To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. From the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 × 10⁻⁶ ± 0.2 × 10⁻⁶ per-base per-infection cycle (mean across both lineages of SARS-CoV-2 ± 2SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates—an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators—viral populations where mutations occur at a significantly faster rate.

Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen.

In response to the EFSA call New approaches in identifying and characterizing microbial and chemical hazards, the project INNUENDO (https://sites.google.com/site/theinnuendoproject/) aimed to design an analytical platform and standard procedures for the use of whole‐genome sequencing in surveillance and outbreak investigation of food‐borne pathogens. The project firstly attempted to identify existing flaws and needs, and then to provide applicable cross‐sectorial solutions. The project focused in developing a platform for small countries with limited economical and personnel resources. To achieve these goals, we applied a user‐centered design strategy involving the end‐users, such as microbiologists in public health and veterinary authorities, in every step of the design, development and implementation phases. As a result, we delivered the INNUENDO Platform V1.0 (https://innuendo.readthedocs.io/en/latest/), a stand‐alone, portable, open‐source, end‐to‐end system for the management, analysis, and sharing of bacterial genomic data. The platform uses Nextflow workflow manager to assemble analytical software modules in species‐specific protocols that can be run using a user‐friendly interface. The reproducibility of the process is ensured by using Docker containers and throught the annotation of the whole process using an ontology. Several modules, available at https://github.com/TheInnuendoProject, have been developed including: genome assembly and species confirmation; fast genome clustering; in silico typing; standardized species‐specific phylogenetic frameworks for Campylobacter jejuni, Yersinia enterocolitica, Salmonella enterica and Escherichia coli based on an innovative gene‐by‐gene methodology; quality control measures from raw reads to allele calling; reporting system; a built‐in communication protocols and a strain classification system enabling smooth communication during outbreak investigation. As proof‐of‐concepts, the proposed solutions have been thoroughly tested in simulated outbreak conditions by several public health and veterinary agencies across Europe. The results have been widely disseminated through several channels (web‐sites, scientific publications, organization of workshops). The INNUENDO Platform V1.0 is effectively one of the models for the usage of open‐source software in genomic epidemiology.

We show that the SARS-CoV-2 B.1.1.7 lineage is highly disseminated in Portugal, with the odds of B.1.1.7 proportion increasing at an estimated 89% (95% confidence interval: 83–95%) per week until week 3 2021. RT-PCR spike gene target late detection (SGTL) can constitute a useful surrogate to track B.1.1.7 spread, besides the spike gene target failure (SGTF) proxy. SGTL/SGTF samples were associated with statistically significant higher viral loads, but not with substantial shift in age distribution compared to non-SGTF/SGTL cases.

Probiotics are living microorganisms used as nutritional additives that confer health benefits on the host. Their use in food products is very attractive, especially if they could also inhibit important foodborne pathogens. In this study, antimicrobial activity against several foodborne pathogens was screened for 280 lactic acid bacteria (LAB) isolated from different food products and the probiotic characteristics of bacteriocinogenic isolates were evaluated. Seven out of 280 LAB isolates were selected due to their bacteriocinogenic properties and identified by 16S rRNA gene sequence analysis as Pediococcus pentosaceus (n = 6) and Lactobacillus plantarum (n = 1). Virulence factors and antibiotic resistances were not detected for any of the isolates. Except for L. plantarum R23, all the isolates were able to survive through the simulated gastrointestinal tract conditions. Only P. pentosaceus CFF4 was able to adhere to Caco-2 cells after the simulated gastrointestinal tract passage. In conclusion, even though in vivo studies should be performed, P. pentosaceus CFF4, which was also able to inhibit the growth of foodborne pathogens in vitro, seems to be a potential probiotic to be used in the food industry.