TBEV Infection Research Articles

Chapter 12c: Risk map for TBEV

*This map may be different from “official” TBE-risk maps from local authorities as shown in chapter 9b for individual countries. Differences between the map above and country maps are explained by the fact that Public Health officials try to quantify the risk for TBE and thus indicate only those areas as “TBE risk areas” where a certain incidence threshold of TBE case numbers or TBE incidence is reached. This approach, however, does neither take into account the (in-) completeness of testing of all encephalitis cases for TBE as well as vaccine uptake nor the existence of TBEV in regions with low risk of exposition to humans (i.e. high TBE risk areas, however only rarely visited by humans). The goal of the map is to give a more real impression of the distribution of the TBEV, according to available virological/serological data – since all other data are biased by incomplete surveillance.Moreover, the map presented here may not entirely be complete, and very likely TBEV infections and thus TBE may occur in additional (“new”) areas.

Chapter 8: TBE in animals

TBE can cause clinical symptomatic disease in dogs and horses Diagnosis of TBEV infection in animals is similar to diagnosis in humans Animals can be used as sentinels for human exposure

Chapter 5: TBE in Adults

TBE is the most important tick-borne arbovirus disease of humans. Epidemiological data indicate a trend towards an increasing severity with higher age. A number of possible genetic and non-genetic risk factors have been identified, which might have an impact on the manifestation and severity of human disease. Different TBEV strains seem to cause differing clinical courses of disease. While the TBE-EU mainly causes a biphasic course, the clinical course of TBEV-FE and TBEV-SIB are mainly monophasic. The diagnosis of TBE is based on serological tests. So far there is no effective treatment of TBEV infections.

Survival of Tick-Borne Encephalitis Virus in Goat Cheese and Milk.

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, 30min) and fast (72°C, 15s) pasteurization conditions destroyed viable virus particles. A small amount of infective virus could be detected only for 5‒10days from milk, and from unsalted cheese. From milk inoculated with a higher amount of virus, infectious viral particles were detectable for 20‒25days and from unsalted cheese samples for 10‒15days, 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.

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection

BackgroundTick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis.MethodHuman neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype.ResultsWe showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV.ConclusionOur results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.

Transcriptomic analysis of human neurons and astrocytes infected with TBEV strains of different virulence

Tick-borne encephalitis virus (TBEV; Flaviviridae) can cause serious infections in humans which may result in encephalitis/meningoencephalitis. It has been previously reported that TBEV infects both, neurons and astrocytes, however, with a different outcome. So far, the principle of this cell type-specific response to TBEV is not fully understood. In order to gain more insight into this phenomenon, we described new in vitro infection model utilizing human neural stem cells (hNSCs) and two strains of Western European TBEV subtype varying in the pathogenicity - mild Neudoerfl and severe Hypr. In detail, neurons and astrocytes were artificially differentiated from hNSCs and presence of CNS markers was checked. TBEV infection in both cell types was characterised afterwards. As expected, both cell types proved to be susceptible to TBEV infection. Viability was negatively affected only in infected neurons. In order to identify possible effectors responsible for different susceptibility of neurons and astrocytes, the analyses of changes in poly-(A) and small RNA transcriptome upon TBEV infection were performed. Preliminary results from poly-(A) RNA transcriptome revealed that in both cell types mainly interferon-stimulated genes (ISGs) were up-regulated. However, the expression kinetics of particular ISGs varied. In addition, the vast spectrum of long non-coding RNAs was described to be differentially expressed upon infection. Surprisingly, U1 snRNA was found to be the most down-regulated RNA species among almost all infected samples. Further analyses are in progress in order to get a complete description of virus-induced changes on the transcriptomic level.

Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication.

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.

Chapter 12a: Epidemiology by country – an overview

• TBE is a flavivirus infection of the central nervous system (CNS), transmitted by ticks and in some rare instances by ingestion of unpasteurized milk. • TBE is diagnosed in the forested belts of Northern Eurasia ranging from eastern France and Norway down to northern Italy through central and Eastern Europe, Russia, Kazakhstan, and China to the northern Japanese island of Hokkaido. • About 10,000 cases of TBE are reported annually. • The European Centers for Disease Control (E-CDC) have put TBE on their list of notifiable diseases; their case definition requires clinical symptoms of CNS infection plus microbiolog-ical confirmation of the infection, usually by detection of specific immunoglobulins, IgG and IgM. • Despite this, surveillance of the TBEV in both ticks and humans is incomplete. Reported incidences do not reflect actual risk since this fluctuates annually as a result of changes in exposure, vaccine uptake, intensity of case finding and reporting, and climate, just to men-tion the most relevant factors. • For largely unknown reasons (including human behavior, improved diagnostics, or climate change) TBEV appears to be spreading north, east, and even south to areas that were pre-viously believed to be free of the virus. • The country reports in Chapter 12b provide a current and official ‘state-of-the-art’ survey on TBE, with available information about the virus, vectors, modes of transmission, case reporting, vaccine and prevention efforts, burden of disease over time, age and gender distribution of cases, virus isolation data, and risk area distribution. • Chapter 12c provides a risk map for TBEV based on documented TBE cases, TBEV infection, as well as on the detection of TBEV in nature. This map does not reflect the incidence of the disease or the prevalence of the virus in a given area. As the intensity and complete-ness of epidemiological surveillance varies between different countries, the map presented here may not be entirely complete, and very likely TBEV infections and thus TBE may occur in additional (‘new’) areas.

Chapter 8: TBE in animals

• TBEV infection via routes other than tick bites • Animal hosts of the TBEV • Diagnosis of TBEV infection in animals • Implications for human TBE transmission

Chapter 5: TBE in adults

• TBE is the most important tick-borne arbovirus disease of humans. Epidemiological data indicate a trend towards an increasing severity with higher age. • A number of possible genetic and non-genetic risk factors have been identified, which might have an impact on the manifestation and severity of human disease. • Different TBEV strains seem to cause differing clinical courses of disease. While the TBE-Eu mainly causes a biphasic course, the clinical course of TBEV-FE and TBEV-Sib are mainly monophasic. • The diagnosis of TBE is based on serological tests. • So far there is no effective treatment of TBEV infections.

Seroprevalence of TBEV in bank voles from Poland—a long-term approach

Rodents are known to play a significant role as reservoir hosts for TBEV. During three sequential expeditions at 4-year intervals to three ecologically similar study sites in NE Poland, we trapped bank voles (Myodes glareolus) and then tested their blood for the presence of specific antiviral antibodies to TBEV. The strongest effects on seroprevalence were the extrinsic factors, site of capture of voles and year of sampling. Seroprevalence increased markedly with increasing host age, and our analysis revealed significant interactions among these three factors. Seroprevalence did not differ between the sexes. Therefore, based on the seroprevalence results, the dynamics of TBEV infection differ significantly in time, between local sub-populations of bank voles and with increasing host age. To fully understand the circulation of the virus among these reservoir hosts and in the environment, long-term monitoring is required and should employ a multi-site approach, such as the one adopted in the current study.

Breadth and Dynamics of HLA-A2- and HLA-B7-Restricted CD8+ T Cell Responses against Nonstructural Viral Proteins in Acute Human Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8+ T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2- and HLA-B7-restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2- and HLA-B7-restricted Ag-specific CD8+ T cell response during the acute stages of human TBEV infection. HLA-A2- and HLA-B7-restricted TBEV epitope-specific effector cells predominantly displayed a CD45RA-CCR7-CD27+CD57- phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2- and HLA-B7-restricted TBEV-specific CD8+ T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2-restricted CD8+ T cell populations was CD45RA-CCR7-CD27+CD57+, whereas the HLA-B7-restricted CD8+ T cell population was predominantly CD45RA+CCR7-CD27+CD57+ Almost all TBEV epitope-specific CD8+ T cells expressed α4 and β1 integrins at days 7 and 21, whereas the bulk CD8+ T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2- and HLA-B7-restricted TBEV epitope-specific CD8+ T cells.

A new hot spot for tick-borne encephalitis (TBE): A marked increase of TBE cases in France in 2016

ObjectivesTick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis. In 2016, in Northeastern France, we faced a TBEV infection increase, leading to a warning from the Regional Health Agency. Here, we report the confirmed TBE cases diagnosed between January 2013 and December 2016, with particular emphasis on the year 2016. MethodsA total of 1643 blood and cerebrospinal fluid (CSF) samples from everywhere in France, corresponding to 1460 patients, were prospectively tested for anti-TBEV-specific IgM and IgG antibodies by ELISA. Additional 39 blood and CSF samples from patients with suspected Lyme neuroborreliosis were retrospectively investigated. ResultsThe TBEV seropositivity rate was estimated to 5.89% and 54 patients were diagnosed as TBE-confirmed cases. A significant increase in TBE cases was observed during the year 2016 with 29 confirmed cases, instead of a mean of eight cases during the three previous years (p=0.0006). Six imported cases and 48 autochthonous cases, located in the Alsace region (n=43) and in the Alpine region (n=5) were reported. Forty-six patients experienced neurological impairment. Nine patients showed an incomplete recovery at last follow-up (from 15days to eight months post-infection). TBE diagnosis was performed earlier for patients taken in charge in the Alsace region than those hospitalized elsewhere in France (p=0.0087). Among the 39 patients with suspected Lyme neuroborreliosis retrospectively investigated, one showed a TBEV recent infection. ConclusionThe TBE increase that occurred in France in 2016 highlights the need to improve our knowledge about the true burden of TBEV infection and subsequent long-term outcomes.

No molecular or serological evidence of Zikavirus infection among healthy blood donors living in or travelling to regions where Aedes albopictus circulates.

BackgroundPrevious studies have shown that Zika virus can infect and be transmitted by A. albopictus. The World Health organization (WHO) has raised concerns of autochthonous transmission of the virus in regions where the vector is endemic. The aim of this pilot study was to assess the occurrence of Zika virus (ZIKV) in western Austria (Tyrol) especially after a history of travel to A. albopictus endemic regions.MethodsThe study participants were healthy blood donors at randomly selected donation sites in the west Austrian region Tyrol. Rest blood (plasma) samples were tested for the presence of ZIKV nucleic acid and antibodies against the virus.ResultsMean age of the study participants was 44.6 (SD = 12.9) and 58.8% were men. Eighty percent reported to have received vaccine against TBEV, whereas only 4.9 and 0.9% had received YFV and JEV vaccines. Three out of 1001 (0.03%) participants tested positive solely for ZIKV IgM antibody but not for other flaviviruses. Only one individual had ZIKV IgG antibody. All four donors were negative in the neutralization (confirmation) assay. No viral RNA was detected in any of the samples.ConclusionThe null finding of our study refutes WHO’s initial fear of global expansion of ZIKV infection including its occurrence in Europe. There appears to be no urgent need to introduce universal screening of donated blood for ZIKV in central Europe at least until the next warm season. Further, Euroimmun anti-Zika ELISA proved to be a highly suitable and reliable test system in populations with high prevalence of TBEV infection and/or immunization.

NK Cell Responses to Human Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV) is a flavivirus that is transferred to humans by infected ticks. The virus causes tick-borne encephalitis, a severe infection of the CNS with a high risk for long-lasting sequelae. Currently, no treatment exists for the disease. Understanding the cellular immune response to this infection is important to gain further understanding into the pathogenesis, treatment, and prevention of the disease. NK cells are known to participate in the control of viral infections. We performed a longitudinal analysis of the human NK cell response to TBEV infection in a cohort of infected individuals from the onset of severe clinical symptoms to the convalescence phase. NK cell activation, as measured by expression of Ki67, was apparent at the time of hospitalization. By 3 wk after hospitalization, it decreased to levels seen in healthy controls. Concomitant with the increase in NK cell activation, augmented levels of IL-12, IL-15, IL-18, IFN-γ, and TNF were detected in patient plasma. This TBEV-induced NK cell activation was restricted predominantly to differentiated CD57(+)CD56(dim) NK cells. Functionally, CD56(dim) NK cells responded poorly to target cells at the time of hospitalization, but they recovered functional capacity to control levels during the convalescent phase. In contrast, the responsiveness of NK cells to cytokine stimulation remained intact throughout the disease. These findings demonstrate that NK cells respond to TBEV infection with characteristics that are distinct from those of other human viral infections and provide insights into the NK cell response to clinical TBEV infection.

Seroprevalence of tick-borne-encephalitis virus in wild game in Mecklenburg-Western Pomerania (north-eastern Germany)

Mecklenburg-Western Pomerania, a federal state in the north east of Germany, has never been a risk area for TBEV infection, but a few autochthonous cases, along with TBEV-RNA detection in ticks, have shown a low level of activity in natural foci of the virus in the past. As wild game and domestic animals have been shown to be useful sentinels for TBEV we examined sera from wild game shot in Mecklenburg-Western Pomerania for the prevalence of TBEV antibodies.A total of 359 sera from wild game were investigated. All animals were shot in Mecklenburg-Western Pomerania in 2012. Thirteen of 359 sera tested positive or borderline for anti-TBEV-IgG with ELISA and four samples tested positive using NT.The four TBEV-positive sera confirmed by NT constitute the first detection of TBEV-antibodies in sera of wild game in Mecklenburg-Western Pomerania since 1986–1989. This underlines that the serological examination of wild game can be a useful tool in defining areas of possible TBEV infection, especially in areas of low TBEV-endemicity.

Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell-Mediated Viral Clearance.

Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5(-/-) mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5(-/-) mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control-treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.

Tick-borne encephalitis in north-east Italy: a 14-year retrospective study, January 2000 to December 2013

Italy is considered at low incidence of tick-borne encephalitis (TBE), and the occurrence of human cases of TBE appears to be geographically restricted to the north east of the country. However, most information to date derives from case series, with no systematic data collection. To estimate incidence rates (IR) and spatial distribution of TBE cases, we conducted a retrospective study in north-eastern Italy. Data were collected through the infectious disease units and public health districts of three regions (Friuli Venezia Giulia, Trentino Alto Adige and Veneto) between 2000 and 2013. Overall, 367 cases were identified (IR: 0.38/100,000). The cases' median age was 56 years and 257 (70%) were male. Central nervous system involvement was reported in 307 cases (84%). Annual fluctuations in case numbers occurred, with peaks in 2006 and in 2013, when 44 and 42 cases were respectively observed. A strong seasonality effect was noted, with the highest number of cases in July. In terms of geographical location, three main endemic foci with high TBE IR (>10/100,000) were identified in three provinces, namely Belluno (Veneto region), Udine (Friuli Venezia Giulia) and Trento (Trentino Alto-Adige). When investigating the whole study area in terms of altitude, the IR between 400 and 600 m was greater (2.41/100,000) than at other altitudes (p<0.01). In conclusion, the incidence of TBE in Italy is relatively low, even considering only the three known affected regions. However, three endemic foci at high risk were identified. In these areas, where the risk of TBEV infection is likely high, more active offer of TBE vaccination could be considered.

Nucleoside inhibitors of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2'-CMA, 7.1 ± 1.2 μM for 2'-CMA, and 14.2 ± 1.9 μM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses. Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the tick-borne encephalitis serocomplex, genus Flavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in nonstructural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of tick-borne flavivirus pathogenesis.