Characterization of SARS-CoV-2 Mutational Signatures from 1.5+ Million Raw Sequencing Samples.

We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned to the Omicron variant.

Humoral and cellular immune response in patients with hematological disorders after two doses of BNT162b2 mRNA COVID‐19 vaccine: A single‐center prospective observational study (NCT05074706)

Hematological patients at higher risk of severe COVID‐19 were excluded from the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccine trials. In this single‐center observational prospective study (NCT05074706), we evaluate immune response in the hematological patients followed at the Hematological Division of San Gerardo Hospital, Monza (Italy) deemed to be severely immunosuppressed after vaccination with two doses of the BNT162b2 vaccine. Anti‐SARS‐CoV‐2 immunoglobulin G titers above the cutoff value of 33.8 BAU/ml were detected in 303 (80.2%) out of the 378 patients enrolled. Patients with lymphoproliferative disorders had a significant lower probability of immunization (43.2% vs. 88.4%, p < 0.001). Patients treated with anti‐CD20 showed a significantly lower probability of immunization compared to all other treatments (21.4%, p < 0.0001). Among 69 patients who failed seroconversion, 15 patients (22.7%) showed a positive T‐cell response. Patients previously treated with anti‐CD20 were 2.4 times more likely to test positive for T‐cell responses (p = 0.014). Within a follow‐up of 9 months from the second COVID‐19 vaccination, symptomatic SARS‐CoV‐2 infections were reported by 20 patients (5.3%) and four of them required hospitalization. Successful serological or T‐cell‐mediated immunization conferred protection from symptomatic COVID‐19. Patients treated with anti‐CD20 who were not seroconverted after vaccination might still be protected from COVID‐19 due to the T‐cell immune response.

Early detection and improved genomic surveillance of SARS-CoV-2 variants from deep sequencing data.

A key task of genomic surveillance of infectious viral diseases lies in the early detection of dangerous variants. Unexpected help to this end is provided by the analysis of deep sequencing data of viral samples, which are typically discarded after creating consensus sequences. Such analysis allows one to detect intra-host low-frequency mutations, which are a footprint of mutational processes underlying the origination of new variants. Their timely identification may improve public-health decision-making with respect to traditional approaches exploiting consensus sequences. We present the analysis of 220,788 high-quality deep sequencing SARS-CoV-2 samples, showing that many spike and nucleocapsid mutations of interest associated to the most circulating variants, including Beta, Delta, and Omicron, might have been intercepted several months in advance. Furthermore, we show that a refined genomic surveillance system leveraging deep sequencing data might allow one to pinpoint emerging mutation patterns, providing an automated data-driven support to virologists and epidemiologists.

Large-scale analysis of SARS-CoV-2 synonymous mutations reveals the adaptation to the human codon usage during the virus evolution.

Many large national and transnational studies have been dedicated to the analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome, most of which focused on missense and nonsense mutations. However, approximately 30 per cent of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence. By performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, we show that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome overtime. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations. One-Sentence Summary. Synonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to the human codon usage.

VERSO: A comprehensive framework for the inference of robust phylogenies and the quantification of intra-host genomic diversity of viral samples.

We introduce VERSO, a two-step framework for the characterization of viral evolution from sequencing data of viral genomes, which is an improvement on phylogenomic approaches for consensus sequences. VERSO exploits an efficient algorithmic strategy to return robust phylogenies from clonal variant profiles, also in conditions of sampling limitations. It then leverages variant frequency patterns to characterize the intra-host genomic diversity of samples, revealing undetected infection chains and pinpointing variants likely involved in homoplasies. On simulations, VERSO outperforms state-of-the-art tools for phylogenetic inference. Notably, the application to 6,726 amplicon and RNA sequencing samples refines the estimation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution, while co-occurrence patterns of minor variants unveil undetected infection paths, which are validated with contact tracing data. Finally, the analysis of SARS-CoV-2 mutational landscape uncovers a temporal increase of overall genomic diversity and highlights variants transiting from minor to clonal state and homoplastic variants, some of which fall on the spike gene. Available at: https://github.com/BIMIB-DISCo/VERSO.

Mutational signatures and heterogeneous host response revealed via large-scale characterization of SARS-CoV-2 genomic diversity.

To dissect the mechanisms underlying the inflation of variants in the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) genome, we present a large-scale analysis of intra-host genomic diversity, which reveals that most samples exhibit heterogeneous genomic architectures, due to the interplay between host-related mutational processes and transmission dynamics. The decomposition of minor variants profiles unveils three non-overlapping mutational signatures related to nucleotide substitutions and likely ruled by APOlipoprotein B Editing Complex (APOBEC), Reactive Oxygen Species (ROS), and Adenosine Deaminase Acting on RNA (ADAR), highlighting heterogeneous host responses to SARS-CoV-2 infections. A corrected-for-signatures dN/dS analysis demonstrates that such mutational processes are affected by purifying selection, with important exceptions. In fact, several mutations appear to transit toward clonality, defining new clonal genotypes that increase the overall genomic diversity. Furthermore, the phylogenomic analysis shows the presence of homoplasies and supports the hypothesis of transmission of minor variants. This study paves the way for the integrated analysis of intra-host genomic diversity and clinical outcomes of SARS-CoV-2 infections.