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.

• Keywords
• CP: Immunology, antibodies, flaviviruses, tick diseases,
• MeSH
• Epitopes immunology   MeSH
• Flavivirus immunology   MeSH
• Ticks virology   immunology   MeSH
• Humans MeSH
• Antibodies, Monoclonal immunology   MeSH
• Antibodies, Neutralizing * immunology   MeSH
• Antibodies, Viral immunology   MeSH
• Encephalitis Viruses, Tick-Borne immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Geographicals
• Brazil MeSH
• Mexico MeSH
• Names of Substances
• Epitopes MeSH
• Antibodies, Monoclonal MeSH
• Antibodies, Neutralizing * MeSH
• Antibodies, Viral MeSH

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

• Keywords
• capsid, nucleolus, nucleus, protein structure, tick-borne flaviviruses, translational shutoff,
• MeSH
• Capsid metabolism   MeSH
• RNA, Viral metabolism   MeSH
• Viral Nonstructural Proteins metabolism   MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH
• Viral Nonstructural Proteins MeSH
• Capsid Proteins MeSH

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

• Keywords
• A3SS, alternative 3′ splice site, A5SS, alternative 5′ splice site, ACACA, Acetyl-CoA Carboxylase Alpha, AKR1C2, Aldo-Keto Reductase Family 1 Member C2, ANKS1A, Ankyrin Repeat And Sterile Alpha Motif Domain Containing 1A, ANOS1, Anosmin 1, AOX1, Aldehyde Oxidase 1, APOBEC3G, Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3G, APOL1/6, Apolipoprotein L1/6, ARID2, AT-Rich Interaction Domain 2, AUTS2, Activator Of Transcription And Developmental Regulator AUTS2, Alternative splicing, Astrocytes, BCL11B, BAF Chromatin Remodeling Complex Subunit BCL11B, BCL9L, BCL9 Transcription Coactivator-like, BDKRB2, Bradykinin Receptor B2, BDNF, Brain Derived Neurotrophic Factor, BEND3, BEN Domain Containing 3, BSA, bovine serum albumin, BST2, Bone Marrow Stromal Cell Antigen 2, CALB1, Calbindin 1, CAMK2A, Calcium/Calmodulin Dependent Protein Kinase II Alpha, CD, complement determinant, CDKN1C, Cyclin Dependent Kinase Inhibitor 1C, CFAP61, Cilia And Flagella Associated Protein 61, CHRNA3, Cholinergic Receptor Nicotinic Alpha 3 Subunit, CHRNB4, Cholinergic Receptor Nicotinic Beta 4 Subunit, CLIC5, Chloride Intracellular Channel 5, CMPK2, Cytidine/Uridine Monophosphate Kinase 2, CNS, central nervous system, CNTN2, Contactin 2, CREG2, Cellular Repressor Of E1A Stimulated Genes 2, CXADR, Coxsackievirus B-Adenovirus Receptor, CYYR1, Cysteine And Tyrosine Rich 1, DACH1, Dachshund Family Transcription Factor 1, DAPI, diamidino-2-phenylindole, DCC, Netrin 1 Receptor, DCX, Doublecortin, DDX60, DExD/H-Box Helicase 60, DDX60L, DExD/H-Box 60 Like, DE, differentially expressed, DENV, Dengue virus, DIRAS2, DIRAS Family GTPase 2, DLX1/5/6, Distal-Less Homeobox 1/5/6, DNMT3B, DNA Methyltransferase 3 Beta, DPYSL2, Dihydropyrimidinase Like 2, EBF1, EBF Transcription Factor 1, EGF, Epidermal Growth Factor, ELAVL2/4, ELAV Like RNA Binding Protein 2/4, EPHB1, EPH Receptor B1, EPSTI1, Epithelial Stromal Interaction 1, ERBB4, Erb-B2 Receptor Tyrosine Kinase 4, ES, exon skipping, ESRRG, Estrogen Related Receptor Gamma, FGFb, Fibroblast Growth Factor 2, FPKM, Fragments Per Kilobase of transcript per Million mapped reads, FUT9, Fucosyltransferase 9, G2E3, G2/M−Phase Specific E3 Ubiquitin Protein Ligase, GABRG2, Gamma-Aminobutyric Acid Type A Receptor Subunit Gamma 2, GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase, GAS2L3, Growth Arrest Specific 2 Like 3, GAS7, Growth Arrest Specific 7, GATAD2B, GATA Zinc Finger Domain Containing 2B, GFAP, Glial Fibrillary Acidic Protein, GIPC2, GIPC PDZ Domain Containing Family Member 2, GLRA2, Glycine Receptor Alpha 2, GNG2, G Protein Subunit Gamma 2, GO, gene ontology, GOLGA4, Golgin A4, GRIN2A, Glutamate Ionotropic Receptor NMDA Type Subunit 2A, GSEA, gene set enrichment analysis, HERC5/6, HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 5/6, HEYL, Hes Related Family BHLH Transcription Factor With YRPW Motif Like, HPRT1, Hypoxanthine Phosphoribosyltransferase 1, HS, hot-spot, HSPA6, Heat Shock Protein Family A (Hsp70) Member 6, HUDD (ELAV4), Hu-Antigen D/ELAV Like Neuron-Specific RNA Binding Protein 4, IFI6, Interferon Alpha Inducible Protein 6, IFIH1 (MDA5), Interferon Induced With Helicase C Domain 1/Melanoma Differentiation-Associated Protein 5, IFIT1-3, Interferon Induced Protein With Tetratricopeptide Repeats 1–3, IFITM1/2, Interferon Induced Transmembrane Protein 1/2, IFN, interferon, IGB, Integrated Genome Browser, IL6, Interleukin 6, IR, intron retention, ISG20, Interferon Stimulated Exonuclease Gene 20, ISGF3, Interferon-Stimulated Gene Factor 3 Gamma, ISGs, interferon-stimulated genes, JEV, Japanese encephalitis virus, KCND2, Potassium Voltage-Gated Channel Subfamily D Member 2, KCNK10, Potassium Two Pore Domain Channel Subfamily K Member 10, KCNS2, Potassium Voltage-Gated Channel Modifier Subfamily S Member 2, KIT, KIT Proto-Oncogene, Receptor Tyrosine Kinase, KLHDC8A, Kelch Domain Containing 8A, KLHL13, Kelch Like Family Member 13, KRR1, KRR1 Small Subunit Processome Component Homolog, LCOR, Ligand Dependent Nuclear Receptor Corepressor, LEKR1, Leucine, Glutamate And Lysine Rich 1, LGI1, Leucine Rich Glioma Inactivated 1, LRRTM3, Leucine Rich Repeat Transmembrane Neuronal 3, LSV, local splicing variation, LUZP2, Leucine Zipper Protein 2, MAN1A1, Mannosidase Alpha Class 1A Member 1, MAP2, Microtubule Associated Protein 2, MBNL2, Muscleblind Like Splicing Regulator 2, MCTP1, Multiple C2 And Transmembrane Domain Containing 1, MMP13, Matrix Metallopeptidase 13, MN1, MN1 Proto-Oncogene, Transcriptional Regulator, MOI, multiplicity of infection, MTUS2, Microtubule Associated Scaffold Protein 2, MX2, MX Dynamin Like GTPase 2, MYCN, MYCN Proto-Oncogene, BHLH Transcription Factor, NAV1, Neuron Navigator 1, NCAM1, Neural Cell Adhesion Molecule 1, NDRG4, N-Myc Downstream-Regulated Gene 4 Protein, NEK7, NIMA Related Kinase 7, NFASC, Neurofascin, NKAIN1, Sodium/Potassium Transporting ATPase Interacting 1, NMI, N-Myc And STAT Interactor 2, NRAP, Nebulin Related Anchoring Protein, NRARP, NOTCH Regulated Ankyrin Repeat Protein, NREP, Neuronal Regeneration Related Protein, NRN1, Neuritin 1, NS3, flaviviral non-structural protein 3, NXPH2, Neurexophilin 2, NYNRIN, NYN Domain And Retroviral Integrase Containing, Neurons, Neuropathogenesis, OAS, 2′-5′-Oligoadenylate Synthetase, OASL, 2′-5′-Oligoadenylate Synthetase Like, ONECUT2, ONECUT-2 Homeodomain Transcription Factor, OPCML, Opioid Binding Protein/Cell Adhesion Molecule Like, OTX2, Orthodenticle Homeobox 2, PBS, phosphate buffer saline, PBX1, Pre-B-Cell Leukemia Transcription Factor 1, PCDH18/20, Protocadherin 18/20, PFKFB3, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3, PIK3C2B, Phosphatidylinositol-4-Phosphate 3-Kinase Catalytic Subunit Type 2 Beta, PIP4P2, Phosphatidylinositol-4,5-Bisphosphate 4-Phosphatase 2, PLCH1, Phospholipase C Eta 1, POU3F4, Brain-Specific Homeobox/POU Domain Protein 4, PPM1L, Protein Phosphatase, Mg2+/Mn2+ Dependent 1L, PPP1R17, Protein Phosphatase 1 Regulatory Subunit 17, PRDM12, PR Domain Zinc Finger Protein 12, PSI, percent selective index, PSRC1, Proline And Serine Rich Coiled-Coil 1, PTPN5, Protein Tyrosine Phosphatase Non-Receptor Type 5, PTPRH, Protein Tyrosine Phosphatase Receptor Type H, RAPGEF5, Rap Guanine Nucleotide Exchange Factor 5, RBFOX1, RNA Binding Fox-1 Homolog 1, RIG-I (DDX58), Retinoic Acid-Inducible Gene 1 Protein, RNF212, Ring Finger Protein 212, RNVU1, RNA, Variant U1 Small Nuclear, RSAD2, Radical S-Adenosyl Methionine Domain Containing 2, RTL8B, Retrotransposon Gag Like 8B, Response to infection, SAMD9, Sterile Alpha Motif Domain Containing 9, SEMA3E, Semaphorin 3E, SH3TC2, SH3 Domain And Tetratricopeptide Repeats 2, SHF, Src Homology 2 Domain Containing F, SHISAL1, Shisa Like 1, SIAH3, Siah E3 Ubiquitin Protein Ligase Family Member 3, SIRPA, Signal Regulatory Protein Alpha, SLITRK5, SLIT And NTRK Like Family Member 5, SNP, single-nucleotide polymorphism, SOGA1, Suppressor Of Glucose, Autophagy Associated 1, SPSB4, SplA/Ryanodine Receptor Domain And SOCS Box Containing 4, ST6GAL1, ST6 Beta-Galactoside Alpha-2,6-Sialyltransferase 1, TBC1D30, TBC1 Domain Family Member 30, TBEV, Tick-borne encephalitis virus, TFAP2A, Transcription Factor AP-2 Alpha, TFAP2B, Transcription Factor AP-2 Beta, THSD7A, Thrombospondin Type 1 Domain Containing 7A, THUMPD2, THUMP Domain-Containing Protein 2/SAM-Dependent Methyltransferase, TIPARP, TCDD Inducible Poly(ADP-Ribose) Polymerase, TM4SF18, Transmembrane 4 L Six Family Member 18, TMC8, Transmembrane Channel Like 6, TMEM229B, Transmembrane Protein 229B, TMTC1, Transmembrane O-Mannosyltransferase Targeting Cadherins 1, TNFSF10, TNF Superfamily Member 10, TRHDE, Thyrotropin Releasing Hormone Degrading Enzyme, TRIM38, Tripartite Motif Containing 38, TSHZ1, Teashirt Zinc Finger Homeobox 1, Tick-borne encephalitis virus, Transcriptomics, USP18, Ubiquitin Specific Peptidase 18/ISG15-Specific-Processing Protease, UTR, untranslated region, UTS2R, Urotensin 2 Receptor, WNV, West Nile virus, XAF1, XIAP Associated Factor 1, XRN1, 5′-3′ Exoribonuclease 1, ZIKV, Zika virus, ZMAT3, Zinc Finger Matrin-Type 3, ZMYM5, Zinc Finger MYM-Type Containing 5, ZNF124, Zinc Finger Protein 124, ZNF730, Zinc Finger Protein 730, gRNA, genomic TBEV RNA, hNSC, human neural stem cells, lncRNA, long non-coding RNA, mRNA, messenger RNA, miRNA, miRNA, micro RNA, ncRNA, non-coding RNA, pc-mRNA, protein-coding mRNA, qRT-PCR, quantitative reverse transcription real-time PCR, snRNP, small nuclear ribonucleoproteins, vd-sRNA, virus-derived small RNA,
• Publication type
• Journal Article 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

A highly virulent strain (Hypr) of tick-borne encephalitis virus (TBEV) was serially subcultured in the mammalian porcine kidney stable (PS) and Ixodes ricinus tick (IRE/CTVM19) cell lines, producing three viral variants. These variants exhibited distinct plaque sizes and virulence in a mouse model. Comparing the full-genome sequences of all variants, several nucleotide changes were identified in different genomic regions. Furthermore, different sequential variants were revealed to co-exist within one sample as quasispecies. Interestingly, the above-mentioned nucleotide changes found within the whole genome sequences of the new variants were present alongside the nucleotide sequence of the parental strain, which was represented as a minority quasispecies. These observations further imply that TBEV exists as a heterogeneous population that contains virus variants pre-adapted to reproduction in different environments, probably enabling virus survival in ticks and mammals.

• Keywords
• TBEV, flavivirus adaptation, genome mutation, host alternation, neuroinvasiveness, quasispecies, tick cell line,
• MeSH
• Cell Line MeSH
• Adaptation, Physiological genetics   MeSH
• Genetic Variation MeSH
• Genome, Viral MeSH
• Ixodes cytology   virology   MeSH
• Encephalitis, Tick-Borne virology   MeSH
• Kidney cytology   virology   MeSH
• Mutation MeSH
• Mice MeSH
• Swine MeSH
• Quasispecies * MeSH
• Virulence MeSH
• Encephalitis Viruses, Tick-Borne genetics   pathogenicity   physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

• MeSH
• Glycoproteins chemistry   metabolism   MeSH
• Glycosylation MeSH
• Ticks virology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Viral Envelope Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Encephalitis Viruses, Tick-Borne growth & development   metabolism   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
• Glycoproteins MeSH
• Viral Envelope Proteins MeSH

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.

• MeSH
• Transcription, Genetic MeSH
• Encephalitis, Tick-Borne genetics   metabolism   virology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• RNA Precursors MeSH
• Protein Biosynthesis genetics   MeSH
• RNA, Ribosomal genetics   metabolism   MeSH
• RNA Polymerase I genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne physiology   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
• RNA Precursors MeSH
• RNA, Ribosomal MeSH
• RNA Polymerase I 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

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'-C-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'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-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'-C-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'-C-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.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-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'-C-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.

• Keywords
• antiviral agents, antiviral therapy, escape mutant, tick-borne encephalitis virus, tick-borne pathogens,
• Publication type
• Journal Article MeSH

BACKGROUND: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule. RESULTS: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established. CONCLUSIONS: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

• MeSH
• Animal Structures immunology   MeSH
• Anti-Infective Agents isolation & purification   pharmacology   MeSH
• Defensins genetics   immunology   isolation & purification   MeSH
• Erythrocytes drug effects   MeSH
• Gram-Negative Bacteria drug effects   MeSH
• Gram-Positive Bacteria drug effects   MeSH
• Ixodes genetics   immunology   MeSH
• Yeasts drug effects   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Guinea Pigs MeSH
• Protein Isoforms genetics   immunology   isolation & purification   MeSH
• Gene Expression Profiling MeSH
• Viruses drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Guinea Pigs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Infective Agents MeSH
• Defensins MeSH
• Protein Isoforms MeSH