An increased ratio of SARS-CoV-2 positive to negative sense genomic and subgenomic RNAs within routine diagnostic upper respiratory tract swabs may be a marker of virion shedding [preprint]

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly in the global population since its emergence in humans in late 2019. Replication of SARS-CoV-2 is characterised by transcription and replication of genomic length RNA and shorter subgenomic RNAs to produce virus proteins and ultimately progeny virions. Here we explore the pattern of both genome-length and subgenomic RNAs and positive and negative strand SARS-CoV-2 RNAs in diagnostic nasopharyngeal swabs using sensitive probe based PCR assays as well as Ampliseq panels designed to target subgenomic RNAs. Using these assays, we measured the ratios of genomic to subgenomic RNAs as well as the ratios of positive to negative strand RNAs in SARS-CoV-2 positive nasopharyngeal swab samples. We found that while subgenomic RNAs and negative strand RNA can be readily detected in swab samples taken up to 19 and 17 days post symptom onset respectively, and therefore their detection alone is not likely an indicator of active SARS-CoV-2 replication. However, the ratios of genomic-length to subgenomic RNA and also of positive to negative strand RNA were elevated in some swabs, particularly those collected around the onset of clinical symptoms or in an individual with decreasing PCR Cts in successive swab samples. We tentatively conclude that it may be possible to refine such molecular assays to help determine if active replication of virus is occurring and progeny virions likely present in a SARS-CoV-2 positive individual. Assays targeting subgenomic N or ORF7a RNAs as well as strand specific ORF7a total genome-length and subgenomic RNAs may be the most sensitive for this purpose as these targets were consistently the most abundant in the swab samples.