Doctoral Thesis
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- Abteilung für Mikrobiologie und Molekularbiologie (2) (remove)
Bats are ancient mammals that evolved more than 50 million years ago. There are 1,240 different species (> 20% of mammalian species) described so far, which represent one of the most abundant, diverse and widely distributed mammalian groups. Bats are the only mammals which actively fly and therefore can migrate to different areas of the world. It has been increasingly recognized that bats are reservoirs for more than 100 virus species, and several are associated with animal and human epidemics. As natural hosts of rabies virus (RABV) and related lyssaviruses, bats have become a focus of research not only in South America and Africa, but also in Europe and North America. Bats are also considered to be unique in their potential to host emerging and re-emerging zoonotic viruses. To evaluate and reduce the potential risk of rabies transmission to humans or carnivore hosts (like fox, raccoon and dog etc.), active and passive surveillance studies of bat have been performed. Using these approaches diverse lyssaviruses have been detected in bats. However, these studies did not explain the rarely discovered epidemics and the underlying resistance or immune mechanisms in bats as natural hosts for lyssaviruses. Probably, bats are more resistant to lyssavirus infections than other animals. This hypothesis is introducing the research questions of the present thesis: (1) How do the innate immune responses protect bats from fatal outcome of lyssavirus infections? Interferon (IFN) responses which can be induced by the recognition of viruses by pattern recognition receptors act as the first line of defense against lyssavirus infections. Therefore, type I and type III IFNs from European bats were cloned and functionally characterized in this thesis (Chapter 2 and 4). (2) How do the lyssaviruses adapt to escape the host defenses by counteracting the IFN-mediated immune responses? And how do the bats control the viral replication via the IFN responses? To explore the complicated interactions and understand how European bats (Eptesicus serotinus, Myotis myotis and Nyctalus noctula) interact with European bat lyssaviruses (EBLV-1 and 2), a natural host related model for investigations of the bat´s immune system and the virus-host interactions has to be established. Since all of 52 identified European bats species are endangered and strictly protected, stable cell lines from different tissues of M. myotis for in vitro studies were developed and used for molecular and functional studies (Chapter 3 and 4). The data obtained from this thesis revealed that: (a) European bat IFNs do have similar but also distinct molecular characteristics compared with other mammalian IFNs (Chapter 2 and 4); (b) Both investigated bat type I IFNs, IFN-Kappa; and IFN-Omega; present potent anti-lyssaviral activities and display a pathogen associated pattern in the tested cell line (Chapter 2); (c) The established immortalized M. myotis cell lines are differently susceptible to lyssaviruses and contain a functional IFN-mediated signaling cascade (Chapter 3); (d) Bat type III IFN-Lambda;s display cell-type specific functions due to the distinct expression of the IFN-Lambda; receptor (Chapter 4); (e) In bat cell lines a possible evasive strategy of lyssavirus is based on the counteraction of IFN production and/or IFN-mediated defensive pathways (Chapter 3); (f) The higher resistance of brain derived cell line MmBr compared to other cell lines to lyssavirus infection indicates the natural ability of bat´s central nervous system to control the growth of neurotropic virus, which might be an essential reason for the nonclinical outcome (Chapter 3). Overall, the present thesis provides first insights into IFN-mediated innate immune responses against RABV and EBLVs infection in their natural reservoir hosts and an useful toolbox for comparative analysis of virus-host interactions.
In this study the potential of molecular RT-PCR based methods for diagnostic or epidemiological investigations concerning negative-sense RNA viruses should be demonstrated exemplary for orthobunyaviruses (segmented genome) and lyssaviruses (non segmented genome). The recent discovery of a novel orthobunyavirus from the Simbu serogroup, Schmallenberg virus (SBV), via next generation sequencing and metagenome analysis led to the development of novel molecular detection methods. Due to the potential emergence of further orthobunyaviruses from the Simbu serogroup, a generic pan-Simbu real-time RT-PCR system was developed. This system was able to detect all tested Simbu serogroup viruses. As additional feature a species classification via sequencing is possible. Moreover, the novel pan-Simbu real-time RT-PCR system seems to offer a broad detection spectrum for orthobunyaviruses in general. Hence, this protocol allows a broad screening of samples predominantly for Simbu serogroup virus genomes but also might allow the identification of some related orthobunyaviruses in mammalian or insect samples. A comparison of the pan-Simbu real-time RT-PCR system with diagnostic real-time RT-PCRs revealed an overall higher sensitivity of the diagnostic assays for SBV detection. The diagnostic SBV-S3 assay convinced with the highest sensitivity and reliability for SBV detection. Additionally, the SBV-M1 assay turned out as highly specific for SBV and therefore is a valuable tool for a precise diagnosis in geographical regions where multiple orthobunyaviruses are endemic. Furthermore, the SBV genome diversity in Germany was investigated using a molecular epidemiological approach. Genome variability was extremely high in the N-terminal region of the putative envelope glycoprotein Gc which might have an impact on immunogenicity or host-cell infection. Phylogenetic analyses indicated that sequence variation is independent of host species and geographical distribution. In contrast to SBV as a novel pathogen, rabies encephalitis (caused by the prototype lyssavirus Rabies virus) is known for more than 4000 years. Thus numerous molecular techniques have been developed for lyssavirus detection, considering the diversity of this genus they all have certain limitations as regards their diagnostic range. Results of a lyssavirus ring trial among European laboratories indicate that RT-PCR could be a highly reliable diagnostic tool if at least two independent tests with broad diagnostic range are applied. Another approach suggested that a change from two-step to one-step PCR strategy or a variation of the RT-chemistry may have a remarkable influence on assay performance. However, no ultimate approach or strategy has been found yet, that would facilitate rabies routine diagnosis or epidemiological surveys on molecular grounds. Thus, there is a need for a potent, reliable and practical system for lyssavirus diagnosis and characterization, suitable as a second diagnostic line next to classical techniques like the fluorescent antibody test. For this purpose a diagnostic two level cascade protocol was developed with emphasis on the most relevant European lyssaviruses. On a first level two independent generic pan-lyssavirus screening assays, targeting different genomic regions, were applied. On a second level two probe-based species-specific multiplex PCR systems for the rapid classification of European lyssaviruses were used. All applied assays displayed an overall highly sensitive and specific detection with an excellent reproducibility and repeatability. Moreover, the diagnostic cascade protocol combines all known advantages of the real-time PCR technology including speed and reduced risk of cross-contamination with improved safety of molecular testing based on a double-check strategy for the screening as well as the confirmatory assays. In the frame of the second Bokeloh bat lyssavirus case in a German bat, the capability of real-time PCR for the quantification of viral loads was demonstrated. Another convenient example for the potential of molecular RT-PCR based methods is the epidemiological investigation of the rabies epizootic in Namibian kudu antelopes. Phylogenetic analyses of a 602 bp fragment of the nucleoprotein gene indicated a separate grouping of the Rabies virus (RABV) isolates from kudu apart from RABV isolates from jackals. Full genome sequencing revealed unique mutations in the glycoprotein gene of RABV isolates from kudu, suggesting an independent rabies cycle in Namibian kudu antelopes. All given examples were used to illustrate the application spectrum of molecular RT-PCR based methods for diagnostic or epidemiological purposes. The advantages of molecular techniques were emphasized and in particular real-time RT-PCR systems proved their fitness for purpose and appear to represent standard techniques for the next decade.