biomedicines Article Different Within-Host Viral Evolution Dynamics in Severely Immunosuppressed Cases with Persistent SARS-CoV-2 Laura Pérez-Lago 1,2,† , Teresa Aldámiz-Echevarría 1,2,†, Rita García-Martínez 2,3,† , Leire Pérez-Latorre 1,2,†, Marta Herranz 1,2,4, Pedro J. Sola-Campoy 1,2, Julia Suárez-González 2,5, Carolina Martínez-Laperche 2,6, Iñaki Comas 7,8, Fernando González-Candelas 8,9 , Pilar Catalán 1,2,4, Patricia Muñoz 1,2,4,10 , Darío García de Viedma 1,2,4,* and on behalf of Gregorio Marañón Microbiology-ID COVID 19 Study Group ‡ 1 Clinical Microbiology and Infectious Diseases Department, Gregorio Marañón General University Hospital, 28007 Madrid, Spain; [email protected] (L.P.-L.); [email protected] (T.A.-E.); [email protected] (L.P.-L.); [email protected] (M.H.); [email protected] (P.J.S.-C.); [email protected] (P.C.); [email protected] (P.M.) 2 Gregorio Marañón Health Research Institute (IiSGM), 28007 Madrid, Spain; [email protected] (R.G.-M.); [email protected] (J.S.-G.); [email protected] (C.M.-L.) 3 Internal Medicine Department, Gregorio Marañón General University Hospital, 28007 Madrid, Spain 4 CIBER Respiratory Diseases (CIBERES), 28029 Madrid, Spain 5 Genomics Unit, Gregorio Marañón General University Hospital, 28007 Madrid, Spain 6 Oncohematology Service, Gregorio Marañón General University Hospital, 28007 Madrid, Spain 7 Tuberculosis Genomics Unit, Instituto de Biomedicina de Valencia-CSIC, 46010 Valencia, Spain; [email protected] Citation: Pérez-Lago, L.; 8 CIBER Salud Pública (CIBERESP), 28029 Madrid, Spain; [email protected] 9 Aldámiz-Echevarría, T.; Joint Research Unit “Infection and Public Health”, Institute for Integrative Systems Biology (I2SysBio), García-Martínez, R.; Pérez-Latorre, L.; FISABIO-University of Valencia, 46980 Valencia, Spain 10 Departamento de Medicina, Universidad Complutense, 28040 Madrid, Spain Herranz, M.; Sola-Campoy, P.J.; * Correspondence: [email protected]; Tel.: +34-914265104 Suárez-González, J.; Martínez-Laperche, † These authors contributed equally to this work. The order of the author was determined by agreement C.; Comas, I.; González-Candelas, F.; between the authors. et al. Different Within-Host Viral ‡ On behalf of the Gregorio Marañón’s Microbiology-ID COVID 19 Study Group members are provided in the Evolution Dynamics in Severely Acknowledgments. Immunosuppressed Cases with Persistent SARS-CoV-2. Biomedicines Abstract: A successful Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant, 2021, 9, 808. https://doi.org/ B.1.1.7, has recently been reported in the UK, causing global alarm. Most likely, the new variant 10.3390/biomedicines9070808 emerged in a persistently infected patient, justifying a special focus on these cases. Our aim in this study was to explore certain clinical profiles involving severe immunosuppression that may help Academic Editor: Marjorie Pion explain the prolonged persistence of viable viruses. We present three severely immunosuppressed cases (A, B, and C) with a history of lymphoma and prolonged SARS-CoV-2 shedding (2, 4, and Received: 22 May 2021 Accepted: 7 July 2021 6 months), two of whom finally died. Whole-genome sequencing of 9 and 10 specimens from Cases Published: 13 July 2021 A and B revealed extensive within-patient acquisition of diversity, 12 and 28 new single nucleotide polymorphisms, respectively, which suggests ongoing SARS-CoV-2 replication. This diversity was Publisher’s Note: MDPI stays neutral not observed for Case C after analysing 5 sequential nasopharyngeal specimens and one plasma with regard to jurisdictional claims in specimen, and was only observed in one bronchoaspirate specimen, although viral viability was published maps and institutional affil- still considered based on constant low Ct values throughout the disease and recovery of the virus iations. in cell cultures. The acquired viral diversity in Cases A and B followed different dynamics. For Case A, new single nucleotide polymorphisms were quickly fixed (13–15 days) after emerging as minority variants, while for Case B, higher diversity was observed at a slower emergence: fixation pace (1–2 months). Slower SARS-CoV-2 evolutionary pace was observed for Case A following the Copyright: © 2021 by the authors. administration of hyperimmune plasma. This work adds knowledge on SARS-CoV-2 prolonged Licensee MDPI, Basel, Switzerland. shedding in severely immunocompromised patients and demonstrates viral viability, noteworthy This article is an open access article acquired intra-patient diversity, and different SARS-CoV-2 evolutionary dynamics in persistent cases. distributed under the terms and conditions of the Creative Commons Keywords: COVID-19; SARS-CoV-2; persistence; immunosuppressed; diversity; viral viability; Attribution (CC BY) license (https:// genomics creativecommons.org/licenses/by/ 4.0/). Biomedicines 2021, 9, 808. https://doi.org/10.3390/biomedicines9070808 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 808 2 of 12 1. Introduction A recent review [1] established that the mean duration of SARS-CoV-2 in upper respiratory tract viral shedding is 17 days, with a maximum of 83 days, although certain studies report SARS-CoV-2 positive RT-PCRs up to 101 days [2]. With the identification of prolonged persistence, it was necessary to clarify whether these cases were still infectious or if they corresponded to the presence of residual genomic RNA. The standard approach to assess viability has relied on cell cultures, and most studies fail to detect live viruses beyond Day 9 [1]. The detection of subgenomic viral RNA as a way to infer viral replication has been used as an alternative to cell cultures to assess viability [2]. The dynamics of SARS-CoV-2 are a balance between its replication and the host’s immune response to control it. Thus, immunocompromised patients may offer the virus better opportunities to persist. This subgroup of patients may constitute a suitable model to dissect the ability of SARS-CoV-2 to acquire intrapatient diversity, determine the magnitude of intrahost evolution, evaluate the potential role of acquired mutations, and extend our understanding on the dynamics of SARS-CoV-2 infection. Whole genome sequencing of SARS-CoV-2 is key to understand the global expansion of the virus [3], track current transmission dynamics [4] or outbreaks [5], and determine reinfections [6]. However, the genomic analysis has scarcely been applied to better un- derstand the evolutionary dynamics of SARS-CoV-2 along persistent infections [7–9]. The interest of understanding within-patient evolution of SARS-CoV-2 in prolonged infections has recently increased due to the identification of a successful variant in the UK, B.1.1.7, which has been hypothesized to have emerged from one persistently infected case [10]. In this study, we present a detailed clinical description of three severely immunocom- promised subjects with SARS-CoV-2 prolonged shedding, in-depth genomic viral analysis on multiple sequential specimens, and an assessment of viability in cell cultures. 2. Materials and Methods 2.1. Diagnostic RT-PCR RNA was extracted and purified from 300 µL of a nasopharyngeal exudate with the KingFisher (Thermo Fisher Scientific, Waltham, MA, USA), and the TaqPath COVID-19 CE-IVD RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA) was applied. 2.2. Whole Genome Sequencing Viral sequencing was done using the Artic_nCov-2019_V3 panel of primers (Integrated DNA Technologies, Inc., Coralville, IA, USA) (artic.network/ncov-2019, 24 March 2020), and libraries were prepared using the Nextera Flex DNA Library Preparation Kit (Illumina lnc, San Diego, CA, USA) and sequenced on the Miseq system (Illumina Inc, San Diego, CA, USA). Details for WGS and bioinformatic analysis are specified in Supplementary Materials. Fasta files above the GISAID thresholds were deposited in GISAID (accession numbers in Supplementary Materials). The following frequencies were considered for variant calling: minority variants (MVs; frequency < 20%), intermediate variants (IVs; frequency 20–80%), and fixed single nucleotide polymorphisms (SNPs; frequency > 80%). 2.3. Short Tandem Repeat Analysis Human identity testing analysis was performed by short tandem repeat (STR) PCR (Mentype® Chimera® Biotype, Dresden, Germany) on the same specimens used to perform SARS-CoV-2 RT-PCRs and sequencing. Details are included in Supplementary Materials. 2.4. Cell Cultures For viral amplification, Vero E6 cells (ATCC CRL1586) were inoculated with positive SARS-CoV-2 samples. Details are included in Supplementary Materials. Cultures were considered positive when fulfilling at least one of the following criteria: (i) appearance of CPE, (ii) positive immunofluorescence detected, or (iii) a decrease of ≥3 Biomedicines 2021, 9, 808 3 of 12 (equivalent to a 1 log increase in virus quantity) between the Ct of the original sample and final culture supernatant. 2.5. Subgenomic RNA RNA remnants purified (KingFisher, Thermo Fisher Scientific, Waltham, MA, USA) from diagnostic nasopharyngeal swab specimens (300 µL of UTM swabs, COPAN, Biomerieux, Marcy-l’Étoile, France) were used as templates for the PCR design described elsewhere [11] for specific detection of the SG E gene RNA (+strand). 3. Results 3.1. Case A 3.1.1. Clinical Case A 52-year-old male with rituximab treatment every three months for a follicular lym- phoma in remission was admitted in June 2020. SARS-CoV-2 infection was diagnosed on 23 March (Day 0) 2020, a few days after receiving