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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Published on: June 16, 2011
Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability
Beatriz Pacin-Ruiz1,2, María Francesca Cortese1,2, David Tabernero1,2
1Liver Pathology Unit, Departments of Biochemistry and Microbiology, Vall d'Hebron University Hospital, 08035 Barcelona, Spain.
Abstract:
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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