Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology.

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.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Susceptibility of Tick-Borne Encephalitis Virus to Inactivation by Heat, Acidic pH, Chemical, or UV Treatment.

Tick-borne encephalitis virus (TBEV) is a single-stranded, positive-sense RNA virus in the family Flaviviridae that is endemic in parts of Europe and Asia and can cause meningitis or encephalitis. Due to the disease severity, TBEV requires handling under heightened biosafety measures. The establishment and validation of inactivation procedures is a prerequisite for downstream analyses and management of occupational exposure. Therefore, different procedures for TBEV inactivation were tested. Our results suggest that TBEV is susceptible to inactivation by heat, acidic pH, different concentrations of alcohol, formaldehyde, or detergents, and exposure to UV irradiation, which may depend on sample size and composition.

Effect of immature tick-borne encephalitis virus particles on antiviral activity of 5-aminoisoxazole-3-carboxylic acid adamantylmethyl esters.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its ß-n-octyl-d-glucoside binding pocket (ß-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessed in vivo and in vitro infectivity of the residual mature virions in the immature virus samples. Analysis of the ß-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.

The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice.

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.

Simplistic perylene-related compounds as inhibitors of tick-borne encephalitis virus reproduction.

Rigid amphipathic fusion inhibitors are potent broad-spectrum antivirals based on the perylene scaffold, usually decorated with a hydrophilic group linked via ethynyl or triazole. We have sequentially simplified these structures by removing sugar moiety, then converting uridine to aniline, then moving to perylenylthiophenecarboxylic acids and to perylenylcarboxylic acid. All these polyaromatic compounds, as well as antibiotic heliomycin, still showed pronounced activity against tick-borne encephalitis virus (TBEV) with limited toxicity in porcine embryo kidney (PEK) cell line. 5-(Perylen-3-yl)-2-thiophenecarboxylic acid (5a) showed the highest antiviral activity with 50% effective concentration of approx. 1.6 nM.

Inhibitory Activity of Scutellaria baicalensis Flavonoids against Tick-Borne Encephalitis Virus.

We studied virus-inhibiting activity of Baikal skullcap (Scutellaria baicalensis) flavonoids against tick-borne encephalitis virus using various model schemes. The half-maximum cytotoxic concentration (CC50) for the plant extract was found (363.9±58.6 µg/ml). Based on the CC50 and IC50, selective index (SI) was calculated for viricidal (53.4), preventive (50.5), and direct antiviral actions (39.1) and for-intracellular replication of the virus (40.4). Suppression of virus reproduction ≥2.0 lg TCID50 was observed at extract concentration ≥5 µg/ml (viricidal effect), ≥11.2 µg/ml (preventive and direct antiviral effects), and ≥9 µg/ml (intracellular replication). Flavonoids of Baikal skullcap extract produced an in vitro inhibitory effect on tick-borne encephalitis virus due to their direct viricidal activity and direct inhibition of adsorption and intracellular replication of tick-borne encephalitis virus, which determines their value as highly effective antiviral drugs.

Molecular and Genetic Bases of Inhibition of Tick-Borne Encephalitis Virus Replication by Eprosartan and Ribavirin.

The antiviral activity of eprosartan (compound selected in silico) towards highly and low-virulent strains of tick-borne encephalitis virus was compared in vitro with activity of ribavirin. Study of the cytopathogenic activity of the virus on SPEV cells by ELISA, IFAT, and PCR showed similar results: both substances (eprosartan and ribavirin) promoted elimination of tick-borne encephalitis virus. Ribavirin exhibited intracellular inhibition towards both strains: the selectivity index for highly virulent Dal'negorsk strain was 160, for low-virulent Primorye-437 strain - 113. Eprosartan inhibited intracellular replication of Dal'negorsk strain (13.7) and less so that of Primorye-437 strain (2.9). The efficiency of virtual screening of the ligand (eprosartan) was demonstrated for highly virulent, but not low virulent tick-borne encephalitis strain.

Tick-borne flavivirus reproduction inhibitors based on isoxazole core linked with adamantane.

Infections caused by flaviviruses pose a huge threat for public health all over the world. The search for therapeutically relevant compounds targeting tick-borne flaviviruses requires the exploration of novel chemotypes. In the present work a large series of novel polyfunctionalized isoxazole derivatives bearing substituents with various steric and electronic effects was obtained by our unique versatile synthetic procedure and their antiviral activity against tick-borne encephalitis, Omsk hemorrhagic fever, and Powassan viruses was studied in vitro. The majority of studied isoxazoles showed activity in low micromolar range. No appreciable cytotoxicity was observed for tested compounds. The lead compounds, 5-aminoisoxazole derivatives containing adamantyl moiety, exhibited strong antiviral activity and excellent therapeutic index.

Antiviral Activity of Uridine Derivatives of 2-Deoxy Sugars against Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.

Effect of Glycosaminoglycans on Pathogenic Properties Far-Eastern Tick-Borne Encephalitis Virus.

We studied the effect of sulfated glycosaminoglycan on the infection properties of high-virulence Dal'negorsk strain and low-virulence Primorye-437 of tick-borne encephalitis virus. Differences in reproductive activity of these strains and their tropism to the target cells were revealed. Glycosaminoglycan reduced pathogenetic activity of high-virulence strain in vitro, but had no effect on low-virulence strain. The interaction of imperfect virus particles of non-pathogen strain with the glycosaminoglycan led to their accumulation in cell, but in the culture medium of SPEV cells infected with experimental and control samples, accumulation of virus particles did not differ. The results on activity of glycosaminoglycan binding with strains differing by their biological and molecular genetic characteristics can be used to assess their pathogenic potential.

Antiviral and Antioxidant Properties of Echinochrome A.

The aim of this study was to examine the in vitro antioxidant and antiviral activities of echinochrome A and echinochrome-based antioxidant composition against tick-borne encephalitis virus (TBEV) and herpes simplex virus type 1 (HSV-1). The antioxidant composition, which is a mixture of echinochrome A, ascorbic acid, and α-tocopherol (5:5:1), showed higher antioxidant and antiviral effects than echinochrome A. We suppose that echinochrome A and its composition can both directly affect virus particles and indirectly enhance antioxidant defense mechanisms in the hosting cell. The obtained results allow considering the echinochrome A and the composition of antioxidants on its basis as the promising agents with the both antioxidant and antiviral activities.

Probing chemical space of tick-borne encephalitis virus reproduction inhibitors with organoselenium compounds.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is the leading cause of arboviral neuroinfections in Europe. Only a few classes of the nucleoside and non-nucleoside inhibitors were investigated against TBEV reproduction. Paving the way to previously unexplored areas of anti-TBEV chemical space, we assessed the inhibition of TBEV reproduction in the plaque reduction assay by various compounds derived from cyanothioacetamide and cyanoselenoacetamide. Compounds from seven classes, including 4-(alkylthio)-2-aryl-3-azaspiro[5.5]undec-4-ene-1,1,5-tricarbonitriles, 3-arylamino-2-(selenazol-2-yl)acrylonitriles, ethyl 6-(alkylseleno)-5-cyano-2-oxo-1,2-dihydropyridine-3-carboxylates, 6-(alkylseleno)-2-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles, 2-(alkylseleno)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carbonitriles, 8-selenoxo-3,5,7,11-tetraazatricyclo[7.3.1.02,7 ]tridec-2-ene-1,9-dicarbonitriles, and selenolo[2,3-b]quinolines, inhibited TBEV reproduction with EC50 values in the micromolar range while showing moderate cytotoxicity and no inhibition of enterovirus reproduction. Thus, new scaffolds with promising anti-TBEV activity were found.

Mechanisms of Antiviral Activity of the Polyphenol Complex from Seagrass of the Zosteraceae Family against Tick-Borne Encephalitis Virus.

Antiviral activity of the polyphenol complex from seagrass of the Zosteraceae family against highly pathogenic strain of the tick-borne encephalitis virus was studied on passaged culture of porcine embryo kidney cells. The antiviral effect of the test compound manifested in a decrease in the infectious titer of the virus and depended on the concentration and application schemes. Polyphenol complex in a concentration of 100 µg/ml suppressed accumulation of the pathogen in the cell culture: pretreatment of the virus reduced its titer by 4 log, pretreatment of cells by 1.4 log, and application of the compound 1 h after cell infection by 2.8 log. Antiviral action of the test compound is determined by direct inactivation of the virus and inhibition of virus replication at the early stage, which attests to potential of this compound in the treatment of tick-borne encephalitis.

New tools in nucleoside toolbox of tick-borne encephalitis virus reproduction inhibitors.

Design and development of nucleoside analogs is an established strategy in the antiviral drug discovery field. Nevertheless, for many viruses the coverage of structure-activity relationships (SAR) in the nucleoside chemical space is not sufficient. Here we present the nucleoside SAR exploration for tick-borne encephalitis virus (TBEV), a member of Flavivirus genus. Promising antiviral activity may be achieved by introduction of large hydrophobic substituents in the position 6 of adenosine or bulky silyl groups to the position 5'. Introduction of methyls to the ribose moiety does not lead to inhibition of TBEV reproduction. Possible mechanisms of action of these nucleosides include the inhibition of viral entry or interaction with TBEV non-structural protein 5 methyltransferase or RNA-dependent RNA polymerase domains.

Emerging Cases of Powassan Virus Encephalitis in New England: Clinical Presentation, Imaging, and Review of the Literature.

BACKGROUND: Powassan virus (POWV) is a rarely diagnosed cause of encephalitis in the United States. In the Northeast, it is transmitted by Ixodes scapularis, the same vector that transmits Lyme disease. The prevalence of POWV among animal hosts and vectors has been increasing. We present 8 cases of POWV encephalitis from Massachusetts and New Hampshire in 2013-2015. METHODS: We abstracted clinical and epidemiological information for patients with POWV encephalitis diagnosed at 2 hospitals in Massachusetts from 2013 to 2015. We compared their brain imaging with those in published findings from Powassan and other viral encephalitides. RESULTS: The patients ranged in age from 21 to 82 years, were, for the most part, previously healthy, and presented with syndromes of fever, headache, and altered consciousness. Infections occurred from May to September and were often associated with known tick exposures. In all patients, cerebrospinal fluid analyses showed pleocytosis with elevated protein. In 7 of 8 patients, brain magnetic resonance imaging demonstrated deep foci of increased T2/fluid-attenuation inversion recovery signal intensity. CONCLUSIONS: We describe 8 cases of POWV encephalitis in Massachusetts and New Hampshire in 2013-2015. Prior to this, there had been only 2 cases of POWV encephalitis identified in Massachusetts. These cases may represent emergence of this virus in a region where its vector, I. scapularis, is known to be prevalent or may represent the emerging diagnosis of an underappreciated pathogen. We recommend testing for POWV in patients who present with encephalitis in the spring to fall in New England.

Use of hydrogen peroxide vapour & plasma irradiation in combination for quick decontamination of closed chambers.

BACKGROUND & OBJECTIVES: Various conventional methods such as gaseous, vapour and misting systems, fogging, manual spray and wipe techniques employing a number of chemical agents are used for decontamination of enclosed spaces. Among all these methods, use of aerosolized formaldehyde is the most preferred method due to cost-effectiveness and practical aspects. However, being extremely corrosive in nature generating very irritating fumes and difficulty in maintaining a high level of gas concentration, many laboratories prefer the vaporization of hydrogen peroxide (H 2 O 2 ) as an alternative. We present here the results of using H 2 O 2 vapour in combination with plasma irradiation for quick decontamination of closed chambers. METHODS: The present study describes a decontamination method, using plasma irradiation in combination with H 2 O 2 (5%). Effect of plasma irradiation and H 2 O 2 on the viability of bacterial spores (Bacillus subtilis), Chikungunya and Kyasanur Forest Disease viruses was assessed. RESULTS: Data suggest that with the combination of H 2 O 2 vapour and plasma irradiation, within short time (three minutes), decontamination of surfaces and space volume could be achieved. Although it showed damage of spores present on the strips, it did not show any penetration power. INTERPRETATION & CONCLUSIONS: The results were encouraging, and this method was found to be efficient for achieving surface sterilization in a short time. This application may be useful in laboratories and industries particularly, those working on clean facility concept following good laboratory and manufacturing practices.

Nucleoside inhibitors of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 µM for 7-deaza-2'-CMA, 7.1 ± 1.2 µM for 2'-CMA, and 14.2 ± 1.9 µM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 µM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 µM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

Synthesis and assessment of 4-aminotetrahydroquinazoline derivatives as tick-borne encephalitis virus reproduction inhibitors.

Tick-borne encephalitis virus (TBEV) belonging to Flavivirus genus causes severe infection in humans. The search for therapeutically relevant compounds targeting TBEV requires the exploration of novel chemotypes. A versatile synthesis of previously unknown 4-aminopyrimidines and 4-aminopyrimidine N-oxides based on a fluorosubstituted heterocyclic core is described. A representative series of 4-aminotetrahydroquinazoline derivatives, containing aliphatic and aromatic substituents as well as the adamantane framework, was obtained and their activity against tick-borne encephalitis virus reproduction was studied. Nine compounds were found to inhibit TBEV entry into the host cells. A bulky hydrophobic adamantyl group was identified to be important for the antiviral activity. The developed synthetic route allowed an easy access to a consistent compound library for further structure-activity relationship studies.