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Case Reports
. 2020 Feb 5;9(1):313-319.
doi: 10.1080/22221751.2020.1725399. eCollection 2020.

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

Liangjun Chen  1   2 , Weiyong Liu  3 , Qi Zhang  1 , Ke Xu  1 , Guangming Ye  2 , Weichen Wu  4 , Ziyong Sun  3 , Fang Liu  1 , Kailang Wu  1 , Bo Zhong  1 , Yi Mei  1 , Wenxia Zhang  1 , Yu Chen  1 , Yirong Li  2 , Mang Shi  4 , Ke Lan  1 , Yingle Liu  1
Affiliations
Case Reports

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

Liangjun Chen et al. Emerg Microbes Infect. .

Abstract

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.

Keywords: 2019-nCoV; Wuhan pneumonia; metagenomic next-generation sequencing; phylogenetic analyses; virus evolution.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
1st-round of RT-PCR assay, amplification and sequence analysis of unusual pneumonia outbreak in Wuhan. (A) RNA samples were subjected to SARS-CoV specific RT-PCR primer sets as indicated, only the SAR1-s/as set showed obvious band. Lane 1, 6, 11, 16, 21 are samples of patient 1. Lane 2, 7, 12, 17, 22 are samples of patient 2. Other lanes are samples of other patients who are irrelevant to this study. (B) The Blast result of PCR products of patient 1 and 2.
Figure 2.
Figure 2.
2nd-round of identification of unusual pneumonia. (A) RNA samples were subjected to multiple primer sets for different genes as indicated. Lane 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 21, 23, 25, 27 are samples of patient 1. Lane 2, 4, 6, 8, 10, 16, 17, 18, 19, 20, 22, 24, 26, 28 are samples of patient 2. (B) The PCR product of patient 1 and 2 were sequenced and the Blast result is shown.
Figure 3.
Figure 3.
Origin and evolutionary history of newly identified CoVs. A. the position of 2019-nCoV in the context of all reference CoVs. The phylogeny is constructed based on ORF1b protein alignment. For clarity, names were only shown for human-associated viruses. Bat associated diversity is shaded with blue and green boxes for alpha- and beta-CoVs respectively. B. genome structure of newly identified viruses and its sequence similarity against bat-SL-CoVZC45 and SARS-CoV in a 1000bp sliding window across the entire genome. Recombination breakpoints are shown as dashed vertical lines. C. the relationship of WHU viruses with the other SARS-like CoVs. Phylogeny is reconstructed based on the nucleotide sequence of four genes: namely 1a, 1b, S, and N. Those grouped with WHU at S gene are marked red, and those grouped with SARS CoVs at S gene are marked blue.
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Grants and funding

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].