nanoDoc: RNA modification detection using Nanopore raw reads with Deep One-Class Classification (preprint)

Advances in Nanopore single-molecule direct RNA sequencing (DRS) have presented the possibility of detecting comprehensive post-transcriptional modifications (PTMs) as an alternative to experimental approaches combined with high-throughput sequencing. It has been shown that the DRS method can detect the change in the raw electric current signal of a PTM; however, the accuracy and reliability still require improvement. Here, we presented a new software, called nanoDoc, for detecting PTMs from DRS data using a deep neural network. Current signal deviations caused by PTMs are analyzed via Deep One-Class Classification with a convolutional neural network. Using a ribosomal RNA dataset, the software archive displayed an area under the curve (AUC) accuracy of 0.96 for the detection of 23 different kinds of modifications in Escherichia coli and Saccharomyces cerevisiae. We also demonstrated a tentative classification of PTMs using unsupervised clustering. Finally, we applied this software to severe acute respiratory syndrome coronavirus 2 data and identified commonly modified sites among three groups. nanoDoc is open source (GPLv3) and available at https://github.com/uedaLabR/nanoDoc

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings (preprint)

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate, SDS, and Triton-X containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In addition, the stability of the virus in cell culture media at 4{degrees}C and on surfaces used in laboratory environment was analyzed. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 disease severity (preprint)

Angiotensin-converting enzyme 2 (ACE2) maintains cardiovascular and renal homeostasis but also serves as the entry receptor for the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), the causal agent of novel coronavirus disease 2019 (COVID-19)1. COVID-19 disease severity, while highly variable, is typically lower in pediatric patients than adults (particularly the elderly), but increased rates of hospitalizations requiring intensive care are observed in infants than in older children. The reasons for these differences are unknown. To detect potential age-based correlates of disease severity, we measured ACE2 protein expression at the single cell level in human lung tissue specimens from over 100 donors from [~]4 months to 75 years of age. We found that expression of ACE2 in distal lung epithelial cells generally increases with advancing age but exhibits extreme intra- and inter-individual heterogeneity. Notably, we also detected ACE2 expression on neonatal airway epithelial cells and within the lung parenchyma. Similar patterns were found at the transcript level: ACE2 mRNA is expressed in the lung and trachea shortly after birth, downregulated during childhood, and again expressed at high levels in late adulthood in alveolar epithelial cells. Furthermore, we find that apoptosis, which is a natural host defense system against viral infection, is also dynamically regulated during lung maturation, resulting in periods of heightened apoptotic priming and dependence on pro-survival BCL-2 family proteins including MCL-1. Infection of human lung cells with SARS-CoV-2 triggers an unfolded protein stress response and upregulation of the endogenous MCL-1 inhibitor Noxa; in juveniles, MCL-1 inhibition is sufficient to trigger apoptosis in lung epithelial cells - this may limit virion production and inflammatory signaling. Overall, we identify strong and distinct correlates of COVID-19 disease severity across lifespan and advance our understanding of the regulation of ACE2 and cell death programs in the mammalian lung. Furthermore, our work provides the framework for potential translation of apoptosis modulating drugs as novel treatments for COVID-19.