SARS-CoV-2 Subgenomic RNA Kinetics in Longitudinal Clinical Samples

Background: Given the persistence of viral RNA in clinically recovered coronavirus disease 2019 (COVID-19) patients, subgenomic RNAs (sgRNAs) have been reported as potential molecular viability markers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples. Methods: We analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of peginterferon Lambda-1a (Lambda; n=177) and favipiravir (n=359). Nasal swabs were collected at 3 time points in the Lambda (days 1, 4, and 6) and favipiravir (days 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by quantitative reverse transcription polymerase chain reaction. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or dimethylsulfoxide control. Results: At 6 days in the Lambda trial and 10 days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R2 = 0.83), and the rate of increase did not differ significantly in the Lambda trial (1.36 cycles/d vs 1.36 cycles/d; P=.97) or the favipiravir trial (1.03 cycles/d vs 0.94 cycles/d; P=.26). From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2-infected A549ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA. Conclusions: In clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.