Method for generating high-titer hepatitis E virus stocks and titration assay for hepatitis E virus
Inventors
Buno, Brett • JOURNIGAN, TERRI • Hotta, JoAnn • Burdick, Michael
Assignees
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Publication Number
US-10208291-B2
Publication Date
2019-02-19
Expiration Date
Abstract
Polybrene as an additive in cell culture medium is used in methods for the generation of high-titer hepatitis E virus stocks and assays for titration of hepatitis E virus. A cell culture medium containing polybrene is used for high-titer HEV generation, a method for determining the presence and/or the level of HEV in a sample, and an HEV titration assay using polybrene.
Core Innovation
The invention describes culturing a cell line in vitro in a medium comprising a concentration of polybrene and infecting the cell line with Hepatitis E Virus (HEV) to produce a titer of HEV. The cell line is HepG2 (ATCC number HB-8065) or HepG2/C3A (ATCC number CRL-10741), and the concentration of polybrene is about 1 μg/ml to about 5 μg/ml.
A further aspect of the invention provides a method of determining a presence and/or a level of HEV in a sample. The method includes providing the sample to a mixture comprising a cell line and a culture medium, where the culture medium comprises polybrene, incubating the mixture to allow for propagation of HEV if present, collecting a portion comprising propagated HEV, and measuring the presence and/or the level of a biological substance associated with HEV in the collected portion.
The disclosed measurement approach includes measuring a biological substance associated with HEV, including workflows in which HEV RNA is exposed, complementary cDNA is generated, a sequence is amplified using a pair of polynucleotides, and a concentration of the amplified sequence is determined. In particular embodiments, the pair of polynucleotides is defined by SEQ ID NO: 1 and SEQ ID NO: 2, and an agent comprises the oligonucleotide sequence identified as SEQ ID NO: 3 with labeled groups including 5’-FAM, ZEN, and 3IABkFQ.
Claims Coverage
The document includes two independent claims, each directed to a core inventive concept: producing an HEV titer using polybrene-supplemented culture of HepG2 or HepG2/C3A, and determining the presence and/or level of HEV in a sample using polybrene-containing culture followed by measurement of an HEV-associated biological substance. Across the independent claims and their dependents, inventive features include specified cell lines, defined polybrene concentration ranges, propagation and collection in a polybrene-containing mixture, and an HEV-associated measurement workflow using defined polynucleotide sequences and labeled agents.
Polybrene-supplemented HEV production in HepG2 or HepG2/C3A
Culturing HepG2 (ATCC number HB-8065) or HepG2/C3A (ATCC number CRL-10741) in vitro in a medium comprising a concentration of polybrene and infecting the cell line with HEV.
Defined polybrene concentration for HEV culturing
Using a polybrene concentration of about 1 μg/ml to about 5 μg/ml in the medium comprising polybrene for culturing the cell line prior to HEV infection.
Polybrene-containing propagation mixture for HEV detection
Providing a sample to a mixture comprising a cell line and a culture medium, where the culture medium comprises polybrene, and incubating the mixture to allow for propagation of HEV if present.
Collecting propagated HEV and measuring an HEV-associated biological substance
Collecting a portion of the incubated mixture comprising HEV if present and propagated, and measuring the presence and/or the level of a biological substance associated with HEV in the collected portion.
RNA-to-amplified-sequence workflow for HEV level determination
Measuring the biological substance by exposing HEV RNA, generating complementary cDNA, amplifying using a pair of polynucleotides, and determining a concentration of the amplified sequence.
Defined primer pair by SEQ ID NO: 1 and SEQ ID NO: 2
Using a pair of polynucleotides comprising SEQ ID NO: 1 and SEQ ID NO: 2.
Labeled HEV-specific agent by SEQ ID NO: 3
Employing an agent comprising the oligonucleotide sequence identified as SEQ ID NO: 3 with labeled groups 5’-FAM, ZEN, and 3IABkFQ.
Cell line selection for HEV propagation
Selecting the cell line from HepG2 (ATCC number HB-8065) and HepG2/C3A (ATCC number CRL-10741).
Overall, claim coverage centers on two complementary polybrene-enabled concepts: producing an HEV titer by infecting polybrene-supplemented cultures of HepG2 or HepG2/C3A, and determining HEV presence and/or level in samples by propagating HEV in a polybrene-containing mixture and measuring an HEV-associated biological substance using defined nucleic-acid components (including SEQ ID NO: 1, SEQ ID NO: 2, and an agent defined by SEQ ID NO: 3).
Stated Advantages
Produces high-titer HEV suitable for virus clearance studies.
Enables attachment and monolayer growth of HepG2/HepG2-C3A cells using polybrene instead of extracellular plate coatings.
Provides a polybrene-enabled presence/level determination workflow for HEV.
Documented Applications
Production of high-titer HEV stocks for virus clearance studies.
Virus clearance-related experiments relying on propagation of HEV in cell culture and subsequent titration.
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