SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing

Abstract

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Keywords: SARS-CoV-2; co-infection; genomic surveillance; long-read sequencing; quasispecies; recombination.

FIG 1

SARS-CoV-2 lineages in France in 2022. Colored areas show the relative abundance of each SARS-CoV-2 clade, calculated from our sequencing results (left axis). The black line represents the percentage of positive SARS-CoV-2 tests (7-day moving average), illustrating the successive waves of infection (right axis). Stars on the x axis indicate the 17 cases of co-infections.

FIG 2

Nucleotide mutation frequencies on the complete genome. Patient 2 (top panel) and Patient 17 (bottom panel): relative abundance of mutations along the whole genome, compared to the Wuhan reference sequence (left axis). Gray bars represent polymorphisms common to the two infecting SARS-CoV-2 lineages and colored bars represent clade/lineage-defining polymorphisms (yellow: 21A/I/J_B.1.617.2, blue: 21K_BA.1, purple: 22B_BA.5, light green: 22E_BQ.1). Black lines show the coverage depth (right axis).

FIG 3

Spike amplicon haplotypes for the 4 patients with recombinant strains. Plots show nucleotide positions that differ from the Wuhan reference on the Spike A6 amplicon (genome positions: 21563-23823). Colors highlight the different clades/lineages (blue: 21K_BA.1, green: 21L_BA.2, red: 22A_BA.4, purple: 22B_BA.5, maroon: 22C_BA.1.12.1). The red boxes indicate the recombination regions. The number of reads indicated for each haplotype is the result of 1of the 2 (concordant) sequencing performed.