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

Abstract

The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1-8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.

Keywords: A549; HEV; HEV-3; ORF2; cell culture; isolate; subtype; swine; zoonosis.

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.

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).

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.

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.

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.