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

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in China 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. We here describe detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection, and provide a likely explanation not only for extended PCR positivity of such samples, but also for discrepancies in results of different PCR methods described by others. Overall, we present evidence that subgenomic RNAs may not be an indicator of active coronavirus replication/infection, but that these RNAs, similar to the virus genome RNA, may be rather stable, and thus detectable for an extended period, most likely due to their close association with cellular membranes.

periscope: sub-genomic RNA identification in SARS-CoV-2 Genomic Sequencing Data

“…We were able to detect sgRNAs with a high leader alignment score from all canonical ORFs in multiple samples (Figure 2, Supplementary Table S1). As shown in Figure 2A, sgRNA from the N & M ORFs were the most abundant sgRNA (dependent on normalisation method), with N being found in 97.3% of Sheffield samples, consistent with published reports in vitro (Alexandersen, Chamings, and Bhatta 2020;Finkel et al 2020;Kim et al 2020). To demonstrate that the levels of sgRNA detected in the Sheffield dataset were not site specific, we applied periscope to an independent dataset of 55 ARTIC Network Nanopore sequenced SARS-CoV-2 samples from Glasgow, UK (Figure 2A).…”

“…Like previously published reports (Taiaroa et al 2020;Alexandersen, Chamings, and Bhatta 2020;Finkel et al 2020;Kim et al 2020), we were unable to find strong evidence of sgRNA supporting the presence of ORF10 (Supplementary Table 2, Figure 2A) with only 0.95% of samples containing high (HQ) or low quality (LQ) sgRNA calls at this ORF. We aligned the 12 reads from these samples to a reference composing of ORF10 and leader (Supplementary Figure S3).…”

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

“…Genome variation in the amplicon primer region may also impact sequence assembly. Transcriptome characterization can further contribute to our knowledge of mutation within the SARS-CoV-2 genome, and direct RNA long read sequencing, both alone and in combination with short read sequencing, have been described 1,9,10 . Unfortunately, these analyses are equally hampered by sample quality limitations and necessitate use of cultured cell lines.…”

A nanobody-functionalized organic electrochemical transistor for the rapid detection of SARS-CoV-2 and MERS antigens at the physical limit

“…We here introduced a label-free electrochemical immunosensing platform with single protein molecule sensitivity and large dynamic range. Nasal swab samples protein may, in fact, be a better indicator of infectious virus than RT-PCR, as the latter is also detecting membrane-associated viral RNA fragments long after the infection has been cleared 47 .…”