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Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5' RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Kyasanur Forest disease virus (KFDV), a tick-borne flavivirus prevalent in India, presents a serious threat to human health. KFDV NS3 helicase (NS3hel) is considered a potential drug target due to its involvement in the viral replication complex. Here, we resolved the crystal structures of KFDV NS3hel apo and its complex with three phosphate molecules, which indicates a conformational switch during ATP hydrolysis. Our data revealed that KFDV NS3hel has a higher binding affinity for dsRNA, and its intrinsic ATPase activity was enhanced by dsRNA while being inhibited by DNA. Through mutagenesis analysis, several residues within motifs I, Ia, III, V, and VI were identified to be crucial for NS3hel ATPase activity. Notably, the M419A mutation drastically reduced NS3hel ATPase activity. We propose that the methionine-aromatic interaction between residues M419 and W294, located on the surface of the RNA-binding channel, could be a target for the design of efficient inhibitor probes. Moreover, epigallocatechin gallate (EGCG), a tea-derived polyphenol, strongly inhibited NS3hel ATPase activity with an IC 50 value of 0.8 μM. Our computational docking data show that EGCG binds at the predicted druggable hotspots of NS3hel. Overall, these findings contribute to the development and design of more effective and specific inhibitors., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response., (© 2023 The Authors.)

Kyasanur Forest Disease virus (KFDV), a neglected human pathogenic virus, is a Flavivirus that causes severe hemorrhagic fever in humans. KFDV is transmitted to humans by the bite of the hard tick ( Haemaphysalis spinigera ), which acts as a reservoir of KFDV. The recent expansion of the endemic area of KFDV is of concern and requires the development of new preventive measures against KFDV. Currently, there is no antiviral therapy against KFDV, and the existing vaccine has limited efficacy. To develop a new antiviral therapy against KFDV, we focused on the nonstructural proteins NS2B and NS3 of KFDV, which are responsible for serine protease activity. Viral proteases have shown to be suitable therapeutic targets in the development of antiviral drugs against many diseases. However, success has been limited in flaviviruses, mainly because of the important features of the active site, which is flat and highly charged. In this context, the present study focuses on the dynamics of NS2B and NS3 to identify potential allosteric sites in the NS2B/NS3 protease of KDFV. To our knowledge, there are no reports on the dynamics of NS2B and NS3 in KFDV, and the crystal structure of the NS2B/NS3 protease of KFDV has not yet been solved. Overall, we created the structure of the NS2B/NS3 protease of KFDV using AlphaFold and performed molecular dynamics simulations with and without NS2B cofactor to investigate structural rearrangements due to cofactor binding and to identify alternative allosteric sites. The identified allosteric site is promising due to its geometric and physicochemical properties and druggability and can be used for new drug development. The applicability of the proposed allosteric binding sites was verified for the best-hit molecules from the virtual screening and MD simulations.

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar -/- ) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar -/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar -/- , the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism., (© 2023. The Author(s).)

Flaviviruses are enveloped viruses causing high public concerns. Their maturation spans several cellular compartments having different pH. Thus, complex control mechanisms are in place to avoid premature maturation. Here we report the dynamical behavior at neutral and acidic pH of the precursor of the membrane fusion protein E of tick-borne encephalitis, showing the different stabilizations of the E dimer and the role played by the small fusion-assisting protomer (pr). The comprehension, at atomic resolution, of the fine regulation of viral maturation will be fundamental to the development of efficient strategies against emerging viral threats.

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is typically transmitted upon tick bite and can cause meningitis and encephalitis in humans. In TBEV-infected mice, mitochondrial antiviral-signaling protein (MAVS), the downstream adaptor of retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling, is needed to induce early type I interferon (IFN) responses and to confer protection. To characterize the brain-resident cell subset that produces protective IFN-β in TBEV-infected mice, we isolated neurons, astrocytes, and microglia from mice and exposed these cell types to TBEV in vitro. Under such conditions, neurons showed the highest percentage of infected cells, whereas astrocytes and microglia were infected to a lesser extent. In the supernatant (SN) of infected neurons, IFN-β was not detectable, while infected astrocytes showed high and microglia low IFN-β expression. Transcriptome analyses of astrocytes implied that MAVS signaling was needed early after TBEV infection. Accordingly, MAVS-deficient astrocytes showed enhanced TBEV infection and significantly reduced early IFN-β responses. Nevertheless, at later time points, moderate amounts of IFN-β were detected in the SN of infected MAVS-deficient astrocytes. Transcriptome analyses indicated that MAVS deficiency negatively affected the induction of early anti-viral responses, which resulted in significantly increased TBEV replication. Treatment with MyD88 and TRIF inhibiting peptides reduced only late IFN-β responses of TBEV-infected WT astrocytes and blocked entirely IFN-β responses of infected MAVS-deficient astrocytes. Thus, upon TBEV exposure of brain-resident cells, astrocytes are important IFN-β producers showing biphasic IFN-β induction that initially depends on MAVS and later on MyD88/TRIF signaling., (© 2021 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called "stem" region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Tick-borne encephalitis virus (TBEV) causes 5-7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

Tick-borne encephalitis virus (TBEV) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia and causes encephalitis in humans. In this study, we demonstrate TBEV appears to activate the interferon (IFN)-β dependent on RIG-I/MDA5. Both the IFN-β accumulation and the IFN stimulated genes (ISGs) transcription greatly delay. Further studies reveal that TBEV NS4A could block the phosphorylation and dimerization of STAT1/STAT2 to affect type I and II IFN-mediated STAT signalling. Additional data indicate that the residue at K132 of TBEV NS4A could be modified by ubiquitination and this modification is necessary for the interaction of NS4A with STAT1. Dynamic ubiquitination of the NS4 protein during TBEV infection might account for delayed activation of the ISGs. These results define the TBEV NS4A as an antagonist of the IFN response, by demonstrating a correlation between the association and STAT interference. Our findings provide a foundation for further understanding how TBEV evade innate immunity and a potential viral target for intervention.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 μg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1β. Phosprenyl, on the contrary, increased production of IL-1β and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1β, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

Background: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection., Method: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated., Results: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains., Conclusions: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man 5 GlcNAc 2 ), a complex biantennary galactosylated structure with core fucose (Gal 2 GlcNAc 2 Man 3 GlcNAc 2 Fuc), and a group of hybrid glycans with the composition Gal 0-1 GlcNAc 1 Man 3-5 GlcNAc 2 Fuc 0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man 3-4 GlcNAc 2 Fuc 0-1 ) and high-mannose structures with five and six mannoses (Man 5-6 GlcNAc 2 ) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

Powassan virus lineage II (POWV), also known as deer tick virus, is an emerging tick-borne pathogen transmitted by Ixodes scapularis , the natural vector for the organisms that causes Lyme disease, babesiosis, and anaplasmosis. POWV is the only tick-borne flavivirus in North America known to cause disease in humans. We present a suspected pediatric case of POWV infection in northern Wisconsin., (© 2020 Marshfield Clinic.)

Background and objectives: Tick-borne encephalitis virus (TBEV) infections have been the cause of threatening outbreaks for many years. Apart from several physical and chemical methods to prevent tick bites, active vaccination of people highly exposed to infection is still the most important strategy of prevention. However, in some subjects, the lack of or low response to TBEV antigens is observed. The aim of the current study was to assess the prevalence of seronegative rate for anti-TBEV antibodies and the risk factors for waning immunity. Materials and Methods: 2315 at least primary vaccinated subjects from the high risk group for TBEV infections participated in this study. A commercial enzyme-linked immunosorbent assay (ELISA) test was used for the assessment of anti-TBEV IgG serum level. Results: Data showed that 86.2% of subjects who underwent vaccination were positive for anti-TBEV antibodies within 5 years. As much as 13.8% of subjects that underwent primary or primary and booster vaccination were barely protected after vaccination. Women and subjects under 60 years underwent more effective protection but sex and older age was not a risk factor for being a subject of waning immunity. A logistic regression showed that both a longer time since the vaccination and a lower number of booster doses constantly increased the chance of lost anti-TBEV antibodies. Conclusions: This study demonstrates that the vaccination schedule should be reevaluated. The extension of the interval of booster immunization is risky and all subjects should be surrounded by care consisting of more frequent monitoring of serum antibodies by personalized schedule to adjust the frequency of subsequent doses of booster vaccination.

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEV-SVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29- and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ulf Landegren is a founder, shareholder and board member of Navinci, having rights to the in situ PLA (isPLA) technology., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required. IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level., (Copyright © 2019 Yau et al.)

Tick-borne encephalitis infections have increased the last 30 years. The mortality associated to this viral infection is 0.5 to 30% with a risk of permanent neurological sequelae, however, no therapeutic is currently available. The first steps of virus-cell interaction, such as attachment and entry, are of importance to understand pathogenesis and tropism. Several molecules have been shown to interact with tick-borne encephalitis virus (TBEV) at the plasma membrane surface, yet, no studies have proven that these are specific entry receptors. In this study, we set out to characterize the cellular attachment receptor(s) for TBEV using the naturally attenuated member of the TBEV complex, Langat virus (LGTV), as a model. Inhibiting or cleaving different molecules from the surface of A549 cells, combined with inhibition assays using peptide extracts from high LGTV binding cells, revealed that LGTV attachment to host cells is dependent on plasma membrane proteins, but not on glycans or glycolipids, and suggested that LGTV might use different cellular attachment factors on different cell types. Based on this, we developed a transcriptomic approach to generate a list of candidate attachment and entry receptors. Our findings shed light on the first step of the flavivirus life-cycle and provide candidate receptors that might serve as a starting point for future functional studies to identify the specific attachment and/or entry receptor for LGTV and TBEV., Competing Interests: The authors have declared that no competing interests exist.

Local translation in neurites is considered as an important mechanism to modulate synaptic plasticity of neurons. However, it is hard to specifically express a protein-coding gene in neurites. Recently, the 5'-UTR of Tick-borne encephalitis virus (TBEV) is reported to be able to drive its RNA to the dendrites of infected neurons, as a cis-acting RNA element. To construct a neurite specific gene expression system, present study tested the ability of 5'-UTR of TBEV to bring a mRNA (mCherry CDS) to the neurites for targeted expression. We showed that both the 5'-UTR of TBEV and the 3'-UTR of Actb gene could bring the protein coding mRNA to neurites, and the TBEV 5'-UTR bearing mRNA was more robust targeted into neurites. About the safety of the TBEV 5'-UTR, there was no obvious cytotoxicity to the neurons when adding either cis-acting RNA element to the protein-expressing plasmid vectors. Given the short length and high efficiency of the TBEV 5'-UTR, the 5'-UTR of TBEV were assemble into an AAV plasmid to produce virus particles for expressing protein-coding gene in vivo. After two weeks infection, the TBEV 5'-UTR infected neurons expressed more mCherry protein in their neurites. In conclusion, as a short while high efficient cis-acting RNA element, TBEV 5'-UTR could be useful in neural system research and locally express synaptic proteins more precisely., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tick-borne encephalitis (TBE) is a widespread, dangerous infection. Unfortunately, all attempts to create safe anti-TBE subunit vaccines are still unsuccessful due to their low immunogenicity. The goal of the present work was to investigate the immunogenicity of a recombinant chimeric protein created by the fusion of the EIII protein, comprising domain III and a stem region of the tick-borne encephalitis virus (TBEV) E protein, and the OmpF porin of Yersinia pseudotuberculosis (OmpF-EIII). Adjuvanted antigen delivery systems, the tubular immunostimulating complexes (TI-complexes) based on the monogalactosyldiacylglycerol from different marine macrophytes, were used to enhance the immunogenicity of OmpF-EIII. Also, the chimeric protein incorporated into the most effective TI-complex was used to study its protective activity. The content of anti-OmpF-EIII antibodies was estimated in mice blood serum by enzyme-linked immunosorbent assay (ELISA). To study protective activity, previously immunized mice were infected with TBEV strain Dal'negorsk (GenBank ID: FJ402886). The animal survival was monitored daily for 21 days. OmpF-EIII incorporated into the TI-complexes induced about a 30⁻60- and 5⁻10-fold increase in the production of anti-OmpF-EIII and anti-EIII antibodies, respectively, in comparison with the effect of an individual OmpF-EIII. The most effective vaccine construction provided 60% protection. Despite the dramatic effect on the specific antibody titer, the studied TI-complex did not provide a statistically significant increase in the protection of OmpF-EIII protein. However, our results provide the basis of the future search for approaches to design and optimize the anti-TBEV vaccine based on the OmpF-EIII protein.

Tick-borne encephalitis virus (TBEV) is one of the flaviviruses that targets the CNS and causes encephalitis in humans. The mechanism of TBEV that causes CNS destruction remains unclear. It has been reported that RANTES-mediated migration of human blood monocytes and T lymphocytes is specifically induced in the brain of mice infected with TBEV, which causes ensuing neuroinflammation and may contribute to brain destruction. However, the viral components responsible for RANTES induction and the underlying mechanisms remain to be fully addressed. In this study, we demonstrate that the NS5, but not other viral proteins of TBEV, induces RANTES production in human glioblastoma cell lines and primary astrocytes. TBEV NS5 appears to activate the IFN regulatory factor 3 (IRF-3) signaling pathway in a manner dependent on RIG-I/MDA5, which leads to the nuclear translocation of IRF-3 to bind with RANTES promoter. Further studies reveal that the activity of RNA-dependent RNA polymerase (RdRP) but not the RNA cap methyltransferase is critical for TBEV NS5-induced RANTES expression, and this is likely due to RdRP-mediated synthesis of dsRNA. Additional data indicate that the residues at K359, D361, and D664 of TBEV NS5 are critical for RdRP activity and RANTES induction. Of note, NS5s from other flaviviruses, including Japanese encephalitis virus, West Nile virus, Zika virus, and dengue virus, can also induce RANTES expression, suggesting the significance of NS5-induced RANTES expression in flavivirus pathogenesis. Our findings provide a foundation for further understanding how flaviviruses cause neuroinflammation and a potential viral target for intervention., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, we present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.

Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.

Background: Tick-borne encephalitis virus (TBEV) is one of the most important flaviviruses that targets the central nervous system (CNS) and causes encephalitides in humans. Although neuroinflammatory mechanisms may contribute to brain tissue destruction, the induction pathways and potential roles of specific chemokines in TBEV-mediated neurological disease are poorly understood., Methods: BALB/c mice were intracerebrally injected with TBEV, followed by evaluation of chemokine and cytokine profiles using protein array analysis. The virus-infected mice were treated with the CC chemokine antagonist Met-RANTES or anti-RANTES mAb to determine the role of RANTES in affecting TBEV-induced neurological disease. The underlying signaling mechanisms were delineated using RANTES promoter luciferase reporter assay, siRNA-mediated knockdown, and pharmacological inhibitors in human brain-derived cell culture models., Results: In a mouse model, pathological features including marked inflammatory cell infiltrates were observed in brain sections, which correlated with a robust up-regulation of RANTES within the brain but not in peripheral tissues and sera. Antagonizing RANTES within CNS extended the survival of mice and reduced accumulation of infiltrating cells in the brain after TBEV infection. Through in vitro studies, we show that virus infection up-regulated RANTES production at both mRNA and protein levels in human brain-derived cell lines and primary progenitor-derived astrocytes. Furthermore, IRF-3 pathway appeared to be essential for TBEV-induced RANTES production. Site mutation of an IRF-3-binding motif abrogated the RANTES promoter activity in virus-infected brain cells. Moreover, IRF-3 was activated upon TBEV infection as evidenced by phosphorylation of TBK1 and IRF-3, while blockade of IRF-3 activation drastically reduced virus-induced RANTES expression., Conclusions: Our findings together provide insights into the molecular mechanism underlying RANTES production induced by TBEV, highlighting its potential importance in the process of neuroinflammatory responses to TBEV infection.

Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication are believed to trigger the innate immune response through interaction with cytoplasmic cellular sensors. Therefore, understanding the subcellular distribution and dynamics of Flavivirus RNAs is of paramount importance to understand the interaction of the virus with its cellular host, which could be of insect, tick or mammalian, including human, origin. Recent advances on the visualization of Flavivirus RNA in living cells together with the development of methods to measure the dynamic properties of viral RNA are reviewed and discussed in this essay. In particular the application of bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are analysed in the context of tick-borne encephalitis virus replication. Conclusions driven by this approached are discussed in the wider context Flavivirus infection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Infection with tick-borne encephalitis virus (TBEV) causes pathological changes in the central nervous system. However, the possible redox alterations in the infected cells that can contribute to the virus pathogenicity remain unknown., Objective: In the current study we explored the ability of TBEV nonstructural protein 1 (NS1) to induce oxidative stress and activate antioxidant defense via the nuclear factor (erythroid-derived-2)-like 2/antioxidant response element (Nrf2/ARE) pathway., Methods: HEK 293T cells were transfected with plasmid encoding NS1 protein, and the production of reactive oxygen species (ROS) was measured using oxidation-sensitive dyes, the activation of the ARE promoter was estimated using a reporter plasmid, and the expression of phase II detoxifying enzymes was quantified by measuring their mRNA levels using RT-qPCR., Results: A high level of ROS production was detected in cells transfected with NS1-expressing plasmid. In addition, this protein activated the promoter with an ARE and upregulated the transcription of ARE-dependent genes that encode phase II enzymes., Conclusion: TBEV NS1 protein both triggers ROS production and activates a defense Nrf2/ARE pathway. These data suggest that a role of redox-mediated processes in TBEV-induced damage of the central nervous system should also be explored. These data can contribute to a better understanding of TBEV pathogenicity, further improvement of TBE treatment, and the development of vaccine candidates against this infection., (© 2016 S. Karger AG, Basel.)

Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

Introduction: Bioterrorism is a terrorist action involving the intentional release or dissemination of a biological warfare agent (BWA), which includes some bacteria, viruses, rickettsiae, fungi or biological toxins. BWA is a naturally occurring or human-modified form that may kill or incapacitate humans, animals or plants as an act of war or terrorism. BWA is a weapon of choice for mass destruction and terrorism, because of the incubation period, less effective amount than chemical warfare agents, easily distribution, odorless, colorless, difficult to detect, no need of specialized equipment for production and naturally distribution which can easily be obtained. BWA may be disseminating as an aerosol, spray, explosive device, and by food or water., Classification: Based on the risk for human health, BWAs have been prioritized into three categories of A, B and C. Category A includes microorganisms or toxins that easily spread, leading to intoxication with high death rates such as Anthrax, Botulism, Plague, Smallpox, Tularemia and Viral hemorrhagic fevers. Category B has lower toxicity with wider range, including Staphylococcal Entrotoxin type B (SEB), Epsilon toxin of Clostridium perfringens, Ricin, Saxotoxins, Abrin and Trichothecene mycotoxins. The C category includes emerging pathogens that could also be engineered for mass spread such as Hanta viruses, multidrug-resistant tuberculosis, Nipah virus, the tick-borne encephalitis viruses, hemorrhagic fever viruses and yellow fever. CLINICAL MANIFESTATIONS OF BIOTOXINS IN HUMAN: Clinical features and severity of intoxication depend on the agent and exposed dose, route of entry, individual variation and environmental factors. Onset of symptoms varies from 2-24 h in Ricin to 24-96 h in Botulism. Clinical manifestations also vary from irritation of the eyes, skin and mucus membranes in T2 toxin to an acute flaccid paralysis of bilateral cranial nerve impairment of descending manner in botulism. Most of the pyrogenic toxins such as SEB produce the same signs and symptoms as toxic shock syndrome including a rapid drop in blood pressure, elevated temperature, and multiple organ failure., Management: There is no specific antidote or effective treatment for most of the biotoxins. The clinical management is thus more supportive and symptomatic. Fortunately vaccines are now available for most of BWA. Therefore, immunization of personnel at risk of exposure is recommended., Conclusion: Biotoxins are very wide and bioterrorism is a heath and security threat that may induce national and international problems. Therefore, the security authorities, health professional and even public should be aware of bioterrorism., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report the finding of tick-borne encephalitis (TBE)-virus in indigenous Ixodes ricinus (L.), 'Candidatus Neoehrlichia mikurensis' in exotic Ixodes frontalis (Panzer) and Rickettsia aeshlimannii in exotic Hyalomma marginatum Koch subadult ticks detached from 18.5% (107/577) infested migratory birds in the Baltic region of Russia. This is the first record of human pathogenic 'Candidatus N. mikurensis' in I. frontalis ticks. Moreover, seven other pathogens were identified in I. ricinus ticks. Spotted Fever Group rickettsiae were the predominant pathogen group and were detected only in nymphs. Future investigations are warranted to further characterize the role of birds in the epizootiology of tick-borne pathogens in this region., (© 2012 The Authors. Medical and Veterinary Entomology © 2012 The Royal Entomological Society.)

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3(pro) processing., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000-3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3' untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tick-borne encephalitis virus (TBEV) causes extensive CNS disease in humans known as TBE, however, relatively little is known of the molecular mechanisms for its progress. Here, we now show that TBEV produces defects in neuronal development of PC12 cells through a function of the viral NS5 protein. The methyltransferase domain of NS5 is critical and sufficient for restriction of nerve growth factor induced neurite outgrowth. This effect is reversed by expression of NS5 mutants unable to bind Scribble and unexpectedly, in Scribble depleted cells with binding-competent NS5. Furthermore, we also demonstrate that the Rho GTPase Rac1 and the guanine nucleotide-exchange factor, betaPIX are outcompeted by NS5 for binding to Scribble, linking to effects on neurite outgrowth by TBEV. Together, these findings provide the first experimental evidence that Rac1 and betaPIX are indirect targets of NS5 acting through the multifunctional polarity protein Scribble to oppose neuronal differentiation. In conclusion, our results offer a potential mechanism by which TBEV alters neuronal circuitry and opens new avenues for therapeutic interventions.

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tickborne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 --> Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 --> Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alpha- and flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187-210, and MAB 4F6 interacts with the fragment of residues 263-278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187-210 and 263-278) as well as the central beta-folded region turning into the alpha-helical site including residues 200-216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.

The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsid cleavage site in tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) by the 2A protein of foot-and-mouth disease virus (TBEV-2A and WNV-2A). This obviated the need for NS2B/3 processing at the C terminus of mature protein C while simultaneously producing a 19-amino-acid extension on protein C. Infectious virions were generated with both viruses; the phenotype depended on the host cell. TBEV-2A replicated well in BHK-21 cells but was essentially incapable of replication in tick cells. In contrast, WNV-2A replicated well in mosquito cells but showed a small-plaque phenotype in Vero cells, with frequent production of larger plaques. Sequencing of viral RNA from the larger plaques showed substitutions in the signal sequence for prM, presumably improving coordinated protein processing at the C-prM junction. Furthermore, both TBEV-2A and WNV-2A were also defective in unpackaging and/or early RNA synthesis. Together, these results indicate a role for flavivirus protein C in both viral assembly and RNA replication, possibly by interacting with host cell factors required to set up the cell for RNA replication.

Homology modeling of NS3 helicase in Tick-borne encephalitis virus using known protein crystal structure was done. Its 3-D structure was evaluated and validated using PROCHECK comprising amino acid residues in favored region of Ramachandran plot. Helicase forms a large family of proteins which ubiquitously distributes in wide variety of organisms. It plays crucial role in transcription and replication of single-stranded viral RNA genomes. Consequently, NS3 represents an interesting target for the development of specific antiviral inhibitors. Several helicase inhibiting effective drugs and analogs were selected and the active amino acid residues were targeted. Levovirin, Ribamidine and Ribavirin were found more potent to inhibit TBEV on the basis of robust binding affinity between protein-drug interactions. This finding may help to understand the nature of helicase and development of specific anti-TBEV therapies.

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.

The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Site-specific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.

The tick-borne encephalitis (TBE) complex of viruses, genus Flavivirus, can cause severe encephalitis, meningitis, and/or hemorrhagic fevers. Effective interferon (IFN) responses are critical to recovery from infection with flaviviruses, and the mosquito-borne flaviviruses can inhibit this response. However, little is known about interactions between IFN signaling and TBE viruses. Langat virus (LGTV), a member of the TBE complex of viruses, was found to be highly sensitive to the antiviral effects of IFN. However, LGTV infection inhibited IFN-induced expression of a reporter gene driven by either IFN-alpha/beta- or IFN-gamma-responsive promoters. This indicated that LGTV can inhibit the IFN-mediated JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway of signal transduction. The mechanism of inhibition was due to blocks in the phosphorylation of both Janus kinases, Jak1 and Tyk2, during IFN-alpha signaling and at least a failure of Jak1 phosphorylation following IFN-gamma stimulation. To determine the viral protein(s) responsible, we individually expressed all nonstructural (NS) proteins and examined their ability to inhibit signal transduction. Expression of NS5 alone inhibited STAT1 phosphorylation in response to IFN, thus identifying NS5 as a potential IFN antagonist. Examination of interactions between NS5 and cellular proteins revealed that NS5 associated with IFN-alpha/beta and -gamma receptor complexes. Importantly, inhibition of JAK-STAT signaling and NS5-IFN receptor interactions were demonstrated in LGTV-infected human monocyte-derived dendritic cells, important target cells for early virus replication. Because NS5 may interfere with both innate and acquired immune responses to virus infection, this protein may have a significant role in viral pathogenesis.

The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 3' non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20- and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression.

Enveloped viruses enter cells by fusion of their own membrane with a cellular membrane. Incorporation of inverted-cone-shaped lipids such as lysophosphatidylcholine (LPC) into the outer leaflet of target membranes has been shown previously to impair fusion mediated by class I viral fusion proteins, e.g., the influenza virus hemagglutinin. It has been suggested that these results provide evidence for the stalk-pore model of fusion, which involves a hemifusion intermediate (stalk) with highly bent outer membrane leaflets. Here, we investigated the effect of inverted-cone-shaped LPCs and the cone-shaped oleic acid (OA) on the membrane fusion activity of a virus with a class II fusion protein, the flavivirus tick-borne encephalitis virus (TBEV). This study included an analysis of lipid mixing, as well as of the steps preceding or accompanying fusion, i.e., binding to the target membrane and lipid-induced conformational changes in the fusion protein E. We show that the presence of LPC in the outer leaflet of target liposomes strongly inhibited TBEV-mediated fusion, whereas OA caused a very slight enhancement, consistent with a fusion mechanism involving a lipid stalk. However, LPC also impaired the low-pH-induced binding of a soluble form of the E protein to liposomes and its conversion into a trimeric postfusion structure that requires membrane binding at low pH. Because inhibition is already observed before the lipid-mixing step, it cannot be determined whether impairment of stalk formation is a contributing factor in the inhibition of fusion by LPC. These data emphasize, however, the importance of the composition of the target membrane in its interactions with the fusion peptide that are crucial for the initiation of fusion.

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.