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. 2021 Jan 7;13(1):75. doi: 10.3390/v13010075

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Presence of ORF4 from gt1 enhances the gt3 HEV and gt1 HEV replication. (A) Workflow of HEV transfection of target cells. ORF4+, ORF4− and Huh7 cell lines were transfected with in vitro-transcribed capped HEV mRNA (P1, P6 and Sar55). Each cell line was grown in DMEM with 10% heat-inactivated fetal bovine serum (HI-FBS). After 48 h, cells were passed 1:3. At 5 and 7 dpt, cells were harvested and used to detect HEV ORF2-positive cells. (B) Immunofluorescence detection of HEV ORF2 antigen in methanol-fixed ORF4+, ORF4−, Huh7 cell lines and respective mock cells after 5 dpt. Cells are stained with goat anti-rabbit IgG H&L combined with anti-rabbit Alexa fluor 594 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images serve to validate antibody staining specificity and are not necessarily representative images as most fields are devoid of antigen staining. (C,D) Flow cytometry quantification of ORF4+, ORF4− and Huh7 cells transfected with the capped RNA transcripts of the HEV gt3 (P1 and P6) strains and HEV gt1 (Sar55) strain, performed at 5 and 7 dpt. Samples were fixed in methanol and probed with rabbit anti-ORF2 followed by goat anti-rabbit PE antibodies. Each bar (mean ± SEM) represents separate transfections stained in parallel and display the mean of three independent biological experiments with nine replicates per sample (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.001 t-test). Statistically significant difference exists between ORF4+ cell lines transfected with HEV gt3 (P6, P1) and gt1 (Sar55) strains verses other cell lines respectively transfected with similar strains.

Presence of ORF4 from gt1 enhances the gt3 HEV and gt1 HEV replication. (A) Workflow of HEV transfection of target cells. ORF4+, ORF4− and Huh7 cell lines were transfected with in vitro-transcribed capped HEV mRNA (P1, P6 and Sar55). Each cell line was grown in DMEM with 10% heat-inactivated fetal bovine serum (HI-FBS). After 48 h, cells were passed 1:3. At 5 and 7 dpt, cells were harvested and used to detect HEV ORF2-positive cells. (B) Immunofluorescence detection of HEV ORF2 antigen in methanol-fixed ORF4+, ORF4−, Huh7 cell lines and respective mock cells after 5 dpt. Cells are stained with goat anti-rabbit IgG H&L combined with anti-rabbit Alexa fluor 594 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images serve to validate antibody staining specificity and are not necessarily representative images as most fields are devoid of antigen staining. (C,D) Flow cytometry quantification of ORF4+, ORF4− and Huh7 cells transfected with the capped RNA transcripts of the HEV gt3 (P1 and P6) strains and HEV gt1 (Sar55) strain, performed at 5 and 7 dpt. Samples were fixed in methanol and probed with rabbit anti-ORF2 followed by goat anti-rabbit PE antibodies. Each bar (mean ± SEM) represents separate transfections stained in parallel and display the mean of three independent biological experiments with nine replicates per sample (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.001 t-test). Statistically significant difference exists between ORF4+ cell lines transfected with HEV gt3 (P6, P1) and gt1 (Sar55) strains verses other cell lines respectively transfected with similar strains.