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Coronavirus induced changes in mouse pathophysiology
Patrizia Gibler
Kathrin Kober-Rychli
Alice Assinger
Philipp Hohensinner
Master thesis - University of Veterinary Medicine Vienna - 2022
The main research topic of this master thesis is the analysis of host-pathogen interactions during a coronavirus infection. C57BL/6 mice were intranasally infected with the murine coronavirus (MCoV), which belongs to the same subgroup of betacoronaviruses as the human severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and is therefore a great model to translate findings in our mouse model to the human situation. Moreover, the model was sensitive enough that even a low dose virus infection resulted in a rapid influx of T-cells as well as macrophages in the lung, even though virus burden was not any more detectably. The hypothesis was, that the interaction between the murine coronavirus and its host results in changes in the body temperature, microbiome and a prolonged prothrombotic and inflammatory response. We observed differences in virus burden and presence of neutrophil extracellular trap (NET) derived thrombi between the lung and the liver. Lung virus burden was highest at day 2 with thrombi at day 10, whereas in the liver the virus burden was highest at day 4 with high thrombus numbers at day 2 and 10. qPCR analysis of inflammation markers of liver mRNA, like tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), or intracellular adhesion molecule 1 (ICAM-1) revealed that the highest inflammatory response could be seen at day 4. Moreover, collagen deposits in the lung were highest at day 4, whereas liver collagen was highest at day 10. As NET formation is paralleled by coagulation, also the coagulation factors and fibrinolytic players were investigated. Plasminogen activator inhibitor 1 (PAI-1) and urokinase plasminogen activator (uPA) mRNA were upregulated on day 4, which coincided with high plasma protein levels of TNFα and IFNγ. The induction of uPA and PAI-1 by these cytokines was confirmed in an ex vivo liver stimulation model. In line with the in vivo findings, PAI-1 and uPA mRNA were again increased in comparison to untreated organ culture, indicating that their expression was directly regulated by hepatic inflammation. Another aim was to identify if the outer body temperature, measured with an infrared camera, is a better indicator than body weight regarding the wellbeing. It could be shown that body weight was more suited as an early marker for wellbeing than the outer body temperature. However, a clear point of no return could be set with the infrared camera, which may support the decision if the animal should be euthanized or not. The last part of the thesis focused on alterations in the Altered Schaedler Flora (ASF) microbiota, which includes eight specific bacteria represented in the gut microbiome. Significant differences between control and MCoV infected mice as well as between mice from the Anna Spiegel Animal facility, which had SPF conditions, and the Quarantine, where mice were infected with Murine Norovirus, Pasteurella sp., Helicobacter sp. and protozoa were detected. In conclusion, we were able to demonstrate differences between the lung and the liver during an infection with MHV, observing the NET formation and inflammatory response. Furthermore our in vivo results were in line with ex vivo organ culture and we observed changes of the ASF during a virus infection.
Masterarbeit - Veterinärmedizinische Universität Wien - 2022
Diese Arbeit umfasst Ergebnisse von C57Bl/6 Mäuse welche nasal mit einem Mäusecoronavirus, welcher ein gutes Modell für eine Infektion mit SARS-CoV-2 darstellt, infiziert worden sind. Der Mäusecoronavirus ist ein Betacoronavirus und mit SARS-CoV-1 und 2 verwandt. Die Ergebnisse dieser Arbeit zeigen organspezifische Unterschiede in der Viruslast, da man die höchsten Werte am Tag 2 in der Lunge und an Tag 4 in der Leber sehen konnte. Die NET Bildung in der Lunge war am Tag 4 am höchsten, wobei in der Leber Thromben am Tag 2 und 10 gefunden werden konnten. Am Tag 4 war die Anzahl der Thromben in der Leber signifikant geringer. Diese Beobachtung führte zur Untersuchung von Interaktionspartnern in dem Gerinnungssystem, bei der hohe Werte von PAI-1 und uPA am Tag 4 beobachtet wurden. Weiters wurde der Entzündungsprozess in der Leber untersucht und eine Organkultur etabliert, welche die Resultate der in vivo Experimente ex vivo bestätigte. Die äußere Körpertemperatur wurde mit einer Infrarotkamera gemessen und mit dem Gewichtsverlust im Laufe einer Infektion verglichen. Das Mikrobiom, vor allem die ASF, von MCoV infizierten Mäusen wurde mit Kontrollmäusen verglichen, genauso wie das ASF von Mäusen aus der Quarantäne mit dem von Mäusen aus der SPF Haltung verglichen wurden. Die Ergebnisse zeigten, dass mithilfe einer qPCR und einem definierten sauberen Mikrobiom, Statusänderungen der ASF auf eine Infektion in Mäusen hinweisen können.
Englisch
2022
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