A SARS-CoV-2 mutant from B.1.258 lineage with ∆H69/∆V70 deletion in the Spike protein circulating in Central Europe in the fall 2020

Abstract

SARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged independently in at least six lineages of the virus (namely, B.1.1.7, B.1.1.298, B.1.160, B.1.177, B.1.258, B.1.375). We analyzed SARS-CoV-2 samples collected from various regions of Slovakia between November and December 2020 that were presumed to contain B.1.1.7 variant due to drop-out of the Spike gene target in an RT-qPCR test caused by this deletion. Sequencing of these samples revealed that although in some cases the samples were indeed confirmed as B.1.1.7, a substantial fraction of samples contained another ∆H69/∆V70 carrying mutant belonging to the lineage B.1.258, which has been circulating in Central Europe since August 2020, long before the import of B.1.1.7. Phylogenetic analysis shows that the early sublineage of B.1.258 acquired the N439K substitution in the receptor-binding domain (RBD) of the Spike protein and, later on, also the deletion ∆H69/∆V70 in the Spike N-terminal domain (NTD). This variant was particularly common in several European countries including the Czech Republic and Slovakia but has been quickly replaced by B.1.1.7 early in 2021.

Keywords: B.1.1.7; B.1.258; Deletion; SARS-CoV-2; Spike; Variant.

Fig. 1

A Points of recurrent emergence of the ∆H69/∆V70 mutation. Nextclade lineages [20] in color. Pangolin lineages [12] in brackets. B.1.258∆ denotes B.1.258 with ∆H69/∆V70 deletion. B Origins of B.1.258 ∆H69/∆V70 variant. Mutations S:N439K, S:∆H69/∆V70, NSP9:M101I, NSP12:V720I, and NSP13:A598S are marked. Collection dates near to the important branching points are shown. The samples shown in the phylogenetic trees were selected from GISAID database [20] to cover significant lineages of interest (B.1.258 ∆H69/∆V70 variant in different countries and its outgroups and lineages containing ∆H69/∆V70 mutation and their outgroups). Phylogenetic trees were built using Augur v. 6 [21]. The prevalence was assessed based on all samples in GISAID (downloaded on April 29, 2021) for a particular country based on PANGO lineage classification [12] provided in GISAID metadata. C Prevalence of B.1.258 and B.1.1.7 variants in selected countries out of GISAID samples collected between September 2020 and March 2021. The highest monthly prevalence of both B.1.258 and B.1.1.7 is shown for each country. B.1.258 counts include all sublineages, regardless of the presence of ∆H69/∆V70 mutation. Only months where at least 20 samples were sequenced in the country are shown. Note that the samples may not be representative, as the sampling strategy differs from country to country, and it also changes over time. D Ct values in the swab specimens from the city of Trenčín (Slovakia) mass testing grouped according to the identified lineages. Ct values from routine RT-qPCR assay targeting RdRp, E, and human RNase P genes (used as a control to exclude possible impact of the sample quality) are shown. Classification of samples marked in red was confirmed by sequencing. B.1.258∆ denotes B.1.258 with ∆H69/∆V70 deletion. RT-qPCR assays were performed on RNA extracted by the Biomek i5 Automated Workstation using the RNAdvance Viral kit (Beckman Coulter, Indianapolis, Indiana, USA) from swab samples previously collected for the primary diagnostics. Besides rTEST COVID-19 RT-qPCR Allplex kit (MultiplexDX, Bratislava, Slovakia) targeting the RNA-dependent RNA polymerase (RdRp) and Envelope (E) genes, the newly developed rTEST COVID-19 qPCR B.1.1.7 kit (MultiplexDX, Bratislava, Slovakia) was used to differentiate B.1.1.7 and B.1.258 ∆H69/∆V70 variants [17]. The real-time PCR was performed on a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Foster City, California, USA). The SARS-CoV-2 sequences were determined on a MinION sequencer (Oxford Nanopore Technologies) using a protocol based on PCR-tiling of 2-kb long amplicons [22]. The horizontal lines represent mean values. The differences in Ct values were statistically evaluated by unpaired t test using GraphPad Prism version 8.4.0