Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

doi:10.1016/J.CELL.2020.04.021

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Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

Journal Article•DOI•

Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

Joseph R. Fauver¹, Mary E. Petrone¹, Emma B. Hodcroft², Emma B. Hodcroft³, Kayoko Shioda¹, Hanna Y. Ehrlich¹, Alexander Watts, Chantal B.F. Vogels¹, Anderson F. Brito¹, Tara Alpert¹, Anthony Muyombwe⁴, Jafar Razeq⁴, Randy Downing⁴, Nagarjuna R. Cheemarla¹, Anne L. Wyllie¹, Chaney C. Kalinich¹, Isabel M. Ott¹, Joshua Quick⁵, Nicholas J. Loman⁵, Karla M. Neugebauer¹, Alexander L. Greninger⁶, Alexander L. Greninger⁷, Keith R. Jerome⁷, Keith R. Jerome⁶, Pavitra Roychoudhury⁷, Pavitra Roychoudhury⁶, Hong Xie⁶, Lasata Shrestha⁶, Meei Li Huang⁷, Meei Li Huang⁶, Virginia E. Pitzer¹, Akiko Iwasaki¹, Akiko Iwasaki⁸, Saad B. Omer, Kamran Khan⁹, Kamran Khan¹⁰, Isaac I. Bogoch¹⁰, Richard A. Martinello¹, Ellen F. Foxman¹, Marie L. Landry¹, Richard A. Neher², Richard A. Neher³, Albert I. Ko¹, Nathan D. Grubaugh¹ - Show less +40 more•Institutions (10)

Yale University¹, University of Basel², Swiss Institute of Bioinformatics³, United States Department of State⁴, University of Birmingham⁵, University of Washington⁶, Fred Hutchinson Cancer Research Center⁷, Howard Hughes Medical Institute⁸, St. Michael's Hospital⁹, University of Toronto¹⁰

07 May 2020-Cell (Elsevier)-Vol. 181, Iss: 5

TL;DR: It is shown that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions, and the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of the estimated effects of federal travel restrictions.

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About: This article is published in Cell.The article was published on 2020-05-07 and is currently open access. It has received 307 citations till now.

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Journal Article•DOI•

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

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Bette T. Korber¹, Will Fischer¹, Sandrasegaram Gnanakaran¹, Hyejin Yoon¹, James Theiler¹, Werner Abfalterer¹, Nick Hengartner¹, Elena E. Giorgi¹, Tanmoy Bhattacharya¹, Brian T. Foley¹, Kathryn M. Hastie², Matthew Parker³, David G Partridge, Cariad Evans, Timothy M. Freeman³, Thushan I de Silva³, Adrienne Angyal⁴, Rebecca Brown⁴, Laura Carrilero⁴, Luke R. Green⁵, Luke R. Green², Luke R. Green⁶, Danielle C. Groves⁶, Katie Johnson⁶, Alexander J Keeley³, Benjamin B Lindsey⁴, Paul J. Parsons⁴, Mohammad Raza, Sarah Rowland-Jones, Nikki Smith, Rachel Tucker, Dennis Wang, Matthew Wyles, Charlene McDanal⁴, Lautaro G. Perez⁴, Haili Tang⁴, Alex Moon-Walker⁶, Alex Moon-Walker², Alex Moon-Walker⁵, Sean P. J. Whelan⁶, Celia C. LaBranche⁴, Erica Ollmann Saphire², David C. Montefiori⁴ - Show less +39 more•Institutions (6)

Los Alamos National Laboratory¹, La Jolla Institute for Allergy and Immunology², University of Sheffield³, Durham University⁴, Harvard University⁵, Washington University in St. Louis⁶

20 Aug 2020-Cell

TL;DR: A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic, and it is found that the G614 variant grows to higher titer as pseudotyped virions.

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3,302 citations

Cites background from "Coast-to-Coast Spread of SARS-CoV-2..."

..., 2013), and SARS-CoV-2 sequence diversity is very low (Fauver et al., 2020)....
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Journal Article•DOI•

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.

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Tyler N. Starr¹, Allison J. Greaney², Allison J. Greaney¹, Sarah K Hilton², Sarah K Hilton¹, Daniel Ellis², Katharine H.D. Crawford¹, Katharine H.D. Crawford², Adam S. Dingens¹, Mary Jane Navarro², John E. Bowen², M. Alejandra Tortorici², Alexandra C. Walls², Neil P. King², David Veesler², Jesse D. Bloom², Jesse D. Bloom¹, Jesse D. Bloom³ - Show less +14 more•Institutions (3)

Fred Hutchinson Cancer Research Center¹, University of Washington², Howard Hughes Medical Institute³

03 Sep 2020-Cell

TL;DR: It is found that a substantial number of mutations to the RBD are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses.

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1,517 citations

Cites background from "Coast-to-Coast Spread of SARS-CoV-2..."

...These genomic sequences have been useful for understanding viral emergence and spread (Andersen et al., 2020; Bedford et al., 2020; Fauver et al., 2020), but the lack of corresponding high-throughput...
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Journal Article•DOI•

Spike mutation D614G alters SARS-CoV-2 fitness.

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Jessica A. Plante¹, Yang Liu¹, Jianying Liu¹, Hongjie Xia¹, Bryan A. Johnson¹, Kumari G. Lokugamage¹, Xianwen Zhang¹, Antonio E. Muruato¹, Jing Zou¹, Camila R. Fontes-Garfias¹, Divya Mirchandani¹, Dionna Scharton¹, John P. Bilello, Zhiqiang Ku², Zhiqiang An², Birte Kalveram¹, Alexander N. Freiberg, Vineet D. Menachery¹, Xuping Xie¹, Kenneth S. Plante¹, Scott C. Weaver, Pei Yong Shi - Show less +18 more•Institutions (2)

University of Texas Medical Branch¹, University of Texas Health Science Center at Houston²

01 Apr 2021-Nature

TL;DR: Hamsters infected with SARS-CoV-2 expressing spike D614G (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission.

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Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

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1,285 citations

Journal Article•DOI•

Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater.

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Artem Nemudryi¹, Anna Nemudraia¹, Tanner Wiegand¹, Kevin Surya¹, Murat Buyukyoruk¹, Calvin Cicha¹, Karl K Vanderwood, Royce A. Wilkinson¹, Blake Wiedenheft¹ - Show less +5 more•Institutions (1)

Montana State University¹

22 Sep 2020

TL;DR: It is shown that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results, and how genome sequencing can be used for genotyping viral strains circulating in a community.

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Abstract: SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.

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415 citations

Journal Article•DOI•

Introductions and early spread of SARS-CoV-2 in the New York City area.

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Ana S. Gonzalez-Reiche¹, Matthew M. Hernandez¹, Mitchell J. Sullivan¹, Brianne Ciferri¹, Hala Alshammary¹, Ajay Obla¹, Shelcie Fabre¹, Giulio Kleiner¹, Jose Polanco¹, Zenab Khan¹, Bremy Alburquerque¹, Adriana van de Guchte¹, Jayeeta Dutta¹, Nancy Francoeur¹, Betsaida Salom Melo¹, Irina Oussenko¹, Gintaras Deikus¹, Juan C. Diaz Soto¹, Shwetha Hara Sridhar¹, Ying-Chih Wang¹, Kathryn Twyman², Andrew Kasarskis¹, Andrew Kasarskis², Deena R. Altman¹, Melissa Smith¹, Robert Sebra, Judith A. Aberg¹, Florian Krammer¹, Adolfo García-Sastre, Marta Luksza¹, Gopi Patel¹, Alberto Paniz-Mondolfi¹, Melissa R. Gitman¹, Emilia Mia Sordillo¹, Viviana Simon², Viviana Simon¹, Harm van Bakel¹ - Show less +33 more•Institutions (2)

Icahn School of Medicine at Mount Sinai¹, Mount Sinai Health System²

17 Jul 2020-Science

TL;DR: To identify the early transmission events underlying the rapid spread of the virus in the NYC metropolitan area, the virus that causes coronavirus disease 2019 (COVID-19) was sequenced in patients seeking care at the Mount Sinai Health System.

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Abstract: New York City (NYC) has emerged as one of the epicenters of the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To identify the early transmission events underlying the rapid spread of the virus in the NYC metropolitan area, we sequenced the virus that causes coronavirus disease 2019 (COVID-19) in patients seeking care at the Mount Sinai Health System. Phylogenetic analysis of 84 distinct SARS-CoV-2 genomes indicates multiple, independent, but isolated introductions mainly from Europe and other parts of the United States. Moreover, we found evidence for community transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different neighborhoods of the city.

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365 citations

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References

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Journal Article•

R: A language and environment for statistical computing.

[...]

R Core Team

01 Jan 2014-MSOR connections

TL;DR: Copyright (©) 1999–2012 R Foundation for Statistical Computing; permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and permission notice are preserved on all copies.

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Abstract: Copyright (©) 1999–2012 R Foundation for Statistical Computing. Permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and this permission notice are preserved on all copies. Permission is granted to copy and distribute modified versions of this manual under the conditions for verbatim copying, provided that the entire resulting derived work is distributed under the terms of a permission notice identical to this one. Permission is granted to copy and distribute translations of this manual into another language, under the above conditions for modified versions, except that this permission notice may be stated in a translation approved by the R Core Team.

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272,030 citations

Journal Article•DOI•

A pneumonia outbreak associated with a new coronavirus of probable bat origin

[...]

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 - Show less +25 more•Institutions (3)

Chinese Academy of Sciences¹, Wuhan Jinyintan Hospital², Centers for Disease Control and Prevention³

03 Feb 2020-Nature

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.

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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.

...read moreread less

16,857 citations

"Coast-to-Coast Spread of SARS-CoV-2..." refers background in this paper

...A novel coronavirus, known as SARS-CoV-2, was identified as the cause of an outbreak of pneumonia in Wuhan, China, in December 2019 (Gorbalenya et al., 2020; Wu et al., 2020; Zhou et al., 2020)....
[...]

Journal Article•DOI•

IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies

[...]

Lam Tung Nguyen¹, Heiko A. Schmidt¹, Arndt von Haeseler¹, Bui Quang Minh¹•Institutions (1)

Medical University of Vienna¹

01 Jan 2015-Molecular Biology and Evolution

TL;DR: It is shown that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented and found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space.

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Abstract: Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3-97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.

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13,668 citations

"Coast-to-Coast Spread of SARS-CoV-2..." refers methods in this paper

...Specifically, we aligned sequences using mafft (Katoh et al., 2002), masked sites at the 50 and 30 ends of the alignment as well as a small number of sites that likely vary due to assembly artifacts (see https://github.com/nextstrain/ncov), and reconstructed a phylogeny using IQ-Tree (Nguyen et al., 2015)....
[...]
...…NCBI https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA614976 Software and Algorithms R CRAN https://cran.r-project.org/ IQ-Tree http://www.iqtree.org/ Nguyen et al., 2015 augur toolkit https://bedford.io/projects/augur/ Hadfield et al., 2018 MAFFT https://mafft.cbrc.jp/alignment/software/ Katoh et…...
[...]
...4.1 Flow cell Oxford Nanopore Tech FLO-MIN106D Blunt/TA Ligase Master Mix New England BioLabs MO367L NEBNext Ultra II End Repair/dA-Tailing Module New England BioLabs E7546S NEBNext Quick Ligation Module New England BioLabs E6056S Deposited Data International COVID-19 cases ECDC https://ourworldindata.org/ coronavirus-source-data U.S. COVID-19 cases JHU Dong et al., 2020 Air passenger volumes (commercial) IATA https://www.iata.org/pages/default.aspx SARS-CoV-2 Genomes GISAID (EPI_ISL_416416-416424) https://www.gisaid.org/ SARS-CoV-2 Sequencing Data SRA, NCBI https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA614976 Software and Algorithms R CRAN https://cran.r-project.org/ IQ-Tree http://www.iqtree.org/ Nguyen et al., 2015 augur toolkit https://bedford.io/projects/augur/ Hadfield et al., 2018 MAFFT https://mafft.cbrc.jp/alignment/software/ Katoh et al., 2002 treetime https://github.com/neherlab/treetime Sagulenko et al., 2018 RAMPART ARTIC Network https://github.com/artic-network/rampart ARTIC Network Bioinformatic protocol ARTIC Network https://artic.network/ncov-2019/ ncov2019-bioinformatics-sop.html Nextstrain https://nextstrain.org/ Hadfield et al., 2018 Other Amplicon sequencing protocol PrimalSeq Quick et al., 2017...
[...]
...…we aligned sequences using mafft (Katoh et al., 2002), masked sites at the 50 and 30 ends of the alignment as well as a small number of sites that likely vary due to assembly artifacts (see https://github.com/nextstrain/ncov), and reconstructed a phylogeny using IQ-Tree (Nguyen et al., 2015)....
[...]

Journal Article•DOI•

MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform

[...]

Kazutaka Katoh¹, Kazuharu Misawa, Kei-ichi Kuma¹, Takashi Miyata¹•Institutions (1)

Kyoto University¹

15 Jul 2002-Nucleic Acids Research

TL;DR: A simplified scoring system is proposed that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length.

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Abstract: A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homologous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.

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12,003 citations

"Coast-to-Coast Spread of SARS-CoV-2..." refers background or methods in this paper

...Specifically, we aligned sequences using mafft (Katoh et al., 2002), masked sites at the 50 and 30 ends of the alignment as well as a small number of sites that likely vary due to assembly artifacts (see https://github.com/nextstrain/ncov), and reconstructed a phylogeny using IQ-Tree (Nguyen et…...
[...]
...…Nguyen et al., 2015 augur toolkit https://bedford.io/projects/augur/ Hadfield et al., 2018 MAFFT https://mafft.cbrc.jp/alignment/software/ Katoh et al., 2002 treetime https://github.com/neherlab/treetime Sagulenko et al., 2018 RAMPART ARTIC Network…...
[...]

Journal Article•DOI•

A new coronavirus associated with human respiratory disease in China.

[...]

Fan Wu¹, Su Zhao², Bin Yu³, Yan-Mei Chen¹, Wen Wang³, Zhi gang Song¹, Yi Hu², Zhao Wu Tao², Jun Hua Tian³, Yuan Yuan Pei¹, Ming Li Yuan², Yu Ling Zhang¹, Fa Hui Dai¹, Yi Liu¹, Qi Min Wang¹, Jiao Jiao Zheng¹, Lin Xu¹, Edward C. Holmes⁴, Edward C. Holmes¹, Yong-Zhen Zhang³, Yong-Zhen Zhang¹ - Show less +17 more•Institutions (4)

Fudan University¹, Huazhong University of Science and Technology², Centers for Disease Control and Prevention³, University of Sydney⁴

03 Feb 2020-Nature

TL;DR: Phylogenetic and metagenomic analyses of the complete viral genome of a new coronavirus from the family Coronaviridae reveal that the virus is closely related to a group of SARS-like coronaviruses found in bats in China.

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Abstract: Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health1–3. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China5. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans. Phylogenetic and metagenomic analyses of the complete viral genome of a new coronavirus from the family Coronaviridae reveal that the virus is closely related to a group of SARS-like coronaviruses found in bats in China.

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9,231 citations

"Coast-to-Coast Spread of SARS-CoV-2..." refers background in this paper

...A novel coronavirus, known as SARS-CoV-2, was identified as the cause of an outbreak of pneumonia in Wuhan, China, in December 2019 (Gorbalenya et al., 2020; Wu et al., 2020; Zhou 990 Cell 181, 990–996, May 28, 2020 ª 2020 Elsevier Inc. et al., 2020)....
[...]
...A novel coronavirus, known as SARS-CoV-2, was identified as the cause of an outbreak of pneumonia in Wuhan, China, in December 2019 (Gorbalenya et al., 2020; Wu et al., 2020; Zhou et al., 2020)....
[...]