Genome-wide association study between SARS-CoV-2 single nucleotide polymorphisms and virus copies during infections

Abstract

Significant variations have been observed in viral copies generated during SARS-CoV-2 infections. However, the factors that impact viral copies and infection dynamics are not fully understood, and may be inherently dependent upon different viral and host factors. Here, we conducted virus whole genome sequencing and measured viral copies using RT-qPCR from 9,902 SARS-CoV-2 infections over a 2-year period to examine the impact of virus genetic variation on changes in viral copies adjusted for host age and vaccination status. Using a genome-wide association study (GWAS) approach, we identified multiple single-nucleotide polymorphisms (SNPs) corresponding to amino acid changes in the SARS-CoV-2 genome associated with variations in viral copies. We further applied a marginal epistasis test to detect interactions among SNPs and identified multiple pairs of substitutions located in the spike gene that have non-linear effects on viral copies. We also analyzed the temporal patterns and found that SNPs associated with increased viral copies were predominantly observed in Delta and Omicron BA.2/BA.4/BA.5/XBB infections, whereas those associated with decreased viral copies were only observed in infections with Omicron BA.1 variants. Our work showcases how GWAS can be a useful tool for probing phenotypes related to SNPs in viral genomes that are worth further exploration. We argue that this approach can be used more broadly across pathogens to characterize emerging variants and monitor therapeutic interventions.

Fig 1. Genomic sequences of SARS-CoV-2 infections and associated viral copies from cross-sectional samples collected in Connecticut, US.

(A) The daily number of genomic sequences of SARS-CoV-2 VOCs from February 2021 to March 2023. (B) The summary of viral copies of all samples, expressed as log10(viral copies per milliliter). (C) The summary of viral copies aggregated by month. The data gap in October 2021 is because we were unable to conduct PCR to obtain viral copies during this time.

Fig 2. Viral copies by category and regression analysis results.

Comparison of viral copies stratified by (A) age groups, (B) vaccination statuses, (C) variant of concerns. (D) Association of age, vaccination status, and VOCs with viral copies, expressed as log₁₀(viral copies per milliliter (Genome Equivalents/ml)). The reference groups (in gray) are Age <18 years old, 0 doses of vaccination, and the Other variant, respectively. The positive coefficients indicate the covariate is associated with higher viral copies value compared to the reference group, and vice versa. 0 Vax, 1 Vax, 2 Vax, 2 Vax 1 booster, and 2 vax 2 boosters denote vaccination statuses of 0 doses, 1 dose, 2 doses, 2 doses, and 1 booster, and 2 doses and 2 boosters, respectively, corresponding to the labels in (B). Results are shown as means with 95% confidence intervals. *** p < 0.001.

Fig 3. GWAS analysis identifies several single nucleotide polymorphisms (SNPs) that are associated with the changes in viral copies.

(A) Genome-wide association results of the impact of identified SNPs on viral copies during SARS-CoV-2 infection. The dashed line indicates the permuted threshold for genome-wide significance p = 4.67×10⁻⁶ (0.05/10697 SNPs). Significant SNPs are shown with solid colors. (B) SNPs (with p<1×10⁻¹⁰) that have positive (blue) or negative (red) effects on viral copies. (C) The corresponding synonymous (triangles) and non-synonymous (circles) amino acid changes that associate with increased or decreased viral copies. Data shown as means with 95% confidence intervals. The estimated effective sizes and associated standard deviations are given S1 Table.

Fig 4. The temporal dynamics of non-synonymous amino acid changes in the ORF1ab gene (P3395H, I4915T and L5086I), S gene (T19I, G252V, L452Q, Q498R, N679K, S704L, N856K, Q954H, N969K and L981F), and N gene (R203M) associated with changes in viral copies.

The results are based on the multivariate regression analysis using the sequence clusters (i.e., a categorical variable) inferred from the MDS components. (A) The phylogenetic tree estimated from a representative set of 996 genome sequences showing variant assignments and the locations of amino acid changes that increase (blue) or decrease (red) viral copies. (B) The temporal dynamics of the SNPs from February 2021 to March 2023. The transparency of the color corresponds to the mutation fraction in the daily sequence count: transparent color indicates low fractions, and opaque color indicates high fractions. The temporal dynamics of the SNPs, using MDS-inferred distance as a population control, are shown in S9–S11 Figs.