5a966859cd93cdbdea7d4441e8ab89c5 321350.pdf b05253d61a33d59b59a5edebc32fd58e21c4419c 321350.pdf ba83ee649a118966161258a6f98b4f2b752dfbddb063e4d13f77aa0de050b535 321350.pdf Title: In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments Subject: Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible. Keywords: quasispecies; deep sequencing; variability; rare haplotypes; fitness; mutagens Author: Sergi Colomer-Castell, Josep Gregori, Damir Garcia-Cehic, Mar Riveiro-Barciela, Maria Buti, Ariadna Rando-Segura, Judit Vico-Romero, Carolina Campos, Marta Ibañez-Lligoña, Caroline Melanie Adombi, Maria Francesca Cortese, David Tabernero, Juan Ignacio Esteban, Francisco Rodriguez-Frias and Josep Quer Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Dec 6 10:13:14 2023 CET ModDate: Wed Dec 6 10:22:34 2023 CET 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: 1592307 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IGDFIY+VnURWPalladioL Type 1 Custom yes yes no 10 0 BEBSEE+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 GAWQFQ+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 HUTKMW+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 42 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 45 0 LZKCTS+CMSY10 Type 1 Builtin yes yes no 67 0 CKBPVA+EURM10 Type 1 Builtin yes yes no 71 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 87 0 GJIGAI+PalatinoLinotype CID TrueType Identity-H yes yes yes 90 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 96 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 99 0 GJIGMH+PalatinoLinotype TrueType MacRoman yes yes no 102 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 116 0 GJIGAI+PalatinoLinotype CID TrueType Identity-H yes yes yes 119 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 125 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 128 0 GJIGMH+PalatinoLinotype TrueType MacRoman yes yes no 131 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 145 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 148 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 151 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 163 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 169 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 184 0 GJIGAI+PalatinoLinotype CID TrueType Identity-H yes yes yes 187 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 193 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 196 0 GJIGMH+PalatinoLinotype TrueType MacRoman yes yes no 199 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 213 0 GJIGAI+PalatinoLinotype CID TrueType Identity-H yes yes yes 216 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 222 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 225 0 GJIGMH+PalatinoLinotype TrueType MacRoman yes yes no 228 0 GJILML+Symbol Type 1C Custom yes yes yes 248 0 GJILMM+Arial CID TrueType Identity-H yes yes yes 253 0 GJIGAH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 259 0 GJIGAI+PalatinoLinotype CID TrueType Identity-H yes yes yes 262 0 GJIGAJ+PalatinoLinotype TrueType WinAnsi yes yes no 268 0 GJIGAL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 271 0 GJIGMH+PalatinoLinotype TrueType MacRoman yes yes no 274 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-01-31 04:20:11 CET RepresentationInformation: 321350.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-01-30 13:36:48 CET Size: 1592307 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 492 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Patient Data Destination: subsection.4.1 Item: Title: RNA Extraction and Amplification Destination: subsection.4.2 Item: Title: Library Preparation and Illumina Sequencing Destination: subsection.4.3 Item: Title: Processing the Sequencing Data Destination: subsection.4.4 Item: Title: Bioinformatic Procedures Destination: subsection.4.5 Item: Title: Software and Statistics Destination: subsection.4.6 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments Author: Sergi Colomer-Castell, Josep Gregori, Damir Garcia-Cehic, Mar Riveiro-Barciela, Maria Buti, Ariadna Rando-Segura, Judit Vico-Romero, Carolina Campos, Marta Ibañez-Lligoña, Caroline Melanie Adombi, Maria Francesca Cortese, David Tabernero, Juan Ignacio Esteban, Francisco Rodriguez-Frias and Josep Quer Subject: Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible. Keywords: quasispecies; deep sequencing; variability; rare haplotypes; fitness; mutagens Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Dec 06 10:13:14 CET 2023 ModDate: Wed Dec 06 10:22:34 CET 2023 ID: 0x666485ba385e8ab2c25850ce94c17d71, 0x666485ba385e8ab2c25850ce94c17d71 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LZKCTS+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: LZKCTS+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: GAWQFQ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: GAWQFQ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CKBPVA+EURM10 FontSubset: true FirstChar: 11 LastChar: 11 FontDescriptor: FontName: CKBPVA+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: HUTKMW+URWPalladioL-Ital FontSubset: true 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