Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19 – Nature.com

Ethics statement and patient consent

This study was approved by the Research Ethics Committees of Graduate School of Medicine, Chiba University (M10505). The instructions for patients include the following: Research purpose, research methods, expected effects and risks, not being disadvantaged even if you do not consent, being able to withdraw consent at any time after consent, strict management of personal information, research results being reported in academic journals, research organization and funding sources. Participant gave written informed consent, according to CARE guidelines and in compliance with the Declaration of Helsinki principles.

The patient received three courses of RDV medication. During each course, the patient received an initial dose of 200mg IV, followed by four daily doses of 100mg IV (5 days in total).

SARS-CoV-2 RNA was detected using a real-time RT-PCR kit (Ampdirect 2019-nCoV detection kit; Shimadzu, Kyoto, Japan).

Reverse transcription was performed using a LunaScript RT SuperMix Kit (New England Biolabs, MA, USA) as the following cycling condition: 25C, 2min; 55C, 10min; 95C, 1min. Then, a 2kb tiling PCR was performed using a standard protocol with Tks Gflex DNA polymerase (Takara Bio, Shiga, Japan) and four primer pools (Supplementary Table2, synthesised by Integrated DNA Technologies, IA, USA) as the following cycling condition: 94C, 1min; 40 cycles at 98C, 10s, 60C, 15s, and 68C, 1min. After amplification, a library was prepared using the xGen DNA Library EZ UNI Kit (Integrated DNA Technologies, IA, USA) and sequenced using an iSeq100 instrument (Illumina, CA, USA). Sequencing data were pre-processed using fastp (trimming 1 base in 5- and 3-ends of reads)27 and mapped onto the SARS-CoV-2 genome (NC_045512) using a BWA aligner28. Trimming of primer sequences, variant calling, and consensus sequence generation were performed using iVar with default settings (variants with 3% frequencies were called)29. Used commands are shown in Supplementary information. WGS (Whole Genome Sequencing) data are available in the NCBI Sequence Read Archive (SRA), submission SUB13521440 under BioProject number PRJNA983865 (BioSample accession numbers SAMN35736960SAMN35736967, https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA983865).

SARS-CoV-2 lineage was assigned by both Pangolin (https://cov-lineages.org/index.html)30 and Nextclade (https://clades.nextstrain.org/)31 web applications. In addition, the phylogenetic analysis was performed using whole genome sequence data of current strains and consensus sequence of WHO designated VOC, Alpha, Beta, Gamma, Delta, and Omicron (BA.1, BA.2, BA.4, BA.5, BA.2.75, and XBB.1.5) variants. The multiple sequence alignment was performed by MAFFT32. The phylogenetic tree was inferred by IQ-TREE with 1000 bootstrap resampling33,34. The best-fit substitution model (TIM+F+I) was selected by ModelFinder plus option35. The inferred phylogenetic tree was visualised by iTOL (https://itol.embl.de/) web application36.

TMPRSS2-expressing VeroE6 cells (JCRB1819), a SARS-CoV-2-susceptive cell line11, were obtained from the JCRB (Japanese Collection of Research Bioresources Cell Bank, Tokyo, Japan), and cultured in Dulbeccos modified Eagle medium-high glucose (DMEM; Sigma-Aldrich, London, UK) supplemented with 5% (v/v) fetal bovine serum (FBS; Cytiva, Tokyo, Japan) and an antibiotic mixture containing penicillin G (100 units/ml), streptomycin (100g/ml), and amphotericin B (0.25g/ml) (Nacalai Tesque, Kyoto, Japan). Viral isolation was achieved by inoculating a portion of a saline extract of a nasopharyngeal swab from the patient (Day 22 and thereafter) onto a VeroE6/TMPRSS2 culture in a 6-well plate. The cultures were incubated at 37C/5% CO2 and monitored by daily microscopic observation.

Conspicuous CPE (usually cell rounding) spread throughout the well of the culture plate, usually 24 d.p.i. After low-speed centrifugation (800g, 6min), the culture supernatant was harvested and stored at 70C as isolated virus stock. For further experiments, a working virus solution was prepared by inoculating a portion of the stock onto fresh VeroE6/TMPRSS2 cells cultured in a 25-cm2 flask. The cells were cultured for 23 days, and the supernatants were harvested when full-blown CPE was observed and stored in the same manner as the original stock.

To examine the viral growth properties of respective isolates, we performed the following experiments in triplicate. First, VeroE6/TMPRSS2 cells were seeded in 6-well plates (4ml/well) and allowed to nearly reach confluence within 2 days. The medium was removed, and 1ml of the new medium was added. Then, 100l of virus solution adjusted to a multiplicity of infection (MOI) of 0.01 (~4000 TCID50 of each virus / (4105 cells per well)) was inoculated, and the plates were placed in a CO2 incubator for 1h, with occasional gentle shaking. This virus inoculum solution was removed and washed once with 2ml of the new medium, and 4ml of the fresh medium was added again. These were cultured further at 37C/5% CO2. Aliquots of the culture supernatants were harvested every 24h for 5 days, and their viral titres were quantified as described below.

To investigate the extent of RDV resistance in the various isolates, inhibitor assessment experiments were performed according to the method described by Stevens et al.12. First, VeroE6/TMPRSS2 cells were seeded in 24-well plates (1ml/well) and allowed to nearly reach confluence within 2 days. The medium was removed, and 500l of the new medium containing various concentrations of RDV (GS-5734; Aobious, MA, USA), ranging from 0.125M, was added. Then, 100l of the test specimen virus solution (CH-LT1 to CH-LT3m) was added into each well at a MOI of 0.001 (~100 TCID50 of each virus / (105 cells per well)), and the plates were placed in a CO2 incubator for 1h with occasional gentle shaking. This virus inoculum solution was removed and washed once with 500l of the RDV-containing medium, and 1ml of the medium containing various concentrations of RDV was added again. These were cultured further at 37C/5% CO2. Culture supernatants were harvested 72h after virus inoculation, and viral titres were quantified. Viral infectivity (%) was calculated from the viral titres (expressed as TCID50/ml values) in the harvested culture supernatants 72h after virus inoculation in the presence of various concentrations (ranging from 0.1 to 25M) of RDV divided by the titre without RDV (100). Dose-response analysis and calculation of median effect concentration (EC50) values were performed using the drc package (version 3.0-1) from the R statistical software (version 4.2.2)37.

We did not include CH-LT4 and later isolates in this assay because their growth curves were different from those of earlier isolates. We wanted to focus on the impact of mutation(s) that occurred in nsp12. Alternately, we separately performed the comparison experiments of later isolates with the original isolate CH-LT1 in the presence or absence of 5M RDV at 3 and 4 d.p.i. We used 6-well plates, and the MOI was adjusted to 0.01. Other conditions were the same as described above. All RDV resistance experiments were performed in triplicate and statistical analysis was treated.

VeroE6/TMPRSS2 cells were seeded in 96-well plates (100l/well) in a similar manner as described in the growth kinetics experiment, and allowed to nearly reach confluence within 2 days, with 100l of 10-fold serial dilutions of virus-containing culture supernatants added into each well. The presence of live virus in each well was determined based on the CPE at 4 d.p.i., and the TCID50 values were calculated using the BehrensKrber method. The viral titres are expressed as the TCID50/ml.

Antibody responses against the S and N proteins were analyzed using Anti-SARS CoV-2 S RUO and Elecsys Anti-SARS-CoV-2 RUO (Roche Diagnostics, Switzerland), respectively, on the Cobas 8000 e801 module (Roche Diagnostics). The former system allows for the quantitative detection of antibodies, predominantly IgG, that target the viral S protein receptor-binding domain. The measurement threshold is 0.4U/ml, and values of 0.8U/ml were considered positive. The latter system allows for the quantitative detection of antibodies targeting the viral N antigen, with values of 1.0 considered positive.

The three-dimensional structure of the SARS-CoV-2 RNA-dependent RNA polymerase (SARS-CoV-2 RdRp) harbouring V792I, M794I, and C799F mutations at Domain-II was constructed with MOE, version 2022.02 (CCG Inc, Montreal, Canada), based on the Brookhaven Protein Databank 6XEZ. Docking simulations of SARS-CoV-2 RdRp of V792I, M794I, and C799F mutants with RDV-TP (PubChem CID 56832906) were performed using the Amber99 force field in MOE.

All the software(s) used in our study are freely available through the following sites except Molecular Operating Environment (MOE).

fastp (version 0.23.2, https://github.com/OpenGene/fastp).

bwa (version: 0.7.17-r1188, https://github.com/lh3/bwa).

iVar (version 1.4, https://andersen-lab.github.io/ivar/html/index.html).

MAFFT (version 7, https://mafft.cbrc.jp/alignment/software/).

IQ-TREE (version 2.2.6, http://www.iqtree.org).

iTOL (version 6.8.1, https://itol.embl.de).

R (version 4.2.2, https://www.r-project.org).

drc package (version 3.0-1, https://cran.r-project.org/web/packages/drc/index.html).

Molecular Operating Environment (MOE), version 2016.08 (CCG Inc, Montreal, Canada) is commercially available (https://www.chemcomp.com/index.htm).

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19 - Nature.com