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Post by Admin on Mar 11, 2020 20:42:18 GMT
Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Daniel Wrapp1,*, Nianshuang Wang1,*, Kizzmekia S. Corbett2, Jory A. Goldsmith1, Ching-Lin Hsieh1, Olubukola Abiona2, Barney S. Graham2, Jason S. McLellan1,† Science 19 Feb 2020 Abstract The outbreak of a novel betacoronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development, we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis. The novel coronavirus 2019-nCoV has recently emerged as a human pathogen in the city of Wuhan in China’s Hubei province, causing fever, severe respiratory illness and pneumonia—a disease recently named COVID-19 (1, 2). According to the World Health Organization (WHO) on February 16th, 2020, there had been over 51,000 confirmed cases globally, leading to at least 1,600 deaths. The emerging pathogen was rapidly characterized as a novel member of the betacoronavirus genus, closely related to several bat coronaviruses as well as severe acute respiratory syndrome coronavirus (SARS-CoV) (3, 4). Compared to SARS-CoV, 2019-nCoV appears to be more readily transmitted from human-to-human, spreading to multiple continents and leading to the WHO declaration of a Public Health Emergency of International Concern (PHEIC) on January 30th, 2020 (1, 5, 6). 2019-nCoV makes use of a densely glycosylated spike (S) protein to gain entry into host cells. The S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a dramatic structural rearrangement to fuse the viral membrane with the host-cell membrane (7, 8). This process is triggered when the S1 subunit binds to a host-cell receptor. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the S1 subunit and transition of the S2 subunit to a stable postfusion conformation (9). In order to engage a host-cell receptor, the receptor-binding domain (RBD) of S1 undergoes hinge-like conformational movements that transiently hide or expose the determinants of receptor binding. These two states are referred to as the “down” conformation and the “up” conformation, where down corresponds to the receptor-inaccessible state and up corresponds to the receptor-accessible state, which is thought to be less stable (10–13). Due to the indispensable function of the S protein, it represents a target for antibody-mediated neutralization, and characterization of the prefusion S structure would provide atomic-level information to guide vaccine design and development. Based on the first reported genome sequence of 2019-nCoV (4), we expressed ectodomain residues 1−1208 of 2019-nCoV S, adding two stabilizing proline mutations in the C-terminal S2 fusion machinery based on a previous stabilization strategy that proved effective for other betacoronavirus S proteins (11, 14). Figure 1A shows the domain organization of the expression construct and figure S1 shows the purification process. We obtained roughly 0.5 mg/L of the recombinant prefusion-stabilized S ectodomain from FreeStyle 293 cells, and the protein was purified to homogeneity by affinity chromatography and size-exclusion chromatography (fig. S1). Cryo-EM grids were prepared using this purified, fully glycosylated S protein and preliminary screening revealed a high particle density with little aggregation near the edges of the holes. Fig. 1 Structure of 2019-nCoV S in the prefusion conformation. (A) Schematic of 2019-nCoV S primary structure, colored by domain. Domains that were excluded from the ectodomain expression construct or could not be visualized in the final map are colored white. SS= signal sequence, NTD= N-terminal domain, RBD= receptor-binding domain, SD1= subdomain 1, SD2= subdomain 2, S1/S2= S1/S2 protease cleavage site, S2′= S2′ protease cleavage site, FP= fusion peptide, HR1= heptad repeat 1, CH= central helix, CD= connector domain, HR2= heptad repeat 2, TM= transmembrane domain, CT= cytoplasmic tail. Arrows denote protease cleavage sites. (B) Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the up conformation. The two RBD-down protomers are shown as cryo-EM density in either white or gray and the RBD-up protomer is shown in ribbons, colored corresponding to the schematic in (A). After collecting and processing 3,207 micrograph movies, we obtained a 3.5 Å-resolution 3D reconstruction of an asymmetrical trimer in which a single RBD was observed in the up conformation. (Fig. 1B, fig. S2, and table S1). Due to the small size of the RBD (~21 kDa), the asymmetry of this conformation was not readily apparent until ab initio 3D reconstruction and 3D classification were performed (Fig. 1B and fig. S3). By using the 3D variability feature in cryoSPARC v2 (15), we observed breathing of the S1 subunits as the RBD underwent a hinge-like movement, which likely contributed to the relatively poor local resolution of S1 compared to the more stable S2 subunit (movies S1 and S2). This seemingly stochastic RBD movement has been captured during structural characterization of the closely related betacoronaviruses SARS-CoV and MERS-CoV, as well as the more distantly related alphacoronavirus porcine epidemic diarrhea virus (PEDV) (10, 11, 13, 16). The observation of this phenomenon in 2019-nCoV S suggests that it shares the same mechanism of triggering that is thought to be conserved among the Coronaviridae, wherein receptor-binding to exposed RBDs leads to an unstable 3 RBD-up conformation that results in shedding of S1 and refolding of S2 (11, 12). Because the S2 subunit appeared to be a symmetric trimer, we performed a 3D refinement imposing C3 symmetry, resulting in a 3.2 Å-resolution map, with excellent density for the S2 subunit. Using both maps, we built most of the 2019-nCoV S ectodomain, including glycans at 44 of the 66 N-linked glycosylation sites per trimer (fig. S4). Our final model spans S residues 27–1146, with several flexible loops omitted. Like all previously reported coronavirus S ectodomain structures, the density for 2019-nCoV S begins to fade after the connector domain (CD), reflecting the flexibility of the heptad repeat 2 (HR2) domain in the prefusion conformation (fig. S4A) (13, 16–18). The overall structure of 2019-nCoV S resembles that of SARS-CoV S, with a root mean square deviation (RMSD) of 3.8 Å over 959 Cα atoms (Fig. 2A). One of the larger differences between these two structures (although still relatively minor) is the position of the RBDs in their respective down conformations. Whereas the SARS-CoV RBD in the down conformation packs tightly against the N-terminal domain (NTD) of the neighboring protomer, the 2019-nCoV RBD in the down conformation is angled closer to the central cavity of the trimer (Fig. 2B). Despite this observed conformational difference, when the individual structural domains of 2019-nCoV S are aligned to their counterparts from SARS-CoV S, they reflect the high degree of structural homology between the two proteins, with the NTDs, RBDs, subdomains 1 and 2 (SD1 and SD2) and S2 subunits yielding individual RMSD values of 2.6 Å, 3.0 Å, 2.7 Å and 2.0 Å, respectively (Fig. 2C). Fig. 2 Structural comparison between 2019-nCoV S and SARS-CoV S. (A) A single protomer of 2019-nCoV S with the RBD in the down conformation (left) is shown in ribbons, colored according to Fig. 1. A protomer of 2019-nCoV S in the RBD-up conformation is shown (center) next to a protomer of SARS-CoV S in the RBD-up conformation (right), displayed as ribbons and colored white (PDB ID: 6CRZ). (B) The RBDs of 2019-nCoV and SARS-CoV have been aligned based on the position of the adjacent NTD from the neighboring protomer. The 2019-nCoV RBD is colored green and the SARS-CoV RBD is colored white. The 2019-nCoV NTD is colored blue. (C) The following structural domains from 2019-nCoV S have been aligned to their counterparts from SARS-CoV S: NTD (top left), RBD (top right), SD1 and SD2 (bottom left), and S2 (bottom right). 2019-nCoV S shares 98% sequence identity with the S protein from the bat coronavirus RaTG13, with the most notable variation arising from an insertion in the S1/S2 protease cleavage site that results in an “RRAR” furin recognition site in 2019-nCoV (19), rather than the single arginine in SARS-CoV (fig. S5) (20–23). Notably, in influenza viruses, amino acid insertions that create a polybasic furin site in a related position in influenza hemagglutinin proteins are often found in highly virulent avian and human influenza viruses (24). In the structure reported here, the S1/S2 junction is in a disordered solvent-exposed loop. In addition to this insertion of residues in the S1/S2 junction, 29 variant residues exist between 2019-nCoV S and RaTG13 S, with 17 of these positions mapping to the RBD (figs. S5 and S6). We also analyzed the 61 available 2019-nCoV S sequences in the Global Initiative on Sharing All Influenza Data (GISAID, gisaid.org) database and found that there were only 9 amino acid substitutions among all deposited sequences. Most of these substitutions are relatively conservative and are not expected to have a dramatic effect on the structure or function of the 2019-nCoV S protein (fig. S6). Recent reports demonstrating that 2019-nCoV S and SARS-CoV S share the same functional host-cell receptor—angiotensin-converting enzyme 2 (ACE2) (22, 25–27)—prompted us to quantify the kinetics of this interaction by surface plasmon resonance (SPR). ACE2 bound to 2019-nCoV S ectodomain with ~15 nM affinity, which is approximately 10- to 20-fold higher affinity than ACE2 binding to SARS-CoV S (Fig. 3A and fig. S7) (14). We also formed a complex of ACE2 bound to the 2019-nCoV S ectodomain and observed it by negative-stain EM, where it strongly resembled the complex formed between SARS-CoV S and ACE2, which has been observed at high-resolution by cryo-EM (Fig. 3B) (14, 28). The high affinity of 2019-nCoV S for human ACE2 may contribute to the apparent ease with which 2019-nCoV can spread from human-to-human (1), however, additional studies are needed to investigate this possibility.
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Post by Admin on Mar 12, 2020 0:10:05 GMT
Watch NBC News NOW special coverage hosted by Dr. John Torres - Coronavirus Crisis: Your Questions Answered. Fig. 3 2019-nCoV S binds human ACE2 with high affinity. (A) SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red. (B) Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein protomers colored tan, pink and green. The overall structural homology and shared receptor usage between SARS-CoV S and 2019-nCoV S prompted us to test published SARS-CoV RBD-directed monoclonal antibodies (mAbs) for cross-reactivity to the 2019-nCoV RBD (Fig. 4A). A 2019-nCoV RBD-SD1 fragment (S residues 319–591) was recombinantly expressed, and appropriate folding of this construct was validated by measuring ACE2 binding using biolayer interferometry (BLI) (Fig. 4B). Cross-reactivity of the SARS-CoV RBD-directed mAbs S230, m396 and 80R was then evaluated by BLI (12, 29–31). Despite the relatively high degree of structural homology between the 2019-nCoV RBD and the SARS-CoV RBD, no binding to the 2019-nCoV RBD could be detected for any of the three mAbs at the concentration tested (1 μM) (Fig. 4C), in contrast to the strong binding we observed to the SARS-CoV RBD (fig. S8). Although the epitopes of these three antibodies represent a relatively small percentage of the surface area of the 2019-nCoV RBD, the lack of observed binding suggests that SARS-directed mAbs will not necessarily be cross-reactive and that future antibody isolation and therapeutic design efforts will benefit from using 2019-nCoV S proteins as probes. Fig. 4 Antigenicity of the 2019-nCoV RBD. (A) The SARS-CoV RBD is shown as a white molecular surface (PDB ID: 2AJF), with residues that vary in the 2019-nCoV RBD colored red. The ACE2 binding site is outlined with a black dotted line. (B) A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red. (C) Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line. The rapid global spread of 2019-nCoV, prompting the PHEIC declaration by WHO signals the urgent need for coronavirus vaccines and therapeutics. Knowing the atomic-level structure of the 2019-nCoV spike will allow for additional protein engineering efforts that could improve antigenicity and protein expression for vaccine development. The structural data also facilitates the evaluation of 2019-nCoV spike mutations that will occur as the virus undergoes genetic drift and help define whether those residues have surface exposure and map to sites of known antibody epitopes for other coronavirus spike proteins. In addition, the structure provides assurance that the protein produced by this construct is homogeneous and in the prefusion conformation, which should maintain the most neutralization-sensitive epitopes when used as candidate vaccine antigens or B cell probes for isolating neutralizing human monoclonal antibodies. Furthermore, the atomic-level detail will enable the design and screening of small molecules with fusion-inhibiting potential. This information will support precision vaccine design and discovery of anti-viral therapeutics, accelerating medical countermeasure development.
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Post by Admin on Mar 12, 2020 21:06:08 GMT
Watch live coverage as New York City Mayor de Blasio gives an update on how the city is handling the COVID-19 spread.
Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China
Prof Chaolin Huang, MD et al.
Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients.
Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not.
Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα.
Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies.
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Post by Admin on Mar 13, 2020 21:27:34 GMT
Results By Jan 2, 2020, 41 admitted hospital patients were identified as laboratory-confirmed 2019-nCoV infection in Wuhan. 20 [49%]) of the 2019-nCoV-infected patients were aged 25–49 years, and 14 (34%) were aged 50–64 years (figure 1A). The median age of the patients was 49·0 years (IQR 41·0–58·0; table 1). In our cohort of the first 41 patients as of Jan 2, no children or adolescents were infected. Of the 41 patients, 13 (32%) were admitted to the ICU because they required high-flow nasal cannula or higher-level oxygen support measures to correct hypoxaemia. Most of the infected patients were men (30 [73%]); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Figure 1 Date of illness onset and age distribution of patients with laboratory-confirmed 2019-nCoV infection 27 (66%) patients had direct exposure to Huanan seafood market (figure 1B). Market exposure was similar between the patients with ICU care (nine [69%]) and those with non-ICU care (18 [64%]). The symptom onset date of the first patient identified was Dec 1, 2019. None of his family members developed fever or any respiratory symptoms. No epidemiological link was found between the first patient and later cases. The first fatal case, who had continuous exposure to the market, was admitted to hospital because of a 7-day history of fever, cough, and dyspnoea. 5 days after illness onset, his wife, a 53-year-old woman who had no known history of exposure to the market, also presented with pneumonia and was hospitalised in the isolation ward. The most common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38; table 1). More than half of patients (22 [55%] of 40) developed dyspnoea. The median duration from illness onset to dyspnoea was 8·0 days (IQR 5·0–13·0). The median time from onset of symptoms to first hospital admission was 7·0 days (4·0–8·0), to shortness of breath was 8·0 days (5·0–13·0), to ARDS was 9·0 days (8·0–14·0), to mechanical ventilation was 10·5 days (7·0–14·0), and to ICU admission was 10·5 days (8·0–17·0; figure 2). Figure 2 Timeline of 2019-nCoV cases after onset of illness The blood counts of patients on admission showed leucopenia (white blood cell count less than 4 × 109/L; ten [25%] of 40 patients) and lymphopenia (lymphocyte count <1·0 × 109/L; 26 [63%] patients; table 2). Prothrombin time and D-dimer level on admission were higher in ICU patients (median prothrombin time 12·2 s [IQR 11·2–13·4]; median D-dimer level 2·4 mg/L [0·6–14·4]) than non-ICU patients (median prothrombin time 10·7 s [9·8–12·1], p=0·012; median D-dimer level 0·5 mg/L [0·3–0·8], p=0·0042). Levels of aspartate aminotransferase were increased in 15 (37%) of 41 patients, including eight (62%) of 13 ICU patients and seven (25%) of 28 non-ICU patients. Hypersensitive troponin I (hs-cTnI) was increased substantially in five patients, in whom the diagnosis of virus-related cardiac injury was made. Most patients had normal serum levels of procalcitonin on admission (procalcitonin <0·1 ng/mL; 27 [69%] patients; table 2). Four ICU patients developed secondary infections. Three of the four patients with secondary infection had procalcitonin greater than 0·5 ng/mL (0·69 ng/mL, 1·46 ng/mL, and 6·48 ng/mL). On admission, abnormalities in chest CT images were detected among all patients. Of the 41 patients, 40 (98%) had bilateral involvement (table 2). The typical findings of chest CT images of ICU patients on admission were bilateral multiple lobular and subsegmental areas of consolidation (figure 3A). The representative chest CT findings of non-ICU patients showed bilateral ground-glass opacity and subsegmental areas of consolidation (figure 3B). Later chest CT images showed bilateral ground-glass opacity, whereas the consolidation had been resolved (figure 3C). Figure 3 Chest CT images Initial plasma IL1B, IL1RA, IL7, IL8, IL9, IL10, basic FGF, GCSF, GMCSF, IFNγ, IP10, MCP1, MIP1A, MIP1B, PDGF, TNFα, and VEGF concentrations were higher in both ICU patients and non-ICU patients than in healthy adults (appendix pp 6–7). Plasma levels of IL5, IL12p70, IL15, Eotaxin, and RANTES were similar between healthy adults and patients infected with 2019-nCoV. Further comparison between ICU and non-ICU patients showed that plasma concentrations of IL2, IL7, IL10, GCSF, IP10, MCP1, MIP1A, and TNFα were higher in ICU patients than non-ICU patients. All patients had pneumonia. Common complications included ARDS (12 [29%] of 41 patients), followed by RNAaemia (six [15%] patients), acute cardiac injury (five [12%] patients), and secondary infection (four [10%] patients; table 3). Invasive mechanical ventilation was required in four (10%) patients, with two of them (5%) had refractory hypoxaemia and received extracorporeal membrane oxygenation as salvage therapy. All patients were administered with empirical antibiotic treatment, and 38 (93%) patients received antiviral therapy (oseltamivir). Additionally, nine (22%) patients were given systematic corticosteroids. A comparison of clinical features between patients who received and did not receive systematic corticosteroids is in the appendix (pp 1–5). As of Jan 22, 2020, 28 (68%) of 41 patients have been discharged and six (15%) patients have died. Fitness for discharge was based on abatement of fever for at least 10 days, with improvement of chest radiographic evidence and viral clearance in respiratory samples from upper respiratory tract.
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Post by Admin on Mar 14, 2020 1:35:47 GMT
Discussion We report here a cohort of 41 patients with laboratory-confirmed 2019-nCoV infection. Patients had serious, sometimes fatal, pneumonia and were admitted to the designated hospital in Wuhan, China, by Jan 2, 2020. Clinical presentations greatly resemble SARS-CoV. Patients with severe illness developed ARDS and required ICU admission and oxygen therapy. The time between hospital admission and ARDS was as short as 2 days. At this stage, the mortality rate is high for 2019-nCoV, because six (15%) of 41 patients in this cohort died.
The number of deaths is rising quickly. As of Jan 24, 2020, 835 laboratory-confirmed 2019-nCoV infections were reported in China, with 25 fatal cases. Reports have been released of exported cases in many provinces in China, and in other countries; some health-care workers have also been infected in Wuhan. Taken together, evidence so far indicates human transmission for 2019-nCoV. We are concerned that 2019-nCoV could have acquired the ability for efficient human transmission.19 Airborne precautions, such as a fit-tested N95 respirator, and other personal protective equipment are strongly recommended. To prevent further spread of the disease in health-care settings that are caring for patients infected with 2019-nCoV, onset of fever and respiratory symptoms should be closely monitored among health-care workers. Testing of respiratory specimens should be done immediately once a diagnosis is suspected. Serum antibodies should be tested among health-care workers before and after their exposure to 2019-nCoV for identification of asymptomatic infections.
Similarities of clinical features between 2019-nCoV and previous betacoronavirus infections have been noted. In this cohort, most patients presented with fever, dry cough, dyspnoea, and bilateral ground-glass opacities on chest CT scans. These features of 2019-nCoV infection bear some resemblance to SARS-CoV and MERS-CoV infections.20, 21 However, few patients with 2019-nCoV infection had prominent upper respiratory tract signs and symptoms (eg, rhinorrhoea, sneezing, or sore throat), indicating that the target cells might be located in the lower airway. Furthermore, 2019-nCoV patients rarely developed intestinal signs and symptoms (eg, diarrhoea), whereas about 20–25% of patients with MERS-CoV or SARS-CoV infection had diarrhoea.21 Faecal and urine samples should be tested to exclude a potential alternative route of transmission that is unknown at this stage.
The pathophysiology of unusually high pathogenicity for SARS-CoV or MERS-CoV has not been completely understood. Early studies have shown that increased amounts of proinflammatory cytokines in serum (eg, IL1B, IL6, IL12, IFNγ, IP10, and MCP1) were associated with pulmonary inflammation and extensive lung damage in SARS patients.22 MERS-CoV infection was also reported to induce increased concentrations of proinflammatory cytokines (IFNγ, TNFα, IL15, and IL17).23 We noted that patients infected with 2019-nCoV also had high amounts of IL1B, IFNγ, IP10, and MCP1, probably leading to activated T-helper-1 (Th1) cell responses. Moreover, patients requiring ICU admission had higher concentrations of GCSF, IP10, MCP1, MIP1A, and TNFα than did those not requiring ICU admission, suggesting that the cytokine storm was associated with disease severity. However, 2019-nCoV infection also initiated increased secretion of T-helper-2 (Th2) cytokines (eg, IL4 and IL10) that suppress inflammation, which differs from SARS-CoV infection.22 Further studies are necessary to characterise the Th1 and Th2 responses in 2019-nCoV infection and to elucidate the pathogenesis. Autopsy or biopsy studies would be the key to understand the disease.
In view of the high amount of cytokines induced by SARS-CoV,22, 24 MERS-CoV,25, 26 and 2019-nCoV infections, corticosteroids were used frequently for treatment of patients with severe illness, for possible benefit by reducing inflammatory-induced lung injury. However, current evidence in patients with SARS and MERS suggests that receiving corticosteroids did not have an effect on mortality, but rather delayed viral clearance.27, 28, 29 Therefore, corticosteroids should not be routinely given systemically, according to WHO interim guidance.30 Among our cohort of 41 laboratory-confirmed patients with 2019-nCoV infection, corticosteroids were given to very few non-ICU cases, and low-to-moderate dose of corticosteroids were given to less than half of severely ill patients with ARDS. Further evidence is urgently needed to assess whether systematic corticosteroid treatment is beneficial or harmful for patients infected with 2019-nCoV.
No antiviral treatment for coronavirus infection has been proven to be effective. In a historical control study,31 the combination of lopinavir and ritonavir among SARS-CoV patients was associated with substantial clinical benefit (fewer adverse clinical outcomes). Arabi and colleagues initiated a placebo-controlled trial of interferon beta-1b, lopinavir, and ritonavir among patients with MERS infection in Saudi Arabia.32 Preclinical evidence showed the potent efficacy of remdesivir (a broad-spectrum antiviral nucleotide prodrug) to treat MERS-CoV and SARS-CoV infections.33, 34 As 2019-nCoV is an emerging virus, an effective treatment has not been developed for disease resulting from this virus. Since the combination of lopinavir and ritonavir was already available in the designated hospital, a randomised controlled trial has been initiated quickly to assess the efficacy and safety of combined use of lopinavir and ritonavir in patients hospitalised with 2019-nCoV infection.
Our study has some limitations. First, for most of the 41 patients, the diagnosis was confirmed with lower respiratory tract specimens and no paired nasopharyngeal swabs were obtained to investigate the difference in the viral RNA detection rate between upper and lower respiratory tract specimens. Serological detection was not done to look for 2019-nCoV antibody rises in 18 patients with undetectable viral RNA. Second, with the limited number of cases, it is difficult to assess host risk factors for disease severity and mortality with multivariable-adjusted methods. This is a modest-sized case series of patients admitted to hospital; collection of standardised data for a larger cohort would help to further define the clinical presentation, natural history, and risk factors. Further studies in outpatient, primary care, or community settings are needed to get a full picture of the spectrum of clinical severity. At the same time, finding of statistical tests and p values should be interpreted with caution, and non-significant p values do not necessarily rule out difference between ICU and non-ICU patients. Third, since the causative pathogen has just been identified, kinetics of viral load and antibody titres were not available. Finally, the potential exposure bias in our study might account for why no paediatric or adolescent patients were reported in this cohort. More effort should be made to answer these questions in future studies. Both SARS-CoV and MERS-CoV were believed to originate in bats, and these infections were transmitted directly to humans from market civets and dromedary camels, respectively.35 Extensive research on SARS-CoV and MERS-CoV has driven the discovery of many SARS-like and MERS-like coronaviruses in bats. In 2013, Ge and colleagues36 reported the whole genome sequence of a SARS-like coronavirus in bats with that ability to use human ACE2 as a receptor, thus having replication potentials in human cells.37 2019-nCoV still needs to be studied deeply in case it becomes a global health threat. Reliable quick pathogen tests and feasible differential diagnosis based on clinical description are crucial for clinicians in their first contact with suspected patients. Because of the pandemic potential of 2019-nCoV, careful surveillance is essential to monitor its future host adaption, viral evolution, infectivity, transmissibility, and pathogenicity.
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