Heterogeneity of SARS-CoV-2 virus produced in cell culture revealed by shotgun proteomics and supported by genome sequencing.

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics.

Evaluation of the Biotoxis qPCR Detection Kit for Francisella tularensis Detection in Clinical and Environmental Samples.

Rapid and reliable detection and identification of Francisella tularensis (a tier 1 select agent) are of primary interest for both medical and biological threat surveillance purposes. The Biotoxis qPCR detection kit is a real-time quantitative PCR (qPCR) assay designed for the detection of Bacillus anthracis, Yersinia pestis, and F. tularensis in environmental or biological samples. Here, we evaluated its performance for detecting F. tularensis in comparison to previously validated qPCR assays. The Biotoxis qPCR was positive for 87/87 F. tularensis subsp. holarctica (type B) strains but also for F. tularensis subsp. novicida It was negative for Francisella philomiragia and 24/24 strains belonging to other bacterial species. For 31 tularemia clinical specimens, the Biotoxis qPCR displayed a sensitivity between 90.32% and 96.55%, compared to qPCR tests targeting ISFtu2 or a type B-specific DNA sequence, respectively. All 30 nontularemia clinical specimens were Biotoxis qPCR negative. For water samples, the Biotoxis qPCR limit of detection was 1,000 CFU/liter of F. tularensis For 57 environmental water samples collected in France, the Biotoxis qPCR was positive for 6/15 samples positive for ISFtu2 qPCR and 4/4 positive for type B qPCR. In conclusion, the Biotoxis qPCR detection kit demonstrated good performances for F. tularensis detection in various biological and environmental samples, although cross-amplification of F. tularensis subsp. novicida must be considered. This plate format assay could be useful to test a large number of clinical or environmental specimens, especially in the context of natural or intentional tularemia outbreaks.

Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.