Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus that invades the central nervous system, leading to severe neurological manifestations. In this study, we developed a reporter virus comprising TurboGFP-expressing TBEV (tGFP-TBEV) as a versatile tool for advancing TBEV research. The tGFP-TBEV facilitates quantitative measurement of viral replication, enables precise tracking of individual infected cells, and supports high-throughput screening of potential antiviral compounds and virus-neutralization assays. Furthermore, tGFP-TBEV proved effective as a model for studying TBEV infection in rat organotypic cerebellar slices cultured ex vivo and for visualizing TBEV infection in the mouse brain. Using tissue-clearing protocols and light-sheet fluorescence microscopy, we achieved high-resolution, three-dimensional mapping of the TBEV distribution in the mouse brain. This analysis uncovered distinct patterns of TBEV tropism, with infections concentrated in regions associated with neurogenesis, olfactory processing, and specific neuroanatomical pathways. The ability to visualize infection at both the cellular and whole-organ level provides a new tool for detailed investigations into viral tropism, replication, and interactions with host tissues, paving the way for deeper insights into TBEV biology and the pathogenesis of tick-borne encephalitis.

• Keywords
• TBEV, light-sheet microscopy, neurotropism, organotypic cerebellar slices, reporter viruses, tissue clearing,
• MeSH
• Encephalitis, Tick-Borne * virology   MeSH
• Rats MeSH
• Humans MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Brain * virology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Virus Replication MeSH
• Genes, Reporter MeSH
• Viral Tropism MeSH
• Encephalitis Viruses, Tick-Borne * genetics   physiology   MeSH
• Imaging, Three-Dimensional MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Luminescent Proteins 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

Ultraviolet-C (UV-C) radiation and ozone gas are potential mechanisms employed to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), each exhibiting distinct molecular-level modalities of action. To elucidate these disparities and deepen our understanding, we delve into the intricacies of SARS-CoV-2 inactivation via UV-C and ozone gas treatments, exploring their distinct molecular-level impacts utilizing a suite of advanced techniques, including biological atomic force microscopy (Bio-AFM) and single virus force spectroscopy (SVFS). Whereas UV-C exhibited no perceivable alterations in virus size or surface topography, ozone gas treatment elucidated pronounced changes in both parameters, intensifying with prolonged exposure. Furthermore, a nuanced difference was observed in virus-host cell binding post-treatment: ozone gas distinctly reduced SARS-CoV-2 binding to host cells, while UV-C maintained the status quo. The results derived from these methodical explorations underscore the pivotal role of advanced Bio-AFM techniques and SVFS in enhancing our understanding of virus inactivation mechanisms, offering invaluable insights for future research and applications in viral contamination mitigation.

• Keywords
• binding activity, infectivity test, sterilization mechanisms, structural characteristics, topographical characteristics,
• MeSH
• Chlorocebus aethiops MeSH
• COVID-19 * MeSH
• Virus Inactivation * drug effects   radiation effects   MeSH
• Humans MeSH
• Microscopy, Atomic Force * MeSH
• Ozone * chemistry   pharmacology   MeSH
• Plasma Gases chemistry   pharmacology   MeSH
• SARS-CoV-2 * drug effects   MeSH
• Sterilization methods   MeSH
• Ultraviolet Rays * MeSH
• Vero Cells MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ozone * MeSH
• Plasma Gases MeSH

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.

• Keywords
• SARS-CoV-2, antivirals, perylene, photosensitizers, singlet oxygen,
• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 * MeSH
• Cats MeSH
• Membranes MeSH
• SARS-CoV-2 MeSH
• Singlet Oxygen * MeSH
• Animals MeSH
• Check Tag
• Cats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH
• Singlet Oxygen * 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

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.

• Keywords
• Flaviruses, RNA-dependent RNA-polymerase, SARS-CoV-2, antiviral agents, helquat-like compound,
• MeSH
• COVID-19 * MeSH
• Humans MeSH
• Pandemics MeSH
• RNA-Dependent RNA Polymerase MeSH
• SARS-CoV-2 MeSH
• Encephalitis Viruses, Tick-Borne * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA-Dependent RNA Polymerase 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

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.

• Keywords
• COVID-19, RNA-dependent RNA polymerase, SAR-CoV-2, antiviral agents, non-nucleotide inhibitor,
• MeSH
• Adenosine Monophosphate analogs & derivatives   pharmacology   MeSH
• Alanine analogs & derivatives   pharmacology   MeSH
• Antiviral Agents pharmacology   MeSH
• Benzothiazoles pharmacology   MeSH
• Cell Line MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Coronavirus RNA-Dependent RNA Polymerase antagonists & inhibitors   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Pyridones pharmacology   MeSH
• Virus Replication drug effects   MeSH
• SARS-CoV-2 drug effects   enzymology   physiology   MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Monophosphate MeSH
• Alanine MeSH
• Antiviral Agents MeSH
• Benzothiazoles MeSH
• HeE1-2Tyr MeSH Browser
• Enzyme Inhibitors MeSH
• Coronavirus RNA-Dependent RNA Polymerase MeSH
• NSP12 protein, SARS-CoV-2 MeSH Browser
• Pyridones MeSH
• remdesivir MeSH Browser

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.

• Keywords
• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), antiviral activity, cleistanthin B, cytotoxicity, diphyllin, helioxanthin, vacuolar ATPase blocker,
• Publication type
• Journal Article MeSH

Emerging flaviviruses are causative agents of severe and life-threatening diseases, against which no approved therapies are available. Among the nucleoside analogues, which represent a promising group of potentially therapeutic compounds, fluorine-substituted nucleosides are characterized by unique structural and functional properties. Despite having first been synthesized almost 5 decades ago, they still offer new therapeutic opportunities as inhibitors of essential viral or cellular enzymes active in nucleic acid replication/transcription or nucleoside/nucleotide metabolism. Here, we report evaluation of the antiflaviviral activity of 28 nucleoside analogues, each modified with a fluoro substituent at different positions of the ribose ring and/or heterocyclic nucleobase. Our antiviral screening revealed that 3'-deoxy-3'-fluoroadenosine exerted a low-micromolar antiviral effect against tick-borne encephalitis virus (TBEV), Zika virus, and West Nile virus (WNV) (EC50 values from 1.1 ± 0.1 μM to 4.7 ± 1.5 μM), which was manifested in host cell lines of neural and extraneural origin. The compound did not display any measurable cytotoxicity up to concentrations of 25 μM but had an observable cytostatic effect, resulting in suppression of cell proliferation at concentrations of >12.5 μM. Novel approaches based on quantitative phase imaging using holographic microscopy were developed for advanced characterization of antiviral and cytotoxic profiles of 3'-deoxy-3'-fluoroadenosine in vitro In addition to its antiviral activity in cell cultures, 3'-deoxy-3'-fluoroadenosine was active in vivo in mouse models of TBEV and WNV infection. Our results demonstrate that fluoro-modified nucleosides represent a group of bioactive molecules with excellent potential to serve as prospective broad-spectrum antivirals in antiviral research and drug development.

• Keywords
• 3′-deoxy-3′-fluoroadenosine, antiviral activity, cytotoxicity, flavivirus, mouse model, nucleoside analogue, tick-borne encephalitis virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Deoxyadenosines pharmacology   MeSH
• Zika Virus Infection * MeSH
• Mice MeSH
• Prospective Studies MeSH
• Virus Replication MeSH
• Zika Virus * MeSH
• Encephalitis Viruses, Tick-Borne * 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
• 3'-fluoro-3'-deoxyadenosine MeSH Browser
• Antiviral Agents MeSH
• Deoxyadenosines MeSH