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biBackground:/i/bRats are a major carrier of several pathogens, including zoonotic pathogens that can cause fatal diseases in humans. Indonesia has one of the fastest growing populations, with high infestation of rats in urban areas. Therefore, this study aims to assess the seropositivity of zoonotic pathogens in rats from four markets in Bogor, Indonesia.biMaterials and Methods:/i/bA total of 80 brown rats (iRattus norvegicus/i) were captured from the markets and screened for the presence of some zoonotic pathogens, specifically hantavirus,iLeptospira/ispp.,iOrientia tsutsugamushi/i, tick-borne encephalitis virus (TBEV), and lymphocytic choriomeningitis virus (LCMV) antibodies, using indirect fluorescence assay or enzyme-linked immunosorbent assay.biResults:/i/bAmong the 80 rats, 40% were seropositive for hantavirus, 36.3% foriLeptospira/ispp., 11.3% foriO. tsutsugamushi/i, 6.3% for TBEV, and 0% for LCMV.biConclusion:/i/bOverall, these results indicate that rats in Bogor pose a potential zoonotic risk to humans.

Omsk hemorrhagic fever was first described in the early 1940s and is a natural focal infection, spread exclusively in four regions of Western Siberia, and associated with muskrat (Ondatra zibethicus). The etiological agent of this disease is the Omsk hemorrhagic fever virus (OHFV) which is closely related to the tick-borne encephalitis virus (TBEV), and its range entirely lies within the TBEV area. OHFV belongs to the mammalian tick-borne flaviviruses and the ecological group of arboviruses. The problem concerning the origin of OHFV remains unresolved to date. This study analyzed all nucleotide sequences of the OHFV genome obtained in the present study and available in GenBank, including the E gene fragment and the amino acid sequences of the surface glycoprotein encoded by it. The conclusions, based on the clusteron approach, suggest that OHFV originated directly from the TBEV of the Far Eastern subtype due to the host-jump phenomenon, that is, through a rapid change from an arthropod host, Ixodes persulcatus, to a rodent, O. zibethicus. The muskrat was introduced to Western Siberia in the second half of the 1930s. The peculiarities of the biology and ecology of the muskrat in the new habitat became the reason for the TBEV cross-species transmission. Calculations show that host-jumping occurred between 1931 and 1947 and accompanied a cascade of adaptive amino acid substitutions in protein E. As a result, the virus changed its transmission to contact, alimentary, and airborne routes. Based on the data obtained, OHFV would be more correctly attributed to zoonotic viruses transmitted by rodents and, accordingly, to the ecological group of roboviruses.

Significance Kyasanur forest disease (KFD), a re-emerging tick-borne viral disease causes severe hemorrhagic fever in humans and nonhuman primates. KFD virus (KFDV) is a member of genus Flavivirus. KFD is now increasingly reported outside its endemic zone in India. Rapid and specific detection of the KFDV plays a critical role in containment of the outbreak. The diagnosis of KFD currently relies on real-time RT-PCR, nested RT-PCR, end point RT-PCR, and serodiagnostic assay. These assays are tedious, time-consuming, and cannot be used as a routine screening platform. Objective The present study was aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for molecular diagnosis of KFD. Design The gene amplification reaction was accomplished by incubation at a constant temperature of 63 °C for 60 min. Results The limit of detection of RT-LAMP assay was 10 copies. KFD RT-LAMP assay was successfully evaluated with diverse host samples including humans, monkeys, and tick. The assay correctly picked up different KFD isolates indicating its applicability for divergent strains. Comparative evaluation of RT-LAMP assay with quantitative TaqMan real-time RT-PCR revealed 100% concordance. No cross-reaction with related flavi and other hemorrhagic fever viruses was observed, indicating its high specificity. Conclusion and relevance : The RT-LAMP test developed in this study will serve as a rapid, sensitive alternate detection method for KFDV infection and would be useful for high throughput screening of clinical samples in resource limited areas during outbreaks.

Abstract Tick-borne diseases (TBDs) are bacterial, viral, and parasitic diseases transmitted by ticks. Viral TBDs have increased in prevalence over the last decade with many new pathogenic viruses being discovered. Doxycycline is often empirically prescribed by clinicians to treat symptomatic patients following tick bites due to suspicions of bacterial TBDs such as Rocky Mountain spotted fever, anaplasmosis, and ehrlichiosis. However, viral TBDs are included in the differential diagnosis if patients do not clinically improve following antibiotic therapy. Several viral TBDs present with dermatological manifestations. Recognizing the differences in clinical presentations of TBDs, particularly of newly emerging viral TBDs in the United States, can help physicians identify the viral TBD, and possibly rule out viral illnesses with different clinical presentations. Therefore, this review discusses clinical manifestations, with an emphasis on dermatologic manifestations of Heartland Virus, Bourbon Virus, Powassan Virus, Deer Tick Virus and Colorado Tick Fever Virus. Key points Viral tick-borne diseases have increased in prevalence over the last decade and often have similar clinical manifestations to other tick-borne diseases, including bacterial infections. Here, we review the dermatologic manifestations of Heartland Virus (HRTV), Bourbon Virus (BRBV), Powassan Virus (POWV), Deer Tick Virus (DTV) and Colorado Tick Fever Virus (CTFV) that are important for clinicians.

Tick-borne encephalitis virus (TBEV) is the most important tick-borne zoonotic virus in Europe. In Belgium, antibodies to TBEV have already been detected in wildlife and domestic animals, but up-to-date prevalence data for TBEV are lacking, and no studies have assessed its seroprevalence in sheep. Serum samples of 480 sheep from all over Belgium and 831 wild boar hunted in Flanders (northern Belgium) were therefore screened for TBEV antibodies by ELISA and plaque reduction neutralization test (PRNT), respectively. The specificity of positive samples was assessed by PRNTs for TBEV and the Louping Ill, West Nile, and Usutu viruses. TBEV seroprevalence was 0.42% (2/480, CI 95%: 0.11–1.51) in sheep and 9.27% (77/831, CI 95%: 7.48–11.43) in wild boar. TBEV seroprevalence in wild boar from the province of Flemish Brabant was significantly higher (22.38%, 15/67) compared to Limburg (7.74%, 34/439) and Antwerp (8.61%, 28/325). Oud-Heverlee was the hunting area harboring the highest TBEV seroprevalence (33.33%, 11/33). In an attempt to obtain a Belgian TBEV isolate, 1983 ticks collected in areas showing the highest TBEV seroprevalence in wild boars were tested by real-time qPCR. No TBEV-RNA-positive tick was detected. The results of this study suggest an increase in TBEV prevalence over the last decade and highlight the need for One-Health surveillance in Belgium.

Tick-borne encephalitis virus (TBEV) is an important human arthropod-borne virus that causes tick-borne encephalitis (TBE) in humans. TBEV acutely infects the central nervous system (CNS), leading to neurological symptoms of various severity. No therapeutics are currently available for TBEV-associated disease. Virus strains of various pathogenicity have been described, although the basis of their diverse clinical outcome remains undefined. Work with infectious TBEV requires high-level biocontainment, meaning model systems that can recapitulate the virus life cycle are highly sought. Here, we report the generation of a self-replicating, noninfectious TBEV replicon used to study properties of high (Hypr) and low (Vs) pathogenic TBEV isolates. Using a Spinach2 RNA aptamer and luciferase reporter system, we perform the first direct comparison of Hypr and Vs in cell culture. Infectious wild-type (WT) viruses and chimeras of the nonstructural proteins 3 (NS3) and 5 (NS5) were investigated in parallel to validate the replicon data. We show that Hypr replicates to higher levels than Vs in mammalian cells, but not in arthropod cells, and that the basis of these differences map to the NS5 region, encoding the methyltransferase and RNA polymerase. For both Hypr and Vs strains, NS5 and the viral genome localized to intracellular structures typical of positive-strand RNA viruses. Hypr was associated with significant activation of IRF-3, caspase-3, and caspase-8, while Vs activated Akt, affording protection against caspase-mediated apoptosis. Higher activation of stress-granule proteins TIAR and G3BPI were an additional early feature of Vs but not for Hypr. These findings highlight novel host cell responses driven by NS5 that may dictate the differential clinical characteristics of TBEV strains. This highlights the utility of the TBEV replicons for further virological characterization and antiviral drug screening.

Background Tick-borne encephalitis virus (TBEV) is an important causative agent that causes neurological infections in humans and animals. In recent years, only few epidemiological surveys on TBEV have been conducted in China. The purpose of this study was to determine the prevalence and subtype of TBEV in ticks in northeastern (NE) China. Methods A total of 3799 questing ticks were collected in NE China between April 2015 and June 2016. Ticks were pooled and tested for TBEV RNA using semi-nested reverse transcriptase polymerase chain reaction. Positive pools were used to isolate the virus and amplify complete sequences, followed by sequence identity and phylogenetic analysis. Results TBEV RNA was detected in Ixodes persulcatus ticks at a total prevalence of 2.9% (6/143; 95% confidence interval: 1.2-5.9%). Three TBEV strains were isolated (JL-T75, HLB-T74, and DXAL-T83) and showed 93.9-99.1% nucleotide identities and 97.1-99.5% amino acid identities in Far Eastern (FE) TBEV subtypes, and 82.9-87.6% nucleotide identities and 92.9-96.4% amino acid identities in other subtypes. For polyprotein, the JL-T75, HLB-T74, and DXAL-T83 strains showed 29, 50, and 55 amino acid residues, respectively, different from those in the TBEV vaccine (Senzhang) strain in China. Phylogenetic analysis revealed that these viruses were clustered in the FE-TBEV branch but formed distinct clades depending on the natural foci. Conclusions The results of this study suggest that the FE-TBEV subtype is still endemic in I. persulcatus ticks in NE China, and the viruses in different natural foci in NE China are more likely to have genetic differences. This article is protected by copyright. All rights reserved.

Background: New risk areas for tick-borne encephalitis (TBE) are emerging and the spread of disease and vaccine coverage is unclear in Sweden. We wanted to study the prevalence and levels of TBE-virus (TBEV) antibodies in southern Sweden, and to investigate whether there were individuals with undiagnosed TBE. Materials and Methods: Two cohorts of sera were collected: One group of anonymous individuals in rural areas (AIRA) in Skane and one group of volunteers who often got tick-bites (tick-bitten individuals [TBI]). An enzyme-linked immunosorbent assay for TBEV IgM and IgG was performed, as well as a TBEV neutralization test (NT) in selected individuals. Results: In the AIRA group, there was an IgG seropositivity of 5.3%. There were individuals with high antibody levels both in areas previously considered as risk areas (Bromolla and Knislinge), as well as in another area (Tyringe). In the TBI group, 45% of the individuals were vaccinated according to the questionnaires and IgG seropositivity was 28%. A lower seroprevalence and levels of antibodies were seen in the middle-aged group (50-69 years) compared with younger or elderly study participants. A positive NT revealed several individuals with suspected undiagnosed episodes of TBE. Conclusion: Subclinical or misdiagnosed cases have probably occurred in Skane. Middle-aged individuals had lower levels of IgG, which could indicate either less tick exposure or a lower vaccine response. Less than half of the TBI were vaccinated, an indication that more information about the disease and vaccine might be needed. We conclude that the study motivates an increased awareness of TBEV in the region.

Kyasanur forest disease virus (KFDV) causing tick-borne hemorrhagic fever which was earlier endemic to western Ghats, southern India, it is now encroaching into new geographic regions, but there is no approved medicine or effective vaccine against this deadly disease. In this study, we did in-silico design of multi-epitope subunit vaccine for KFDV. B-cell and T-cell epitopes were predicted from conserved regions of KFDV envelope protein and two vaccine candidates (VC1 and VC2) were constructed, those were found to be non-allergic and possess good antigenic properties, also gives cross-protection against Alkhurma hemorrhagic fever virus. The 3D structures of vaccine candidates were built and validated. Docking analysis of vaccine candidates with toll-like receptor-2 (TLR-2) by Cluspro and PatchDock revealed strong affinity between VC1 and TLR2. Ligplot tool was identified the intermolecular hydrogen bonds between vaccine candidates and TLR-2, iMOD server confirmed the stability of the docking complexes. JCAT sever ensured cloning efficiency of both vaccine constructs and in-silico cloning into pET30a (+) vector by SnapGene showed successful translation of epitope region. IMMSIM server was identified increased immunological responses. Finally, multi-epitope vaccine candidates were designed and validated their efficiency, it may pave the way for up-coming vaccine and diagnostic kit development.

Objectives Due to the remote forest area locations of sporadic cases and outbreaks of Kyasanur forest disease (KFD), rapid diagnosis poses a significant challenge. This study aimed to evaluate the diagnostic performance of Truenat KFD, a simple, rapid and user-friendly point-of-care test for detection of KFD and compare diagnostic accuracy with conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) testing. Truenat KFD can be deployed in a field laboratory setting. Methods The study involved 145 clinical specimens, including human serum, monkey necropsy tissues and tick pool, to validate Truenat KFD (Molbio Diagnostics Pvt.Ltd.) for KFD diagnosis. Results We have optimized and validated the microchip-based Truenat KFD (Molbio Diagnostics Pvt.Ltd.) for KFD diagnosis. Point-of-care testing was highly sensitive and specific, with a detection limit of up to 10 copies of KFD viral RNA. Results were comparable with the gold-standard TaqMan and commercially available Altona RealStar AHFV / KFDV real-time RT-PCR assays. Screening results for human, monkey and tick specimens were 100% concordant across the assays. Conclusion Truenat KFD(Molbio Diagnostics Pvt.Ltd.) was found to be highly sensitive and specific with a significant limit of detection. This point-of-care test would be useful in rapid diagnosis of KFD in remote and/or field settings, quick patient management and control of virus spread.

The beginning decades of the 21st century have been marked by multiple emergence and re-emergence phenomena of viral diseases [...]

The positive-sense flavivirus RNA genome bears a cap 1 structure essential for RNA stability and viral protein translation, and the formation of cap 1 requires the virally encoded nonstructural protein NS5 harboring guanylyltransferase (GTase), cap guanine N7 methyltransferase (N7 MTase), and 5′-nucleotide ribose 2′-O MTase activities in its single-domain MTase module. Despite numerous MTase-containing structures reported, the structural evidence for a critical GMP-enzyme intermediate formation and RNA repositioning when transitioning among different reactions is missing. Here, we report 10 high-resolution MTase crystal structures of Omsk hemorrhagic fever virus (OHFV), a representative high-consequence tick-borne flavivirus, capturing previously unidentified GMP-arginine adduct structures and a rarely observed capped RNA conformation. These structures help us thread capping events in the canonical model with a structure-based hypothesis involving the flipping of the 5′ nucleotide, while the observation of an m(7)GMP-arginine adduct is compatible with an alternate capping model that decouples the N7 and 2′-O methylation steps. IMPORTANCE The methyltransferase (MTase) domain of flavivirus NS5 is unique in harboring guanylyltransferase (GTase), N7 MTase, and 2′-O MTase activities, playing a central role in viral RNA capping. However, the detailed mechanisms of the multistep capping process remain elusive. Here, we report 10 crystal structures of a flavivirus MTase to help understand the guanylyl transfer from GTP to the GTase itself and the transition between guanylyl transfer and methylation steps. In particular, a previously unobserved GMP-arginine covalent intermediate was captured multiple times in MTase crystal soaking trials with GTP present in the soaking solution, supporting its role in bridging the guanylyl transfer from GTP to the GTase and subsequent transfer to the 5′-diphosphate RNA.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes tick-borne encephalitis (TBE) in humans. Infections of Sapporo-17-Io1 (Sapporo) and Oshima 5-10 (Oshima) TBEV strains showed different pathogenic effects in mice. However, the differences between the two strains are unknown. In this study, we examined neuronal degeneration and death, and activation of glial cells in mice inoculated with each strain to investigate the pathogenesis of TBE. Viral growth was similar between Sapporo and Oshima, but neuronal degeneration and death, and activation of glial cells, was more prominent with Oshima. In human neuroblastoma cells, apoptosis and pyroptosis were not observed after TBEV infection. However, the expression of the necroptosis marker, mixed lineage kinase domain-like (MLKL) protein, was upregulated by TBEV infection, and this upregulation was more pronounced in Oshima than Sapporo infections. As necroptosis is a pro-inflammatory type of cell death, differences in necroptosis induction might be involved in the differences in neuropathogenicity of TBE.

Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12250-021-00396-6.

Objectives This review article summarizes what has been published on Alkhumra hemorrhagic fever virus (AHFV), a novel flavivirus that was discovered in Saudi Arabia in 1995. Methods PubMed was used to search for studies published from January 1995 to June 2019 using the key words Alkhumra virus, Alkhurma virus, novel flavivirus, and tick-borne encephalitis virus. Additionally, records of the Saudi Ministry of Health were reviewed. Results Thirty-two articles on AHFV were identified. Acute febrile flu-like illness, hepatitis, hemorrhagic manifestations, and, less commonly, encephalitis are the main clinical features. The virus seems to be transmitted from livestock animals to humans by direct contact with these animals or their raw meat, or perhaps by tick or mosquito bites. The ability of ticks and mosquitoes to serve as vectors for AHFV needs to be confirmed by biological studies. The exact role of animals such as sheep, goats, camels, and other mammals in the transmission and maintenance of the virus remains to be elucidated. Preventive measures require an interdisciplinary approach involving the human and veterinary health sectors, the municipality, the ministry of agriculture, the vector control sector, and academic and research institutes. Conclusions AHFV has been well characterized; nevertheless, some aspects remain to be elucidated.

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

Tick-borne encephalitis (TBE) usually has a biphasic course which begins with unspecific febrile illness, followed by central nervous system involvement. Because TBE is not yet suspected during the initial phase, knowledge of early TBE pathogenesis is incomplete. Herein we evaluated laboratory and immune findings in the initial and second (meningoencephalitic) phase of TBE in 88 well-defined adult patients. Comparison of nine laboratory blood parameters in both phases of TBE revealed that laboratory abnormalities, consisting of low leukocyte and platelet counts and increased liver enzymes levels, were predominately associated with the initial phase of TBE and resolved thereafter. Assessment of 29 immune mediators in serum during the initial phase, and in serum and cerebrospinal fluid (CSF) during the second phase of TBE revealed highly distinct clustering patterns among the three groups. In the initial phase of TBE, the primary finding in serum was a rather heterogeneous immune response involving innate (CXCL11), B cell (CXCL13, BAFF), and T cell mediators (IL-27 and IL-4). During the second phase of TBE, growth factors associated with angiogenesis (GRO-α and VEGF-A) were the predominant characteristic in serum, whereas innate and Th1 mediators were the defining feature of immune responses in CSF. These findings imply that distinct immune processes play a role in the pathophysiology of different phases of TBE and in different compartments.

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study’s overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.

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.

BackgroundTick-borne encephalitis virus (TBEv) is a flavivirus that circulates in a complex cycle involving small mammals as amplifying hosts and ticks as vectors and reservoirs. The current study aimed to investigate the presence of TBEv in Tunisian sheep. A sample of 263 adult sheep were selected from 6 localities whereIxodes ricinusis well established. Sera were screened using ELISA for TBEv IgG detection, then the doubtful and positive sera were tested by the seroneutralisation test (SNT) and screened for West Nile Virus (WNv) IgG for cross-reaction assessment.ResultsThe ELISA for TBEv IgG detected one positive serum and 17 borderlines. The SNT showed one positive serum among the 18 tested, giving an overall antibody prevalence of 0.38% (95% CI = 0.07–2.12%). All but one serum tested negative to WNv ELISA. None of the sheep farmers reported neurological signs among sheep or humans in their households.ConclusionsThe results may indicate the circulation of TBEv for the first time in Tunisia and in North Africa. Further studies based on either virus isolation or RNA detection, are needed to confirm the presence of TBEv in North Africa.

Background: Tick-borne encephalitis (TBE) constitutes a public health concern in Europe. Certain coastal municipalities in southern Norway are considered TBE risk areas and in the last two years, there have been increasing numbers of TBE cases. Since the majority of infections are claimed to be asymptomatic, the aim of the current study was to assess the seroprevalence of antibodies to tick-borne encephalitis virus (TBEV) among unvaccinated adults living in a TBE endemic area in Norway. Methods: One thousand one hundred and twenty-three blood donors living in Vestfold and Telemark county were included and associated sera were analysed for TBEV IgG antibodies. Information regarding tick bites, previous flavivirus exposure and knowledge regarding TBE and TBE prevention were obtained through a questionnaire. Results: Fifty-eight samples were reactive by ELISA, of which 21 (36.2%) were confirmed by a TBEV-specific serum neutralization test. Of the 21 blood donors with neutralizing TBEV antibodies detected, 17 reported previous TBE vaccination.Thus, only four blood donors (0.4%) had TBEV neutralizing antibodies consistent with previously undergone TBEV infection. Regarding TBE awareness, half of the blood donors were familiar with TBE, but only 35% were aware of a preventiveTBE vaccine. Conclusions: Our study indicates low prevalence of subclinical TBEV infections among blood donors living in Vestfold and Telemark county and there is a lack of awareness among general public.

There is a considerable increase in vector-borne zoonotic diseases around the world, including Turkey, such as Crimean-Congo hemorrhagic fever (CCHF), tick borne encephalitis (TBE), Rift Valley fever (RVF), and West Nile fever (WNF), causing disease and death in humans and animals and significant economical losses. Hence, the aim of this study was to investigate the presence of CCHF virus (CCHFV) and TBE virus (TBEV) in ticks and RVF virus (RVFV) and WNF virus (WNV) in mosquitos, as well as in sheep and cattle, in the Thrace district of the Marmara region, which borders Bulgaria and Greece. Buffy-coat samples from 86 cattle and 81 sheep, as well as 563 ticks and 7390 mosquitos, were collected and examined by quantitative real-time RT-PCR for the presence of CCHFV, TBEV, RVFV, and WNV. All buffy-coat samples from cattle and sheep were negative for these viruses. Similarly, all tick samples were negative for CCHFV-RNA and TBEV-RNA. Among 245 pools representing 7390 mosquitos, only 1 pool sample was found to be positive for WNV-RNA and was confirmed by sequencing. Phylogenetic analysis revealed that it was WNV lineage-2. No RVFV-RNA was detected in the 245 mosquito pools. In conclusion, results of this study indicate that CCHFV, TBEV, and RVFV are not present in livestock and respective vectors in the Thrace district of Marmara region of Turkey, whereas WNV-RNA was found in mosquitos from this region.

The protective mechanisms of specific antibodies against tick-borne encephalitis virus were demonstrated on in vitro model. The effect of specific IgG on tick-borne encephalitis virus was comprehensively assessed in virucidal, preventive, direct antiviral, and intracellular actions by ELISA and virus titration results. The IC50 values were obtained for virucidal (3.8±0.7 U/ml), preventive (42.8±9.9 U/ml), direct antiviral (7.2±0.9 U/ml), and intracellular action (1.7±0.4 U/ml). During titration of the samples, complete elimination of the virus was observed at IgG concentration of 16 U/ml (virucidal), 320 U/ml (preventive), 32 U/ml (direct antiviral), and 8 U/ml (intracellular action). It was demonstrated that specific IgG produces a complex inhibitory effect on tick-borne encephalitis virus: it possesses both direct neutralizing activity on the virus and reduces its adsorption and intracellular replication.

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 (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 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 (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) 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.

Tick‐borne encephalitis virus (TBEV), a member of the family Flaviviridae, is the most important tick‐transmitted arbovirus in Europe. It can cause severe illnesses in humans and in various animal species. The main mechanism for the spread of TBEV into new areas is considered to be the translocation of infected ticks. To find out whether ducks can function as a natural virus reservoir in addition to serving as passive transport vectors, we carried out an experimental TBEV challenge study to reveal their susceptibility and resulting pathogenesis. Nineteen ducks were inoculated subcutaneously with TBEV strain “Neudoerfl” and monitored for 21 days. Blood, oropharyngeal and cloacal swabs were collected throughout the experiment and organ samples upon necropsy at the end of the study. All samples were tested for TBEV‐RNA by real‐time polymerase chain reaction. TBEV specific antibodies were determined by virus neutralization test and ELISA. Organ samples were examined histopathologically and by immunohistochemistry. The inoculated ducks did not show any clinical symptoms. TBEV‐specific RNA was detected in all brain samples as well as in a few blood and swab samples. Moreover, all challenged birds produced TBEV antibodies and showed a mild to severe acute to subacute necrotizing encephalitis. TBEV specific antigen was detected in the brain of 14 ducks by immunohistochemistry. The short and low viremic phases, as well as the low virus load in tissues suggest that ducks should not be considered as reservoir hosts. However, due to the high antibody levels, ducks can serve as sentinel species for the detection of natural TBEV foci.

Tick-borne encephalitis virus (TBEV) is a medically important representative of the Flaviviridae family. The TBEV genome encodes a single polyprotein, which is co/post-translationally cleaved into three structural and seven non-structural proteins. Of the non-structural proteins, NS5, contains an RNA-dependent RNA polymerase (RdRp) domain that is highly conserved and is responsible for the genome replication. Screening for potential antivirals was done using a hybrid receptor and ligand-based pharmacophore search likely targeting the RdRp domain. For the identification of pharmacophores, a mixture of small probe molecules and nucleotide triphosphates were used. The ligand/receptor interaction screenings of structures from the ZINC database resulted in five compounds. Zinc 3677 and 7151 exhibited lower cytotoxicity and were tested for their antiviral effect against TBEV in vitro. Zinc 3677 inhibited TBEV at micromolar concentrations. The results indicate that Zinc 3677 represents a good target for structure-activity optimizations leading potentially to a discovery of effective TBEV antivirals.

Tick-borne encephalitis virus (TBEV) is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus in Flaviviridae. It can cause the server infectious diseases named tick-borne encephalitis (TBE), which is characterized by paralysis and epilepsy. However, no effective treatment for TBE has been developed targeting TBEV. The NS3 helicase from TBEV plays an essential role in viral replication, which makes it an important target for drug design. In this study, the crystal structure of TBEV NS3 helicase has been determined to the resolution of 2.14 A. Subsequent alignment with homologous structures reveals that the NTP binding site and RNA-binding sites are located in motifs Ⅱ and Ⅵ of NS3 and the critical residues for binding are conserved across species in the genus, while the distinct conformation transition implies that the TBEV helicase need a different local rearrangement. This study demonstrates the key atomic-level features of TBEV helicase and provides basis for the design of antiviral drugs targeting TBEV helicase.

The aim of our study was to compare the course of TBE in children and adults. A retrospective analysis of the medical records of 669 patients was performed. The patients were categorized into 2 groups: Group I with 68 children and group II with 601 adults. TBE symptoms in children were milder compared with adults, with meningitis in 97% of cases. In adults, meningoencephalitis and meningoencephalomyelitis made up 49.26% of cases. Nausea and vomiting are more frequent in children, while neurological manifestations are more frequent in adults. There were no differences in CSF pleocytosis at the onset of disease in both groups, while CSF protein concentration was higher in adults. Children treated with corticosteroids over 7 days had higher checkup pleocytosis than pleocytosis at the onset of disease compared with adults. Corticosteroid use prolongs the disease duration but does not influence the development of TBE sequelae. Children had more favourable outcomes than adult patients.

Background Tick-borne encephalitis (TBE) is the most common viral CNS infection with incidences much higher than all other virus infections together in many risk areas of central and eastern Europe. The Odenwald Hill region (OWH) in southwestern Germany is classified as a TBE risk region and frequent case numbers but also more severe infections have been reported within the past decade. The objective of the present study was to survey the prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus and to associate TBEV genetic findings with TBE infections in the OWH. Methods Ticks were collected by the flagging methods supported by a crowdsourcing project implementing the interested public as collectors to cover completely and collect randomly a 3532 km2 area of the OWH TBE risk region. Prevalence of TBEV in I. ricinus was analysed by reversed transcription quantitative real-time PCR. Phylogeographic analysis was performed to classify OWH TBEV isolates within a European network of known TBEV strains. Mutational sequence analysis including 3D modelling of envelope protein pE was performed and based on a clinical database, a spatial association of TBE case frequency and severity was undertaken. Results Using the crowd sourcing approach we could analyse a total of 17,893 ticks. The prevalence of TBEV in I. ricinus in the OWH varied, depending on analysed districts from 0.12% to 0% (mean 0.04%). Calculated minimum infection rate (MIR) was one decimal power higher. All TBEV isolates belonged to the European subtype. Sequence analysis revealed a discontinuous segregation pattern of OWH isolates with two putative different lineages and a spatial association of two isolates with increased TBE case numbers as well as exceptional severe to fatal infection courses. Conclusions TBEV prevalence within the OWH risk regions is comparatively low which is probably due to our methodological approach and may more likely reflect prevalence of natural TBEV foci. As for other European regions, TBEV genetics show a discontinuous phylogeny indicating among others an association with bird migration. Mutations within the pE gene are associated with more frequent, severe and fatal TBE infections in the OWH risk region.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

Survival of tick-borne encephalitis virus was studied from pasteurized and unpasteurized goat milk and from salted/unsalted and spiced/unspiced cheese made from goat milk inoculated with low and high litres of infective virus. Both soft (63 °C, 30 min) and fast (72 °C, 15 s) pasteurization conditions destroyed viable virus particles. A small amount of infective virus could be detected only for 5‒10 days from milk, and from unsalted cheese. From milk inoculated with a higher amount of virus, infectious viral particles were detectable for 20‒25 days and from unsalted cheese samples for 10‒15 days, independently of the use of spices. Pasteurization and salt treatment made goat milk and cheese safely consumable. These two methods must be used when making any human food from goat milk to avoid milk-borne human TBEV infections.

BackgroundIxodes ricinusis the most common tick species in Europe and the main vector forBorrelia burgdorferi(sensu lato) and tick-borne encephalitis virus (TBEV). It is involved also in the transmission ofBorrelia miyamotoi, a relapsing fever spirochete that causes health disorders in humans. Little is known regarding the circulation ofBorreliaspecies and the natural foci of TBEV in north-eastern Germany. The goal of this study was to investigate the infection rates ofBorreliaspp. and of TBEV inI. ricinusticks from north-eastern Germany.MethodsTicks were collected by flagging from 14 forest sites in Mecklenburg-Western Pomerania between April and October 2018. RNA and DNA extraction was performed from individual adult ticks and from pools of 2–10 nymphs. Real time reverse transcription PCR (RT-qPCR) targeted the 3′ non-coding region of TBEV, while DNA ofBorreliaspp. was tested by nested PCR for the amplification of 16S-23S intergenic spacer. Multilocus sequence typing (MLST) was performed onB. miyamotoiisolates.ResultsIn total, 2407 ticks were collected (239 females, 232 males and 1936 nymphs). Female and maleI. ricinusticks had identical infection rates (both 12.1%) forBorreliaspp., while nymphal pools showed a minimum infection rate (MIR) of 3.3%. Sequencing revealed fourBorreliaspecies:B. afzelii,B. garinii,B. valaisianaandB. miyamotoi.Borrelia afzeliihad the highest prevalence in adult ticks (5.5%) and nymphs (MIR of 1.8%).Borrelia miyamotoiwas identified in 3.0% of adults and registered the MIR of 0.8% in nymphs.Borrelia valaisianawas confirmed in 2.5% adult ticks and nymphs had the MIR of 0.7%, whileB. gariniiwas present in 1.1% of adults and showed a MIR of 0.1% in nymphs. The MLST ofB. miyamotoiisolates showed that they belong to sequence type 635. No tick sample was positive after RT-qPCR for TBEV RNA.ConclusionsThe prevalence ofB. miyamotoiinI. ricinusticks registered similar levels to other reports from Europe suggesting that this agent might be well established in the local tick population. The detection ofB. burgdorferi(s.l.) indicates a constant circulation in tick populations from this region.

During February 2018–January 2019, we conducted large-scale surveillance for the presence and prevalence of tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) in sentinel animals and ticks in the United Kingdom. Serum was collected from 1,309 deer culled across England and Scotland. Overall, 4% of samples were ELISA-positive for the TBEV serocomplex. A focus in the Thetford Forest area had the highest proportion (47.7%) of seropositive samples. Ticks collected from culled deer within seropositive regions were tested for viral RNA; 5 of 2,041 ticks tested positive by LIV/TBEV real-time reverse transcription PCR, all from within the Thetford Forest area. From 1 tick, we identified a full-length genomic sequence of TBEV. Thus, using deer as sentinels revealed a potential TBEV focus in the United Kingdom. This detection of TBEV genomic sequence in UK ticks has important public health implications, especially for undiagnosed encephalitis.

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.

Here we report the first detection and confirmation of Powassan virus (POWV) (family: Flaviridae) in Ixodes scapularis ticks collected from Appalachian Virginia. Ixodes scapularis ticks were collected from vegetation across field sites in eight counties of western Virginia from June 2019 to April 2021. From these collections, one nymph and one adult male I. scapularis were determined to be positive for POWV using real-time RT-PCR and Sanger sequencing. Both positive ticks were collected from Floyd county, VA, at residential sites; the nymph in June 2020 and the adult male in April 2021. The presence of POWV in Virginia in its natural tick vector is crucial knowledge in beginning to understand the movement and transmission of this pathogen into new geographical areas and the risk it poses to medical and veterinary health.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3' untranslated region of the TBEV genome. This study analyzed the role of a Y-shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus-infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR–Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Ticks represent significant risks for human and animal health. Because they are obligate hematophagous ectoparasites and feed on diverse vertebrate hosts, they are considered as one of the most important vectors of zoonotic pathogens. Ticks can transmit a wide variety of bacteria, parasitesand viruses. In human and veterinary medicine, most tick-borne pathogens are transmitted by various hard ticks belonging to genera Ixodes, Haemaphysalis, Dermacentor, Hyalomma and Rhipicephalus and by certain soft ticks belonging to genera Argas and Ornithodoros. Among tick-borne pathogens, 170 tick-borne viruses (TBVs) were identified and belong to nine virus families and twelve virus genera. Viruses transmitted via tick bites can cause various symptoms in humans and animals, ranging from mild febrile illness to neurological disorders or even haemorrhagic fevers. Theoversite and existing gaps in our knowledge of ticks and TBV are partly due to the difficulty of setting effective experimental models to assess vector competence or study virus-tick interactions in general. To overcome this gap of knowledge, it is essential to reproduce transmission cycles undercontrolled laboratory conditions. In our study, we used viruses belonging to genera Flavivirus or Orbivirus to infect I. ricinus. TBEV is known to be transmitted by I. ricinus and responsible for severe neurological illness in humans in Europe and Asia. It was used as a positive control to assess theefficacy of both artificial feeding system (AFS) and immersion technique (IT) as infection methods.Kemerovo virus (KEMV, genus Orbivirus) is suspected to be the causative agent of encephalitis cases in humans in central Europe and Russia. AFS and IT were used to assess for the first time vector competence of I. ricinus for KEMV. The virus has been isolated/detected in I. persulcatus and I. ricinus.Assessing the efficacy of both infection techniques was based on the three criteria of vector competence: (i) virus acquisition by ticks, (ii) trans-stadial transmission, and (iii) transmission of the viruses to a vertebrate host. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR-/- or BALB/c).Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.

Background Tick-Borne Encephalitis (TBE) is a viral infection of the Central Nervous System (CNS) caused by Tick-Borne Encephalitis Virus (TBEV). It might take several clinical courses such as: meningitis, meningoencephalitis or meningoencephalomyelitis. The aim this study was to compare the YKL-40 concentration in cerebrospinal fluid (CSF) of patients with different clinical presentations of TBE and patients with excluded meningitis (control group). Methods The concentration of YKL-40 in CSF was determined using Fujirebio tests (Ghent, Belgium) in 32 patients with TBE: group I—patients with meningoencephalitis (n = 16); group II—patients with meningitis (n = 16). The control group (CG) consisted of 17 patients in whom inflammatory process in central nervous system was excluded. Results The concentration of YKL-40 was significantly higher in encephalitis group than in CG after 7 days from the last dose of treatment. The concentration in patients with neuroinflammation had significantly different concentration of YKL-40 compared to patients with no neuroinflammation control groups. ROC curve analysis indicates that: CSF YKL-40 concentration at cut off 783.87 differentiated TBE patients from CG with 100% specificity and 70% sensitivity and CSF YKL-40 concentration at cut off 980.11 differentiated meningitis from meningoencephalitis with 87.5% specificity and 62.5% sensitivity. Conclusions YKL-40 takes part in TBE pathogenesis, its concentration is the highest at the early stage of Central Nervous System involvement and decreases in the convalescent period. As YKL-40 is significantly higher in meningitis than in meningoencephalitis, it might be used as biomarker in differentiation of these clinical forms of TBE.

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilizing the membrane protein arrangement in the virion. The residues responsible for the M protein inter-actions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein, which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions.

Autophagy is a lysosomal degradative pathway responsible for recycling cytosolic proteins and organelles and also functions as an innate defense mechanism that host cells use against viral infection. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. For flaviviruses, both the positive and negative role of autophagy in virus replication has been reported. The interplay between autophagy and tick-borne encephalitis virus (TBEV) in innate immune cells is largely unknown. Here we report the relationship between an autophagy and TBEV replication in mouse macrophage cell line PMJ2-R using Hypr strain of TBEV. First, we examined the effect of Hypr infection on the autophagy pathway. We detected a mild and a temporary increase of autophagy marker LC3-II in Hypr-infected cells. The role of autophagy in TBEV replication was evaluated in autophagy related gene 5 (Atg5) knockdown cells (shAtg5). Our results showed that during an early stage of Hypr infection the viral titers were increased, while later on, at 72 hpi, the titers have declined in shAtg5 cells compared to control. Moreover, the higher number of virus-positive cells was observed in shAtg5 cells in early stage of infection and correlated with enhanced virus entry. Finally, we found an increased production of IFN-β in Hypr-infected shAtg5 cells in comparison to control at 48 and 72 hpi implicating that autophagy restricts the amount of IFN produced by TBEV-infected macrophages. To conclude, in mouse macrophages TBEV replication is controlled by autophagy in time dependent manner, having temporally an antiviral and then a pro-viral role during infection. Our study points out to a delicate and complex involvement of autophagy machinery at level of virus entry and IFN-β production when controlling TBEV infection.

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.

Tick-borne encephalitis virus (TBEV) causes serious the neurological disease, tick-borne encephalitis (TBE). TBEV can be transmitted to humans by ticks as well as by the alimentary route, which is mediated through the consumption of raw milk products from infected ruminants such as sheep, goats, and cows. The alimentary route of TBEV was recognized in the early 1950s and many important experimental studies were performed shortly thereafter. Nowadays, alimentary TBEV infections are recognized as a relevant factor contributing to the overall increase in TBE incidences in Europe. This review aims to summarize the history and current extent of alimentary TBEV infections across Europe, to analyze experimental data on virus secretion in milk, and to review possible alimentary infection preventive measures.

Powassan virus lineage 2 (deer tick virus) is an emergent threat to American public health, causing severe neurologic disease. Its life cycle in nature remains poorly understood. We use a host-specific retrotransposon-targeted real time PCR assay to test the hypothesis that white-footed mice, considered the main eastern U.S. reservoir of the coinfecting agent of Lyme disease, is the reservoir for deer tick virus. Of 20 virus-infected host-seeking nymphal black-legged ticks 65% fed on shrews and none on mice. The proportion of ticks feeding on shrews at a site is positively associated with prevalence of viral infection, but not the Lyme disease agent. Viral RNA is detected in the brain of one shrew. We conclude that shrews are a likely reservoir host for deer tick virus and that host bloodmeal analysis can provide direct evidence to incriminate reservoir hosts, thereby promoting our understanding of the ecology of tick-borne infections., Goethert and colleagues use a host-specific retrotransposon targeted real time PCR assay on questing nymphal ticks to identify a reservoir for Powassan virus lineage 2 (or deer tick virus). Infected ticks were found to have preferentially fed on shrews, as opposed to white-footed mice as was expected, suggesting that they may be a reservoir host for this virus.

International audience; Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, Flavivirus genus, is responsible for neurological symptoms that may cause permanent disability or death. With an incidence on the rise, it is the major arbovirus affecting humans in Central/Northern Europe and North-Eastern Asia. Neuronal death is a critical feature of TBEV infection, yet little is known about the type of death and the molecular mechanisms involved. In this study, we used a recently established pathological model of TBEV infection based on human neuronal/glial cells differentiated from fetal neural progenitors and transcriptomic approaches to tackle this question. We confirmed the occurrence of apoptotic death in these cultures and further showed that genes involved in pyroptotic death were up-regulated, suggesting that this type of death also occurs in TBEV-infected human brain cells. On the contrary, no up-regulation of major autophagic genes was found. Furthermore, we demonstrated an up-regulation of a cluster of genes belonging to the extrinsic apoptotic pathway and revealed the cellular types expressing them. Our results suggest that neuronal death occurs by multiple mechanisms in TBEV-infected human neuronal/glial cells, thus providing a first insight into the molecular pathways that may be involved in neuronal death when the human brain is infected by TBEV.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the Northeastern U.S., with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted in as little as 15 minutes of a tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of spread cell to cell, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9, -LI41 and Lineage I POWV-LB, productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-β (IFNβ) and IFN-stimulated genes, with delayed IFNβ secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion a subset of POWV infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments.ImportanceWe isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculating VeroE6 cells with POWV containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell to cell spread. POWV-LI9, -LI41, and Lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower chamber pericytes, suggesting a mechanism for POWV transmission across BBB. POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB, and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Powassan virus (POWV) is a neuroinvasive flavivirus transmitted to mammals by the bite of ixodid ticks. In this study, we sought to investigate the impact of tick salivary gland extract (SGE) on POWV neuroinvasion. BALB/c mice were footpad inoculated with either a high dose or a low dose of POWV, with and without Ixodes scapularis salivary gland extract. Brain and spinal cord were extracted daily, and immunohistochemical techniques were used for temporal tracking of POWV antigen. The temporal pattern of POWV staining showed a caudal to rostral spread of POWV in the brains of mice from both high dose infection groups. For the high dose infection groups, the presence of tick SGE did not influence the spread of POWV in the brain. Mice infected with the low dose of virus alone did not present POWV staining in the brain; however, in the presence of SGE, low dose infected mice presented scattered foci of POWV-infected cells throughout the brain. This study shows that tick SGE facilitates POWV neuroinvasion when mice are infected with the lower dose of POWV. We also found two patterns of central nervous system invasion that were directly influenced by the dose of POWV administered.

Tick-borne encephalitis virus (TBEV) is a major dangerous human pathogen, as TBEV infection can cause serious illness that can lead to irreversible neurological sequelae and even death. Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serogroup, belongs to the family Flaviviridae, genus Flavivirus. Its nonstructural protein 5 (NS5) protein contains a methyltransferase (MTase) domain that can methylate RNA cap structures, which is critical for viral replication. We determined the structure of LGTV NS5 methyltransferase bound to S-adenosyl-L-homocysteine (SAH) at a 1.70 Å resolution. Sequence analysis and structural comparison of homologous MTases suggests that folds and structures are closely conserved throughout Flavivirus species and play important roles. This study provides the key structural information on LGTV MTase and the foundation for research on antiviral drugs targeting LGTV MTase.

Tick-borne encephalitis virus (TBEV), a member of the genusFlavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its β-n-octyl-d-glucoside binding pocket (β-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessedin vivoandin vitroinfectivity of the residual mature virions in the immature virus samples. Analysis of the β-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.