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Flaviviruses, including Zika virus (ZIKV) and Dengue virus (DENV), rely on their non-structural protein 5 (NS5) for both replication of viral genome and suppression of host IFN signaling. DENV and ZIKV NS5s were shown to facilitate proteosome-mediated protein degradation of human STAT2 (hSTAT2). However, how flavivirus NS5s have evolved for species-specific IFN-suppression remains unclear. Here we report structure-function characterization of the DENV serotype 2 (DENV2) NS5-hSTAT2 complex. The MTase and RdRP domains of DENV2 NS5 form an extended conformation to interact with the coiled-coil and N-terminal domains of hSTAT2, thereby promoting hSTAT2 degradation in cells. Disruption of the extended conformation of DENV2/ZIKV NS5, but not the alternative compact state, impaired their hSTAT2 binding. Our comparative structural analysis of flavivirus NS5s further reveals a conserved protein-interaction platform with subtle amino-acid variations likely underpinning diverse IFN-suppression mechanisms. Together, this study uncovers a conformational selection mechanism underlying species-specific hSTAT2 inhibition by flavivirus NS5.

Dengue virus (DENV) is a mosquito-borne virus that causes dengue fever, a significant public health concern in many tropical and subtropical regions. Dengue is endemic in more than 100 countries, primarily in tropical and subtropical regions of the world. Each year, up to 400 million people get infected with dengue. Approximately 100 million people get sick from infection, and 40,000 die from severe dengue. Unfortunately, dengue vaccine development is also marred with various complicating factors, as the forefront candidate vaccine performed unsatisfactorily. Moreover, the only licensed vaccine (Dengvaxia) for children 9 through 16 years of age is available in just a few countries. The treatment difficulties are compounded by the absence of an effective antiviral agent. Exploring plant-based therapeutics for dengue from the laboratory to clinical application involves a multi-stage process, encompassing various scientific disciplines. Individual investigators have screened a wide range of plant extracts or compounds for potential antiviral activity against DENV. In vitro studies help identify candidates that exhibit inhibitory effects on viral replication. Some of the most promising medicinal plants showing in vitro activity against DENV include Andrographis paniculate, Acorus calamus, and Cladogynos orientalis. Further laboratory studies, both in vitro and in animal models (in vivo), elucidate the mechanisms of action by which the identified compounds exert antiviral effects. Medicinal plants such as Carica papaya, Cissampelos pareira, and Ipomea batata exhibited potent platelet-enhancing activities while Azadirachta indica and Curcuma longa showed promising effects in both in vitro and in vivo studies. Based on positive preclinical results, researchers design clinical trials. This involves careful planning of trial phases, patient recruitment criteria, ethical considerations, and endpoints. The most important medicinal plants showing efficacy and safety in clinical trials include Carica papaya and Cissampelos pareira. This review suggests that several promising medicinal plants exist that have the potential to be turned into clinical drugs to treat dengue infection. However, in addition to developing synthetic and plant-based therapies against dengue infection, vector management strategies should be made robust, emphasizing the need to focus on reducing disease incidence. [ABSTRACT FROM AUTHOR]

The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionsUsutu virus (USUV) and West Nile virus (WNV) are zoonotic arthropod-borne orthoflaviviruses. The enzootic transmission cycles of both include Culex mosquitoes as vectors and birds as amplifying hosts. For more than 10 years, these viruses have been monitored in birds in Germany by a multidisciplinary network. While USUV is present nationwide, WNV used to be restricted to the central-east.In 2021 and 2022, over 2300 live bird blood samples and organs from over 3000 deceased birds were subjected to molecular and serological analysis regarding the presence of WNV and USUV. The samples were collected at sites all over Germany.Circulation of both viruses increased in 2022. For USUV, the nationwide presence of lineages Africa 3 and Europe 3 reported in previous years was confirmed. Lineage Europe 2, formerly restricted to the German east, was able to expand westward. Nonetheless, USUV neutralizing antibody (nAb) detection rates remained low (< 9%). Years 2021 and 2022 were characterized by stable enzootic circulation of WNV lineage 2, dominated by one previously identified subcluster (95% of generated sequences). In 2022, >20% of birds in the endemic region in eastern Germany carried nAb against WNV. Serological data also indicate expanding WNV circulation west and south of the known hotspots in Germany.USUV circulates enzootically nationwide. Emergence of WNV at several new locations in Germany with a potential increase in human infections may be imminent. In this context, wild bird monitoring serves as a capable early warning system in a One Health setting. [ABSTRACT FROM AUTHOR]

The diversity and abundance of vectors are essential parameters in the transmission dynamics of West Nile virus (WNV) between its avian reservoirs and clinically susceptible mammalian species. Knowing the determinants of vector abundance could be thus useful in preventing West Nile fever (WNF) cases and associated socio‐economic impact. We designed a survey at the wildlife–livestock interface to test the hypothesis that variations in environmental favourability between anthropized and wild scenarios modulate WNV vector abundance and transmission risk. In a continental Mediterranean region where WNF has recently emerged, we selected nine sampling sites and allocated three areas to every site with a decreasing gradient of wildlife–livestock interaction: A1—a horse farm where interaction is maximal; A2—a zone of intermediate interaction 500–1000 m from the farm; and A3—an entirely wild zone of low interaction 1–5 km from the farm. At a fortnightly frequency, we estimated mosquito abundance at each of the 27 study sites in May–December 2018 and April–July 2019. We estimated bird and mammal abundance, collected meteorological information and characterised mosquito habitat at the site scale. Thereafter, we studied the determinants of Culex spp., Culex pipiens sensu lato (s.l.) Linnaeus, 1758 (Diptera: Culicidae) and Culex theileri  Theobald, 1903 abundance by constructing negative binomial generalised linear mixed models. We identified 20 mosquito species, with a notable predominance of Culex spp. and, particularly, of Cx. pipiens s.l. We found differences in the spatiotemporal distribution of Culex spp. abundance and confirmed our hypothesis by finding important effects of local environmental variations in abundance. The accumulated rainfall in fortnights 4–14 and the mean temperature of the two fortnights before sampling were positively and statistically significantly associated with the abundance of Cx. pipiens s.l. (Z = 13.09, p < 0.001, and Z = 9.91, p < 0. 001, respectively) and Culex spp. (Z = 13.35, p < 0.001, and Z = 6.99, p < 0.001, respectively), while the mean temperature of the two previous fortnights was a positive statistically significant predictor (Z = 14.69, p < 0.001) of the abundance of Cx. theileri. The farm environment was the most conducive predictor to hosting Culex spp. compared with wild settings. Our results indicate that continental Mediterranean environments are favourable for WNV circulation and maintenance, especially the environment of anthropized rural settings such as farms. These results will have an impact on the spatiotemporal risk prediction of WNF emergence in continental Mediterranean environments. [ABSTRACT FROM AUTHOR]

Recent studies on viral diseases showed that neutrophils exist in various phenotypes and functional statuses, that may play a role either in viral clearance or in immunopathology. Emerging evidence suggests that dengue virus can activate neutrophils, leading to the release of myeloperoxidase and the formation of neutrophil extracellular traps; these were shown to enhance vascular permeability and mediate dengue-associated cardiac impairment, contributing to severe dengue. Neutrophils are recruited during early dengue virus infection, and they release soluble molecules such as olfactomedin 4 and soluble urokinase plasminogen activator receptor into the circulation; these may be useful biomarkers to predict disease progression in dengue. Given that neutrophils may mediate severe dengue, attenuating the neutrophil response can be an attractive pathway to ameliorate disease severity. Dengue is a mosquito-borne viral disease which causes significant morbidity and mortality each year. Previous research has proposed several mechanisms of pathogenicity that mainly involve the dengue virus and host humoral immunity. However, innate immune cells, such as neutrophils, may also play an important role in dengue, albeit a much less defined role. In this review, we discuss the emerging roles of neutrophils in dengue and their involvement in pathologies associated with severe dengue. We also describe the potential use of several neutrophil proteins as biomarkers for severe dengue. These studies suggest that neutrophils are important players in dengue, and a better understanding of neutrophil-dengue biology is urgently needed. [ABSTRACT FROM AUTHOR]

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2‐mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin‐proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild‐type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265‐type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs. [ABSTRACT FROM AUTHOR]

Though interferons (IFNs) were once heralded as panaceas to numerous diseases, how cells decode varying IFN stimuli and subsequently produce (in)appropriate signaling remain unclear. Our labs recently engineered novel erythropoietin receptor-IFN chimeric receptors, and we highlight their utility in two cases uncovering different ial genetic determinants of type I (IFN-α/β) and type III (IFN-λ) IFN signaling. These and other types of synthetic (cytokine) receptors could be expanded to real-time signaling dynamics and in vivo studies. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects of a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated, and the s pecific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection. [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]

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion. Brief summary: West Nile virus, but not Usutu virus, can productively infect human motor neurons as a possible route of neuroinvasion. [ABSTRACT FROM AUTHOR]

Emerging infectious diseases are a major threat to biodiversity and an important public health issue. Flaviviruses are the cause of several emerging vector-borne zoonotic arboviruses whose distribution is currently increasing in Europe. The evidence that West Nile virus (WNV) circulates in resident and migratory species has implications for both animal and public health and should therefore be studied in depth. USUTU (USUV), Bagaza (BAGV) and tick-borne encephalitis virus (TBEV) are other viruses that are beginning to spread more widely. An integrated surveillance program, namely in birds, is essential for reducing the risk of infection in human populations within the One Health principles. In the present study, wild birds admitted to wildlife rehabilitation centers in Portugal were sampled. Two hundred eight blood samples were assayed serologically for antibodies to flaviviruses by using a commercial ELISA kit. An overall seroprevalence of 19.6% (95% confidence interval [CI]: 13.7–26.7%) was observed. Antibodies against flaviviruses were detected in 13 (35.1%) different species of wild birds. Accipitriformes (26.7%; 95% CI: 18.5–36.2%) and Strigiformes (26.7%; 95% CI: 14.6–42.0%) were the orders with the highest seroprevalence rates recorded. There were no statistically significant differences (p = 0.725) between the geographical regions (NUTS II) studied, but a statistically significant difference (p = 0.017) was found between sex (male: 34.4%; female: 4.8%). A higher seroprevalence was detected in adults (32.1%) than in juvenile birds (9.3%) (p = 0.014), and age was considered a risk factor for flavivirus infection in wild birds (odds ratio 1.4; 95% CI: 0.5–4.0). More epidemiological studies are needed in Portugal since the actual spread of the genus Flavivirus throughout the country is unknown. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the clinical usefulness of VirClia IgM/IgG single-assay chemiluminescence tests for the diagnosis of tick-borne encephalitis (TBE) in an endemic part of Norway. Patients hospitalized at Vestfold or Telemark Hospitals with suspected infection in the central nervous system (CNS) in the period between May 2021 and December 2023 were included, with 85 TBE cases identified. The VirClia IgM assay was positive in the initial serum sample in 75/85 cases, giving a sensitivity of 88.2% (95% CI, 79.4–94.2). The ReaScan TBE IgM rapid test was positive in 80/85 cases, with an estimated sensitivity of 94.1% (95% CI, 86.8–98.1). Vaccine breakthrough infections were the predominant cause of non-reactive IgM cases. The calculated specificity for the VirClia IgM was 95.8% (95% CI, 92.5–98.0). In conclusion, the sensitivity of the VirClia IgM was non-inferior to the ReaScan TBE IgM rapid test. However, isolated IgM reactive results must be interpreted with caution, since false-reactive results occur. [ABSTRACT FROM AUTHOR]

Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called "superinfection exclusion", whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control. [ABSTRACT FROM AUTHOR]

Flavivirus infection capitalizes on cellular lipid metabolism to remodel the cellular intima, creating a specialized lipid environment conducive to viral replication, assembly, and release. The Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is responsible for significant morbidity and mortality in both humans and animals. Currently, there are no effective antiviral drugs available to combat JEV infection. In this study, we embarked on a quest to identify anti-JEV compounds within a lipid compound library. Our research led to the discovery of two novel compounds, isobavachalcone (IBC) and corosolic acid (CA), which exhibit dose-dependent inhibition of JEV proliferation. Time-of-addition assays indicated that IBC and CA predominantly target the late stage of the viral replication cycle. Mechanistically, JEV nonstructural proteins 1 and 2A (NS1 and NS2A) impede 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation by obstructing the liver kinase B1 (LKB1)–AMPK interaction, resulting in decreased p-AMPK expression and a consequent upsurge in lipid synthesis. In contrast, IBC and CA may stimulate AMPK by binding to its active allosteric site, thereby inhibiting lipid synthesis essential for JEV replication and ultimately curtailing viral infection. Most importantly, in vivo experiments demonstrated that IBC and CA protected mice from JEV-induced mortality, significantly reducing viral loads in the brain and mitigating histopathological alterations. Overall, IBC and CA demonstrate significant potential as effective anti-JEV agents by precisely targeting AMPK-associated signaling pathways. These findings open new therapeutic avenues for addressing infections caused by Flaviviruses. IMPORTANCE This study is the inaugural utilization of a lipid compound library in antiviral drug screening. Two lipid compounds, isobavachalcone (IBC) and corosolic acid (CA), emerged from the screening, exhibiting substantial inhibitory effects on the Japanese encephalitis virus (JEV) proliferation in vitro. In vivo experiments underscored their efficacy, with IBC and CA reducing viral loads in the brain and mitigating JEVinduced histopathological changes, effectively shielding mice from fatal JEV infection. Intriguingly, IBC and CA may activate 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) by binding to its active site, curtailing the synthesis of lipid substances, and thus suppressing JEV proliferation. This indicates AMPK as a potential antiviral target. Remarkably, IBC and CA demonstrated suppression of multiple viruses, including Flaviviruses (JEV and Zika virus), porcine herpesvirus (pseudorabies virus), and coronaviruses (porcine deltacoronavirus and porcine epidemic diarrhea virus), suggesting their potential as broad-spectrum antiviral agents. These findings shed new light on the potential applications of these compounds in antiviral research. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV; family, Flaviviridae), which causes congenital Zika syndrome, Guillain-Barré Syndrome, and other severe diseases, is transmitted mainly by mosquitoes; however, the virus can be transmitted through other routes. Among the three structural and seven nonstructural proteins, the surface envelope (E) protein of ZIKV plays a critical role in viral entry and pathogenesis, making it a key target for the development of effective entry inhibitors. This review article describes the life cycle, genome, and encoded proteins of ZIKV, illustrates the structure and function of the ZIKV E protein, summarizes E protein-targeting entry inhibitors (with a focus on those based on natural products and small molecules), and highlights challenges that may potentially hinder the development of effective inhibitors of ZIKV infection. Overall, the article will provide useful guidance for further development of safe and potent ZIKV entry inhibitors targeting the viral E protein. [ABSTRACT FROM AUTHOR]

Arthropod‐borne viruses, such as dengue virus (DENV), pose significant global health threats, with DENV alone infecting around 400 million people annually and causing outbreaks beyond endemic regions. This study aimed to enhance serological diagnosis and discover new drugs by identifying immunogenic protein regions of DENV. Utilizing a comprehensive approach, the study focused on peptides capable of distinguishing DENV from other flavivirus infections through serological analyses. Over 200 patients with confirmed arbovirus infection were profiled using high‐density pan flavivirus peptide arrays comprising 6253 peptides and the computational method matrix of local coupling energy (MLCE). Twenty‐four peptides from nonstructural and structural viral proteins were identified as specifically recognized by individuals with DENV infection. Six peptides were confirmed to distinguish DENV from Zika virus (ZIKV), West Nile virus (WNV), Yellow Fever virus (YFV), Usutu virus (USUV), and Chikungunya virus (CHIKV) infections, as well as healthy controls. Moreover, the combination of two immunogenic peptides emerged as a potential serum biomarker for DENV infection. These peptides, mapping to highly accessible regions on protein structures, show promise for diagnostic and prophylactic strategies against flavivirus infections. The described methodology holds broader applicability in the serodiagnosis of infectious diseases. [ABSTRACT FROM AUTHOR]

Dengue is a disease caused by a flavivirus (DENV) and transmitted by the bite of a mosquito, primarily the Aedes aegypti and Aedes albopictus species. Previous studies have demonstrated a relationship between the host gut microbiota and the evolution of dengue. It seems to be a bidirectional relationship, in which the DENV can affect the microbiota by inducing alterations related to intestinal permeability, leading to the release of molecules from microbiota dysbiosis that can influence the evolution of dengue. The role of angiotensin II (Ang II) in the microbiota/dengue relationship is not well understood, but it is known that the renin‐angiotensin system (RAS) is present in the intestinal tract and interacts with the gut microbiota. The possible effect of Ang II on the microbiota/Ang II/dengue relationship can be summarised as follows: the presence of Ang II induced hypertension, the increase in angiotensinogen, chymase, and microRNAs during the disease, the induction of vascular dysfunction, the production of trimethylamine N‐oxide and the brain/microbiota relationship, all of which are elements present in dengue that could be part of the microbiota/Ang II/dengue interactions. These findings suggest the potential use of Ang II synthesis blockers and the use of AT1 receptor antagonists as therapeutic drugs in dengue. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV), a mosquito‐borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain–Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very‐low density‐lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto‐inhibitory conformation due to a disordered N‐terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development. [ABSTRACT FROM AUTHOR]

Abstract Dengue virus (DENV) is a mosquito-borne virus that causes dengue fever, a significant public health concern in many tropical and subtropical regions. Dengue is endemic in more than 100 countries, primarily in tropical and subtropical regions of the world. Each year, up to 400 million people get infected with dengue. Approximately 100 million people get sick from infection, and 40,000 die from severe dengue. Unfortunately, dengue vaccine development is also marred with various complicating factors, as the forefront candidate vaccine performed unsatisfactorily. Moreover, the only licensed vaccine (Dengvaxia) for children 9 through 16 years of age is available in just a few countries. The treatment difficulties are compounded by the absence of an effective antiviral agent. Exploring plant-based therapeutics for dengue from the laboratory to clinical application involves a multi-stage process, encompassing various scientific disciplines. Individual investigators have screened a wide range of plant extracts or compounds for potential antiviral activity against DENV. In vitro studies help identify candidates that exhibit inhibitory effects on viral replication. Some of the most promising medicinal plants showing in vitro activity against DENV include Andrographis paniculate , Acorus calamus, and Cladogynos orientalis. Further laboratory studies, both in vitro and in animal models (in vivo), elucidate the mechanisms of action by which the identified compounds exert antiviral effects. Medicinal plants such as Carica papaya, Cissampelos pareira, and Ipomea batata exhibited potent platelet-enhancing activities while Azadirachta indica and Curcuma longa showed promising effects in both in vitro and in vivo studies. Based on positive preclinical results, researchers design clinical trials. This involves careful planning of trial phases, patient recruitment criteria, ethical considerations, and endpoints. The most important medicinal plants showing efficacy and safety in clinical trials include Carica papaya and Cissampelos pareira. This review suggests that several promising medicinal plants exist that have the potential to be turned into clinical drugs to treat dengue infection. However, in addition to developing synthetic and plant-based therapies against dengue infection, vector management strategies should be made robust, emphasizing the need to focus on reducing disease incidence.

Abstract The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.

Abstract Hepatitis C virus (HCV) infection poses a significant public health challenge and often leads to long-term health complications and even death. Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a proposed viral etiology. HCV infection and PD have been previously suggested to be related. This work aimed to identify potential biomarkers and pathways that may play a role in the joint development of PD and HCV infection. Using BioOptimatics-bioinformatics driven by mathematical global optimization-, 22 publicly available microarray and RNAseq datasets for both diseases were analyzed, focusing on sex-specific differences. Our results revealed that 19 genes, including MT1H, MYOM2, and RPL18, exhibited significant changes in expression in both diseases. Pathway and network analyses stratified by sex indicated that these gene expression changes were enriched in processes related to immune response regulation in females and immune cell activation in males. These findings suggest a potential link between HCV infection and PD, highlighting the importance of further investigation into the underlying mechanisms and potential therapeutic targets involved.

Introduction. West Nile Virus (WNV), a member of Flaviviridae family, is one of the most widely distributed arboviruses in the world. In developing countries like Nigeria, fever resulting from the WNV infection is often presumptively ascribed to malaria or typhoid due to misdiagnosis and low-level awareness of the viral infection. This study determined the prevalence of WNV IgM and IgG antibodies among febrile patients in the Ilorin metropolis. Materials and methods. A total of two hundred (200) blood samples were collected from consenting patients and each serum was screened for anti-WNV IgM and IgG antibodies using indirect enzyme-linked immunosorbent assay (ELISA). Statistical correlation and logistic regression analysis were conducted. Results. Overall, 6% (12/200) anti-WNV IgM seropositivity rate was recorded amongst the acute febrile patients with higher prevalence (6.30%) in females than in males (5.45%). Anti-WNV IgG positivity rate of 52% (104/200) was recorded, with 50.67% positivity rate in males and 38.95% in female participants. The convalescence phase posited by the 5.4% (11/200) co-detection of anti-WNV IgG and IgM antibodies among the participants was recorded. A statistical correlation was noticed with the age and religion of respondents to WNV serological positivity while gender, occupation, use of mosquito nets and formal education had no positive correlation at p 0.05. However, based on odd ratio at 95% CI and logistic regression coefficients, the evaluated risk factors such as blood transfusion, residency, malaria parasite, and proximity to stagnant water and bush were significant to anti-WNV IgG and IgM positivity. Conclusion. The findings of this study show the circulation of WNV in the study area. There is an urgent need for clinicians/physicians to include screening for the West Nile virus in cases of febrile patients before the commencement of treatment.

Abstract Emerging infectious diseases are a major threat to biodiversity and an important public health issue. Flaviviruses are the cause of several emerging vector-borne zoonotic arboviruses whose distribution is currently increasing in Europe. The evidence that West Nile virus (WNV) circulates in resident and migratory species has implications for both animal and public health and should therefore be studied in depth. USUTU (USUV), Bagaza (BAGV) and tick-borne encephalitis virus (TBEV) are other viruses that are beginning to spread more widely. An integrated surveillance program, namely in birds, is essential for reducing the risk of infection in human populations within the One Health principles. In the present study, wild birds admitted to wildlife rehabilitation centers in Portugal were sampled. Two hundred eight blood samples were assayed serologically for antibodies to flaviviruses by using a commercial ELISA kit. An overall seroprevalence of 19.6% (95% confidence interval [CI]: 13.7–26.7%) was observed. Antibodies against flaviviruses were detected in 13 (35.1%) different species of wild birds. Accipitriformes (26.7%; 95% CI: 18.5–36.2%) and Strigiformes (26.7%; 95% CI: 14.6–42.0%) were the orders with the highest seroprevalence rates recorded. There were no statistically significant differences (p = 0.725) between the geographical regions (NUTS II) studied, but a statistically significant difference (p = 0.017) was found between sex (male: 34.4%; female: 4.8%). A higher seroprevalence was detected in adults (32.1%) than in juvenile birds (9.3%) (p = 0.014), and age was considered a risk factor for flavivirus infection in wild birds (odds ratio 1.4; 95% CI: 0.5–4.0). More epidemiological studies are needed in Portugal since the actual spread of the genus Flavivirus throughout the country is unknown.

Abstract West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.

One of the most recent advances in the analysis of viral RNA-cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized ‘ZAP-independent’ and ‘ZAP-dependent’ cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.

ABSTRACTAs positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

Abstract Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2′C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of all arboviruses, while 2′C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection.

Abstract 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.

Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2′C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of all arboviruses, while 2′C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection. [ABSTRACT FROM AUTHOR]

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development. [ABSTRACT FROM AUTHOR]

Zika virus infection is a mosquito-borne viral disease that has become a global health concern recently. Zika virus belongs to the Flavivirus genus and is primarily transmitted by Aedes mosquitoes. Prevention of Zika virus infection involves avoiding mosquito bites by using repellent, wearing protective clothing, and staying in screened areas, especially for pregnant women. Treatment focuses on managing symptoms with rest, fluids, and acetaminophen, with close monitoring for pregnant women. Currently, there is no specific antiviral treatment or vaccine for the Zika virus, highlighting the importance of prevention strategies to control its spread. Therefore, in this study, the Zika virus non-structural protein NS3 was targeted to inhibit Zika infection by identifying the novel inhibitor through an in silico approach. Here, 2864 natural compounds were screened using a machine learning-based QSAR model, and later docking was performed to select the potential target. Subsequently, Tanimoto similarity and clustering were performed to obtain the potential target. The three most potential compounds were obtained: (a) 5297, (b) 432449, and (c) 85137543. The protein–ligand complex's stability and flexibility were then investigated by dynamic modelling. The 300 ns simulation showed that 5297 exhibited the steadiest deviation and constant creation of hydrogen bonds. Compared to the other compounds, 5297 demonstrated a superior binding free energy (ΔG = −20.81 kcal/mol) with the protein when the MM/GBSA technique was used. The study determined that 5297 showed significant therapeutic potential and justifies further experimental investigation as a possible inhibitor of the NS2B-NS3 protease target implicated in Zika virus infection. [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]

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10–20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5–15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2–4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFβ, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3′-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3′-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication. IMPORTANCE Japanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3′-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding. [ABSTRACT FROM AUTHOR]

Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3′ untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5′−3′ exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer. [ABSTRACT FROM AUTHOR]

Flaviviruses, including dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), yellow fever (YFV), and tick-borne encephalitis (TBEV) viruses, pose a significant global emerging threat. With their potential to cause widespread outbreaks and severe health complications, the development of effective vaccines and antiviral therapeutics is imperative. The flaviviral non-structural protein 5 (NS5) is a highly conserved and multifunctional protein that is crucial for viral replication, and the NS5 protein of many flaviviruses has been shown to be a potent inhibitor of interferon (IFN) signalling. In this review, we discuss the functions of NS5, diverse NS5-mediated strategies adopted by flaviviruses to evade the host antiviral response, and how NS5 can be a target for the development of vaccines and antiviral therapeutics. [ABSTRACT FROM AUTHOR]

Despite the availability of tick-borne encephalitis (TBE) vaccines, the incidence of TBE is increasing. To understand the historical patterns of infection, we conducted a global meta-analysis of studies before December 2023 reporting human antibody prevalence against TBEV (TBE virus) among general or high-risk population groups stratified by country, collection year, serological method, and vaccination status. Pooled data were compared within groups over time by random-effects modeling. In total, 2403 articles were retrieved; 130 articles published since 1959 were included. Data were extracted from 96 general populations (117,620 participants) and 71 high-risk populations (53,986 participants) across 33 countries. Germany had the most population groups (21), and Poland had the most participants (44,688). Seven serological methods were used; conventional IgG/IgM ELISAs were the most common (44%). Four studies (1.7%) used NS1-ELISA serology. Between 1956–1991 and 1992–2022, anti-TBEV seroprevalence remained at ~2.75% across all population groups from "high-risk" areas (p = 0.458) but decreased within general populations (1.7% to 1%; p = 0.001) and high-risk populations (5.1% to 1.3%; p < 0.001), possibly due to differences in the study methodologies between periods. This global summary explores how serological methods can be used to assess TBE vaccination coverage and potential exposure to TBEV or measure TBE burden and highlights the need for standardized methodology when conducting TBE seroprevalence studies to compare across populations. [ABSTRACT FROM AUTHOR]

AbstractThe yellow fever (YF) vaccine is usually produced with egg-based methods, which has limitations, including potential adverse effects and low production yields. Alternatively, producing the vaccine using Vero cells or HEK 293 cells can overcome some of these issues, but these methods are significantly more expensive. In the current study, the YF vaccine candidate 17DD virus was produced in primary chicken embryo fibroblast (CEF) cells. The primary CEF cells isolation from eggs was optimized through a two-step process. In the first step, the important parameters that contribute to the development of the egg embryo, such as egg position, relative humidity (RH), and incubation time are optimized. In second step, primary CEF release parameters namely; trypsin volume and incubation temperature are optimized. Both steps were optimized using statistical methods. Further, the seeding cell density of isolated CEF was also optimized. It was observed that 5 x 104 cells/cm2 gave the highest virus titer of 3.89 million PFU/ml. The 17DD yields achieved in primary CEFs are much higher than egg-based production and it is an economically viable method. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is an arthropod-borne, positive-sense RNA virus that poses an increasing global threat due to warming climates and lack of effective therapeutics. Like other enzootic viruses, little is known about how host context affects the structure of the full-length RNA genome. Here, we report a complete secondary structure of the entire WNV genome within infected mammalian and arthropod cell lines. Our analysis affords structural insights into multiple, conserved aspects of flaviviral biology. We show that the WNV genome folds with minimal host dependence, and we prioritize well-folded regions for functional validation using structural homology between hosts as a guide. Using structure-disrupting, antisense locked nucleic acids, we then demonstrate that the WNV genome contains riboregulatory structures with conserved and host-specific functional roles. These results reveal promising RNA drug targets within flaviviral genomes, and they highlight the therapeutic potential of ASO-LNAs as both WNV-specific and pan-flaviviral therapeutic agents. [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]

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. [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]

Introduction: Widespread transmission of Japanese encephalitis virus (JEV) genotype four (GIV) occurred across mainland Australia in 2022. This resulted in forty-five human cases, including seven deaths, and the identification of JEV infection in over 80 commercial piggeries. Materials and Methods: We collected mosquitoes which were trapped using CO2-baited light traps deployed near piggeries reporting disease or in regions linked to human cases in the Wide Bay region in the state of Queensland. Mosquitoes from four traps yielded JEV RNA by real-time RT-PCR. Pools containing RNA positive mosquitoes were inoculated onto mosquito cell monolayers. Discussion: A single isolate of JEV was obtained from a pool of mixed mosquito species. Near whole genome sequencing and phylogenetic analysis of the JEV isolate demonstrated its high genomic relatedness with JEV GIV pig sequences sampled from Queensland and the state of New South Wales in 2022. Conclusion: We report the first isolation of JEV GIV from mosquitoes collected in Australia. With only a few JEV GIV isolates available globally, the isolate we report will be essential for future research of JEV host interactions, evolution and disease markers, and development of effective therapies, vaccines, diagnostic assays, and mosquito control strategies. [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]

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]

West Nile Virus (WNV) is an arbovirus endemic to many countries and has caused over 56,000 cases, with 2776 deaths in the U.S. from 1999 to 2022. WNV occurs most often in the fall, typically affecting elderly populations in states like Nebraska and Arizona. Currently, supportive care is the only management for WNV. Our case is a female patient in her mid-70s in an intermountain state who presented in the fall with WNV meningoencephalitis and experienced a delay in care due to the unique clinical presentation. This demonstrates the importance of early inclusion of WNV in the differential for altered mental status, especially with WNV risk factors, and expedition of supportive care. Doing so could potentially reduce antibiotic duration and hospital costs. [ABSTRACT FROM AUTHOR]