f4a487823379dbc62ba9ccae395b2052 pmc_35215809.pdf 0ddb5670897a4ebf88e4dd147cf537f944a6e39c pmc_35215809.pdf 21d92777b592df28acb9fe71bc4221cae3c9868ba9aef60982f5830596d8d77e pmc_35215809.pdf Title: Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability Subject: The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688–771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715–745. No difference in QS was observed over time. The most variable region was between nt 739–769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence. Keywords: hepatitis delta virus; ribozyme; next-generation sequencing; quasispecies; conservation; variability; viral fitness; persistence; target; gene silencing Author: Beatriz Pacin-Ruiz, María Francesca Cortese, David Tabernero, Sara Sopena, Josep Gregori, Selene García-García, Rosario Casillas, Adrián Najarro, Unai Aldama, Adriana Palom, Ariadna Rando-Segura, Anna Galán, Marta Vila, Mar Riveiro-Barciela, Josep Quer, Gloria González-Aseguinolaza, María Buti and Francisco Rodríguez-Frías Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Jan 22 12:58:14 2022 CET ModDate: Sat Jan 22 13:03:27 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1523368 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CIQLLF+VnURWPalladioL Type 1 Custom 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1.12.1 (2019-04-17) LastModified: 2022-03-06 18:36:43 CET Size: 1523368 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 451 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Patients and Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Serologic and Molecular Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Next-Generation Sequencing of the Ribozyme Region Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Quasispecies Analysis and Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: In Vitro Test of Mutations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Patients and Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Ribozyme Conservation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Ribozyme Quasispecies (QS) Evolution and Variability during Follow-Up Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Analysis of Mutations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: In Vitro Test of Mutations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Info: Title: Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability Author: Beatriz Pacin-Ruiz, María Francesca Cortese, David Tabernero, Sara Sopena, Josep Gregori, Selene García-García, Rosario Casillas, Adrián Najarro, Unai Aldama, Adriana Palom, Ariadna Rando-Segura, Anna Galán, Marta Vila, Mar Riveiro-Barciela, Josep Quer, Gloria González-Aseguinolaza, María Buti and Francisco Rodríguez-Frías Subject: The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688 771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715 745. No difference in QS was observed over time. The most variable region was between nt 739 769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence. 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