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Infectious diseases remain a significant threat to the wellbeing of humans and animals
worldwide. Thus, infectious disease outbreaks should be investigated to understand the
emergence of these pathogens, leading to prevention and mitigation strategies for future
outbreaks. High-throughput sequencing (HTS) and bioinformatic analysis tools are reshaping
the surveillance of viral infectious diseases through genome-based outbreak investigations. In
particular, analyzing generic HTS datasets using a metagenomic analysis pipeline enable
simultaneous identification, characterization, and discovery of pathogens.
In this thesis, generic HTS datasets derived from the 2018-19 WNV epidemic and USUV
epizooty in Germany were evaluated using a unified pipeline for outbreak investigation and an
early warning system (EWS). This pipeline obtained 34 West Nile virus (WNV) whole-genome
sequences and detected several sequences of Usutu virus (USUV) and other potential
pathogens. A few WNV and USUV genome sequences were completed using targeted HTS
approaches. Phylogenetic and phylogeographic inferences, reconstructed using WNV wholegenome sequences, revealed that Germany experienced at least six WNV introduction events.
The majority of WNV German variants clustered into the so-called “Eastern German clade
(EGC),” consisting of variants derived from birds, mosquitoes, a horse, and human cases. The
progenitors of the EGC subclade probably circulated within Eastern Europe around 2011. These
flavivirus genome sequences also provided substantial evidence for the first reported cases of
WNV and USUV co-infection in birds. Phylogenetic inferences of USUV genome sequences
showed the further spread of the USUV lineage Africa 3 and might indicate the overwintering
of the USUV lineage Europe 2 in Germany. Among viral sequences reported in the EWS, Hedwig
virus (HEDV; a novel peribunyavirus) and Umatilla virus (UMAV; detected in Europe for the
first time) were investigated using genome characterization, molecular-based screening, and
virus cultivation since these viruses were suspected of causing co-infections in WNV-infected
birds. The EWS detected overall 8 HEDV-positive and 15 UMAV-positive birds in small sets of
samples, and UMAV could be propagated in a mosquito cell culture Future studies are necessary
to investigate the pathogenicity of these viruses and their role in the health of wild and captive
birds.
In conclusion, this study provided a proof-of-concept that the developed unified and
generic pipeline is an effective tool for outbreak investigation and pathogen discovery using the
same generic HTS datasets derived from outbreak and surveillance samples. Therefore, this
thesis recommends incorporating the unified pipeline in the key response to viral outbreaks to
enhance outbreak preparedness and response.
The Flavivirus genus (Flaviviridae family) comprises the most important arboviruses in the world such as dengue virus, West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus and yellow fever virus (YFV). Every year, several outbreaks caused by flaviviruses are reported worldwide (i.e.: ZIKV and YFV outbreaks in South America) with a huge impact on economy and public health. In the last few decades, many aspects of the flavivirus biology and the interaction of flaviviruses with host cells have been elucidated. However, many underlying mechanisms concerning receptor usage, entry process and viral interaction with host cell factors are still not completely understood. Integrins, the major class of cell adhesion molecules have been implicated in the infectious cycle of different viruses including flaviviruses. A previous report proposed that a particular integrin, the αVβ3 integrin, might act as a cellular receptor for WNV. However, this hypothesis was not confirmed by other groups. In the present study, murine cell lines lacking the expression of one or more integrin subunits were used to evaluate the involvement of different integrins in the flavivirus infection cycle. Mouse fibroblasts lacking the expression of β1 integrin (MKF-β1-/-) or β3 integrin (MEF-β3-/-) subunits or αVβ3 integrin (MEF-αVβ3-/-) as well as their corresponding wild-type cells were utilized. A second model using Chinese hamster ovary cells (CHO-K1), a cell line that has been described to be refractory to some flaviviruses, were modified to express either αV (CHO-αV+/+) or β3 (CHO-β3+/+) integrin subunits. All cell lines were first characterized by confocal laser microscopy, flow cytometry and functional assays prior to infection to assess their integrin expression. The cell lines were then inoculated with different flaviviruses of public health relevance: WNV, YFV-17D, Usutu virus (USUV), Langat virus (LGTV) and ZIKV. Infection assays were designed in order to evaluate whether integrins influence i) cell susceptibility; ii) binding; iii) internalization and iv) replication of the investigated flaviviruses. Our findings clearly demonstrate that β1, β3 and αVβ3 integrins do not act as flavivirus cellular receptor or attachment factor since their ablation does not completely abrogate flavivirus infection in the investigated cell lines. Flavivirus binding to the cell surface of MEFs, MKFs and CHO cells was not disturbed by the genomic deletion of the above-mentioned integrins. The deletion of β1 and β3 integrin subunit did not affect internalization of any of the flaviviruses tested. In contrast to that, loss of αVβ3 integrin in the MEF-αVβ3-/- cells showed a statistically significant decrease in WNV and USUV internalization while ZIKV, YFV-17D and LGTV internalization remained unaffected suggesting that αVβ3 integrin might be involved in the internalization process of at least some flaviviruses. On the other hand, flavivirus replication was substantially impaired in the integrin-deficient cell lines in comparison to their corresponding wild-type cells. Both, MEF-β3-/- and MKF-β1-/- cells showed a statistically significant reduction on viral load for all flaviviruses tested in comparison to their respective wild-type cells. The MEF-αVβ3-/- cells in particular, showed a strong inhibition of flavivirus replication with a reduction of up to 99% on viral loads for all flaviviruses tested. Levels of flavivirus negative-strand RNA were substantially decreased in MEF-αVβ3-/- cells indicating that integrins might influence flavivirus RNA replication. The ectopic expression of either αV or β3 integrin subunits in CHO cells slightly increased the replication of all flaviviruses tested. Taken together, this is the first report highlighting the involvement of integrins in ZIKV, USUV, LGTV and YFV infection. The results strongly indicate that the investigated integrins play an important role in flavivirus infection and might represent a novel host cell factor that enhances flavivirus replication. Although the exact mechanism of interaction between integrins and flaviviruses is currently unknown, the results provided in this study deepen our insight into flavivirus - host cell interactions and open doors for further investigations.