BACKGROUND: Tick-borne encephalitis (TBE) is the most common tick-borne viral infection in Eurasia. Outcomes range from asymptomatic infection to fatal encephalitis, with host genetics likely playing a role. BALB/c mice have intermediate susceptibility to TBE virus (TBEV) and STS mice are highly resistant, whereas the recombinant congenic strain CcS-11, which carries 12.5% of the STS genome on the BALB/c background, is more susceptible than BALB/c mice. In the present study, we employed these genetically distinct mouse models to investigate the host response to TBEV infection in both peripheral macrophages, one of the initial target cell populations, and the brain, the terminal target organ of the virus. METHODS: TBEV growth and the production of key cytokines and chemokines were measured and compared in macrophages derived from BALB/c, CcS-11, and STS mice. In addition, brains from these TBEV-infected mouse strains underwent in-depth transcriptomic analysis. RESULTS: Virus production in BALB/c and CcS-11 macrophages exhibited similar kinetics 24 and 48 h post-infection (hpi), but CcS-11 macrophages yielded significantly higher titers 72 hpi. Macrophages from both sensitive strains demonstrated elevated chemokine and proinflammatory cytokine production upon infection, whereas the resistant strain, STS, showed no cytokine/chemokine activation. Transcriptomic analysis of brain tissue demonstrated that the genetic background of the mouse strains dictated their transcriptional response to infection. The resistant strain exhibited a more robust cell-mediated immune response, whereas both sensitive strains showed a less effective cell-mediated response but increased cytokine signaling and signs of demyelination, with loss of oligodendrocytes. CONCLUSIONS: Our findings suggest that variations in susceptibility linked to host genetic background correspond with distinct host responses, both in the periphery upon virus entry into the organism and in the brain, the target organ of the virus. These results provide insights into the influence of host genetics on the clinical trajectory of TBE.

• Klíčová slova
• Genetics, Macrophages, Mouse model, Neuroinflammation, Tick-borne encephalitis, Tick-borne encephalitis virus, Transcriptomics,
• MeSH
• cytokiny * metabolismus   genetika   MeSH
• genotyp MeSH
• klíšťová encefalitida * imunologie   virologie   genetika   MeSH
• makrofágy * imunologie   virologie   MeSH
• mozek * virologie   imunologie   MeSH
• myši inbrední BALB C * MeSH
• myši MeSH
• viry klíšťové encefalitidy * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny * MeSH

Tick-borne encephalitis virus (TBEV) is a leading cause of vector-borne viral encephalitis with expanding endemic regions across Europe. In this study we tested in mice the efficacy of preinfection with a closely related low-virulent flavivirus, Langat virus (LGTV strain TP21), or a naturally avirulent TBEV strain (TBEV-280) in providing protection against lethal infection with the highly virulent TBEV strain (referred to as TBEV-Hypr). We show that prior infection with TP21 or TBEV-280 is efficient in protecting mice from lethal TBEV-Hypr challenge. Histopathological analysis of brains from nonimmunized mice revealed neuronal TBEV infection and necrosis. Neuroinflammation, gliosis, and neuronal necrosis was however also observed in some of the TP21 and TBEV-280 preinfected mice although at reduced frequency as compared to the nonimmunized TBEV-Hypr infected mice. qPCR detected the presence of viral RNA in the CNS of both TP21 and TBEV-280 immunized mice after TBEV-Hypr challenge, but significantly reduced compared to mock-immunized mice. Our results indicate that although TBEV-Hypr infection is effectively controlled in the periphery upon immunization with low-virulent LGTV or naturally avirulent TBEV 280, it may still enter the CNS of these animals. These findings contribute to our understanding of causes for vaccine failure in individuals vaccinated with TBE vaccines.

• Klíčová slova
• CNS, Langat virus, neuronal damage, tick-borne encephalitis virus, virus induced immunity,
• Publikační typ
• časopisecké články MeSH

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

• Klíčová slova
• BCX4430, attenuation, drug resistance, galidesivir, mutation, tick-borne encephalitis virus,
• MeSH
• adenin analogy a deriváty   chemie   farmakologie   MeSH
• adenosin analogy a deriváty   MeSH
• alely MeSH
• antibiotická rezistence MeSH
• antivirové látky chemie   farmakologie   MeSH
• buněčné linie MeSH
• genotyp MeSH
• klíšťová encefalitida farmakoterapie   virologie   MeSH
• modely nemocí na zvířatech MeSH
• mutace * MeSH
• myši MeSH
• pyrrolidiny chemie   farmakologie   MeSH
• substituce aminokyselin * MeSH
• virová léková rezistence * MeSH
• virové nestrukturální proteiny genetika   MeSH
• viry klíšťové encefalitidy účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• adenosin MeSH
• antivirové látky MeSH
• galidesivir MeSH Prohlížeč
• pyrrolidiny MeSH
• virové nestrukturální proteiny MeSH

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.

• Klíčová slova
• 2-deoxy sugars, antivirals, glycosylation inhibition, tick-borne encephalitis, uridine,
• MeSH
• antivirové látky chemie   farmakologie   MeSH
• deoxycukry chemie   farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• plakové testy MeSH
• proteosyntéza účinky léků   MeSH
• uridin analogy a deriváty   chemie   farmakologie   MeSH
• viry klíšťové encefalitidy účinky léků   fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• deoxycukry MeSH
• uridin MeSH

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

• Klíčová slova
• antiviral agents, antiviral therapy, escape mutant, tick-borne encephalitis virus, tick-borne pathogens,
• Publikační typ
• časopisecké články MeSH

Tick-borne encephalitis (TBE) is an acute febrile illness with neurological manifestations that is prevalent in forested areas of moderate climate in Europe and Asia. TBE virus is transmitted by ticks and rarely by unpasteurized milk and dairy products. The disease burden is attributed mainly to resulting long-term disability, especially in individuals over 50 y of age. Currently, there is no causative treatment, but a very effective vaccination is available with a good safety profile. The vaccination requires 3 basic doses to be fully effective and regular boosters afterwards. An accelerated vaccination schedule enables a patient to reach reasonably protective titres within 3 to 4 weeks from the first injection. The risk of travel-related TBE is estimated to be less than the risk of acquiring typhoid fever while visiting highly endemic regions in South Asia, but more than the risk of acquiring Japanese encephalitis, meningococcal invasive disease, or rabies. The pre-travel risk assessment of acquiring TBE should consider known risk factors which include 1) the country and regions to be visited; 2) April to November season; 3) altitude less than 1500 m above the sea level; 4) duration of stay; 5) the extent of tick-exposure associated activities including leisure and professional outdoor activities within the endemic area; and 6) age and comorbidities of the traveler. A major challenge, however, is the very low awareness of the risk of contracting TBE in those who travel to industrialized European countries.

• Klíčová slova
• endemic country, flavivirus, tick-borne encephalitis, tick-borne encephalitis virus, travel medicine, vaccination,
• MeSH
• cestování * MeSH
• endemické nemoci * MeSH
• klíšťová encefalitida epidemiologie   prevence a kontrola   MeSH
• lidé MeSH
• virové vakcíny aplikace a dávkování   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Asie epidemiologie   MeSH
• Evropa epidemiologie   MeSH
• Názvy látek
• virové vakcíny MeSH

A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

• Klíčová slova
• Immunoblotting, Immunofluorescence, TBEV, TuORF, uORF,
• MeSH
• biosyntéza peptidů genetika   imunologie   MeSH
• buněčné linie MeSH
• fylogeneze MeSH
• genom virový * MeSH
• glioblastom virologie   MeSH
• klíště virologie   MeSH
• klíšťová encefalitida virologie   MeSH
• lidé MeSH
• meduloblastom virologie   MeSH
• mutace MeSH
• neuroblastom virologie   MeSH
• otevřené čtecí rámce * MeSH
• viry klíšťové encefalitidy genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• antivirové látky chemie   farmakologie   MeSH
• buněčné linie MeSH
• cytidin analogy a deriváty   chemie   farmakologie   MeSH
• lidé MeSH
• nukleosidy chemie   farmakologie   MeSH
• prasata MeSH
• replikace viru účinky léků   MeSH
• tubercidin analogy a deriváty   chemie   farmakologie   MeSH
• viry klíšťové encefalitidy účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2'-C-methyladenosine MeSH Prohlížeč
• 2'-C-methylcytidine MeSH Prohlížeč
• 7-deaza-2'-C-methyladenosine MeSH Prohlížeč
• adenosin MeSH
• antivirové látky MeSH
• cytidin MeSH
• nukleosidy MeSH
• tubercidin MeSH

Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.

• MeSH
• autofagie účinky léků   genetika   MeSH
• benzylaminy farmakologie   MeSH
• chinazoliny farmakologie   MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   virologie   MeSH
• lidé MeSH
• mikrotubuly účinky léků   ultrastruktura   virologie   MeSH
• nádorové buněčné linie MeSH
• neurony účinky léků   ultrastruktura   virologie   MeSH
• nokodazol farmakologie   MeSH
• primární buněčná kultura MeSH
• replikace viru účinky léků   MeSH
• sirolimus farmakologie   MeSH
• tomografie elektronová MeSH
• virion účinky léků   růst a vývoj   ultrastruktura   MeSH
• viry klíšťové encefalitidy účinky léků   růst a vývoj   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzylaminy MeSH
• chinazoliny MeSH
• nokodazol MeSH
• sirolimus MeSH
• spautin-1 MeSH Prohlížeč

Tick-borne encephalitis (TBE) is a substantial public health problem in many parts of Europe and Asia. To assess the effect of increasing TBE vaccination coverage in Austria, we compared incidence rates over 40 years for highly TBE-endemic countries of central Europe (Czech Republic, Slovenia, and Austria). For all 3 countries we found extensive annual and longer range fluctuations and shifts in distribution of patient ages, suggesting major variations in the complex interplay of factors influencing risk for exposure to TBE virus. The most distinctive effect was found for Austria, where mass vaccination decreased incidence to ≈16% of that of the prevaccination era. Incidence rates remained high for the nonvaccinated population. The vaccine was effective for persons in all age groups. During 2000-2011 in Austria, ≈4,000 cases of TBE were prevented by vaccination.

• MeSH
• dítě MeSH
• hromadná vakcinace * MeSH
• incidence MeSH
• klíšťová encefalitida epidemiologie   imunologie   prevence a kontrola   virologie   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• riziko MeSH
• věkové faktory MeSH
• virové vakcíny aplikace a dávkování   imunologie   MeSH
• viry klíšťové encefalitidy imunologie   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Rakousko epidemiologie   MeSH
• Slovinsko epidemiologie   MeSH
• Názvy látek
• virové vakcíny MeSH