Molecular bases of tick-borne flavivirus transmission

Lead Research Organisation: University of Reading
Department Name: Sch of Biological Sciences

Abstract

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.

Technical Summary

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.
 
Description International Joint Projects scheme 2010, Royal Society
Amount £10,087 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 01/2010 
End 01/2013
 
Title Final GM plasmids and viruses 
Description Totally 115 new recombinant plasmids have been created. Totally 14 new GM viruses have been engineered Totally another 50 recombinant viruses are planned to be recovered from available plasmids. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact New GM plasmids will be used to construct other GM viruses to study the pathogenic characteristics of tick-borne encephalitis virus New GM viruses will comprise the basis for a new project designed to investigate the role of the E protein and capsid (C) protein in tick-borne encephalitis virus transmission in ticks. 
 
Title GM plasmids and viruses 
Description New bacterially-based plasmids have been created. Using these plasmids, new genetically modified viruses have been engineered. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact New viruses will be used for studies on the molecular basis of tick-borne encephalitis virus transmission and pathogenesis. Plasmids will be used for further mutagenesis studies of the virus genome 
 
Title GM plasmids and viruses 
Description New recombinant plasmids and GM viruses that contain fragments of tick-borne encephalitis virus genome have been constructed. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact New GM viruses will comprise the basis for a new project designed to investigate the role of the envelope (E) protein on the efficiency of tick-borne encephalitis virus transmission in ticks. 
 
Title GM plasmids and viruses 
Description New recombinant plasmids that contain fragments of tick-borne encephalitis virus genome have been constructed. These plasmids will be used to recover new mutant viruses. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact The impact of these research tools will be evaluated during the further progression of the project 
 
Title New plasmids and GM viruses 
Description Experiments are now in progress to evaluate the effect of defined mutations that change virus characteristics 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Experiments now are in progress to evaluate the effect of mutations that change virus characteristics. 
 
Description Institute of Microbiology, Irkutsk, Russia 
Organisation Russian Academy of Sciences
Department Institute of Epidemiology and Microbiology, Irkutsk, Russia
Country Russian Federation 
Sector Public 
PI Contribution We provided Dr. Maxim Khasnatinov with engineered chimaeric viruses that he is using to study tick-borne encephalitis virus transmission in ticks.
Collaborator Contribution Dr. M Khasnatinov has initiated the experiments on virus transmission in Ixodes persulcatus ticks. He also completed all the experiments that provide characteristics of engineered chimaeric viruses in cell culture. He is expected to be a 1st author of the paper we are preparing to publish.
Impact The papers were published in collaboration with Dr. Khasnatinov 1. 2010 Gould, EA, Coutard, B, Malet, H, Morin, B, Jamal, S, Weaver, S, Gorbalenya, A, Moureau, G, Baronti, C, Delogu, I, Forrester, N, Khasnatinov, M, Gritsun, T, de Lamballerie, X and Canard, B. Understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control. Antiviral Res, 87, 111-24. 2. 2009 Khasnatinov, MA, Ustanikova, K, Frolova, TV, Pogodina, VV, Bochkova, NG, Levina, LS, Slovak, M, Kazimirova, M, Labuda, M, Klempa, B, Eleckova, E, Gould, EA and Gritsun, TS. Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks. PLoS One, 4, e7295.
Start Year 2007
 
Description Institute of Virology, Bratisalva 
Organisation Slovak Academy of Sciences
Department Institute of Virology
Country Slovakia 
Sector Academic/University 
PI Contribution I provided the collaborators with research data that became the subject for collaborative studies
Collaborator Contribution Dr. Boris Klempa supervises the experimental work in Bratislava, Slovakia
Impact Collaborative publications 1. 2014 Slovak, M, Kazimirova, M, Siebenstichova, M, Ustanikova, K, Klempa, B, Gritsun, T, Gould, EA and Nuttall, PA. Survival dynamics of tick-borne encephalitis virus in Ixodes ricinus ticks. Ticks and tick-borne diseases. 10.1016/j.ttbdis.2014.07.019 2. 2009 Khasnatinov, MA, Ustanikova, K, Frolova, TV, Pogodina, VV, Bochkova, NG, Levina, LS, Slovak, M, Kazimirova, M, Labuda, M, Klempa, B, Eleckova, E, Gould, EA and Gritsun, TS. Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks. PLoS One, 4, e7295.
Start Year 2006
 
Description University of Marseille, France 
Organisation Aix-Marseille University
Country France 
Sector Academic/University 
PI Contribution I provided the collaborators with ideas and research data that became the subject for collaborative studies
Collaborator Contribution Professor Gould provides the research with research funds and contributes to overall development of projects
Impact The collaborative papers (totals over 30) have been published, with the latest in the scope of the MRC-funded project. 1. 2014 Slovak, M, Kazimirova, M, Siebenstichova, M, Ustanikova, K, Klempa, B, Gritsun, T, Gould, EA and Nuttall, PA. Survival dynamics of tick-borne encephalitis virus in Ixodes ricinus ticks. Ticks and tick-borne diseases. 10.1016/j.ttbdis.2014.07.019 2. 2014 Gritsun, DJ, Jones, IM, Gould, EA and Gritsun, TS. Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution. PLoS One, 9, e92056. 3. 2011 Tuplin, A, Evans, DJ, Buckley, A, Jones, IM, Gould, EA and Gritsun, TS. Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus. Nucleic Acids Res, 39, 7034-48. 4. 2010 Gould, EA, Coutard, B, Malet, H, Morin, B, Jamal, S, Weaver, S, Gorbalenya, A, Moureau, G, Baronti, C, Delogu, I, Forrester, N, Khasnatinov, M, Gritsun, T, de Lamballerie, X and Canard, B. Understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control. Antiviral Res, 87, 111-24. Some publications are still in preparation (to be added following publication)
Start Year 2006
 
Description University of Warwick 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided the collaborators with research data that became the subject for collaborative studies
Collaborator Contribution Professor Evans and Dr. Tuplin introduced new statistical methods to analyse the secondary RNA structures of Tick-borne encephalitis virus. This will extend our knowledge of RNA structures that might have an impact on virus transmission in ticks.
Impact The paper has been published in a high impact journal 2011 Tuplin, A, Evans, DJ, Buckley, A, Jones, IM, Gould, EA and Gritsun, TS. Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus. Nucleic Acids Res, 39, 7034-48.
Start Year 2008
 
Title Chimaeric viruses on the basis of tick-borne encephalitis virus 
Description New recombinant viruses, chimaeras between virulent and attenuated strains of tick-borne encephalitis virus (TBEV) have been produced. The comparison of their biological properties will enable to identify virus genes responsible for the different virus pathogenic properties in humans. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2011
Development Status Under active development/distribution
Impact New information has been obtained about the ant-apoptotic mechanisms of tick-borne encephalitis virus 
 
Title Viruses with point mutations in the capsid gene of tick-borne encephalitis virus 
Description Viruses with point mutations in the capsid gene of tick-borne encephalitis virus (TBEV) has been produced and analysed. The TBEV replication enhancer element has been identified using these viruses as a tool for the research. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Closed
Impact A new fundamental knowledge has been produced that proves the proposed concept about functional relationships between promoter and enhancer elements of flaviviruses