Full Genome Nobecovirus Sequences From Malagasy Fruit Bats Define a Unique Evolutionary History for This Coronavirus Clade

  1. Gwenddolen Kettenburg
  2. Amy Kistler
  3. Hafaliana Christian Ranaivoson
  4. Vida Ahyong
  5. Angelo Andrianiaina
  6. Santino Andry
  7. Joseph L. DeRisi
  8. Anecia Gentles
  9. Vololoniaina Raharinosy
  10. Tsiry Hasina Randriambolamanantsoa
  11. Ny Anjara Fifi Ravelomanantsoa
  12. Cristina M. Tato
  13. Philippe Dussart
  14. Jean-Michel Heraud
  15. Cara E. Brook

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Abstract

Bats are natural reservoirs for both Alpha - and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat ( Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis ), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis . Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent ancestral sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats.

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  1. Version published to 10.3389/fpubh.2022.786060
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    SciScore for 10.1101/2021.09.29.462406: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Additional unrelated total RNA samples (a dilution series of total RNA isolated from cultured HeLa cells) and a set of local lab water samples were included on each 384 well plate to serve as library preparation controls.
    HeLa
    suggested: None
    Software and Algorithms
    SentencesResources
    An initial equivolume library pool was generated, and the quality and quantity of that pool was assessed via electrophoresis (High-Sensitivity DNA Kit and Agilent Bioanalyzer; Agilent Technologies, Santa Clara, CA, USA), real-time quantitative polymerase chain reaction (qPCR) (
    Agilent Bioanalyzer
    suggested: None
    Samples were deemed “positive” for coronavirus infection if IDseq successfully assembled at least two contigs with an average read depth >2 reads/nucleotide that showed significant nucleotide or protein BLAST alignment(s) (alignment length >100 nt/aa and E-value < 0.00001 for nucleotide BLAST/ bit score >100 for protein BLAST) to any CoV reference present in NCBI NR/NT database (version 12-01-2019).
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    NCBI NR/NT
    suggested: None
    To verify that no positives were missed from IDseq, all non-host contigs assembled in IDseq underwent directed, offline BLASTn and BLASTx (75) against a reference database constructed from all available full-length nucleotide and protein reference sequences for Alpha- and Betacoronavirus available in NCBI Virus (last access: August 15, 2021).
    BLASTn
    suggested: (BLASTN, RRID:SCR_001598)
    We then used NCBI BLAST and BLASTx to query identity of our full length recovered genomes and their respective translated proteins to publicly available sequences in NCBI (75).
    BLASTx
    suggested: (BLASTX, RRID:SCR_001653)
    Finally, we aligned representative sequences from each major Nobecovirus clade and visually examined the region of p10 orthoreovirus insertion from the RoBat-CoV GCCDC1 lineage in the newly described sequences from Madagascar. Phylogenetic Analysis: Contigs returned from IDseq were combined with publicly available coronavirus sequences in NCBI to perform phylogenetic analysis.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    After compiling sequences for each phylogenetic analysis, sequence subsets for the full-length, RdRp, and four amino acid phylogenies were aligned in MAFFT v.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    We constructed the Bayesian timetree using the Bayesian Skyline Coalescent (82) model in BEAST2 (83), assuming a constant population prior, and the best fit nucleotide substitution model as indicated by ModelTest-NG.
    BEAST2
    suggested: (BEAST2, RRID:SCR_017307)
    Markov chain Monte Carlo (MCMC) sample chains were run for 1 billion iterations, convergence was checked using TRACER v1.7 (84), and trees were averaged after 10% burn-in using TreeAnnotater v2.6.3 (85) to visualize mean posterior densities at each node.
    TRACER
    suggested: (Tracer, RRID:SCR_019121)
    After alignment, genomes were analyzed for recombination in the program SimPlot (v.3.5.1)
    SimPlot
    suggested: None

    Results from OddPub: Thank you for sharing your code and 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.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.09.29.462406v1 on bioRxiv