The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus is highly transmissible, and quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. Research groups around the world are strongly focused on this, and the susceptibility of different cell lines to SARS-CoV-2 infection has already been demonstrated by molecular techniques. However, data on the biology of SARS-CoV-2 at the ultrastructural level in these in vitro models is still scarce. In this study, we documented, by transmission electron microscopy and real-time RT-PCR, the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

BACKGROUNDNo therapeutics have yet been proven effective for the treatment of severe illness caused by SARS-CoV-2. METHODSWe conducted a randomized, controlled, open-label trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the respiratory illness Covid-19, and an oxygen saturation (Sao 2 ) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao 2 ) to the fraction of inspired oxygen (Fio 2 ) of less than 300 mm Hg. Patients were randomly assigned in a 1:1 ratio to receive either lopinavir-ritonavir (400 mg and 100 mg, respectively) twice a day for 14 days, in addition to standard care, or standard care alone. The primary end point was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a seven-category ordinal scale or discharge from the hospital, whichever came first. RESULTSA total of 199 patients with laboratory-confirmed SARS-CoV-2 infection underwent randomization; 99 were assigned to the lopinavir-ritonavir group, and 100 to the standard-care group. Treatment with lopinavir-ritonavir was not associated with a difference from standard care in the time to clinical improvement (hazard ratio for clinical improvement, 1.24; 95% confidence interval [CI], 0.90 to 1.72). Mortality at 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% vs. 25.0%; difference, −5.8 percentage points; 95% CI, −17.3 to 5.7). The percentages of patients with detectable viral RNA at various time points were similar. In a modified intention-to-treat analysis, lopinavir-ritonavir led to a median time to clinical improvement that was shorter by 1 day than that observed with standard care (hazard ratio, 1.39; 95% CI, 1.00 to 1.91). Gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. Lopinavir-ritonavir treatment was stopped early in 13 patients (13.8%) because of adverse events. CONCLUSIONS

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To face SARS-CoV-2 pandemic various attempts are made to identify potential effective treatments by repurposing available drugs. Among them, indomethacin, an anti-inflammatory drug, was shown to have potent in-vitro antiviral properties on human SARS-CoV-1, canine CCoV, and more recently on human SARS-CoV-2 at low micromolar range. Our objective was to show that indomethacin could be considered as a promising candidate for the treatment of SARS-CoV-2 and to provide criteria for comparing benefits of alternative dosage regimens using a model-based approach. A multi-stage model-based approach was developed to characterize % of recovery and viral load in CCoVinfected dogs, to estimate the PK of indomethacin in dog and human using published data after administration of immediate (IR) and sustained-release (SR) formulations, and to estimate the expected antiviral activity as a function of different assumptions on the effective exposure in human. Different dosage regimens were evaluated for IR formulation (25 mg and 50 mg three-times-a-day, and 25 mg four-times-a-day), and SR formulation (75 mg once and twice-a-day). The best performing dosing regimens were: 50 mg three-times-a-day for the IR formulation, and 75 mg twice-a-day for the SR formulation. The treatment with the SR formulation at the dose of 75 mg twice-a-day is expected to achieve a complete response in three days for the treatment in patients infected by the SARS-CoV-2 coronavirus. These results suggest that indomethacin could be considered as a promising candidate for the treatment of SARS-CoV-2 whose potential therapeutic effect needs to be further assessed in a prospective clinical trial.

Since the outbreak of coronavirus disease 2019 (COVID-19), clinicians have tried every effort to understand the disease, and a brief portrait of its clinical features have been identified. In clinical practice, we noticed that many severe or critically ill COVID-19 patients developed typical clinical manifestations of shock, including cold extremities and weak peripheral pulses, even in the absence of overt hypotension. Understanding the mechanism of viral sepsis in COVID-19 is warranted for exploring better clinical care for these patients. With evidence collected from autopsy studies on COVID-19 and basic science research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, we have put forward several hypotheses about SARS-CoV-2 pathogenesis after multiple rounds of discussion among basic science researchers, pathologists, and clinicians working on COVID-19. We hypothesise that a process called viral sepsis is crucial to the disease mechanism of COVID-19. Although these ideas might be proven imperfect or even wrong later, we believe they can provide inputs and guide directions for basic research at this moment.

Background The global numbers of confirmed cases and deceased critically ill patients with COVID-19 are increasing. However, the clinical course, and the 60-day mortality and its predictors in critically ill patients have not been fully elucidated. The aim of this study is to identify the clinical course, and 60-day mortality and its predictors in critically ill patients with COVID-19. Methods Critically ill adult patients admitted to intensive care units (ICUs) from 3 hospitals in Wuhan, China, were included. Data on demographic information, preexisting comorbidities, laboratory findings at ICU admission, treatments, clinical outcomes, and results of SARS-CoV-2 RNA tests and of serum SARS-CoV-2 IgM were collected including the duration between symptom onset and negative conversion of SARS-CoV-2 RNA. Results Of 1748 patients with COVID-19, 239 (13.7%) critically ill patients were included. Complications included acute respiratory distress syndrome (ARDS) in 164 (68.6%) patients, coagulopathy in 150 (62.7%) patients, acute cardiac injury in 103 (43.1%) patients, and acute kidney injury (AKI) in 119 (49.8%) patients, which occurred 15.5 days, 17 days, 18.5 days, and 19 days after the symptom onset, respectively. The median duration of the negative conversion of SARS-CoV-2 RNA was 30 (range 6–81) days in 49 critically ill survivors that were identified. A total of 147 (61.5%) patients deceased by 60 days after ICU admission. The median duration between ICU admission and decease was 12 (range 3–36). Cox proportional-hazards regression analysis revealed that age older than 65 years, thrombocytopenia at ICU admission, ARDS, and AKI independently predicted the 60-day mortality. Conclusions Severe complications are common and the 60-day mortality of critically ill patients with COVID-19 is considerably high. The duration of the negative conversion of SARS-CoV-2 RNA and its association with the severity of critically ill patients with COVID-19 should be seriously considered and further studied.

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Few data are available on the incidence of deep vein thrombosis (DVT) in critically ill COVID-19 with thrombosis prophylaxis. This study retrospectively included 88 patients in the ICU with critically ill COVID-19 at Jinyintan Hospital in Wuhan, China. All patients underwent compression ultrasonography for identifying DVT. Firth logistic regression was used to examine the association of DVT with sex, age, hypoalbuminemia, D-dimer, and SOFA score. The median (interquartile range [IQR]) age and SOFA score of 88 patients were 63 (55-71) years old and 5 (4-6), respectively. Despite all patients receiving guideline-recommended low-molecular-weight heparin (LMWH) thromboprophylaxis, the incidence of DVT was 46% (95% CI 35-56%). Proximal DVT was recognized in 9% (95% CI 3-15%) of the patients, while 46% (95% CI 35-56%) of patients had distal DVT. All of the proximal DVT combined with distal DVT. Risk factors of DVT extension occurred in all distal DVT patients. As Padua score ≥ 4 or IMPROVE score ≥ 2, 53% and 46% of patients had DVT, respectively. Mortality was higher in patients with acute DVT (30%) compared with non-DVT (17%), but did not reach statistical significance. Hypoalbuminemia (odds ratio [OR], 0.17; 95% CI 0.06-0.05, P = 0.001), higher SOFA score (OR per IQR, 2.07; 95% CI 1.38-3.39, P = 0.001), and elevated D-dimer (OR per IQR, 1.04; 95% CI 1.03-1.84, P = 0.029) were significant DVT risk factors in multivariable analyses. High incidence of DVT was identified in patients with critically ill COVID-19, despite the use of guideline-recommended pharmacologic thromboprophylaxis. The presence of hypoalbuminemia, higher SOFA score, and elevated D-dimer were significantly independent risk factors of DVT. More effective VTE prevention and management strategies may need to be addressed. Keywords Deep vein thrombosis • Hypoalbuminemia • D-dimer • SOFA score • Coronavirus Highlights • The incidence of DVT in patients with critically ill COVID-19 was 46% despite the use of guideline-recommended thromboprophylaxis. • The presence of hypoalbuminemia, SOFA score, and elevated D-dimer were predictors of DVT. • More effective VTE prevention strategies are necessary for patients with critically ill COVID-19.