0daf477bf65a368f537044e5610855e6 pmc_11124460.pdf 0f6cd28dd91f79c037514f07821ec8f8ac6bf5e7 pmc_11124460.pdf 8d32df20be813a51c597d390b2a9ceb7ab5500f5897911aa2cfa025ecd8a6c73 pmc_11124460.pdf Title: In-Host Flat-like Quasispecies: Characterization Methods and Clinical Implications Subject: The repeated failure to treat patients chronically infected with hepatitis E (HEV) and C (HCV) viruses, despite the absence of resistance-associated substitutions (RAS), particularly in response to prolonged treatments with the mutagenic agents of HEV, suggests that quasispecies structure may play a crucial role beyond single point mutations. Quasispecies structured in a flat-like manner (referred to as flat-like) are considered to possess high average fitness, occupy a significant fraction of the functional genetic space of the virus, and exhibit a high capacity to evade specific or mutagenic treatments. In this paper, we studied HEV and HCV samples using high-depth next-generation sequencing (NGS), with indices scoring the different properties describing flat-like quasispecies. The significance of these indices was demonstrated by comparing the values obtained from these samples with those from acute infections caused by respiratory viruses (betacoronaviruses, enterovirus, respiratory syncytial viruses, and metapneumovirus). Our results revealed that flat-like quasispecies in HEV and HCV chronic infections without RAS are characterized by numerous low-frequency haplotypes with no dominant one. Surprisingly, these low-frequency haplotypes (at the nucleotide level) exhibited a high level of synonymity, resulting in much lower diversity at the phenotypic level. Currently, clinical approaches for managing flat-like quasispecies are lacking. Here, we propose methods to identifying flat-like quasispecies, which represents an essential initial step towards exploring alternative treatment protocols for viruses resistant to conventional therapies. Keywords: viral treatment failures; resistance-associated mutations (RAS); mutagens; enhanced fitness; flat-like quasispecies; near-flat; regular quasispecies Author: Josep Gregori, Sergi Colomer-Castell, Marta Ibañez-Lligoña, Damir Garcia-Cehic, Carolina Campos, Maria Buti, Mar Riveiro-Barciela, Cristina Andrés, Maria Piñana, Alejandra González-Sánchez, Francisco Rodriguez-Frias, Maria Francesca Cortese, David Tabernero, Ariadna Rando-Segura, Tomás Pumarola, Juan Ignacio Esteban, Andrés Antón and Josep Quer Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri May 17 11:17:48 2024 CEST ModDate: Fri May 24 17:07:59 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1253275 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 GFEWDD+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 PACVRA+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 TLTZJW+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 OHKNFJ+VnURWPalladioL Type 1 Custom yes yes yes 32 0 SIXRFS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 48 0 YGOXWR+CMSY10 Type 1 Builtin yes yes yes 68 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 76 0 IKXQUG+PazoMath Type 1 Builtin yes yes yes 84 0 LRMGWC+PazoMath-Italic Type 1 Builtin yes yes yes 89 0 CZKGAS+CMR10 Type 1 Builtin yes yes yes 94 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 110 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 114 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 118 0 BCDLEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 122 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 135 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 139 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 143 0 BCDLEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 147 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 163 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 167 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 171 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 175 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-06-14 02:39:39 CEST RepresentationInformation: pmc_11124460.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-06-13 02:35:34 CEST Size: 1253275 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 368 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Regular Quasispecies Samples Destination: subsection.2.1 Item: Title: HEV Flat-Like Quasispecies Samples Destination: subsection.2.2 Item: Title: HCV Near-Flat Quasispecies Samples Destination: subsection.2.3 Item: Title: Sample Preparation Destination: subsection.2.4 Item: Title: Deep Sequencing Destination: subsection.2.5 Item: Title: Data Treatment of Fastq Files to Amplicon Haplotypes Destination: subsection.2.6 Item: Title: Quasispecies Fitness Partition Destination: subsection.2.7 Item: Title: Hill Numbers Destination: subsection.2.8 Item: Title: Indices of Evenness and Flatness Destination: subsection.2.9 Item: Title: Substitution Load Destination: subsection.2.10 Item: Title: Synonymity Destination: subsection.2.11 Item: Title: Rarefaction Destination: subsection.2.12 Item: Title: Results Destination: section.3 Children: Item: Title: Sequencing Coverage Destination: subsection.3.1 Item: Title: Singletons Destination: subsection.3.2 Item: Title: Hill Numbers Profile and Hill Evenness Profile Destination: subsection.3.3 Item: Title: Indices of Evenness and Flatness Destination: subsection.3.4 Item: Title: Genetic Space Destination: subsection.3.5 Item: Title: Diversities, Evenness, and Sample Size Destination: subsection.3.6 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: In-Host Flat-like Quasispecies: Characterization Methods and Clinical Implications Author: Josep Gregori, Sergi Colomer-Castell, Marta Ibañez-Lligoña, Damir Garcia-Cehic, Carolina Campos, Maria Buti, Mar Riveiro-Barciela, Cristina Andrés, Maria Piñana, Alejandra González-Sánchez, Francisco Rodriguez-Frias, Maria Francesca Cortese, David Tabernero, Ariadna Rando-Segura, Tomás Pumarola, Juan Ignacio Esteban, Andrés Antón and Josep Quer Subject: The repeated failure to treat patients chronically infected with hepatitis E (HEV) and C (HCV) viruses, despite the absence of resistance-associated substitutions (RAS), particularly in response to prolonged treatments with the mutagenic agents of HEV, suggests that quasispecies structure may play a crucial role beyond single point mutations. Quasispecies structured in a flat-like manner (referred to as flat-like) are considered to possess high average fitness, occupy a significant fraction of the functional genetic space of the virus, and exhibit a high capacity to evade specific or mutagenic treatments. In this paper, we studied HEV and HCV samples using high-depth next-generation sequencing (NGS), with indices scoring the different properties describing flat-like quasispecies. The significance of these indices was demonstrated by comparing the values obtained from these samples with those from acute infections caused by respiratory viruses (betacoronaviruses, enterovirus, respiratory syncytial viruses, and metapneumovirus). Our results revealed that flat-like quasispecies in HEV and HCV chronic infections without RAS are characterized by numerous low-frequency haplotypes with no dominant one. Surprisingly, these low-frequency haplotypes (at the nucleotide level) exhibited a high level of synonymity, resulting in much lower diversity at the phenotypic level. Currently, clinical approaches for managing flat-like quasispecies are lacking. Here, we propose methods to identifying flat-like quasispecies, which represents an essential initial step towards exploring alternative treatment protocols for viruses resistant to conventional therapies. 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