Evaluation of antivirals against alphaviruses using the example of Chikungunya Virus

Alphaviruses are enveloped single-stranded RNA arboviruses of the Togaviridae family and are geographically widely distriguted [1, 2]. They cause various diseases in humans and animals such as encephalitis, arthritis fever, rash and arthralgia [1]. Among the medically relevant members of the alphaviruses are Venezuelan, Western, and Eastern Equine Encephalitis viruses (VEEV, WEEV, and EEEV), Ross River virus (RRV) and Chikungunya virus (CHIKV). The Equine Encephalitis viruses are categoriesed as potential agents for bioterrorism since they can all be transmitted via aerosols, causing severe disease [3-7]. Chikungunya virus (CHIKV) is categorised as a(n) (re)emerging disease and is mainly transmitted by Aedes spp. mosquitoes [8]. CHIKV is the causative agent of chikungunya fever (CHIKF) which is characterised by high fever, headache and myalgia and polyarthralgia [9]. Especially the polyarthralgia may last for months or even years and leave patients with a severely deteriorated quality of life. CHIKV has repeatedly been responsible for outbreaks that caused serious economic and public health problems in the affected countries [8]. To date, no vaccine or specific antiviral therapies are available. This thesis focusses on in vitro antiviral testing against a wild type CHIKV isolate and selecting possible hit to lead compounds. Climate change leads to the introduction of vectors in more temperate zones and thus it is possible that new diseases emerge with these vectors [10]. Consequently, the need for specific antivirals to treat such emerging diseases like CHIKV is current. Most of these in vitro antiviral assays are conducted in Vero cells, a cell line that originated from the kidney of an African green monkey [11]. Although this cell line is the model cell line to probagate CHIKV in, it lacks the clinical relevance of the disease and is not of human origin. Therefore, another goal of the thesis was to identify a human cell line with clinical relevance (especially for neurogenic CHIKV disease) to test antivirals in. This study was the first to describe the human glioblastoma cell line U138 in extensive antiviral tests against CHIKV. Furthermore, different assay methods were compared for their usefulness in antiviral tests against CHIKV in Vero-B4 and U138 cells

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

<p>Abstract</p> <p>Background</p> <p>The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SR<it>β </it>subunit is an integral membrane protein, which tethers the SRP-interacting SR<it>α </it>subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SR<it>β </it>subunit and consists of only the SR<it>α </it>homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of <it>Escherichia coli</it>. So far, the exact function of the soluble SR in <it>E. coli </it>is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.</p> <p>Results</p> <p>In the current study we have determined the contribution of soluble FtsY to co-translational targeting <it>in vitro </it>and have re-analysed the localization of FtsY <it>in vivo </it>by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs) in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in <it>E. coli</it>. In agreement with this observation, our <it>in vivo </it>analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species <it>in vivo</it>.</p> <p>Conclusion</p> <p>The exact function of the SRP receptor (SR) in bacteria has so far been enigmatic. Our data show that the bacterial SR is almost exclusively membrane-bound <it>in vivo</it>, indicating that the presence of a soluble SR is probably an artefact of cell fractionation. Thus, co-translational targeting in bacteria does not involve the formation of a soluble SR-signal recognition particle (SRP)-ribosome nascent chain (RNC) intermediate but requires membrane contact of FtsY for efficient SRP-RNC recruitment.</p

CHIKV strains Brazil (wt) and Ross (lab-adapted) differ with regard to cell host range and antiviral sensitivity and show CPE in human glioblastoma cell lines U138 and U251

Chikungunya virus (CHIKV), a (re)emerging arbovirus, is the causative agent of chikungunya fever. To date, no approved vaccine or specific antiviral therapy are available. CHIKV has repeatedly been responsible for serious economic and public health impacts in countries where CHIKV epidemics occurred. Antiviral tests in vitro are generally performed in Vero-B4 cells, a well characterised cell line derived from the kidney of an African green monkey. In this work we characterised a CHIKV patient isolate from Brazil (CHIKVBrazil) with regard to cell affinity, infectivity, propagation and cell damage and compared it with a high-passage lab strain (CHIKVRoss). Infecting various cell lines (Vero-B4, A549, Huh-7, DBTRG, U251, and U138) with both virus strains, we found distinct differences between the two viruses. CHIKVBrazil does not cause cytopathic effects (CPE) in the human hepatocarcinoma cell line Huh-7. Neither CHIKVBrazil nor CHIKVRoss caused CPE on A549 human lung epithelial cells. The human astrocyte derived glioblastoma cell lines U138 and U251 were found to be effective models for lytic infection with both virus strains and we discuss their predictive potential for neurogenic CHIKV disease. We also detected significant differences in antiviral efficacies regarding the two CHIKV strains. Generally, the antivirals ribavirin, hydroxychloroquine (HCQ) and T-1105 seem to work better against CHIKVBrazil in glioblastoma cells than in Vero-B4. Finally, full genome analyses of the CHIKV isolates were done in order to determine their lineage and possibly explain differences in tissue range and antiviral compound efficacies