Journal: Nature Communications

Article Title: Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

doi: 10.1038/s41467-024-49219-8

Figure Lengend Snippet: The identified insertions were cloned into the HEV reporter or full genome of the Kernow-C1-p6 strain, thereby replacing the HVR with insertion containing HVRs as indicated in Fig. . a Replication kinetics of HVR constructs with Kernow-C1-p6 (p6) and Kernow-C1-p1 (p1) as references are shown. Plotted is the time post electroporation as well as mean (+/- SD) relative light units (RLU) normalized to the four-hour value of n = 7 biologically independent experiments for p1 h.TRIM22, n = 3 for dup constructs and n = 6 for other constructs. b The HEV replicon system was used to analyse the ribavirin (RBV) sensitivity by treating the cells for five days post-electroporation with RBV concentrations ranging from 0.19 µM to 100 µM. Plotted is the mean (+/- SD) HEV replication as a percentage of untreated controls in n = 3 biologically independent experiments for dup constructs, n = 6 for other constructs. Lines represent dose-response curves of four-parameter log-logistic analysis. c The full-length system was used to produce infectious particles, which were titrated onto HepG2/C3A cells to determine the achieved viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column. Plotted are the means with standard deviation (+/- SD) of n = 8 biologically independent experiments for p1 and p6, n = 3 for other constructs. Source data are provided in the Source Data file.

Article Snippet: The HepG2 subclone C3A (HepG2/C3A, also kindly provided by Charles Rice, last cell line authentication 2023/07/05, Microsynth) was used for infection experiments since greater infection efficiencies are achieved.

Techniques: Clone Assay, Construct, Electroporation, Immunofluorescence, Infection, Staining, Standard Deviation

Journal: Nature Communications

Article Title: Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

doi: 10.1038/s41467-024-49219-8

Figure Lengend Snippet: a The insertions reported by Lhomme et al. were analysed for their insertion site in relation to the strain Kernow-C1-p1. The insertion site as well as their similarity (dots) to the strain Kernow-C1-p1 are shown. Mismatches to the reference are indicated by the used amino acid as a single letter code. The inserted amino acid sequence is indicated in blue. The name of created constructs is mentioned in front of each row. b The replicon system was used to determine their impact on replication versus the reference strain Kernow-C1-p6 (p6) and Kernow-C1-p1 (p1). Plotted is the time post-electroporation as well as the mean (+/- SD) relative light units (RLU) normalized to the four-hour value of n = 4 biologically independent experiments for p1, n = 5 for other constructs. c The replicon was used to analyse the ribavirin (RBV) sensitivity by treating the cells for five days post-electroporation with RBV concentrations ranging from 0.19 µM to 100 µM. A non-linear regression and the IC 50 values were calculated using GraphPad Prism. Depicted are the respective IC 50 values and confidence intervals (CI 95%) of n = 3 biologically independent experiments. d The HEV full-length system was used to produce infectious particles, which were titrated onto HepG2/C3A cells to determine viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column. Plotted are the means with standard deviation (+/- SD) as well as individual data points (circle) of n = 6 biologically independent experiments for p1 and p6, n = 3 for other constructs. Source data are provided in the Source Data file.

Article Snippet: The HepG2 subclone C3A (HepG2/C3A, also kindly provided by Charles Rice, last cell line authentication 2023/07/05, Microsynth) was used for infection experiments since greater infection efficiencies are achieved.

Techniques: Sequencing, Construct, Electroporation, Immunofluorescence, Infection, Staining, Standard Deviation

Journal: Nature Communications

Article Title: Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

doi: 10.1038/s41467-024-49219-8

Figure Lengend Snippet: a Depicted is the Kernow-C1-p1 (p1) ORF1 encoding sequences with mutations that differentiate it from Kernow-C1-p6 (p6) ORF1. Additionally, the RPS17 insertion site is indicated on the genome. The mutations over the genome were cloned separately into p1, while the thirteen variants near the RPS17 (see Fig. ) were cloned together with the RPS17 RNA and were termed RPS17+flanking regions (RPS17/FR). b Replication kinetics were measured with p1 and p6 as reference (grey triangles) while constructs of interest are depicted in red. Plotted are mean relative light units (RLU) normalized to the four-hour value over time (hours post electroporation) of n = 3 biologically independent experiments, n = 6 for A220T and RPS17/FR constructs. c , f The 72-hour replication values were plotted as a column diagram. The left red/green area depicts the p1 replication, while the right red/green area depicts the p6 replication level. d The HEV full-length system was used to produce infectious particles with indicated constructs, which were titrated onto HepG2/C3A cells to determine viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column (mean, +/- SD). Dots represent individual data points of n = 5 individual experiments. e The RPS17 insertion alone or with a flanking region was cloned into the HEV3-83-2-27-Gluc replicon. Replication kinetics were measured with p1 and p6 as reference (grey triangles) while constructs of interest are depicted in green. Depicted are mean (+/- SD) relative light units (RLU) normalized to the four-hour value over time (hours post electroporation) of n = 3 biologically independent experiments for p1 and p6, n = 6 for other constructs. g The HEV full-length system was used to produce infectious particles with indicated constructs, which were titrated onto HepG2/C3A cells to determine viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column (mean, +/- SD). Dots represent individual data points of n = 3 individual experiments. Source data are provided in the Source Data file.

Article Snippet: The HepG2 subclone C3A (HepG2/C3A, also kindly provided by Charles Rice, last cell line authentication 2023/07/05, Microsynth) was used for infection experiments since greater infection efficiencies are achieved.

Techniques: Clone Assay, Construct, Electroporation, Immunofluorescence, Infection, Staining