Flavivirus assembly is driven by the envelope glycoproteins pre-membrane (prM) and envelope (E) in the neutral pH environment of the endoplasmic reticulum. Newly budded, spiky particles are exported through the Golgi apparatus, where mildly acidic pH induces a major surface rearrangement. The glycoproteins reorganize into (prM/E)\₂ complexes at the surface of smooth particles, with prM trapped at the E dimer interface, thereby exposing a furin cleavage site (FCS) for proteolytic maturation into infectious virions. Here, we show that in the absence of furin, immature tick-borne flavivirus particles-tick-borne encephalitis virus, Langat virus, and Louping ill virus-remain fully infectious and pathogenic in female BALB/c mice, in contrast to mosquito-borne flaviviruses such as Usutu, West Nile, and Zika viruses. We further show that the FCS in tick-borne viruses remains exposed at neutral pH, allowing furin at the surface of target cells to activate viral fusogenicity, while mosquito-borne counterparts require acidic re-exposure. Mutations increasing the dynamic behavior of the E dimer mimic the mosquito-borne phenotype, with retracted FCS at neutral pH and loss of infectivity. Our multidisciplinary approach-combining virological assays, targeted mutagenesis, structural modeling, and molecular dynamics simulations-highlights the role of E dimer dynamics in regulating flavivirus maturation and infectivity.

• Publication type
• Journal Article MeSH

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.

• Keywords
• Antivirals, Immunofluorescence, Monoclonal antibodies, Mouse infection, Neutralization test, Plaque assay, Tick-borne encephalitis virus,
• MeSH
• Antiviral Agents * pharmacology   MeSH
• Cell Line MeSH
• Encephalitis, Tick-Borne * virology   drug therapy   immunology   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Antibodies, Monoclonal immunology   MeSH
• Mice MeSH
• Neutralization Tests methods   MeSH
• Antibodies, Neutralizing * immunology   pharmacology   MeSH
• Viral Plaque Assay MeSH
• Antibodies, Viral * immunology   MeSH
• Encephalitis Viruses, Tick-Borne * drug effects   immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents * MeSH
• Antibodies, Monoclonal MeSH
• Antibodies, Neutralizing * MeSH
• Antibodies, Viral * 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.

• Keywords
• CP: Immunology, CP: Microbiology, escape mutant, monoclonal antibody, neutralization, tick-borne encephalitis, tick-borne encephalitis virus,
• MeSH
• Epitopes MeSH
• Encephalitis, Tick-Borne * MeSH
• Humans MeSH
• Antibodies, Monoclonal MeSH
• Antibodies, Viral MeSH
• Encephalitis Viruses, Tick-Borne * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Epitopes MeSH
• Antibodies, Monoclonal MeSH
• Antibodies, Viral 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
• Virology * MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Editorial MeSH
• Geographicals
• Czech Republic MeSH

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

• MeSH
• Antiviral Agents * pharmacology   therapeutic use   MeSH
• G-Quadruplexes * MeSH
• Encephalitis, Tick-Borne drug therapy   genetics   MeSH
• Humans MeSH
• Ligands MeSH
• RNA, Viral genetics   MeSH
• RNA-Dependent RNA Polymerase genetics   MeSH
• Encephalitis Viruses, Tick-Borne * drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents * MeSH
• Ligands MeSH
• RNA, Viral MeSH
• RNA-Dependent RNA Polymerase MeSH

Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. 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.

• MeSH
• Arachnid Vectors physiology   virology   MeSH
• Host-Pathogen Interactions MeSH
• Ixodes physiology   virology   MeSH
• Encephalitis, Tick-Borne transmission   virology   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Orbivirus physiology   MeSH
• Reoviridae Infections transmission   virology   MeSH
• Virology methods   MeSH
• Encephalitis Viruses, Tick-Borne physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

The aim of this review is to follow the history of studies on endemiv arboviruses and the diseases they cause which were detected in the Czech lands (Bohemia, Moravia and Silesia (i.e., the Czech Republic)). The viruses involve tick-borne encephalitis, West Nile and Usutu flaviviruses; the Sindbis alphavirus; Ťahyňa, Batai, Lednice and Sedlec bunyaviruses; the Uukuniemi phlebovirus; and the Tribeč orbivirus. Arboviruses temporarily imported from abroad to the Czech Republic have been omitted. This brief historical review includes a bibliography of all relevant papers.

• Keywords
• arthropods, birds, mammals, mosquitoes, ticks,
• MeSH
• Arbovirus Infections history   MeSH
• Arboviruses physiology   MeSH
• History, 20th Century MeSH
• History, 21st Century MeSH
• Humans MeSH
• Animals MeSH
• Check Tag
• History, 20th Century MeSH
• History, 21st Century MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Review MeSH
• Geographicals
• Czech Republic epidemiology   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.

• Keywords
• BCX4430, attenuation, drug resistance, galidesivir, mutation, tick-borne encephalitis virus,
• MeSH
• Adenine analogs & derivatives   chemistry   pharmacology   MeSH
• Adenosine analogs & derivatives   MeSH
• Alleles MeSH
• Drug Resistance, Microbial MeSH
• Antiviral Agents chemistry   pharmacology   MeSH
• Cell Line MeSH
• Genotype MeSH
• Encephalitis, Tick-Borne drug therapy   virology   MeSH
• Disease Models, Animal MeSH
• Mutation * MeSH
• Mice MeSH
• Pyrrolidines chemistry   pharmacology   MeSH
• Amino Acid Substitution * MeSH
• Drug Resistance, Viral * MeSH
• Viral Nonstructural Proteins genetics   MeSH
• Encephalitis Viruses, Tick-Borne drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Adenosine MeSH
• Antiviral Agents MeSH
• galidesivir MeSH Browser
• Pyrrolidines MeSH
• Viral Nonstructural Proteins MeSH