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

ABSTRACT 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.

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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

10.1101/563544 ◽

2019 ◽

Cited By ~ 2

Author(s):

Ludek Eyer ◽

Antoine Nougairède ◽

Marie Uhlířová ◽

Jean-Sélim Driouich ◽

Darina Zouharová ◽

...

Keyword(s):

Cell Culture ◽

Ebola Virus ◽

Rna Virus ◽

Encephalitis Virus ◽

Viral Fitness ◽

Rna Dependent Rna Polymerase ◽

Virus Inhibitor ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Ebola Virus Infection

AbstractThe 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 virus infection. 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. Stochastic molecular simulations indicate that the steric freedom caused by the E460D substitution increases close electrostatic interactions between the inhibitor and the interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an incorrect/modified nucleotide. 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 only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. 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.ImportanceTick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in large areas of 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 virus infection, 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 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.

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Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.00587-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8272-8282 ◽

Cited By ~ 6

Author(s):

Wolfgang Fischl ◽

Sigrid Elshuber ◽

Sabrina Schrauf ◽

Christian W. Mandl

Keyword(s):

Cell Culture ◽

Cleavage Site ◽

Encephalitis Virus ◽

Wild Type Virus ◽

Sequence Motif ◽

Amino Acid Residues ◽

Tick Borne Encephalitis ◽

Protease Specificity ◽

Tick Borne Encephalitis Virus ◽

Cleavage Motif

ABSTRACT The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.

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Tick-borne encephalitis virus infections in Germany. Seasonality and in-year patterns. A retrospective analysis from 2001-2018

PLoS ONE ◽

10.1371/journal.pone.0224044 ◽

2019 ◽

Vol 14 (10) ◽

pp. e0224044

Author(s):

Johannes P. Borde ◽

Klaus Kaier ◽

Philip Hehn ◽

Merle M. Böhmer ◽

Teresa M. Kreusch ◽

...

Keyword(s):

Retrospective Analysis ◽

Encephalitis Virus ◽

Virus Infections ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

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Flavivirus-induced antibody cross-reactivity

Journal of General Virology ◽

10.1099/vir.0.031641-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2821-2829 ◽

Cited By ~ 143

Author(s):

Karen L. Mansfield ◽

Daniel L. Horton ◽

Nicholas Johnson ◽

Li Li ◽

Alan D. T. Barrett ◽

...

Keyword(s):

Yellow Fever Virus ◽

Cross Reactivity ◽

Encephalitis Virus ◽

Antigenic Relationship ◽

Tick Borne Encephalitis ◽

History Of ◽

Louping Ill ◽

Tick Borne Encephalitis Virus ◽

Antigenic Cartography ◽

The Individual

Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.

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Tick-borne encephalitis virus infections in Lithuanian domestic animals and ticks

Scandinavian Journal of Infectious Diseases ◽

10.1080/00365540510012134 ◽

2005 ◽

Vol 37 (10) ◽

pp. 742-746 ◽

Cited By ~ 16

Author(s):

Aurita Juceviciene ◽

Milda Zygutiene ◽

Pauli Leinikki ◽

Henrikki Brummer-Korvenkontio ◽

Mika Salminen ◽

...

Keyword(s):

Domestic Animals ◽

Encephalitis Virus ◽

Virus Infections ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

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Experience of application of IgY-technology for laboratory diagnostics of viral infections

Voprosy Virusologii ◽

10.36233/0507-4088-2020-65-1-21-26 ◽

2020 ◽

Vol 65 (1) ◽

pp. 21-26 ◽

Cited By ~ 2

Author(s):

A. P. Ivanov ◽

T. D. Klebleeva ◽

O. E. Ivanova

Keyword(s):

Yellow Fever ◽

Viral Infections ◽

Yellow Fever Virus ◽

Laboratory Diagnosis ◽

Encephalitis Virus ◽

Fever Virus ◽

Laboratory Animals ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

Introduction. The well-known advantages of class Y antibodies (IgY) from egg yolks of immunized hens in comparison with class G antibodies (IgG) of laboratory animals traditionally used in laboratory diagnosis of infectious diseases determine the stable interest of researchers in using IgY for these purposes (IgY technology) . Over the past 20 years, the obvious benefits of IgY technology have been demonstrated for a number of viral and bacterial infections. Goals and objectives. Construction of ELISA systems based on specific IgY for laboratory diagnosis of infections caused by tick-borne encephalitis virus, yellow fever virus, poliovirus.Material and methods. Obtaining yolk preparations of immunized chickens, obtaining highly purified IgY preparations (salting out, affinity chromatography), constructing ELISA systems for determining virus-specific antigens, testing the parameters of ELISA systems.Results and discussion. For the first time in laboratory practice, ELISA systems based on the use of specific polyclonal IgY were designed for laboratory diagnosis of topical human viral infections caused by flaviviruses and enteroviruses: determination of antigens of tick-borne encephalitis virus, yellow fever virus, 3 types of poliovirus. It was experimentally shown that these ELISA systems have high sensitivity and specificity, which allows them to be used for the semiquantitative and quantitative determination of antigens of these viruses in various materials (infected cell cultures, vaccines, etc.).Conclusion. The ELISA systems developed on the basis of specific IgY for determination of viral antigens can be effectively used for laboratory diagnosis of a number of viral infections, for the validation and control of vaccine preparations.

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

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa405 ◽

2020 ◽

Author(s):

Laura Wiesner ◽

Carla Schmutte ◽

Imke Steffen

Keyword(s):

Occupational Exposure ◽

Sample Size ◽

Rna Virus ◽

Encephalitis Virus ◽

Acidic Ph ◽

Uv Treatment ◽

Tick Borne Encephalitis ◽

The Family ◽

Positive Sense ◽

Tick Borne Encephalitis Virus

Abstract 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.

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Astrocytes in Flavivirus Infections

International Journal of Molecular Sciences ◽

10.3390/ijms20030691 ◽

2019 ◽

Vol 20 (3) ◽

pp. 691 ◽

Cited By ~ 14

Author(s):

Maja Potokar ◽

Jernej Jorgačevski ◽

Robert Zorec

Keyword(s):

Cell Types ◽

Encephalitis Virus ◽

Neural Cells ◽

Virus Infections ◽

Neurotropic Viruses ◽

Neuronal Synapses ◽

Tick Borne Encephalitis ◽

The Central Nervous System ◽

Tick Borne Encephalitis Virus ◽

New Strategies

Virus infections of the central nervous system (CNS) can manifest in various forms of inflammation, including that of the brain (encephalitis) and spinal cord (myelitis), all of which may have long-lasting deleterious consequences. Although the knowledge of how different viruses affect neural cells is increasing, understanding of the mechanisms by which cells respond to neurotropic viruses remains fragmented. Several virus types have the ability to infect neural tissue, and astrocytes, an abundant and heterogeneous neuroglial cell type and a key element providing CNS homeostasis, are one of the first CNS cell types to get infected. Astrocytes are morphologically closely aligned with neuronal synapses, blood vessels, and ventricle cavities, and thereby have the capacity to functionally interact with neurons and endothelial cells. In this review, we focus on the responses of astrocytes to infection by neurotropic flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), which have all been confirmed to infect astrocytes and cause multiple CNS defects. Understanding these mechanisms may help design new strategies to better contain and mitigate virus- and astrocyte-dependent neuroinflammation.

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