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 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.

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 The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell

1994 ◽  
Vol 75 (12) ◽  
pp. 3453-3460 ◽  
Author(s):  
A. J. Crooks ◽  
J. M. Lee ◽  
L. M. Easterbrook ◽  
A. V. Timofeev ◽  
J. R. Stephenson
Keyword(s):  
Host Cell ◽  
Encephalitis Virus ◽  
Ns1 Protein ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

scholarly journals Functional Analysis of Potential Carboxy-Terminal Cleavage Sites of Tick-Borne Encephalitis Virus Capsid Protein

2007 ◽  
Vol 82 (5) ◽  
pp. 2218-2229 ◽  
Author(s):  
Sabrina Schrauf ◽  
Petra Schlick ◽  
Tim Skern ◽  
Christian W. Mandl
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Protein C ◽  
Terminal Region ◽  
Encephalitis Virus ◽  
Viral Protease ◽  
Tick Borne Encephalitis ◽  
Carboxy Terminal Region ◽  
Tick Borne Encephalitis Virus ◽  
Carboxy Terminal

ABSTRACT The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Site-specific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

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