Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR

Dong Kyun Ryu, Yeji Ahn, Wang Shick Ryu, Marc P. Windisch

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation.

Original languageEnglish
Pages (from-to)287-293
Number of pages7
JournalBioTechniques
Volume59
Issue number5
DOIs
Publication statusPublished - 2015 Nov

Fingerprint

Nucleocapsid
Viruses
Hepatitis B virus
Assays
RNA
Viral RNA
Hepatitis B Antibodies
Immunosorbents
Agar Gel Electrophoresis
Transcription
Ribonucleases
Electrophoresis
Isotopes
Sepharose
Reverse Transcription
Purification
Antiviral Agents
Screening
Genes
Gels

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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abstract = "After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation.",
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Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR. / Ryu, Dong Kyun; Ahn, Yeji; Ryu, Wang Shick; Windisch, Marc P.

In: BioTechniques, Vol. 59, No. 5, 11.2015, p. 287-293.

Research output: Contribution to journalArticle

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