A highly virulent strain (Hypr) of tick-borne encephalitis virus (TBEV) was serially subcultured in the mammalian porcine kidney stable (PS) and Ixodes ricinus tick (IRE/CTVM19) cell lines, producing three viral variants. These variants exhibited distinct plaque sizes and virulence in a mouse model. Comparing the full-genome sequences of all variants, several nucleotide changes were identified in different genomic regions. Furthermore, different sequential variants were revealed to co-exist within one sample as quasispecies. Interestingly, the above-mentioned nucleotide changes found within the whole genome sequences of the new variants were present alongside the nucleotide sequence of the parental strain, which was represented as a minority quasispecies. These observations further imply that TBEV exists as a heterogeneous population that contains virus variants pre-adapted to reproduction in different environments, probably enabling virus survival in ticks and mammals.

• Klíčová slova
• TBEV, flavivirus adaptation, genome mutation, host alternation, neuroinvasiveness, quasispecies, tick cell line,
• MeSH
• buněčné linie MeSH
• fyziologická adaptace genetika   MeSH
• genetická variace MeSH
• genom virový MeSH
• klíště cytologie   virologie   MeSH
• klíšťová encefalitida virologie   MeSH
• ledviny cytologie   virologie   MeSH
• mutace MeSH
• myši MeSH
• prasata MeSH
• quasispecies * MeSH
• virulence MeSH
• viry klíšťové encefalitidy genetika   patogenita   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

• MeSH
• elektronová kryomikroskopie MeSH
• exprese genu MeSH
• fúze membrán genetika   MeSH
• imunoglobuliny - Fab fragmenty biosyntéza   chemie   MeSH
• internalizace viru MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• nádorové buněčné linie MeSH
• neurony patologie   virologie   MeSH
• neutralizující protilátky biosyntéza   chemie   MeSH
• proteinové domény MeSH
• protilátky virové biosyntéza   chemie   MeSH
• virion genetika   metabolismus   ultrastruktura   MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy genetika   metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - Fab fragmenty MeSH
• neutralizující protilátky MeSH
• protilátky virové MeSH
• virové proteiny 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