Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

Shukla, Priyanka; Nguyen, Hanh; Torian, Udana; Engle, Ronald E.; Faulk, Kristina; Dalton, Harry R.; Bendall, Richard; Keane, Frances; Purcell, Robert H.; Emerson, Suzanne U.

doi:10.1073/pnas.1018878108

Cited by 281 publications

(347 citation statements)

References 27 publications

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“…AF444002, with insertion of an S17 sequence in the hypervariable region) and one the Kernow‐C1 p6, were used to generate HEV in vitro transcripts as described 22, 23, 24. Capping was performed using Ribom7G Cap Analog (Promega, Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis E virus replication and interferon responses in human placental cells

Knegendorf

Drave

Thi

et al. 2018

Hepatology Communications

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Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV‐infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental‐derived cells (JEG‐3). Following transfection of JEG‐3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver‐derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon‐α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon‐stimulated gene expression levels demonstrated an efficient HEV‐dependent restriction in JEG‐3. Conclusion: We showed differential tissue‐specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell‐culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated. (Hepatology Communications 2018;2:173–187)

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Section: Methodsmentioning

confidence: 99%

Hepatitis E virus replication and interferon responses in human placental cells

Knegendorf

Drave

Thi

et al. 2018

Hepatology Communications

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“…The RPS17 insertion mutant was further selected during serial passages of HEV-containing fecal materials in HepG2C3A cells and exhibited an expanded host range, infecting cells from pig, deer, chicken, cat, dog, mouse, and hamster in vitro (26). In the other case, the HVR of HEV had a 117-nucleotide in-frame insertion derived from the human RPS19 gene, inserting amino acids 93 to 132 (25), and the RSP19 insertion bestowed an enhanced cell culture replication efficiency of HEV, although not to the same degree as that with the RPS17 insertion.…”

mentioning

confidence: 99%

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

Kenney

Meng

2015

J Virol

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Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCEHEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future. Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in many parts of the world (1, 2). Major outbreaks and epidemics are most common in developing countries, where a lack of proper sanitation conditions leads to waterborne spread of the disease. In industrialized countries, HEV infection is typically sporadic and endemic. Recently, chronic HEV infection has become an important clinical problem in immunosuppressed individual...

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“…4 Subsequent analysis showed that the HEV genotype-3 strain recovered from this patient's stool contained an insertion of 58 amino acids from a human ribosomal protein gene, 61 confirming that a human/hepatitis E recombinant virus exists. However, HEV co-infection in patients with HIV is not common and, to date, less than 20 cases have been documented in the literature, and only a handful have had chronic infection, the majority of these cases become cleared of HEV spontaneously.…”

Section: Hev Infection In Hiv Patientsmentioning

confidence: 80%