Genomic Surveillance Identifies SARS-CoV-2 Transmission Patterns in Local University Populations, Wisconsin, USA, 2020-2022 (preprint)

Novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge as the current coronavirus disease 2019 (COVID-19) pandemic extends into its third year. Understanding SARS-CoV-2 circulation in university populations is vital for effective interventions in a higher education setting that will inform pubic health policy during pandemics. In this study, we performed whole-genome sequencing of 537 of 1,717 SARS-CoV-2 positive nasopharyngeal/nasal swab samples collected for nearly 20 months from the two university populations in Wisconsin, United States. We observed that the viral sequences were distributed into 57 lineages/sub-lineages belonging to 15 clades of which the majority were from 21K (Omicron, 36.13%) and 21J (Delta, 30.91%). Nearly 40% (213) of the sequences were Omicron of which BA.1 and its eight descendent lineages account for 91%, while the remaining belong to BA.2 and its six descendent lineages. The independent analysis of these two universities sequences revealed significant differences in circulating the SARS-CoV-2 variants. The genome-based analysis of closely-related strains along with phylogenetic clusters had identified that potential virus transmission occurred within as well as between universities, and between the university and local community. Although this study improves our understanding of distinct transmission patterns of circulating variants in local universities, expanding the genomic surveillance capacity will aid local jurisdictions in identifying emerging SARS-CoV-2 variants like BA.4 and BA.5, and improve data-driven public health mitigation and policy efforts.

Hippo Signaling Pathway Activation during SARS-CoV-2 Infection Contributes to Host Antiviral Response (preprint)

SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling (preprint)

ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.

Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication (preprint)

Emergence of a highly contagious novel coronavirus, SARS-CoV-2 that causes COVID-19, has precipitated the current global health crisis with over 479,000 deaths and more than 9.3 million confirmed cases. Currently, our knowledge of the mechanisms of COVID-19 disease pathogenesis is very limited which has hampered attempts to develop targeted antiviral strategies. Therefore, we urgently need an effective therapy for this unmet medical need. Viruses hijack and dysregulate cellular machineries in order for them to replicate and infect more cells. Thus, identifying and targeting dysregulated signaling pathways that have been taken over by viruses is one strategy for developing an effective antiviral therapy. We have developed a high-throughput drug screening system to identify potential antiviral drugs targeting SARS-CoV-2. We utilized a small molecule library of 430 protein kinase inhibitors, which are in various stages of clinical trials. Most of the tested kinase antagonists are ATP competitive inhibitors, a class of nucleoside analogs, which have been shown to have potent antiviral activity. From the primary screen, we have identified 34 compounds capable of inhibiting viral cytopathic effect in epithelial cells. Network of drug and protein relations showed that these compounds specifically targeted a limited number of cellular kinases. More importantly, we have identified mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response (DDR) pathways as key cellular signaling pathways critical for SARS-CoV-2 infection. Subsequently, a secondary screen confirmed compounds such as Berzosertib (VE-822), Vistusertib (AZD2014), and Nilotinib with anti SARS-CoV-2 activity. Finally, we found that Berzosertib, an ATR kinase inhibitor in the DDR pathway, demonstrated potent antiviral activity in a human epithelial cell line and human induced pluripotent stem cell (hIPSC)-derived cardiomyocytes. These inhibitors are already in clinical trials of phase 2 or 3 for cancer treatment, and can be repurposed as promising drug candidates for a host-directed therapy of SARS-CoV-2 infection. In conclusion, we have identified small molecule inhibitors exhibiting anti SARS-CoV-2 activity by blocking key cellular kinases, which gives insight on important mechanism of host-pathogen interaction. These compounds can be further evaluated for the treatment of COVID-19 patients following additional in vivo safety and efficacy studies. DisclosuresNone declared.

Insights into Cross-species Evolution of Novel Human Coronavirus 2019-nCoV and Defining Immune Determinants for Vaccine Development (preprint)

Novel Coronavirus (nCoV) outbreak in the city of Wuhan, China during December 2019, has now spread to various countries across the globe triggering a heightened containment effort. This human pathogen is a member of betacoronavirus genus carrying 30 kilobase of single positive-sense RNA genome. Understanding the evolution, zoonotic transmission, and source of this novel virus would help accelerating containment and prevention efforts. The present study reported detailed analysis of 2019-nCoV genome evolution and potential candidate peptides for vaccine development. This nCoV genotype might have been evolved from a bat-CoV by accumulating non-synonymous mutations, indels, and recombination events. Structural proteins Spike (S), and Membrane (M) had extensive mutational changes, whereas Envelope (E) and Nucleocapsid (N) proteins were very conserved suggesting differential selection pressures exerted on 2019-nCoV during evolution. Interestingly, 2019-nCoV Spike protein contains a 39 nucleotide sequence insertion relative to SARS-like bat-SL-CoVZC45/2017. Furthermore, we identified eight high binding affinity (HBA) CD4 T-cell epitopes in the S, E, M and N proteins, which can be commonly recognized by HLA-DR alleles of Asia and Asia-Pacific Region population. These immunodominant epitopes can be incorporated in universal subunit CoV vaccine. Diverse HLA types and variations in the epitope binding affinity may contribute to the wide range of immunopathological outcomes of circulating virus in humans. Our findings emphasize the requirement for continuous surveillance of CoV strains in live animal markets to better understand the viral adaptation to human host and to develop practical solutions to prevent the emergence of novel pathogenic CoV strains.