SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication

  1. Soren Alexandersen
  2. Anthony Chamings
  3. Tarka Raj Bhatta

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Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first detected in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. Here we show detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection and provide evidence for their nuclease resistance and protection by cellular membranes suggesting that detection of subgenomic RNAs in such samples may not be a suitable indicator of active coronavirus replication/infection.

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  1. Version published to 10.1038/s41467-020-19883-7
  2. Version published to 10.1101/2020.06.01.20119750v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.01.20119750: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study complied with all relevant ethical regulations and has been approved by the Barwon Health Human Research Ethics Committee (Ref HREC 20/56) and participants provided opt-out consent.
    Consent: The study complied with all relevant ethical regulations and has been approved by the Barwon Health Human Research Ethics Committee (Ref HREC 20/56) and participants provided opt-out consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    As described previously 28, generated sequence reads were then mapped to a SARS-CoV-2 reference genome (Wuhan-Hu-1-NC_045512/MN908947.3) using the TMAP software included in the Torrent Suite 5.10.1 37, and virus genomic consensus sequences generated using additional Torrent Suite plugins supplied by Thermofisher Scientific, and visualized in Integrative Genomic Viewer 38 (IGV 2.6.3) (Broad Institute, Cambridge, MA, USA).
    TMAP
    suggested: (TMAP, RRID:SCR_000687)
    Near complete and partial SARS-CoV-2 genomes were aligned using Clustal-W 39 in MEGA 7 software 40 and near full length sequences submitted to the Global Initiative on Sharing All influenza Database (GISAID) 41,42 (https://www.gisaid.org/) as described in our previous study 28.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    Analyses for the detection of SARS-CoV-2 subgenomic mRNAs in the NGS reads: Although the SARS-CoV-2 Ampliseq panel used for the NGS has been designed to generate near full length SARS-CoV-2 genomic sequences, it uses simultaneous amplification of sample cDNA with a total of 242 primer pairs of which 237 primer pairs cover the near full genome of SARS-CoV-2 and an additional 5 amplicons targeting cellular genes in two primer pools 28.
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    This final composite reference used for mapping then included the first 21500 nucleotides of the SARS-CoV-2 genome and the 10 subgenomic RNA specific sequences, each including the leader and gene specific sequences and having a length of 233-364 nucleotides (Supplementary Information S1 [file: Wuhan-Hu-1-NC_045512-21500-and-subgenomics-SA4.fasta] and also available at NCBI Sequence Read Archive (SRA): PRJNA636225)
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    Reads within the mapped reads BAM files were filtered on whether they had a MAPQ of 32 or higher and contained the partial leader sequence GTAGATCTGTTCTCT, using a custom script written in BASH 4.4.20 and AWK 4.1.4 (GNU project, www.gnu.org) using samtools 1.7 43.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    To assign the reads to the corresponding subgenomic RNA, the reads were grouped by the nucleotide position at the end of the leader sequence and tallied in Excel.
    Excel
    suggested: None
    SAM files from 15 SRA accessions were downloaded with the NCBI SRAtoolkit sam-dump 2.8.2 and mapped to the Ampliseq SARS-CoV-2 reference MN908947.3 2 using NCBI Magic-BLAST 1.3.0 with a minimum alignment score of 50 and percentage identity of 90% or higher.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    A base calling Phred score reflecting the signal quality at each base is then assigned and reads which have poor quality 3’ ends are trimmed by scanning using a 30nt window until the average base calling quality drops to 15.
    Phred
    suggested: (Phred, RRID:SCR_001017)
    Counting/abundance of reads mapped to the individual amplicons were then done using BEDTools 44 with a minimum mapping quality (MAPQ) of 20 and requiring the mapped reads to cover more than 90% of an amplicon, and for the amplicons to cover no less than 70% of the read to be included in the count.
    BEDTools
    suggested: (BEDTools, RRID:SCR_006646)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The caveat is that samples from even relatively early infection in vivo, as assessed by upper respiratory swab samples, are more alike to a late infection cell culture supernatant or partly purified virion preparation and less like what is found for early intracellular coronavirus RNAs in cell culture. Consequently, when looking at what is known for other coronaviruses and cell culture studies, intracellular subgenomic RNAs may dominate over genomic RNA very early on, with 8-70 times more intracellular subgenomic than genomic RNA at 6-8 hours after infection for infectious bronchitis virus, a gammacoronavirus, and bovine coronavirus, a betacoronavirus, and with at least 10 times more plus sense than minus sense RNA 13,16,27. In contrast, the same authors found that extracellular and partly purified coronavirus virion preparations from late cell culture infection, while being RNase resistant and susceptible to detergents, have a much higher genomic to subgenomic RNA ratio of 10-30 or higher and at least 100-fold more, positive rather than negative sense RNA13,16,27. Consequently, our findings based on NGS, specific PCR assays and fractionation together with nuclease and detergent treatment, is fully consistent with what has been shown from cell culture infection and fractionation of coronavirus replication/transcription complexes in cellular membrane structures, most likely double-membrane vesicles (DMVs). Thus, SARS-CoV-2 RNA in diagnostic swab samples are likely found as a m...

    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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  4. Version published to 10.1101/2020.06.01.20119750v1 on medRxiv