Supplementary methods
Sample collection
Naso-pharyngeal swab (NPS) were collected using 3mL universal transport medium tubes. Positive SARS-CoV-2 specimen were frozen at -80 upon result reception. Serum samples were collected in SST II plastic tubes for antibody assays and neutralizing antibodies assessment. Cell-preparation tubes with sodium citrate were used for collection of whole blood and separation of peripheral blood mononuclear cells (PBMC) to assess B cell responses on day 12 post onset of symptoms during reinfection.
Antibody assays
Qualitative detection of total immunoglobulin against the nucleocapsid (N) by
electrochemiluminescence assay (ELISA) (Elecsys® Anti-SARS-CoV-2 anti-N, Roche, Switzerland) was performed according to manufacturer’s instructions on serum samples, as well as quantitative assessment of total immunoglobulin against the receptor binding domain of the spike (S) protein (Elecsys® Anti-SARS-CoV-2 anti-S, Roche, Switzerland). Results are expressed in U/ml, and cut-off for positivity is 1 for the N and 0.8 for the RBD assay.
S1 domain-specific IgG antibody responses were measured using a commercially available ELISA (Euroimmun AG, Lubeck, Germany; EI 2606-9601) according to manfacturer’s instructions.
Plaque reduction Neutralisation Test (PRNT)
Vero E6 cells were seeded at a density of 4x10^5 cells/ml in 24-well cell culture plates 1 day before and incubated over night at 37°C, 5% CO2. All sera were inactivated at 56°C for 30min and 2-fold dilution series in Opti-Pro serum free medium (volume: 220ul for each dilution) were prepared starting with a 1:5 (final dilution 1:10 after addition of virus) dilution. Recombinant SARS-CoV-2 strain Wuhan-Hu-1 (kindly provided by V. Thiel, University of Bern) was diluted to a concentration of 500
PFU/ml in Opti-Pro and 220ul of virus was added to the serum dilutions. Virus serum mixtures were incubated at 37°C for 1h. Virus without serum was used as an infection control and medium without virus was used as a negative control. All samples were run in duplicate and for each neutralization experiment, a reference serum was used to insure reproducibility between different experiments.
Medium was removed and Vero E6 cells were washed 1x with PBS before inoculation with virus serum mixture (200ul/well) for 1h at 37°C, 5% CO2. Afterwards, the inoculum was removed and 500ul of a high viscosity overlay medium (DMEM + 1% Glutamax + 1% Penicillin Streptamycin +10% FBS +1.2% Avicel) was applied to each well. After incubation for 40h at 37°C, 5% CO2, the overlay medium was removed, cells were fixed in 6% formaldehyde solution for 1h, plates were washed 1x with PBS and finally stained with crystal violet for 20min. Plaques were counted in wells inoculated with virus- serum mixtures and compared to plaque counts in infection control wells (virus without serum).
Serum dilutions that reach at least 50% or 90% reduction of viral plaques were recorded as PRNT50 and PRNT90 titers for each serum. All sera were assessed twice in independent experiments and values are displayed as the geometric mean of endpoint titers.
Blood cell phenotyping and B cell responses assessment:
ELISpot was performed as previously described [1] using as antigens SARS-CoV-2 S1 protein (Sino biological, #40591-V08H,) and N-protein (PROSPEC, Cat sars-040-c).
RT-PCR testing for SARS-CoV-2 and other respiratory viruses and viral load measurement
NPS were tested for SARS-CoV-2 by real-time RT-PCR using the Cobas 6800 SARS CoV2 RT-PCR asssay (Roche, Switzerland) which includes the ORF1/a and E-gene targets.
Viral load was estimated from the cycle threshold values (Ct values) retrieved for the E-gene, using a standard curve obtained by quantified supernatant from a cell culture isolate of SARS-CoV-2 as previously described [2].
Nasopharyngeal swabs were screened by acid nucleic detection for the presence of influenza A and B virus, respiratory syncytial virus (RSV) A and B, parainfluenza 1 to 4, human metapneumovirus, rhinoviruses, enteroviruses, bocavirus 1, adenovirus and human coronaviruses 229E, OC43, HKU1 and NL63 using an in-house RT-PCR panel as previously described [3].
Whole-genome sequencing and phylogenetic analysis:
Nucleic acids were extracted from each NPS individually using the NucliSENS easyMAG (BioMérieux, Switzerland) protocol. Whole-genome sequencing was done using an updated version of the nCoV- 2019 sequencing protocol (https://www.protocols.io/view/ncov-2019-sequencing-protocol- bbmuik6w) (Microsynth, Switzerland) on a MiSeq instrument (Illumina) using a 2x250-bp protocol.
After removal of duplicate sequences (cd-hit v4.6.8) and trimming of low-quality and adapter sequences (Trimmomatic v0.33), reads were mapped (snap-aligner v1.0beta.18) to SARS-CoV-2 reference genome "NC_045512". Consensus sequences were then obtained for regions covered ≥ 10- fold. Sequence alignment was performed with MUSCLE (v3.8.31). The sequences obtained for the first and the second episodes were submitted to GISAID database (GISAID accession ID:
EPI_ISL_708381 and EPI_ISL_708380, respectively). The raw sequence data were deposited in the NCBI Sequence Read Archive under the BioProject accession number PRJNA697927.
The Evolutionary analyses were conducted in MEGA X [4] using the Maximum Likelihood method and Tamura 3-parameter model [5]. The tree includes also SARS-CoV-2 complete genomes sequenced by our laboratory and submitted to GISAID from COVID-19 positive patients presenting to our institute or others medical center in Geneva, Switzerland, during the 1st and 2nd episodes.
S gene Sanger sequencing
To fill the residual gaps in the S gene after whole-genome sequencing, SARS-CoV-2 was lysed and viral RNA extracted from patient samples using the automated NucliSENS easyMAG nucleic acid kit according to the manufacturer’s instructions. The cDNA synthesis reaction for PCR amplification was
performed using SuperScript III Platinum one step cDNA synthesis kit (Invitrogen, USA), in accordance to the manufacturer’s instructions with primers flanking the S gene gaps (Supplementary Table 2).
Amplification conditions of the chosen primers included 30 min at 48°C and 5 min at 95°C, followed by 35 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min with a final extension step at 72°C for 5 min. If needed, PCR products were then used for the nested PCR amplification using the according primers combination (Supplementary Table 2). Cycling conditions were 3 min at 95°C, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min with a final extension step at 72°C for 5 min. Outcomes from PCR fragments were purified using the preparative gel electrophoresis GeneJET PCR Purification Kit (Thermo Fisher Scientific Ltd., United Kingdom) following manufacturer’s instructions and sequenced by the Sanger sequencing method (Microsynth, Switzerland). Obtained sequences were edited using Geneious Prime software (2021.0.3). [6]
Supplementary Figure 1: Phylogenetic tree constructed on the basis of SARS-CoV-2 genome
sequences. Sequences corresponding to the first and the subsequent infection are shown in bold. The tree is rooted using the "NC 045512.2" sequence, and includes SARS-CoV-2 complete genome
sequences (all submitted to GISAID) obtained from samples collected in Geneva, Switzerland during each of the corresponding episodes.
Supplementary Figure 2: S gene sequences obtained by Sanger sequencing during the first and
second infection. The S477N (highlighted in blue) and results from a single nucleotide change (underlined).
S gene sequence (5’-3’): 1st SARS-CoV-2 episode
ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACAC TAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCT TTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCAT TTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGA CCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGT GTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATAT GTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGAT GGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGG TAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCT TCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGA ACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGG AATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTG AAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCC TATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATG CAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAAT TACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTAT AGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAAT GGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAG TAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAAT GTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTG GCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTG GTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCT GTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTT TAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGA CTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTG CTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACA TCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAAT TAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAA AACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTAT TGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGC TAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACAC TTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAAT GGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGC TATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAA GCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACA AAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATT AGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGA TTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCT GCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTC AAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTA ACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAG ATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAA AGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGT
ATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTAT GACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTG CTCAAAGGAGTCAAATTACATTACACATAA
S gene sequence (5’-3’): 2nd SARS-CoV-2 episode
ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACAC TAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCT TTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCAT TTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGA CCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGT GTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATAT GTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGAT GGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGG TAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCT TCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGA ACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGG AATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTG AAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCC TATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATG CAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAAT TACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTAT AGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGAtATTTCAACTGAAATCTATCAGGCCGGTAACACACCTTGTAAT GGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAG TAGTAgTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATG TGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGG CAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGG TGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGT TGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTA ATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACT AATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCT TACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATC AGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTA AACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAA CACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTG AAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTA GAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTT CTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGG CTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTAT TGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCT TTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAG TTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGA GCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTT TTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCA CAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAA TGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTG TGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAA ATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAA ATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATA AAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCA GTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAA GGAGTCAAATTACATTACACATAA
Supplementary Figure 3: Representative ELISpot illustrating the number of anti-S1 (left) and anti-N
(middle) memory B cells after in vitro stimulation, where each counted spot correspond to one antigen-specific cell secreting SARS-CoV-2 specific IgG antibodies. The right well shows the number of all IgG-secreting cells. The numbers indicate the number of counted spots, corresponding to the number of cells, per total plated cells.
Supplementary Table 1:
Nucleotide and amino acid changes observed between the 1st and 2nd infection. The differences from the reference sequence NC_045512 are shown in bold. N.A. : not available.
1st infection 2nd infection Reference: NC_045512
nucleotide positions nucleotide amino acid nucleotide amino acid nucleotide amino acid regions
241 N.A. T C non coding
region
2358 C A T V C A ORF1ab
2939 C P T S C P ORF1ab
3037 T T T T C S ORF1ab
4543 C T T T C T ORF1ab
5239 T Y C Y C Y ORF1ab
5512 C N T N C N ORF1ab
9526 G M T I G M ORF1ab
14408 T L T L C P ORF1ab
11497 C Y T Y C Y ORF1ab
12088 A L G L A L ORF1ab
13669 C L T F C L ORF1ab
13993 G A T S G A ORF1ab
15324 T N C N C N ORF1ab
15766 G V T L G V ORF1ab
16889 A K G R A K ORF1ab
17019 G E T D G E ORF1ab
17058 G M T I G M ORF1ab
18877 C L T L C L ORF1ab
22992 G S A N G S S
23403 G G G G A D S
25563 G Q T H G Q ORF3a
25710 C L T L C L ORF3a
26735 C Y T Y C Y M
26876 T I C I T I M
28677 C T T I C T N
28975 G M C I G M N
29179 G P T P G P N
29399 G A A T G A N
Supplementary Table 2
Primer sets used for PCR, nested PCR and sequencing.
PCR Primer
Sequence (5' - 3')
assay name product length (bp)
1st infection [EPI_ISL_708381] Simple PCR F46 CCTTCACTGTAGAAAAAGGAATC R47 CATATGAGTTGTTGACATGTTCAG
2nd infection [EPI_ISL_708380]
Simple PCR F45 TTGTAATGATCCATTTTTGGGTGT R47 CATATGAGTTGTTGACATGTTCAG Nested PCR F45 TTGTAATGATCCATTTTTGGGTGT
R45 CTAACAATAGATTCTGTTGGTTG Nested PCR F46 CCTTCACTGTAGAAAAAGGAATC
R47 CATATGAGTTGTTGACATGTTCAG
References
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3. Vetter, P., et al., Daily Viral Kinetics and Innate and Adaptive Immune Response Assessment in COVID-19: a Case Series. mSphere, 2020. 5(6).
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5. Tamura, K., Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol, 1992. 9(4): p. 678-87.
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