In Vitro Replication of Swine Hepatitis E Virus (HEV): Production of Cell-Adapted Strains

Figures

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  Figure 1. Propagation of HEV-3 strain (HEV-3-e, HEV-3-e, HEV-3f and HEV-3c) on A549 monolayers for replicate r.1 (red) and r.2 (blue). HEV-RNA present in the supernatant of cultured cells obtained during 9 weeks of observation period after inoculation with faecal samples (P0) and the following passages (P1, P2). HEV-RNA titres are measured as Log10 copies of genome per mL.

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  Figure 2. Transmission electron micrographs of infected cell supernatants, positive for HEV-RNA. Representative images of assembled virions (A,B) and protein aggregates (C,D).

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  Figure 3. Immunofluorescence analysis of mock (A) and infected cells stained with anti-ORF-2 (B) and ORF-3 (C) primary antibodies. A goat anti-mouse IgG FITC antibody was used to bind primary antibodies. Cell nuclei were stained with DAPI (blue staining). Arrows indicate HEV-infected cells expressing viral proteins. Fluorescent images were taken using a Leica DM4000 fluorescence microscope.

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  Figure 4. Estimation of minimum infectious dose (MID) of the IT-13 strain. The HEV-RNA in culture supernatants of A549 cells inoculated with serial ten-fold dilutions of HEV stocks was estimated as Log10 genome copies/mL for 28 days p.i. The r.1 and r.2 in legend represent the replicates.

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  Figure 5. Maximum Likelihood phylogenetic tree built with 15 HEV ORF2 partial sequences obtained from this study (indicated in bold), 18 HEV-3 reference sequences, 21 HEV from strains isolated on cells and one HEV-4 sequence as outgroup. 3*: unassigned subtypes.