Isolation of 15 hepatitis E virus strains lacking ORF1 rearrangements from wild boar and pig organ samples and efficient replication in cell culture

Abstract

As a zoonotic pathogen, the hepatitis E virus (HEV) leads to numerous infections in humans with different clinical manifestations. Especially genotype 3, as causative agent of a foodborne zoonosis, is transmitted to humans by ingestion of undercooked or raw meat containing liver from HEV-infected animals. Although the virus' prevalence and dissemination in hosts like wild boar and pig have been well characterized, HEV is greatly understudied on a molecular level and reliable cell culture models are lacking. For this reason, the present study concentrated on the isolation and subsequent characterization of porcine HEV from tissue samples derived from wild boar and domestic pigs: 222 wild boars hunted in Northern Germany were investigated for the presence of HEV RNA with a detection rate of 5.9%. Three additional HEV-positive wild boar liver samples as well as an HEV-positive spleen and a positive kidney from domestic pigs were included. After inoculation of positive samples onto the human hepatoma cell line PLC/PRF/5, cells were grown for several weeks. Successful isolation was confirmed by RT-qPCR, virus passage, immunofluorescence staining and titration. Overall, 15 strains from a total of 18 RNA-positive organ samples could be obtained and viral loads >10 9 RNA copies/ml were measured in cell culture supernatants. Accordingly, 83.3% of the HEV RNA-positive samples contained infectious hepatitis E viral particles and therefore must be considered as a potential source for human infections. Phylogenetic analyses revealed that all isolated strains belong to genotype 3. Further genetic characterization showed a high degree of sequence variability, but no sequence insertions, in the hypervariable region within the open reading frame 1.

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

“…The cultivation of HEV-3 and HEV-4 strains from pigs and wild boar was obtained using caecum faecal content and other organs on both hepatoma (PLC/PRF/5 cells) and non-hepatoma human cells (A549) [27][28][29].…”

A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment

“…We observed a significant increase in viral RNA in supernatants within just five days for A549-D3 cells and six days for HuH7-S10-3 cells by cultivating cells in MEM supplemented with 10% FBS and 2% DMSO during the infection period. It is noteworthy to mention that many established cell culture models for HEV typically employ more extended periods, ranging from seven days to even several months [ 29 , 68 , 75 , 86 , 87 ]. Moreover, these models often exhibit a slow initial phase of viral replication before substantial increases.…”

Development of a Sensitive and Specific Quantitative RT-qPCR Method for the Detection of Hepatitis E Virus Genotype 3 in Porcine Liver and Foodstuff

“…A total of 46 samples containing HEV RNA from different subtypes of HEV genotypes 3 and 4 were tested to determine the inclusivity of the JBH4-HEV RT-qPCR assay. The sample eluates tested were HEV reference material from the Paul Ehrlich Institute [36], RNA isolated from wild boar liver in previous HEV prevalence studies by Schotte et al [41], RNA isolated from liver samples from the study by Wist et al [42], and RNA isolated from field samples of wild boar and domestic pig liver by Gremmel et al [43]. The selected subtypes were primarily chosen due to their association with foodborne transmission and their significance for human infections, covering HEV subtypes 3c, 3f, 3i, 3e, 3h, 3-i-like, 3b, 3ra, 4c and 4g [44].…”