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Coronavirus Research

Validation of current PCR-based methods
In March 2020, the Max von Pettenkofer Institute initiated a multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2. Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Spearheaded by Dr. Maximilian Münchhoff, we compared 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic (This study was recently published: Euro Surveill 2020 Jun;25(24).)

 

Assisting high-throughput PCR diagnostics for SARS-CoV-2
A bottleneck in SARS-CoV-2 PCR diagnostics that many laboratories were facing in the first months of the pandemic, was the extraction of viral nucleic acids from nasopharyngeal swabs and other respiratory material for subsequent PCR analysis. To meet these increasing demands, we teamed up with the laboratory of Prof. Veit Hornung from LMU’s Gene Center in Munich. Automation specialist Jochen Rech from the Hornung lab together with Dr. Paul R. Wratil and Niklas Teichert and Katharina Hofmann from the Pettenkofer Institute have implemented a high- throughput nucleic acid extraction methodology that allows the simultaneous processing of 96 samples in 30 minutes. With this setup at hand, the PCR testing capacities can be increased by up to 800 samples per day. Testings are currently being performed in the context of SARS-CoV-2 research studies and will be implemented into quality-controlled routine diagnostics of clinical samples.

 
Robotics to assist automated nucleic and acid extraction and antibody analyses.

 

Deciphering SARS-CoV-2 transmissions in the second largest university hospital in Germany
The major aim of this project seeks to understand cluster outbreaks among patients and health care workers in the largest university hospital in Bavaria using a combination of anamnestic reporting of interpersonal interactions, perceived relevant exposures and next generation sequencing (NGS)-based phylogenetic analyses of SARS-CoV-2 isolates from cluster members to establish likely scenarios and time lines for transmissions. In collaboration with the laboratory of Dr. Hubert Blum of LMU’s Gene Center Munich, we have established a fast and cost-effective method for sequencing of SARS-Cov-2 genomes. Dr. Maximilian Münchhoff (Pettenkofer & Gene Center), Dr. Alexander Graf and Dr. Stefan Krebs (both Gene Center) are driving this project. From throat swabs of COVID-19 patients the virus genome is first amplified in a multiplex PCR and then characterized by nanopore sequencing. These results are made available to the scientific community via the freely accessible platforms GISAID and Nextstrain. We will elucidate whether and how infections in cluster outbreaks in a hospital setting were either community-acquired or rather transmitted in the hospital, which is of high relevance for risk stratification and potential optimization of preventive measures for health care workers and patients.


Phylogenetic tree of near-full-length SARS-CoV-2 sequences. Sequences were derived from the clinical samples of patients (blue) and staff (green) of the LMU University Hospital shown against a selection of sequences from all parts of the world.

 

Development of potent SARS-CoV-2 antibody detection systems
Highly sensitive as well as specific serological detection of SARS-CoV-2 in sick and recovered patients is a worldwide need in the context of COVID-19. These antibody tests should for example not detect closely related corona viruses that cause the common cold and should ideally identify 100% of COVID-19 patients a few weeks after infection. In collaboration with the laboratories of Prof. Veit Hornung, Prof. Karl-Peter Hopfner (both Gene Center Munich) and Dr. Sabine Suppmann from the Max-Planck-Institute of Biochemistry, we are developing different SARS-CoV-2 antibody ELISA systems. These are currently being validated in comparison to several commercially available test systems.

 

Generation of SARS-CoV-2 antibodies
The research group of Prof. Conzelmann of the Max von Pettenkofer Institute & Gene Center Munich develops presentation systems for SARS-CoV-2 antigen based on self-replicating RNAs for immunization and serological investigations. In collaboration with Helmholtz Munich (HMGU) monoclonal antibodies (MAbs) are generated which shall be used for detection of virus in acute infections and which could complement the widely used PCR tests. An immunofluorescence test using SARS-1 and SARS-2 specific antigens is employed for demonstration of COVID-19 specific antibodies of diseased and recovered patients and useful for determining infection rates.

 

Development of novel antiviral strategies
The need for innovative antiviral strategies for the treatment of COVID-19 patients is high. We have established several cell-based assays to quantify the infection of SARS-CoV-2 and evaluate specific antivirals. These biosafety level 3 assays are being performed by Dr. Marcel Stern and Dr. Manuel Albanese from the Keppler laboratory in the P3 laboratory of the Institute of Virology of our sister university in Munich, under the directorship of Prof. Ulrike Protzer. Collaborators for the advancement of novel therapeutic strategies include Prof. Thomas Carell (LMU Munich) and Eisbach Bio (http://www.eisbach.bio/). 


Dr. Marcel Stern (pictures taken and kindly provided by Moritz Knolle)


SARS-CoV-2's cytopathic effect can be prevented in tissue culture. Human cells were either left untreated (left image) or pretreatead with a specific antiviral intervention (right image), challenged with a SARS-CoV-2 patient isolate and cultivated for 48 hours before images were taken.

 

The All-Corona-Care Study (ACC Study) with 10,000 employees of the LMU University Hopital is not only one of the largest antibody studies on SARS-CoV-2 in Germany, but also aims to determine how high the stress level of employees was during the peak phase of the coronavirus pandemic. Among other things, this study will also be used to derive measures to improve the training and protection of healthcare workers during future pandemic waves.

Zur All-Corona-Care-Studie des LMU Klinikums

 

From virus-host protein-protein interactions to broad-spectrum antivirals
Human coronaviruses HCoV-NL63, HCoV-HKU-1, HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-MERS, and SARS-CoV-2 cause respiratory tract illness ranging from mild common cold infections in immune-competent individuals to deadly virus-associated pneumonia and further organ failure. The SARS-CoV-2 pandemic urges the development of effective compounds broadly active against this and ideally also against other viruses. Usually inhibitors aim at individual viral structures.
The research group of PD Dr. Dr. Albrecht von Brunn identifies cellular checkpoint proteins required for virus replication, which promises to lead to inhibitors with a broader antiviral range. The group screens orfeomes of mildly and highly pathogenic coronaviruses, including SARS-CoV-2, for interaction with important cellular proteins by utilizing yeast and mammalian protein-protein interaction assay systems. A number of cellular factors, whose normal function might be hampered in the presence of virus infection, are identified.
The approach has led to the discovery of immunosuppressive (cyclosporin A, CsA) and non-immunosuppressive (Alisporivir and others) cyclophilin inhibitors as broad-range blockers of the replication of human and animal coronaviruses. It needs to be resolved whether the dual role of CsA might be advantageous in seriously ill COVID-19 patients as the drug displays antiviral activities and prevents an overshoot of the immune system. A small but increasing number of publications on immunosuppressed transplantation patients point into direction.

Research Group von Brunn