Page 103 - TBE_Book_V2_2019

Chapter 4: Pathogenesis of TBE

This DNA clone enables the introduction of importance of different regions in the 3’NCR.
mutations into the genome, and subsequent Neurovirulence in mice was shown to be
analysis of the resulting phenotype. Reverse dependent on specific amino acid residues in
genetics have been used to study virulence, the upper lateral surface of domain III in the
replication, host range, vaccines, and envelope (E) protein of TBEV (residues E308,
functions of the coding and non-coding E310 and E311), possibly due to disruption of
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regions. However, these clones are laborious the receptor binding. The residues S267L,
and difficult to generate due to instability and K315E, N389D in LGTV E protein and K46E in
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toxicity of some viral sequences in bacteria. the NS3 protein, were shown to be crucial for
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neuroinvasiveness in immunodeficient mice.
For TBEV 2 separate approaches were used in The 5’ and the 3’ NCR contain complementary
the beginning; plasmid-based infectious sequences that help genomic cyclization to
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clones and the PCR-based methods for form panhandle structures. The NCRs have
constructing recombinant virus. 93,94 Both rely several conserved structural stem loops that
on in vitro transcription and transfection of are important for replication, translation
RNA. The most recent technique for initiation and packaging. 99,100 At the beginning
generating TBEV clones is the infectious- of the flavivirus 3’ NCR, a secondary structure
subgenomic-amplicon (ISA) method. Three forms a pseudoknot that protects the terminal
PCR amplicons are produced that have a CMV 300- to 500-bases from exoribonuclease XRN1
promoter at the 5′ non-coding region (NCR) degradation, generating a subgenomic
and 70-100 bp overlapping regions; the flavivirus RNA (sfRNA). 101-103 The sfRNA has
hepatitis delta ribozyme is followed by the been shown to be critical for WNV induced
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simian virus 40 polyadenylation signal. The cytopathic effects and pathogenicity in
amplicons are mixed and introduced into the mice, and is involved in viral subversion of
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cells where they recombine and produce type I IFN response by a yet unknown
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infectious virus. mechanism. 105 The TBEV sfRNA has been
shown to specifically interfere with the RNAi
Infectious clone systems have been very system of ticks. The 3’ NCR of TBEV can be
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useful in studying determinants of replication
divided into a highly conserved core element
and biological characteristics as well as to
and a variable region that is both heterogenic
identify pathogenicity factors of TBEV. Two in length and sequence. 107 Several European
advantages of this approach are that the TBEV strains contain an internal poly(A) tract
genome is defined and can be manipulated. In in the variable region of the 3’ NCR, which was
contrast, natural viral isolates of positive-
considered dispensable for replication and
stranded RNA viruses are present as a virulence in mice. 108,109
population of different viral types also called
quasispecies. This is due to the error-prone However, studies recently showed that the
RNA-dependent RNA polymerase. In addition, variable region and the poly(A) tract can
manipulating natural viral isolates with modulate virulence of the Far Eastern
specific mutagenesis-inducing drugs is a very TBEV. 110,111 We have also detected different
nonspecific approach. lengths of the poly(A) tract in a blood-feeding
tick indicating that the poly(A) might be
With this technique, several determinates of important for the switch between inverte-
pathogenicity have been identified. Specifi- 112
brate to vertebrate. To investigate this
cally, the envelope protein responsible for further a long-poly(A) Torö-38A and a TBEV
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receptor-mediated entry, the function of the Torö with a short-poly(A) were cloned and
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membrane protein in virus budding, and the
rescued. We were able to show that the

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