scholarly journals Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus

2004 ◽  
Vol 85 (4) ◽  
pp. 1007-1018 ◽  
Author(s):  
Daisuke Hayasaka ◽  
Tamara S. Gritsun ◽  
Kentarou Yoshii ◽  
Tomotaka Ueki ◽  
Akiko Goto ◽  
...  
Keyword(s):  
Cell Culture ◽  
Amino Acid ◽  
Cdna Clone ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Infectious Cdna Clone ◽  
Amino Acid Substitutions ◽  
Parent Virus ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

A stable full-length infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus (Far-Eastern subtype) was developed by a long high-fidelity RT-PCR and one-step cloning procedure. The infectious clone (O-IC) had four amino acid substitutions and produced smaller plaques when compared with the parent Oshima 5-10 strain. Using site-directed mutagenesis, the substitutions were reverted to restore the parent virus sequence (O-IC-pt). Although genetically identical, parent virus Oshima 5-10 and virus recovered from O-IC-pt demonstrated some biological differences that are possibly explained by the presence of quasispecies with differing virulence characteristics within the original virus population. These observations may have implications for vaccines based on modified infectious clones. It was also demonstrated that the amino acid substitution E-S40→P at position 40 in the envelope (E) glycoprotein was responsible for plaque size reduction, reduced infectious virus yields in cell culture and reduced mouse neurovirulence. Additionally, two amino acid substitutions in the non-structural (NS)5 protein (virus RNA-dependent RNA polymerase) NS5-V378→A and NS5-R674→K also contributed to attenuation of virulence in mice, but did not demonstrate a noticeable biological effect in baby hamster kidney cell culture. Comparative neurovirulence tests revealed how the accumulation of individual mutations (E-S40→P, NS5-V378→A and NS5-R674→K) can result in the attenuation of a virus.

Construction of a replicon and an infectious cDNA clone of the Sofjin strain of the Far-Eastern subtype of tick-borne encephalitis virus

2011 ◽  
Vol 156 (11) ◽  
pp. 1931-1941 ◽  
Author(s):  
Ayako Takano ◽  
Kentaro Yoshii ◽  
Yuki Omori-Urabe ◽  
Kana Yokozawa ◽  
Hiroaki Kariwa ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Encephalitis Virus ◽  
Infectious Cdna Clone ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Far Eastern

scholarly journals Characterization of a Siberian Virus Isolated from a Patient with Progressive Chronic Tick-Borne Encephalitis

2003 ◽  
Vol 77 (1) ◽  
pp. 25-36 ◽  
Author(s):  
T. S. Gritsun ◽  
T. V. Frolova ◽  
A. I. Zhankov ◽  
M. Armesto ◽  
S. L. Turner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Encephalitis Virus ◽  
Plaque Morphology ◽  
Amino Acid Substitutions ◽  
Ns1 Protein ◽  
Nonstructural Proteins ◽  
Dimerization Domain ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

ABSTRACT A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T277→V and E279→G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

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

2008 ◽  
Vol 82 (17) ◽  
pp. 8272-8282 ◽  
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.

scholarly journals Spontaneous Mutations Restore the Viability of Tick-Borne Encephalitis Virus Mutants with Large Deletions in Protein C

2003 ◽  
Vol 77 (1) ◽  
pp. 443-451 ◽  
Author(s):  
Regina M. Kofler ◽  
Agnes Leitner ◽  
Gabriel O'Riordain ◽  
Franz X. Heinz ◽  
Christian W. Mandl
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Deletion Mutant ◽  
Hydrophobic Interactions ◽  
Encephalitis Virus ◽  
Sequence Element ◽  
Hydrophobic Domain ◽  
Large Deletions ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

ABSTRACT The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these “double mutants” provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.

scholarly journals 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

2019 ◽  
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.

scholarly journals 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

2019 ◽  
Vol 93 (16) ◽  
Author(s):  
Ludek Eyer ◽  
Antoine Nougairède ◽  
Marie Uhlířová ◽  
Jean-Sélim Driouich ◽  
Darina Zouharová ◽  
...  
Keyword(s):  
Cell Culture ◽  
Yellow Fever Virus ◽  
Rna Virus ◽  
Encephalitis Virus ◽  
Viral Fitness ◽  
Virus Infections ◽  
Rna Dependent Rna Polymerase ◽  
Virus Inhibitor ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

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.

Sign in / Sign up

Export Citation Format

Share Document