Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

Influence of Nasopharyngeal Viral Load on the Spread of the Omicron BA.2 Variant.

We used variant typing polymerase chain reaction to describe the evolution of severe acute respiratory syndrome coronavirus 2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

A frantic confusion: beyond rabies and anti-N-methyl-D-aspartate encephalitis.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. In rare cases, HEV may generate neurologic lesions such as neuralgic amyotrophy, Guillain-Barré syndrome, and meningoencephalitis. Thirteen cases of HEV meningoencephalitis have been reported over 20 years. The clinical landscape varied from mild symptoms to coma and seizures. Most of patients were immunocompetent adults and spontaneously recovered. We report here the case of a 44-year-old immunocompetent adult with HEV meningoencephalitis presenting with aggressiveness and then coma. The evolution was spontaneously favorable without any specific treatment. This clinical case aims to draw attention on this emerging and probably under-recognized cause of meningoencephalitis.

Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection.

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as "quasi-enveloped" particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2S results from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2S secretion via the secretory pathway. Unlike ORF2C, ORF2S is glycosylated and exists as a dimer. Nonetheless, ORF2S exhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

Hepatitis E virus replication in human intestinal cells.

OBJECTIVE: Hepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by eating raw or undercooked infected meat for HEV genotype 3 (HEV3) and HEV4. However, little is known about how the ingested HEV reaches the liver or its ability to replicate in intestinal cells. DESIGN: We developed human primary cultures of small intestine epithelial cells and intestinal explants obtained from small bowel resections. The epithelial cells were also polarised on transwells. Cells were infected with Kernow-p6 strain or clinically derived virions. RESULTS: Primary intestinal cells supported the growth of Kernow-p6 strain and HEV1 and HEV3 clinically derived virions. Polarised enterocytes infected with HEV1 and HEV3 strains released HEV particles vectorially: mostly into the apical compartment with a little basally. Iodixanol density gradient centrifugation of enterocyte-derived HEV virions gave bands at a density of 1.06-1.08 g/cm3, corresponding to that of quasi-enveloped HEV particles. Ribavirin therapy inhibited HEV excretion from the basal surface but not from the apical side of infected human enterocytes. HEV virions also infected intestinal tissue explants. Lastly, HEV RNA and antigen were detected in the intestinal crypts of a chronically infected patient. CONCLUSION: HEV can replicate in intestinal cells and reaches the liver as quasi-enveloped virions.

Risk for Hepatitis E Virus Transmission by Solvent/Detergent-Treated Plasma.

Hepatitis E has emerged as a major transfusion-transmitted infectious risk. Two recipients of plasma from 2 lots (A and B) of pooled solvent/detergent-treated plasma were found to be infected by hepatitis E virus (HEV) that was determined to have been transmitted by the solvent/detergent-treated plasma. HEV RNA viral loads were 433 IU in lot A and 55 IU in lot B. Retrospective studies found that 100% (13/13) of evaluable lot A recipients versus 18% (3/17) of evaluable lot B recipients had been infected by HEV (p<0.001), albeit not necessarily at time of transfusion. Among evaluable recipients, 86% with a transfused HEV RNA load >50,000 IU were infected, most likely by the HEV-containing solvent/detergent-treated plasma, versus only 7% with a transfused HEV RNA load <50,000 IU (p<0.001). Overall, solvent/detergent-treated plasma might harbor HEV. Such an occurrence might result in a dose-dependent risk for transfusion-transmitted hepatitis E.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes.

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

Does HEV-3 subtype play a role in the severity of acute hepatitis E?

Hepatitis E virus genotype 3 (HEV-3) is a major aetiologic agent of acute hepatitis in industrialized countries. Two main HEV-3 subtypes are found in Europe: subtypes 3c and 3f. We have analysed the clinical and biological parameters from 100 French immunocompetent patients with an HEV subtype 3f or subtype 3c infection, included in a prospective multicentre study. Stepwise regression analysis found that infections with HEV subtype 3f were associated with fever (OR: 6.1 95%CI: 1.4-26.1), have a greater virus load (OR: 7.4; 95%CI: 1.3-42.2) and require more frequent hospitalization (OR: 7.6; 95%CI: 1.1-51.4) than those infected with subtype 3c. The directed acyclic graph strengthens the multivariate analyses indicating a direct link between the HEV subtype, HEV RNA concentration, fever and hospitalization. Further studies on patients in other European countries are needed to confirm this relationship and determine the underlying mechanism.

Hepatitis E virus persists in the presence of a type III interferon response.

The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs.

Viral load and clinical manifestations of hepatitis E virus genotype 3 infections.

A fraction of plasma donations undergoes hepatitis E virus (HEV) RNA screening since late 2014 in France. The aim of this study was to determine the frequency of HEV RNA as well as the viral load and the evolution of genotype distribution over a 3-year period (2015-2017) in asymptomatic blood donors in comparison with symptomatic patients routinely diagnosed. The overall detection rate of HEV RNA in plasma donations was 0.10% during the 3-year period, with a median viral load of 717 IU/mL (range: <60-168 000 IU/mL) in the 189 samples found HEV RNA positive. One hundred and twenty samples (64.4%) were successfully HEV genotyped. Most strains were HEV3f (n = 54; 44.3%) and HEV3c (n = 46; 37.7%). The genotype distribution was not different throughout the 3-year period and we found no association between the genotype and where the blood donors lived (P = 0.96). The HEV genotype distributions in infected blood donors and symptomatic patients were similar. However, the symptomatic patients had a higher viral load (median 282 000 IU/mL; range: <60-136 000 000 IU/mL; P < 0.01) than the blood donors. Overall, asymptomatic blood donors and patients with symptomatic hepatitis E had similar genotype distributions but the blood donors had lower viral loads.

Diversity of hepatitis E virus genotype 3.

Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.

No evidence of occult hepatitis C or E virus infections in liver-transplant patients with sustained virological response after therapy with direct acting agents.

BACKGROUND AND AIMS: It has been recently suggested that occult hepatitis C virus (HCV) infection and hepatitis E virus (HEV) reactivation might occur after direct acting antiviral agent-induced (DAA-induced) sustained virological response (SVR). The aim of our study was to identify occult HCV and HEV infection in a cohort of organ transplant patients who had achieved SVR and had persistent elevation in liver-enzyme levels. PATIENTS AND METHOD: Sixty-six liver and/or kidney transplant patients were treated with DAAs. All but one achieved SVR12. Twenty-nine (8-39) months post-SVR12, 8 of the 65 patients (12.3%) who achieved SVR12 had persistently elevated liver enzyme levels. In 1 patient, this was related to hepatitis B virus reactivation. In the 7 remaining patients, blood samples (n = 7), liver biopsies (n = 4), and peripheral blood mononuclear cells (PBMCs) (n = 7) were collected simultaneously in order to identify occult HCV or HEV infection. RESULTS: Hepatitis C virus RNA and HEV RNA were not detected in serum, liver tissues, or PBMCs. No HEV reactivation was observed after HCV clearance in patients who had anti-HEV IgG. CONCLUSION: Our study suggests that there is no occult HCV or HEV infection in transplant patients after successful treatment of HCV infection with DAAs, even in patients with a persistent elevation of liver enzyme levels. However, due to the small number of patients included in our study, this finding should be confirmed in a larger cohort.

Chronic Genotype 3 Hepatitis E in Pregnant Woman Receiving Infliximab and Azathioprine.

Acute hepatitis E virus infection during pregnancy has a high fatality rate in developing countries. Little data are available on chronic infection in pregnant women. We report a case of chronic hepatitis E during treatment with infliximab and azathioprine, without adverse event during pregnancy and with spontaneous resolution after delivery.

Hepatitis E virus-associated cryoglobulinemia in solid-organ-transplant recipients.

BACKGROUND & AIMS: An association between hepatitis E virus (HEV) infection and cryoglobulinemia has been suggested. The aims of this study were to assess the prevalence of cryoglobulinemia during HEV infection in solid-organ-transplant (SOT) recipients, to describe its outcomes under ribavirin therapy and to evaluate its effects on kidney function and histology. METHODS: Between November 2005 and June 2016, 128 cases of HEV infection were diagnosed among SOT recipients followed in our institution. Cryoglobulinemia data obtained from 66 patients during acute-phase HEV and 51 patients during chronic-phase HEV were compared to a historical control group of 89 SOT recipients without HEV markers. Cryoglobulins were also monitored in a group of 43 patients treated by ribavirin. RESULTS: The prevalence of cryoglobulinemia was increased in HEV-infected SOT patients during a chronic phase (52.9%) compared to HEV-infected SOT patients at acute phase (36.4%) (P = .1) and to HEV-negative SOT patients (23.6%) (P < .001). HEV infection was identified as an independent predictive factor for cryoglobulinemia (OR 2.3, CI 95%: 1.17-4.55, P = .02). After ribavirin therapy and HEV clearance, the prevalence of cryoglobulin was significantly decreased from 53.5% to 20.9% (P = .003). Kidney function was significantly worse and proteinuria tended to be higher in chronically HEV-infected patients with cryoglobulinemia compared to those without cryoglobulinemia. Membranoproliferative glomerulonephritis was diagnosed in 2 patients, of which 1 had detectable cryoglobulinemia. CONCLUSIONS: In conclusion, a relationship between HEV and cryoglobulin formation seems to exist. However, the clinical impact of cryoglobulinemia in SOT patients infected with HEV has to be confirmed.

[Hepatitis E virus: from the infected organism to the cellular response]. / Virus de l'hépatite E : de l'organisme infecté à la réponse cellulaire.

Hepatitis E virus (HEV) presents a worldwide distribution. In developing countries, hepatitis E, related to HEV1 and HEV2, is a waterborne disease. In developed countries, hepatitis E is a zoonotic disease due to HEV3 and HEV4. It is mainly transmitted through meat consumption from animal reservoirs such as pig, boar, deer and rabbit. New clinical forms include neurological manifestations that are now clearly associated with HEV3 infection. Recent studies showed that ORF1 polyprotein was able to disrupt the innate immune response. It was also shown that ORF2 protein exists at least in two forms: a free, glycosylated form and a non-glycosylated form, which assembles to form the capsid. Lastly, it was shown that ORF3 protein, involved in the virus egress, acts as a viroporin. New culture systems and animal models have been developed recently, and will be very helpful to complete our understanding of HEV life cycle and pathogenesis.

Hepatitis E Virus Infection in Solid Organ Transplant Recipients, France.

The rate of transfusion-transmitted hepatitis E virus (HEV) in transplant recipients is unknown. We identified 60 HEV-positive solid organ transplant patients and retrospectively assessed their blood transfusions for HEV. Seven of 60 patients received transfusions; 3 received HEV-positive blood products. Transfusion is not the major route of infection in this population.

Rabbit Hepatitis E Virus Infections in Humans, France.

Hepatitis E virus (HEV) has been detected in rabbits, but whether rabbit HEV strains can be transmitted to humans is not known. Of 919 HEV-infected patients in France during 2015-2016, five were infected with a rabbit HEV strain. None of the patients had direct contact with rabbits, suggesting foodborne or waterborne infections.

Transfusion-acquired hepatitis E infection misdiagnosed as severe critical illness polyneuromyopathy in a heart transplant patient.

This is the case of a 56-year-old man who underwent heart transplantation. Within the first postoperative days, his respiratory and limb muscles weakened, which was attributed to critical illness polyneuromyopathy (CIPM). At day 70 post transplantation, he had increased liver enzyme levels and acute hepatitis E virus (HEV) infection was diagnosed. HEV RNA was found in the serum, stools, and cerebrospinal fluid. Results of further investigations suggested a possible HEV-related polyradiculoneuropathy. At transplantation, the patient was negative for immunoglobulin (Ig)G, IgM, and HEV RNA. A trace-back procedure identified the source of infection and concluded that HEV infection was contracted from blood transfusion 12 days prior to transplantation from an HEV RNA-positive donor. Tests of the organ donor for HEV were negative. Phylogenetic analysis revealed sequence homology between the HEV-3 strain of the patient and the HEV-3 strain of the blood donor. Despite ribavirin treatment, the patient died on day 153 post transplantation from multiorgan failure. In conclusion, patients with hepatitis or neuropathic illness who have received blood products should be screened for HEV.