Simplified point-of-care full SARS-CoV-2 genome sequencing using nanopore technology (preprint)

Background: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment a global decentralized surveillance and warning system to recognize local outbreaks and the emergence of novel variants-of-concern. Among the available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, since it is mobile, scalable and acquisition investments are comparably low. Therefore, streamlined and efficient nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification, in particular for smaller hospital laboratories with lower throughput. Results: We adapted and simplified existing workflows using the midnight 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. Conclusions: The adapted protocol contains less processing steps. Diagnostic CT values are the principal criteria for specimen selection. Subsequent to diagnostic qRT-PCR, multiplex library preparation, quality controls, nanopore sequencing and the bioinformatic pipeline can be completely conducted within one working-day.

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process (preprint)

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Herein, we used X-ray crystallography to characterize an immature form of the main protease. We propose that this form preludes the cis-cleavage of N-terminal residues within the dimer, leading to the mature active site. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the main protease bound to its endogenous N and C-terminal residues during the formation of the tetramer. This quaternary form is also present in solution, suggesting a transitional state during the C-terminal trans-cleavage.

Combined RT-qPCR and Pyrosequencing of a SARS-CoV-2 Spike Glycoprotein Polybasic Cleavage Motif Uncovers Rare Pediatric COVID-19 Spectrum Diseases of Unusual Presentation (preprint)

Background: Surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results. Results: We successfully established a combined RT-qPCR and S-gene pyrosequencing method. This method can be optionally exploited after routine diagnostics or for epidemiologic studies allowing a more reliable interpretation of conflicting RT-qPCR results. This may occur in specimens with relatively low viral loads and close to the detection limits of qPCR, practically for CT values >30. After laboratory implementation and characterization of a best practice protocol we tested the combined method in a field study on a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover previously unrecognized cases of pediatric COVID-19 spectrum diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment when routinely confirmed SARS-CoV-2-positive specimens were used we did not observe any case of false-positive diagnosis. Conclusions: The proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.

Elucidating the antiviral mechanism of different MARCH proteins (preprint)

The Membrane Associated RING-CH (MARCH) proteins belong to a family of E3 ubiquitin ligases, whose main function is to remove transmembrane proteins from the plasma membrane. Recent work has shown that the human MARCH1, 2 and 8 are antiretroviral factors that target the Human Immunodeficiency virus-1 (HIV-1) envelope glycoproteins by reducing their incorporation in the budding virions. Nevertheless, the dearth of information regarding the antiviral mechanism of this family of proteins necessitates further examination. In this study, using both the human MARCH proteins and their mouse homologues, we provide a comprehensive analysis of the antiretroviral mechanism of this family of proteins. Moreover, we show that human MARCH proteins restrict to varying degrees the envelope glycoproteins of a diverse number of viruses. This report sheds light on the important antiviral function of MARCH proteins and their significance in cell intrinsic immunity.

Identification of COVID-19-relevant transcriptional regulatory networks and associated kinases as potential therapeutic targets (preprint)

Identification of transcriptional regulatory mechanisms and signaling networks involved in the response of host to infection by SARS-CoV-2 is a powerful approach that provides a systems biology view of gene expression programs involved in COVID-19 and may enable identification of novel therapeutic targets and strategies to mitigate the impact of this disease. In this study, we combined a series of recently developed computational tools to identify transcriptional regulatory networks involved in the response of epithelial cells to infection by SARS-CoV-2, and particularly regulatory mechanisms that are specific to this virus. In addition, using network-guided analyses, we identified signaling pathways that are associated with these networks and kinases that may regulate them. The results identified classical antiviral response pathways including Interferon response factors (IRFs), interferons (IFNs), and JAK-STAT signaling as key elements upregulated by SARS-CoV-2 in comparison to mock-treated cells. In addition, comparing SARS-Cov-2 infection of airway epithelial cells to other respiratory viruses identified pathways associated with regulation of inflammation (MAPK14) and immunity (BTK, MBX) that may contribute to exacerbate organ damage linked with complications of COVID-19. The regulatory networks identified herein reflect a combination of experimentally validated hits and novel pathways supporting the computational pipeline to quickly narrow down promising avenue of investigations when facing an emerging and novel disease such as COVID-19.