Identification of Various Recombinants in a Patient Coinfected With the Different SARS‐CoV‐2 Variants

ABSTRACT Background Viral recombination that occurs by exchanging genetic materials between two viral genomes coinfecting the same host cells is associated with the emergence of new viruses with different virulence. Herein, we detected a patient coinfected with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Delta and Omicron variants and identified various recombinants in the SARS‐CoV‐2 full‐length spike gene using long‐read and Sanger sequencing. Methods Samples from five patients in Japan with household transmission of coronavirus disease 2019 (COVID‐19) were analyzed using molecular assays for detection and identification of SARS‐CoV‐2. Whole‐genome sequencing was conducted using multiplex PCR with short‐read sequencing. Results Among the five SARS‐CoV‐2‐positive patients, the mutation‐specific assay identified the Delta variant in three, the Omicron variant in one, and an undetermined in one. The undermined patient was identified as Delta using whole‐genome sequencing, but samples showed a mixed population of Delta and Omicron variants. This patient was analyzed for viral quasispecies by long‐read and Sanger sequencing using a full‐length spike gene amplicon. In addition to the Delta and Omicron sequences, the viral quasispecies analysis identified nine different genetic recombinant sequences with various breakpoints between Delta and Omicron sequences. The nine detected recombinant sequences in the spike gene showed over 99% identity with viruses that were detected during the Delta and Omicron cocirculation period from the United States and Europe. Conclusions This study demonstrates that patients coinfected with different SARS‐CoV‐2 variants can generate various viral recombinants and that various recombinant viruses may be produced during the cocirculation of different variants.

strains, alteration of transmission vector specificities, increases in virulence and pathogenesis, modification of tissue tropisms, evasion of host immunity, and evolution of resistance to antivirals [1,3,4].Viral recombination has been observed in several viruses, including members of the Coronaviridae family, to which the human coronaviruses belong [5].
Coronavirus disease 2019 (COVID- 19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Wuhan, China, in December 2019 [6].Since then, SARS-CoV-2 has evolved into phylogenetically distinct lineages, some of which have been designated as variants of concern (VOC) [7,8].These variants differ in terms of transmissibility, virulence, and immune escape capability against humoral immunity [9,10].Particularly, the SARS-CoV-2 spike gene is crucial for vaccine development and treatment using monoclonal antibodies [11].SARS-CoV-2 recombination has been identified in some strains through whole-genome sequencing (WGS) [12,13].In addition, the number of infections with SARS-CoV-2 recombinant viruses, including XBB.1.5,XBB.1.16,and EG.5.1, has increased worldwide from late 2022 (GISAID, https:// gisaid.org/ ).However, only a few studies have analyzed SARS-CoV-2 recombination in patients coinfected with different variants.Particularly, the identification of viral quasispecies using long-read sequencing has been poorly documented [14,15].Recombinant viruses play a significance role in the evolution of the novel viruses, contributing to the generation of genetic diversity and the emergence of novel viral strains.Therefore, it is important to analyze recombinants in patients coinfected with different SARS-CoV-2 variants.Thus, this study aimed to identify recombination sequences by long-read and Sanger sequencing using a full-length spike gene amplicon from a patient coinfected with the Delta and Omicron variants.Our findings provide novel insights into recombination events in SARS-CoV-2.

| Sample Collection, Screening Tests, and WGS
In February 2022, nasopharyngeal swabs were collected from 12 patients in Miyagi Prefecture, Japan, with COVID-19 in the household.Laboratory molecular diagnosis confirmed that 11 of the 12 patients were positive for SARS-CoV-2.We conducted further studies using samples from 5 of the 11 patients with SARS-CoV-2 infection.Samples were not available from the other six patients.RNA was extracted using RNA extraction kits (Promega Maxwell RSC Viral Total Nucleic Acid Purification kit, Promega, Madison, WI, USA; QIAamp Viral RNA Mini Kit, Qiagen, Hilden, Germany).The extracted RNA was screened for SARS-CoV-2 detection using a reverse-transcription realtime PCR assay targeting the SARS-CoV-2 nucleocapsid (N) gene [16].Positive samples were further tested for VOC-specific amino-acid substitution by targeting the single nucleotide polymorphism (SNP) spike L452R [17].Amplification and genotyping results were analyzed using QuantStudio 5 (ThermoFisher, Waltham, MA, USA).Complementary (c) DNA was synthesized using a LunaScript RT Super Mix kit (New England Biolabs, Ipswich, MA, USA), and WGS of SARS-CoV-2 was performed by multiplexed PCR amplification using ItokawaK primer set ver_ N4 [18].PCR products were purified and subjected to Illumina library construction using Illumina DNA Prep Tagmentation and Nextera DNA CD indices (Illumina, San Diego, CA, USA).The MiniSeq platform (Illumina) was used to sequence the indexed libraries using the MiniSeq High Output Reagent Kit (300 cycles).Data analyses were processed using the Dragen Lineage Pipeline 3.5.3(Illumina), with each parameter set to default.This pipeline provided the percentage of non-N bases (coverage ≥ 10), median coverage, Pango lineage, Clade, and FASTA consensus sequences.

| Clinical Information of Patients
The five patients included in this study were 26-44 years old, immunocompetent, and unvaccinated for COVID-19 (Table 1).They developed COVID-19 symptoms between January 31 and February 2, 2022.Three (H540, H543, and H544) of the five patients required hospitalization.

| Molecular Test and WGS Results
We detected samples from the five patients (H540-H544) using PCR targeting of the N gene (Table 1).Using an SNP   spike L452R assay, the Omicron variant was identified in one patient (H540), and the Delta variant was identified in three patients (H541, H543, and H544).However, the variant in the sample from the remaining patient (H542) could not be determined using the SNP PCR assay.SARS-CoV-2 whole-genome analysis using short-read sequencing yielded similar results in patient H540 (infected with Omicron variant BA.1.1.2,21K) and patients H541, H543, and H544 (all infected with Delta variant AY29.2, 21J).Patient H542, in whom the variant was undetermined by the SNP PCR assay, was found to be detected with the Delta variant (AY29.2,21J) using WGS.However, clade-defining mutation analysis showed the presence of both Delta and Omicron variants in this patient (H542; Figure 1), suggesting coinfection with both variants.The Delta variant exhibited a higher population than the Omicron variant in the patient with coinfection.
In the remaining two (H542-S4 and H542-S5) clones, we detected different recombinant sequences that show Delta-Omicron sequences in the full-length spike gene.Finally, we conducted a Basic Local Alignment Search Tool (BLAST, https:// blast.ncbi.nlm.nih.gov/ Blast.cgi) analysis using each recombinant sequence obtained in this study.The detected recombinants showed a high level of identity (99.74%-99.99%)with virus strains that were detected during the Delta and Omicron cocirculation period from the United States and Germany (Table S1).These virus strains were detected as putative recombinants using SimPlot analysis (Figure S4).In this study, we identified Delta and Omicron sequences and at least nine distinct recombinant sequences in a patient coinfected with the SARS-CoV-2 Delta and Omicron variants.This coinfected patient was identified to be infected with the Delta variant by whole-genome analysis using short-read sequencing; however, the analysis also showed a mixed population of Delta and Omicron variants.Previous studies have reported 0.13%-0.25%detection rates of SARS-CoV-2 coinfection during the period of Delta and Omicron cocirculation [14,15,20].The detection rates of recombinants among samples from patients with in these studies were 1/7 (14%) and 2/18 (11%) [14,15], suggesting that the detection rate of patients with coinfection and recombinants is very low.In this study, among the 11 patients positive for SARS-CoV-2 infection, we could not analyze SARS-CoV-2 variants in six patients owing to the lack of available samples.One of the five patients in whom we conducted variant analysis was identified as having Delta and Omicron coinfection, which is a higher recombinant detection rate than that reported in previous studies.Further studies with larger sample sizes are required to analyze the frequency of coinfection in patients during periods of cocirculation of different SARS-CoV-2 variants and the recombinants generated in patients with coinfection.
In the present study, we identified one patient (H542) who was infected with the Delta and Omicron variants and various recombinant viruses.This patient, aged 26 and nonvaccinated for COVID-19, showed fever (>37.5°C),cough, headache, joint muscle ache, and taste disturbance dysosmia.However, the patient was not hospitalized because, in Japan, samples were not used to determine the requirement for hospitalization according to the severity of illness at that time.Consequently, we could not analyze the severity of infection in the patients enrolled in this study.It was reported that patients infected with SARS-CoV-2 recombinant viruses showed low clinical severity until infection with the XD strain, a recombinant of the Delta and Omicron variants [21].Thus, recombinant viruses originating from the Delta and Omicron variants may cause clinical symptoms with low severity.
This study identified two variants (Delta and Omicron) of SARS-CoV-2 and nine recombinants with distinct breakpoints using full-length spike gene amplicons by long-read and Sanger sequencing.Deep sequencing investigation of the viral population revealed that the Delta variant (in H542-L1 and H542-S1) was predominant.This result is consistent with the WGS results, which showed that the viral population of the patient with coinfection comprised a higher number of Delta than Omicron sequences.Other studies of patients with coinfection with different SARS-CoV-2 variants have also found that the size of the viral population of each variant differed [22].The virus population in patients with coinfection might be determined by several factors, such as host immunocompetence, vaccination history, and virus phenotype.This study identified nine recombinants in the patient with coinfection.A previous study reported two types of recombination sequences in a patient with Delta-Omicron coinfection using long-read sequencing of the partial spike gene [14].Our results confirmed that patients with coinfection can generate various types of recombination sequences.The patient with coinfection in this study had not been vaccinated against SARS-CoV-2.In unvaccinated patients, SARS-CoV-2 shows high within-host diversity [23].Therefore, various recombinants might have been generated in the patient with coinfection in this study.We further detected recombinants not detected by long-read sequencing by Sanger sequencing.Sanger sequencing is only able to detect minority variants at frequencies between 10% and 40%, has limited power to sequence genomes, and involves high costs compared with next generation sequencing, including long-read sequencing [24].In contrast, for long-read sequencing, many cycles of amplification are needed to add tagged PCR amplicons using PCR.Analysis of recombinant by both methods using samples from coinfected individuals is not well understood.Thus, future analyses using samples from coinfected patients should be performed to examine and ascertain the efficacy of these methods.This study identified that the nine recombinants with various breakpoints (at 216-348, 454-479, 549-657, 683-766, and 798-858) in the spike gene amino acids.However, the breakpoints detected in this study differed from those in XD.SARS-CoV-2 recombination breakpoints have been reported to occur disproportionately in the 3′ region of the genome containing the spike gene [25].Viral recombinant breakpoints have been suggested to occur in various sites of these regions.In addition, BLAST analysis of the recombinant sequences detected in this study in Japan revealed their high identity with those of viruses detected in the United States and Germany.These deposited samples in Genebank were collected during December 2021 to January 2022, the period of Delta and Omicron variant cocirculation in these countries.Thus, these results suggest that various recombinants are generated in patients with coinfection, particularly during periods of contemporaneous cocirculation of different variants.
Viral recombination, including that of SARS-CoV-2, generates new variants with unpredictable epidemic or pathogenic characteristics; in particular, the recombination of Delta and Omicron variants may lead to the acquisition of immune evasion capabilities [26].Recombinant XD originating from Delta and Omicron variants was identified.However, this strain was transmitted within a local area only and did not spread more extensively [21,27].In contrast, XBB sublineages, such as XBB1.16 and EG.5.1, are currently the primary variants circulating worldwide, according to GISAID (https:// gisaid.org/ ).Furthermore, XBB.1.16has several advantages over other variants, such as a relatively effective reproduction number (R e ) and resistance to neutralization antibodies [28].These properties could enable XBB sublineages to circulate worldwide and outcompete previous recombinants.In addition, the number of recombinants identified has rapidly increased, possibly due to high levels of cocirculation between Delta and BA.1 or among Omicron subvariants, and improved genomic surveillance systems in several countries since 2022 [26,29].Currently, various Omicron subvariants are circulating worldwide.New recombinants may be generated in patients coinfected with different subvariants.Thus, future emerging SARS-CoV-2 strains, including recombinants from coinfected patients that develop into new circulating strains, should be monitored carefully, particularly during periods of cocirculation of multiple variants.
This study has a few limitations.First, we could not conduct variant analysis in six of the 11 patients in the household with SARS-CoV-2 infection owing to lack of available samples.Moreover, multiple samples could not be collected from the patient with coinfection; thus, we could not confirm any changes over the course of the infection.The recombination analysis in this study was limited to the full-length spike gene.Previous studies have reported recombination breakpoints not only in the spike gene, but also in open-reading-frame 1a and other regions [23,26].Additionally, different viral recombinant sequences were detected by each of the two employed sequencing methods.Future studies should analyze changes in the virus population and recombination in samples from coinfected patients using other genes and sequencing methods (long-read and Sanger sequencing).
The present study demonstrated the generation of at least nine different viral recombinants in a patient coinfected with the Delta and Omicron variants.The detected recombinants showed a high identity with the virus collected during the cocirculation period in other areas.Currently, various strains, including Omicron subvariants (such as JN.1), are in cocirculation worldwide; thus, novel recombinants in addition to the XBB lineage may be generated.The prevalence of recombinant virus strains, such as XDK and XDD, originating from the JN.1 and XBB/ EG.5.sublineages, respectively, has increased worldwide, especially in Europe (GISAID).Thus, the effective genomic surveillance of SARS-CoV-2 is also of great clinical importance because viral recombinants can generate viral strains with unpredictable infectivity, virulence, and immune escape characteristics.

FIGURE 2 |
FIGURE 2 | Description of 13 viral quasispecies amino acids identified via long-read and Sanger sequencing using a full-length spike gene amplicon.Each color is specific for a variant and insertion/deletion: blue (Delta), magenta (Omicron), and gray (insertion and deletion).H542-L1 to L11 obtained based on long-read sequencing.H542-S1 to S6 obtained based on Sanger sequencing.The amino acids of H542-L5 were located at 970-980 because of a deletion at 2904 and an insertion at 2939 of thymidine in the thymidine-repeating region in the nucleotide.

FIGURE 3 |
FIGURE 3 | SimPlot analysis for putative SARS-CoV-2 recombinants.Comparisons of genetic similarity between recombinant and Delta (H542-L1/blue) and Omicron (H542-L3/magenta) sequences were made using the SimPlot software.The results are shown for the viral quasispecies sequences H542-L2 (A), H542-L4 (B), H542-L6 (C), H542-L7 (D), H542-L8 (E), H542-L10 (F), and H542-L11 (G).The vertical axis represents the percent nucleotide sequence similarity between the putative recombinant and each strain used for comparison, and the horizontal axis shows the relative nucleotide position along the Spike gene.In each analysis, a window size of 200 nucleotides and the Kimura distance model (2-parameter) were used.

TABLE 1 |
Demographic, clinical, and virological characteristics of the five patients in this study.