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

  1. Renu Verma
  2. Eugene Kim
  3. Giovanny Joel Martinez-Colón
  4. Prasanna Jagannathan
  5. Arjun Rustagi
  6. Julie Parsonnet
  7. Hector Bonilla
  8. Chaitan Khosla
  9. Marisa Holubar
  10. Aruna Subramanian
  11. Upinder Singh
  12. Yvonne Maldonado
  13. Catherine A. Blish
  14. Jason R. Andrews
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Abstract

Background

Given the persistence of viral RNA in clinically recovered COVID-19 patients, subgenomic RNAs (sgRNA) have been reported as potential molecular viability markers for 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 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 A549 ACE2+ 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 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 (Pearson’s r=0.87) and the 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 A549 ACE2+ 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.

Summary

We observed prolonged detection of subgenomic RNA in nasal swabs and equivalent decay rates to genomic RNA in both longitudinal nasal swabs and in remdesivir-treated A549 ACE2+ cells infected with SARS-CoV-2. Taken together, these findings suggest that subgenomic RNA from SARS-CoV-2 is comparably stable to genomic RNA and that its detection is therefore not a more reliable indicator of replicating virus.

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