ABSTRACT
A one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of prevalence of the N501Y positive variants because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.
ABSTRACT
The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus within the Orthocoronavirinae causing an emergent infectious disease in humans, the ongoing coronavirus disease 2019 pandemic (COVID-19). Due to the high zoonotic potential of these viruses, it is critical to unravel their evolutionary history of host species shift, adaptation and emergence. Only such knowledge can guide virus discovery, surveillance and research efforts to identify viruses posing a pandemic risk in humans. We present a comprehensive analysis of the composition and codon usage bias of the 82 Orthocoronavirinae members, infecting 47 different avian and mammalian hosts. Our results clearly establish that synonymous codon usage varies widely among viruses and is only weakly dependent on the type of host they infect. Instead, we identify mutational bias towards AT-enrichment and selection against CpG dinucleotides as the main factors responsible of the codon usage bias variation. Further insight on the mutational equilibrium within Orthocoronavirinae revealed that most coronavirus genomes are close to their neutral equilibrium, the exception is the three recently-infecting human coronaviruses, which lie further away from the mutational equilibrium than their endemic human coronavirus counterparts. Finally, our results suggest that while replicating in humans SARS-CoV-2 is slowly becoming AT-richer, likely until attaining a new mutational equilibrium.