Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

Abstract

In coronaviridae such as SARS-CoV-2, subgenomic RNAs (sgRNA) are replicative intermediates, therefore, their abundance and structures could infer viral replication activity and severity of host infection. Here, we systematically characterized the sgRNA expression and their structural variation in 81 clinical specimens collected from symptomatic and asymptomatic individuals with a goal of assessing viral genomic signatures of disease severity. We demonstrated the highly coordinated and consistent expression of sgRNAs from individuals with robust infections that results in symptoms, and found their expression is significantly repressed in the asymptomatic infections, indicating that the ratio of sgRNAs to genomic RNA (sgRNA/gRNA) is highly correlated with the severity of the disease. Using long read sequencing technologies to characterize full-length sgRNA structures, we also observed widespread deletions in viral RNAs, and identified unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Furthermore, based on the sgRNA structures, the frequently occurred structural variants in SARS-CoV-2 genomes serves as a mechanism to further induce SARS-CoV-2 proteome complexity. Taken together, our results show that differential sgRNA expression and structural mutational burden both appear to be correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers

“…To date, previous studies reported evidence regarding the detection of viral sgRNAs using sequencing, RT-PCR and ddPCR, and all these findings were based on non-vaccinated cases [ 8 , 23 , 24 ]. The detection of viral sgRNA coupled with an increased viral load has been shown to be indicative of an aggravation of the disease [ 11 ]. Conversely, a reduction in viral load combined with a decrease in sgRNA might reflect a reduced risk of transmission to other susceptible contacts.…”

“…Thus, the rapid and accurate detection of SARS-CoV-2, as well as of viral infectivity, are essential in controlling the outbreak of infections [ 6 ]. Based on the literature, the detection of SARS-CoV-2 subgenomic RNA (sgRNA) in diagnostic samples can indicate active virus replication/transcription and recent infection [ 6 , 7 , 8 , 9 , 10 , 11 ]. Indeed, in the Coronaviridae family, including SARS-CoV-2, sgRNAs are replicative intermediates, and their abundance may reflect viral replication activity and the severity of host infection.…”

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis

“…SgmRNAs can be considered markers of ongoing active viral replication and, likely, of viral infectivity, as they are synthesized exclusively inside infected cells and not packaged into virions 20,34,35 . Although our data are not related to clinical data and/or to infectiousness data on cell culture, we believe that this study provides evidence for the application of ddPCR and meta-transcriptomics for the study of the SARS-CoV-2.…”

Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples

“…There are conflicting reports regarding the clinical utility of using the presence of sgRNA as a predictor of infectivity. Some studies promote its use [10, 11] while others (including those using the Wölfel-sgE primer-probe set) deemed it unsuitable for predicting the presence of infectious virus [5, 13, 14]. However, the presence of sgRNA has been used to successfully distinguish input challenge virus from actively replicating virus, particularly in non-human primate models of SARS-CoV-2 vaccination and challenge [15-19].…”