Journal: PNAS Nexus

Article Title: Rat hepatitis E virus cross-species infection and transmission in pigs

doi: 10.1093/pnasnexus/pgae259

Figure Lengend Snippet: Rat HEV is replication competent in cells. A) Workflow of HEV capping and transfection of target cells. LMTK, A549, Huh7 S10-3, and ST cell lines were transfected with in vitro transcribed capped HEV RNA (rat HEV). Huh7 S10-3 cells were transfected with Kernow-C1 genotype 3 P6 strain and are used as a positive control. B) Immunofluorescence detection of HEV ORF2 antigen in methanol-fixed LMTK, A549, Huh7 S10-3, and ST cells after 5 dpt. Cells are stained with goat antirabbit IgG H&L combined with antirabbit Alexa Fluor 594 (red) and 4′, 6-DAPI (blue). C) Flow cytometry quantification of LMTK, A549, Huh7 S10-3, and ST cells transfected with capped RNA transcripts of rat HEV; Huh7 S10-3 transfected with cell culture-adaptive Kernow-C1 genotype 3 P6 strain and noncell culture-adaptive genotype 1 Sar55 and Kernow-C1 genotype 3 P1 strain. Sar55, P6, and P1 belong to Paslahepevirus which is used as the control to determine the replication ability of rat HEV. The assay was performed in the cells harvested on day 5 post-transfection. Samples were fixed in methanol and probed with rabbit anti-ORF2 followed by goat antirabbit Alexa Fluor 594 antibodies. Each bar (mean ± SD) represents separate transfections stained in parallel and displays the mean of two independent biological experiments with three replicates per sample. D) RT-qPCR data from the supernatants collected from day 5 of replication assay. E) RT-qPCR data from the cell lysates collected from day 5 of replication assay. F) ST cells were transfected with rat HEV LCK-3110 and left untreated or incubated with 50 µM ribavirin (RBV). Samples were collected every 3 days, and viral RNA was measured.

Article Snippet: 16 μL of 1 × 10 5 viral RNA copies/ml was utilized for RNA transfection using a Mirus Trans-IT mRNA transfection kit.

Techniques: Transfection, In Vitro, Positive Control, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Control, Quantitative RT-PCR, Incubation

Journal: PNAS Nexus

Article Title: Rat hepatitis E virus cross-species infection and transmission in pigs

doi: 10.1093/pnasnexus/pgae259

Figure Lengend Snippet: Rat liver cells (clone 9) susceptibility to LCK-3110 strain. A) Flow cytometry quantification of rat liver (clone 9) cells transfected with capped RNA transcripts of rat HEV. The assay was performed in the cells harvested on day 5 post-transfection. Samples were fixed in methanol and probed with rabbit anti-ORF2 followed by goat antirabbit Alexa Fluor 594 antibodies. Each bar (mean ± SD) represents separate transfections stained in parallel and displays the mean of two independent biological experiments with four replicates per sample. Rat HEV RNA loads in B) culture supernatant (S) and C) cell lysates (CL) of rat liver cell lines after rat HEV RNA transfection and inoculation of filtered fecal suspension from rat HEV-inoculated pigs. Independent biological experiments, mean ± SD of 4 replicates, are presented. Dotted line represents the cutoff value demonstrating the background referring to the attachments of the virus to the cell surface. D) Immunofluorescence detection of HEV ORF2 antigen in methanol-fixed rat liver cells after 5 dpt. Cells are stained with goat antirabbit IgG H&L combined with antirabbit Alexa Fluor 594 (red) and 4′, 6-DAPI (blue).

Article Snippet: 16 μL of 1 × 10 5 viral RNA copies/ml was utilized for RNA transfection using a Mirus Trans-IT mRNA transfection kit.

Techniques: Flow Cytometry, Transfection, Staining, Suspension, Virus, Immunofluorescence