Quantitative detection and typing of hepatitis D virus in human serum by real-time polymerase chain reaction and melting curve analysis

Hofmann, Jörg; Frenzel, Katrin; Minh, Bùi Quang; Haeseler, Arndt von; Edelmann, Anke; Roß, Stefan; Berg, Thomas; Krüger, Detlev H.; Meisel, Helga

doi:10.1016/j.diagmicrobio.2010.02.003

Cited by 22 publications

(17 citation statements)

References 34 publications

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“…Several in-house methods based on quantitative reverse transcription-PCR (qRT-PCR) and targeting the HDAg or the ribozyme domain have been described and have proven effective for quantifying all HDV genotypes. These techniques involve the use of a one-step qRT-PCR procedure (8)(9)(10)(11)(12)(13), the incorporation of heat shock treatment before the RT step (10,(14)(15)(16), or the addition of heterologous internal-control RNAs (11,12). However, all these methods rely on the use of a specific in-house standard; therefore, a comparison between studies is impossible.…”

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confidence: 99%

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

et al. 2014

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

et al. 2014

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“…PCRs to detect WHV DNA and HDV RNA in woodchuck sera were performed as described earlier (7,15,18). Sera of woodchucks which tested positive for HDV RNA in the nested PCR were quantified on the LightCycler 2.0 instrument (Roche, Basel, Switzerland) using a recently described protocol (19). Markers of infection and immune response were monitored weekly after challenge.…”

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confidence: 99%

Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Fiedler

Kosinska

Schumann

et al. 2013

J Virol

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Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8 ؉ and CD4 ؉ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven. H epatitis delta virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes the most severe hepatitis in humans.Nearly all patients develop chronic HDV infection that has a high probability of progressing to liver cirrhosis and hepatocellular carcinoma (1, 2). Worldwide, approximately 15 million patients are affected with HDV. About 8% of HBV surface antigen (HBsAg)-positive patients in several European countries have tested positive for antibodies against HDV (2). Therapeutic options for HBV/HDV carriers are limited. Only in about 25% of the patients does alpha interferon therapy result in sustained viral clearance (3).HBV carriers are at risk of being superinfected with HDV. Therefore, a vaccine protecting HBV carriers from HDV superinfection would be eligible. A main obstacle for the design of a vaccine against HDV infection is the fact that antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HDV particle. The HDV protein/RNA complex is covered by the envelope protein of HBV (HBsAg). Therefore, classical vaccines which induce neutralizing antibodies cannot be expected to prevent HDV infection.Immunizations with nucleoproteins of, e.g., influenza A virus-, HBV-, or woodchuck hepatitis virus (WHV) induced virus-specific T cells and were able to suppress replication, e.g., by cytokine secretion. In a second step, these virus-specific T cells are able to eliminate infected cells by their cytolytic activity and thus prevent the spread of the virus (4-7). T cell vaccines may not provide sterile immunity, because they do not induce neutralizing antibodies. However, T cell vaccines may stop infection via the cellular immune response at a very early phase of infection. Most conventional vaccines for humans induce sufficient amounts of neutralizing antibodies which prevent infection. ...

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“…Currently, a few commercial tests are available for HDV RNA quantification, but they perform poorly, at least for non-genotype 1 HDV samples (13). Numerous in-house assays have been developed with very different protocols (11,(14)(15)(16)(17)(18)(19)(20). To our knowledge, these assays have not been evaluated on a large panel of clinical samples of various genotypes and viral loads (VL).…”

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Serological and Molecular Diagnosis of Hepatitis Delta Virus Infection: Results of a French National Quality Control Study

et al. 2014

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A French national quality control study for the serological and molecular diagnosis of hepatitis delta virus (HDV) was organized. Total HDV antibodies were properly detected by all laboratories; 8/14 laboratories failed to detect low titers of IgM, and 6/11 failed to quantify and/or underestimated the RNA viral load in several samples. These discrepancies are likely related to the molecular diversity of HDV.

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Quantitative detection and typing of hepatitis D virus in human serum by real-time polymerase chain reaction and melting curve analysis

Cited by 22 publications

References 34 publications

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

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

Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Serological and Molecular Diagnosis of Hepatitis Delta Virus Infection: Results of a French National Quality Control Study

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