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Genome Med ; 12(1): 57, 2020 06 30.
Article in English | MEDLINE | ID: covidwho-618232


BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.

Betacoronavirus/genetics , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Whole Genome Sequencing/methods , Coronavirus Infections , Genetic Variation/genetics , Host-Pathogen Interactions/genetics , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pneumonia, Viral , RNA, Viral/genetics
Emerg Microbes Infect ; 9(1): 761-770, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-29222


Circulating in China and 158 other countries and areas, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 (HCoV-19) infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signalling pathway in lymphocytes may be the cause of patients' lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response.

Bronchoalveolar Lavage Fluid , Chemokines/analysis , Coronavirus Infections/genetics , Cytokines/analysis , Leukocytes, Mononuclear , Pneumonia, Viral/genetics , Transcriptome , Apoptosis , Betacoronavirus , Coronavirus Infections/blood , Coronavirus Infections/immunology , Humans , Lymphopenia , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/immunology , RNA-Seq , Signal Transduction , Tumor Suppressor Protein p53
Lancet ; 395(10224): 565-574, 2020 02 22.
Article in English | MEDLINE | ID: covidwho-80


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.

Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Genome, Viral , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Receptors, Virus/metabolism , Betacoronavirus/metabolism , Bronchoalveolar Lavage Fluid/virology , China/epidemiology , Coronavirus Infections/diagnosis , Coronavirus Infections/transmission , DNA, Viral/genetics , Disease Reservoirs/virology , Genomics/methods , High-Throughput Nucleotide Sequencing/methods , Humans , Phylogeny , Pneumonia, Viral/diagnosis , Pneumonia, Viral/transmission , Sequence Alignment