Sensitive reverse transcription-digital droplet PCR-based discrimination of hepatitis delta virus RNAs reveals differential effects of interferon-α on viral RNA species

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.