In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments
Abstract
:1. Introduction
2. Results
Traits in Quasispecies Evolution
3. Discussion
4. Materials and Methods
4.1. Patient Data
4.2. RNA Extraction and Amplification
4.3. Library Preparation and Illumina Sequencing
4.4. Processing the Sequencing Data
- Obtain Fastq files with Illumina 2 × 300-bp paired-end reads;
- Recover full amplicon reads with FLASH [46] (minimum 20-bp overlap, maximum 10% mismatches). The 300-bp reads, when overlapped, result in reads covering complete ~400–500 bp amplicons;
- Remove full reads with 5% or more bases below a Phred score of Q30;
- Demultiplex and trim primers (max three differences accepted);
- Collapse reads (molecules) to haplotypes (amplicon-genomes) and their frequencies (read counts);
- Multiple alignment of all haplotypes in each sample/amplicon;
- Remove all haplotypes that are not common to both DNA strands and supported at least by 5 reads;
- Remove insertions in master haplotypes and repair single gaps in remaining haplotypes. Recollapse to haplotypes and vectors of frequencies;
- The haplotypes in each sample/amplicon are translated to phenotypes and recollapsed to obtain the set of phenotypes with corresponding frequencies (read counts).
4.5. Bioinformatic Procedures
4.6. Software and Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Intervention * | ID a | Date | Days b | Weeks c | CumDays d | Viral Load |
---|---|---|---|---|---|---|
Infection | October 2012 | |||||
1st evidence | S01 | 13 November 2012 | 0 | 0.0 | 0 | 5.04 × 106 |
S02 | 15 December 2012 | 34 | 4.9 | 34 | 6.84 × 106 | |
RBV 200 mg | 3 February 2014 | 413 | 59.0 | 447 | ||
S03 | 4 February 2014 | 1 | 0.1 | 448 | 4.00 × 106 | |
S04 | 6 February 2014 | 2 | 0.3 | 450 | 2.78 × 106 | |
S05 | 12 February 2014 | 6 | 0.9 | 456 | 1.17 × 106 | |
EOT | 3 May 2014 | 80 | 11.4 | 536 | RNA− | |
Relapse | 1 January 2015 | 243 | 34.7 | 779 | RNA+ | |
S06 | 5 November 2015 | 308 | 44.0 | 1087 | 3.56 × 106 | |
RBV 200/400 mg | S07 | 19 January 2016 | 75 | 10.7 | 1162 | 1.30 × 107 |
EOT | 5 July 2016 | 168 | 19.7 | 1300 | RNA+ | |
S08 | 2 August 2016 | 28 | 8.3 | 1358 | 2.00 × 104 | |
S09 | 27 September 2016 | 56 | 8.0 | 1414 | 6.93 × 105 | |
RBV 400 mg | 9 April 2018 | 559 | 79.9 | 1973 | 1.27 × 107 | |
S13 | 10 July 2018 | 92 | 13.1 | 2065 | 1.51 × 105 | |
S15 | 10 August 2018 | 31 | 4.4 | 2096 | 8.22 × 104 | |
S18 | 18 September 2018 | 39 | 5.6 | 2135 | 7.50 × 104 | |
EOT | 27 November 2018 | 70 | 10.0 | 2205 | 8.20 × 104 | |
2 March 2019 | 95 | 13.6 | 2300 | 7.00 × 106 |
WT | Var | N | Fitch | Grantham | qGrantham |
---|---|---|---|---|---|
T | N | 27 | 1 | 65 | 0.2579 |
T | A | 20 | 1 | 58 | 0.2105 |
G | S | 3 | 1 | 56 | 0.2000 |
V | I | 3 | 1 | 29 | 0.0789 |
A | T | 2 | 1 | 58 | 0.2105 |
E | K | 2 | 1 | 56 | 0.2000 |
I | M | 2 | 1 | 10 | 0.0105 |
L | F | 2 | 1 | 22 | 0.0368 |
A | V | 1 | 1 | 64 | 0.2421 |
F | Y | 1 | 1 | 22 | 0.0368 |
I | V | 1 | 1 | 29 | 0.0789 |
S | A | 1 | 1 | 99 | 0.5368 |
S | P | 1 | 1 | 74 | 0.2947 |
V | A | 1 | 1 | 64 | 0.2421 |
Y | H | 1 | 1 | 83 | 0.3421 |
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Colomer-Castell, S.; Gregori, J.; Garcia-Cehic, D.; Riveiro-Barciela, M.; Buti, M.; Rando-Segura, A.; Vico-Romero, J.; Campos, C.; Ibañez-Lligoña, M.; Adombi, C.M.; et al. In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments. Int. J. Mol. Sci. 2023, 24, 17185. https://doi.org/10.3390/ijms242417185
Colomer-Castell S, Gregori J, Garcia-Cehic D, Riveiro-Barciela M, Buti M, Rando-Segura A, Vico-Romero J, Campos C, Ibañez-Lligoña M, Adombi CM, et al. In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments. International Journal of Molecular Sciences. 2023; 24(24):17185. https://doi.org/10.3390/ijms242417185
Chicago/Turabian StyleColomer-Castell, Sergi, Josep Gregori, Damir Garcia-Cehic, Mar Riveiro-Barciela, Maria Buti, Ariadna Rando-Segura, Judit Vico-Romero, Carolina Campos, Marta Ibañez-Lligoña, Caroline Melanie Adombi, and et al. 2023. "In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments" International Journal of Molecular Sciences 24, no. 24: 17185. https://doi.org/10.3390/ijms242417185