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.

We collected information on demographic characteristics, exposure history, and illness timelines of laboratory-confirmed cases of NCIP that had been reported by January 22, 2020. We described characteristics of the cases and estimated the key epidemiologic time-delay distributions. In the early period of exponential growth, we estimated the epidemic doubling time and the basic reproductive number. RESULTSAmong the first 425 patients with confirmed NCIP, the median age was 59 years and 56% were male. The majority of cases (55%) with onset before January 1, 2020, were linked to the Huanan Seafood Wholesale Market, as compared with 8.6% of the subsequent cases. The mean incubation period was 5.2 days (95% confidence interval [CI], 4.1 to 7.0), with the 95th percentile of the distribution at 12.5 days. In its early stages, the epidemic doubled in size every 7.4 days. With a mean serial interval of 7.5 days (95% CI, 5.3 to 19), the basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9). CONCLUSIONSOn the basis of this information, there is evidence that human-to-human transmission has occurred among close contacts since the middle of December 2019. Considerable efforts to reduce transmission will be required to control outbreaks if similar dynamics apply elsewhere. Measures to prevent or reduce transmission should be implemented in populations at risk. (Funded by the Ministry of Science and Technology of China and others.) a bs tr ac t Early Transmission Dynamics

Background In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. MethodsWe did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus.Findings The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues.Interpretation 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensinconverting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation.

the final date of follow-up was February 15, 2020. All consecutive inpatients with laboratory-confirmed COVID-19 were included in this study.MAIN OUTCOMES AND MEASURES Clinical laboratory, radiological, and treatment data were collected and analyzed. Outcomes of patients with and without cardiac injury were compared. The association between cardiac injury and mortality was analyzed. RESULTS A total of 416 hospitalized patients with COVID-19 were included in the final analysis; the median age was 64 years (range, 21-95 years), and 211 (50.7%) were female. Common symptoms included fever (334 patients [80.3%]), cough (144 [34.6%]), and shortness of breath (117 [28.1%]). A total of 82 patients (19.7%) had cardiac injury, and compared with patients without cardiac injury, these patients were older (median [range] age, 74 [34-95] vs 60 [21-90] years; P < .001); had more comorbidities (eg, hypertension in 49 of 82 [59.8%] vs 78 of 334 [23.4%]; P < .001); had higher leukocyte counts (median [interquartile range (IQR)], 9400 [6900-13 800] vs 5500 [4200-7400] cells/μL) and levels of C-reactive protein (median [IQR], 10.2 [6.4-17.0] vs 3.7 [1.0-7.3] mg/dL), procalcitonin (median [IQR], 0.27 [0.10-1.22] vs 0.06 [0.03-0.10] ng/mL), creatinine kinase-myocardial band (median [IQR], 3.2 [1.8-6.2] vs 0.9 [0.6-1.3] ng/mL), myohemoglobin (median [IQR], 128 [68-305] vs 39 [27-65] μg/L), high-sensitivity troponin I (median [IQR], 0.19 [0.08-1.12] vs <0.006 [<0.006-0.009] μg/L), N-terminal pro-B-type natriuretic peptide (median [IQR], 1689 [698-3327] vs 139 [51-335] pg/mL), aspartate aminotransferase (median [IQR], 40 [27-60] vs 29 [21-40] U/L), and creatinine (median [IQR], 1.15 [0.72-1.92] vs 0.64 [0.54-0.78] mg/dL); and had a higher proportion of multiple mottling and ground-glass opacity in radiographic findings (53 of 82 patients [64.6%] vs 15 of 334 patients [4.5%]). Greater proportions of patients with cardiac injury required noninvasive mechanical ventilation (38 of 82 [46.3%] vs 13 of 334 [3.9%]; P < .001) or invasive mechanical ventilation (18 of 82 [22.0%] vs 14 of 334 [4.2%]; P < .001) than those without cardiac injury. Complications were more common in patients with cardiac injury than those without cardiac injury and included acute respiratory distress syndrome (48 of 82 [58.5%] vs 49 of 334 [14.7%]; P < .001), acute kidney injury (7 of 82 [8.5%] vs 1 of 334 [0.3%]; P < .001), electrolyte disturbances (13 of 82 [15.9%] vs 17 of 334 [5.1%]; P = .003), hypoproteinemia (11 of 82 [13.4%] vs 16 of 334 [4.8%]; P = .01), and coagulation disorders (6 of 82 [7.3%] vs 6 of 334 [1.8%]; P = .02). Patients with cardiac injury had higher mortality than those without cardiac injury (42 of 82 [51.2%] vs 15 of 334 [4.5%]; P < .001). In a Cox regression model, patients with vs those without cardiac injury were at a higher risk of death, both during the time from symptom onset (hazard ratio, 4.26 [95% CI, 1.92-9.49]) and from admission to end point (hazard ratio, 3.41 [95% CI, 1.62-7.16]).CONCLUSIONS AND RELEVANCE Cardiac injury is a commo...

Background Although the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in respiratory specimens has been widely used to diagnose coronavirus disease 2019 (COVID-19), it is undeniable that serum SARS-CoV-2 nucleic acid (RNAemia) could be detected in a fraction of COVID-19 patients. However, it is not clear whether testing for RNAemia is correlated with the occurrence of cytokine storms or with the specific class of patients. Methods This study enrolled 48 patients with COVID-19 admitted to the General Hospital of Central Theater Command, People’s Liberation Army, a designated hospital in Wuhan, China. The patients were divided into 3 groups according to the Diagnosis and Treatment of New Coronavirus Pneumonia (sixth edition) guidelines issued by the National Health Commission of China. Clinical and laboratory data were collected, and the serum viral load and interleukin 6 (IL-6) level were determined. Results Analysis of clinical characteristics of 48 cases of COVID-19 showed that RNAemia was diagnosed only in the critically ill group and seemed to reflect the severity of the disease. Furthermore, the level of the inflammatory cytokine IL-6 in critically ill patients increased significantly, almost 10 times that in other patients. More importantly, the extremely high IL-6 level was closely correlated with the detection of RNAemia (R = 0.902). Conclusions Detectable serum SARS-CoV-2 RNA (RNAemia) in patients with COVID-19 was associated with elevated IL-6 concentration and poor prognosis. Because elevated IL-6 may be part of a larger cytokine storm that could worsen outcome, IL-6 could be a potential therapeutic target for critically ill patients with an excessive inflammatory response.

Aims To investigate the characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019 (COVID-19). Methods and results We enrolled 671 eligible hospitalized patients with severe COVID-19 from 1 January to 23 February 2020, with a median age of 63 years. Clinical, laboratory, and treatment data were collected and compared between patients who died and survivors. Risk factors of death and myocardial injury were analysed using multivariable regression models. A total of 62 patients (9.2%) died, who more often had myocardial injury (75.8% vs. 9.7%; P &lt; 0.001) than survivors. The area under the receiver operating characteristic curve of initial cardiac troponin I (cTnI) for predicting in-hospital mortality was 0.92 [95% confidence interval (CI), 0.87–0.96; sensitivity, 0.86; specificity, 0.86; P &lt; 0.001]. The single cut-off point and high level of cTnI predicted risk of in-hospital death, hazard ratio (HR) was 4.56 (95% CI, 1.28–16.28; P = 0.019) and 1.25 (95% CI, 1.07–1.46; P = 0.004), respectively. In multivariable logistic regression, senior age, comorbidities (e.g. hypertension, coronary heart disease, chronic renal failure, and chronic obstructive pulmonary disease), and high level of C-reactive protein were predictors of myocardial injury. Conclusion The risk of in-hospital death among patients with severe COVID-19 can be predicted by markers of myocardial injury, and was significantly associated with senior age, inflammatory response, and cardiovascular comorbidities.

Background:Although the SARS-CoV-2 viral load detection of respiratory specimen has been widely used for novel coronavirus disease diagnosis, it is undeniable that serum SARS-CoV-2 nucleic acid (RNAaemia) could be detected in a fraction of the COVID-19 patients. However, it is not clear that if the incidence of RNAaemia could be correlated with the occurrence of cytokine storm or with the specific class of patients. Methods:This study enrolled 48 patients with COVID-19 admitted to the General Hospital of Central Theater Command, PLA, a designated hospital in Wuhan, China. The patients were divided into three groups according to the Dia gnosis and Treatment of New Coronavirus Pneumonia (version 6) published by the National Health Commission of China. The clinical and laboratory data were collected. The serum viral load detection and serum IL-6 levels were determined. Except for routine statistical analysis, Generalized Linear Models (GLMs) analysis was used to establish a patient status prediction model based on real-time RT-PCR Ct value. Findings:The Result showed that cases with RNAaemia were exclusively confirmed in critically ill patients group and appeared to reflect the illness severity. Further more, the inflammatory cytokine IL-6 levels were significantly elevated in critically ill patients, which is almost 10-folds higher than those in other patients. More importantly, the extremely high IL-6 level was closely correlated with the incidence of RNAaemia (R=0.902) and the vital signs of COVID-19 patients (R= -0.682). Interpretation:Serum SARS-CoV-2 viral load (RNAaemia) is strongly associated with cytokine storm and can be used to predict the poor prognosis of COVID-19 patients.Moreover, our results strongly suggest that cytokine IL-6 should be considered as a therapeutic target in critically ill patients with excessive inflammatory response. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged in Wuhan City, Hubei Province, China in December 2019. By Feb. 11, 2020, the World Health Organization (WHO) officially named the disease resulting from infection with SARS-CoV-2 as coronavirus disease 2019 (COVID-19). COVID-19 represents a spectrum of clinical manifestations that typically include fever, dry cough, and fatigue, often with pulmonary involvement. SARS-CoV-2 is highly contagious and most individuals within the population at large are susceptible to infection. Wild animal hosts and infected patients are currently the main sources of disease which is transmitted via respiratory droplets and direct contact. Since the outbreak, the Chinese government and scientific community have acted rapidly to identify the causative agent and promptly shared the viral gene sequence, and have carried out measures to contain the epidemic. Meanwhile, recent research has revealed critical aspects of SARS-CoV-2 biology and disease pathogenesis; other studies have focused on epidemiology, clinical features, diagnosis, management, as well as drug and vaccine development. This review aims to summarize the latest research findings and to provide expert consensus. We will also share ongoing efforts and experience in China, which may provide insight on how to contain the epidemic and improve our understanding of this emerging infectious disease, together with updated guidance for prevention, control, and critical management of this pandemic.