SARS-CoV-2 N501Y introductions and transmissions in Switzerland from beginning of October 2020 to February 2021 â implementation of Swiss-wide diagnostic screening and whole genome sequencing

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations by the European Center for Disease Prevention and Control (ECDC) and World Health Organization (WHO) for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological definitions based on travel history and the S gene dropout in certain diagnostic systems. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 3492 VoCs have been identified since the detection of the first Swiss case in October 2020, with 1370 being B1.1.7, 61 B.1.351, and none P.1. The remaining 2061 cases of VoCs have been described without further lineage specification. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Yspecific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint


Introduction
Since December 2020, three emerging SARS-CoV-2 lineages -B.1.1.7 (N501Y.V1), B.1.351 (N501Y.V2), and P.1 (B.1.1.28.1; N501Y.V3) -have generated concern in public and scientific communities. All three lineages show a rapid spread and displacement of locally established SARS-CoV-2 lineages, in the United Kingdom (UK), South Africa (ZA), and Brazil (BR), respectively, where they were first detected [1][2][3][4][5][6][7][8] . The B.1.1.7 and B. 1.351 lineages have subsequently been reported in many countries around the globe, including Switzerland. Most recently the P.1 lineage, exhibiting the N501Y and E484K mutations, among others, was described in Brazil 9-11 and has also been found in Japan 12 . It is hypothesized that the viral variants B.1.1.7, B.1.351, and P.1 are more transmissible compared to other circulating variants, due to a higher affinity towards the angiotensinconverting enzyme 2 (ACE2) receptor resulting from the N501Y mutation 13 and were defined as variants of concern (VoC). In the last week of December 2020, the B.1.1.7 lineage accounted for more than 25% of overall published genomes from the UK (according to the Global Initiative on Sharing Avian Influenza Data (GISAID) as of 19 January 2021), but it is estimated to account for up to 70% of transmission events in specific areas of the UK 14 .
Waste water screening in Switzerland suggests that the B.1.1.7 lineage was present in Switzerland in early December 15 . In South Africa, no reliable prevalence data on the B.1.351 lineage is available, but published data suggests that this VoC is also spreading more rapidly 6,16 ).
The first genome belonging to the B.1.1.7 lineage was detected in September 2020 in the UK (according to the GISAID database) and showed 17 lineage specific polymorphisms, eight of which are located in the 1273 amino acid spike glycoprotein (nucleotide position 21563 to 25384, [17][18][19] Table S1). The spike glycoprotein is crucial for viral infection of host cells and is an important target for neutralizing antibodies 20 . Some of the B.1.1.7 polymorphisms may modulate the protein's function, such as the N501Y mutation in the receptor binding domain, the HV 69-70 deletion, and the P681H mutation in the furin cleavage site 21,22 . The HV 69-70 deletion at nucleotide position 21765-21770 of the SARS-CoV-2 genome results in a dropout of the spike glycoprotein (S) gene diagnostic target in some commercial PCR assays. Although the S gene dropout is not specific for the B.1.1.7 lineage, it may nevertheless be a good first approach to screen for B.1.1.7 variants 23,24 .
This HV 69-70 deletion in the spike glycoprotein might favour immune escape 17 . The B.1.1.7 variant also carries several lineage specific mutations in the ORF8 gene (Table S1), which might also be associated with decreased host immunity against SARS-CoV-2. Indeed, the ORF8 protein disrupts antigen presentation and reduces the recognition and the elimination of virus-infected cells by cytotoxic T-cells 25 .
The B.1.351 lineage was first detected in October 2020 in ZA (according to the GISAID database) and also shares the N501Y mutation, but has otherwise different lineagedetermining polymorphisms (Table S1) and does not show a characteristic S gene dropout due to lack of the HV 69-70 deletion. Of particular concern is the spike glycoprotein E484K mutation, which has been shown to reduce binding affinities towards neutralizing antibodies 6,26,27 . Some of the polymorphisms that the viral variants described here possess are also present in other SARS-CoV-2 lineages (Table S2)  lineage. Third, N501Y-specific PCRs were established in several laboratories. Suspected cases were confirmed by amplicon sequencing or whole genome sequencing for accurate lineage determination. This screening strategy was rapidly and sequentially implemented . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

Methods
Ethical statement. This study was conducted in close collaboration with the FOPH and part of an epidemiological assessment (Communicable Diseases Legislation -Epidemics Act). In addition, the study was approved as a multi-center study by the leading ethical committee  . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint As a first step, an epidemiological case definition with recent travel history to the UK or ZA was used to identify potential carriers of the VoC. In mid-January 2021 this was expanded to BR. Both direct and indirect contacts of people travelling from these areas were considered.
Patient travel history was recorded on mandatory FOPH reporting forms by clinical and laboratory institutions 31 , as well as by cantonal physicians during contact tracing. In Switzerland, quarantine upon arrival was made mandatory for travellers from the UK and ZA from 28th December 2020; and from BR from 21st January 2021 32 .
As a second step, a microbiological case definition was used. LZM Risch AG using the TaqPath™ COVID-19 Combo Kit diagnostic assay (Thermo Fisher) noted a significant increase in S gene dropouts through November and December 2020. This multi-target PCR assay target sequences within the SARS-CoV-2 genes ORF1ab, N and S. A geographical distinction was observed: S gene dropouts were mainly noted in the eastern region of Switzerland, whereas other laboratories using the same assay in the western region of During the Christmas holidays 2020, personnel and technical resources were limited, and focusing on these first two steps provided an initial screening program (from 22nd December,). In January 2021 the screening strategy was modified with a third step: several N501Y-specific PCR protocols were validated and established in laboratories throughout the country. All diagnostic laboratories in Switzerland (Table S3). Most centers used the commercial assay SARS . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint Spike N501Y (53-0780-96; TIB MOLBIOL, Germany). In addition to the N501Y-specific PCR, at the University Hospital Lausanne, ORF8 PCR/sequencing was used for the initial 12 samples received, in order to rapidly obtain results based on the presence/absence of the B.1.1.7 specific mutations C27972T, G28048T and A28111G 17 , while waiting for the results of whole genome sequencing and the implementation of the S dropout and N501Y-specific PCR.
Included samples for sequencing and reporting. The initial identified samples, from 22nd December 2020, were strongly biased towards the epidemiological and microbiological case definition (S gene dropout). From the beginning of January 2021, an increasing number of laboratories have joined the incentive and implemented N501Y-specific protocols.
Meanwhile, older samples collected from September to December 2020 have also been sequenced. All VoC were reported to the FOPH via an electronic reporting form. Lausanne. Briefly, specific primers were used to generate an amplicon for Sanger sequencing. All sequences were then compared to available sequences on GISAID.
Whole genome sequencing protocols. For this study whole genome sequencing data was produced using Illumina and Oxford Nanopore Technologies (ONT, Oxford, UK) sequencing.
SARS-CoV-2 genomes were generally amplified following the amplicon sequencing strategy CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint flow cell preparation were as described previously 39,40 . ONT sequencing was performed on a GridION X5 instrument (Oxford Nanopore Technologies) with real-time base calling enabled (ont-guppy-for-gridion v.4.2.3; fast base calling mode). Sequencing runs were terminated after production of at least 100,000 reads per sample. Bioinformatic analyses followed the workflow described (https://artic.network/ncov-2019/ncov2019-bioinformaticssop.html) using artic version 1.1.3. Consensus sequences were generated using medaka (https://github.com/nanoporetech/medaka) and bcftools 41 .
Each center used individual bioinformatic pipelines to check for sequencing quality and generate the consensus sequences details are shared in GISAID (e.g. 37 ; https://gitlab.com/RKIBioinformaticsPipelines/ncov_minipipe/). The consensus sequence data were either directly shared between diagnostic laboratories or via GISAID. The resulting alignment of focal and contextual genomes was used to infer clusters with zero single nucleotide mutations (SNPs) using a custom python script (https://github.com/appliedmicrobiologyresearch). Identified clusters were investigated regarding cantonal origin of the sample as well as known travel history.  . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Figure 2. Distribution of absolute numbers as epidemiological curves according to canton and time.
Absolute numbers reflect a biased sample set due to the initial case definitions, higher usage of antigen test in some regions, and distribution of diagnostic capacities. This does not reflect the prevalence of cases. Also the current amount of specific lineages is biased due to different sequencing capacities.
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The copyright holder for this preprint this version posted February 12, 2021. ; This initial screening was implemented in a few centers in the last two weeks of December 2020, during which time there was a selection bias due to the case definition and limited sequencing capacity over the Christmas holidays. Complementary to these cases, two additional datasets were analysed using whole genome sequencing: the first included 545 randomly selected SARS-CoV-2 samples from throughout Switzerland (Viollier AG, a large private laboratory) from 18th to 24th December 2020. Within this first dataset, no VoC was found. This corresponds to 0.06% of the overall SARS-CoV-2 positive cases.
Since the second week of January 2021, increasing numbers of SARS-CoV-2 positive samples were analysed using an N501Y-specific PCR. However, at this stage our data does not allow the reliable determination of a Swiss-wide prevalence, as not all PCR positive cases are fully re-analysed with the N501Y-specific PCR. However, some laboratories reanalyse every SARS-CoV-2 positive case and thereby individual prevalence rates for VoCs could be determined for the last two weeks: The University Hospital Geneva reported 40% positivity for VoCs (25th to 31th of January); University of Zurich reported 13.3% (18th to 24th January), 20.2% (25th to 31th of January), and 28.4% (1st to 5th of February); Viollier AG reported 15% (25th to 31th of January) and 19.9% (1st to 5th of February); University Hospital Basel reported 29% (25th to 31th January) and 45% (1st to 4th of February); University of Bern reported 10.2% (25th to 31st January) and 30% (1st to 3rd February); Bioanalytica reported 6% (25th to 31st January) and 21.6% (1st to 5th February). LMZ Dr.
Risch reported 18.5% (25th to 31st January) and 21% (1st to 3rd February). Within the upcoming weeks, we expect that a Swiss wide prevalence determination for VoC is established and reported. Of note, the detection rate has been going down in some centers and also overall, albeit slowly. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint biased due to the fact that certain laboratories may receive samples more predominantly from paediatric physicians or hospitals.
Phylogenetic relatedness of first cases. A total of 260 N501Y-carrying (B.1.1.7 n=249, and B.1.351 n=11) high quality genomes from Switzerland were available for phylogenetic analysis. For 58 cases (known for University Hospitals Basel and Lausanne) a travel history to an endemic country or known contact to a traveller was available, however, for most cases the risk exposure was not available. For 213 cases the canton of residence was known. Using a 0 SNP threshold, we infer 9 out of the 11 B.1.351 cases to be single introduction events, two cases (from BS) are genetically (0 SNPs) and epidemiologically linked and trace back to a ZA travel returner and a transmission to a family member ( Figure   S3). The phylogenetic analysis of B.1.1.7 cases shows at least 116 single introductions into 11 cantons (Figure 2A), 106 without immediate links (0 SNP distance) to other genomes in the sub-sampled global dataset, 11 of which were known risk contacts or travellers ( Figure   2B). Ten further single introductions had genetic links to genomes from UK samples, two of which had known travel history or risk contact. We identified 45 clusters (0 SNP distance) comprising 133 (range 2-10) genomes. 18 clusters contain samples with known travel links to the UK or risk contacts. Of interest, ten of these clusters contained cases from different cantons -suggesting outside of household transmission (Figure 2C).
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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint were documented. However, they could also be due to multiple introductions of identical variants from the UK to Switzerland and is a possibility that we cannot rule out.
Our sampling strategy focusing first on the epidemiological risk and a microbiological case definition including the S gene dropout, and the initial lack of diagnostic capabilities to confirm the VoC introduced a strong selection bias of samples. Thus our findings should be interpreted with care. Currently, our data does not allow us to properly determine the prevalence of VoCs in Switzerland. However, some laboratories have established a workflow . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted February 12, 2021. VoC identification based on the S gene dropout is not specific and sensitive enough to identify VoC lineages (see Table S1). Our WGS data of samples collected in December 2020 showed that most of the S gene dropout samples were due to the B. it was available, with a specific PCR, allowed to rapidly screen isolates and identify the N501Y mutation as a surrogate marker for a potentially more transmissible variant. The subsequent confirmation with sequencing provides an efficient way to rapidly identify certain . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint VoCs. We feel it is strongly recommended to further sequence the VoCs and not stop at the identification of the N501Y mutations. Lineage or also whole genome resolution provides highly valuable information for public health management 48 . The development of our screening system, with all attached pre-to post-analytical aspects of diagnostics, may be valuable in the search for future upcoming variants such as vaccine escape mutants.
However, it is clearly time for nations to seriously consider implementing national surveillance programs with an unbiased sequencing approach, incorporating sustainable elements for other key pathogens and potential future pandemics 49  . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
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(which was not certified by peer review)
The copyright holder for this preprint this version posted February 12, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint Table S4. GISAID database identifier of N501Y-carrying genomes from Switzerland included into phylogenetic analysis.
. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint    is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint Figure S3. Phylogeny of sequenced B.1.351 cases in Switzerland.
. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251589 doi: medRxiv preprint