|Abstract||Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) emerged in the end of 2019 in Wuhan, China and spread all over the world causing the coronavirus disease 2019 (COVID-19) pandemic. In my thesis, I established methods and protocols to investigate SARS-CoV-2 biology, infection and inhibition. Virus replication was quantified by SARS-CoV-2 genome or titer determination by RT-qPCR, plaque assay and tissue culture infectious dose 50 (TCID50) endpoint titration which also allowed studying cell-to-cell spread and antiviral mechanisms. In order to increase throughput but also specificity and sensitivity of screening assays for the identification and characterization of antiviral compounds, an MTS-based cell death, an enzyme-based immunodetection, and a flow cytometry-based immunodetection assay were developed. All can be performed in 96 well microtiter format and allow quantification of antiviral potency of drugs (50% inhibitory concentrations) after one to three days with good high-throughput performance. However, assay choice depends on cell types, work effort, time expenditure, specificity, sensitivity, equipment and costs.
Application of these assays contributed to the identification and characterization of several antivirals, including heparin and carrageenan-containing nasal and oral sprays. The antiviral activity of the sprays was confirmed in physiologically relevant human airway epithelial cells that resemble the entry site for SARS-CoV-2 and was attributed to ι- and κ-carrageenans. Heparin and carrageenan are both sulfated polysaccharides already in clinical use, which may allow fast therapeutic usage. They block viral attachment to cellular receptors preventing virus entry. Heparin has additional anti-inflammatory and anticoagulative properties, making it a unique therapy option for COVID-19, as it covers both the pathogen and the ensuing disease. Oral and nasal administration of the sprays may prevent infection or transmission between individuals. Thus, repurposing heparin and carrageenan-containing nasal and oral sprays may be advisable to treat COVID-19.
Studying the virucidal activity of common rapid antigen assay buffers revealed that most commercially available extraction buffers do not inactivate SARS-CoV-2. This may pose a risk of infection during performance of point-of-care tests. Therefore, in cooperation with an industrial partner, we developed a virus-inactivating antigen extraction buffer that allows downstream detection of antigen as well as viral RNA. Thus, ready-to-use extraction buffers with virucidal activity and unchanged analytical performance should replace non-virucidal buffers to allow save handling of the point-of-care tests.
Since early in the pandemic, SARS-CoV-2 transmission routes were unclear, human milk was studied to evaluate the potential for spread via breastfeeding. Indeed, we found SARS-CoV-2 RNA in milk for the first time, but infectious virus could not be detected, suggesting a low risk of vertical transmission via breast milk. To assure safety of potentially SARS-CoV-2 containing milk, Holder pasteurization is an effective sterilization measure, as it completely inactivated SARS-CoV-2. It can be used by milk banks or mothers, who might consider discontinuing breastfeeding, but is not suitable as a routine measure.
Furthermore, human breast milk has some intrinsic virucidal activity that is generated during milk storage varying with time, temperature and between donors. Studying this antiviral mechanism in detail for Zika virus (ZIKV) revealed that the effect is mediated by the lipid fraction and that lipases are the responsible factor. This suggests that this milk inactivation property on ZIKV and other enveloped viruses such as SARS-CoV-2 is due to membrane-disrupting fatty acids which are generated by lipolysis of triglycerides released from milk fat globules. Probably, this explains why ZIKV transmission via breastfeeding is hardly observed despite the presence of infectious virus in breast milk and why infectious SARS-CoV-2 has never been isolated from human milk despite the presence of viral RNA. Altogether, breast milk seems safe and breastfeeding should be continued in case of ZIKV as well as SARS-CoV-2 infection as it provides many benefits for the newborns.
Several non-pulmonary manifestations of COVID-19 have been described, such as new-onset diabetes. Our analysis of its molecular basis showed for the first time that SARS-CoV-2 can directly infect and replicate in ex vivo cultured pancreatic islets, specifically in β-cells. The infection was associated with hormone loss and morphological and functional changes in those cells, supporting a mechanism in which direct infection impairs β-cells leading to decreased insulin production and autoantibody-negative type 1 diabetes. This is in line with histological examinations of patient tissue as well as further laboratory studies, confirming that SARS-CoV-2 infection impairs pancreatic cell survival and/or function. However, the mechanism of β-cells impairment and whether these changes underlie the development of diabetes needs to be further studied.
In summary, this work advanced our understanding of the safety of SARS-CoV-2 rapid antigen diagnostics, risk of transmission by breastfeeding, pancreatic tropism and treatment options, thus contributing to the containment of the pandemic.||dc.description.abstract