Hepatitis E virus: has anything changed?

PURPOSE OF REVIEW: Infection with hepatitis E virus (HEV) is a global health concern, yet a clinically underdiagnosed cause of acute and chronic hepatitis. The WHO estimates that 20 million people are infected with HEV annually, yet the epidemiology, diagnosis and prevention remain elusive in many clinical settings. RECENT FINDINGS: Orthohepevirus A (HEV-A) genotypes 1 and 2 cause acute, self-limited hepatitis through faecal-oral transmission. In 2022, the first-ever vaccine campaign was implemented as a response to an HEV outbreak in an endemic region. HEV-A genotypes 3 and 4 are zoonotic infections that primarily cause chronic HEV infection in immunosuppressed populations. Pregnant women and immunocompromised persons are at high risk for severe illness in some settings. Another recent advance in our knowledge of HEV is the zoonotic transmission of Orthohepevirus C (HEV-C) to humans, presumably from contact with rodents and/or their excrement. Previously, HEV infection in humans was presumed to be limited to HEV-A only. SUMMARY: Clinical recognition and accurate diagnosis are essential to the management of HEV infection and understanding the global burden of the disease. Epidemiology affects clinical presentations. Targeted response strategies in HEV outbreaks are needed for the prevention of disease, and vaccine campaigns may prove to be an effective part of these strategies.

Optimization of the molecular diagnosis of the acute hepatitis E virus infection.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Integrative Multiomics and Regulatory Network Analyses Uncovers the Role of OAS3, TRAFD1, miR-222-3p, and miR-125b-5p in Hepatitis E Virus Infection.

The hepatitis E virus (HEV) is a long-ignored virus that has spread globally with time. It ranked 6th among the top risk-ranking viruses with high zoonotic spillover potential; thus, considering its viral threats is a pressing priority. The molecular pathophysiology of HEV infection or the underlying cause is limited. Therefore, we incorporated an unbiased, systematic methodology to get insights into the biological heterogeneity associated with the HEV. Our study fetched 93 and 2016 differentially expressed genes (DEGs) from chronic HEV (CHEV) infection in kidney-transplant patients, followed by hub module selection from a weighted gene co-expression network (WGCN). Most of the hub genes identified in this study were associated with interferon (IFN) signaling pathways. Amongst the genes induced by IFNs, the 2'-5'-oligoadenylate synthase 3 (OAS3) protein was upregulated. Protein-protein interaction (PPI) modular, functional enrichment, and feed-forward loop (FFL) analyses led to the identification of two key miRNAs, i.e., miR-222-3p and miR-125b-5p, which showed a strong association with the OAS3 gene and TRAF-type zinc finger domain containing 1 (TRAFD1) transcription factor (TF) based on essential centrality measures. Further experimental studies are required to substantiate the significance of these FFL-associated genes and miRNAs with their respective functions in CHEV. To our knowledge, it is the first time that miR-222-3p has been described as a reference miRNA for use in CHEV sample analyses. In conclusion, our study has enlightened a few budding targets of HEV, which might help us understand the cellular and molecular pathways dysregulated in HEV through various factors. Thus, providing a novel insight into its pathophysiology and progression dynamics.

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors.

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 µg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 µg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 µg per gram of fresh leaf tissue; the yield after purification was up to 20 µg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1−1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

Expression and Immunogenicity of SARS-CoV-2 Virus-Like Particles based on Recombinant Truncated HEV-3 ORF2 Capsid Protein.

COVID-19 is an emerging disease that poses a severe threat to global public health. As such, there is an urgent demand for vaccines against SARS-CoV-2, the virus that causes COVID-19. Here, we describe a virus-like nanoparticle candidate vaccine against SARS-CoV-2 produced by an E. coli expression system. The fusion protein of a truncated ORF2-encoded protein of aa 439~608 (p170) from hepatitis E virus CCJD-517 and the receptor-binding domain of the spike protein from SARS-CoV-2 were expressed, purified and characterized. The antigenicity and immunogenicity of p170-RBD were evaluated in vitro and in Kunming mice. Our investigation revealed that p170-RBD self-assembled into approximately 24 nm virus-like particles, which could bind to serum from vaccinated people (p < 0.001) and receptors on cells. Immunization with p170-RBD induced the titer of IgG antibody vaccine increased from 14 days post-immunization and was significantly enhanced after a booster immunization at 28 dpi, ultimately reaching a peak level on 42 dpi with a titer of 4.97 log10. Pseudovirus neutralization tests showed that the candidate vaccine induced a strong neutralizing antibody response in mice. In this research, we demonstrated that p170-RBD possesses strong antigenicity and immunogenicity and could be a potential candidate for use in future SARS-CoV-2 vaccine development.

Hepatitis E Virus Seroprevalence and Associated Risk Factors in Pregnant Women Attending Antenatal Consultations in Senegal.

In West Africa, research on the hepatitis E virus (HEV) is barely covered, despite the recorded outbreaks. The low level of access to safe water and adequate sanitation is still one of the main factors of HEV spread in developing countries. HEV infection induces acute or sub-clinical liver diseases with a mortality rate ranging from 0.5 to 4%. The mortality rate is more alarming (15 to 25%) among pregnant women, especially in the last trimester of pregnancy. Herein, we conducted a multicentric socio-demographic and seroepidemiological survey of HEV in Senegal among pregnant women. A consecutive and non-redundant recruitment of participants was carried out over the period of 5 months, from March to July 2021. A total of 1227 consenting participants attending antenatal clinics responded to a standard questionnaire. Plasma samples were collected and tested for anti-HEV IgM and IgG by using the WANTAI HEV-IgM and IgG ELISA assay. The overall HEV seroprevalence was 7.8% (n = 96), with 0.5% (n = 6) and 7.4% (n = 91) for HEV IgM and HEV IgG, respectively. One of the participant samples was IgM/IgG-positive, while four were declared indeterminate to anti-HEV IgM as per the manufacturer's instructions. From one locality to another, the seroprevalence of HEV antibodies varied from 0 to 1% for HEV IgM and from 1.5 to 10.5% for HEV IgG. The data also showed that seroprevalence varied significantly by marital status (p < 0.0001), by the regularity of income (p = 0.0043), and by access to sanitation services (p = 0.0006). These data could serve as a basis to setup national prevention strategies focused on socio-cultural, environmental, and behavioral aspects for a better management of HEV infection in Senegal.

Seroprevalence of Anti-Hepatitis E Virus Antibodies among Patients from a Tertiary Hospital from Northeast Romania.

Background and Objectives. Being an enterically transmitted pathogen with a growing prevalence in developed countries, hepatitis E virus (HEV) infection remains an underdiagnosed disease in Eastern Europe. As far as Romania is concerned, only a few studies address this issue. Our goal was to estimate the prevalence of serum anti-HEV IgA/IgM/IgG antibodies in a group of patients admitted to the Clinical Hospital for Infectious Diseases "St. Parascheva" Iasi. Materials and Methods. The cross-sectional study consisted of enrollment of 98 patients admitted to the clinic for COVID-19 over a period of three months in 2020. Results. The median age in our study was 73 years, with an equal gender ratio and with a predominance of people from the urban environment (75%). The overall HEV antibody seroprevalence was 12.2%. The main risk factors associated with HEV infection were consumption of water from unsafe sources (58.3% HEV-positive patients vs. 26.7% HEV-negative patients, p = 0.026) and improperly cooked meat (58.3% HEV-positive patients vs. 23.2% HEV-negative patients, p = 0.01). Zoonotic transmission was an important criterion in our study, with patients reporting contact with pigs, poultry, rats, or other farms animals, but no significant differences were found between HEV antibody positive and negative groups. Conclusions. The seroprevalence rate of HEV antibodies was similar to other previous reports from our area but higher than in most European countries. The fact that HEV antibodies were detected in patients without identifiable risk factors for hepatitis E is evidence of subclinical infection as a silent threat.

Genetic diversity of hepatitis E virus (HEV) in imported and domestic camels in Saudi Arabia.

Camels gained attention since the discovery of MERS-CoV as intermediary hosts for potentially epidemic zoonotic viruses. DcHEV is a novel zoonotic pathogen associated with camel contact. This study aimed to genetically characterize DcHEV in domestic and imported camels in Saudi Arabia. DcHEV was detected by RT-PCR in serum samples, PCR-positive samples were subjected to sequencing and phylogenetic analyses. DcHEV was detected in 1.77% of samples with higher positivity in domestic DCs. All positive imported dromedaries were from Sudan with age declining prevalence. Domestic DcHEV sequences clustered with sequences from Kenya, Somalia, and UAE while imported sequences clustered with one DcHEV isolate from UAE and both sequences clustered away from isolates reported from Pakistan. Full-genome sequences showed 24 amino acid difference with reference sequences. Our results confirm the detection of DcHEV in domestic and imported DCs. Further investigations are needed in human and camel populations to identify DcHEV potential zoonosis threat.

Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

Nosocomial transmission of hepatitis E virus and development of chronic infection: The wider impact of COVID-19.

BACKGROUND: Transmission of hepatitis E virus (HEV) within the healthcare setting is extremely rare. Additionally, the development of chronic HEV infection in association with severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) infection and/or its immunomodulatory therapy has not been reported previously. AIMS: To describe the investigation and management of a nosocomial HEV transmission incident during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Epidemiological and molecular investigation of two individuals hospitalised with COVID-19 who were both diagnosed with HEV infection. RESULTS: Findings from our investigation were consistent with transmission of HEV from one patient with a community-acquired HEV infection to another individual (identical HEV sequences were identified in the two patients), most likely due to a breach in infection control practices whilst both patients shared a bed space on the intensive care unit (ICU). Chronic HEV infection requiring treatment with ribavirin developed in one patient with prolonged lymphopaenia attributable to COVID-19 and/or the immunomodulators received for its treatment. Further investigation did not identify transmission of HEV to any other patients or to healthcare workers. CONCLUSIONS: The extraordinary demands that the COVID-19 pandemic has placed on all aspects of healthcare, particularly within ICU settings, has greatly challenged the ability to consistently maintain optimal infection prevention and control practices. Under the significant pressures of the COVID-19 pandemic a highly unusual nosocomial HEV transmission incident occurred complicated further by progression to a chronic HEV infection in one patient.

Hepatitis E in Bangladesh: Insights From a National Serosurvey.

BACKGROUND: Hepatitis E virus (HEV) genotypes 1 and 2 are a major cause of avoidable morbidity and mortality in South Asia. Despite the high risk of death among infected pregnant women, scarce incidence data has been a contributing factor to global policy recommendations against the introduction of licensed hepatitis E vaccines, one of the only effective prevention tools. METHODS: We tested serum from a nationally representative serosurvey in Bangladesh for anti-HEV immunoglobulin G and estimated seroprevalence. We used Bayesian geostatistical models to generate high-resolution maps of seropositivity and examined variability in seropositivity by individual-level, household-level, and community-level risk factors using spatial logistic regression. RESULTS: We tested serum samples from 2924 individuals from 70 communities representing all divisions of Bangladesh and estimated a national seroprevalence of 20% (95% confidence interval [CI], 17%-24%). Seropositivity increased with age and male sex (odds ratio, 2.2 male vs female; 95% CI, 1.8-2.8). Community-level seroprevalence ranged widely (0-78%) with higher seroprevalence in urban areas, including Dhaka, with a 3.0-fold (95% credible interval, 2.3-3.7) higher seroprevalence than the rest of the country. CONCLUSIONS: Hepatitis E infections are common throughout Bangladesh. Strengthening surveillance for hepatitis E, especially in urban areas, can provide additional evidence to appropriately target interventions.

Natural antiviral compound silvestrol modulates human monocyte-derived macrophages and dendritic cells.

Outbreaks of infections with viruses like Sars-CoV-2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona-, Ebola-, Zika-, Picorna-, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down-regulated several pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, CCL2, CCL18) and increased TNF-α during differentiation and activation of M1-macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti-inflammatory potential of M2-macrophages by increasing expression of anti-inflammatory surface markers CD206, TREM2 and reducing release of pro-inflammatory IL-8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down-regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.