I have attached my instruction file and scientific file below
Instructions for the paper 1. Write 5 critiques about the scientific paper that I have uploaded. 2. Below are the list of uncertainties I studied in the class. Sources of uncertainty about phylogenetic trees. Please critique based on the information below. 1. Uncertainty about common ancestry 2. Uncertainty about the topology 3. Uncertainty about the branch lengths 4. Uncertainty about the substitution model 5. Uncertainty about the tree estimation method 6. Uncertainty about the statistical method • Uncertainty about prior probabilities Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding Articles www.thelancet.com Vol 395 February 22, 2020 565 Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding Roujian Lu*, Xiang Zhao*, Juan Li*, Peihua Niu*, Bo Yang*, Honglong Wu*, Wenling Wang, Hao Song, Baoying Huang, Na Zhu, Yuhai Bi, Xuejun Ma, Faxian Zhan, Liang Wang, Tao Hu, Hong Zhou, Zhenhong Hu, Weimin Zhou, Li Zhao, Jing Chen, Yao Meng, Ji Wang, Yang Lin, Jianying Yuan, Zhihao Xie, Jinmin Ma, William J Liu, Dayan Wang, Wenbo Xu, Edward C Holmes, George F Gao, Guizhen Wu¶, Weijun Chen¶, Weifeng Shi¶, Wenjie Tan¶ Summary 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. Methods We 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 angiotensin- converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. Funding National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University. Copyright © 2020 Elsevier Ltd. All rights reserved. Introduction Viruses of the family Coronaviridae possess a single- strand, positive-sense RNA genome ranging from 26 to 32 kilobases in length.1 Coronaviruses have been identified in several avian hosts,2,3 as well as in various mammals, including camels, bats, masked palm civets, mice, dogs, and cats. Novel mammalian coronaviruses are now regularly identified.1 For example, an HKU2- related coronavirus of bat origin was responsible for a fatal acute diarrhoea syndrome in pigs in 2018.4 Among the several coronaviruses that are pathogenic to humans, most are associated with mild clinical symptoms,1 with two notable exceptions: severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), a novel betacoronavirus that emerged in Guangdong, southern China, in November, 2002,5 and resulted in more than 8000 human infections and 774 deaths in 37 countries during 2002–03;6 and Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), which was first detected in Saudi Arabia in 20127 and was responsible Lancet 2020; 395: 565–74 Published Online January 29, 2020 https://doi.org/10.1016/ S0140-6736(20)30251-8 *Contributed equally ¶Contributed equally NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China (Prof R Lu MSc, X Zhao MD, P Niu PhD, Prof W Wang PhD, B Huang PhD, N Zhu PhD, Prof X Ma PhD, Prof W Zhou MD, L Zhao PhD, Y Meng PhD, J Wang PhD, Prof W J Liu PhD, Prof D Wang PhD, Prof W Xu MD, Prof G F Gao DPhil, Prof G Wu MD, Prof W Tan MD); Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Universities of Shandong, Shandong First Medical University and Shandong Academy of Medical Sciences, Tai’an, China (J Li PhD, T Hu MSc, H Zhou PhD, Prof W Shi PhD); Division for Viral Disease Detection, Hubei Provincial Center for Disease Control and Prevention, Wuhan, China (B Yang MSc, Prof F Zhan PhD); BGI PathoGenesis Pharmaceutical Technology, Shenzhen, China (H Wu MSc, Y Lin BS, J Yuan MSc, Z Xie BS, J Ma PhD, Prof W Chen PhD); Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China (H Song PhD); Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (Prof Y Bi PhD, L Wang PhD, Prof G F Gao); Center for Influenza Research http://crossmark.crossref.org/dialog/?doi=10.1016/S0140-6736(20)30251-8&domain=pdf Articles 566 www.thelancet.com Vol 395 February 22, 2020 for 2494 laboratory-confirmed cases of infection and 858 fatalities since September, 2012, including 38 deaths following a single introduction into South Korea.8,9 In late December, 2019, several patients with viral pneumonia were found to be epidemiologically associ- ated with the Huanan seafood market in Wuhan, in the Hubei province of China, where a number of non-aquatic animals such as birds and rabbits were also on sale before the outbreak. A novel, human-infecting corona- virus,10,11 provisionally named 2019 novel corona virus (2019-nCoV), was identified with use of next-generation sequencing. As of Jan 28, 2020, China has reported more than 5900 confirmed and more than 9000 suspected cases of 2019-nCoV infection across 33 Chinese prov- inces or municipalities, with 106 fatalities. In addition, 2019-nCoV has now been reported in Thailand, Japan, South Korea, Malaysia, Singapore, and the USA. Infec- tions in medical workers and family clusters were also reported and human-to-human transmission has been confirmed.12 Most of the infected patients had a high fever and some had dyspnoea, with chest radiographs revealing invasive lesions in both lungs.12,13 We report the epidemiological data of nine inpatients, from at least three hospitals in Wuhan, who were diagnosed with viral pneumonia of unidentified cause. Using next-generation sequencing of bronchoalveolar lavage fluid samples and cultured isolates from these patients, 2019-nCoV was found. We describe the genomic characterisation of ten genomes of this novel virus, providing important information on the origins and cell receptor binding of the virus. Methods Patients and samples Nine patients with viral pneumonia and negative for common respiratory pathogens, who presented to at least three hospitals in Wuhan, were included in this study. Eight of the patients had visited the Huanan seafood market before the onset of illness, and one patient (WH04) did not visit the market but stayed in a hotel near the market between Dec 23 and Dec 27, 2019 (table). Five of the patients (WH19001, WH19002, WH19004, WH19008, and YS8011) had samples collected by the Chinese Center for Disease Control and Prevention (CDC) which were tested for 18 viruses and four bac- teria using the RespiFinderSmart22 Kit (PathoFinder, Maastricht, Netherlands) on the LightCycler 480 Real- Time PCR system (Roche, Rotkreuz, Switzerland). Presence of SARS-CoV and MERS-CoV was tested using a previously reported method.14 All five CDC samples were negative for all common respiratory pathogens screened for. Four of the patients (WH01, WH02, WH03, and WH04) had samples collected by BGI (Beijing, China), and were tested for five viruses and one bacterium using the RespiPathogen 6 Kit (Jiangsu Macro & Micro Test, Nantong, China) on the Applied Biosystems ABI 7500 Real-Time PCR system (ThermoFisher Scientific, Foster City, CA, USA). All four samples were negative for the targeted respiratory pathogens. Virus isolation Special-pathogen-free human airway epithelial (HAE) cells were used for virus isolation. Briefly, bronchoalveolar lavage fluids or throat swabs from the patients were inoculated into the HAE cells through the apical surfaces. HAE cells were maintained in an air–liquid interface incubated at 37°C. The cells were monitored daily for cytopathic effects by light microscopy and the cell supernatants were collected for use in quantitative RT-PCR assays. After three passages, apical samples were collected for sequencing. BGI sequencing strategy All collected samples were sent to BGI for sequencing. 140 μL bronchoalveolar lavage fluid samples (WH01 to WH04) were reserved for RNA extraction using the QIAamp Viral RNA Mini Kit (52904; Qiagen, Heiden, Germany), according to the manufacturer’s recom- mendations. A probe-captured technique was used to remove human nucleic acid. The remaining RNA was