SARS-CoV-2 entry into and evolution within a skilled nursing facility

Abstract

SARS-CoV-2 belongs to the family Coronaviridae which includes multiple human pathogens that have an outsized impact on aging populations. As a novel human pathogen, SARS-CoV-2 is undergoing continuous adaptation to this new host species and there is evidence of this throughout the scientific and public literature. However, most investigations of SARS-CoV-2 evolution have focused on large-scale collections of data across diverse populations and/or living environments. Here we investigate SARS-CoV-2 evolution in epidemiologically linked individuals within a single outbreak at a skilled nursing facility beginning with initial introduction of the pathogen. The data demonstrate that SARS-CoV-2 was introduced to the facility multiple times without establishing an interfacility transmission chain, followed by a single introduction that infected many individuals within a week. This large-scale introduction by a single genotype then persisted in the facility. SARS-CoV-2 sequences were investigated at both the consensus and intra-host variation levels. Understanding the variability in SARS-CoV-2 during transmission chains will assist in understanding the spread of this disease and can ultimately inform best practices for mitigation strategies.

Figure 1

Timeline of positive samples identified in Facility G over time. Staff and residents were swabbed and tested for presence of SARS-CoV-2 by qRT-PCR at least weekly. Positive samples are graphed by individual across time.

Figure 2

Multiple separate introductions of SARS-CoV-2 to Facility G, with one intrafacility outbreak. PHYML Timetrees colored by (A) time with blue for earliest sample emergence and transitioning to red for the latest, or (B) by facility G (orange), H (blue), I (green), J (red) or reference sequences (gray). (C) Distance to root graph over time for samples present in the phylogenetic analysis.

Figure 3

Facility G infections belong to two sequence groups with few mutations present between samples. SARS-CoV-2 sequences mapped to reference genome. Mutations that differ from the consensus sequence of the core Facility G outbreak shown as a dark line. Reference sequences shown in blue. The number of unique individuals carrying shown sequences and the facility those individuals worked or lived in are shown (#—Facility Letter, i..e. 17-G is 17 individuals in facility G with identical sequences).

Figure 4

Analysis of consensus mutations present across all samples with the phylogenetic analysis. (A) Types of consensus variants observed: synonymous, nonsynonymous, stop codon, deletions, insertion, or non-coding. (B) Mutation frequencies observed from vertical nucleotide to horizontal nucleotide. (C) Transitions vs. transversion fixed in the population sequenced. Each dot represents a specific transition (e.g., X → Y) or transversion (e.g., X → Y). Error bars SD. (D) Number of times specific mutations were likely introduced in the data set determined by TimeTree analysis.

Figure 5

Analysis of minority variants present across facility G samples. (A) Types of minority variants observed: synonymous, nonsynonymous, stop codon, deletions, insertion, or non-coding. (B) Specific minority variant mutations from vertical nucleotide to horizontal nucleotide. (C) Observed transitions vs. transversions across samples. Each dot represents a specific transition (e.g., X → Y) or transversion (e.g., X → Y). Error bars SD. (D) Variants by frequency across the genome. Log scale x-axis starts at 0.03 (3%).

Figure 6

Within facility G two mutations are found together across individuals: C3796U and C17766U. Frequency of mutations in individual’s virus population by weeks post first SARS-CoV-2 introduction (A) 0, (B) 7, (C) 10, and (D) 13. Lines link minority variant found within the same sample. Frequency starts at 0.03 (3%). (E) Presence of WT vs double mutant in sequences by subject over time. (F) Percent of WT vs. double mutant present in sequences.