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Rabies virus (RABV) is an ancient, highly neurotropic rhabdovirus that causes lethal encephalitis. Most RABV pathogenesis determinants have been identified with laboratory-adapted or attenuated RABVs, but details of natural RABV pathogenesis and attenuation mechanisms are still poorly understood. To provide a deeper insight in the cellular mechanism of pathogenies of field RABV, this work was performed to assess virus strain specific differences in intra-neuronal virus transport, to identify cell culture adaptive mutations in recombinant field viruses and to explore shRNA-expressing RABVs as research tools for targeted host manipulation in infected cells.
Comparison of chimeric RABVs with glycoprotein (G) ecto-domains of different lyssaviruses, together with field RABVs from dog and fox in dorsal root ganglion (DRG) neurons revealed no detectable differences in the axonal accumulation of the viruses. This indicates that previously described G-dependent transport of newly formed RABV in axons can occur both in laboratory-adapted and field RABV. Moreover, partial overlap of nucleoprotein (N) and G protein particles in field virus infected DRG axons supported the hypothesis of the “separate model” for anterograde RABV transport.
Serial passages of recombinant dog and fox field clones in different cell lines led to the identification of general (D266N) and cell line specific (K444N) adaptive mutations in the G ecto-domain of both viruses. In BHK cells, synergistic effects of D226N, K444N and A417T on field dog virus G protein surface localization led to the loss of endoplasmic reticulum (ER) retention of G and increased virus titers in the supernatant, indicating that limited virus release by ER retention is a major bottleneck in cell culture adaptation. In addition, selection of mutations within the C-terminus of the RABV phosphoprotein (P) (R293H and R293C in fox and dog viruses, respectively) led to the hypothesis of altered binding affinities to nucleoprotein and RNP complexes. Identification of the above mentioned amino acid substitutions together with alterations in a suboptimal transcription stop signal in the P/M gene border indicated that adaptation to cell culture replication occurs on both levels, RNA transcription/replication and virus release.
To evaluate the possibility of an expression of a functional microRNA-adapted short-hairpin RNAs (miR-shRNA) expressing RABV, recombinant RABVs encoding miR-shRNAs against cellular Dynein Light Chain 1 (DYNLL1) and Acidic Nuclear Phosphoprotein 32 family member B (ANP32B) were generated. In spite of cytoplasmic transcription of the respective mRNAs, downregulation of DYNLL1 and ANP32B mRNA and respective protein levels in infected cells revealed correct processing to functional shRNAs. Specific downregulation of the cellular genes at 2, 3 and 4 days post infection further demonstrated feasibility of the approach in standard cell lines. However, it remained open whether miR-shRNA expressing RABV can be used to study neuro-infection in vivo. Since first attempts in primary rat neuron cultures failed, it has to be clarified in further experiments whether this strategy can be used in mature, non-dividing neurons or whether breakdown of the nucleus in the course of cell division is a requirement for the processing of cytoplasmically expressed miR-RNA by nuclear RNases.
By providing novel insights in axonal RABV transport and cell culture adaptive mutations this work extends the current understanding of RABV pathogenesis in natural and non-natural cell environments. Moreover, it provides a basis for further pathogenicity studies in which the impact of cell culture adaptation through increased virus release on RABV virulence can be investigated. With successful expression of functional miR-shRNAs from RABV vectors, this work also provides a tool for RABV gene targeting in infected cell lines and thus may contribute to the further investigation of RABV-host-cell-interactions.
Objektive Eingruppierung sequenzierter Tollwutisolate mithilfe des Affinity Propagation Clusterings.
(2018)
Das International Committee on Taxonomy of Viruses (ICTV) reguliert die Nomenklatur von Viren sowie die Entstehung neuer Taxa (dazu gehören: Ordnung, Familie, Unterfamilie, Gattung und Art/Spezies). Dank dieser Anstrengungen ist die Einteilung für verschiedenste Viren in diese Kategorien klar und transparent nachvollziehbar. In den vergangenen Jahrzehnten sind insgesamt mehr als 21.000 Datensätze der Spezies „rabies lyssavirus“ (RABV) sequenziert worden. Eine weiterführende Unterteilung der sequenzierten Virusisolate dieser Spezies ist bislang jedoch nicht einheitlich vorgeschlagen. Die große Anzahl an sequenzierten Isolaten führte auf Basis von phylogenetischen Bäumen zu uneindeutigen Ergebnissen bei der Einteilung in Cluster. Inhalt meiner Dissertation ist daher ein Vorschlag, diese Problematik mit der Anwendung einer partitionierenden Clusteringmethode zu lösen. Dazu habe ich erstmals die Methodik des affinity propagation clustering (AP) für solche Fragestellungen eingesetzt. Als Datensatz wurden alle verfügbaren sequenzierten Vollgenomisolate der Spezies RABV analysiert. Die Analysen des Datensatzes ergaben vier Hauptcluster, die sich geographisch separieren ließen und entsprechend als „Arctic“, „Cosmopolitain“, „Asian“ und „New World“ bezeichnet wurden. Weiterführende Analysen erlaubten auch eine weitere Aufteilung dieser Hauptcluster in 12-13 Untercluster. Zusätzlich konnte ich einen Workflow generieren, der die Möglichkeit bietet, die mittels AP definierten Cluster mit den Ergebnissen der phylogenetischen Auswertungen zu kombinieren. Somit lassen sich sowohl Verwandtschaftsverhältnisse erkennen als auch eine objektive Clustereinteilung vornehmen. Dies könnte auch ein möglicher Analyseweg für weitere Virusspezies oder andere vergleichende Sequenzanalysen sein.