RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak
Authors: Fang Liu
Ke Lan
Yirong Li
Bo Zhong
Weiyong Liu
Weichen Wu
Ke Xu
Ziyong Sun
Yi Mei
Yingle Liu
Mang Shi
Guangming Ye
Yu Chen
Qi Zhang
Wenxia Zhang
Kailang Wu
Liangjun Chen
Journal: Emerging microbes & infections (2020)
Discipline: MICROBIOLOGY
Keywords: 2019-nCoV; Wuhan pneumonia; metagenomic next-generation sequencing; phylogenetic analyses; virus evolution.
Affiliations: State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China., Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan, People's Republic of China., Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China., School of Medicine, Sun yat-Sen University, Guangzhou, People's Republic of China.

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. 

Retracted: No

PubPeer comments:
 How do they [miss out on Prevotella] ? Just detected Capnocytophaga sp and Veillonella sp - non-pathogenic bacteria? In [patient 1] and [patient 2] All 3 studies in [Wuhan have Prevotella. Cant be random] , 
 #1 #1 In our study, Prevotella was not detected with Metaphlan2 program, suggest it must be of relatively low abundance if it exists. That being said, Prevotella do appear in other libraries we sequenced before (including controls). And if you found all three studies contain Prevotella, we might need to consider the possibility that all three studies used a same reagent (reagent contamination) , 
 I will send you the abundance % soon - but you think [this is reagent contamination] in the other studies? 1 4 of 26 millions reads? And even they dont mention it - at least say it secondary infection, if not anything. Others will check for this then ![file] ![file] , 
 #3 Interesting observation (but please send the table to my email account because the resolution is poor)! And could you please specify which are the three studies with high Prevotella prevalence, and I will have a look at their methodology? In our case, we do have Prevotella hits but they were not dominant ones, and we normally don t rely on blasting result for quantification of bacteria pathogens. , 
 #4 [SI Table] of the other Chinese paper. And Prevotella is equal numbers as Veillonella - so contamination doesnt hold here either. 1. Patient1 [3500] (Veillonella) 1500(Prevotella) 2. Patient2 [4100] (Veillonella) 4300(Prevotella) The sequences are in the link (Prevotella was in the first post). I predict that the first patient got better faster, w o knowing details. , 
 #5 We did not count bacteria rRNA for fear that rRNA depletion would introduce bias, and hence the difference from your result. I am not insisting on contamination theory, but we don t have the right experimental design to prove either. I d be cautious with the interpretation though: (i) Prevotella is not known as respiratory pathogen causing pneumonia, (ii) Prevotella do appear in healthy controls whereas 2019-nCoV does not, (iii) I don t know about the other study but in our study Prevotella has a much lower abundance level in comparison to 2019-nCoV. , 
 Its proteins. And I give you sample of reads matching to elongation factor Tu. 1. SRR10903402.53581.1_4 matched=49 outof=50 iden=98 108 2. SRR10903402.53581.2_3 matched=49 outof=49 iden=100 106 3. SRR10903402.574944.1_3 matched=43 outof=50 iden=86 84.7 4. SRR10903402.574944.2_1 matched=38 outof=39 iden=97 78.6 5. SRR10903402.203306.1_5 matched=44 outof=49 iden=90 76.3 The same protein which is the most expressed in the other China study. And is known for immune evasion. Please raise the alarm - and check for Prevotella ![file] , 
 Also, to answer your 3 points. 1. That what I am saying - Prevotella is not known. Thats why its not being tested. But the other China study, with the high loads. You really should know about that study. How did the patients do? Please find out. You are in Wuhan. I am not. 2. What occurs in healthy patients is not the one that has nCoV integrated. So, that doesnt count. This is different. Dont go by conventional wisdom. This is not normal. 3. Yes, Prevetolla is low in your study. Thats why both your patients recovered. As I said above, please raise the alarm. Test for Prevotella. Its a test. Prove me wrong. Please. I will quit twitter, pubpeer and all social media - and still be thankful to you. , 
 There are reads here encoding [beta-lactamases from plasmids] - need to understand the source and implications of that. Also there in the [HK study] . But not the other China study , 
 #9 Thanks for your suggestion, we do have plans to look into opportunistic infections in these patients, but this will be based on a better experimental design and larger dataset. , 
 Thanks ! Please update here when you know. , 
 China has just published RNA-seq data for [5 patients] The main takeaway is that viral load is negligible, no wonder the tests are giving so many false negatives. Something else is happening, if these patients had symptoms IMO. ![file] , 
 Done the full [metagenomic analysis] for this paper (sequences in the SI) - and also for [four other studies - 2 China, 1 San Diego, 1 Brazil] ... ![file] ,