SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.
TL;DR: The sensitivity of the two viruses to three established inhibitors of coronavirus replication is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha.
Abstract: The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays
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TL;DR: The predominant pattern of lung lesions in patients with COVID-19 patients is diffuse alveolar damage, as described in patients infected with severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses.
Abstract: Summary Background COVID-19 is characterised by respiratory symptoms, which deteriorate into respiratory failure in a substantial proportion of cases, requiring intensive care in up to a third of patients admitted to hospital. Analysis of the pathological features in the lung tissues of patients who have died with COVID-19 could help us to understand the disease pathogenesis and clinical outcomes. Methods We systematically analysed lung tissue samples from 38 patients who died from COVID-19 in two hospitals in northern Italy between Feb 29 and March 24, 2020. The most representative areas identified at macroscopic examination were selected, and tissue blocks (median seven, range five to nine) were taken from each lung and fixed in 10% buffered formalin for at least 48 h. Tissues were assessed with use of haematoxylin and eosin staining, immunohistochemical staining for inflammatory infiltrate and cellular components (including staining with antibodies against CD68, CD3, CD45, CD61, TTF1, p40, and Ki-67), and electron microscopy to identify virion localisation. Findings All cases showed features of the exudative and proliferative phases of diffuse alveolar damage, which included capillary congestion (in all cases), necrosis of pneumocytes (in all cases), hyaline membranes (in 33 cases), interstitial and intra-alveolar oedema (in 37 cases), type 2 pneumocyte hyperplasia (in all cases), squamous metaplasia with atypia (in 21 cases), and platelet–fibrin thrombi (in 33 cases). The inflammatory infiltrate, observed in all cases, was largely composed of macrophages in the alveolar lumina (in 24 cases) and lymphocytes in the interstitium (in 31 cases). Electron microscopy revealed that viral particles were predominantly located in the pneumocytes. Interpretation The predominant pattern of lung lesions in patients with COVID-19 patients is diffuse alveolar damage, as described in patients infected with severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. Hyaline membrane formation and pneumocyte atypical hyperplasia are frequent. Importantly, the presence of platelet–fibrin thrombi in small arterial vessels is consistent with coagulopathy, which appears to be common in patients with COVID-19 and should be one of the main targets of therapy. Funding None.
1,027 citations
TL;DR: In this article, structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the spike (S) protein with ACE2, engagement of the receptor-binding domain of the S protein with ACS, proteolytic activation of S protein, endocytosis and membrane fusion are provided.
Abstract: The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.
988 citations
Beata Turoňová1, Beata Turoňová2, Mateusz Sikora1, Christoph Schürmann3, Wim J. H. Hagen2, Sonja Welsch1, Florian Blanc1, Sören von Bülow1, Michael Gecht1, Katrin Bagola3, Cindy Hörner3, Ger van Zandbergen4, Ger van Zandbergen3, Jonathan J M Landry2, Nayara Trevisan Doimo de Azevedo2, Shyamal Mosalaganti2, Shyamal Mosalaganti1, Andre Schwarz2, Roberto Covino5, Roberto Covino1, Michael D. Mühlebach3, Gerhard Hummer1, Gerhard Hummer6, Jacomine Krijnse Locker3, Martin Beck1, Martin Beck2 •
TL;DR: The stalk domain of S contains three hinges, giving the head unexpected orientational freedom and it is proposed that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat.
Abstract: The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
461 citations
TL;DR: It is proposed that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs. Here the authors visualize SARS-CoV-2 infected cells by in situ cryo-electron tomography, delineating the structural organization and conformational changes that occur during virus replication and budding; and provide insight into vRNP architecture and RNA networks in double membrane vesicles.
452 citations
References
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TL;DR: Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
Abstract: As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
37,898 citations
Na Zhu1, Dingyu Zhang, Wenling Wang1, Xingwang Li2, Bo Yang1, Jingdong Song1, Xiang Zhao1, Baoying Huang1, Weifeng Shi, Roujian Lu1, Peihua Niu1, Faxian Zhan1, Xuejun Ma1, Dayan Wang1, Wenbo Xu1, Wenbo Xu3, Guizhen Wu1, George F. Gao, Wenjie Tan1 •
TL;DR: Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily, which is the seventh member of the family of coronaviruses that infect humans.
Abstract: In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).
21,455 citations
Peng Zhou1, Xing-Lou Yang1, Xian Guang Wang2, Ben Hu1, Lei Zhang1, Wei Zhang1, Hao Rui Si1, Yan Zhu1, Bei Li1, Chao Lin Huang2, Hui-Dong Chen2, Jing Chen1, Yun Luo1, Hua Guo1, Ren Di Jiang1, Meiqin Liu1, Ying Chen1, Xu Rui Shen1, Xi Wang1, Xiao Shuang Zheng1, Kai Zhao1, Quanjiao Chen1, Fei Deng1, Lin Lin Liu3, Bing Yan1, Fa Xian Zhan3, Yan-Yi Wang1, Gengfu Xiao1, Zhengli Shi1 •
TL;DR: Identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China, and it is shown that this virus belongs to the species of SARSr-CoV, indicates that the virus is related to a bat coronav virus.
Abstract: Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1–4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5–7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV. Characterization of full-length genome sequences from patients infected with a new coronavirus (2019-nCoV) shows that the sequences are nearly identical and indicates that the virus is related to a bat coronavirus.
16,857 citations
"SARS-coronavirus-2 replication in V..." refers background in this paper
...This sequence constitutes a (potential) processing site that is present in a subset of CoVs (including SARSCoV-2 and MERS- CoV) but is lacking in others, like SARSCoV and certain bat CoVs [61, 63]....
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...On the one hand, there are four endemic human CoVs (HCoVs), the first of which were identified in the 1960s, annually causing a substantial number of common colds [3, 4]....
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...On the other hand, we now know of (at least) three zoonotic CoVs that recently have caused outbreaks in the human population: severe acute respiratory syndrome coronavirus (SARS- CoV) [5, 6] in 2002–2003, Middle East respiratory syndrome- coronavirus (MERS- CoV) [7, 8] since 2012 (and probably earlier) and the current pandemic SARS- CoV-2 [9, 10]....
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...This deletion maps to a previously described ‘hypervariable region’ in the otherwise conserved 3′ UTR, and in particular to the so- called s2m motif [72] that is conserved among CoVs and also found in several other virus groups [73, 74]....
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...To date, seven CoVs that can infect humans have been identified, which segregate into two classes....
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TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.
15,362 citations
"SARS-coronavirus-2 replication in V..." refers background in this paper
...Many reports posted over the past months have described such similarities, including the common affinity of the two viruses for the angiotensinconverting enzyme 2 (ACE2) receptor [9, 31]....
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Roujian Lu1, Xiang Zhao1, Juan Li2, Peihua Niu1, Bo Yang3, Honglong Wu, Wenling Wang1, Hao Song4, Baoying Huang1, Na Zhu1, Yuhai Bi4, Xuejun Ma1, Faxian Zhan3, Liang Wang4, Tao Hu2, Hong Zhou2, Zhenhong Hu, Weimin Zhou1, Li Zhao1, Jing Chen5, Yao Meng1, Ji Wang1, Yang Lin, Jianying Yuan, Zhihao Xie, Jinmin Ma, William J. Liu1, Dayan Wang1, Wenbo Xu1, Edward C. Holmes6, George F. Gao4, George F. Gao1, Guizhen Wu1, Weijun Chen, Weifeng Shi2, Wenjie Tan4, Wenjie Tan1 •
TL;DR: The 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.
9,474 citations
"SARS-coronavirus-2 replication in V..." refers background in this paper
...The newly emerged SARS- CoV-2 was rapidly identified as a CoV that is relatively closely related to the 2003 SARSCoV [9, 29, 30]....
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...Cross- reactivity of antisera to SARSCoV-2 targets was evaluated microscopically by immunofluorescence assay (IFA) and for some antisera (nsp3 and N protein) also by Western blot analysis....
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...Based on the close ancestral relationship between SARSCoV-2 and SARS- CoV [98], one might expect that the patterns and modes of interaction with host antiviral defence mechanisms would be similar....
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...Screening of other available SARSCoV-2 genome sequences indicated that the presence of this deletion apparently is unique for this particular isolate, and likely represents an additional adaptation acquired during cell- culture passaging....
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...During the early stages of infection (until 8 h p.i.), the growth curves of the two viruses were similar, but subsequently cells infected with SARS- CoV clearly produced more infectious progeny (about 50- fold more) than SARSCoV-2- infected cells, with both viruses reaching their plateau by about 14 h p.i....
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