Monitoring SARS-CoV-2 variant transitions using differences in diagnostic cycle threshold values of target genes (preprint)

Monitoring the emergence of new SARS-CoV-2 variants is important to detect potential risks of increased transmission or disease severity. We investigated the identification of SARS-CoV-2 variants from real-time reverse transcriptase polymerase chain reaction (RT-PCR) routine diagnostics data. Cycle threshold (Ct) values of positive samples were collected from April 2021 to January 2022 in the Northern Metropolitan Area of Barcelona (n = 15,254). Viral lineage identification from whole-genome sequencing (WGS) was available for 4,618 (30.3%) of these samples. Pairwise differences in the Ct values between gene targets (ΔCt) were analyzed for variants of concern or interest circulating in our area. A specific delay in the Ct of the N-gene compared to the RdRp-gene (ΔCtNR) was observed for Alpha, Delta, Eta and Omicron. Temporal differences in ΔCtNR correlated with the dynamics of viral replacement of Alpha by Delta and of Delta by Omicron according to WGS results. Using ΔCtNR, prediction of new variants of concern at early stages of circulation was achieved with high sensitivity and specificity (91.1% and 97.8% for Delta; 98.5% and 90.8% for Omicron). Thus, tracking population-wide trends in ΔCt values obtained from routine diagnostics testing in combination with WGS could be useful for real-time management and response to local epidemics.

Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients (preprint)

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site, generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We propose that natural selection has favored a "Dont burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.