Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed ddPCR

  1. Hyon S. Hwang
  2. Che-Min Lo
  3. Michael Murphy
  4. Tanner Grudda
  5. Nicholas Gallagher
  6. Chun Huai Luo
  7. Matthew L. Robinson
  8. Agha Mirza
  9. Madison Conte
  10. Abigail Conte
  11. Ruifeng Zhou
  12. Christopher B. Brooke
  13. Andrew Pekosz
  14. Heba H. Mostafa
  15. Yukari C. Manabe
  16. Chloe L. Thio
  17. Ashwin Balagopal

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Abstract

Control of SARS-CoV-2 (SCV-2) transmission is a major priority that requires understanding SCV-2 replication dynamics. We developed and validated novel droplet digital PCR (ddPCR) assays to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from full-length genomic RNAs (gRNAs) in a multiplexed format. We applied this multiplex ddPCR assay to 144 cross-sectional nasopharyngeal samples. sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was remarkably stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Interestingly, adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Longitudinal daily testing of 6 persons for up to 14 days revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. These data indicate stable viral transcription during infection. More work is needed to understand why cultures are negative despite persistence of viral RNAs.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.18.22271199: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: We then converted the copy number to copies/µl based on the VTM volume in which nasal swab specimens were collected.
    Consent: Study approval: Informed consent was obtained from the participants in the UIUC cohort.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Statistics: For each sample, Poisson statistics were used to calculate the total copy numbers of N sgRNA and gRNA (along with 95% CI) using Bio-Rad QuantaSoft Analysis Pro software.
    Bio-Rad QuantaSoft Analysis Pro
    suggested: None
    QuantaSoft
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    There are several limitations to our study. The sensitivity of RNA detection in NP samples may not reflect the presence of RNA in deeper and more sequestered compartments of the body. However, by using ddPCR to improve our sensitivity by at least an order of magnitude, and by quantifying human RNA using housekeeping genes, we have at least addressed sensitivity in clinically relevant specimens. A related limitation, however, is that we did not perform a comprehensive analysis of human RNAs in clinical samples, which may have revealed which cells were infected or contributing to COVID-19 pathology, as has been done previously (28). With regards to viral culture, it is well-appreciated that there are limits to its sensitivity. Indeed, as we speculate above, culture negativity does not reveal whether virions are replication incompetent or are adsorbed to antibodies and thus neutralized in the culture experiment. Unfortunately, samples for antibody testing were not available, but it is possible that antibody testing may have explained why some culture samples were negative despite deriving from NP swabs that had abundant amounts of sgRNA, gRNA, and total viral RNA. Nevertheless, culture negativity does correspond well with when COVID-19 patients are no longer likely to transmit to others, and thus is still a useful tool for research, if challenging to implement for widespread clinical use. In addition, we note here that while there was considerable homogeneity in our findings in ...

    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

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  2. Version published to 10.1101/2022.02.18.22271199v1 on medRxiv