Year-long COVID-19 infection reveals within-host evolution of SARS-CoV-2 in a patient with B cell depletion

Abstract

Background: B-cell depleting therapies may lead to protracted disease and prolonged viral shedding in individuals infected with SARS-CoV-2. Viral persistence in the setting of immunosuppression raises concern for viral evolution.

Methods: Amplification of sub-genomic transcripts for the E gene (sgE) was done on nasopharyngeal samples over the course of 355 days in a patient infected with SARS-CoV-2 who had previously undergone CAR T cell therapy and had persistently positive SARS-CoV-2 nasopharyngeal swabs. Whole genome sequencing was performed on samples from the patient's original presentation and 10 months later.

Results: Over the course of almost a year, the virus accumulated a unique in-frame 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. Also, minority variants that were identified in the early samples-reflecting the heterogeneity of the initial infection-were found to be fixed late in the infection. Remdesivir and high-titer convalescent plasma treatment were given, and the infection was eventually cleared after 335 days of infection.

Conclusions: The unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts, and the implication of these mutations in the emergence of viral variants.

Summary: We report an immunocompromised patient with persistent symptomatic SARS-CoV-2 infection for 335 days. During this time, the virus accumulated a unique in-frame deletion in the spike, and a complete deletion of ORF7b and ORF8 which is the first report of its kind in an immunocompromised patient.

Figure 1.. Timeline of diagnostic tests for SARS-CoV-2 and treatment.

Nasopharyngeal or oropharyngeal (upper respiratory) specimens were collected for detection of SARS-CoV-2 RNA, except when indicated by the following: ^# indicates BAL sample, * indicates days when sputum specimens were collected and † indicates days when saliva was collected. Specimens with Ct values over 40 were considered negative for SARS-CoV-2 RNA. PCR for sub-genomic 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. Treatments administered are indicated with a black arrow and labeled.

Figure 2.. Phylogeny of sequenced samples.

Maximum likelihood timed strain tree reconstructed from 266 local sequences from GISAID (Table S1). Samples sequenced from the patient in this case study are colored in purple and labeled with the day of infection from first diagnosis.

Figure 3.. 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: 2020-05-01, day 1), color-coded by residue. (B) Schematic showing 9 nt deletion in Spike region (ntpos: 22290–22299, spike aa positions shown). (C) Coverage plot, tile plot and schematic showing the 497nt deletion in the ORF7b and ORF8 coding regions. Coverage plot represents log10 raw coverage. Dotted line is at 5X coverage. Tile plot represents amino acid changes in this region, as compared to initial infectious sample (day 1), colored by residue as above in 3A. 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 amino acid in another sample, colored by residue as in 3A. ORFs and aa positions within the encoded protein are listed.