A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020

Abstract

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Keywords: B.1.1 variant; COVID-19; SARS-CoV-2; Sicily; new sequence; novel SARS-CoV-2 genome sequence; virus monitorization.

Figure 1

Deepness of sequencing (DS) along the viral genome and sequence variations identified. In the lower part, SARS-CoV-2 genomic coverage obtained by next-generation sequencing (NGS). The horizontal axis corresponds to positions in the SARS-CoV-2 Wuhan-Hu-1 consensus genome sequence (GenBank ID: NC_045512.2) and the vertical one to the depth of the sequence (DS) at a given position. The dashed horizontal line marks the NGS filter (DS ≥ 50). Above this, aligned over the nucleotide positions, the viral genome is schematized as a horizontal bar, with identification of the genes hosting nucleotide substitutions in our reported genome. Gene boundaries are marked with vertical lines crossing the bar, giving their corresponding nucleotide position in the sequence. Gene names are inscribed into the bar or placed under it, with indication with horizontal short lines or double-pointed arrows, of their spans (including the one of ORF1ab, which is a polyprotein-encoding gene). The part of the S gene that encodes the Receptor Binding Domain (RBD) is marked within the genome bar using transversal broken lines to indicate its boundaries. The amplicons used in Sanger sequencing (Table 1) are marked as magenta-colored rectangles within the genome bar, with their abbreviated names given below in magenta. Blue and red vertical lines emerging up from the genome bar, respectively, mark synonymous and non-synonymous sequence variants, described in the corresponding banners (equally colored). For non-synonymous changes the banners inscribe the nucleotide change (nucleotide numbering is that for the complete genome) and the amino acid substitution (amino acid numbering is that for the sequence of the mature protein), also numbering in black the amino acid changes from 1 to 11 as they occur along the genome from 5′ to 3′. A double frame in black and red indicates that the amino acid change belongs to the canonical set for the B.1.1. lineage. The background color of the banner is orange for amino acid changes reported in millions of sequences, deep yellow for tens of thousands, light yellow for thousands, and white for smaller numbers. These numbers are given in Table 2.

Figure 2

Maximum Likelihood Trees based on complete SARS-CoV-2 genomes. Evolutionary analyses were conducted in MEGA (see Methods), inferring the histories using the neighbor-joining method. Optimal trees are shown. The percentage of replicate trees in which the associated taxa clustered together is shown next to the branches. The scale bars measure phylogenetic distances as the indicated nucleotide substitutions per site. Optimal trees are shown. Trees are drawn to scale, with branch length in the same units as those of the evolutionary distance used to infer the phylogenetic tree. (A) Phylogenetic relations of our new B.1.1 sequence in the context of the most relevant SARS-CoV-2 variants, including variants which during the pandemic have been considered at least once as VOCs (Alpha, Beta, Gamma, Delta and Omicron) or VOIs (Epsilon, Mu, Iota, Eta, Kappa, Zeta, Theta, and Lambda), and also including the most prevalent variants (B.1, B.1.177, B.1.160, and B.1.258) that were circulating in Europe before the collection date of our study's sample. This analysis involved 21 nucleotide sequences, with a total of 29,919 position in the final dataset. (B) Phylogenetic relations of our new B.1.1 sequence with the seven previously reported SARS-CoV-2 sequences from Sicily in the time spanning between September and December 2020. Sequence lineage and GISAID ID are given for each genome, giving it an arbitrary number (1–8). Our new B.1.1 sequence is highlighted in bold in (A, B).