SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

Verma, R.; Kim, E.; Martinez-Colon, G. J.; Jagannathan, P.; Rustagi, A.; Parsonnet, J.; Bonilla, H.; Khosla, C.; Holubar, M.; Subramanian, A.; Singh, U.; Maldonado, Y.; Blish, C. A.; Andrews, J. R.

Abstract Background In light of numerous reports during the COVID-19 pandemic demonstrating an inexplicable persistence of viral RNA in clinically recovered patients, subgenomic RNAs (sgRNA) have recently been reported as potential molecular viability markers for SARS-CoV-2. However, few data are available on the longitudinal kinetics of sgRNA, 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 three time points in the Lambda (Day 1, 4 and 6) and favipiravir (Day 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by RT-qPCR. 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 DMSO control. Results At six days in the Lambda trial and ten days in Favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold values for gRNA and sgRNA were highly linearly correlated (Pearsons r= 0.87) and the daily rate of increase did not differ significantly in Lambda (1.36 cycles/day vs 1.36 cycles/day; p = 0.97) or favipiravir (1.03 cycles/day vs 0.94 cycles/day; p=0.26) trials. 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 molecular viability marker.