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.

Keywords: SARS‐CoV‐2; coinfection; recombination; spike gene; viral quasispecies.

FIGURE 1

Representation of variant frequency of clade‐defining mutations of SARS‐CoV‐2, including Delta and Omicron variants, using whole‐genome sequencing via short‐read sequencing. Each clade‐defining mutation based on WT (Accession no. NC_045512) along the genome of the coinfected patient is shown. The variant allele frequency shows mutations on the left Y‐axis, and the depth of each genomic position is shown on the right Y‐axis. ORF, open reading frame.

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

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.