Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection (preprint)

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain to be determined. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells’ self-nucleic acids can be employed as a “danger signal” to alarm the immune system.

Augmentation of anti-MDA5 antibody implies severe disease in COVID-19 patients (preprint)

Recent studies have provided insights into the autoinflammation triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection, which is associated with high mortality of coronavirus disease 2019 (COVID-19). Striking similarities has been noted between COVID-19 and anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab)-related dermatomyositis (DM), implying a shared autoinflammatory aberrance. However, it is unclear whether anti-MDA5 Ab is present in COVID-19 and correlates with the severity and adverse outcome of COVID-19 patients. Here, we found that the positive rate of anti-MDA5 Ab in patients with COVID-19 was 48.2% and the anti-MDA5 Ab positive patients tended to develop severe disease (88.6% vs 66.9%, P<0.0001). In particular, the titer of anti-MDA5 Ab was increased in the non-survivals (5.95{+/-}5.16 vs 8.22{+/-}6.64, P=0.030) and the positive rate was also higher than that in the survivals (23.5% vs 12.0%, P=0.012). Regarding to severe COVID-19 patients, we found that high titer of anti-MDA5 Ab ([≥]10.0 U/mL) was more prevalent in the non-survivals (31.2% vs 14.0%, P=0.006). Moreover, early profiling of anti-MDA5 Ab could distinguish severe patients from those with non-severe ones. Overall, our data reveal that anti-MDA5 Ab is prevalent in the COVID-19 patients and high titer of this antibody is correlated with severe disease and unfavorable outcomes.

Identification of potent and safe antiviral therapeutic candidates against SARS-CoV-2 (preprint)

COVID-19 pandemic has infected millions of people with mortality exceeding 300,000. There is an urgent need to find therapeutic agents that can help clear the virus to prevent the severe disease and death. Identifying effective and safer drugs can provide with more options to treat the COVID-19 infections either alone or in combination. Here we performed a high throughput screen of approximately 1700 US FDA approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 24 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, and merfloquine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating wide therapeutic window compared to hydroxychloroquine which had safety index of 22 in similar experiments. Mechanistically, five of the effective compounds were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA approved compounds can provide much needed therapeutic options that we urgently need in the midst of the pandemic.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Single-cell transcriptional atlas of the Chinese horseshoe bat (Rhinolophus sinicus) provides insight into the cellular mechanisms which enable bats to be viral reservoirs (preprint)

Bats are a major "viral reservoir" in nature and there is a great interest in not only the cell biology of their innate and adaptive immune systems, but also in the expression patterns of receptors used for cellular entry by viruses with potential cross-species transmission. To address this and other questions, we created a single-cell transcriptomic atlas of the Chinese horseshoe bat (Rhinolophus sinicus) which comprises 82,924 cells from 19 organs and tissues. This atlas provides a molecular characterization of numerous cell types from a variety of anatomical sites, and we used it to identify clusters of transcription features that define cell types across all of the surveyed organs. Analysis of viral entry receptor genes for known zoonotic viruses showed cell distribution patterns similar to that of humans, with higher expression levels in bat intestine epithelial cells. In terms of the immune system, CD8+ T cells are in high proportion with tissue-resident memory T cells, and long-lived effector memory nature killer (NK) T-like cells (KLRG1, GZMA and ITGA4 genes) are broadly distributed across the organs. Isolated lung primary bat pulmonary fibroblast (BPF) cells were used to evaluate innate immunity, and they showed a weak response to interferon {beta} and tumor necrosis factor- compared to their human counterparts, consistent with our transcriptional analysis. This compendium of transcriptome data provides a molecular foundation for understanding the cell identities, functions and cellular receptor characteristics for viral reservoirs and zoonotic transmission.

Characterization of spike glycoprotein of 2019-nCoV on virus entry and its immune cross-reactivity with spike glycoprotein of SARS-CoV (preprint)

Since beginning of this century, there have already been three zoonotic outbreaks caused by beta coronaviruses (CoV), SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly identified 2019-nCoV in late 2019, Wuhan, China. As to Feb 10th, 2020, there are over 40,000 confirmed cases and over 900 deaths. However, little is known about the biology of this newly emerged virus. Here we developed a lentiviral based pseudovirus system for S protein of 2019-nCoV to study virus entry in BSL2 settings. First, we confirmed that human angiotensin converting enzyme 2 (hACE2) is the main entry receptor for 2019-nCoV. Second, we found that 2019-nCoV S protein mediated entry on 293/hACE2 cells was mainly through endocytosis, and PIKfyve, TPC2, and cathepsin L are critical for virus entry. Third, 2019-nCoV S protein is less stable than SARS-CoV, and it could trigger protease-independent and receptor dependent cell-cell fusion, which might help virus rapidly spread from cell to cell. Finally and more importantly, polyclonal anti-SARS S1 antibodies T62 effectively inhibited entry of SARS-CoV S pseudovirions, but almost had no effect on entry of 2019-nCoV S pseudovirions. Further studies using sera from one recovered SARS-CoV patient and five 2019-nCoV patients showed that there was only limited cross-neutralization activities between SARS-CoV and 2019-nCoV sera, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for 2019-nCoV.