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      RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak


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          From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881 nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into β-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370 nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.

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          Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

          ABSTRACT A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.
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            Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.

            The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.
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              An emerging coronavirus causing pneumonia outbreak in Wuhan, China: calling for developing therapeutic and prophylactic strategies

              In December of 2019, an outbreak of pneumonia caused by an unknown aetiology occurred in Wuhan, China and most patients were linked to a single seafood market, which reportedly sold seafood and some live animals, including poultry, bats, marmots and other wild animals, suggesting that the pathogen may be transmitted from an animal to human. The pathogen was soon identified to be a novel coronavirus, 2019-nCoV denoted by WHO [1]. On 19 January 2020, Wuhan Health Commission reported that a total 198 cases in the 25–89-year-old range were confirmed positive for 2019-nCoV, including 25 being discharged and 3 having died. Among the 170 patients under treatment in hospitals, 126, 35, and 9 are in mild, severe, and critical condition, respectively (http://www.thatsmags.com/china/post/30618/new-coronavirus-spreads-to-over-130-in-china-death-toll-rises). In addition, two patients in Thailand, one in Japan, and one in South Korea, were detected positive for 2019-nCoV. They did not visit the specific seafood market, but might have close contact with some pneumonia patients during their trip in Wuhan, raising the concern of limited human-to-human transmission of 2019-nCoV (http://www.thatsmags.com/china/post/30618/new-coronavirus-spreads-to-over-130-in-china-death-toll-rises). Research scientists have released the full genomic sequence of 2019-nCoV, such as Wuhan-Hu-1 (GenBank, accession no. MN908947). The phylogenetic analysis revealed that the gene sequence of 2016-nCoV is 89% identical to that of bat SARS-like coronavirus ZXC21 (bat-SL-CoVZXC21, accession no. MG772934.1) and ZC45 (MG772933.1), and 82% identical to that of SARS-CoV Tor2 (JX163927), suggesting that 2019-nCoV also belongs to betacoronavirus Lineage B, but has closer homology to bat-SL-CoVZC45 and bat-SL-CoVZXC21 than SARS-CoV [2] (Figure 1). Both bat-SL-CoV ZC45 and ZXC21 were found in Chinese horseshoe bats (Rhinolopus sinicus) in Zhoushan city of Zhejiang Province, China between 2015 and 2017 [3], which can infect suckling rats and cause disease. Given that there were some bats and live animals in the seafood market, 2019-nCoV may be originated from bats or live animals exposure to the materials contaminated with bat droppings in the seafood market or surrounding area. Figure 1. Analysis of the functional domains in 2019-nCoV spike protein and its gene. (A) Phylogenetic analysis of S gene of 2019-nCoV (Wuhan-Hu-1), bat-SL-CoVZXC21, bat-SL-CoVZXC45, SARS-CoV and other coronaviruses using Neighbor-Joining method. (B) The representative scheme of functional domains in S protein of 2019-nCoV. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide, HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CP, cytoplasmic domain. (C) The target sites in 2019-nCoV S for development of vaccines, antibodies and fusion/entry inhibitors. The rapid identification of this novel coronavirus is attributed to recent advances in the detection of respiratory virus infection, including reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (rRT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-LAMP as well as multiplex nucleic acid amplification and microarray-based assays [4]. These methods are useful for detecting novel coronaviruses not only in humans, but also in animals for identification of animal reservoir or intermediate host of 2019-nCoV. WHO recommended that if there is no clue about the putative pathogen from the pneumonia outbreak, a pan-coronavirus assay should be used for amplification followed by sequencing of the amplicon for characterization and confirmation (https://apps.who.int/iris/bitstream/handle/10665/330374/WHO-2019-nCoV-laboratory-2020.1-eng.pdf). By aligning 2019-nCoV S protein sequence with those of SARS-CoV and several bat-SL-CoVs, we predicted that the cleavage site for generating S1 and S2 subunits is located at R694/S695 (Figure 1). S1 subunit contains two functional domains, the N-terminal domain (NTD) and a receptor-binding domain (RBD), both of which are responsible for the binding of the virion to the receptor on the host cell. They also contain several conformational neutralizing epitopes, serving as a target for developing neutralizing antibodies and vaccines [5]. S2 subunit contains three functional domains, fusion peptide (FP), and heptad repeat (HR) 1 and 2. After binding of RBD in S1 to the receptor, the S2 subunit changes conformation by inserting the FP into the host cell membrane and association between HR1 and HR2 to form six-helical bundle (6-HB), resulting in the fusion between viral and cellular membranes. The viral genetic materials enter into the host cell through the fusion pore for replication in the cell [5]. A peptide derived from the HR2 domain of SARS-CoV S protein (SC-1) can interact with HR1 region in viral S protein to form heterologous 6-HB, resulting in the inhibition of homologous 6-HB formation between HR1 and HR2 domains in viral S protein and thus blocking the viral fusion with the host cell [6]. Since 2019-nCoV S-HR2 sequence is 100% identical to that of SARS-CoV, while there are only a few mutations of non-critical amino acids in S-HR1 region, SC-1 peptide is expected to be also effective against 2019-nCoV infection. We have recently designed and engineered a pan-CoV fusion inhibitor, EK1 peptide, which could inhibit infection of five human coronaviruses, including SARS-CoV and MERS-CoV, and three bat-SL-CoVs [7]. Intranasal application of EK1 peptide before or after viral challenge, EK1 peptide can protect human DPP4-transgenic mice from MERS-CoV infection, suggesting its potential prophylactic and therapeutic effect against 2019-nCoV infection. Once confirmed, we will develop EK1 peptide as a t prophylactic or therapeutic for intranasal application to prevent or treat infection by 2019-nCoV and other emerging coronaviruses in the future. The RBDs of SARS-CoV and MERS-CoV contain multiple conformation-dependent neutralizing epitopes that induce more potent neutralizing antibodies and protective efficacy against SARS-CoV and MERS-CoV infections, respectively, than other regions in S protein [5,8,9]. Modification of MERS-CoV S-RBD amino acid residues based on the structure design could improve its protection against MERS-CoV infection [9], suggesting that 2019-nCoV S-RBD or modified S-RBD of other coronavirus may be applied for developing 2019-nCoV vaccines. Of course, the RBD-containing S and S1 of a coronavirus, e.g. 2019-nCoV, can also be used for vaccine development [8]. The recently developed SARS-CoV and MERS-CoV neutralizing monoclonal antibodies (mAbs) and nanobodies with protective efficacy are specific to the S1 subunit of S protein, particularly the RBD [5,8 9 –10]. Therefore, the 2019-nCoV S-RBD is anticipated to be a key target for developing 2019-nCoV neutralizing mAbs. The neutralizing mAbs targeting non-RBD regions, including NTD and S2 of SARS-CoV and/or MERS-CoV S could also be identified [5,8,11,12], although their neutralizing potency is generally lower than that of RBD-specific mAbs. It may take several months or even years for researching and developing neutralizing antibodies against 2019-nCoV infection. One of the rapid approaches is to evaluate the currently available SARS-CoV neutralizing antibodies with cross-neutralizing and protection activity against 2019-nCoV infection. We have shown that SARS-CoV S-RBD-specific neutralizing mAbs and sera could cross-neutralize bat-SL-CoVs, such as bat-SL-CoV-W1V1 and bat-SL-CoV-SHC014 [13], suggesting that they may also cross-neutralize 2019-nCoV. Once identified, these cross-neutralizing antibodies can be promptly developed for urgent prevention and treatment of 2019-nCoV infection.

                Author and article information

                Emerg Microbes Infect
                Emerg Microbes Infect
                Emerging Microbes & Infections
                Taylor & Francis
                5 February 2020
                : 9
                : 1
                : 313-319
                [a ]State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University , Wuhan, People’s Republic of China
                [b ]Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University , Wuhan, People’s Republic of China
                [c ]Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, People’s Republic of China
                [d ]School of Medicine, Sun yat-Sen University , Guangzhou, People’s Republic of China
                Author notes
                [CONTACT ] Yingle Liu mvlwu@ 123456whu.edu.cn

                These authors have equal contribution to this study.

                Supplemental data for this article can be accessed https://doi.org/10.1080/22221751.2020.1725399

                Author information
                © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                : 27 January 2020
                : 29 January 2020
                : 30 January 2020
                Page count
                Figures: 3, Tables: 2, Equations: 0, References: 13, Pages: 7
                Funded by: National Mega Project on Major Infectious Disease Prevention
                Award ID: 2017ZX10103005
                Funded by: National Key Research and Development Program of China
                Award ID: 2018YFE0204500
                Funded by: National Science and Technology Major Project
                Award ID: 2018ZX10733403
                This work was supported by the National Mega Project on Major Infectious Disease Prevention under [grant number 2017ZX10103005]; National Key Research and Development Program of China under [grant number 2018YFE0204500]; and National Science and Technology Major Project under [grant number 2018ZX10733403].
                Original Article

                2019-ncov,wuhan pneumonia,metagenomic next-generation sequencing,phylogenetic analyses,virus evolution


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