key: cord-1031847-oa9lm3c6
authors: Fonager, Jannik; Bennedbæk, Marc; Bager, Peter; Wohlfahrt, Jan; Ellegaard, Kirsten Maren; Ingham, Anna Cäcilia; Edslev, Sofie Marie; Stegger, Marc; Sieber, Raphael Niklaus; Lassauniere, Ria; Fomsgaard, Anders; Lillebaek, Troels; Svarrer, Christina Wiid; Møller, Frederik Trier; Møller, Camilla Holten; Legarth, Rebecca; Sydenham, Thomas Vognbjerg; Steinke, Kat; Paulsen, Sarah Juel; Castruita, José Alfredo Samaniego; Schneider, Uffe Vest; Schouw, Christian Højte; Nielsen, Xiaohui Chen; Overvad, Maria; Nielsen, Rikke Thoft; Marvig, Rasmus L; Pedersen, Martin Schou; Nielsen, Lene; Nilsson, Line Lynge; Bybjerg-Grauholm, Jonas; Tarpgaard, Irene Harder; Ebsen, Tine Snejbjerg; Lam, Janni Uyen Hoa; Gunalan, Vithiagaran; Rasmussen, Morten
title: Molecular epidemiology of the SARS-CoV-2 variant Omicron BA.2 sub-lineage in Denmark, 29 November 2021 to 2 January 2022
date: 2022-03-10
journal: Euro Surveill
DOI: 10.2807/1560-7917.es.2022.27.10.2200181
sha: 1c99e9619b28d83b43986808eb47717c3cb1376a
doc_id: 1031847
cord_uid: oa9lm3c6

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sublineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Following the discovery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern Omicron (Phylogenetic Assignment of Named Global Outbreak (Pango) lineage designation B.1.1.529) on 19 November 2021 (week 46) in South Africa [1] , this variant with immune evasive properties has spread rapidly worldwide [2] [3] [4] . Since the Omicron emergence, sub-lineages within Omicron have been described, notably BA.1, BA.1.1 and BA.2 [5, 6] . The first Omicron sub-lineage BA.1 expanded rapidly and replaced the Delta variant (Pango lineage designation B.1.617.2) [7] . However, an increasing number of SARS-CoV-2 cases with the Omicron sub-lineage BA.2 have been reported in several countries, especially in Denmark [8] . Here, we provide a molecular epidemiological characterisation of the first BA.2 cases identified in Denmark.

The Danish national SARS-CoV-2 genomic surveillance system [9] identified the first two cases of Omicron sub-lineage BA.1 in samples from 22 

In total, 16,137 BA.1 and 2,623 BA.2 cases were identified among 55,273 SARS-CoV-2-positive cases confirmed by RT-PCR tests performed at both community testing centres and hospitals between 29 November 2021 and 2 January 2022 (weeks 48-52) with usable consensus genomes (≤ 3,000 Ns) obtained through whole genome sequencing (WGS), as previously described [10] . During the period from 29 November to 16 December 2021, all samples that indicated an Omicron variant based on variant-specific PCR (S:WT452) were selected for WGS. From 17 December 2021 to 2 January 2022, samples from community testing centres were randomly selected for WGS by an algorithm from all positive samples with cycle threshold (Ct) values below 35. Samples from Omicron screening after 20 December 2021 were limited to specific patient groups at some hospitals across the country and were selected based on variant-specific PCR indicating Omicron variants during the study period. The proportion of samples from hospitals were similar for both BA.1 and BA.2 (16% and 18%, respectively, in 4/5 regions). Whole genome sequences have been submitted to the Global Initiative on Sharing All Influenza Data (GISAID) sequence database [11] .

Risk ratios (RRs) of hospitalisation with BA.2 vs BA.1 were estimated using a log-linear Poisson regression model, adjusted for sex, age, vaccination status, time period, geographic region, comorbidities and SARS-CoV-2 reinfection, as described previously [12] . No significant differences were observed between individuals infected with BA. 1 

BA.2 genomes were aligned using MAFFT version 7.310 with Wuhan-Hu-1 (GenBank accession number: NC_045512.2) as a reference and maximum likelihood (ML) phylogenetic inference was performed using IQ-TREE2, with the transition model, empirical base frequencies and a free rate model with four categories [13, 14] . The ML tree (Supplementary data S2: Phylogenetic tree) was rooted with Wuhan-Hu-1 as an outgroup, timescaled and outlier tips removed (seven tips) using Rlsd2 (version 1.10) [15] . Ancestral character reconstruction was performed using PastML (version 1.9.34), with MPPA and F81, annotated to Danish regions [16] . The first introduction of BA.2 was in the Capital Region of Denmark which includes Copenhagen, from where multiple introductions were made to the other four Danish regions in which ten clusters had ten or more samples ( Figure 1A ). We further delineated clusters with more than 10 samples and visualised them as density plots by sample date using R and ggplot2 ( Figure 1B ) [17, 18] . Three of the clusters (C.J. clusters 1 and 3 and N.J. cluster 2) were characterised by a rapid expansion on 2 January 2022 and one (C.J. cluster 1) also had an expansion on 27 December 2021 ( Figure  1B ).

Lineage-specific mutations were derived from analysis of WGS consensus genomes with Nextclade CLI 1.9.0 [19] relative to the Wuhan-Hu-1 reference genome and filtered for substitutions and deletions which comprise at least 50% of one lineage and less than 5% of the other. Thirty-nine substitutions and deletions differed between BA.1 and BA.2 and were distributed across the genome (Figure 2A ). BA.2-specific spike mutations were clustered in the N-terminal domain (NTD) and in the receptor-binding domain (RBD). BA.1 and BA.2 diverged at spike residue 371 (L and F, respectively) and 142-145 in the RBD. The prevalence of these mutations was close to 100%, except for a few sites with amplicon Figure 2B ). BA.2 substitutions S371F, T376A, D405N and R408S were seen in the interior RBD surface in both closed and open spike conformations, within the cavity formed by the trimer and also close to an adjacent monomer.

This study was conducted using data from the Danish COVID-19 surveillance. According to Danish law, ethical approval is not needed for this type of research.

The SARS-CoV-2 variant Omicron BA.2 sub-lineage has spread rapidly since its first detection in Denmark, while the BA.1 sub-lineage, which appeared 2 weeks earlier, has decreased in numbers. However, we do not find BA.2 cases to be significantly different from BA.1 cases with respect to age, sex, SARS-CoV-2 reinfection, hospitalisation or mortality. These findings are consistent with reports from Norway [24] suggesting that BA.2 leads to an equally mild course of disease with COVID-19 as BA.1 compared with the Delta variant.

Our phylogenetic analyses showed that BA.2 spread from the Capital Region in eastern Denmark to the western parts of the country, mainly through 10 transmission clusters with between 19 and 208 people. No large outbreaks were identified among the clusters from available data sources. Three clusters expanded in a manner suggesting association with travel patterns States (US)) or 12% Spikevax (mRNA-1273, Moderna, Cambridge, Massachusetts, US), and less than 2% received the non-replicating viral vector vaccines Vaxzevria (ChAdOx1 nCoV-19, Oxford-AstraZeneca, Cambridge, United Kingdom) or Janssen vaccine (Ad26.COV2-S, Janssen-Cilag International NV, Beerse, Belgium). A COVID-19 hospitalisation was defined as admission 14 days after or 48 hours before the primary RT-PCR SARS-CoV-2 positive test. All cases were followed for more than 14 days for hospitalisation by using complete admission data extracted on 22 February 2022. Adjusted RRs were adjusted for the basic (a priori) covariates sex, age (10-year groups) [10] . vaccination status (if not stratified by), period as a continuous variable (week 50, 51, 52), region (five groups), comorbidities in the preceding 5 years (none or one or more), and previous SARS-CoV-2 infection within the past 60 days. In a sub-analysis, not shown in the during the Christmas holiday and have led to the seeding of BA.2 in the population within different geographical regions. This pattern of expansion is in contrast to the initial descriptions of BA.1 in Denmark and Norway, where large single outbreaks were seeding events for the transmission and spread of BA.1 [9, 25] .

The ability of Omicron sub-lineage BA.1 to replace the previously dominant Delta variant has been attributed to immune escape rather than a higher intrinsic transmissibility [26, 27] , but BA.2 has been shown to be even more transmissible than BA.1 [10] . Our analysis of the mutation profiles showed different constellations of mutations in BA.1 compared with BA.2, and the structural mapping suggests different effects on receptor binding or changes in interaction with adjacent spike monomers. At the NTD, the BA.2-specific substitution T19I abrogates a glycosylation site at N17 [28, 29] . Furthermore, deletions from amino acid positions 24-26 (BA.2), 69-70 (BA.1), 142-144 (BA.1) as well as an A27S substitution (BA.2) are situated in or close to a known NTD antigenic site [30] and are associated with resistance to neutralising monoclonal antibodies [30] .

A limitation of this study is that only WGS could be used to identify BA.2, but not all samples were sequenced during the study period because of the high incidence of Omicron in the population at the time. In addition, variant-specific PCR was implemented at different local hospitals but used differentially over time in the community testing centres, which might have affected the pre-selection of samples sent for WGS to some degree. Furthermore, some of the hospital cases might have been admitted for other reasons than COVID-19 and incidentally been detected as part of routine screening of hospital admissions.

SARS-CoV-2 variant Omicron BA.2 has quickly become the dominant sub-lineage in Denmark, but based on data available on 10 January 2022, BA.2 is not associated with increased severity of disease or hospitalisation. The initial spread of BA.2 in Denmark was characterised by an initial increase in the Capital Region followed by transmission and expansion to the rest of Denmark. The mutation profiles of BA.1 and BA.2 differ in the spike gene in regions associated with receptor binding, glycosylation and resistance to monoclonal antibodies. This study provides novel information about molecular and epidemiological aspects of BA.2 severity, possible national transmission patterns and mutational profile, which can help to inform public health decisions regarding the handling of this Omicron sub-lineage.

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License, supplementary material and copyright

We would like to thank The Danish Covid-19 Genome Consortium (DCGC, www.covid19genomics.dk). Also, we would like to thank all laboratory technicians in the Virus Research and Development Laboratory at Statens Serum Institut, Copenhagen, Denmark for their extraordinary effort during the emergence of Omicron.Funding statement: No particular funding was obtained for this work as part of the Danish national health response to the COVID-19 pandemic.

None declared. 

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