Mühlemann Kathrin

Molecular epidemiology of tick-borne encephalitis viruses in Switzerland

Project Number: CH-4825
Project Type: Dissertation
Project Duration: 05/01/2008 - 04/30/2010 project completed
Funding Source: other ,
Leading Institution: Universität Bern
Project Leader: Prof. Kathrin Mühlemann

related to this project.
for which the project has a relevance.


Research Areas:
Biodiversity

Disciplines:
biology and medicine

Keywords:
Tick-born encephalitis, endemic foci, Ixodes ricinus, reverse transcription-PCR, QIAsymphony SP system

Abstract:
Tick-borne encephalitis (TBE) is a zoonotic arbovirus infection of the central nervous system. The disease is caused by the tick-born encephalitis virus (TBEV), which is endemic in an area ranging from western Europe to China and Japan. Within endemic foci, the virus is maintained in cycles involving ixodid ticks and wild vertebrate animals. During the last 30 years, the incidence of TBE has increased significantly. In Switzerland, about 110 to 120 annual human disease cases are presently reported. However, only very limited information on the endemicity of TBEV has been available so far. This thesis focused on the molecular epidemiology of TBEV in our country. Using a self-developed PCR-based test we performed a national tick surveillance study, the results of which significantly contribute to an improved risk assessment of TBE in Switzerland. Furthermore, the circulating viruses were characterized with respect to their basic biological and genetic properties.
For the purpose of large-scale surveillance studies, we developed a protocol involving an automated, high-throughput nucleic acid (NA) extraction method (QIAsymphony SP system) and a one-step duplex real-time reverse transcription-PCR (RT-PCR) for the detection of TBEV in ticks, including an internal process control. Establishment of the automated NA extraction procedure included optimization of a standard protocol and its evaluation against a modified guanidinium thiocyanate-phenol-chloroform extraction protocol. High usability, reproducibility, and equivalent performance for virus concentrations down to 5 x 103 viral genome equivalents/µl favor the automated protocol compared to the precipitation procedure. The validated real-time RT-PCR allows fast, sensitive (limit of detection: 10 RNA copies/µl), and specific (no false-positive test results for other TBEV subtypes, other flaviviruses, or other tick-transmitted pathogens) detection of European subtype TBEV. The established analytical system permits an extensive characterization of the true virus prevalence in natural TBE reservoirs and should be implemented in national surveillance systems in any European country where TBE is endemic.
We applied the new molecular test procedure in a national tick surveillance study, in which 62,343 ticks were collected at 165 collection sites throughout Switzerland by flagging low vegetation. Thirty-eight endemic foci could be identified, with a mean virus prevalence of 0.46%. The foci do not fully agree with those defined by geographic mapping of clinical cases. Therefore, our data are a unique complement of human disease case mapping and significantly improve risk assessment of TBE in Switzerland.
Sixty-four of 72 TBE viruses detected by real-time RT-PCR could successfully be recovered using porcine kidney stable or Ixodes ricinus tick cell culture systems. These isolates were characterized in vitro with respect to their plaque phenotype, since this has been associated with virulence. Two thirds (67%) of isolates produced a mixture of plaques of different sizes, reflecting a heterogeneous population of virus variants. Isolates consistently forming plaques of small size, indicating low virulence, were associated with recently detected endemic foci with no or only sporadic reports of clinical cases. All of six virus isolates investigated in an in vivo mouse model were highly neurovirulent (100% mortality) but exhibited a relatively low level of neuroinvasiveness, with mouse survival rates ranging from 50 to 100%. Thus, both in vitro and in vivo surrogates of virulence suggest a high proportion of isolates with relatively low neuropathogenic potential, which is in agreement with a hypothesized high proportion of subclinical or mild TBE cases.
The envelope (E) gene sequences and phylogenetic classification of all 72 TBE viruses were analysed. Although the E protein is an important determinant of TBEV virulence, we could not clearly correlate any individual substitutions in this protein with attenuation of virus virulence, suggesting the presence of additional determinants of virulence in other genome regions. Phylogenetically, all isolates belonged to the European subtype and were highly related (mean pairwise sequence identity of 97.8% at the nucleotide and 99.6% at the amino acid level of the E protein). Isolates from our cantons formed clearly separated lineages, indicating a high degree of isolation of the respective foci.
Future research focusing on whole genome sequence analyses will permit more profound phylogenetic classification of Swiss TBEV. Besides, these studies will allow for the identification of additional mutations possibly affecting the pathogenic potential of TBE viruses. The biological significance of these substitutions will require confirmation by cDNA clone studies. Finally, characterization of viruses isolated from human TBE patients will provide important information on the clinical relevance of the different viruses circulating among the tick population.


Leading questions:
The aims of the project are:
  • to establish and validate a high-througput sample preparation and real-time RT-PCR method for the identification of TBEV in tick samples

  • to assess the virus prevalence in Ixodes ricinus ticks collected in endemic and non-endemic areas throughout Switzerland

  • to recover TBEV field isolates from tick samples using a mammalian cell culture system

  • to determine the genetic properties and phylogenetic classification of Swiss TBEV by analyses of the viral envelope gene sequence

  • to characterize the isolates with respect to their virulence using an in vitro mammalian cell culture system and an in vivo mouse model of TBE

    Publications:
  • Gäumann, R. 2010. Molecular epidemiology of tick-borne encephalitis viruses in Switzerland. Ph.D. Thesis, Institute for Infectious Diseases, Medical Faculty of the University of Bern, Switzerland.

  • Gäumann R., Mühlemann K., Strasser M., Beuret, CM., 2010. High-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in Switzerland. Appl. Environ. Microbiol. 2010, 76(13):4241

  • Gäumann R., R??ek D., Mühlemann K., Strasser M., Beuret C.M., 2011. Phylogenetic and virulence analysis of tick-borne encephalitis virus field isolates from Switzerland. Journal of Medical Virology 83:853–863 (2011)



    Last update: 1/19/17
    Source of data: ProClim- Research InfoSystem (1993-2024)
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