for neuroinvasion and dissemination within the CNS. This is clinically relevant as the development of treatments and therapeutics needs to be evaluated for these virus lineages. Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus. [ABSTRACT FROM AUTHOR]

Pyridine-containing amides of rimantadine (1-(1-adamantyl)ethylamine) were synthesized by the amidation of rimantadine hydrochloride by the corresponding acids in the NEt3—N,N′-dicyclohexylcarbodiimide (DCC)—4-(N,N-dimethylamino)pyridine (DMAP) system as the analogues of previously described antiviral compounds 1-adamantylmethyl-5-aminoisoxazole-3-carboxylate and N-{[5-(thien-2-yl)isoxazol-3-yl]methyl}adamantane-1-amine. The spectrum of antiviral activity against the rimantadine-resistant A/PR/8/34 strain of influenza A virus, tick-borne encephalitis virus (TBEV) strain Absettarov (European subtype), yellow fever virus (YFV) vaccine strain 17D, and West Nile virus (WNV) strain Strix nebulosa-12 was studied. The structure—activity relationships were discussed. Synthesized compounds efficiently inhibited the reproduction of TBEV and WNV. N-[1-(Adamantan-1-yl)ethyl]picolinamide efficiently inhibited the reproduction of enveloped viruses with both type I (influenza) and type II (flaviviruses) fusion proteins. The antiviral activity against the rimantadine-resistant influenza virus A/PR/8/34 and a low cytotoxicity (high selectivity index) were revealed for N-[1-(adamantan-1-yl)ethyl]-isonicotinamide. [ABSTRACT FROM AUTHOR]

Arthropod-borne diseases (ABDs) refer to a group of viral pathogens that affect a wide range of vertebrate hosts, including humans and non-human primates. In addition to being transmitted by mosquitoes and ticks, arthropods can also spread pathogens that cause severe human diseases. On the other hand, extracellular vesicles (EVs) can serve as cross-placental drug delivery vehicles (DDVs) to the fetus and even as antigen-presenting cells (APCs). To this end, the current review aimed to examine the role of small EVs (sEVs) in the transmission and inhibition of arthropod-borne viruses, also known as arboviruses. First, a deeper understanding of the mechanistic aspects of how these vesicles function during insect-pathogen interactions is required. Next, scalability and yield optimization must be addressed while introducing EV-based therapeutics on an industrial scale in order to implement them effectively. Finally,it is recommended to consider that sEV-mediated transfer plays a crucial role in the spread of ABDs. This is because it transfers pathogenic agents between cells within vectors, resulting in subsequent transmission to hosts. Consequently, sEVs provide potential targets for the development of novel therapies that inhibit pathogen replication or reduce arthropod vector populations. Future research in this area should emphasize how these vesicles function within host-vector systems, using advanced imaging techniques – such as high-resolution microscopy (HRM) – and cost-effective methods, in order to produce sufficient quantities for large-scale implementation.

BackgroundYellow fever virus (YFV) and dengue virus (DENV) are among the major re-emerging arboviruses that pose a significant threat to public health. Their associated burden and prevalence can be substantially underestimated due to insufficient surveillance and inadequate diagnosis. This study aimed to determine evidence of dengue, yellow and related flaviviruses circulation among the rural human populations residing in Nguruman (Kajiado County) and Kerio Valley (Baringo County), two dryland ecosystems in the Kenyan Rift Valley.MethodsSerum samples obtained from febrile patients between 5 and 85 years through a hospital-based cross-sectional survey from July 2020 – May 2023, were screened for neutralizing antibodies to YFV, DENV-2 and related flaviviruses, West Nile virus (WNV) and Zika virus (ZIKV) via Plaque reduction neutralization test (PRNT). The study sites and important demographic characteristics were obtained using a structural questionnaire and the data analyzed and seroprevalence compared. A multinomial logistic regression model was done to predict risk for each of the most prevalent viruses with covariates; age, gender, and occupation.ResultsOverall, 54.5% (50.1–59.0% 95% confidence interval (CI) of the samples tested positive for at least one of the four Flaviviruses. The percentage was significantly higher in Kerio Valley (64.34%, 184/286) than in Nguruman (40.2%, 78/194) (P

Flaviviruses are enveloped, single-stranded RNA viruses that are largely arthropod-borne, circulating globally and infecting up to 400 million people annually. Their widespread distribution, shared antigenicity, and evolutionary relationships lead to immune responses that cross-react across multiple flaviviruses. This presents challenges for accurate serological diagnosis and vaccine development, especially in regions where multiple flaviviruses are endemic. Such challenges can result in direct and indirect adverse effects, including antibody-dependent enhancement (ADE) and the production of sub-neutralizing antibodies. To fully understand the mechanism of cross-reactivity among medically important flaviviruses, it is crucial to explore the underlying factors contributing to this phenomenon. These factors encompass the conservation of the envelope protein (E protein), pre-existing immunity, and co-infection of different flaviviruses. While previous reviews have often discussed these factors separately, our review aims to elucidate their interconnectedness and their combined effects on cross-reactivity across clinically significant flaviviruses.

In flaviviruses such as Dengue or Zika, non-structural (NS) NS4A protein forms homo-oligomers, participates in membrane remodelling and is critical for virulence. In both viruses, mature NS4A has the same length and three predicted hydrophobic domains. The oligomers formed by Dengue NS4A are reported to be small (n = 2, 3), based on denaturing SDS gels, but no high-resolution structure of a flavivirus NS4A protein is available, and the size of the oligomer in lipid membranes is not known. Herein we show that crosslinking Zika NS4A protein in lipid membranes results in oligomers at least up to hexamers. Further, sedimentation velocity shows that NS4A in mild detergent C14-betaine appears to be in fast equilibrium between at least two species, where one is smaller, and the other larger, than a trimer or a tetramer. Consistently, sedimentation equilibrium data was best fitted to a model involving an equilibrium between dimers (n = 2) and hexamers (n = 6). Overall, the large, at least hexameric, oligomers obtained herein in liposomes and in mild detergent are more likely to represent the forms of NS4A present in cell membranes. [ABSTRACT FROM AUTHOR]