key: cord-0866594-stx7ltoh
authors: Nussenblatt, Veronique; Roder, Allison E; Das, Sanchita; de Wit, Emmie; Youn, Jung-Ho; Banakis, Stephanie; Mushegian, Alexandra; Mederos, Christopher; Wang, Wei; Chung, Matthew; Pérez-Pérez, Lizzette; Palmore, Tara; Brudno, Jennifer N; Kochenderfer, James N; Ghedin, Elodie
title: Year-long COVID-19 infection reveals within-host evolution of SARS-CoV-2 in a patient with B cell depletion
date: 2021-12-23
journal: J Infect Dis
DOI: 10.1093/infdis/jiab622
sha: 7ebdbab0c5ef8c02bb8f8e588fa35bfc8ec83334
doc_id: 866594
cord_uid: stx7ltoh

B-cell depleting therapies may lead to prolonged disease and viral shedding in individuals infected with SARS-CoV-2 and this viral persistence raises concern for viral evolution. We report on the 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. Overall, 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.

M a n u s c r i p t 4 BACKGROUND Cell-mediated and humoral immunity are necessary to clear SARS-CoV-2 infection [1, 2] . Individuals receiving B-cell depleting therapies can have protracted disease and prolonged viral shedding [3, 4] . Persistent shedding of viral RNA for weeks to months after onset of symptoms has been reported, however viable virus is often not detected after 9 days post illness onset [5] . In contrast, viral replication has been detected in immunocompromised patients for several months after initial infection [3, 6, 7] . Persistent viral replication in these patients is likely the result of profound lymphocyte defects due to B-and T-cell depleting therapies or underlying hematologic disease. Viral persistence in the setting of immunosuppression has raised concern for viral evolution and emergence of variants, especially during treatment with convalescent plasma [4] . In addition to single nucleotide variants, recent studies have demonstrated that SARS-CoV-2 in immunocompromised hosts is prone to deletion mutations in the spike protein, especially in the S1 region [3, 6, 7] . Deletions across the genome can reflect virus-host interactions and are found in both immunocompetent and immunosuppressed hosts.

Here, we report on a patient with persistent symptomatic viral infection over a period of 335 days. Viral genome sequencing revealed the emergence of two unique deletions and showed fixation of early minority variants, displaying viral evolution, a concern in the context of immunosuppression.

Written consent was obtained for human research subjects, as approved by the NIH Institutional Review Board (protocol # NCT02659943).

A c c e p t e d M a n u s c r i p t 5 RNA and sgRNA qPCR Detection of the N gene or ORF1a/b was performed on all specimens collected. Amplification of sub-genomic transcripts for the E gene (sgE) was done prospectively on samples after day 275, and retrospectively on samples before, as described previously [6] .

Amplification of viral genomes, library preparation, and genome analysis was done according to the protocols available at https://github.com/GhedinSGS/SARS-CoV-2_analysis. Libraries were sequenced on the Illumina NextSeq500 using the 2x300 bp paired end protocol. Adapters and primers were trimmed, reads were aligned to the Wuhan/Hu-1 strain (NC_045512.2) and the two libraries for each sample were merged. Consensus sequences and variants were identified using the timo variant calling pipeline.

Phylogenetic trees containing 266 background sequences from Maryland (obtained from GISAID, Table S1 ) were generated using Nextstrain with default parameters [8] . Lineages were called using Pangolin [9] .

Case presentation -A 48-year-old woman with type II diabetes mellitus and in complete remission from past diffuse large B-cell lymphoma (DLBCL) presented with fever, headache, nasal congestion, and productive cough on April 27, 2020. The patient's history is relevant for treatment with multiple lines of therapy and anti-CD19 chimeric antigen receptor-modified T-cell A c c e p t e d M a n u s c r i p t 6 (CAR-T) therapy [10] in December 2017, resulting in ongoing B-cell aplasia, hypogammaglobulinemia, CD4 lymphopenia, and recurrent upper respiratory infections. was not yet available, thus neither were administered at the time.

The patient was discharged a month later but continued to have temperatures of 99-100F, intermittent episodes of worsening cough and to require 3L NC supplemental oxygen.

Testing for SARS-CoV-2 by PCR on NP swabs was performed monthly for 3 months and every 3 months, thereafter. These were positive intermittently with Ct values above 37 (Fig. 1) . Due to the patient's overall mild to absent symptoms, positive SARS-CoV-2 tests during this period were thought to probably reflect shedding of non-viable virus particles. Chest CTs over the A c c e p t e d M a n u s c r i p t 7 same period showed bilateral increasing multifocal ground-glass opacities with crazy paving pattern and mixed changes and, therefore, organizing pneumonia and superimposed bacterial or fungal infection were considered. The patient preferred conservative management and declined bronchoscopy to rule out a superimposed infection. Induced sputum was negative for bacterial, fungal, or mycobacterial pathogens.

On Day 242, prednisone 50mg daily was initiated for the treatment of COVID-19-related cryptogenic organizing pneumonia and resulted in moderate symptom and slight radiographic improvement. SARS-CoV-2 PCR from a NP sample on day 284 was positive with a Ct value of 27.5, a marked decrease from the previous Ct value, indicating a substantial increase in viral load. This increase in the setting of steroids and only modest decrease in symptoms was concerning for COVID-19 relapse. A Ct value of 32.7 from sub-genomic RNA (sgRNA) real-time PCR indicated recent virus replication [6] (Fig. 1) . SARS-CoV-2 antibody testing was negative.

Shortly after, the patient reported worsening respiratory symptoms and required increased Global surveillance of SARS-CoV-2 genomes reveals that B.1.332 was circulating in March/April of 2020 but was no longer prevalent by March 2021 [9] . Consensus sequences were mapped onto a phylogenetic tree containing 266 background samples from Maryland collected between May 2020 and March 2021 (Table S1), using the publicly available Nextstrain software package ( Fig. 1B) [8] . All samples from this patient clustered on the same branch of the tree, with no intermixed background samples, indicative of a prolonged infection over 335 days, rather than a re-infection in March of 2021 (Fig. 1B) . Fig. 2A) . More interestingly, the March 2021 samples contained 2 deletions: a gap at nt 22290 to 22298 that led to a unique S:del244-246 and, consequently, a A243G substitution (Fig. 2B) ; and a 497nt deletion spanning the entire length of the ORF7b coding region and all but two amino acids of ORF8 (Fig. 2C) . Of note, some amino acid changes identified in the March 2021 samples were present as minority variants in the initial samples, suggesting a heterogeneous infection early on (Fig. 2D , smaller circles) with eventual fixation, as observed for ORF1a: A3070V, ORF7a: S37F, and N: P365L. Conversely, a consensus change present in the early samples also existed as a minority variant in the last sample in March 2021 (Fig. 2D) . The observed number of consensus changes in the March 2021 specimens from the initial sample indicates that the virus acquired the expected number of A c c e p t e d M a n u s c r i p t 9 mutations based on the error rate of the polymerase and evolved within this patient at approximately the same evolutionary rate that has been reported for SARS-CoV-2 in the global population [11] , estimated to be around 2 fixed mutations per month. ORF7b and ORF8 regions. The specific spike NTD deletion, del244-246, would impact the supersite and could induce resistance against NTD-directed antibodies [12] . This type of deletion has also been observed in variant B.1.351 (Beta), which contains NTD deletion 242-244 and a R246I mutation [12] . The appearance of this deletion in this patient supports previous observations where chronic SARS-CoV-2 infection in severely immunocompromised hosts receiving convalescent plasma, as was the case for this patient, can lead to variant emergence and reduced sensitivity to neutralizing antibodies [4] .

A c c e p t e d M a n u s c r i p t 10 The 497nt deletion in the ORF7b and ORF8 genes is the longest deletion reported in this region of the genome, and the first in an immunocompromised patient. Other reported ORF7b/ORF8 deletions range from 62nt to 382nt in length, with the first instance identified in Singapore in January of 2020 [13, 14] . In vitro analyses of similar deletions indicated mutants replicated to slightly higher levels than WT following infection with equal MOI, but showed similar levels of cytopathic effect. This same study further showed that deletion mutant viruses are transmissible, but may be less effective at establishing infection in a new host due to loss of immune evasion features of ORF8 [14] . ORF8 has been established as a key antagonist of innate immunity, eliciting a robust and highly specific antibody response during infection, suggesting that the deletion in competent hosts may be due to immune driven selection [15] . In our case, it is possible that the immunocompromised nature of this patient removes a need for ORF8 during infection. This is supported by data showing ORF8 is particularly tolerant to mutation, acquiring many missense and nonsense mutations, and is dispensable in cell culture [16] . A retrospective cohort study performed on patients in Singapore found that the deletion mutant virus was able to outcompete the WT in some patients that carried a mix of WT and a 382nt ORF7-ORF8 deletion viruses [17] . We found evidence of a few WT reads in the Days 313-314 samples, indicating a possible mixed infection, suggesting that the same competition may have occurred in this patient (Fig. 2C) .

This case demonstrates that severely immunocompromised patients may experience L293F  T814I  A3070V  L3606F  A4067T  L3915F  K1883T  S50L  A653V  I1115L  T30I I121L E121A 

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We thank the Department of Laboratory Medicine staff for processing clinical specimens and providing technical assistance. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov).

Data is available in NCBI GenBank under the following accession numbers: MZ385697-MZ385702 and MW990333 and raw sequence reads are available on SRA under bioproject PRJNA784993.

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