key: cord-0288291-8usmp0o4
authors: Sekizuka, T.; Itokawa, K.; Saito, M.; Shimatani, M.; Matsuyama, S.; Hasegawa, H.; Saito, T.; Kuroda, M.; Japan, COVID-19 Genomic Surveillance Network in
title: Genome Recombination between Delta and Alpha Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
date: 2021-10-14
journal: nan
DOI: 10.1101/2021.10.11.21264606
sha: c7a132b8064a963bfaba8162d73f0119305d49f4
doc_id: 288291
cord_uid: 8usmp0o4

Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from clinical specimens in Japan. It is necessary to intensively study such marked genetic variations and characterize the emerging variants after careful verification of their lineage and clade assignment.

specimens in Japan. It is necessary to intensively study such marked genetic variations and 27 characterize the emerging variants after careful verification of their lineage and clade 28 assignment. 29 30 Text 31

The study protocol was approved by the National Institute of Infectious Diseases, Japan 33 (Approval no. 1091). The ethics committee waived the requirement for written consent with 34 respect to research on viral genome sequences. 35

We conducted a genome surveillance of the severe acute respiratory syndrome coronavirus 2 37 (SARS-CoV-2) with help from the local public health centers, laboratories in research institutes, 38 commercial laboratories (1, 2), and airport quarantine stations (3). The coronavirus disease-19 39 (COVID-19) Genomic Surveillance Network in Japan (COG-JP) has consistently monitored the 40 prevalence of the Phylogenetic Assignment of Named Global Outbreak (PANGO) lineages of 41 SARS-CoV-2 from the first COVID-19 case (January 15, 2020) to recent cases (September 30, 42 2021) . Until now, 120,476 domestic and 2,018 quarantine isolates have been deposited in the 43 Global Initiative on Sharing All Influenza Data (GISAID) EpiCoV database (1, 2). Whole-44 genome sequences were assigned to the SARS-CoV-2 isolates (≥ 29 kb genome size) obtained 45 . CC-BY-NC-ND 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.

The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10.11.21264606 doi: medRxiv preprint from domestic COVID-19-positive patients in Japan (n = 120,476), according to the PANGO 46 lineage definition (v3.1.14, 2021-09-28) (4).

During the surveillance, we monitored several variants of concern (VOCs) of the PANGO 49 lineage. We found six unique specimens ( Fig. 1 ) among the 21A (Delta) clade isolates that 50 exhibited a low quality assignment ("not determined" or "none") in the PANGO lineage, even 51 though their genome sequences had been completely determined with high read coverage 52 throughout the whole genome region. A detailed genome alignment by Nextclade (5) suggested 53 that despite being clonal isolates of the 21A (Delta) clade, these six isolates show identical 54 mutation profiles with 20I (Alpha, V1), particularly between the ORF6 and N genes, located 55 towards the latter part of the SARS-CoV-2 genome (Fig. 1) . Additionally, the alignment clearly 56 revealed a possible recombination spot between the ORF6 and ORF7a genes ( Fig. 1) . However, 57 the next generation sequencing (NGS)-based read mapping analysis did not indicate the presence 58 of any heterogeneous mix of alleles in the clinical specimens, suggesting that these patients had 59 not acquired multiple variant infections with 21A (Delta) and 20I (Alpha, V1) clades at the acute 60 stage of the infection (Fig. 2) . 61

Interestingly, all six clinical isolates that exhibit the abovementioned recombination were 62 detected around mid-August 2021. Even though the mutation profiles point towards the clonal 63 nature of these six isolates (Fig. 1) , there is no epidemiological link among the patients. 64

Incidentally, the two variants, namely 21A (Delta) and 20I (Alpha), were detected in 93% and 65 5% of the total specimens, respectively, in mid-August 2021 in Japan (6), suggesting the 66 possibility of multi-variant infection in a single patient leading to such a recombination event. 67

However, we have not yet identified a potential patient with mixed infection, i.e., one who has 68 . CC-BY-NC-ND 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) creating the coronavirus diversity, and RNA proofreading exoribonuclease (nsp14-ExoN) is 74 responsible for generating the recombination frequency as well as the altered recombination 75 products in the in vitro culture experiments (7). The report has also described eight potential 76 recombination hotspots at the microhomologous sequence of the SARS-CoV-2 genome. In fact, 77 one of the hotspots, which lies between ORF6 and ORF7a, could be the target site responsible 78 for the recombination event observed in the current study. Interestingly, the recombination 79 variant detected in this study carries a spike protein identical to the one in the domestic Delta 80 variant, thereby suggesting that further risks would not be concerned with infectivity and 81 immune escape. 82

The detection of SARS-CoV-2 variants with ORF7a, ORF7b, can 83 explain the occurrence of the abovementioned recombination event. In fact, the hotspots around 84

ORF7a can facilitate the generation of a novel isolate that exhibits different genetic profiles 85 owing to co-infection by distinct variants in the COVID-19 patient. In addition to the six isolates 86 mentioned in this study, we also investigated the total deposits in GISAID (by 2021-09-30) for 87 other possible genomic recombinations. We found a USA isolate [hCoV-19/USA/MO-CDC-88 LC0213262/2021 (EPI_ISL_4164992|2021-08-07)] portraying a recombination between the 89 Delta and Alpha variants in ORF7a, but we could not reconfirm the validity of the event because 90

we were unable to obtain the raw NGS sequencing reads for analysis. 91 . CC-BY-NC-ND 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.

The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10.11.21264606 doi: medRxiv preprint

In conclusion, this is the first identification of a novel recombination SARS-CoV-2 variant 93 between the 21A (Delta) and 20I (Alpha, V1) clades in domestic clinical specimens. As 94 suggested by previous in vitro culture experiments (7), such a recombination can possibly be 95 generated in the real world. Therefore, the simple PANGO and clade assignment might mis-96 identify notable variants based upon ordinary genome surveillance, and we must intensively CC-BY-NC-ND 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 October 14, 2021. ; https://doi.org/10.1101/2021.10.11.21264606 doi: medRxiv preprint

Raw NGS sequencing reads have been deposited in the DNA Data Bank of Japan (DDBJ; 116 accession number: DRA012825), as shown in the Table.  117 118

Dr. Tsuyoshi Sekizuka is a chief at the National Institute of Infectious Diseases in 120

Shinjuku-ku, Tokyo, Japan. His main research interest is pathogen genomics. 121

Disentangling primer 124 interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR

Genome Epidemiological Study of SARS-CoV-2 Introduction into Japan

. CC-BY-NC-ND 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) CC-BY-NC-ND 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) . CC-BY-NC-ND 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.

The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CC-BY-NC-ND 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.

The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10.11.21264606 doi: medRxiv preprint (A: red, G: yellow, C: blue, and T: green) on the read mapping area in dark gray. Co-infection 179 samples with Alpha and Delta variants were prepared virtually for comparison. 180 . CC-BY-NC-ND 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 October 14, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021