Tick-borne encephalitis (TBE) represents one of the most serious arboviral neuro-infections in Europe and northern Asia. As no specific antiviral therapy is available at present, there is an urgent ...need for efficient drugs to treat patients with TBE virus (TBEV) infection. Using two standardised in vitro assay systems, we evaluated a series of 29 nucleoside derivatives for their ability to inhibit TBEV replication in cell lines of neuronal as well as extraneural origin. The series of tested compounds included 2′-C- or 2′-O-methyl substituted nucleosides, 2′-C-fluoro-2′-C-methyl substituted nucleosides, 3′-O-methyl substituted nucleosides, 3′-deoxynucleosides, derivatives with 4′-C-azido substitution, heterobase modified nucleosides and neplanocins. Our data demonstrate a relatively stringent structure-activity relationship for modifications at the 2′, 3′, and 4′ nucleoside positions. Whereas nucleoside derivatives with the methylation at the C2′ position or azido modification at the C4′position exerted a strong TBEV inhibition activity (EC50 from 0.3 to 11.1 μM) and low cytotoxicity in vitro, substitutions of the O2′ and O3′ positions led to a complete loss of anti-TBEV activity (EC50 > 50 μM). Moreover, some structural modifications of the heterobase moiety resulted in a high increase of cytotoxicity in vitro. High antiviral activity and low cytotoxicity of C2′ methylated or C4′ azido substituted pharmacophores suggest that such compounds might represent promising candidates for further development of potential therapeutic agents in treating TBEV infection.
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•A series of 29 nucleosides was evaluated for anti-TBEV/cytotoxicity effects using standardised in vitro assay systems.•A relatively stringent structure-activity relationship exists for modifications at the 2′, 3′, and 4′ nucleoside positions.•Some structure-activity relationship flexibility was found for changes of the purine/pyrimidine heterobase identity.•Structural modifications of the heterobase were accompanied by either inefficacy against TBEV or cytotoxicity.•C2′ methyl or C4′ azido substituents are crucial for a highly selective TBEV inhibition and a low cytotoxicity in vitro.
Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European-Asian continent. Although more than 12,000 ...clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure-activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.
During 2008-2018, we collected samples from rodents and patients throughout the Czech Republic and characterized hantavirus isolates. We detected Dobrava-Belgrade and Puumala orthohantaviruses in ...patients and Dobrava-Belgrade, Tula, and Seewis orthohantaviruses in rodents. Increased knowledge of eco-epidemiology of hantaviruses will improve awareness among physicians and better outcomes of patients.
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'-
-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'-
-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-
-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'-
-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'-
-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.
This study found that the nucleoside analog 7-deaza-2'-
-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'-
-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.
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