Dataset Information

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.

SUBMITTER: Ianiro G

PROVIDER: S-EPMC9854997 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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

Ianiro Giovanni G Monini Marina M Ammendolia Maria Grazia MG De Sabato Luca L Ostanello Fabio F Vaccari Gabriele G Di Bartolo Ilaria I

Animals : an open access journal from MDPI 20230113 2

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; eve ...[more]

PMID: 36670816

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Project description:HEV is the most causative agent of acute viral hepatitis globally. HEV causes acute, chronic, and extrahepatic manifestations. Chronic HEV infection develops in immunocompromised patients such as organ transplant patients, HIV-infected patients, and leukemic patients. The source of chronic HEV infection is not known. Also, the source of extrahepatic manifestations associated with HEV infection is still unclear. Hepatotropic viruses such as HCV and HBV replicate in peripheral blood mononuclear cells (PBMCs) and these cells become a source of chronic reactivation of the infections in allograft organ transplant patients. Herein, we reported that PBMCs and bone marrow-derived macrophages (BMDMs), isolated from healthy donors (n = 3), are susceptible to HEV in vitro. Human monocytes (HMOs), human macrophages (HMACs), and human BMDMs were challenged with HEV-1 and HEV-3 viruses. HEV RNA was measured by qPCR, the marker of the intermediate replicative form (ds-RNA) was assessed by immunofluorescence, and HEV capsid protein was assessed by flow cytometry and ELISA. HEV infection was successfully established in primary HMOs, HMACs, and human BMDMs, but not in the corresponding cells of murine origin. Intermediate replicative form (ds RNA) was detected in HMOs and HMACs challenged with HEV. The HEV load was increased over time, and the HEV capsid protein was detected intracellularly in the HEV-infected cells and accumulated extracellularly over time, confirming that HEV completes the life cycle inside these cells. The HEV particles produced from the infected BMDMs were infectious to naive HMOs in vitro. The HEV viral load was comparable in HEV-1- and HEV-3-infected cells, but HEV-1 induced more inflammatory responses. In conclusion, HMOs, HMACs, and human BMDMs are permissive to HEV infection and these cells could be the source of chronic and recurrent infection, especially in immunocompromised patients. Replication of HEV in human BMDMs could be related to hematological disorders associated with extrahepatic manifestations.

Project description:BackgroundTo fulfill epidemiological data and investigate possible interspecies transmission, this study shall attempt to sequence representative HEV strains of human, swine and wild boar origin collected from 2010 to 2017 in Croatia.MethodsIn total, 174 anti-HEV antibody positive human sera samples; 1419 blood or faeces samples of swine, as well as 720 tissue and/or blood samples of wild boar originating from different counties (18 in total) in Croatia were tested for the presence of HEV RNA.ResultsHEV RNA was detected in 26 human sera samples (14.9%; 95% CI 10.4-21.0%). HEV RNA was detected in 216 tested swine (15.2%; 95% CI 13.5-17.1%), regardless of age, farm breeding system or geographical origin. Viral RNA was also detectable in faeces samples which prove that swine actively participate in shedding HEV into the environment. Of the total of 720 tested wild boar samples, 83 were HEV RNA positive (11.5, 95% CI 9.4-14.1%) originating from six counties. According to the sequence analysis all strains have shown to be members of Orthohepevirus A genotype HEV-3, regardless of host. The genotyping results confirm grouping of sequences into four subtypes of HEV strains of which subtypes 3a and 3c belong to the general cluster 3abchij, and were predominately detected during the study, while subtypes 3e and 3f fall within cluster 3efg. Strains within subtypes 3a and 3e were found in humans, swine and wild boars; subtype 3c strains were derived from humans and swine, whereas subtype 3f strains were found only in humans. Strains belonging to subtypes 3a and 3c were derived during the entire investigated period and may be considered endemic in Croatia, whereas strains within subtypes 3e and 3f were detected sporadically indicating the possibility of newly imported infections.ConclusionsAll detected strains show to be genetically highly related to strains found in humans and/or animals from other European Countries, indicating that trade of live animals or wild boar movement increases the risk of HEV infection spread. Furthermore, homologous strains found in different investigated species within this study indicate interspecies transmission of HEV and/or an existence of an accessible mutual source of infection.

Project description:Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

Dataset Information

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

Publications

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

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