Isolation and Characterization of 2019-nCoV-like Coronavirus from Malayan Pangolins

  1. Kangpeng Xiao
  2. Junqiong Zhai
  3. Yaoyu Feng
  4. Niu Zhou
  5. Xu Zhang
  6. Jie-Jian Zou
  7. Na Li
  8. Yaqiong Guo
  9. Xiaobing Li
  10. Xuejuan Shen
  11. Zhipeng Zhang
  12. Fanfan Shu
  13. Wanyi Huang
  14. Yu Li
  15. Ziding Zhang
  16. Rui-Ai Chen
  17. Ya-Jiang Wu
  18. Shi-Ming Peng
  19. Mian Huang
  20. Wei-Jun Xie
  21. Qin-Hui Cai
  22. Fang-Hui Hou
  23. Yahong Liu
  24. Wu Chen
  25. Lihua Xiao
  26. Yongyi Shen

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Abstract

The outbreak of 2019-nCoV in the central Chinese city of Wuhan at the end of 2019 poses unprecedent public health challenges to both China and the rest world 1 . The new coronavirus shares high sequence identity to SARS-CoV and a newly identified bat coronavirus 2 . While bats may be the reservoir host for various coronaviruses, whether 2019-nCoV has other hosts is still ambiguous. In this study, one coronavirus isolated from Malayan pangolins showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N and S genes, respectively. In particular, the receptor-binding domain of the S protein of the Pangolin-CoV is virtually identical to that of 2019-nCoV, with one amino acid difference. Comparison of available genomes suggests 2019-nCoV might have originated from the recombination of a Pangolin-CoV-like virus with a Bat-CoV-RaTG13-like virus. Infected pangolins showed clinical signs and histopathological changes, and the circulating antibodies reacted with the S protein of 2019-nCoV. The isolation of a coronavirus that is highly related to 2019-nCoV in the pangolins suggests that these animals have the potential to act as the intermediate host of 2019-nCoV. The newly identified coronavirus in the most-trafficked mammal could represent a continuous threat to public health if wildlife trade is not effectively controlled.

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    SciScore for 10.1101/2020.02.17.951335: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationThe cell line was tested free of mycoplasma contamination using LookOut Mycoplasma PCR Detection Kit ( SIGMA)

    Table 2: Resources

    Antibodies
    SentencesResources
    Serological test Plasma samples from eight Malayan pangolins were tested for anti-2019-nCoV antibodies using a double-antigen ELISA kit for the detection of antibodies against 2019-nCoV by Hotgen ( Beijing , China) , following manufacturer-recommended procedures .
    anti-2019-nCoV
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To isolate the virus, supernatant from homogenized lung tissue from one dead animal was inoculated into Vero E6 cells.
    Vero E6
    suggested: CVCL_XD71
    Software and Algorithms
    SentencesResources
    As the S proteins of both SARS-CoV and 2019-nCoV have been shown to specifically recognize angiotensin converting enzyme II (ACE2) during the entry of host cells2,11, we conducted a comparative analysis of the interaction of the S proteins of the four closely related SARSr-CoV viruses with ACE2 proteins from humans, civets and pangolins.
    SARS-CoV
    suggested: (RayBiotech Cat# MD-05-0425, AB_951740)
    Materials and methods Metagenomic analysis and viral Genome assembly We collected viromic, metagenomic, and transcriptomic data of different mammals and birds in public databases, including NCBI Sequence Read Archive (SRA) and European Nucleotide Archive (ENA), for searching potential coronavirus sequences.
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA), SCR_004891)
    The clean reads were mapped to the 2019-nCoV reference sequence (MN908947) using BWA-MEM (v0.7.17)17 with > 30% matches.
    BWA-MEM
    suggested: (Sniffles, SCR_017619)
    Contigs were de novo assembled using Megahit (v1.0.3)18 and identified as 2019-nCoV-related using BLASTn with Evalues < 1e5 and sequence identity > 90%.
    BLASTn
    suggested: (BLASTN, SCR_001598)
    Phylogenetic analysis Multiple sequence alignment of all sequence data was done using MAFFT v7.22119.
    MAFFT
    suggested: (MAFFT, SCR_011811)
    The phylogenetic relationship of the samples was constructed using RAxML v.
    RAxML
    suggested: (RAxML, SCR_006086)
    The MM-GBSA28 approach was used to calculate the binding free energy of each ACE2 protein to the RBD of CoV S-protein, using the python script MMPBSA.py29 in the build-in procedure of AMBER 18 suite.
    python
    suggested: (IPython, SCR_001658)

    Results from OddPub: Thank you for sharing your data.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.02.17.951335v1 on bioRxiv