Relevance of a Full-Length Genomic RNA Standard and a Thermal-Shock Step for Optimal Hepatitis Delta Virus Quantification

Abstract

Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to ؊80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification. Hepatitis D virus (HDV) is a small defective RNA virus that requires hepatitis B virus surface antigens (HBsAg) for viral assembly and replication. The HDV genome is a circular, negative, and single-stranded RNA composed of 1,672 to 1,697 nucleotides. HDV virions present a nucleocapsid-like structure formed by genomic HDV-RNA, which is associated with the hepatitis delta antigen (HDAg) protein and surrounded by the three envelope proteins of the hepatitis B virus (1). The replication of HDV occurs intrahepatically through a double-rolling circle mechanism that leads to an antigenomic RNA intermediate that is the exact complement sequence of the genome. The genomic and antigenomic molecules both present 70% internal base pairing, enabling self-assembly of the molecule and leading to a rod-like structure. An additional RNA form, the 800-nucleotide mRNA responsible for expression of the hepatitis delta antigen (HDAg), is also known to be produced during replication (2).HDV infection causes acute or chronic liver disease in patients with hepatitis B virus (HBV) infection. HDV infection is widespread, and the prevalence differs between areas, but it is estimated that among the 350 million individuals with chronic HBV infection, approximately 15 million are also exposed to HDV (2). Eight HDV genotypes with specific geographic patterns have been reported (3). Nonetheless, population migration has led to changes in the distributions of HDV infection and genotypes (4-6).The diagnosis of HDV infection relies on the detection of specific antibodies (anti...

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“…Another main point was the use of DNA, rather than RNA, as the standard of quantification. This point had been raised by Homs et al, who demonstrated that a full‐length genomic RNA and a strong thermal‐shock denaturation step were indicated for optimal HDVL quantification . A strong denaturation step has also been a key point for optimal results in our own experience .…”

“…This point had been raised by Homs et al, who demonstrated that a full-length genomic RNA and a strong thermal-shock denaturation step were indicated for optimal HDVL quantification. (34) A strong denaturation step has also been a key point for optimal results in our own experience. (19) It could be argued that RNA should be more appropriate, like in human immunodeficiency virus or hepatitis C virus commercial assays, to properly quantify an RNA target.…”

“…Alternatively, another region could be chosen, as long as the overall genetic variability of HDV is considered. Furthermore, an RNA, rather than a DNA plasmid, should be used as the standard of quantification, and the HDV‐WHO‐IS should be used to standardize and express the results in IU/mL . As discussed above, one issue remains unresolved, that is, whether or not international standards for the different genotypes should be provided.…”

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“…Given the wide genetic variability of the viral RNA, quantification of non-genotype 1 or African-specific genotype 1 samples has yet to be optimized (158). The number of published qRT-PCR techniques has increased over the last years, based on both two-steps and one-step protocols (159)(160)(161)(162)(163)(164). Significant technical challenges associated with HDV amplification arise from the high CG content and complementarity of viral RNA (that may limit the RT efficiency) and the high genetic variability of HDV that requires a careful design of primers (and probes) (160).…”

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“…There is no robust commercially available assay for HDV RNA detection 14 . The use of a plasmid-standard curve in PCR assays, as it was the case in the HIDIT-1 study 15 , is a recognized cause of decreased sensitivity [16][17][18] . Thus, some samples with very low serum HDV RNA levels in the HIDIT-1 study might have been considered falsely undetectable.…”