Journal: JHEP Reports

Article Title: A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

doi: 10.1016/j.jhepr.2021.100296

Figure Lengend Snippet: Establishment of an HBV infection assay to monitor the entire HBV life cycle. (A) HBV life cycle scheme. Early life cycle steps of infection establishment (1–5, green arrows) inhibited by reference inhibitors MyrB, MA18/07 (Step 1, receptor binding), and CCC-0975 (Step 5, cccDNA formation). Late HBV life cycle steps of morphogenesis and egress (6–12, blue arrows) inhibited by TDF and HID (Step 9, reverse transcription, RNase H). (B) Experimental setup of the 2-step HBV supernatant transfer infection assay. HepG2-NTCPsec+ p1 cells were seeded at 8,000 cells/well in 384-well plates and pre-incubated with the early or late life cycle phase reference inhibitor MyrB, MA18/07, CCC-0975, TDF, or HID for 2 h prior to inoculation with HBV for 18 h. After cells had been repeatedly washed at 1 dpi, they were treated with reference inhibitors and cultured until 6 dpi. Supernatants were harvested and transferred to naïve HepG2-NTCPsec+ p2 cells. Cells p1 and p2 were analysed using IFA (HBc) and counterstained with Hoechst (nuclei). Infected cells were identified microscopically and analysed as described in Fig. 1 . (C) Infection with serially diluted HBV under untreated conditions (phenotype A). (D) HBV infection under treatment with early HBV life cycle inhibitor (phenotype B, top panel) or late HBV life cycle inhibitor (phenotype C, lower panel). HBc (green) and nuclei (blue); dose-response curve regression: red line = % cell viability (right y-axis); black line = % inhibition of infection (left y-axis). cccDNA, covalently closed circular DNA; dpi, days post-infection; HID, N -hydroxyisoquinolinedione; IFA, immunofluorescence analysis; MyrB, myrcludex B; pgRNA, pregenomic RNA; TDF, tenofovir disoproxil fumarate.

Article Snippet: Next, utilizing stable HBV-replicating cells, we evaluated the MoA of the newly identified late HBV life cycle inhibitors fludarabine (Fludara; Selleckchem, Houston, TX, USA) and dexmedetomidine (Precedex; Selleckchem, Houston, TX, USA), which are an FDA-approved nucleic acid synthesis inhibitor used as a chemotherapy medication for the treatment of leukaemia and lymphoma and an anxiety-reducing sedative and pain medication, respectively.

Techniques: Infection, Binding Assay, Reverse Transcription, Incubation, Cell Culture, Inhibition, Immunofluorescence

Journal: JHEP Reports

Article Title: A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

doi: 10.1016/j.jhepr.2021.100296

Figure Lengend Snippet: HCS overview and representative inhibitor confirmation. (A) Screening campaign summary of hit identification and confirmation. Number of hits, hit selection, and confirmation criteria are shown. Confirmation of selected early (B) (pranlukast and cytochalasin D) and late (C) (fludarabine and dexmedetomidine) HBV life cycle phase inhibitors by DRC analysis. %Imax, maximum inhibition; CC 50 , 50% cytotoxic concentration; dpi, days post-infection; DRC, dose–response curve; FDA, Food and Drug Administration; HCS, high-content screening; HTS, high-throughput screening; IFA, immunofluorescence analysis; TI, therapeutic index.

Article Snippet: Next, utilizing stable HBV-replicating cells, we evaluated the MoA of the newly identified late HBV life cycle inhibitors fludarabine (Fludara; Selleckchem, Houston, TX, USA) and dexmedetomidine (Precedex; Selleckchem, Houston, TX, USA), which are an FDA-approved nucleic acid synthesis inhibitor used as a chemotherapy medication for the treatment of leukaemia and lymphoma and an anxiety-reducing sedative and pain medication, respectively.

Techniques: Selection, Inhibition, Concentration Assay, Infection, High Content Screening, High Throughput Screening Assay, Immunofluorescence

Journal: JHEP Reports

Article Title: A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

doi: 10.1016/j.jhepr.2021.100296

Figure Lengend Snippet: Antiviral efficacies and mechanisms of re-identified early and late HBV life cycle inhibitors .

Article Snippet: Next, utilizing stable HBV-replicating cells, we evaluated the MoA of the newly identified late HBV life cycle inhibitors fludarabine (Fludara; Selleckchem, Houston, TX, USA) and dexmedetomidine (Precedex; Selleckchem, Houston, TX, USA), which are an FDA-approved nucleic acid synthesis inhibitor used as a chemotherapy medication for the treatment of leukaemia and lymphoma and an anxiety-reducing sedative and pain medication, respectively.

Techniques:

Journal: JHEP Reports

Article Title: A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

doi: 10.1016/j.jhepr.2021.100296

Figure Lengend Snippet: DRC analysis of reference inhibitors and confirmed novel early and late HBV life cycle inhibitors .

Article Snippet: Next, utilizing stable HBV-replicating cells, we evaluated the MoA of the newly identified late HBV life cycle inhibitors fludarabine (Fludara; Selleckchem, Houston, TX, USA) and dexmedetomidine (Precedex; Selleckchem, Houston, TX, USA), which are an FDA-approved nucleic acid synthesis inhibitor used as a chemotherapy medication for the treatment of leukaemia and lymphoma and an anxiety-reducing sedative and pain medication, respectively.

Techniques: Binding Assay, DNA Synthesis

Journal: JHEP Reports

Article Title: A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

doi: 10.1016/j.jhepr.2021.100296

Figure Lengend Snippet: Mechanism of action studies with early and late HBV life cycle inhibitors. (A) Time-of-addition experiment schedule. The reference inhibitors MyrB, pranlukast, and cytochalasin D were either pretreated and cotreated, or treated post-HBV inoculation in time-of-addition experiments. (B) DRC analysis of time-of-addition experiments. (C) Binding assay of HBV surface protein-derived Alexa633-labelled preS1 peptide under Mock (DMSO) or MyrB, pranlukast, cytochalasin D treatment. (D) Live-cell imaging of HBV surface protein-derived Alexa633-labelled preS1 peptide internalization (red) under cytochalasin D treatment. (E) Evaluation of HBV late step inhibitors in stably HBV replicating HepAD38 cells treated with serial dilutions of LMV, fludarabine, or dexmedetomidine. Supernatants were analysed by quantitative PCR at 7 days post-treatment. Cell viability was determined by automated nuclei counting. (F) Evaluation of late step inhibitors with HBVpt. HepG2-NTCPsec+ cells were pre-treated with 3-fold serially diluted fludarabine or dexmedetomidine (both starting from 50 μM) and infected with HBVpt genotype C. At 10 dpi, HBV inhibition of p1 cells (black squares) was analysed as described above, and supernatants were transferred to naïve p2 cells and analysed at 10 days post transfer (blue squares). Cell viability was determined as described before (red circles). Percentages of HBV inhibition and cell viability are indicated on the left and right y-axes, respectively. %Imax, percent maximum inhibition; CC 50 , 50% cytotoxic concentration; dpi, days post-infection; DRC, dose–response curve; GEq, genome equivalents; HBVpt, patient-derived HBV; IFA, immunofluorescence analysis; LMV, lamivudine; MyrB, myrcludex B; Ti, therapeutic index.

Article Snippet: Next, utilizing stable HBV-replicating cells, we evaluated the MoA of the newly identified late HBV life cycle inhibitors fludarabine (Fludara; Selleckchem, Houston, TX, USA) and dexmedetomidine (Precedex; Selleckchem, Houston, TX, USA), which are an FDA-approved nucleic acid synthesis inhibitor used as a chemotherapy medication for the treatment of leukaemia and lymphoma and an anxiety-reducing sedative and pain medication, respectively.

Techniques: Binding Assay, Derivative Assay, Live Cell Imaging, Stable Transfection, Real-time Polymerase Chain Reaction, Infection, Inhibition, Concentration Assay, Immunofluorescence

Journal: Blood Transfusion

Article Title: Viral safety of APOSECTM: a novel peripheral blood mononuclear cell derived-biological for regenerative medicine

doi: 10.2450/2019.0249-18

Figure Lengend Snippet: List of tested viruses and corresponding indicator cells

Article Snippet: The master cell bank was characterised, e.g. by purity, identity, and stability and then used to prepare a working cell bank of the indicator/propagation cell lines. table ft1 table-wrap mode="anchored" t5 caption a7 Bovine viral diarrhoea virus (BVDV) Family Flaviviridae Strain NADL Origin/supplier American Type Culture Collection (ATCC), VR 534 Characteristics Single-stranded RNA virus, enveloped, 40–50 nm Indicator/propagation cells MDBK, bovine kidney Origin/supplier European Collection of Animal Cell Cultures (ECACC), 90050801 Determination of CPE After 5–8 days Human immunodeficiency virus type 1 (HIV-1) Family Retroviridae Strain Bru Origin/supplier Georg-Speyer-Haus, Frankfurt Characteristics Single-stranded RNA virus, enveloped, 80–100 nm Indicator/propagation cells C8166, human T-cell line, HTLV-I transformed Origin/supplier European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, UK (#88051601) Determination of CPE After 7–12 days Pseudorabies virus (PRV) Family Herpesviridae Strain Aujeszky Origin/supplier American Type Culture Collection (ATCC), VR-135 Characteristics Double-stranded DNA virus, enveloped, 120–200 nm Indicator/propagation cells Vero, African green monkey cell line from kidney tissue Origin/supplier European Collection of Animal Cell Cultures (ECACC), 84113001 Determination of CPE After 5–7 days Hepatitis A virus (HAV) Family Picornaviridae Strain HM175 Origin/supplier FDA Feinstone, RKI, Analysis; Robert-Koch-Institute Characteristics Single stranded RNA virus, non-enveloped, 27 nm Indicator/propagation cells FRhK-4, foetal Rhesus monkey kidney cells Origin/supplier European Collection of Animal Cell Cultures (ECACC), 84120701 Determination of CPE After 11- 6 days Porcine parvovirus (PPV) Family Parvoviridae Strain NADL-2 Origin/supplier American Type Culture Collection (ATCC), VR-742 Characteristics Single-stranded DNA virus, non-enveloped, 20–26 nm Indicator/propagation cells PK13, a porcine cell from kidney tissue Origin/supplier American Type Culture Collection (ATCC), CRL-6489 Determination of CPE After 10–14 days Open in a separate window CPE: Cytopath(ogen)ic effect.

Techniques: Virus, Transformation Assay