Yearlong COVID-19 Infection Reveals Within-Host Evolution of SARS-CoV-2 in a Patient With B-Cell Depletion

Abstract

B-cell-depleting therapies may lead to prolonged disease and viral shedding in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and this viral persistence raises concern for viral evolution. We report sequencing of early and late samples from a 335-day infection in an immunocompromised patient. The virus accumulated a unique deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts.

Keywords: COVID-19; SARS-CoV-2; immunocompromised; prolonged infection; viral evolution.

Figure 1.

Timeline of diagnostic tests for SARS-CoV-2 and treatment, and phylogeny of sequenced samples. A, Nasopharyngeal or oropharyngeal (upper respiratory) specimens were collected for detection of SARS-CoV-2 RNA, except when indicated by the following: ^# BAL sample, ∗ days when sputum specimens were collected, and † days when saliva was collected. Specimens with Ct values over 40 were considered negative for SARS-CoV-2 RNA. PCR for subgenomic RNA was performed only on specimens that tested positive for genomic RNA. Samples that were used for next-generation sequencing are indicated with an orange circle. Red lines indicate periods of hospitalization. Treatments administered are indicated with a black arrow and labeled. B, Maximum likelihood timed strain tree reconstructed from 266 local sequences from GISAID (Supplementary Table 1). Samples sequenced from the patient in this case study are colored in purple and labeled with day of infection from timeline. Abbreviations: BAL, bronchoalveolar lavage; Ct, cycle threshold; IVIG, intravenous immunoglobulin; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; sgPCR, subgenomic polymerase chain reaction.

Figure 2.

Mutations and deletions in sequenced samples over time. A, Tile plot showing consensus changes across the genome as compared to the initial infectious sample (collection date, 1 May 2020, day 1), color-coded by residue. B, Schematic showing 9-nt deletion in spike region (nt positions: 22290–22299, spike aa positions shown). C, Coverage plot, tile plot, and schematic showing the 497-nt deletion in the ORF7b and ORF8 coding regions. Coverage plot represents log₁₀ raw coverage. Dotted line is at 5 × coverage. Tile plot represents aa changes in this region, as compared to initial infectious sample (collection date: 1 May 2020, day 1), colored by residue as above in (A). Deletions identified in previous studies are shown as schematics and labeled by their country of origin. D, Circle plot showing major (larger, outer circle) and minor (smaller, inner circle) at locations where a minority variant in one sample exists as a major aa in another sample, colored by residue as in (A). ORFs and aa positions within the encoded protein are listed. Abbreviations: aa, amino acid; nt, nucleotide; ORF, open reading frame.