Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

  1. Davida S. Smyth
  2. Monica Trujillo
  3. Devon A. Gregory
  4. Kristen Cheung
  5. Anna Gao
  6. Maddie Graham
  7. Yue Guan
  8. Caitlyn Guldenpfennig
  9. Irene Hoxie
  10. Sherin Kannoly
  11. Nanami Kubota
  12. Terri D. Lyddon
  13. Michelle Markman
  14. Clayton Rushford
  15. Kaung Myat San
  16. Geena Sompanya
  17. Fabrizio Spagnolo
  18. Reinier Suarez
  19. Emma Teixeiro
  20. Mark Daniels
  21. Marc C. Johnson
  22. John J. Dennehy

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Abstract

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

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  1. Version published to 10.1038/s41467-022-28246-3
  2. ScreenIT

    SciScore for 10.1101/2021.07.26.21261142: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Antibody Neutralization Assay: All blood collection and processing were performed under the approved protocols (MU Study of Serology for SARS-CoV-2 and MU COVID19 Vaccine study) by the Institutional Review Board of the University of Missouri.
    IRB: Antibody Neutralization Assay: All blood collection and processing were performed under the approved protocols (MU Study of Serology for SARS-CoV-2 and MU COVID19 Vaccine study) by the Institutional Review Board of the University of Missouri.
    Consent: Written consent was received from all human subjects prior to being enrolled in the study.
    Sex as a biological variablenot detected.
    RandomizationSequences with poor mapping to sequences in the index and a random selection of sequences with good mapping were checked by Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify the organism match.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Subjects were requested to provide a date of positive PCR test for SARS-CoV-2 and subsequently had laboratory-based serologic tests to confirm the presence of antibody against SARS-CoV-2 S1 RBD protein.
    SARS-CoV-2 S1 RBD protein.
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Monoclonal antibody synthesis: Transfections of 10cm dishes of 293FT cells were performed 5 mg each of heavy and light chain vectors and 40 mg polyethyleneimine (PEI)57.
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Recombinant DNA
    SentencesResources
    The ACE2 cell lines were generated by transfecting 293FT cells with 500 ng HIV GagPol expression vector, 400 ng of pscALPSpuro-MmACE2 (Mouse) or pscALPSpuro-RnACE2 (Rat), and 100 ng of VSV-G expression vector.
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    Secondary PCR (25 µl) was performed on RBD amplifications using 5 ul of the primary PCR as template with MiSeq nested gene specific primers containing 5’ adapter sequences (Table 1) (0.5 µM each), dNTPs (100 µM each) and Q5 DNA polymerase (New England Biolabs).□Secondary PCR amplification was performed as follows: 95°C(2:00) + [95°C(0:15) + 55°C(0:30) + 72°C(1:00)] x 20 cycles.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Variants present at frequencies of 1% or above were called using the Annotate and Predict Find Variations/SNPs in Geneious and verified by using the V-PIPE SARS-CoV-2 application49.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    For sequencing from rRNA templates, dereplicated reads were mapped with Bowtie2 and Minimap2 to a collected reference index of mitochondrial and rRNA related animal sequences from NCBI’s nucleotide and refseq databases (https://www.ncbi.nlm.nih.gov/).
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    https://www.ncbi.nlm.nih.gov/
    suggested: (GENSAT at NCBI - Gene Expression Nervous System Atlas, RRID:SCR_003923)
    Sequences with poor mapping to sequences in the index and a random selection of sequences with good mapping were checked by Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify the organism match.
    https://blast.ncbi.nlm.nih.gov/Blast.cgi
    suggested: (TBLASTX, RRID:SCR_011823)
    Neutralization IC50 titers were calculated using nonlinear regression (Inhibitor vs normalized response—variable slope) in GraphPad Prism 9.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

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

    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 checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.07.26.21261142 on medRxiv