RESUMO
High transmissibility is a hallmark of the Omicron variant of SARS-CoV-2. Understanding the molecular determinants of Omicrons transmissibility will impact development of intervention strategies. Here we map the electrostatic potential surface of the Spike protein to show that major SARS-CoV-2 variants have accumulated positive charges in solvent-exposed regions of the Spike protein, especially its ACE2-binding interface. Significantly, the Omicron Spike-ACE2 complex has complementary electrostatic surfaces. In contrast, interfaces between Omicron and neutralizing antibodies tend to have similar positively charged surfaces. Structural modeling demonstrates that the electrostatic property of Omicrons Spike receptor binding domain (S RBD) plays a role in enhancing ACE2 recognition and destabilizing Spike-antibody complexes. Collectively, our structural analysis implies that Omicron S RBD interaction interfaces have been optimized to simultaneously promote access to human ACE2 receptors and evade antibodies. These findings suggest that electrostatic interactions are a major contributing factor for increased Omicron transmissibility relative to other variants.
RESUMO
BackgroundA major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used standard RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. MethodsWe implement a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification and nano-scale qPCR based on the Fluidigm 192.24 microfluidic chip. We validate the method using both positive controls and nasopharyngeal swab samples. ResultsUsing SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of the Fluidigm method by 1,000-fold, enabling detection below 1 copy/l. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/l). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/l) in 17 samples with negative clinical diagnosis, indicating a potential false negative rate of 18.7% by clinical diagnostic procedures. ConclusionThe three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/l) and has the potential to reduce the false negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections. SummaryWe test, implement and report the results of a microfluidic RT-qPCR assay system involving sequential RT, preamplification and nano-scale qPCR that can robustly detect SARS-CoV-2 in clinical samples with viral loads less than 1 copy/ul.
