A SARS-CoV-2-Related Virus from Malayan Pangolin Causes Lung Infection without Severe Disease in Human ACE2-Transgenic Mice.

Coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most severe emerging infectious disease in the current century. The discovery of SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins in South Asian countries indicates that SARS-CoV-2 likely originated from wildlife. To date, two SARSr-CoV-2 strains have been isolated from pangolins seized in Guangxi and Guangdong by the customs agency of China, respectively. However, it remains unclear whether these viruses cause disease in animal models and whether they pose a transmission risk to humans. In this study, we investigated the biological features of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin (Manis javanica) captured by the Guangxi customs agency, termed MpCoV-GX, in terms of receptor usage, cell tropism, and pathogenicity in wild-type BALB/c mice, human angiotensin-converting enzyme 2 (ACE2)-transgenic mice, and human ACE2 knock-in mice. We found that MpCoV-GX can utilize ACE2 from humans, pangolins, civets, bats, pigs, and mice for cell entry and infect cell lines derived from humans, monkeys, bats, minks, and pigs. The virus could infect three mouse models but showed limited pathogenicity, with mild peribronchial and perivascular inflammatory cell infiltration observed in lungs. Our results suggest that this SARSr-CoV-2 virus from pangolins has the potential for interspecies infection, but its pathogenicity is mild in mice. Future surveillance among these wildlife hosts of SARSr-CoV-2 is needed to monitor variants that may have higher pathogenicity and higher spillover risk. IMPORTANCE SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health. Several SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins have been identified since the SARS-CoV-2 outbreak. It is therefore important to assess their potential of crossing species barriers for better understanding of their risk of future emergence. In this work, we investigated the biological features and pathogenicity of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin, named MpCoV-GX. We found that MpCoV-GX can utilize ACE2 from 7 species for cell entry and infect cell lines derived from a variety of mammalian species. MpCoV-GX can infect mice expressing human ACE2 without causing severe disease. These findings suggest the potential of cross-species transmission of MpCoV-GX, and highlight the need of further surveillance of SARSr-CoV-2 in pangolins and other potential animal hosts.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model.

BACKGROUND: Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those institutions without such facilities or 2019-nCoV. This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV-related coronavirus model. METHODS: A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described. Whether GX_P2V uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA)-mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection. The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively. RESULTS: The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs, including cepharanthine (CEP), selamectin, and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture at 10 µmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 µmol/L. The viral RNA yield in cells treated with 10 µmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ±â€Š0.02] × 10vs. 1.00 ±â€Š0.12, t = 150.38, P < 0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 µmol/L CEP at 48 h p.i. Furthermore, we found CEP had potent anti-viral activities against both viral entry (0.46 ±â€Š0.12, vs.1.00 ±â€Š0.37, t = 2.42, P < 0.05) and viral replication ([6.18 ±â€Š0.95] × 10vs. 1.00 ±â€Š0.43, t = 3.98, P < 0.05). CONCLUSIONS: Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin, and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and further study of CEP for treatment of 2019-nCoV infection is warranted.

Profile of RT-PCR for SARS-CoV-2: A Preliminary Study From 56 COVID-19 Patients.

A novel coronavirus (COVID-19) pandemic threatens the world. Here, we first studied the dynamics profile of SARS-CoV-2 from 56 recovered patients with COVID-19. We found viral shedding occurred up to 6 weeks after onset of symptoms. A prolonged observation period is necessary for older patients.

Sub10 μm macroporous aramid substrates with a hierarchically structured interface for organic solvent nanofiltration

Compared with traditional methods for elaborately tailoring the active layer of thin-film composite (TFC) membranes, this study focused on building novel substrates for potential applications in developing organic solvent nanofiltration (OSN) membranes. One kind of “three-parts” hierarchically structured interface with nanospheres and macro surface pores was successfully prepared via Michael addition and Schiff's base (M&S) reactions between N-(2-aminoethyl)-3-aminopropyl triethoxysilane (NAE-A) and glycerite (a natural polyphenol) in the aramid substrate. The thickness was reduced to sub10 μm with the aid of the high-speed spin coating process coupling nonsolvent-induced phase separation (HSSC-co-NIPS) method. The composition characterization results demonstrated the introduction of glycerite, and the reactions occurred in/on the substrate. Scanning electron microscopy (SEM) images clearly showed that the sub10 μm substrate contained a “three-level” hierarchically structured interface that included: (1) a “stalk-like” structure;(2) glycerite-NAE-A silane (GNAS) nanospheres;and (3) the substrate surface. These phenomena also resulted in better surface hydrophilicity. All types of organic solvents, including harsh solvents, such as tetrahydrofuran (THF) and N,N-dimethylformamide (DMF), had stable permeability within the substrate, as did apolar n-hexane and isopar™ G. Therefore, the as-prepared TFC OSN membrane, which had an extremely short polymerization time, retained broad-spectrum solvent stability and had the highest solvent permeance in acetonitrile (24.5 ± 0.4 L m−2 h−1·bar−1). In addition, the resultant TFC membrane almost completely rejected the popular macrolide antibiotic azithromycin (AZM, 748.98 g mol−1) in ethanol, which is used to treat COVID-19.

Prevalence and prognostic value of elevated troponins in patients hospitalised for coronavirus disease 2019: a systematic review and meta-analysis.

BACKGROUND: The clinical significance of cardiac troponin measurement in patients hospitalised for coronavirus disease 2019 (covid-19) is uncertain. We investigated the prevalence of elevated troponins in these patients and its prognostic value for predicting mortality. METHODS: Studies were identified by searching electronic databases and preprint servers. We included studies of hospitalised covid-19 patients that reported the frequency of troponin elevations above the upper reference limit and/or the association between troponins and mortality. Meta-analyses were performed using random-effects models. RESULTS: Fifty-one studies were included. Elevated troponins were found in 20.8% (95% confidence interval [CI] 16.8-25.0 %) of patients who received troponin test on hospital admission. Elevated troponins on admission were associated with a higher risk of subsequent death (risk ratio 2.68, 95% CI 2.08-3.46) after adjusting for confounders in multivariable analysis. The pooled sensitivity of elevated admission troponins for predicting death was 0.60 (95% CI 0.54-0.65), and the specificity was 0.83 (0.77-0.88). The post-test probability of death was about 42% for patients with elevated admission troponins and was about 9% for those with non-elevated troponins on admission. There was significant heterogeneity in the analyses, and many included studies were at risk of bias due to the lack of systematic troponin measurement and inadequate follow-up. CONCLUSION: Elevated troponins were relatively common in patients hospitalised for covid-19. Troponin measurement on admission might help in risk stratification, especially in identifying patients at high risk of death when troponin levels are elevated. High-quality prospective studies are needed to validate these findings. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020176747.

Proinflammatory cytokines are associated with prolonged viral RNA shedding in COVID-19 patients.

Since December 2019, Coronavirus Disease 2019 (COVID-19) has emerged as a global pandemic. We aimed to investigate the clinical characteristics and analyzed the risk factors for prolonged viral RNA shedding. We retrospectively collected data from 112 hospitalized COVID-19 patients in a single center in Wuhan, China. Factors associated with prolonged viral RNA shedding (≥28 days) were investigated. Forty-nine (43.8%) patients had prolonged viral RNA shedding. Patients with prolonged viral shedding were older and had a higher rate of hypertension. Proinflammatory cytokines, including interleukin-2R (IL-2R) and tumor necrosis factor-α (TNF-α), were significantly elevated in patients with prolonged viral shedding. Multivariate analysis revealed that hypertension, older age, lymphopenia and elevated serum IL-2R were independent risk factors for prolonged viral shedding. This comprehensive investigation revealed the distinct characteristics between patients with or without prolonged viral RNA shedding. Hypertension, older age, lymphopenia and high levels of proinflammatory cytokines may be correlated with prolonged viral shedding.

Clinical Studies on the Treatment of Novel Coronavirus Pneumonia With Traditional Chinese Medicine-A Literature Analysis.

OBJECTIVE: This study aims to analyze the current situation and characteristics of traditional Chinese medicine for treatment of novel coronavirus pneumonia, clarify its clinical advantages and provide a reference for clinical treatment. METHODS: Clinical randomized controlled trials, clinical control trials and case series research involving the use of Chinese medicine for novel coronavirus pneumonia treatment were selected from PubMed, Chinese Journal Service Platform of CNKI, VIP, and WanFang Data Knowledge Service Platform from the establishment of the library to 11:00 am on April 15, 2020. The published information, research design, intervention measures and research observation index were statistically analyzed. RESULTS: Twenty studies were included. The research methods were mainly clinical controlled trials. The observation indicators were mostly fever improvement time, cough improvement time, shortness of breath improvement time, chest CT and CRP examination. Maxing Ganshi (Ephedrae Herba, Armeniacae Semen Amarum, Glycyrrhizae Radix Et Rhizoma, and Gypsum Fibrosum) decoction was the core prescription. The most frequently used drugs were Glycyrrhizae Radix Et Rhizoma (Gancao), Ephedrae Herba (Mahuang), Armeniacae Semen Amarum (Kuxingren), Atractylodis Rhizoma (Cangzhu), and Scutellariae Radix (Huangqin). The most frequently used drug combination was Ephedrae Herba (Mahuang)-Armeniacae Semen Amarum (Kuxingren). The most frequently used Chinese patent medicine was Lianhua Qingwen capsule/granule. CONCLUSIONS: Traditional Chinese medicine has widely used for novel coronavirus pneumonia in China. It is worthy of global attention. Also, high-quality randomized controlled clinical trials on the effectiveness and safety of traditional Chinese medicine in the treatment of novel coronavirus pneumonia need to carry out.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2.

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Prevalence and Prognostic Value of Elevated Troponins in Patients Hospitalised for Coronavirus Disease 2019: A Systematic Review and Meta-Analysis (preprint)

Background: The clinical significance of cardiac troponin measurement in patients hospitalised for coronavirus disease-2019 (covid-19) is uncertain. We investigated the prevalence of elevated troponins in these patients and its prognostic value for predicting mortality. Methods: : Studies were identified by searching electronic databases and preprint servers. We included studies of hospitalised covid-19 patients that reported the frequency of troponin elevations above the upper reference limit and/or the association between troponins and mortality. Meta-analyses were performed using random-effects models. Results: : Forty-four studies were included. Elevated troponins were found in 21.3% (95% confidence interval [CI] 18.0-24.9 %) of patients who received troponin test on hospital admission. Elevated troponins on admission were associated with a higher risk of subsequent death (risk ratio 2.81, 95% CI 2.01-3.93) after adjusting for confounders in multivariable analysis. The pooled sensitivity of elevated admission troponins for predicting death was 0.64 (95% CI 0.58-0.70), and the specificity was 0.88 (0.82-0.92). The post-test probability of death was about 50% for patients with elevated admission troponins, and was about 7% for those with non-elevated troponins on admission. There were significant heterogeneity and publication bias in the analyses, and many included studies were at risk of selection bias due to the lack of systematic troponin measurement and inadequate follow-up. Conclusion: Elevated troponins were relatively common in patients hospitalised for covid-19. Troponin measurement on admission might help in risk stratification, especially in identifying patients at high risk of death when troponin levels are elevated. High-quality prospective studies are needed to validate these findings. Systematic Review Registration: PROSPERO (CRD42020176747).

Dynamic profile of RT-PCR findings from 301 COVID-19 patients in Wuhan, China: A descriptive study.

BACKGROUND: With the spread of Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, its effect on society is amplified. We aimed to describe the viral detection results across different timepoints throughout the disease course. METHODS: A retrospective study of 301 confirmed COVID-19 patients hospitalized at Tongji Hospital in Wuhan, China, were included. Demographic characteristics of the patients were collected. Upper respiratory specimens (throat and/or nasal swabs) were obtained and analyzed by real-time RT-PCR for SARS-CoV-2 infection. Period of viral infection and the contagious stage were analyzed. RESULTS: Of 301 hospitalized COVID-19 patients, the median age was 58 years and 51.2 % were male. The median period between symptoms presence and positive SARS-CoV-2 RT-PCR results was 16 days (IQR, 10-23, N = 301). The median period between symptoms presence and an effective negative SARS-CoV-2 RT-PCR result was 20 days (IQR, 17-24; N = 216). Infected patient ≥65 years old stayed contagious longer (22 days vs 19 days, p = 0.015). Although two consecutive negative results were confirmed in 70 patients, 30 % of them had positive viral test results for the third time. Using specimens from nasal swabs to run the RT-PCR test showed a higher positive rate than using specimens from throat swabs. CONCLUSIONS: This large-scale investigation with 1113 RT-PCR test results from 301 COVID-19 patients showed that the average contagious period of SARS-CoV-2 infected patients was 20 days. Longer observation period and more than 2 series of negative viral test are necessary for patients ≥65 years.

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.