Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90ß on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90ß in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Human lung organoid: Models for respiratory biology and diseases.

The human respiratory system, consisting of the airway and alveoli, is one of the most complex organs directly interfaced with the external environment. The diverse epithelial cells lining the surface are usually the first cell barrier that comes into contact with pathogens that could lead to deadly pulmonary disease. There is an urgent need to understand the mechanisms of self-renewal and protection of these epithelial cells against harmful pathogens, such as SARS-CoV-2. Traditional models, including cell lines and mouse models, have extremely limited native phenotypic features. Therefore, in recent years, to mimic the complexity of the lung, airway and alveoli organoid technology has been developed and widely applied. TGF-ß/BMP/SMAD, FGF and Wnt/ß-catenin signaling have been proven to play a key role in lung organoid expansion and differentiation. Thus, we summarize the current novel lung organoid culture strategies and discuss their application for understanding the lung biological features and pathophysiology of pulmonary diseases, especially COVID-19. Lung organoids provide an excellent in vitro model and research platform.

A rank-based approach to design and analysis of pretest-posttest randomized trials, with application to COVID-19 ordinal scale data.

Randomized controlled trials with a pretest-posttest design frequently yield ordered categorical outcome data. Focusing on the estimation of the win probability that a treated participant would have a better score than (or win over) a control participant, we developed methods for analysis and sample size planning for such trials. We exploited the analysis of covariance framework with the dependent variable being individual participants' win fractions at posttest and the covariate being the win fractions at pretest. The win fractions were obtained using the mid-ranks of the ordinal data. Simulation evaluation based on a recent randomized trial on COVID-19 suggests that the methods perform very well. A sample SAS code for data analysis is presented.

Non-pharmacological interventions of travel restrictions and cancelation of public events had a major reductive mortality affect during pre-vaccination coronavirus disease 2019 period.

Background: The coronavirus disease 2019 (COVID-19) is a severe acute respiratory disease that poses a continuous threat to global public health. Many non-pharmacological interventions (NPIs) have been implemented to control the COVID-19 pandemic since the beginning. The aim of this study was to assess the impact of various NPIs on COVID-19 mortality during pre-vaccination and vaccination periods. Methods: The COVID-19 data used in this study comes from Our World in Data, we used the Oxford Strict Index (OSI) and its five combination interventions as independent variables. The COVID-19 mortality date (MRT) was defined as a date when daily rate of 0.02 COVID-19 deaths per 100,000 population in a country was reached, and the COVID-19 vaccination date (VRT) was defined as people vaccinated reaching 70%. Linear regression and random forest models were used to estimate the impact of various NPI implementation interventions during pre-vaccination and vaccination periods. The performance of models was assessed among others with Shapley Additive Explanations (SHAP) explaining the prediction capability of the model. Results: During the pre-vaccination period, the various NPIs had strong protective effect. When the COVID-19 MRT was reached, for every unit increase in OSI, the cumulative mortality as of June 30, 2020 decreased by 0.71 deaths per 100,000 people. Restrictions in travel (SHAP 1.68) and cancelation of public events and gatherings (1.37) had major reducing effect on COVID-19 mortality, while staying at home (0.26) and school and workplace closure (0.26) had less effect. Post vaccination period, the effects of NPI reduced significantly: cancelation of public events and gatherings (0.25), staying at home (0.22), restrictions in travel (0.14), and school and workplace closure (0.06). Conclusion: Continued efforts are still needed to promote vaccination to build sufficient immunity to COVID-19 in the population. Until herd immunity is achieved, NPI is still important for COVID-19 prevention and control. At the beginning of the COVID-19 pandemic, the stringency of NPI implementation had a significant negative association with COVID-19 mortality; however, this association was no longer significant after the vaccination rate reached 70%. As vaccination progresses, "cancelation of public events and gatherings" become more important for COVID-19 mortality.

Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is responsible for the COVID-19 pandemic that has caused unprecedented loss of life and economic trouble all over the world, though the mechanism of its replication remains poorly understood. In this study, antibodies were generated and used to systematically determine the expression profile and subcellular distribution of 11 SARS-CoV-2 nonstructural replicase proteins (nsp1, nsp2, nsp3, nsp5, nsp7, nsp8, nsp9, nsp10, nsp13, nsp14, and nsp15) by Western blot and immunofluorescence assay. Nsp3, nsp5, and nsp8 were detected in perinuclear foci at different time points, with diffusion and stronger fluorescence observed over time. In particular, colocalization of nsp8 and nsp13 with different replicase proteins suggested viral protein-protein interaction, which may be key to understanding their functions and potential molecular mechanisms. Viral intermediate dsRNA was detected in perinuclear foci as early as 2-h postinfection, indicating the initiation of virus replication. With the passage of time, these perinuclear dsRNA foci became larger and brighter, and nearly all colocalized with N protein, consistent with viral growth over time. Thus, the development of these anti-nsp antibodies provides basic tools for the further study of replication and diagnosis of SARS-CoV-2. IMPORTANCE The intracellular localization of SARS-CoV-2 replicase nonstructural proteins (nsp) during infection has not been fully elucidated. In this study, we systematically analyzed the expression and subcellular localization of 11 distinct viral nsp and dsRNA over time in SARS-CoV-2-infected cells by using individual antibody against these replicase proteins. The data indicated that nsp gene expression is highly regulated in space and time, which could be useful to understand the function of viral replicases and future development of diagnostics and potential antiviral strategies against SARS-CoV-2.

First evidence that an emerging mammalian alphacoronavirus is able to infect an avian species.

A novel swine enteric alphacoronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), related to Rhinolophus bat CoV HKU2 in the subgenus Rhinacovirus emerged in southern China in 2017, causing diarrhoea in newborn piglets, and critical questions remain about the pathogenicity, cross-species transmission and potential animal reservoirs. Our laboratory's previous research has shown that SADS-CoV can replicate in various cell types from different species, including chickens. Here, we systematically explore the susceptibility of chickens to a cell-adapted SADS-CoV strain both in vitro and in vivo. First, evidence of SADS-CoV replication in primary chicken cells, including cytopathic effects, immunofluorescence staining, growth curves and structural protein expression, was proven. Furthermore, we observed that SADS-CoV replicated in chicken embryos without causing gross lesions and that experimental infection of chicks resulted in mild respiratory symptoms. More importantly, SADS-CoV shedding and viral distribution in the lungs, spleens, small intestines and large intestines of infected chickens were confirmed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The genomic sequence of the original SADS-CoV from the pig source sample in 2017 was determined to have nine nucleotide differences compared to the cell-adapted strain used; among these were three nonsynonymous mutations in the spike gene. These results collectively demonstrate that chickens are susceptible to SADS-CoV infection, suggesting that they are a potential animal reservoir. To our knowledge, this study provides the first experimental evidence of cross-species infection in which a mammalian alphacoronavirus is able to infect an avian species.

Emergency Preparedness and Management of Mobile Cabin Hospitals in China During the COVID-19 Pandemic.

The healthcare systems in China and globally have faced serious challenges during the coronavirus disease (COVID-19) pandemic. The shortage of beds in traditional hospitals has exacerbated the threat of COVID-19. To increase the number of available beds, China implemented a special public health measure of opening mobile cabin hospitals. Mobile cabin hospitals, also called Fangcang shelter hospitals, refer to large-scale public venues such as indoor stadiums and exhibition centers converted to temporary hospitals. This study is a mini review of the practice of mobile cabin hospitals in China. The first part is regarding emergency preparedness, including site selection, conversion, layout, and zoning before opening the hospital, and the second is on hospital management, including organization management, management of nosocomial infections, information technology support, and material supply. This review provides some practical recommendations for countries that need mobile cabin hospitals to relieve the pressure of the pandemic on the healthcare systems.

Association of age distribution with the expression of angiotensin-converting enzyme 2 in lung tissues in severe acute respiratory syndrome coronavirus 2 infection: Reflections from the study of RAS pathway expression in mice

Objective To study the expression of angiotensin-converting enzyme 2 (ACE2) and other key molecules of the RAS pathway in normal mice at different developmental stages, and to provide ideas for understanding the infection mechanism of coronavirus disease 2019 (COVID-19) as well as the diagnosis and treatment of children with COVID-19. Methods The mice at different developmental stages were enrolled, including fetal mice (embryonic days 14.5 and 18.5), neonatal mice (0, 3, 7, 14, and 21 days old), young mice (28 and 42 days old), and adult mice (84 days old). The lung tissues of all fetal mice from 4 pregnant mice were collected at each time point in the fetal group. Four mice were sampled in other age groups at each time point. Whole transcriptome resequencing was used to measure the mRNA expression of AGT, ACE, ACE2, Renin, Agtr1a, Agtr1b, Agtr2, and Mas1 in mouse lung tissue. Results The expression of ACE2 in the lungs showed changes from embryonic stage to adult stage. It increased gradually after birth, reached a peak on day 3 after birth, and reached a nadir on day 14 after birth (P<0.05). The expression of AGT reached a peak on days 0 and 7 after birth and reached a nadir on day 21 after birth (P<0.05). The expression of ACE increased rapidly after birth and reached a peak on day 21 after birth (P<0.05). Agtr1a expression reached a peak on day 21 after birth (P<0.05). Agtr2 expression gradually decreased to a low level after birth. Renin, Agtr1b, and Mas1 showed low expression in lung tissues at all developmental stages. Conclusions At different developmental stages of mice, ACE2 has dynamic expression changes, with high expression in early neonatal and adult mice. The other key molecules of the RAS pathway have their own expression patterns. These suggest that the difference in clinical features between children and adults with COVID-19 might be associated with the different expression levels of ACE2 in the different stages, and further studies are needed for the mechanism.

Clinical Efficacy of Corticosteroids in the Early Stages of Deterioration in COVID-19 Pneumonia.

BACKGROUND: The World Health Organization (WHO) strongly suggests using corticosteroids in patients with severe coronavirus disease 2019 (COVID-19). Similarly, a large randomized controlled clinical trial (RCT) in the UK found that dexamethasone effectively reduced the mortality rate in severe COVID-19 patients. However, the safety profile of corticosteroids has been a controversial area of study. CASE DESCRIPTION: A case of a COVID-19 patient is described and the clinical characteristics are observed as the mildly symptomatic patient progresses into a critically ill patient and during their dramatic improvement with corticosteroid therapy in the early stage of the deterioration process with COVID-19 pneumonia. CONCLUSION: The most suitable timing and dosage for the use of corticosteroids to maximize its effect during the worsening of COVID-19 pneumonia are discussed. One of the main pathophysiological hypotheses for severe COVID-19 patients is related to cytokine storm and virus load, which can be effectively treated with corticosteroid therapy.

The Association of Hypertension With the Severity of and Mortality From the COVID-19 in the Early Stage of the Epidemic in Wuhan, China: A Multicenter Retrospective Cohort Study.

Background: Hypertension may affect the prognosis of COVID-19 illness. We analyzed the epidemiological and clinical characteristics associated with the disease severity and mortality in hypertensive vs. non-hypertensive deceased COVID-19 patients. Methods: We included all the deceased patients with laboratory-confirmed COVID-19 admitted to >200 health facilities in Wuhan between December 1 and February 24, 2020. The median survival time in COVID-19 patients with and without hypertension, the association of hypertension with the disease severity, and the risk factors associated with the COVID-19 mortality stratified by the hypertension status were assessed using the Kaplan-Meier survival analysis, logistic regression, and Cox proportional regression, respectively before and after the propensity score-matching (PS) for age and sex. Results: The prevalence of hypertension in the studied 1,833 COVID-19 patients was 40.5%. Patients with hypertension were more likely to have severe COVID-19 illness than patients without hypertension; the PS-matched multivariable-adjusted odds ratio (95% CI) was 2.44 (1.77-3.08). Moreover, the median survival time in the hypertension group was 3-5 days shorter than the non-hypertension group. There was a 2-fold increased risk of COVID-19 mortality in the hypertension group compared with the non-hypertension group; the PS-matched multivariable-adjusted hazard ratio (HR) = 2.04 (1.61-2.72), and the significant increased risk of COVID-19 mortality in the moderate vs. mild COVID-19 illness was confined to patients with hypertension. Additionally, the history and the number of underlying chronic diseases, occupation, and residential location showed stronger associations with the COVID-19 mortality among patients with hypertension than patients without hypertension. Conclusion: Hypertension was associated with the severity and mortality of COVID-19 illness.

The comparison of epidemiological characteristics between confirmed and clinically diagnosed cases with COVID-19 during the early epidemic in Wuhan, China.

BACKGROUND: To put COVID-19 patients into hospital timely, the clinical diagnosis had been implemented in Wuhan in the early epidemic. Here we compared the epidemiological characteristics of laboratory-confirmed and clinically diagnosed cases with COVID-19 in Wuhan. METHODS: Demographics, case severity and outcomes of 29,886 confirmed cases and 21,960 clinically diagnosed cases reported between December 2019 and February 24, 2020, were compared. The risk factors were estimated, and the effective reproduction number (Rt) of SARS-CoV-2 was also calculated. RESULTS: The age and occupation distribution of confirmed cases and clinically diagnosed cases were consistent, and their sex ratio were 1.0 and 0.9, respectively. The epidemic curve of clinical diagnosis cases was similar to that of confirmed cases, and the city centers had more cumulative cases and higher incidence density than suburbs in both of two groups. The proportion of severe and critical cases (21.5 % vs. 14.0 %, P < 0.0001) and case fatality rates (5.2 % vs. 1.2 %, P < 0.0001) of confirmed cases were all higher than those of clinically diagnosed cases. Risk factors for death we observed in both of two groups were older age, male, severe or critical cases. Rt showed the same trend in two groups, it dropped below 1.0 on February 6 among confirmed cases, and February 8 among clinically diagnosed cases. CONCLUSIONS: The demographic characteristics and spatiotemporal distributions of confirmed and clinically diagnosed cases are roughly similar, but the disease severity and clinical outcome of clinically diagnosed cases are better than those of confirmed cases. In cases when detection kits are insufficient during the early epidemic, the implementation of clinical diagnosis is necessary and effective.

The impact of COVID-19 on short-term prognosis of ST-segment elevation myocardial infarction patients receiving primary percutaneous coronary intervention in Wuhan China.

ST-segment elevation myocardial infarction (STEMI) is a common cardiovascular emergency for which timely reperfusion therapies are needed to minimize myocardial necrosis. The aim of this study was to investigate the impact of the COVID-19 pandemic and reorganization of chest pain centers (CPC) on the practice of primary percutaneous coronary intervention (PPCI) and prognosis of STEMI patients. This single-center retrospective survey included all patients with STEMI admitted to our CPC from January 22, 2020 to April 30, 2020 (during COVID-19 pandemic in Wuhan), compared with those admitted during the analogous period in 2019, in respect of important time points of PPCI and clinical outcomes of STEMI patients. In the present article, we observed a descending trend in STEMI hospitalization and a longer time from symptom onset to first medical contact during the COVID-19 pandemic as compared to the control period (4.35 h versus 2.58 h). With a median delay of 17 minutes in the door to balloon time (D2B), the proportion of in-hospital cardiogenic shock was significantly higher in the COVID-19 era group (47.6% versus 19.5%), and major adverse cardiac events (MACE) tend to increase in the 6-month follow-up period (14.3% versus 2.4%). Although the reorganization of CPC may prolong the D2B time, immediate revascularization of the infarct-related artery could be offered to most patients within 90 minutes upon arrival. PPCI remained the preferred treatment for patients with STEMI during COVID-19 pandemic in the context of timely implementation and appropriate protective measures.

The epidemiological characteristics of deaths with COVID-19 in the early stage of epidemic in Wuhan, China.

OBJECTIVES: To analyze the epidemiological characteristics of COVID-19 related deaths in Wuhan, China and comprehend the changing trends of this epidemic along with analyzing the prevention and control measures in Wuhan. METHODS: Through the China's Infectious Disease Information System, we collected information about COVID-19 associated deaths from December 15, 2019 to February 24, 2020 in Wuhan. We analyzed the patient's demographic characteristics, drew epidemiological curve and made geographic distribution maps of the death toll in each district over time, etc. ArcGIS was used to plot the numbers of daily deaths on maps. Statistical analyses were performed using SPSS and @Risk software. RESULTS: As of February 24, 2020, a total of 1833 deaths were included. Among the deaths with COVID-19, mild type accounted for the most (37.2%), followed by severe type (30.1%). The median age was 70.0 (inter quartile range: 63.0-79.0) years. Most of the deaths were distributed in 50-89 age group, whereas no deaths occurred in 0-9 age group. Additionally, the male to female ratio was 1.95:1. A total of 65.7% of the deaths in Wuhan combined with underlying diseases, and was more pronounced among males. Most of the underlying diseases included hypertension, diabetes and cardiovascular diseases. The peak of daily deaths appeared on February 14 and then declined. The median interval from symptom onset to diagnosis was 10.0 (6.0-14.0) days; the interval from onset to diagnosis gradually shortened. The median intervals from diagnosis to death and symptom onset to deaths were 6.0 (2.0-11.0), 17.0 (12.0-22.0) days, respectively. Most of the disease was centralized in central urban area with highest death rate in Jianghan District. CONCLUSION: COVID-19 poses a greater threat to the elderly people and men with more devastating effects, particularly in the presence of underlying diseases. The geographical distributions show that the epidemic in the central area of Wuhan is more serious than that in the surrounding areas. Analysis of deaths as of February 24 indicates that a tremendous improvement of COVID-19 epidemic in Wuhan has achieved by effective control measures taken by Wuhan Government.

Epidemiological characteristics of patients with severe COVID-19 infection in Wuhan, China: evidence from a retrospective observational study.

BACKGROUND: The new coronavirus (COVID-19) rapidly resulted in a pandemic. We report the characteristics of patients with severe or critical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan city, China, and the risk factors related to infection severity and death. METHODS: We extracted the demographic and clinical data of 7283 patients with severe COVID-19 infection from designated Wuhan hospitals as of 25 February 2020. Factors associated with COVID-19 critical illness and mortality were analysed using logistic- and Cox-regression analyses. RESULTS: We studied 6269 patients with severe COVID-19 illness and 1014 critically ill patients. The median (IQR) age was 64 (53-71) years; 51.2% were male, 38.9% were retirees and 7.4% had self-reported histories of chronic disease. Up to the end of the study, 1180 patients (16.2%) recovered and were discharged, 649 (8.9%) died and the remainder were still receiving treatment. The number of daily confirmed critical cases peaked between 23 January and 1 February 2020. Patients with advanced age [odds ratio (OR), 1.03; 95% confidence intervals (CIs), 1.03-1.04], male sex (OR, 1.57; 95% CI, 1.33-1.86) and pre-existing diabetes (OR, 2.11), hypertension (OR, 2.72), cardiovascular disease (OR, 2.15) or respiratory disease (OR, 3.50) were more likely to be critically ill. Compared with those who recovered and were discharged, patients who died were older [hazard ratio (HR), 1.04; 95% CI, 1.03-1.05], more likely to be male (HR, 1.74; 95% CI, 1.44-2.11) and more likely to have hypertension (HR, 5.58), cardiovascular disease (HR, 1.83) or diabetes (HR, 1.67). CONCLUSION: Advanced age, male sex and a history of chronic disease were associated with COVID-19 critical illness and death. Identifying these risk factors could help in the clinical monitoring of susceptible populations.

Characteristics of myocardial injury in severe and critical coronavirus disease 2019 patients and its effect on prognosis

Objective To analyze the characteristics and related risk factors of myocardial injury in severe and critical coronavirus disease 2019 (COVID-19) patients and their relationship with the prognosis. Methods The clinical data of severe and critical COVID-19 patients treated in General Hospital of Central Theater Command of PLA from Jan. 2020 to Mar. 2020 were collected. The patients were divided into non-myocardial injury group and myocardial injury group. The baseline data, clinical characteristics, auxiliary examination, treatment and prognosis were compared between the two groups, and the risk factors of myocardial injury and the effect on the prognosis of the severe and critical COVID-19 patients were analyzed. Results A total of 56 patients were included, with 22 in the non-myocardial injury group and 34 in the myocardial injury group. Patients were mostly male in both groups, and there was no significant difference in gender composition between the two groups (P>0.05). Compared with the non-myocardial injury group, the age of onset was significantly higher in the myocardial injury group (78.5[ 70.8, 89.0] years vs 56.5[ 50.3, 68.3] years, P<0.01), and the proportions of patients over 65 years old and combined with coronary heart disease were significantly greater (85.3%[ 29/34] vs 31.8%[ 7/22] and 38.2% [13/34] vs 9.1%[ 2/22], both P<0.05). In terms of symptoms, fever (87.5%, 49/56), cough (64.3%, 36/56) and fatigue (46.4%, 26/56) were the most common ones, and there were no significant differences between the two groups (all P>0.05). For the CT findings of the lungs, the proportion of patients having patch-like/plaque-like shadows and ground-glass opacities was significantly greater in the non-myocardial injury group versus the myocardial injury group (72.7%[ 16/22] vs 38.2%[ 13/34], χ2=6.364, P<0.05), and other signs were not significantly different between the two groups (P>0.05). Compared with the non-myocardial injury group, the levels of N-terminal pro-B-type natriuretic peptide, D-dimer, procalcitonin and IL-6 were significantly higher in the myocardial injury group (4 939.5[ 1 817.0, 9 450.3] pg/mL vs 612.5[ 301.0, 1 029.5] pg/mL, 4 386.5 [2 309.5, 9 635.3] ng/mL vs 850.5 [343.5, 2 333.8] ng/mL, 0.46 [0.23, 3.79] ng/mL vs 0.18 [0.13, 0.39] ng/mL, and 138.6 [41.9, 464.8] pg/mL vs 65.1[ 34.7, 99.3] pg/mL, respectively), and the differences were significant (all P<0.01). Multivariate logistic regression analysis showed that age≥65 years old (odds ratio[ OR] =18.62, 95% confidence interval[ CI] 1.61-215.96, P<0.05) and D-dimer level≥3 000 ng/mL (OR=15.48, 95% CI 1.45-164.77, P<0.05) were the independent risk factors for myocardial injury in severe and critical COVID-19 patients. There were no significant differences in the use of antiviral drugs, antibacterial drugs, or glucocorticoids between the two groups (all P>0.05). The mortality rate was significantly higher in the myocardial injury than that in the non-myocardial injury group (58.8% [20/34] vs 9.1% [2/22], P<0.01). Patients who received tracheal intubation, extracorporeal membrane oxygenation, continuous renal replacement therapy (CRRT) and other invasive life support measures were all in the myocardial injury group. Conclusion Older age, male gender, coronary heart disease and (or) cardiac insufficiency, and elevated D-dimer, procalcitonin and IL-6 are the risk factors of myocardial injury in severe and critical COVID-19 patients. Myocardial injury can aggravate the condition and some patients need invasive circulating breathing support, with poor prognosis and high mortality. Therefore, the above indicators need to be observed more closely and dynamically and active treatment should be given according to related factors.