ABSTRACT
Background: SARS-CoV-2 is transcribed as genomic RNA (gRNA) and different subgenomic RNAs (sgRNA), allowing variation in viral gene expression. However, the extent and clinical significance of subgenomic transcription remain unknown. We hypothesized that SARS-CoV-2 RNA levels would vary between genome regions and between patients, tissues, and sample types (cells or fluids). Methods: We designed and validated 7 novel RT-ddPCR assays that target the 5' and 3' untranslated regions (UTR), non-structural genes found only in full length gRNA [Main Proteinase (NSP5) and RNA dependent RNA polymerase (RdRp)], and 3' structural genes [Spike (S), membrane (M), and nucleocapsid (N)] that are also contained in different sgRNAs. Assay efficiencies were measured on standards derived from plasmids and viral stock supernatants. Levels of all 7 RNA regions were measured in nucleic acid extracted by the Abbott m2000 platform from nasopharyngeal (NP) swabs from 3 SARS-CoV-2 infected individuals, and in cells and supernatant from NP, oropharynx (OP), and saliva isolated from 3 additional individuals. Results: In all samples, levels of 3' targets (M, N, and 3'UTR) tended to be higher than 5' targets (5' UTR, NSP5, and RdRp), suggesting an excess of 3' sgRNAs (3'UTR/5'UTR=2.4-6.2 and nucleocapsid/RdRp=1.1-7.5 for NP samples;n=6, p=0.03). All SARS-CoV-2 RNAs were detected in both cells and supernatant from NP, OP, and saliva, but tended to be higher in the NP than OP. In saliva but not NP or OP, levels of gRNA/μL sample were consistently higher in the cells compared to supernatant (cell/sup=2.7-44.8). Surprisingly, the excess of 3' over 5' viral RNAs was even greater in the supernatant (3'UTR/5'UTR=8.2-38.7) compared to cells (3'UTR/5'UTR=1.5-6.2) from NP, OP, and saliva (p=0.016 across all), suggesting a greater excess of sgRNAs in the cell-free fluids. Conclusion: The higher levels of 3' targets suggest an excess of sgRNA in all samples. Assays that target 3' regions found in sgRNAs (N, 3'UTR) may be more sensitive for detecting SARS-CoV-2, but may not indicate infectious virus. The greater excess of 3' transcripts in cell-free fluids suggests that sgRNAs are released from cells and/or persist to a greater degree than gRNAs. Future studies should investigate how levels of sgRNA change over the course of infection in cells and cell-free fluids, and whether sgRNA levels correlate with measures of disease transmission or severity.