The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2.

Coronavirus disease 2019 (COVID-19) patients show lipid metabolic alterations, but the mechanism remains unknown. In this study, we aimed to investigate whether the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs lipid metabolism in host cells. We generated a Spike cell line in HEK293 using the pcDNA vector carrying the Spike gene expression cassette. A control cell line was generated using the empty pcDNA vector. Gene expression profiles related to lipid metabolic, autophagic, and ferroptotic pathways were investigated. Palmitic acid (PA)-overload was used to assess lipotoxicity-induced necrosis. As compared with controls, the Spike cells showed a significant increase in lipid depositions in cell membranes as well as dysregulation of expression of a panel of molecules involving lipid metabolism, autophagy, and ferroptosis. The Spike cells showed an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a multifunctional transcriptional factor, in response to PA. Furthermore, the Spike cells exhibited increased necrosis in response to PA-induced lipotoxicity compared to control cells in a time- and dose-dependent manner via ferroptosis, which could be attenuated by the Nrf2 inhibitor trigonelline. We conclude that the Spike protein impairs lipid metabolic and autophagic pathways in host cells, leading to increased susceptibility to lipotoxicity via ferroptosis which can be suppressed by a Nrf2 inhibitor. This data also suggests a central role of Nrf2 in Spike-induced lipid metabolic impairments.

Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series.

Background: SARS-CoV-2 is a highly contagious virus that causes the disease COVID-19. We have recently reported that androgens regulate the expression of SARS-CoV-2 host entry factors ACE2 and TMPRSS2, and androgen receptor (AR) in lung epithelial cells. We also demonstrated that the transcriptional repression of the AR enhanceosome inhibited SARS-CoV-2 infection in vitro. Methods: To better understand the various sites of SARS-CoV-2 infection, and presence of host entry factors, we extensively characterized the tissue distribution and localization of SARS-CoV-2 virus, viral replication, and host entry factors in various anatomical sites sampled via autopsy. We applied RNA in-situ-hybridization (RNA-ISH), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) approaches. We also assessed histopathological changes in SARS-CoV-2 infected tissues. Results: We detect SARS-CoV-2 virus and viral replication in pulmonary tissues by RNA-ISH and IHC and a variety of non-pulmonary tissues including kidney, heart, liver, spleen, thyroid, lymph node, prostate, uterus, and colon by qRT-PCR. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. In a patient with a history of kidney transplant and under immunosuppressant therapy, we observe an unusually high viral load in lung tissue by RNA-ISH, IHC and qRT-PCR. SARS-CoV-2 virus is also detected in this patent's kidney, liver and uterus. We find ACE2, TMPRSS2 and AR expression to overlap with the infection sites. Conclusions: This study portrays the impact of dispersed SARS-CoV-2 infection in diverse organ systems, thereby facilitating avenues for systematic therapeutic approaches.

Application of refined management in prevention and control of the coronavirus disease 2019 epidemic in non-isolated areas of a general hospital.

OBJECTIVE: This article summarizes the experience in the prevention and control of coronavirus disease 2019(COVID-19) epidemic in non-isolated areas in a general hospital. METHODS: Based on refined management theory, we professionally developed the standards for prevention and control of COVID-19 in non-isolated areas, systematically implemented various prevention and control measures, performed gridding audits, effectively communicated among teams and between medical staff and patients assisted by information techniques, and reported results for quality improvement. RESULTS: There was no hospital-acquired COVID-19 infections among staff in the hospital. The rates of mask-wearing, epidemiological history screening, and the medical supplies disinfection were all 100% in the hospital. The accuracy rate of mask-wearing of patients and their families was 73.79% and the compliance rate of their hand hygiene was 40.78%. CONCLUSION: Refined management strategies for the prevention and control of COVID-19 infection in non-isolated areas of the general hospital are effective. The accuracy rate of mask-wearing and hand hygiene compliance of patients and their families need to be further improved.

An Effective Protocol for Management of International Arrivals at Risk in COVID-19 Pandemic: Experience From the Pre-Hospital Covid-19 Response Teams at Xi'an, China

Background The coronavirus disease 2019 (COVID-19) outbreak within China has been well controlled and stabilized since early April 2020. Therefore, the current major focus in China is to prevent the introduction of COVID into China from international arrivals. To achieve this, pre-Hospital COVID-19 Response Teams (pHCRTs) have been established. Context The pHRCTs were established in Xi'an, China in early 2020. During the 12 months covered in this report, there were 356 international flight arrivals with over 5,000 COVID-19 Nucleic Acid Test (NAT) positive people, 500 of them with symptomatic COVID-19 and requiring admission to special hospitals. All other arrivals were managed in dedicated facilities by pHRCTs. The outcome measure of this report was the number of positive cases among the pHRCT members. Details Four hundred forty-two staff worked in the pHCRTs during the reporting period. Despite multiple throat swab PCR tests during their pHRCTs tour of duty, and the subsequent mandatory 14-day quarantine required before return to the general community, no staff became NAT positive. Conclusion The prevention of community transmission from imported cases is a vital part of the strategy to maintain the low numbers of cases in countries which have achieved control, or suppression of local internal cases. The program of pHCRTs described in this article gives successful protocols for transportation of patients who are infectious based on the minimal transmission of virus and staff safety. The strategies employed may prove useful in future pandemics.

Why are physical restraints still in use? A qualitative descriptive study from Chinese critical care clinicians' perspectives.

OBJECTIVES: To understand why critical care clinicians still implement physical restraints, to prevent unplanned extubation and to explore the driving factors influencing the decision-making of physical restraints use. DESIGN: A qualitative descriptive design was used. The data were collected through one-to-one, semistructured interviews and analysed through the framework of thematic analysis. PARTICIPANTS AND SETTING: The study was conducted from December 2019 to May 2020 at one general intensive care unit (ICU) and one emergency ICU in a general tertiary hospital with 3200 beds in Hangzhou, China. The sampling strategy was combined maximum variation sampling and criterion sampling. RESULTS: A total of 14 clinicians participated in the study. The reason why critical care clinicians implemented physical restraints to prevent unplanned extubation was that the tense healthcare climate was caused by family members' rejection of mismatched expectations. As unplanned extubation was highly likely to create medical disputes, hospitals placed excessive emphasis on unplanned extubation, which resulted in a lack of analysis of the cause of unplanned extubation and strict measures for dealing with unplanned extubation. The shortage of nursing human resources, unsuitable ward environments, intensivists' attitudes, timely extubation for intensivists, nurse experiences and the patient's possibility of unplanned extubation all contributed to the decision-making resulting in the use of physical restraints. CONCLUSIONS: Although nurses played a crucial role in the decision-making process of using physical restraints, changing the healthcare climate and the hospital management mode for unplanned extubation are fundamental measures to reduce physical restraints use.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Spike protein of SARS-CoV-2 activates macrophages and contributes to induction of acute lung inflammation in male mice.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in mediating viral entry into host cells. However, whether it contributes to pulmonary hyperinflammation in patients with coronavirus disease 2019 is not well known. In this study, we developed a spike protein-pseudotyped (Spp) lentivirus with the proper tropism of the SARS-CoV-2 spike protein on the surface and determined the distribution of the Spp lentivirus in wild-type C57BL/6J male mice that received an intravenous injection of the virus. Lentiviruses with vesicular stomatitis virus glycoprotein (VSV-G) or with a deletion of the receptor-binding domain (RBD) in the spike protein [Spp (∆RBD)] were used as controls. Two hours postinfection (hpi), there were 27-75 times more viral burden from Spp lentivirus in the lungs than in other organs; there were also about 3-5 times more viral burden from Spp lentivirus than from VSV-G lentivirus in the lungs, liver, kidney, and spleen. Deletion of RBD diminished viral loads in the lungs but not in the heart. Acute pneumonia was observed in animals 24 hpi. Spp lentivirus was mainly found in SPC+ and LDLR+ pneumocytes and macrophages in the lungs. IL6, IL10, CD80, and PPAR-γ were quickly upregulated in response to infection in the lungs as well as in macrophage-like RAW264.7 cells. Furthermore, forced expression of the spike protein in RAW264.7 cells significantly increased the mRNA levels of the same panel of inflammatory factors. Our results demonstrated that the spike protein of SARS-CoV-2 confers the main point of viral entry into the lungs and can induce cellular pathology. Our data also indicate that an alternative ACE2-independent viral entry pathway may be recruited in the heart and aorta.

Spike Protein of SARS-CoV-2 Activates Macrophages and Contributes to Induction of Acute Lung Inflammations in Mice

Background Coronavirus disease 2019 (COVID-19) patients exhibit multiple organ malfunctions with a primary manifestation of acute and diffuse lung injuries. The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to mediate viral entry into host cells;however, whether it can be cellularly pathogenic and contribute to pulmonary hyper-inflammations in COVID-19 is not well known. Methods and Findings In this study, we developed a Spike protein-pseudotyped (Spp) lentivirus with the proper tropism of SARS-CoV-2 Spike protein on the surface and tracked down the fate of Spp in wild type C57BL/6J mice receiving intravenous injection of the virus. A lentivirus with vesicular stomatitis virus glycoprotein (VSV-G) was used as the control. Two hours post-infection (hpi), Spp showed more than 27-75 times more viral burden in the lungs than other organs;it also exhibited about 3-5 times more viral burden than VSV-G lentivirus in the lungs, liver, kidney and spleen. Acute pneumonia was evident in animals 24 hpi. Spp lentivirus was mainly found in LDLR+ macrophages and pneumocytes in the lungs, but not in MARC1+ macrophages. IL6, IL10, CD80 and PPAR-? were quickly upregulated in response to infection of Spp lentivirus in the lungs in vivo as well as in macrophage-like RAW264.7 cells in vitro. We further confirmed that forced expression of the Spike protein in RAW264.7 cells could significantly increase the mRNA levels of the same panel of inflammatory factors. Conclusions Our results demonstrate that the Spike protein of SARS-CoV-2 alone can induce cellular pathology, e.g. activating macrophages and contributing to induction of acute inflammatory responses.

Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.