key: cord-0853881-ph6hx8vf
authors: Ou, Junxian; Lan, Wendong; Wu, Xiaowei; Zhao, Tie; Duan, Biyan; Yang, Peipei; Ren, Yi; Quan, Lulu; Zhao, Wei; Seto, Donald; Chodosh, James; Wu, Jianguo; Zhang, Qiwei
title: Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events
date: 2022-03-14
journal: bioRxiv
DOI: 10.1101/2022.03.13.484129
sha: 3d66e44d8b2f7a336c918be266f71b7c5066f2b8
doc_id: 853881
cord_uid: ph6hx8vf

The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. Although recombination events among SARS-CoV-1 and MERS-CoV were well-documented, it has been difficult to detect the recombination signatures in SARS-CoV-2 variants due to their high degree of sequence similarity. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, eighteen core mutations of BA.1 variants (frequency >99%) were identified (eight in NTD, five near the S1/S2 cleavage site, and five in S2). BA.2 variants share three additional amino acid deletions with the Alpha variants. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations (del69-70, del144) in BA.1 than BA.2, and therefore BA.1 may be phylogenetically closer to the Alpha variant. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1 subvariant. Most notably, multiple additional amino acid mutations in the Delta spike protein were also identified in the recently emerged Omicron isolates, which implied possible recombination events occurred between the Omicron and Delta variants during the on-going pandemic. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.

The current COVID-19 pandemic is fueled by a more infectious emergent Omicron variant 2 (B.1.1.529), which was first reported in South Africa and quickly spread worldwide 1 . A multitude of 3 mutations (more than 30) in the spike gene of Omicron variant were detected, which when compared 4 to the Alpha and Delta variants (typically less than 15) 2 , raised concerns of enhanced infectivity and 5 immune escape potential 3,4 . Omicron variants is divided into three lineages (BA.1, BA.2, and BA.3) 6 and was classified as the fifth variant of concern (VOC) by the World Health Organization on 7 November 26, 2021. It has been circulating in more than 170 countries/territories. Ongoing concerns of emergent variants includes possible recombinants resulting from different 18 variants replicating simultaneously in a host. Such variants, e.g., "Demicron" or "Deltacron" are 19 controversial that if they are real recombinants or a possible sequencing error 13 . 20 21 Genome recombination is an important evolutionary mechanism for the emergence and 22 re-emergence of human pathogens and a major source of viral evolution, for example, the 23 well-studied "model organism" adenovirus [14] [15] [16] [17] [18] [19] [20] , and also in coronaviruses 21-23 . Recombination Previous research distinguished active recombination events among the SARS-CoV-2 nucleoprotein 9 and ORF1ab genes by using a phylogenetic network strategy based on single nucleotide substitution 10 or SARS-CoV-2 lineage designation 27,28 . More than thirty amino acid mutations have been identified 11 within Omicron spike protein, some of which are shared with other variants 1 . In this study, we 12 demonstrate that the emerging and circulating Omicron subvariants originate in part through 13 recombination with other variants. We first investigated the spike diversity of the Omicron variants 14 along with the shared spike mutations between Omicron and other variants of concern (VOCs) and 15 variants of interest (VOIs). The Omicron spike amino acid sequences archived during the early 16 transmission phase, and released in the GISAID database (submitted before January 15 th , 2022) were 17 accessed, include 52,563 high quality Omicron spike sequences (representing 49,609 BA.1 and 2,954 18 BA.2 sequences). In this study, these were analyzed with Pymol 2.0, TBTools, BioEdit, BioAider, 19 and jvenn 29-35 . The whole genome phylogenetic trees were constructed and annotated using 20 NextClade 36 . Through scanning 52,563 high-quality completed Omicron spike gene sequences, most Omicron 2 spike mutations appear stable (frequency >99%). Eighteen core mutations (frequency >99%) of 3 BA.1 subvariant exist in NTD (A67V, del69-70, T95I, G142D, del143-145), SD (underpinning 4 subdomain) near the S1/S2 cleavage site (T547K, D614G, H655Y, N679K, P681H), and S2 (D796Y, 5 N856K, Q954H, N969K, L981F)( Table1). Table 1 ). 2 In contrast to BA.2 subvariant, BA.1 share three additional amino acid deletions (del69-70, delY144) 3 with the Alpha variants, suggesting a closer relationship between the BA.1 and Alpha variants 4 ( Fig.1A and 1B, and Table 1 ). As a whole, Omicron subvariants have a high number of amino acid 5 mutations in the spike gene (40 in BA.1, and 31 in BA.2), of which some were found in other VOCs: 6 Alpha (10x), Beta (11x), Gamma (12x), and Delta (9x). These mutations mainly occur in NTD and 7 RBD ( Fig.1D and Table 1 ). (G339D, S371L, S373P, S375F, K417N, 440K, G446S, S477N, T478K, E484A, Q493R, G496S,   3 Q498R, N501Y, and Y505H). The frequency of insertions of the amino acids EPE at site 214 in 4 BA.1 decreased during the pandemic from more than 95% on December 1 st , 2021 to 89% on January 5 15 th 2022. However, BA.2 spike protein remained constant (frequency >99%), with the exception of 6 the three amino acid deletion (LPP) found at amino acids 24-26, which decreased from more than 7 95% frequency on December 1 st , 2021 to 85% on January 15 th 2022 (Table 1 ). This may possibly be 8 due to selection pressure on the circulating Omicron strains. France (https://t.co/tVAKmHRYSy). Bootscan analysis of Omicron spike sequences also indicated 1 that the reversion haplotypes (Hap_18, Hap_39, Hap_44) were more similar to Delta variants when 2 compared to typical Omicron haplotypes ( Fig. 3A and 3B ). 3 Furthermore, single nucleotide differences could also originate from recombination events among 4 highly similar strains. Loops detected in phylogenetic networks also indicate possible recombination 5 events among highly similar Omicron variants or subvariants ( Fig. 3A and 3C ). Multiple newly 6 detected or recent mutations in the Omicron spike gene make it possible to trace a putative mutation 7 origin from representative mutations in VOIs or VOCs, especially the Delta variant, which suggests 8 possible recombination events between Omicron and Delta variants (Table 2) . replicates.Wuhan-Hu-1 spike sequences was set as reference, reversion region was annotated. (C) 12 Overview of possible evolution mechanism of reversion haplotypes and haplotypes with mutations 13 from Delta and other variants.

14 15 Except for shared mutations, many other mutations (30 in BA.1 and 25 in BA.2) could not be 5 accounted for among previous dominant variants (Fig. 1) . Because the Omicron variants are believed 6 to have emerged in South Africa 1 , we speculate that some of these spike mutations may have been Table 1 ). The integration of these mutations may lead to changes in phenotype. Five additional 23 typical amino acid mutations in Delta variants were also identified in recently emergent Omicron 24 isolates (before January 15, 2022) ( Table 2) . For example, 899 Omicron sequences of high quality 25 contained L452R mutation reported for the Delta variant (Fig. 4A) . Whole genome analysis also 26 corroborated the diversity among these L452R containing Omicron genomes. The mutation profiles 

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and 13

The N501Y spike substitution enhances SARS-CoV-2 infection and 15 transmission

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R 17 mutation

V367F mutation in SARS-CoV-2 spike RBD emerging during the early 19 transmission phase enhances viral infectivity through increased human ACE2 receptor 20 binding affinity

Functional Effects of Receptor-Binding Domain Mutations of 22 Funding statement

This work was supported by grants from the National Key Research and Development Program of 12

National Natural Science Foundation of China (32170139 and 13 81730061), and Natural Science Foundation of Guangdong Province

We gratefully acknowledge the authors, originating and submitting laboratories of the sequences 7 from GISAID's EpiFlu™ Database on which this research is based. All submitters of data may be 8 contacted directly via www.gisaid.org.