key: cord-0078850-t57jalcj
authors: Caccuri, Francesca; Messali, Serena; Bortolotti, Daria; Di Silvestre, Dario; De Palma, Antonella; Cattaneo, Chiara; Bertelli, Anna; Zani, Alberto; Milanesi, Maria; Giovanetti, Marta; Campisi, Giovanni; Gentili, Valentina; Bugatti, Antonella; Filippini, Federica; Scaltriti, Erika; Pongolini, Stefano; Tucci, Alessandra; Fiorentini, Simona; d’Ursi, Pasqualina; Ciccozzi, Massimo; Mauri, Pierluigi; Rizzo, Roberta; Caruso, Arnaldo
title: Competition for Dominance Within Replicating Quasispecies During Prolonged SARS-CoV-2 Infection in an Immunocompromised Host
date: 2022-05-21
journal: Virus Evol
DOI: 10.1093/ve/veac042
sha: f4eba7391b271dc7386a98a6ba9433dde22ff2cc
doc_id: 78850
cord_uid: t57jalcj

SARS-CoV-2 variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication the original master mutant, named MB61°, was replaced by a minor quasispecies (MB61(222)) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61(222) entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB61°. Interestingly, co-infection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61(222) over MB61° replication. This finding may be explained by a faster replicative activity of MB61(222) as compared to MB61° as well as by the capability of MB61(222) to induce peculiar viral RNA sensing mechanisms leading to an increased production of Interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61(222) may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB61° cannot escape this host antiviral response during MB61(222) co-infection, since it has endocytosis as main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

SARS-CoV-2 positivity have been documented (Choi et al. 2020; Kang et al., 2020) but it is increasingly evident that viral transmission posed by post-convalescent COVID-19 patients may be negligible. For example, in one study of healthcare workers self-isolating due to persistent RT-PCR positivity up to 55 days after the onset of symptoms, no viable virus was recoverable in 29 out of 29 nasopharyngeal/oropharyngeal samples tested (Laferl et al., 2021) . However, there is no evidence whether these results can be applied to special populations such as immunocompromised patients.

Recent reports have, in fact, described cases where the virus can be isolated more than two months after its first detection (Avanzato et al., 2020; Choi et al. 2020 ).

The reasons why some people have productive long-term infection have been not yet completely understood even if this phenomenon has been found to occur in immunocompromised patients only (Camprubí et al., 2020; O'Sullivan et al., 2020; Abdul-Jawad et al., 2021; Baang et al., 2021; Hensley et al., 2021; Siqueira et al., 2021) . It has been suggested that the emergence of VOCs worldwide has likely occurred in immunocompromised patients who allowed intra-host persistent infection and variations in viral population (Al Khatib et al., 2020; Avanzato et al., 2020; Choi et al., 2020; Kemp et al., 2020; Lythgoe et al., 2021; Wang et al., 2021a; Wang et al., 2021b; Valesano et al., 2021) . Indeed, phylogenetic, populational and computational analyses of viral sequences showed that increased diversity was associated with prolonged viral replication (Voloch et al., 2021) . It is worth noting that the RNA virus population in a host is not represented by a single dominating sequence, but rather consists of an ensemble of replicating viruses characterized by closely related sequences termed quasispecies (Sun et al., 2021) . This allows RNA viruses to have a greater possibility to find the best adapting quasispecies to the specific host, usually achieved by changes in functional genes such as the S gene (Zhou et al., 2020) . These findings raise the question about the underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host.

Here we describe the case of an immunocompromised patient affected by B-cell lymphoma where the persistent replication of SARS-CoV-2 led to the accumulation of critical amino acid (aa) changes within the spike protein RBD. These mutations occurred early during infection and reflected the dynamic changes of quasispecies through competitive interactions in the viral population, probably aimed to determine a better replicative activity and overcome selective immune responses. Our results provide new insights into the intra-host evolution highlighting competition among replicating ensembles for dominance.

From April 2 nd to December 28 th , 2020, nasopharyngeal swabs (FLOQSwabs COPAN, Brescia, Italy) were periodically collected in universal transport medium (UTM, COPAN) for diagnostic purposes during hospitalization or at the Brescia community testing site. Samples were transported to the Microbiology Division of the Brescia Civic Hospital (Brescia, Italy). This work was approved by the Brescia Ethical Committee and the patient provided written consent to publish this case study.

Nasopharyngeal samples were processed using the InGenius automatic system (ElitechGroup, Turin, Italy), according to the manufacturer's instructions. The presence of SARS-CoV-2 nucleic acid was evaluated using the GeneFinder™ COVID-19 PLUS RealAmp Kit (Osang Healthcare, Anyang-si, Gyeonggi-do, Republic of Korea) whose reagents detect conserved regions in ORF1ab, E and N genes of the SARS-CoV-2 genome. Cycle threshold (Ct) values and results were automatically calculated by InGenius analysis software.

Vero E6 and Caco-2 cell lines were obtained from Istituto Zooprofilattico Sperimentale (Brescia, Italy) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific).

Calu-3 cell line (HTB-55) was purchased from the American Type Cell Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in complete Eagle's minimum essential medium (MEM;

Gibco, Thermo Fisher Scientific) supplemented with 10% FBS, 1% Sodium Pyruvate (Gibco, Thermo Fisher Scientific) , and 1% L-glutamine (Gibco, Thermo Fisher Scientific). Cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Infection experiments were run as previously described (Caccuri et al., 2020; Caruso et al., 2020) using the clinical SARS-CoV-2 isolates MB61 0 and MB61 222 (Fiorentini et al, 2021) . The viruses were propagated in Vero E6 cells and the viral titer was determined by a standard plaque assay. All the experiments were performed in a biosafety level-3 (BLS-3) laboratory using viruses at a Multiplicity of Infection (MOI) of 0.01. When reported, the cells were infected simultaneously with both MB61 0 and MB61 222 SARS-CoV-2 isolates at a MOI of 0.01 each.

Calu-3 and Caco-2 cells were infected in culture media without FBS containing MB61 0 and MB61 222 SARS-CoV-2 isolates at passage 1, alone or in combination, soon after having confirmed their identity with the original patient's sample by next generation whole-genome sequencing. After a 1 h incubation at 37°C and 5% CO 2 , the inoculum was removed, cells were washed twice with phosphate buffered saline (PBS) and replaced with 3 ml of culture media supplemented with 2% FBS. Supernatant samples were taken at 0, 12, 24, 48, 72, and 96 h post infection (p.i.) . Release of viable virus was evaluated by standard plaque assay and digital droplets polymerase chain reaction (ddPCR).

Total RNA was extracted using the QIAamp DSP Virus kit (Qiagen, Hilden, Germany) following manufacturer's guidelines. DdPCR reaction, was carried out on a QX200 ddPCR system with Automated Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA) using the One-Step SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories). Fluorescence of each droplet was evaluated using the QX 200 Droplet Reader (Bio-Rad Laboratories) equipped with QuantaSoft v1.7 Software. To evaluate the viral load in ddPCR we used the QuantaSoft Analysis Pro v1.0 Software (Bio-Rad Laboratories) that accompanied the droplet reader to calculate the concentration of the target sequences, along with Poisson-based 95% confidence intervals. The positive populations for each probe are identified using SARS-CoV-2 Standard positive control supplied with the One-Step SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories). The declared analytic sensitivity of the ddPCR kit is 0.260 cp/µl to 0.351 cp/µl for N1 and N2 probe sets, respectively. The data generated by the QX 200 droplet reader were rejected from subsequent analysis if a low number of total droplets was measured in 22 μl PCR (<10,000), or if all of the droplets were positive (saturation of the reaction) (Racki et al., 2014) .

An amplicon-based approach, targeting 343 nucleotide-long partially overlapping subgenomic regions that cover the entire SARS-CoV-2 genome, was used. Total RNA was extracted from 200 μl UTM. Full-length SARS-CoV-2 genomes were then generated using Paragon Genomics' CleanPlex multiplex PCR Research and Surveillance Panel, according to the manufacturer's protocol (Al Khabit et al., 2020; Li et al., 2020; Alteri et al. 2021) . Purified library was quantified with Qubit Fluorometer (Qubit DNA HS Assay Kit, Thermo Fisher Scientific). Amplicon library was loaded in a 300-cycle sequencing cartridge and the deep sequencing was performed on MiSeq platform (Illumina, San Diego, CA, USA). Raw data were checked for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then analyzed with the specifically designed software SOPHiA GENETICS' SARS-CoV-2 Panel (SOPHiA GENETICS, Lausanne, Switzerland). To confirmed data analyses, the paired-end reads were trimmed with Trimmomatic ver. 0.38 for quality (Q score > 25) and length (> 36 bp) and then were analyzed with Geneious ® software (version 11.1.5) (Biomatters Ltd, Auckland, New Zealand). Consensus sequence was reconstructed by mapping the reads to the SARS-CoV-2 reference sequence NC_045512 using Bowtie2 in sensitive-local mode with consensus threshold at 65%. The variant calling was carried out by the Variant Finder Tool (Geneious) filtering out variants with a p value greater than 0, using a minimum variant frequency of 0 and default parameters for Maximum Variant p-value (10 -6 ). To minimize false discoveries, a stringent approach to evaluated the presence of quasispecies spectrum in the patient's samples was applied. An intra-host single nucleotide variant (iSNV) was identified at genome positions with > 4000-fold sequencing coverage and with at least four reads supporting the nucleotide substitution. Amino acid substitutions at the identified iSNV sites were measured as the proportion of paired-end mapped reads with the alternative amino acid.

We combined the 12 sequential viral consensus genomes obtained from the patient with 2419 sequences available on GISAID (https://www.gisaid.org/) and representative of the SARS-CoV-2 lineage B.1.1 globally circulating until November 2021. Only genomes > 29,000 bp and < 1% of ambiguities were retrieved, low-quality genomes (> 10% of ambiguous positions), were excluded.

Sequences were aligned using MAFFT (FF-NS-2 algorithm) employing default parameters (Nakamura et al., 2018) . The alignment was manually curated to optimize number and location of gaps using Aliview (Larsson et al, 2014) . Lineage assessment was conducted using the Phylogenetic Assignment of Named Global Outbreak LINeages tool available at https://github.com/hCoV-2019/pangolin (O'Toole et al., 2020) . Phylogenetic analysis was performed using the maximum likelihood (ML) method implemented in IQ-TREE2 employing the GTR+I model of nucleotide substitution (Minh et al., 2020) . The statistical robustness of individual nodes was determined using the SH-aLTR branch support. The branches in the ML-tree were then converted into units of calendar time in TreeTime (Sagulenko et al., 2018) using a constant rate of 0.0008 substitutions/site/year (Wilkinson et al., 2021) with a clock standard deviation of 0.0004 substitutions/site/year.

The starting structure for the wild type hACE2 protein in complex with the SARS-CoV-2 spike protein RBD (PDB ID 6M0J) was obtained from the RCSB Protein Data Bank. Homology modeling SWISS-MODEL web server was used both for adding missing residue and constructing Q493K-N501T model structures (Waterhouse et al., 2018) . The Zinc ion was preserved from the crystal structure. Molecular dynamics simulations (MDs) of the two complexes (WT, Q493K-N501T) were performed using Amber 18 (Case et al., 2018) package and the ff14SB force field parameters was used for protein. Each complex was solvated in a periodic cubic water box using the TIP3P water model with 12 Å between the solutes and the edges of the box, and then a suitable number of Na + and Clions was added to neutralize the whole systems. Each system was energy minimized with 500 steps of steepest descent followed by 500 steps of conjugate gradient, with positional restraints of 2 kcal/mol A 2 on all protein atoms, with a cut-off for non-bonded interactions of 8 Å, this minimization protocol was done four time. The systems were then subjected to heating procedure of 25000 steps, from 0.1 to 300 K in an NVT ensemble with a Langevin thermostat. Bonds involving hydrogen atoms were constrained with the SHAKE algorithm and 2 fs time step was used. The systems were then equilibrated at 300 K in 2 consecutive steps of 500 ps each in the NPT. In the second equilibration step the positional restraints were removed. MDs were performed over 200 ns using the pmemd CUDA program of the Amber18 package and a server Tesla K20 Graphical Processing Unit. The trajectories were analyzed to compare and observe the structural deviation between WT and mutant structure. The conformational ensembles along the last 50 ns of the trajectory were clustered by CPPTRAJ using hierarchical agglomerative algorithm. Hydrogen bonds analysis were carried out using CPPTRAJ obtaining the hydrogen bond frequency for each residue pair during the molecular dynamics. In order to identify salt bridge and VdW interactions of each representative structure PBePISA web server (Krissinel and Henrick, 2007) and RING2.0 web server (Piovesan et al., 2016) were used.

Electrostatic potential maps for the WT and mutant structures were calculated using the Adaptive Poisson Boltzmann Solver (APBS) (Jurrus et al, 2018) .

Endocytosis was inhibited by incubation with 25 M Chlorpromazine (Sigma-Aldrich, Saint Louis, MO, USA). Cathepsin-mediated proteolysis was prevented by the addition of 20 M Cathepsin L inhibitor III (Calbiochem; Sigma-Aldrich). PM fusion was inhibited by treating with 100 M Camostat Mesylate (Sigma-Aldrich). Calu-3 cell pretreatment was performed by incubating for 1 h at 37°C. Then, media were removed and wells were washed twice with PBS. Untreated and treated cells were then infected. Viral titration was determined by Real Time qPCR at 24 and 48 h p.i., as follows. RNA extraction was performed with MagMAX Viral/Pathogen Nuclei Acid Isolation kit (Thermo Fisher Scientific) as previously described (Bortolotti, D., et al., 2020) . SARS-CoV-2 titration was obtained by TaqMan 2019nCoV assay kit v1 RealTime-qPCR (Thermo Fisher Scientific).

RNA sensors pathway genes, cytokines, interferons, IFITMs and LYE6 expression was evaluated on RNA extracted by using the RNeasy kit (Qiagen). DNase treatment was used to check for contaminant DNA presence, using β-actin PCR as a control. RT2 first strand kit (Qiagen) was used for RNA reverse transcription and cDNAs were immediately used or stored at -20°C. Gene expression analysis was performed by real-time quantitative PCR using PowerUp SYBR Green 

The evaluation of RNA sensors expression was performed using specific agonists: RIG-I/MDA5

agonist 5' triphosphate hairpin RNA complexed with transfection reagent Lyovec (1 µg/ml) (Invivogen, San Diego, CA, USA); TLR3 agonist Poly (I:C) (HMW) (2 µg/ml) (Invivogen); TLR7/8 Agonist -Imidazoquinoline compound R848 (2 µg/ml) (Invivogen).

The evaluation of IRF3 and NFκB expression and phosphorylation status was performed using the detection kit human total IRF-3 and phospho-IRF-3 (S386) ELISA kit (RayBiotech, Peachtree

Corners, GA, USA), and total NF-κB p65 and phospho-NF-κB p65 (S536) (Abcam, Cambridge, UK) on cell lysates.

RIG-I, TLR3 and TLR7 protein expression were quantified by Western blot assay. Whole cell lysates were treated with RIPA buffer containing proteinase inhibitor cocktail (Sigma-Aldrich). 

IL-1 α, IL-1 β, IL-4, IL-6, IFN-α, IFN-β, and INF-γ levels were evaluated in cell culture supernatants by single ELISA kit assays (myBio-source, San Diego, CA, USA) following the customer's protocols.

The proteomic analysis described in this study was performed on not infected (NI) Calu-3 cells and on Calu-3 cells infected with SARS-CoV-2 MB61 0 and MB61 222 isolates, collected at 12, 24 and 48 h p.i.. Cells were lysed and proteins were extracted, reduced/alkylated and enzymatically digested using Easy Pep TM Mini MS Sample Prep Kit (Thermo Fisher Scientific). Tryptic and Lys-C digestion was carried out on 80 µg of extracted proteins mixture. Following the kit protocol, peptides were generated, cleaned-up to prepare detergent-free samples and resuspended in 0.1% formic acid (Sigma-Aldrich) for LC-MS/MS analysis in less than 3 h for each examined condition

Peptide mixtures were analyzed using Eksigent nanoLC-Ultra® 2D System (Eksigent, part of AB SCIEX Dublin, CA, USA) combined with cHiPLC-nanoflex system (Eksigent) in trap-elute mode.

Briefly, for each condition three technical replicates were performed, injecting 0.8 µg of proteins on the cHiPLC trap (200 µm x 500 µm ChromXP C18-CL, 3 µm, 120 Å, Eksigent, part of AB SCIEX Dublin, CA, USA) and running the loading pump in isocratic mode with 0.1% formic acid in water for 10 min at a flow of 3 µL/min. The automatic switching of cHiPLC ten-port valve then eluted the trapped mixture on a nano cHiPLC column (75 µm x 15 cm ChromXP C18-CL, 3 µm, 120 Å, Eksigent, part of AB SCIEX Dublin, CA, USA) through a 132 min gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. In depth, gradient was: from 5-10% B in 3 min, 10-35% B in 107 min, 35-95% B in 10 min and holding at 95% B for 12 min.

The eluted peptides were directly analyzed on an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) equipped with EASY-Spray ion source (Thermo Fisher Scientific). Easy spray was achieved using an EASY-Spray Emitter (Thermo Fisher Scientific) (nanoflow 7 µm ID Transfer Line 20 µm x 50 cm) held to 1.6 kV, while the ion transfer capillary was held at 220°C. 

All data generated were searched using the Sequest HT search engine contained in Proteome Discoverer software, version 2.1 (Thermo Fisher Scientific). Experimental MS/MS spectra were compared with the theoretical mass spectra obtained by in silico digestion of 29 SARS-CoV-2 protein sequences obtained from Uniprot (www.uniprot.org) and Homo Sapiens proteome database (74842 entries), downloaded on March 2021. The following criteria were used for the identification of peptide sequences and related proteins: trypsin and Lys-C as enzymes, methionine oxidation, carbamidomethyl cysteine, three missed cleavages per peptide, mass tolerances of ± 10 ppm for precursor ions and ± 0.05 Da for fragment ions. Percolator node was used with a target-decoy strategy to give a final false discovery rates (FDR) at Peptide Spectrum Match (PSM) level of 0.01 (strict) based on q-values, considering maximum deltaCN of 0.05 (Käll et al., 2007) . Only peptides with minimum peptide length of six amino acids, and rank 1 were considered. Protein grouping and strict parsimony principle were applied. Results were then exported to Excel file for further processing.

The 42 LC/MS lists (6 runs for the 7 available conditions) obtained from the Proteome Discoverer software were aligned, normalized using a total signal normalization method (Griffin et al., 2010) and compared using a label-free quantification approach based on aPSMs, as previously reported (Caccuri et al., 2021) . Data matrix dimensionality of 5025 distinct proteins was reduced by linear discriminant analysis (LDA). Following this procedure, we selected and retained proteins with P<0.0001, corresponding to a LDA F ratio > 11. Differentially expressed proteins (DEPs) selected by LDA were processed by Hierarchical Clustering applying the Ward's method and the Euclidean distance metric. All processing was performed by JMP15. (Su et al., 2014) . In particular, DEPs were grouped in functional modules by the support of the GO enrichment tool inserted in STRING (Doncheva et al., 2019) and BINGO (Maere et al., 2005) Cytoscape's apps.

Data were analyzed for statistical significance using the Student's unpaired two-tailed t-test or twoway ANOVA when appropriate. The Bonferroni post-test was used to compare data. Differences were considered significant when p < 0.05. Statistical tests were performed using Prism 8 software (GraphPad Software, La Jolla, CA, USA).

On March 25, 2020, a 59-year-old man with a 24-year history of follicular lymphoma was admitted to the hospital for lymphadenopathies, declining edema and light breathing difficulties. The presence of a bilateral pleural effusion, a segmental pulmonary embolism and a minimal pericardial effusion suggested a transformation of follicular lymphoma toward an aggressive non-Hodgkin lymphoma (NHL). Due to a subsequent development of a bilateral interstitial pneumonia and the presence of a hypoxemic respiratory failure, on April 2, 2020 (day 0) the patient was subjected to nasopharyngeal swab for SARS-CoV-2 testing and was first diagnosed positive for infection. To the best of our knowledge, this is the first described case where an immunocompromised symptomatic individual spontaneously recovers from COVID-19 in the absence of antiviral and/or convalescent plasma treatments.

Using deep sequencing data, we were able to follow the dynamics of the intra-host evolution during a SARS-CoV-2 prolonged infection (Fig. S1) . Genetic screening of all samples revealed 610 intrahost single nucleotide variants (iSNVs) with allele frequency >10%, with nonsynonymous changes representing 408 (85%) out of 482 iSNVs detected in coding regions. The highest abundance of iSNVs were mainly scattered over ORF1ab and S genes. In particular, Orf1ab harbored the majority of the iSNVs detected (n=266) followed by the S (n=156), N (n=36), orf3a (n=21) Concerning nonsynonymous substitutions, genomic analysis of sequences revealed that since day 26 the virus showed non-conserved amino acid (aa) changes in a number of viral proteins. In particular, one aa mutation was observed in nsp3 (D1639N) and in methyltransferase (A6194V) ( Fig. S2A) . At the same time, we observed the occurrence of one deletion (L242del) and one mutation (Q493K) in the spike S1 region ( Fig. S2A and S2B) . These aa changes were represented in the consensus sequence of all consecutive samples until the infection was cleared. It is worth noting that the Q493K mutation has been rarely found elsewhere up to March 15 th 2021, since only 30 out of 784883 (0.004%) genomes available on GISAID (https://www.gisaid.org/) showed the presence of K at the spike position 493. As shown in Fig. S2B and in Fig. 1B , a N501T mutation in the spike S1 region, firstly observed in the consensus sequence at day 48, initially appeared in 11.8% of the total reads at day 34 and then became predominant in all the subsequent sampling and being maintained till viral clearance. Interestingly, at day 125 we found that a N501Y sequence transiently dominated over the N501T (53.7% vs 45.1%, respectively) but disappeared soon after.

In the last viremic sample collected at day 222, other three non-conserved aa substitutions were expressed in helicase (R5661C), exonuclease (V6207I), and orf3a (L108F). The sudden appearance of these nonsynonymous mutations in the master sequence at day 222 is likely to reflect virus evolution more than the emergence of previously concealed quasispecies.

Surprisingly, all dominant non-conserved aa substitutions or deletions we observed over time, even transiently, in the spike S1 region were already present in nasopharyngeal swabs collected as early as at day 0 and 7 (Fig. 1B) . This finding highlights a fast changing also in the genetic characteristics of a key functional gene such as the S gene, further suggesting a dynamic replacement of genome subpopulations of related quasispecies within replicating ensembles.

In order to understand the evolution of SARS-CoV-2 and the dynamics of iSNVs in an immunocompromised host, we performed a phylogenetic analysis using viral consensus sequences obtained from 12 nasopharyngeal swabs collected between day 0 and day 222. The time stamped tree revealed the phylogenetic relationship among the 12 generated strains, showed that all of them belonged to an ancestral SARS-CoV-2 lineage (B.1.1) (Fig. 2) . Our results suggest that sequences at day 0, 7, and 20 scattered on different branches of the phylogenetic tree. At the same time, sequences obtained from day 26 to 222 established an independent cluster. This finding further suggests an intra-host virus evolution, in which genomic diversity is relatively heterogeneous during the early stage of infection, but becoming more homogeneous over time as a consequence of virus adaptation to the immunocompromised host in search for a better infectivity or resistance to innate immune responses.

Replicating ensembles are subjected to episodes of competition. This is mostly due to the biosphere diversity in which viruses are installed and to their need of meeting with unique intracellular environments (Domingo et al., 2021) . To test the hypothesis of a dynamic replacement of SARS-CoV-2 quasispecies due to competition mechanisms in vivo, we first isolated the dominant virus from nasopharyngeal swabs collected at day 0 (MB61 0 ) and at day 222 (MB61 222 ) and then tested its capability to replicate in two different human cancer cell lines as lung adenocarcinoma (Calu-3) and colon carcinoma (Caco-2). Quantitation of viral genomes in cell supernatants collected at 12, 24, 48, 72 and 96 h post infection (p.i.) by digital droplet PCR (ddPCR) did show a significant difference in the replication of the two isolates in Calu-3 cells at 12 h p.i., with a faster growing of MB61 222 than MB61 0 (Fig. 3A) . At the same time, a significantly faster replication of MB61 222 was observed in Caco-2 cells at each time p.i., as compared to MB61 0 (Fig. 3B) . Similar results were obtained by plaque assay quantification of viable virus ( Fig. S3A and B) reads. As shown in Fig. 3C , quantification of RNA released over time shows that viral production is very similar in Calu-3 and Caco-2 cells. This data was also confirmed by plaque assay (Fig. S3C) .

However, as soon as 24 h after coinfection of Calu-3 cells, MB61 222 replication was significantly increased as compared to MB61 0 . Approximately 10% of total virus detected in the supernatant of coinfected cells at the experimental endpoint (96 h) was ascribed to MB61 0 , suggesting that MB61 222 actively replaced its competitor (Fig. 3D) . Even more striking results were obtained in coinfection experiments performed in Caco-2 cells. In fact, a significantly higher replication activity of MB61 222 compared to MB61 0 was evident at as early as 12 h p.i., with MB61 222 representing approximately 70% of the released virus (Fig. 3E) . Results obtained suggest that the capability of MB61 222 to replace MB61 0 in two different target cell lines might be ascribed to mechanisms of interference able to alter viral population dynamics in coinfected cells.

One possible difference in the ability of MB61 0 and MB61 222 to interact with target cells might be ascribed to the cell entry mechanisms. SARS-CoV-2 might enter target cells via endocytosis or PM fusion pathways in the presence of TMPRSS2, the proteolytic process of SARS-CoV-2 is completed at the plasma membrane, but in its absence the virus is endocytosed and sorted into endolysosomes, from which SARS-CoV-2 enters the cytosol via clathrin and cathepsin L. (Jackson et al., 2022) . We investigated the entry mechanisms used by MB61 0 and MB61 222 in Calu-3 cells.

We observed that TMPRSS2 inhibition obtained by Camostat was able to significantly reduce both MB61 0 and MB61 222 replication at 24 h p.i. (Fig. 4A) Since SARS-CoV-2 might also use endocytic processes, we performed the inhibition of endocytosis via Chlorpromazine and Cathepsin L inhibitor III, that block clathrin-mediated endocytosis (Vercauteren, et al., 2010) and cathepsin L, respectively. As shown in Fig. 4A , inhibition of the clathrin-dependent early endosome formation significantly reduced MB61 0 replication. At the same time, cathepsin L significantly reduced MB61 0 replication. On the other hand, clathrin and cathepsin-L inhibitors did not exert any effect on MB61 222 replication. These results support a major involvement of the endocytosis entry mechanism for MB61 0 , while MB61 222 mainly utilizes the PM fusion pathway.

In the effort to elucidate this different behavior of MB61 0 and MB61 222 , we evaluated the expression of Interferon-inducible transmembrane proteins (IFITMs), that are intensely induced during viral infection and play a crucial role in virus restriction of endosomal entry (Winstone, et al., 2021) . Simultaneously, we evaluated the level of LY6E expression, that is implicated in the control of the PM fusion pathway (Pfaender, et al., 2020) . As shown in Comparison at the key interfacial interactions between hACE2 and the RBD of SARS-CoV-2

shows that SARS-CoV-2 WT (Fig. 5A) and SARS-CoV-2 Mut (Fig. 5B) and induces a rearrangement of the interactions in this contact region. The replacement of N501T generates a loss of six hydrogen bonds in CR3, but a new hydrophobic patch is generated among T500, V503 of CR3 and Q325, N330 of hACE2 and these new interactions extend the terminal contact region of CR3 by engaging the helix 18 (H18) close to the beta strands of hACE2 (Fig. 5B) .

The hydrogen networks observed in the representative conformations obtained from the molecular dynamics are in agreement with the frequency of the hydrogen bonds calculated during the simulation (Fig. S5) . Furthermore, Q493K substitution inserts a positive charge and the lysine forms two new saline bridges among K493 of RBD and E35 and D38 of hACE2. A comparison of the surface electrostatic potential identifies a positive charged patch on the SARS-CoV-2 Mut RBD-hACE2 that is absent on the SARS-CoV-2 WT RBD-hACE2 ( Fig. 5C and Fig. 5D ). K493

contributes to form positive electrostatic surface in the SARS-CoV-2 Mut (Fig. 5D) . The present study reveals a reorganization of key interfacial interactions between hACE2 and the SARS-CoV-2

Mut RBD. In addition, Q493K conversion in the SARS-CoV-2 Mut RBD creates a positive electrostatic patch that results in greater electrostatic complementarity than that of the SARS-CoV-2 complex.

A differential pathway for viral entry might induce substantial differences in viral RNA sensing by the infected cells since the different timing and localization for genome released engages different intracellular RNA sensors.

We investigated both cytosolic (RIG-I and MDA5) and endosomal (TLR3, TLR7, and TLR8) RNA sensors to identify possible differences between MB61 0 and MB61 222 SARS-CoV-2 isolates. As shown in Fig. 6 , the cytosolic RIG-I RNA sensor was significantly up-modulated as mRNA (Fig.   6A ) and protein (Fig. 6B ) expression by MB61 222 only. The endosomal TLR3 and TLR7 RNA sensors were triggered at the mRNA (Fig. 6A) and protein (Fig. 6B) The ability of MB61 222 to engage cytosolic RIG-I RNA sensor suggests a differential down-stream transduction as compared to MB61 0 that enhances endosomal TLR3 and TLR7 RNA sensors only.

Among the transcriptional factors involved in RNA sensing by viral infected cells, IRF3 and NF-κB play a central role (Schmitz et al., 2014) . In particular, IRF3 protein is involved in the production of interferons (Yanai et al., 2018) , whereas NF-κB is mainly employed in the induction of the proinflammatory response (Liu et al., 2017) . Even if both IRF3 and NF-κB are reported to be crucial in RNA sensing signaling, they are differentially induced by different RNA sensors. In fact, while endosomal RNA sensors activation leads mainly to NF-κB recruitment, the cytosolic RNA sensor RIG-1 typically activates both NF-κB and IRF3 signal (Nguyen et al, 2020) . As shown in Fig. 6C , a significant activation of IRF3 and NF-kB was observed in MB61 222 -infected cells. On the other hand, MB61 0 -infected cells showed an enhanced NF-kB transcription only. These data agree with the differential RNA sensors induction by the two SARS-CoV-2 isolates. MB61 222 was found to significantly enhance RIG-I, TLR3 and TLR7 RNA sensors and consequently both IRF3

and NF-kB transcription and phosphorylation (Fig. 6D) . On the contrary, MB61 0 induces TLR3 and TLR7 RNA sensors and the down-stream factor NF-kB transcription and phosphorylation (Fig.   6D ).

It is known that depending on the ability of the cell to sense the presence of an infection, different soluble factors, such cytokines and interferons (IFNs), are released. NF-κB and IRF3 are essential to mediate antiviral actions through the expression of virus-induced and IFN-stimulated genes. We

Calu-3 cells infected with MB61 0 or MB61 222 .

As reported in Fig. 6E , MB61 222 infection significantly increased the mRNA transcription of the pro-inflammatory cytokines IL-1β and IL-6 in comparison with uninfected and MB61 0 -infected Calu-3. Similarly, IL-1β and IL-6, as well as TNF-α secretion were significantly increased in MB61 222 -but not in MB61 0 -infected cells (Fig. 6F) . IFN-α, IFN-β, and IFN-γ were significantly induced during MB61 222 infection as mRNA transcription (Fig. 6G) and protein secretion (Fig. 6H) as compared to MB61 0 -infected cells. On the contrary, MB61 0 infection was not found to affect IFN-α and IFN-β transcription and secretion ( Fig. 6G and Fig. 6H ). However, MB61 0 infection was found to significantly induce IFN-γ secretion as compared to uninfected cells (Fig. 6H) . These results are in agreement with the differential effects that the two isolates promote on RNA sensors. and TLR3 RNA sensors, with the consequent activation of NF-kB and IFN-γ secretion.

Proteomics profiles of Calu-3 cells infected or not with MB61 0 or MB61 222 , collected at 12, 24 and 48 h p.i. were analyzed. Through a shotgun label-free platform, based on the coupling of nanoliquid chromatography and high-resolution mass spectrometry (nLC-hrMS/MS), 42 proteomics runs were acquired from the 7 examined conditions. A total of 5113 distinct proteins were identified (Table   S1 ) with at least one unique peptide, molecular weights ranging from 2 to 3990 kDa and isoelectric points from 3.8 to 12.3. The alignment of all protein lists obtained was carried out on the basis of identified proteins and related peptide spectrum match values (PSMs), that represent the number of mass spectra attributed to them and indirectly their abundance in the samples. Then, for each experimental condition three lists were created normalizing and averaging the peptide spectrum match values (aPSMs) of the proteins identified with high confidence with at least one unique peptide using a total signal normalization method. The linear discriminant analysis (LDA) was applied using the proteomics lists and 431 statistically significant proteins presenting a p value  0.0001, were extracted as differentially expressed proteins (DEPs) ( interactions was built. Globally, these proteins were grouped across in 51 functional modules (Fig.   S6 ). The evaluation of the protein pathways showed a differential induction by the two isolates, supporting our results. In particular, as shown in factors; ii) granulin, that binds directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction (Tang et al., 2011) ; iii) SLFN5, which represses IFN-induced STAT1

transcriptional activity (Arslan et al., 2017) ; and iv) nucleobindin 1 (NUCB1), that has been shown to inhibit ATF6 activity (Madden et al., 2019) . On the contrary, MB61 0 was not found to promote synthesis of proteins with antiviral activity.

The enhanced endocytic activity during MB61 0 infection is supported by the up-modulation of:

HIP1R (Chen and Brodsky, 2005) and CLINT1, that interact with clathrin; GATD1 and IGF2R, implicated in intracellular trafficking; and by the focal adhesion proteins (MUCSAC, TLN1, LAMC2, LAMB3, FHL2, GLG1), that might facilitate SARS-CoV-2 adhesion and integrin signaling, which terminates with endosomal trafficking. To facilitate particle uncoating, MB61 0 induced MAPRE2, NUMA1 and MACF1 that sustain the microtubule-associated particle uncoating. The decreased PM fusion pathway during MB61 0 infection might by also determined by the increased expression of SERPINB1 by infected cells, which is predicted to reduce the ability of TMPRSS2 to facilitate SARS-CoV-2 entry into airway epithelial cells (Stanton et al., 2020) .

Interestingly, MB61 0 also enhanced the expression of proteins (GDI1, SEC22B, COPG1, HOOK1, ARFIP1, EMC1, RRBP1, SRP68) involved in the formation of endoplasmic reticulum (ER)-derived perinuclear interconnected membrane structures that contain double-membrane vesicles (DMVs), in which SARS-CoV-2 RNA synthesis occurs (Snijder et al. 2020) . The use of endocytosis also by MB61 222 is supported by the expression of ARPC1B, TES and GC, that facilitate cargo enrichment and budding from the endosomal membrane to generate recycling vesicles. It is worth noting that MB61 222 may require this process for receptor recycling (Al-Saleem et al., 2019). Alternatively, the increased expression of DYNLL2, that controls retrogade vescicle transport, may facilitate virus trafficking to replication sites, or may serve to deliver cargoes to replication sites. The formation of DMVs during MB61 222 infection is also supported by the induction of REEP5, that interacting with SARS-CoV-2 structural M protein, plays a crucial role in viral morphogenesis.

Quasispecies provide an advantage to SARS-CoV-2 for achieving the best fitness during prolonged intra-host evolution by changing the genetic characteristics of key functional genes. Our data show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. After 222 days of active viral replication in vivo, we highlighted the replacement of the original master mutant by a minor mutant, namely MB61 222 , expressing two critical mutations in spike, namely Q493K and N501T. This mutated spike shows a greater affinity to ACE2 than the original spike, which may explain the acquired capability of MB61 222 to gain access into target cells mainly by the PM fusion pathway than endocytosis, which characterizes the entry mechanism used by the previous master mutant named MB61 0 . In this shifting in the entry route preference, we cannot exclude the role played by other spike mutations acquired by MB61 222 during time. The enhanced endocytic activity during MB61 0 infection is supported by the upmodulation of HIP1R (Chen and Brodsky, 2005) and CLINT1, that interact with clathrin, GATD1

and IGF2R, implicated in intracellular trafficking and by the focal adhesion proteins (MUCSAC, TLN1, LAMC2, LAMB3, FHL2, GLG1), that might facilitate MB61 0 adhesion and integrin signaling, which terminates with endosomal trafficking. On the contrary, the decreased PM fusion pathway during MB61 0 infection might by determined by the increased expression of SERPINB1 by infected cells, which is predicted to reduce the ability of TMPRSS2 to facilitate SARS-CoV-2 entry into airway epithelial cells (Stanton et al., 2020) . These findings, in agreement with previous observations (Nelson et al., 2021; Starr et al., 2020) , may support the significant increase in infectivity and replication of MB61 222 as compared to MB61 0 we observed at early time ( Different modality of SARS-CoV-2 entry are known to induce activation of different RNA sensors (Bortolotti et al., 2021; Mdkhana et al, 2021) . The ability of MB61 222 to engage cytosolic RIG-I RNA sensor leads to a differential down-stream transduction as compared to MB61 Kozak context introducing an enhanced transcriptional regulatory sequence (TRS). We evaluated MB61 0 and MB61 222 N gene sequences but, as shown in Fig. S7 , we did not detect any of the above described nucleotide changes, thus excluding their role in establishing different innate immune responses evoked by the two isolates.

It has been previously suggested (Parker et al. 2021 ) that the 3-nucleotide mutation at 28,881-28,883 GGG>AAC found in Alpha, Gamma and Omicron variants creates a novel TRS for N* transcription. Increased N* protein levels are supposed to act as innate immune antagonists by sequestering dsRNA. However, this mechanism cannot explain the different capability of our isolates to trigger innate immunity since both MB61 0 and MB61 222 show these nucleotide changes in their N gene sequence (Fig. S7) .

Interestingly, co-infection of two human cell lines of different origin with our two SARS-CoV-2 isolates highlighted the dramatic predominance of MB61 222 replication over that of MB61 0 . This may be attributed to a faster replication activity of MB61 222 compared to MB61 0 and to the capability of MB61 222 to activate RNA sensors and trigger potent innate immune responses. IFNstimulated gene products are involved in SARS-CoV-2 entry steps, as IFITMs (Winstone et al., 2021; Shi et al., 2021) . Four members of these proteins (IFITM1, IFITM2, IFITM3 and IFITM5) are strongly induced by type I and type II IFNs and known to act as antiviral molecules against Influenza A viruses, Flaviviruses, Filoviruses and SARS-CoV (Szabo and Bugge, 2008; Szabo and Bugge, 2011) . More recently, IFITM2 was found to restrict SARS-CoV-2 entry occurring by endocytosis (Winstone et al, 2021) . Although the mechanisms are still unknown, IFITM2 was found to prevent SARS-CoV-2 to traverse the endosomal membrane to access cellular cytoplasm.

Interestingly, this did not occur if SARS-CoV-2 gains access into the target cells by PM fusion at the plasma membrane (Huang et al, 2011) . We observed an increased expression of IFITM1 and MB61 222 is able to evoke a potent innate immune response, which consists in the production and release of type I and II IFNs and different pro-inflammatory cytokines. Usually, early host innate responses protect airway cells from infection and limit viral dissemination (Sposito et al., 2021) .

The capability of MB61 222 to actively replicate in such an unfavorable microenvironment supports the hypothesis that it can escape host innate responses by activating still unknown antagonists.

Indeed, viral innate resistance is likely to have contributed to prolonged in vivo viral shedding in the immunocompromised host, whose antiviral defenses were essentially based on innate immunity.

This hypothesis is also consistent with reports of enhanced innate evasion and prolonged shedding of the Alpha variant (Calistri et al., 2021; Kissler et al., 2021; Thorne et al., 2021) . It is worth noting that the inflammatory switch operated by MB61 222 , may have been evolved to also enhance viral spreading at the site of infection as already evidenced for the Alpha variant (Davies et al., 2021;  Scientific Advisory Group for Emergencies, 2021). Altogether, our findings highlight the importance of innate immune evasion in the still ongoing process of SARS-CoV-2 evolution aimed to find the optimal adaptation to replicate and disseminate into the human host. However, the role of other mutations than N501T and Q493K acquired by MB61 222 , in increasing viral replication, promoting innate immune evasion and changing the cytokine profile, cannot be completely ruled out.

In conclusion, this study further points to uncontrolled and prolonged infection in immunocompromised patients as the condition that may have shaped the emergence of SARS-CoV-2 variants with a higher capability than their ancestors to rapidly spread around the world.

Interestingly, our data show a competition among virus quasispecies to hold a dominant replicative activity and spreading by manipulating the host cell innate immunity. This finding subverts the idea of quasispecies as an advantage mutation pool for the epidemic variants to find the best genetic fitness during intra-host evolution and introduces a new concept of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of the other competitors in the viral ensemble. This may explain, at least in part, the extraordinary rapid worldwide turnover of a VOC over the original pandemic strain observed in the COVID-19

pandemia. The preexistence of potential fitness mutants calls for the importance of studying the mutation spectra of different hosts, and minor mutants in particular, to predict the emergence of new variants with neutralizing antibody or antiviral drug escape ability. The % of viral fraction was defined as the number of reads endowing the specific signatures of the MB61 222 patient's isolate. Data shown are the mean and the standard deviation for two replicates.

The statistical analysis was performed using a two-way ANOVA and Bonferroni's post-test was used to compare data (**** p < 0.0001). normalized to not infected Calu-3 cells. Data shown are the mean and the standard deviation of the mean for three independent experiments. Student's unpaired two-tailed t-test was used to determine statistical significance between the two isolates for each inhibitor (* p < 0.05; ** p < 0.01). were under the detection limit of the assays. Data shown are the mean and the standard deviation of the mean for three independent experiments. The statistical analysis was performed using the Student's unpaired two-tailed t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). SARS-CoV-2 WT (black) and SARS-CoV-2 Mut (red) RBD. RMSD of CA atoms of RBD, RBM (residues 438-508), hACE2 and H1+H2+H18+β-turn (residues 19-101 + 319-365). RBM region of RBD is highlighted in grey as H1+H2 and H18+β-turn in hACE2 RMSF graph. 

Acute Immune Signatures and Their Legacies in Severe Acute Respiratory Syndrome Coronavirus-2 Infected Cancer Patients

Within-Host Diversity of SARS-CoV-2 in COVID-19 Patients With Variable Disease Severities

HTLV-1 Tax-1 interacts with SNX27 to regulate cellular localization of the HTLV-1 receptor molecule, GLUT1

Genomic epidemiology of SARS-CoV-2 reveals multiple lineages and early spread of SARS-CoV-2 infections in

Human SLFN5 is a transcriptional co-repressor of STAT1-mediated interferon responses and promotes the malignant phenotype in glioblastoma

Case Study: Prolonged Infectious SARS-CoV-2 Shedding from an Asymptomatic Immunocompromised Individual with Cancer

Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised Patient

The alpha/B.1.1.7 SARS-CoV-2 variant exhibits significantly higher affinity for ACE-2 and requires lower inoculation doses to cause disease in K18-hACE2 mice

SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway

TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection

A persistently replicating SARS-CoV-2 variant derived from an asymptomatic individual

SARS-CoV-2 Infection Remodels the Phenotype and Promotes Angiogenesis of Primary Human Lung Endothelial Cells', Microorganisms

Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs

Persistent Replication of SARS-CoV-2 in a Severely Immunocompromised Patient Treated with Several Courses of Remdesivir

Methotrexate inhibits SARS-CoV-2 virus replication "in vitro

SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis

Risk of mortality in patients infected with SARS-CoV-2 variant of concern 202012/1: Matched cohort study

The unfolded protein response in virus infections

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo

Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host

Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7

Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi

Multidimensional protein identification technology for direct-tissue proteomics of heart

Historical Perspective on the Discovery of the Quasispecies Concept

Cytoscape StringApp: Network Analysis and Visualization of Proteomics Data

Rapid Increase of a SARS-CoV-2 Variant with Multiple Spike Protein Mutations Observed in the

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil

First detection of SARS-CoV-2 spike protein N501 mutation in Italy in

Label-free, normalized quantification of complex mass spectrometry data for proteomic analysis

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study', Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus

Mechanisms of SARS-CoV-2 entry into cells

Improvements to the APBS Biomolecular Solvation Software Suite

Semi-supervised learning for peptide identification from shotgun proteomics datasets

Retest positive for SARS-CoV-2 RNA of "recovered" patients with COVID-19: Persistence, sampling issues, or re-infection?

Neutralising Antibodies in Spike Mediated SARS-CoV-2 Adaptation

Densely sampled viral trajectories suggest longer duration of acute infection with B. 1.1. 7 variant relative to non-B. 1.1. 7 SARS-CoV-2', medRxiv

Inference of macromolecular assemblies from crystalline state

An approach to lifting self-isolation for health care workers with prolonged shedding of SARS-CoV-2 RNA

AliView: a fast and lightweight alignment viewer and editor for large data sets

Clinical Course and Molecular Viral Shedding Among Asymptomatic and Symptomatic Patients With SARS-CoV-2 Infection in a Community Treatment Center in the Republic of Korea

Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

NF-κB signaling in inflammation

The basis of a more contagious 501Y.V1 variant of SARS-CoV-2'

SARS-CoV-2 within-Host Diversity and Transmission

The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance

BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks

Nucleic Acid-Sensing Pathways During SARS-CoV-2 Infection: Expectations versus Reality

IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era

Parallelization of MAFFT for large-scale multiple sequence alignments

Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, po tentially resulting in an escape mutant

A Role of Intracellular Toll-Like Receptors (3, 7, and 9) in Response to Mycobacterium tuberculosis and Co-Infection with HIV 1

Prolonged SARS-CoV-2 Viral Shedding in Patients with Chronic Kidney Disease

Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool

Altered Subgenomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections', bioRxiv

Clinical characteristics and risk factors associated with COVID-19 severity in patients with haematological malignancies in Italy: a retrospective, multicentre, cohort study

LY6E impairs coronavirus fusion and confers immune control of viral disease

The RING 2.0 web server for high quality residue interaction networks

Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples

SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity

TreeTime: Maximum-likelihood phylodynamic analysis

The intricate interplay between RNA viruses and NF-κB

Scientific Advisory Group for Emergencies. (2021) NERVTAG: Update Note on B.1.1.7 Severity. UK government

Opposing activities of IFITM proteins in SARS-CoV-2 infection

Distinguishing SARS-CoV-2 Bonafide Re-Infection from Pre-Existing Minor Variant Reactivation

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

The interferon landscape along the respiratory tract impacts the severity of COVID-19

SARS-CoV-2 (COVID-19) and cystic fibrosis

Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding

Biological network exploration with Cytoscape 3

SARS-CoV-2 Quasispecies Provides an Advantage Mutation Pool for the Epidemic Variants

Type II transmembrane serine proteases in development and disease

Membrane-anchored serine proteases in vertebrate cell and developmental biology

The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice

Detection of a SARS-CoV-2 variant of concern in South Africa

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

Evolution of enhanced innate immune evasion by SARS-CoV-2'

N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2

Temporal Dynamics of SARS-CoV-2 Mutation Accumulation within and across Infected Hosts

The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls

Intra-host evolution during SARS-CoV-2 prolonged infection

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

Outcomes of patients with hematologic malignancies and COVID-19: a systematic review and meta-analysis of 3377 patients

IFITs: emerging roles as key anti-viral proteins

Population Bottlenecks and Intra-Host Evolution during Human-to-Human Transmission of SARS-CoV-2

Intra-Host Variation and Evolutionary Dynamics of SARS-CoV-2 Populations in COVID-19 Patients

SWISS-MODEL: homology modelling of protein structures and complexes

CD59 association with infectious bronchitis virus particles protects against antibody-dependent complement-mediated lysis

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa

The polybasic cleavage site in SARS-CoV-2 spike modulates viral sensitivity to type I interferon and IFITM2

A new coronavirus associated with human respiratory disease in China

Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

Persistent Detection and Infectious Potential of SARS-CoV-2 Virus in Clinical Specimens from COVID-19 Patients

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Genomic data reported in this study are available at Global Initiative on Sharing All Influenza Data (GISAID). 

The authors declare no conflict of interest.