Sensitive reverse transcription-digital droplet PCR-based discrimination of hepatitis delta virus RNAs reveals differential effects of interferon-α on viral RNA species
Abstract
Hepatitis delta virus (HDV) replication relies on the production of distinct viral RNA species, including genomic (HDV-G), antigenomic (HDV-AG) and mRNAs (HDV mRNAs), which differ in abundance and function. However, the lack of sensitive and quantitative approaches allowing their simultaneous discrimination has limited the precise analysis of HDV RNA dynamics, particularly under antiviral conditions. Here, we combined nascent RNA and RNA stability assays with a novel reverse transcription digital droplet PCR (RT-ddPCR) approach enabling strand-specific quantification of HDV RNAs. Using this methodology, we confirmed the previously described kinetics of viral RNA production and assessed the ratio of each RNA species in HDV-infected cells. Specific small interfering RNA (siRNA) treatment demonstrated the specificity of our assay in discriminating HDV RNA species. Next, applying this technique to the study of virus-host interactions, we showed that IFN-α treatment decreases the synthesis of HDV-G and may additionally accelerate HDV-AG and HDV-G decay, while HDV mRNA stability remained unaffected in infected HuH7.5-Na+-taurocholate cotransporting polypeptide (NTCP) cells. Immunoprecipitation assays coupled to strand-specific RT-ddPCR revealed no difference in the association of cellular RNA polymerase II (Pol II) with HDV-G and HDV-AG. In parallel, transcriptomic analyses of differentiated HepaRG and HuH7.5-NTCP cells following IFN-α treatment identified IFN-stimulated gene candidates with potential anti-HDV activity. Among them, we showed that HELZ2 overexpression does not affect HDV RNA stability but interferes with HDV-AG and HDV-G RNA synthesis. Altogether, this RT-ddPCR-based approach provides a versatile tool to investigate the effects of current and emerging antiviral strategies on individual HDV RNA species.
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BibTeX
@article{pons2026sensitive,
title = {{Sensitive reverse transcription-digital droplet PCR-based discrimination of hepatitis delta virus RNAs reveals differential effects of interferon-α on viral RNA species}},
author = {Caroline Pons and Emilie Charles and Cassandre Authier and Elise Chabot and Roxanne Fouillé and Faustine Bernardin and Claudie Eber and Léa Corbet and Maud Michelet and Marine Oudot and Charlotte Bach and Michel Rivoire and Guillaume Passot and Thomas F Baumert and Karim Majzoub and Eric Huntzinger and Bertrand Seraphin and David Durantel and Eloi R Verrier and Julie Lucifora},
journal = {Journal of General Virology},
year = {2026},
month = oct,
doi = {10.1099/jgv.0.002324},
url = {https://hal.science/hal-05820755v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Sensitive reverse transcription-digital droplet PCR-based discrimination of hepatitis delta virus RNAs reveals differential effects of interferon-α on viral RNA species
AU - Caroline Pons
AU - Emilie Charles
AU - Cassandre Authier
AU - Elise Chabot
AU - Roxanne Fouillé
AU - Faustine Bernardin
AU - Claudie Eber
AU - Léa Corbet
AU - Maud Michelet
AU - Marine Oudot
AU - Charlotte Bach
AU - Michel Rivoire
AU - Guillaume Passot
AU - Thomas F Baumert
AU - Karim Majzoub
AU - Eric Huntzinger
AU - Bertrand Seraphin
AU - David Durantel
AU - Eloi R Verrier
AU - Julie Lucifora
PY - 2026
DA - 2026/10/07
JO - Journal of General Virology
DO - 10.1099/jgv.0.002324
UR - https://hal.science/hal-05820755v1
AB - Hepatitis delta virus (HDV) replication relies on the production of distinct viral RNA species, including genomic (HDV-G), antigenomic (HDV-AG) and mRNAs (HDV mRNAs), which differ in abundance and function. However, the lack of sensitive and quantitative approaches allowing their simultaneous discrimination has limited the precise analysis of HDV RNA dynamics, particularly under antiviral conditions. Here, we combined nascent RNA and RNA stability assays with a novel reverse transcription digital droplet PCR (RT-ddPCR) approach enabling strand-specific quantification of HDV RNAs. Using this methodology, we confirmed the previously described kinetics of viral RNA production and assessed the ratio of each RNA species in HDV-infected cells. Specific small interfering RNA (siRNA) treatment demonstrated the specificity of our assay in discriminating HDV RNA species. Next, applying this technique to the study of virus-host interactions, we showed that IFN-α treatment decreases the synthesis of HDV-G and may additionally accelerate HDV-AG and HDV-G decay, while HDV mRNA stability remained unaffected in infected HuH7.5-Na+-taurocholate cotransporting polypeptide (NTCP) cells. Immunoprecipitation assays coupled to strand-specific RT-ddPCR revealed no difference in the association of cellular RNA polymerase II (Pol II) with HDV-G and HDV-AG. In parallel, transcriptomic analyses of differentiated HepaRG and HuH7.5-NTCP cells following IFN-α treatment identified IFN-stimulated gene candidates with potential anti-HDV activity. Among them, we showed that HELZ2 overexpression does not affect HDV RNA stability but interferes with HDV-AG and HDV-G RNA synthesis. Altogether, this RT-ddPCR-based approach provides a versatile tool to investigate the effects of current and emerging antiviral strategies on individual HDV RNA species.
SN - 0022-1317
SN - 1465-2099
ER -