8a9304330127df776f4e46f72ab5aac3 321334.pdf d4a87b2dc78ec11625554544bf9d81bb5090d438 321334.pdf 411de62e7f8a14f93e7c16dc0a211a432b701bba29ffa69afb6c82c7f9d4636a 321334.pdf Title: Viral Quasispecies Inference from Single Observations—Mutagens as Accelerators of Quasispecies Evolution Subject: RNA virus populations exist as quasispecies-complex, dynamic clouds of closely related but genetically diverse variants generated by high mutation rates during replication. Assessing quasispecies structure and diversity is crucial for understanding viral evolution, adaptation, and response to antiviral treatments. However, comparing single quasispecies observations from individual biosamples, especially at different infection or treatment time points, presents statistical challenges. Traditional inferential tests are inapplicable due to the lack of replicate observations, and resampling-based approaches such as the bootstrap and jackknife are limited by biases and non-independence, particularly for diversity indices sensitive to rare haplotypes. In this study, we address these limitations by applying the delta method to derive analytical variances for a set of quasispecies structure indicators specifically designed to assess the quasispecies maturation state. We demonstrate the utility of this approach using high-depth next-generation sequencing data from hepatitis C virus (HCV) quasispecies evolving in vitro under various conditions, including free evolution and exposure to antiviral or mutagenic treatments. Our results reveal that with highly fit HCV quasispecies, sofosbuvir inhibits quasispecies genetic diversity, while mutagenic treatments accelerate maturation, compared to untreated controls. We emphasize the interpretation of results through absolute differences, log-fold changes, and standardized effect sizes, moving beyond mere statistical significance. This framework enables robust, quantitative comparisons of quasispecies diversity from single observations, providing valuable insights into viral adaptation and treatment response. The R code and session info with required libraries and versions is provided in the supplementary material. Keywords: inference; quasispecies; single-observation; RNA-virus; mutagens; inhibitors Author: Josep Gregori, Miquel Salicrú, Marta Ibáñez-Lligoña, Sergi Colomer-Castell, Carolina Campos, Alvaro González-Camuesco, and Josep Quer Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Mon Sep 1 06:10:08 2025 CEST ModDate: Mon Sep 1 07:30:59 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 318940 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GPOKSX+URWPalladioL-Ital Type 1 Custom yes yes yes 117 0 XZTLTV+URWPalladioL-Bold Type 1 Custom yes yes yes 118 0 GYOOWY+URWPalladioL-Roma Type 1 Custom yes yes yes 119 0 SOSTRQ+CMR10 Type 1 Builtin yes yes yes 152 0 SYFPBV+CMMI10 Type 1 Builtin yes yes yes 153 0 HHBRNZ+CMSY10 Type 1 Builtin yes yes yes 154 0 VCVGZN+PazoMath-Italic Type 1 Builtin yes yes yes 155 0 NMTTXM+PazoMath Type 1 Builtin yes yes yes 195 0 XRJIOU+CMEX10 Type 1 Builtin yes yes yes 214 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 271 0 Helvetica Type 1 Custom no no no 293 0 Helvetica Type 1 Custom no no no 310 0 Helvetica Type 1 Custom no no no 329 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-02-14 04:22:30 CET RepresentationInformation: 321334.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-02-13 14:19:11 CET Size: 318940 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 459 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Quasispecies Maturity Indicators Item: Title: Delta Method Item: Title: Indicators' Variance and Standard Error by the Delta Method Item: Title: Large Sample Sizes and p-Values Item: Title: Effect Size Item: Title: Z, t, and Cohen's d Item: Title: Quasispeces Samples, Cells, Viruses, and Drugs Item: Title: Implementation Item: Title: Results Children: Item: Title: Maturity Indicator Values on Rarefied Quasispecies Item: Title: Tests Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Viral Quasispecies Inference from Single Observations—Mutagens as Accelerators of Quasispecies Evolution Author: Josep Gregori, Miquel Salicrú, Marta Ibáñez-Lligoña, Sergi Colomer-Castell, Carolina Campos, Alvaro González-Camuesco, and Josep Quer Subject: RNA virus populations exist as quasispecies-complex, dynamic clouds of closely related but genetically diverse variants generated by high mutation rates during replication. Assessing quasispecies structure and diversity is crucial for understanding viral evolution, adaptation, and response to antiviral treatments. However, comparing single quasispecies observations from individual biosamples, especially at different infection or treatment time points, presents statistical challenges. Traditional inferential tests are inapplicable due to the lack of replicate observations, and resampling-based approaches such as the bootstrap and jackknife are limited by biases and non-independence, particularly for diversity indices sensitive to rare haplotypes. In this study, we address these limitations by applying the delta method to derive analytical variances for a set of quasispecies structure indicators specifically designed to assess the quasispecies maturation state. We demonstrate the utility of this approach using high-depth next-generation sequencing data from hepatitis C virus (HCV) quasispecies evolving in vitro under various conditions, including free evolution and exposure to antiviral or mutagenic treatments. Our results reveal that with highly fit HCV quasispecies, sofosbuvir inhibits quasispecies genetic diversity, while mutagenic treatments accelerate maturation, compared to untreated controls. We emphasize the interpretation of results through absolute differences, log-fold changes, and standardized effect sizes, moving beyond mere statistical significance. This framework enables robust, quantitative comparisons of quasispecies diversity from single observations, providing valuable insights into viral adaptation and treatment response. The R code and session info with required libraries and versions is provided in the supplementary material. 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