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

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https://doi.org/10.1007/s11262-021-01866-5 SHORT REPORT

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

Broňa Brejová

¹

· Kristína Boršová

^2,3

· Viktória Hodorová

⁴

· Viktória Čabanová

²

· Lenka Reizigová

^5,6

· Evan D. Paul

⁷

· Pavol Čekan

⁷

· Boris Klempa

²

· Jozef Nosek

⁴

· Tomáš Vinař

¹

Received: 17 May 2021 / Accepted: 13 August 2021 / Published online: 27 August 2021

Abstract

SARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged inde- pendently 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 SARS-CoV-2 · B.1.1.7 · B.1.258 · Variant · Spike · Deletion

The SARS-CoV-2 mutants carrying the Spike ∆H69/∆V70 deletion are easily misidentified in routine RT-qPCR as quickly spreading variant B.1.1.7. Here, we demonstrate that many Slovak samples collected in December 2020, originally presumed to contain B.1.1.7, belong to a sub- lineage of B.1.258, which acquired the Spike N439K sub- stitution followed by the ∆H69/∆V70 deletion. Here, we

denote this sublineage as B.1.258 ∆H69/∆V70 to distin- guish it from sublineages without the ∆H69/∆V70 dele- tion (most samples in sublineages B.1.258.1–B.1.258.3 and B.1.258.14–B.1.258.16).

The variant has been highly prevalent in the Czech Republic (~ 59% sequenced samples between September and December 2020), Slovakia (~ 25% sequenced samples over the same period), and several other countries. The

∆H69/∆V70 deletion is associated with increased infectivity and evasion of the immune response [1] and evidence sug- gests that this mutation has arisen in B.1.258 independently

Edited by Detlev H. Kruger.

Broňa Brejová and Kristína Boršová have contributed equally to this work.

* Boris Klempa boris.klempa@savba.sk

1 Faculty of Mathematics, Physics and Informatics, Comenius University in Bratislava, Mlynská dolina, 842 48 Bratislava, Slovak Republic

2 Institute of Virology, Biomedical Research Center of the Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovak Republic

3 Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, 842 15 Bratislava, Slovak Republic

4 Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, 842 15 Bratislava, Slovak Republic

5 Regional Authority of Public Health, Trenčín, Slovak Republic

6 Department of Laboratory Medicine, Faculty of Healthcare and Social Work, Trnava University, Trnava, Slovak Republic

7 MultiplexDX, s.r.o., Comenius University in Bratislava Science Park, Ilkovičova 8, 841 04 Bratislava, Slovak Republic

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of the B.1.1.7 variant. The deletion is likely to cause a drop- out of the Spike gene target in some RT-qPCR assays [2–5]

and thus its carriers can be easily misidentified as B.1.1.7 (as happened in Slovakia [6]). The B.1.258 ∆H69/∆V70 variant also contains the N439K mutation in the receptor-binding domain (RBD) of the Spike protein, enhancing its affinity to the ACE2 receptor and facilitating escape from monoclonal antibodies and convalescent sera [7]. The lineage B.1.258 is generally characterized by five mutations (one in ORF1a, two in ORF1b, and two in Spike genes) but can be further divided into 21 sublineages differing by various combina- tions of additional 40 mutations. The ∆H69/∆V70 deletion is listed as one of the characteristic mutations for 14 of these sublineages (B.1.258.4–7, 9, 11, 12, 17–23) [8]. Most of the B.1.258 ∆H69/∆V70 samples also include substitutions in the virus replication proteins, namely, NSP9 M101I, NSP12 V720I, and NSP13 A598S.

The ∆H69/∆V70 deletion has arisen independently at least six times (Fig. 1A), frequently co-occurring with mutations in the Spike receptor-binding domain (RBD) such as N439K, Y453F, and N501Y [9–13]. Besides the B.1.258 ∆H69/∆V70 and B.1.1.7 variants, the deletion has been observed in B.1.1.298 (Danish mink farm outbreak [10, 14]) and B.1.375 (USA [15, 16]). Recurrent emergence even within well-established clades (such as EU1 (B.1.177) and EU2 (B.1.160)) suggests that ∆H69/∆V70 can increase overall fitness in concert with mutations that would be oth- erwise neutral or lower the infectivity [7, 9].

The earliest B.1.258 samples with ∆H69/∆V70 deletion were observed in Switzerland and in the UK at the begin- ning of August 2020 (Fig. 1B). The Spike N439K mutation has emerged before the ∆H69/∆V70 deletion (see sample from Romania as early as May 13, 2020); the outgroup that does not contain N439K has been observed in England on March 22, 2020. During the fall of 2020, B.1.258 variant gained significant prevalence in multiple countries, includ- ing Croatia, the Czech Republic, Sweden, Slovakia, Slove- nia, Poland, Denmark, and Austria (Fig. 1C). Analysis of disease incidence and hospitalization data did not reveal any clear trends that would be common to all or most of the examined countries when correlated with B.1.258 preva- lence. Nevertheless, in several countries (e.g., Austria, the Czech Republic, and Slovakia), the increasing incidence and hospitalization trends started already during the fall of 2020 while the B.1.1.7 variant dominance started later, early in 2021. One can therefore speculate that worsening of the epidemiologic situation in those countries could be at least partially attributed to the spread of the B.1.258 variant.

A newly developed RT-qPCR assay differentiating B.1.1.7 from other ∆H69/∆V70 variants [17] indicated that out of 122 clinical samples from a mass testing campaign

50 (41.0%) samples carried the ∆H69/∆V70 deletion but were not B.1.1.7 (selected samples were later confirmed as B.1.258 ∆H69/∆V70 by sequencing). A routine RT-qPCR assay showed significantly lower Ct values in the swab sam- ples for both B.1.1.7 and B.1.258 ∆H69/∆V70 samples, reflecting higher viral loads in patients carrying these vari- ants (Fig. 1D).

Altogether, we have described a B.1.258 ∆H69/∆V70 variant of the SARS-CoV-2 virus that contains the S:N439K mutation shown to enhance the binding affinity of the Spike protein to human ACE2 receptor and facilitating escape from immune response, the S:∆H69/∆V70 mutation, which is known to increase viral infectivity, as well as several other non-synonymous mutations in NSP9, NSP12, and NSP13 likely affecting viral replication. RT-qPCR analysis on ran- dom samples collected during a mass testing campaign indi- cates that B.1.258 ∆H69/∆V70 samples carry higher viral loads compared to other strains, similarly as in the case of the B.1.1.7 variant. Interestingly, the B.1.258.17 sublineage has accumulated a higher number of mutations compared to other B.1.258 samples, including additional substitutions in the Spike protein (L189F, V772I), helicase NSP13 (P53L), and a substitution Q185H in ORF3a involved in apoptosis [18].

B.1.258 ∆H69/∆V70 variant shares many characteristics with quickly spreading B.1.1.7 and is likely responsible for worsening of the epidemiological situation in several coun- tries, including Slovakia and the Czech Republic, in the fall of 2020. While in Slovenia the B.1.258 variant was still one of the major strains as of March 2021, in Slovakia the B.1.1.7 variant has extremely quickly replaced the B.1.258 by the beginning of February 2021 (estimate of ~ 74% of B.1.1.7 and ~ 6% of B.1.258 nationwide on February 3, 2021 by differential RT-qPCR tests [19] compared to 4.1%

of B.1.1.7 and 41% of B.1.258 in the city of Trenčín on December 19–20, 2020). The B.1.258 ∆H69/∆V70 vari- ant, as most of any other variants, was quickly replaced by B.1.1.7 variant early in 2021. Most recently, isolated cases associated with B.1.258 ∆H69/∆V70 variant were reported from Slovenia and Croatia in June 2021 [20].

Our characterization of the B.1.258 ∆H69/∆V70 vari- ant that has been circulating in several European countries and appears to result in higher viral loads highlights the importance of vigilant genomic surveillance in properly identifying and tracking SARS-CoV-2 variants that dis- play the potential to derail worldwide efforts to mitigate the pandemic.

Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11262- 021- 01866-5.

Acknowledgements We gratefully acknowledge the authors from

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A

Wuhan/Hu-1/2019 [B]

Malaysia/UNIMAS-M4061/2020 [B.6]

Uganda/UG089/2020 [A]

Russia/PSK-RII-MH6191S/2020 [B.1.131]

Chile/MA-194116-B/2020 [C.11]

Sweden/20-53002/2020 [B.1.1.302]

Brazil/AM-L70-CD1722/2020 [P.1]

Brazil/RJ-00541/2020 [P.2]

Australia/QLD1140/2020 [D.2]

India/WB-IICB-036/2020 [B.1.1.70]

South Korea/KDCA0108/2020 [B.1.1.130]

Denmark/DCGC-1278/2020 [B.1.1.298]

Denmark/DCGC-19132/2020 [B.1.1.298]

Denmark/DCGC-27186/2020 [B.1.1.298]

Denmark/DCGC-2203/2020 [B.1.1.298]

Russia/MOS-RII-MH9270S/2020 [B.1.1.238]

England/MILK-C2F60D/2020 [B.1.1.7]

England/ALDP-DCB295/2020 [B.1.1.7]

England/ALDP-DC5EC4/2020 [B.1.1.7]

New Zealand/20CV0692/2020 [B.1.1.7]

South Africa/UFS-VIRO-NGS-35/2020 [B.1]

Ireland/CO-NVRL-75IRL32904/2020 [B.1.258.2]

Scotland/QEUH-9B85F0/2020 [B.1.258]

Slovakia/UKBA-404/2020 [B.1.258]

England/SHEF-CA66A/2020 [B.1.258.5]

Denmark/DCGC-4406/2020 [B.1.258.11]

Czech Republic/NRL_10181/2020 [B.1.258]

USA/TX-HMH-5181/2020 [B.1.206]

Norway/5852/2020 [B.1.177]

France/OCC-189/2020 [B.1.177]

England/ALDP-B24B28/2020 [B.1.177.20]

England/MILK-A7DC1F/2020 [B.1.177]

England/CAMC-B1FC1E/2020 [B.1.177]

England/NORW-F2CFE/2020 [B.1.177]

England/CAMC-BBB92C/2020 [B.1.177]

Belgium/ULG-10838/2020 [B.1.389]

Australia/NSW1328/2020 [B.1.517]

USA/MA-MGH-03435/2020 [B.1.2]

South Africa/KRISP-DG01842216/2020 [B.1.351]

USA/NH-UPHL-2012558654/2020 [B.1.375]

USA/AL-CDC-STM-034/2020 [B.1.375]

Tunisia/61627/2020 Malaysia/IMR-WI194/2020 [B.1.36.16]

Norway/3715/2020 [B.1.457]

Switzerland/SG-ETHZ-430703/2020 [B.1.160]

Hungary/UD-90512/2020 [B.1.160]

France/ARA-96089/2020 [B.1.160]

2.0E-4 19A19B 20A.EU2

20I/501.V1 20A 20B

20J/501.V3 20D 20F 20B/S.484K 20A/N.194L 20C20C/S.501T

20E.EU1 Tunisia/61627/2020 [B.1.9]

S: ∆H69/∆V70 S: ∆H69/∆V70

S: ∆H69/∆V70

B.1.258∆

B.1.1.7 B.1.258Δ Others B.1.1.7 B.1.258Δ Others B.1.1.7 B.1.258Δ Others 0

10 20 30 40

Ct value

SARS-CoV-2

RdRp gene SARS-CoV-2

E gene human

RNaseP

p<0.05 p<0.001

B.1.258 B.1.1.7

0%

50%

100%

25%

Austria (n=4479) 80%

11%

Germany (n=94105) 79%

67%

Slovakia (n=3669) 97%

0%

50%

100%

18%

Belgium (n=20781) 76%

13%

Italy (n=25576) 82% 86%

53%

Slovenia (n=12718)

0%

50%

100%

45% 67%

Croatia (n=2167)

12%

Netherlands (n=30319) 88%

31%

Sweden (n=41070) 92%

0%

50%

100%

66% 85%

Czech Republic (n=3837)

17%

Norway (n=8399) 81%

8%

Switzerland (n=34875) 90%

IX X XI XII I II III

0%

50%

100%

15%

50%

Denmark (n=55992)

26%

Poland (n=10871) 91%

4%

United Kingdom (n=509021) 98%

C D

B

Wuhan/Hu-1/2019

England/20130069504/2020[2020-03-22]

Romania/284213/2020 [2020-05-13]

Ireland/KE-NVRL-73IRL40299/2020 England/ALDP-A6F90A/2020 USA/NY-Wadsworth-222417-01/2020 Scotland/QEUH-9B85F0/2020

England/MILK-AB86B0/2020 Switzerland/BL-ETHZ-230022/2020 [2020-08-03]

England/SHEF-CA66A/2020 England/QEUH-9611E3/2020 [2020-08-02]

England/QEUH-96F75C/2020 Switzerland/BL-ETHZ-270140/2020

Czech Republic/NRL-9962/2020 Slovakia/UKBA-403/2020 England/MILK-9AB3C9/2020

Denmark/DCGC-12314/2020 Slovakia/UKBA-404/2020 England/205082085/2020 Wales/PHWC-16C03E/2020 Switzerland/BS-42376604/2020 [2020-08-01]

Czech Republic/NRL_8884/2020 Czech Republic/NRL_10594/2020 Czech Republic/NRL_9840/2020

Czech Republic/NRL_10421/2020

Czech Republic/NRL_10181/2020 Slovakia/UKBA-604/2020 Denmark/DCGC-4406/2020

2.0E-4 B.1B.1.258

S: ∆H69/∆V70

B.1.258∆

S: N439K

NSP9: M101I NSP12: V720I NSP13: A598S

Switzerland/ZH-ETHZ-410010/2020

Denmark/DCGC-23126/2020 Denmark/DCGC-24852/2020

Netherlands/GE-RIVM-20154/2020 Czech Republic/NRL_11782-2/2020

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. Col- lection 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). Phylo- genetic 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 high- est 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 Bio- systems, Foster City, California, USA). The SARS-CoV-2 sequences were determined on a MinION sequencer (Oxford Nanopore Technol- ogies) 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 Graph- Pad Prism version 8.4.0

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generated and shared via GISAID (https:// www. gisaid. org/), on which this research is based (GISAID Acknowledgements in Supplementary Information).

Author contributions BB, TV, JN, and BK conceived the study. BB, KB, BK, and TV analyzed the data. VH, KB, and VČ performed the experimental work. LR and BK procured samples for analysis. PČ, EDP, KB, VČ, and BK conceived and implemented the RT-qPCR study. All authors edited and approved the manuscript.

Funding Our research was supported by Grants from the Slovak Research and Development Agency (APVV-18-0239 to JN, PP- COVID-20-0017 to BK, and PP-COVID-20-0116 to PC and BK), the Scientific Grant Agency (VEGA 1/0463/20 to BB, VEGA 1/0458/18 to TV), and the European Union's Horizon 2020 Research and Innova- tion program (EVA-GLOBAL Project #871029 to BK and PANGAIA Project #872539). The research was also supported in part by OPII project ITMS2014: 313011ATL7.

Data availability All sequences sequenced by the authors have been submitted to GISAID.

Declarations

Conflict of interest MultiplexDX, s.r.o. has developed and manufac- tured the B.1.1.7 differential RT-qPCR assay kit. Biomedical Research Center of the Slovak Academy of Sciences has entered into collabora- tion with MultiplexDX, s.r.o. for development and validation of RT- qPCR tests. All other authors declare no competing interests.

Ethical approval All clinical specimens used within this study were previously collected for the purpose of primary diagnosis of SARS- CoV-2 by the Regional Authority of Public Health, Trenčín, Slovakia and were transferred to the Biomedical Research Center of the Slovak Academy of Sciences, Bratislava, Slovakia while made unidentifiable for the researchers performing this study. The study has been approved by the Ethics committee of Biomedical Research Center of the Slovak Academy of Sciences, Bratislava, Slovakia (Ethics Committee State- ment No. EK/BmV-02/2020).

Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

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