Tick-borne encephalitis (TBE) is a potentially fatal neuroinfection of humans caused by the TBE virus. There is no specific therapy for TBE, as treatment is only supportive. Therefore, efforts to develop an effective specific therapy for TBE are warranted. Small molecule inhibitors and monoclonal antibodies are under intense investigation as potential therapeutics to combat TBE in humans. Here we describe the basic methods that can be used to test small molecule antivirals or monoclonal antibodies against TBEV. These include in vitro methods such as plaque assays, neutralizing assays, immunofluorescence staining of viral antigen, and cell-based antiviral assays, as well as in vivo assays in mouse model of TBE.

• Klíčová slova
• Antivirals, Immunofluorescence, Monoclonal antibodies, Mouse infection, Neutralization test, Plaque assay, Tick-borne encephalitis virus,
• MeSH
• antivirové látky * farmakologie   MeSH
• buněčné linie MeSH
• klíšťová encefalitida * virologie   farmakoterapie   imunologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• neutralizační testy metody   MeSH
• neutralizující protilátky * imunologie   farmakologie   MeSH
• plakové testy MeSH
• protilátky virové * imunologie   MeSH
• viry klíšťové encefalitidy * účinky léků   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• monoklonální protilátky MeSH
• neutralizující protilátky * MeSH
• protilátky virové * MeSH

In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.

In RNA interference (RNAi), the enzyme Dicer cuts long double-stranded RNA into small interfering RNAs that degrade matching RNAs. RNAi is a key antiviral defense in plants and invertebrates but vertebrates evolved a principally different antiviral defense. The authors genetically modified Dicer in mice to activate RNAi in mammals. These modified mice were tested against four RNA viruses but showed no significant antiviral response. However, further increased expression of modified Dicer did suppress one virus (lymphocytic choriomeningitis virus) in embryonic stem cells and in a transgenic mouse model, suggesting that some viruses might be sensitive to increased RNAi activity in mammals.

• MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• malá interferující RNA genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• přirozená imunita * genetika   MeSH
• ribonukleasa III * genetika   metabolismus   MeSH
• RNA interference * MeSH
• virus encefalomyokarditidy genetika   imunologie   MeSH
• virus lymfocytární choriomeningitidy imunologie   genetika   MeSH
• viry klíšťové encefalitidy genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• Dicer1 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• ribonukleasa III * MeSH

Tick-borne encephalitis (TBE) virus (TBEV) is transmitted to humans via tick bites. Infection is benign in >90% of the cases but can cause mild (<5%), moderate (<4%), or severe (<1%) encephalitis. We show here that ∼10% of patients hospitalized for severe TBE in cohorts from Austria, Czech Republic, and France carry auto-Abs neutralizing IFN-α2, -β, and/or -ω at the onset of disease, contrasting with only ∼1% of patients with moderate and mild TBE. These auto-Abs were found in two of eight patients who died and none of 13 with silent infection. The odds ratios (OR) for severe TBE in individuals with these auto-Abs relative to those without them in the general population were 4.9 (95% CI: 1.5-15.9, P < 0.0001) for the neutralization of only 100 pg/ml IFN-α2 and/or -ω, and 20.8 (95% CI: 4.5-97.4, P < 0.0001) for the neutralization of 10 ng/ml IFN-α2 and -ω. Auto-Abs neutralizing type I IFNs accounted for ∼10% of severe TBE cases in these three European cohorts.

• MeSH
• autoprotilátky * imunologie   MeSH
• dospělí MeSH
• interferon typ I * imunologie   MeSH
• klíšťová encefalitida * imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• neutralizující protilátky * imunologie   MeSH
• senioři MeSH
• viry klíšťové encefalitidy imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Rakousko epidemiologie   MeSH
• Názvy látek
• autoprotilátky * MeSH
• interferon typ I * MeSH
• neutralizující protilátky * MeSH

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

• Klíčová slova
• CP: Immunology, antibodies, flaviviruses, tick diseases,
• MeSH
• epitopy imunologie   MeSH
• Flavivirus imunologie   MeSH
• klíšťata virologie   imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• neutralizující protilátky * imunologie   MeSH
• protilátky virové imunologie   MeSH
• viry klíšťové encefalitidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Geografické názvy
• Brazílie MeSH
• Mexiko MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• neutralizující protilátky * MeSH
• protilátky virové MeSH

Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III (EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.

• Klíčová slova
• CP: Immunology, CP: Microbiology, escape mutant, monoclonal antibody, neutralization, tick-borne encephalitis, tick-borne encephalitis virus,
• MeSH
• epitopy MeSH
• klíšťová encefalitida * MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• protilátky virové MeSH
• viry klíšťové encefalitidy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• protilátky virové MeSH

The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• virologie * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Česká republika MeSH