Circulation of hepatitis E virus (HEV) and/or HEV-like agent in non-mixed dairy farms could represent a potential source of infection for Egyptian people

Sayed, Ibrahim M.; Elkhawaga, Amal A.; El‐Mokhtar, Mohamed A.

doi:10.1016/j.ijfoodmicro.2019.108479

Cited by 40 publications

(62 citation statements)

References 40 publications

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“…Quantification of HEV RNA was done as described previously [ 32 , 33 , 34 , 35 ]. Briefly, viral RNA was extracted from cell culture supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro

et al. 2020

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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.

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Section: Methodsmentioning

confidence: 99%

Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro

et al. 2020

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“…The viral load of the inoculums was quantified by qPCR, as described in detail in the Section 4.4. The sequencing and phylogenetic analysis of the isolated viruses was done as previously described and according to Smith et al [4,39,41].…”

Section: Hev Inoculumsmentioning

confidence: 99%

“…The quantification of HEV RNA was done as described previously with slight modifications in the procedure [38,39,41,42]. Briefly, viral RNA was extracted from 10% w/v stool preparations and cell culture supernatants using a QIAamp Viral RNA Mini Kit (Qiagen, Germany) and from the HEV-infected cells using an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions.…”

Section: Quantification Of Hev Rna By Qrt-pcrmentioning

confidence: 99%

Evidence of the Extrahepatic Replication of Hepatitis E Virus in Human Endometrial Stromal Cells

et al. 2020

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Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The tropism of HEV is not restricted to the liver, and the virus replicates in other organs. Not all the extrahepatic targets for HEV are identified. Herein, we found that non-decidualized primary human endometrial stromal cells (PHESCs), which are precursors for the decidua and placenta, are susceptible to HEV infection. PHESCs, isolated from healthy non-pregnant women (n = 5), were challenged with stool-derived HEV-1 and HEV-3. HEV RNA was measured by qPCR, and HEV capsid protein was assessed by flow cytometry, immunofluorescence (IF), and ELISA. HEV infection was successfully established in PHESCs. Intracellular and extracellular HEV RNA loads were increased over time, indicating efficient replication in vitro. In addition, HEV capsid protein was detected intracellularly in the HEV-infected PHESCs and accumulated extracellularly over time, confirming the viral assembly and release from the infected cells. HEV-1 replicated more efficiently in PHESCs than HEV-3 and induced more inflammatory responses. Ribavirin (RBV) treatment abolished the replication of HEV in PHESCs. In conclusion, PHESCs are permissive to HEV infection and these cells could be an endogenous source of HEV infection during pregnancy and mediate HEV vertical transmission.

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“…They are zoonotic, infection is mainly transmitted to humans either by contact with these animals or the ingestion of undercooked meat, sausages, liver, or other products from them [ 8 , 9 , 10 ]. Additionally, HEV-3 and HEV-4 were reported to be excreted in the milk of ruminants [ 11 , 12 , 13 , 14 ]. HEV particles excreted in milk were infectious to in vivo animal models and they could be inactivated by short time boiling but not by pasteurization [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…In these previous reports, we used a molecular qRT-PCR assay targeting the ORF2/3 region, which is highly conserved among all HEV genotypes, to detect HEV RNA in the milk samples. The LoD 95 % (LoD: limit of detection, which is the lowest HEV RNA concentration that could be detected in 95% of replicates) of this assay was 300 IU/mL [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples

et al. 2020

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Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers in milk samples becomes pivotal. However, milk includes inhibitory components that affect HEV detection assays. Previously it was reported that dilution of milk matrix improves the performance of HEV molecular assay, however, the dilution of milk samples is not the best strategy especially when the contaminated milk sample has a low HEV load. Therefore, the objective of this study is to compare the effect of extraction procedures on the efficiency of HEV RNA detection in undiluted milk samples. In addition, we assessed the effect of the removal of milk components such as fats and casein on the performance of the molecular and serological assays of HEV. Phosphate buffered saline (PBS) and different milk matrices (such as whole milk, skim milk, and milk serum) were inoculated with different HEV inoculums and subjected to two different extraction procedures. Method A includes manual extraction using spin column-based extraction, while method B includes silica-based automated extraction. Method A was more sensitive than method B in the whole milk and skim milk matrices with a LoD95% of 300 IU/mL, and virus recovery yield of 47%. While the sensitivity and performance of method B were significantly improved using the milk serum matrix, with LoD95% of 96 IU/mL. Interestingly, retesting HEV positive milk samples using the high sensitivity assay based on method B extraction and milk serum matrix increased the HEV RNA detection rate to 2-fold. Additionally, the performance of HEV serological assays such as anti-HEV IgG and HEV Ag in the milk samples was improved after the removal of the fat globules from the milk matrix. In conclusion, HEV RNA assay is affected by the components of milk and the extraction procedure. Removal of inhibitory substances, such as fat and casein from the milk sample increased the performance of HEV molecular and serological assays which will be suitable for the low load HEV milk with no further dilutions.

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Circulation of hepatitis E virus (HEV) and/or HEV-like agent in non-mixed dairy farms could represent a potential source of infection for Egyptian people

Cited by 40 publications

References 40 publications

Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro

Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro

Evidence of the Extrahepatic Replication of Hepatitis E Virus in Human Endometrial Stromal Cells

Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples

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