RESUMO
The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing for early detection, prevention and surveillance of the disease. Here, we tested if targeted parallel reaction monitoring (PRM) quantification using high resolution Orbitrap instruments can provide the sensitivity and speed required for a high-throughput method that could be used for clinical diagnosis. Here we report a high-throughput and sensitive PRM-MS assay that enables absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times. Concatenated peptides (QconCAT) synthesized using isotopically labelled SARS-CoV-2 were used for absolute quantification. We developed a fast and high-throughput S-trap-based sample preparation method, which was then successfully utilized for testing 25 positive and 25 negative heat-inactivated nasopharyngeal swab samples for SARS-CoV-2 detection. The method was able to differentiate between negative and positive patients accurately within its limits of detection. Moreover, extrapolating from the QconCAT absolute quantification, our data show that patients with Ct values as low as 17.5 have NCAP protein amounts of around 7.5 pmol in swab samples. The present high-throughput method could potentially be utilized in specialized clinics as an alternative tool for detection of SARS-CoV-2.
RESUMO
The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.
