Intra-Host Evolution Provides for the Continuous Emergence of SARS-CoV-2 Variants

Abstract

Variants of concern (VOC) in SARS-CoV-2 refer to viruses whose viral genomes differ from the ancestor virus by ≥3 single-nucleotide variants (SNVs) and that show the potential for higher transmissibility and/or worse clinical progression. VOC have the potential to disrupt ongoing public health measures and vaccine efforts. Still, too little is known regarding how frequently new viral variants emerge and under what circumstances. We report a study to determine the degree of SARS-CoV-2 sequence evolution in 94 patients and to estimate the frequency at which highly diverse variants emerge. Two cases accumulated ≥9 SNVs over a 2-week period and one case accumulated 23 SNVs over 3 weeks, including three nonsynonymous mutations in the spike protein (D138H, E554D, D614G). The remainder of the infected patients did not show signs of intra-host evolution. We estimate that in as much as 2% of hospitalized COVID-19 cases, variants with multiple mutations in the spike glycoprotein emerge in as little as 1 month of persistent intra-host virus replication. This suggests the continued local emergence of variants with multiple nonsynonymous SNVs, even in patients without overt immune deficiency. Surveillance by sequencing for (i) viremic COVID-19 patients, (ii) patients suspected of reinfection, and (iii) patients with diminished immune function may offer broad public health benefits. IMPORTANCE New SARS-CoV-2 variants can potentially disrupt ongoing public health measures and vaccine efforts. Still, little is known regarding how frequently new viral variants emerge and under what circumstances. Based on this study, we estimate that in hospitalized COVID-19 cases, variants with multiple mutations may emerge locally in as little as 1 month, even in patients without overt immune deficiency. Surveillance by sequencing for continuously shedding patients, patients suspected of reinfection, and patients with diminished immune function may offer broad public health benefits.

Keywords: COVID; DNA sequencing; SARS-CoV-2.

FIG 1

Summary characteristics of the cohort and sequencing performance. Red indicates values at baseline (T0), and blue indicates subsequent sampling points. (A) Distribution of observation time for the cohort. (B) Distribution of viral genome copy number as determined by real-time RT-qPCR. This excludes n = 60 samples for which no CT values were available, only a negative/positive determination. (C) Relation between genome copy number as determined by real-time RT-qPCR and fraction of reads mapped/total reads. (D) Relation between the fraction of reads mapped/total reads and coverage at 1×, 10×, and 100×.

FIG 2

Summary of genome diversity. (A) Distribution of SNVs per sample. (B) Distribution of all SNV frequencies in the data set. (C) Count of nonsynonymous SNVs per gene, color-coded by frequency of the SNV (D) Distribution of Hamming distances of all sequenced samples that contained at least 1 SNV of Frequency greater than or equal to 51% (n = 65) representing 2080 comparisons. Blue lines indicated the hamming distance between paired samples. Line A represents LCCC0245 and its preceding sample of <90% coverage. Line B represents LCCC0187 and LCCC0225. Line C represents LCCC0233 and LCCC0239.

FIG 3

(A) Phylogenetic tree obtained from a multiple alignment n = 48 whole SARS-CoV-2 genomes as submitted to GISAID. The tree is under the assumption of unequal evolution rates. Paired samples are indicated by colored lines and labeled by distances in days. (B) Phylogenetic tree based on n = 231 high-quality SNV position for n = 67 complete and partial SARS-CoV-2 genomes. Paired samples are indicated by colored lines and labeled by distances in days.

FIG 4

A phylogenetic tree was obtained using BEAST after a multiple alignment of n = 273 whole SARS-CoV-2 genomes from GISAID that were collected at the same time in the same population and determined by the same technology and bioinformatics pipeline. The initial introductory events are indicated in red, the root Hu-Wu-1 in black, and paired specimen in blue.