Molecular bases of tick-borne flavivirus transmission

The tick-borne encephalitis virus (TBEV) causes annual human infections of tick-borne encephalitis (TBE) at the epidemic scale in forested regions of Europe and Asia. The disease symptoms in people are significantly varied and include asymptomatic infections, severe although recoverable fevers, meningoencephalitis, debilitating or fatal encephalitis and slow developing (for years) progressive encephalitis. The reasons for that are not known although some correlation between the virulent properties of individual strains of TBEV that circulate in different regions and severity of the disease has been observed. Human infections in Far Eastern Asia are the most severe with frequent involvement of the CNS and 20-40% of fatality. TBE in Siberia is usually associated with a less severe disease (7-8% of fatality) but chronic disease is observed more frequently (1-3%). Infections linked to West European strains of the TBEV cause a fatality rate less than 2%. Although the reasons for these differences are not clear, our preliminary research suggests that accidental escape of TBEV into a new tick species might demand the virus to change certain parts of their genome, to speed up virus replication in new tick species and thereby facilitate the virus spread in nature. However these genetic changes, working tothe virus s advantage, might increase the virulent properties of the TBEV and therefore be disadvantageous for humans. The spread of genetically-renewed more virulent TBEV might indeed be the reason for the human epidemics. In this project we suggest to investigate which parts of the TBEV genome are responsible for the adaptation of two different TBEV strains, one called Hypr, to tick I. ricinus and the other, Vs to I. persulcatus. These viruses cannot replicate in ?alien? tick species and they are also different in their pathogenic properties in humans; the Vs was isolated from the patient that recovered from the fever whereas Hypr was associated with fatal case of the TBE. We will use a state of art new molecular technologies such as genetic engineering and DNA microarray analysis to establish whether or not there is a correlation between the parts of the virus genome that determine tick-transmission pattern and those that determine virulent properties of the TBEV. This research contributes to our understanding of the mechanisms of emerging of new pathogens in the environment and will facilitate the design the appropriate strategy to intervene with infections.

Human epidemics emerge in regions where arboviruses circulate harmlessly between vertebrates and invertebrates. The reasons for the human outbreaks of virus infections are unknown. On the basis of the preliminary studies of tick-borne encephalitis virus (TBEV) we hypothesized that the virulent strains might emerge following the TBEV adaptation to new tick hosts. The TBEV is recognised pathogen with ~14 000 human cases annually. Its circulation in nature is maintained by the transmission from uninfected to infected ticks that co-feed on the same rodent. Two TBEV strains Vs and Hypr although are closely related, in nature transmitted by different tick spp. (I. persulcatus and I. ricinus respectively). Using the infectious clones for these viruses we have engineered a range of Vs/Hypr chimaeras, with substituted individual genes. In tick-transmission experiments we showed that Vs and Hypr are incapable of crossing the tick-spp barrier but Vs/Hypr chimaeras revealed different level of transmission, in 70% dependent on the envelope glycoprotein E and in 30% on the other genome regions. On the other hand, our studies of RNA secondary structures in untranslated regions (UTRs) of flavivirus genomes predicted their significant role in virus adaptation to transmission between vertebrates and invertebrates.
In addition to their differences in transmission, the Vs and Hypr show different pathogenic properties; Vs is less virulent in humans (isolated from the recovered patient), cell culture and hamsters, in comparison with Hypr (isolated from fatal case of TBE). The preliminary results with Vs/Hypr chimaeras indicated that polymerase protein NS5 might be responsible for virulent properties of the Hypr, although the precise amino acid involved needs to be determined.
In this project, we propose to investigate the precise molecular mechanisms that determine the ability of TBEV to be transmitted between co-feeding ticks, with emphasis on the genetic modifications within the E and NS5 proteins and UTRs. Secondly using Vs/Hypr chimaeric viruses we also intend to identify amino acids that are responsible for different pathogenic characteristics of Vs and Hypr. The microarray assay will be employed to compare cellular genes that are up- and down-regulated by Vs and Hypr and their chimaeras. All together, these data will establish whether or not there is a correlation between genomic domains (RNA or/and protein) responsible for the TBEV tick-transmission and pathogenicity and estimate the role of ticks in the emergence of virus strains with new pathogenic characteristics in nature, a hypothesis that has never been tested experimentally.