Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

The West Nile virus genome harbors essential riboregulatory elements with conserved and host-specific functional roles.

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.

CD11b maintains West Nile virus replication through modulation of immune response in human neuroblastoma cells.

BACKGROUND: West Nile virus (WNV) is a rapidly spreading mosquito-borne virus accounted for neuroinvasive diseases. An insight into WNV-host factors interaction is necessary for development of therapeutic approaches against WNV infection. CD11b has key biological functions and been identified as a therapeutic target for several human diseases. The purpose of this study was to determine whether CD11b was implicated in WNV infection. METHODS: SH-SY5Y cells with and without MEK1/2 inhibitor U0126 or AKT inhibitor MK-2206 treatment were infected with WNV. CD11b mRNA levels were assessed by real-time PCR. WNV replication and expression of stress (ATF6 and CHOP), pro-inflammatory (TNF-α), and antiviral (IFN-α, IFN-ß, and IFN-γ) factors were evaluated in WNV-infected SH-SY5Y cells with CD11b siRNA transfection. Cell viability was determined by MTS assay. RESULTS: CD11b mRNA expression was remarkably up-regulated by WNV in a time-dependent manner. U0126 but not MK-2206 treatment reduced the CD11b induction by WNV. CD11b knockdown significantly decreased WNV replication and protected the infected cells. CD11b knockdown markedly increased TNF-α, IFN-α, IFN-ß, and IFN-γ mRNA expression induced by WNV. ATF6 mRNA expression was reduced upon CD11b knockdown following WNV infection. CONCLUSION: These results demonstrate that CD11b is involved in maintaining WNV replication and modulating inflammatory as well as antiviral immune response, highlighting the potential of CD11b as a target for therapeutics for WNV infection.

Serosurvey of Blood Donors to Assess West Nile Virus Exposure, South-Central Spain.

We analyzed West Nile Virus (WNV) exposure from 1,222 blood donors during 2017-2018 from an area of south-central Spain. Results revealed WNV seroprevalence of 0.08% (95% CI 0.004%-0.4%) in this population. Our findings underscore the need for continued surveillance and research to manage WNV infection in this region.

Structure of an RNA G-quadruplex from the West Nile virus genome.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

The potential role of the Asian bush mosquito Aedes japonicus as spillover vector for West Nile virus in the Netherlands.

BACKGROUND: In recent years the Asian bush mosquito Aedes japonicus has invaded Europe, including the Netherlands. This species is a known vector for a range of arboviruses, possibly including West Nile virus (WNV). As WNV emerged in the Netherlands in 2020, it is important to investigate the vectorial capacity of mosquito species present in the Netherlands to estimate the risk of future outbreaks and further spread of the virus. Therefore, this study evaluates the potential role of Ae. japonicus in WNV transmission and spillover from birds to dead-end hosts in the Netherlands. METHODS: We conducted human landing collections in allotment gardens (Lelystad, the Netherlands) in June, August and September 2021 to study the diurnal and seasonal host-seeking behaviour of Ae. japonicus. Furthermore, their host preference in relation to birds using live chicken-baited traps was investigated. Vector competence of field-collected Ae. japonicus mosquitoes for two isolates of WNV at two different temperatures was determined. Based on the data generated from these studies, we developed a Susceptible-Exposed-Infectious-Recovered (SEIR) model to calculate the risk of WNV spillover from birds to humans via Ae. japonicus, under the condition that the virus is introduced and circulates in an enzootic cycle in a given area. RESULTS: Our results show that Ae. japonicus mosquitoes are actively host seeking throughout the day, with peaks in activity in the morning and evening. Their abundance in August was higher than in June and September. For the host-preference experiment, we documented a small number of mosquitoes feeding on birds: only six blood-fed females were caught over 4 full days of sampling. Finally, our vector competence experiments with Ae. japonicus compared to its natural vector Culex pipiens showed a higher infection and transmission rate when infected with a local, Dutch, WNV isolate compared to a Greek isolate of the virus. Interestingly, we also found a small number of infected Cx. pipiens males with virus-positive leg and saliva samples. CONCLUSIONS: Combining the field and laboratory derived data, our model predicts that Ae. japonicus could act as a spillover vector for WNV and could be responsible for a high initial invasion risk of WNV when present in large numbers.

The historical ecological background of West Nile virus in Portugal indicates One Health opportunities.

West Nile (WNV) is a zoonotic arbovirus with an expanding geographical range and epidemic activity in Europe. Not having yet experienced a human-associated epidemic, Portugal remains an outlier in the Mediterranean basin. In this study, we apply ecological niche modelling informed by WNV historical evidence and a multitude of environmental variables from across Portugal. We identify that ecological backgrounds compatible with WNV historical circulation are mostly restricted to the south, characterized by a warmer and drier climate, high avian diversity, specific avian species and land types. We estimate WNV ecological suitability across the country, identifying overlaps with the distributions of the three relevant hosts (humans, birds, equines) for public and animal health. From this, we propose a category-based spatial framework providing first of a kind valuable insights for WNV surveillance in Portugal under the One Health nexus. We forecast that near future climate trends alone will contribute to pushing adequate WNV ecological suitability northwards, towards regions with higher human density. This unique perspective on the past, present and future ecology of WNV addresses existing national knowledge gaps, enhances our understanding of the evolving emergence of WNV, and offers opportunities to prepare and respond to the first human-associated epidemic in Portugal.

Exploring the role of temperature and other environmental factors in West Nile virus incidence and prediction in California counties from 2017-2022 using a zero-inflated model.

West Nile virus (WNV) is the most common mosquito-borne disease in the United States, resulting in hundreds of reported cases yearly in California alone. The transmission cycle occurs mostly in birds and mosquitoes, making meteorological conditions, such as temperature, especially important to transmission characteristics. Given that future increases in temperature are all but inevitable due to worldwide climate change, determining associations between temperature and WNV incidence in humans, as well as making predictions on future cases, are important to public health agencies in California. Using surveillance data from the California Department of Public Health (CDPH), meteorological data from the National Oceanic and Atmospheric Administration (NOAA), and vector and host data from VectorSurv, we created GEE autoregressive and zero-inflated regression models to determine the role of temperature and other environmental factors in WNV incidence and predictions. An increase in temperature was found to be associated with an increase in incidence in 11 high-burden Californian counties between 2017-2022 (IRR = 1.06), holding location, time of year, and rainfall constant. A hypothetical increase of two degrees Fahrenheit-predicted for California by 2040-would have resulted in upwards of 20 excess cases per year during our study period. Using 2017-2021 as a training set, meteorological and host/vector data were able to closely predict 2022 incidence, though the models did overestimate the peak number of cases. The zero-inflated model closely predicted the low number of cases in winter months but performed worse than the GEE model during high-transmission periods. These findings suggests that climate change will, and may be already, altering transmission dynamics and incidence of WNV in California, and provides tools to help predict incidence into the future.

Motif-VI loop acts as a nucleotide valve in the West Nile Virus NS3 Helicase.

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.

Phylogenetic analysis and molecular genetic characteristics of West Nile virus lineage 2 isolates circulating in the Russian Federation.

Since its initial detection in Africa, the West Nile virus has disseminated widely across all continents, becoming endemic in numerous countries, including the Russian Federation. A substantial expansion of the West Nile virus range was observed in the European part of the Russian territory in 1999. In light of this epidemiological trend, research endeavours focusing on monitoring West Nile virus circulation activity in endemic regions of the country have gained paramount significance. A substantial dataset has been accrued from 2007 onwards regarding genomic variability and dissemination dynamics across the country throughout the entire monitoring period for the West Nile fever pathogen. The objective of this study was to characterise West Nile virus isolates that have been circulating in the Russian Federation and identify their molecular and genetic characteristics. A phylogenetic analysis of 55 complete genome sequences revealed that the West Nile virus population within the Russian Federation is genetically heterogeneous and is represented by four major clades. One of these clades is currently exhibiting extensive spread into new regions of the country.

Winged Threat on the Offensive: A Literature Review Due to the First Identification of Aedes japonicus in Poland.

Genetic studies preceded by the observation of an unknown mosquito species in Mikolów (Poland) confirmed that it belongs to a new invasive species in Polish fauna, Aedes japonicus (Theobald, 1901), a known vector for numerous infectious diseases. Ae. japonicus is expanding its geographical presence, raising concerns about potential disease transmission given its vector competence for chikungunya virus, dengue virus, West Nile virus, and Zika virus. This first genetically confirmed identification of Ae. japonicus in Poland initiates a comprehensive review of the literature on Ae. japonicus, its biology and ecology, and the viral infections transmitted by this species. This paper also presents the circumstances of the observation of Ae. japonicus in Poland and a methodology for identifying this species.

Lessons Learned from West Nile Virus Infection:Vaccinations in Equines and Their Implications for One Health Approaches.

Humans and equines are two dead-end hosts of the mosquito-borne West Nile virus (WNV) with similar susceptibility and pathogenesis. Since the introduction of WNV vaccines into equine populations of the United States of America (USA) in late 2002, there have been only sporadic cases of WNV infection in equines. These cases are generally attributed to unvaccinated and under-vaccinated equines. In contrast, due to the lack of a human WNV vaccine, WNV cases in humans have remained steadily high. An average of 115 deaths have been reported per year in the USA since the first reported case in 1999. Therefore, the characterization of protective immune responses to WNV and the identification of immune correlates of protection in vaccinated equines will provide new fundamental information about the successful development and evaluation of WNV vaccines in humans. This review discusses the comparative epidemiology, transmission, susceptibility to infection and disease, clinical manifestation and pathogenesis, and immune responses of WNV in humans and equines. Furthermore, prophylactic and therapeutic strategies that are currently available and under development are described. In addition, the successful vaccination of equines against WNV and the potential lessons for human vaccine development are discussed.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

West Nile Virus Subgenomic RNAs Modulate Gene Expression in a Neuronal Cell Line.

Subgenomic flaviviral RNAs (sfRNAs) are small non-coding products of the incomplete degradation of viral genomic RNA. They accumulate during flaviviral infection and have been associated with many functional roles inside the host cell. Studies so far have demonstrated that sfRNA plays a crucial role in determining West Nile virus (WNV) pathogenicity. However, its modulatory role on neuronal homeostasis has not been studied in depth. In this study, we investigated the mechanism of sfRNA biosynthesis and its importance for WNV replication in neuronal cells. We found that sfRNA1 is functionally redundant for both replication and translation of WNV. However, the concurrent absence of sfRNA1 and sfRNA2 species is detrimental for the survival of the virus. Differential expression analysis on RNA-seq data from WT and ΔsfRNA replicon cell lines revealed transcriptional changes induced by sfRNA and identified a number of putative targets. Overall, it was shown that sfRNA contributes to the viral evasion by suppressing the interferon-mediated antiviral response. An additional differential expression analysis among replicon and control Neuro2A cells also clarified the transcriptional changes that support WNV replication in neuronal cells. Increased levels of translation and oxidative phosphorylation, post-translational modification processes, and activated DNA repair pathways were observed in replicon cell lines, while developmental processes such as axonal growth were deficient.

Immunoinformatics assisted profiling of West Nile virus proteome to determine immunodominant epitopes for the development of next-generation multi-peptide vaccine.

Emerging infectious diseases represent a significant threat to global health, with West Nile virus (WNV) being a prominent example due to its potential to cause severe neurological disorders alongside mild feverish conditions. Particularly prevalent in the continental United States, WNV has emerged as a global concern, with outbreaks indicating the urgent need for effective prophylactic measures. The current problem is that the absence of a commercial vaccine against WNV highlights a critical gap in preventive strategies against WNV. This study aims to address this gap by proposing a novel, multivalent vaccine designed using immunoinformatics approaches to elicit comprehensive humoral and cellular immune responses against WNV. The objective of the study is to provide a theoretical framework for experimental scientists to formulate of vaccine against WNV and tackle the current problem by generating an immune response inside the host. The research employs reverse vaccinology and subtractive proteomics methodologies to identify NP_041724.2 polyprotein and YP_009164950.1 truncated flavivirus polyprotein NS1 as the prime antigens. The selection process for epitopes focused on B and T-cell reactivity, antigenicity, water solubility, and non-allergenic properties, prioritizing candidates with the potential for broad immunogenicity and safety. The designed vaccine construct integrates these epitopes, connected via GPGPG linkers, and supplemented with an adjuvant with the help of another linker EAAAK, to enhance immunogenicity. Preliminary computational analyses suggest that the proposed vaccine could achieve near-universal coverage, effectively targeting approximately 99.74% of the global population, with perfect coverage in specific regions such as Sweden and Finland. Molecular docking and immune simulation studies further validate the potential efficacy of the vaccine, indicating strong binding affinity with toll-like receptor 3 (TLR-3) and promising immune response profiles, including significant antibody-mediated and cellular responses. These findings present the vaccine construct as a viable candidate for further development and testing. While the theoretical and computational results are promising, advancing from in-silico predictions to a tangible vaccine requires comprehensive laboratory validation. This next step is essential to confirm the vaccine's efficacy and safety in eliciting an immune response against WNV. Through this study, we propose a novel approach to vaccine development against WNV and contribute to the broader field of immunoinformatics, showcasing the potential to accelerate the design of effective vaccines against emerging viral threats. The journey from hypothesis to practical solution embodies the interdisciplinary collaboration essential for modern infectious disease management and prevention strategies.

Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Impact of climate change on the global circulation of West Nile virus and adaptation responses: a scoping review.

BACKGROUND: West Nile virus (WNV), the most widely distributed flavivirus causing encephalitis globally, is a vector-borne pathogen of global importance. The changing climate is poised to reshape the landscape of various infectious diseases, particularly vector-borne ones like WNV. Understanding the anticipated geographical and range shifts in disease transmission due to climate change, alongside effective adaptation strategies, is critical for mitigating future public health impacts. This scoping review aims to consolidate evidence on the impact of climate change on WNV and to identify a spectrum of applicable adaptation strategies. MAIN BODY: We systematically analyzed research articles from PubMed, Web of Science, Scopus, and EBSCOhost. Our criteria included English-language research articles published between 2007 and 2023, focusing on the impacts of climate change on WNV and related adaptation strategies. We extracted data concerning study objectives, populations, geographical focus, and specific findings. Literature was categorized into two primary themes: 1) climate-WNV associations, and 2) climate change impacts on WNV transmission, providing a clear understanding. Out of 2168 articles reviewed, 120 met our criteria. Most evidence originated from North America (59.2%) and Europe (28.3%), with a primary focus on human cases (31.7%). Studies on climate-WNV correlations (n = 83) highlighted temperature (67.5%) as a pivotal climate factor. In the analysis of climate change impacts on WNV (n = 37), most evidence suggested that climate change may affect the transmission and distribution of WNV, with the extent of the impact depending on local and regional conditions. Although few studies directly addressed the implementation of adaptation strategies for climate-induced disease transmission, the proposed strategies (n = 49) fell into six categories: 1) surveillance and monitoring (38.8%), 2) predictive modeling (18.4%), 3) cross-disciplinary collaboration (16.3%), 4) environmental management (12.2%), 5) public education (8.2%), and 6) health system readiness (6.1%). Additionally, we developed an accessible online platform to summarize the evidence on climate change impacts on WNV transmission ( https://2xzl2o-neaop.shinyapps.io/WNVScopingReview/ ). CONCLUSIONS: This review reveals that climate change may affect the transmission and distribution of WNV, but the literature reflects only a small share of the global WNV dynamics. There is an urgent need for adaptive responses to anticipate and respond to the climate-driven spread of WNV. Nevertheless, studies focusing on these adaptation responses are sparse compared to those examining the impacts of climate change. Further research on the impacts of climate change and adaptation strategies for vector-borne diseases, along with more comprehensive evidence synthesis, is needed to inform effective policy responses tailored to local contexts.

Seroprevalence of West Nile Virus in Tampa Bay Florida Patients Admitted to Hospital during 2020-2021 for Respiratory Symptoms.

West Nile virus (WNV) is an arbovirus spread primarily by Culex mosquitoes, with humans being a dead-end host. WNV was introduced to Florida in 2001, with 467 confirmed cases since. It is estimated that 80 percent of cases are asymptomatic, with mild cases presenting as a non-specific flu-like illness. Currently, detection of WNV in humans occurs primarily in healthcare settings via RT-PCR or CSF IgM when patients present with severe manifestations of disease including fever, meningitis, encephalitis, or acute flaccid paralysis. Given the short window of detectable viremia and requirement for CSF sampling, most WNV infections never receive an official diagnosis. This study utilized enzyme-linked immunosorbent assay (ELISA) to detect WNV IgG antibodies in 250 patient serum and plasma samples collected at Tampa General Hospital during 2020 and 2021. Plaque reduction neutralization tests were used to confirm ELISA results. Out of the 250 patients included in this study, 18.8% of them were IgG positive, consistent with previous WNV exposure. There was no relationship between WNV exposure and age or sex.

Aedes albopictus is a competent vector of five arboviruses affecting human health, greater Paris, France, 2023.

Aedes albopictus collected in 2023 in the greater Paris area (Île-de-France) were experimentally able to transmit five arboviruses: West Nile virus from 3 days post-infection (dpi), chikungunya virus and Usutu virus from 7 dpi, dengue virus and Zika virus from 21 dpi. Given the growing number of imported dengue cases reported in early 2024 in France, surveillance of Ae. albopictus should be reinforced during the Paris Olympic Games in July, when many international visitors including from endemic countries are expected.