Single-cell transcriptional atlas of the Chinese horseshoe bat ( Rhinolophus sinicus ) provides insight into the cellular mechanisms which enable bats to be viral reservoirs

  1. Lili Ren
  2. Chao Wu
  3. Li Guo
  4. Jiacheng Yao
  5. Conghui Wang
  6. Yan Xiao
  7. Angela Oliveira Pisco
  8. Zhiqiang Wu
  9. Xiaobo Lei
  10. Yiwei Liu
  11. Leisheng Shi
  12. Lianlian Han
  13. Hu Zhang
  14. Xia Xiao
  15. Jingchuan Zhong
  16. Hongping Wu
  17. Mingkun Li
  18. Stephen R. Quake
  19. Yanyi Huang
  20. Jianbin Wang
  21. Jianwei Wang

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Abstract

Bats are a major “viral reservoir” in nature and there is a great interest in not only the cell biology of their innate and adaptive immune systems, but also in the expression patterns of receptors used for cellular entry by viruses with potential cross-species transmission. To address this and other questions, we created a single-cell transcriptomic atlas of the Chinese horseshoe bat ( Rhinolophus sinicus ) which comprises 82,924 cells from 19 organs and tissues. This atlas provides a molecular characterization of numerous cell types from a variety of anatomical sites, and we used it to identify clusters of transcription features that define cell types across all of the surveyed organs. Analysis of viral entry receptor genes for known zoonotic viruses showed cell distribution patterns similar to that of humans, with higher expression levels in bat intestine epithelial cells. In terms of the immune system, CD8+ T cells are in high proportion with tissue-resident memory T cells, and long-lived effector memory nature killer (NK) T-like cells ( KLRG1 , GZMA and ITGA4 genes) are broadly distributed across the organs. Isolated lung primary bat pulmonary fibroblast (BPF) cells were used to evaluate innate immunity, and they showed a weak response to interferon β and tumor necrosis factor-α compared to their human counterparts, consistent with our transcriptional analysis. This compendium of transcriptome data provides a molecular foundation for understanding the cell identities, functions and cellular receptor characteristics for viral reservoirs and zoonotic transmission.

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    SciScore for 10.1101/2020.06.30.175778: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The experiments and programs were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableBat, organs and single cell preparation: The two male Chinese horseshoe bat (Rhinolophus sinicus) were obtained in October, 2018 from Anhui province, China.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    , Carlsbad, CA, USA) are characterized by immunofluorescence with antibody specific to fibronectin (Santa Cruz, CA, USA) and Alexa Fluor 488-ligated second antibody (ZSGB-BIO, China).
    fibronectin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The primers targeted to human and bat IFNβ, IL-6, TNFα, RIG-I and MDA5 were used.
    MDA5
    suggested: None
    Software and Algorithms
    SentencesResources
    Clean-reads were mapped to Rhinolophus sinicus genome by hisat2.
    hisat2
    suggested: (HISAT2, RRID:SCR_015530)
    The count matrix was then processed by DESeq2 for normalization and expression profiles.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    The differential expression more than two times were further performed Gene ontology (GO) analysis using clusterprofiler package58 with mouse database (org.Mm.eg.db).
    clusterprofiler
    suggested: (clusterProfiler, RRID:SCR_016884)
    Amino acid identity of viral receptor genes: To analyze the identity of viral receptor genes, all related coding sequences were downloaded from Ensembl and GenBank representing human, bats and mouse.
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    The sequences were manual checked to avoid false annotation or different isoforms, then ClustalW Multiple alignment in BioEdit version 7.0.5.3 was used for amino acids sequence alignment between Chines horseshoe bat (Rhinolophus sinicus) and the other species.
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)
    Adjust the cell density to 1×106 cells/ml.
    Adjust
    suggested: (ADJUST, RRID:SCR_009526)
    The qRT-PCR was performed by using Bio-rad with real-time CFX96 amplifier (Bio-Rad Laboratories, Inc., USA) using the TB Greeen™ Premix Ex Taq™ (TaKaRa, Japan).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.06.30.175778v1 on bioRxiv