Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication.

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

[Research progress on hepatitis E virus-related liver failure].

Hepatitis E virus (HEV) is one of the important causes of acute viral hepatitis worldwide, and its incidence rate is increasing year by year. HEV infection can lead to acute, subacute, or acute-on-chronic liver failure with a high mortality rate among some particular patient population, who are pregnant women, older, chronic liver diseases like chronic hepatitis B and cirrhosis, or immunocompromised. The clinical characteristics of HEV infection, the pathogenesis of HEV-related liver failure, and the progress in diagnosis and treatment will be elaborated upon in this article from these three aspects in order to improve clinicians' ability to identify and prevent HEV-related liver failure and its clinical outcomes.

Infectious hepatitis E virus is associated with the mature sperm head.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV associated pregnancy mortality has been reported as up to 30% in humans. Recent findings suggest HEV may elicit effects directly in the reproductive system with HEV protein found in the testis, viral RNA in semen, and viral replication occurring in placental cell types. Using a natural host model for HEV infection, pigs, we demonstrate infectious HEV within the mature spermatozoa and altered sperm viability from HEV infected pigs. HEV isolated from sperm remained infectious suggesting a potential transmission route via sexual partners. Our findings suggest that HEV should be explored as a possible sexually transmittable disease. Our findings propose that infection routes outside of oral and intravenous infection need to be considered for their potential to contribute to higher mortality in HEV infections when pregnancy is involved and in HEV disease in general.

Hepatitis E Virus in Domestic Ruminants and Virus Excretion in Milk-A Potential Source of Zoonotic HEV Infection.

The hepatitis E virus is a serious health concern worldwide, with 20 million cases each year. Growing numbers of autochthonous HEV infections in industrialized nations are brought on via the zoonotic transmission of HEV genotypes 3 and 4. Pigs and wild boars are the main animal reservoirs of HEV and play the primary role in HEV transmission. Consumption of raw or undercooked pork meat and close contact with infected animals are the most common causes of hepatitis E infection in industrialized countries. However, during the past few years, mounting data describing HEV distribution has led experts to believe that additional animals, particularly domestic ruminant species (cow, goat, sheep, deer, buffalo, and yak), may also play a role in the spreading of HEV. Up to now, there have not been enough studies focused on HEV infections associated with animal milk and the impact that they could have on the epidemiology of HEV. This critical analysis discusses the role of domestic ruminants in zoonotic HEV transmissions. More specifically, we focus on concerns related to milk safety, the role of mixed farming in cross-species HEV infections, and what potential consequences these may have on public health.

Prevalence of Acute Hepatitis E Virus Infections in Swiss Blood Donors 2018-2020.

INTRODUCTION: Hepatitis E virus (HEV) genotype 3 is the major cause of acute viral hepatitis in several European countries. It is acquired mainly by ingesting contaminated pork, but has also been reported to be transmitted through blood transfusion. Although most HEV infections, including those via blood products, are usually self-limiting, they may become chronic in immunocompromised persons. It is thus essential to identify HEV-infected blood donations to prevent transmission to vulnerable recipients. AIMS: Prior to the decision whether to introduce HEV RNA screening for all Swiss blood donations, a 2-year nationwide prevalence study was conducted. METHODS: All blood donations were screened in pools of 12-24 samples at five regional blood donation services, and HEV RNA-positive pools were subsequently resolved to the individual donation index donation (X). The viral load, HEV IgG and IgM serology, and HEV genotype were determined. Follow-up investigations were conducted on future control donations (X + 1) and previous archived donations of the donor (X - 1) where available. RESULTS: Between October 2018 and September 2020, 541,349 blood donations were screened and 125 confirmed positive donations were identified (prevalence 1:4331 donations). At the time of blood donation, the HEV RNA-positive individuals were symptom-free. The median viral load was 554 IU/mL (range: 2.01-2,500,000 IU/mL). Men (88; 70%) were more frequently infected than women (37; 30%), as compared with the sex distribution in the Swiss donor population (57% male/43% female, p < 0.01). Of the 106 genotyped cases (85%), all belonged to genotype 3. Two HEV sub-genotypes predominated; 3h3 (formerly 3s) and 3c. The remaining sub-genotypes are all known to circulate in Europe. Five 3ra genotypes were identified, this being a variant associated with rabbits. In total, 85 (68%) X donations were negative for HEV IgM and IgG. The remaining 40 (32%) were positive for HEV IgG and/or IgM, and consistent with an active infection. We found no markers of previous HEV in 87 of the 89 available and analyzed archive samples (X - 1). Two donors were HEV IgG-positive in the X - 1 donation suggesting insufficient immunity to prevent HEV reinfection. Time of collection of the 90 (72%) analyzed X + 1 donations varied between 2.9 and 101.9 weeks (median of 35 weeks) after X donation. As expected, none of those tested were positive for HEV RNA. Most donors (89; 99%) were positive for anti-HEV lgG/lgM (i.e., seroconversion). HEV lgM-positivity (23; 26%) indicates an often-long persistence of lgM antibodies post-HEV infection. CONCLUSION: The data collected during the first year of the study provided the basis for the decision to establish mandatory HEV RNA universal screening of all Swiss blood donations in minipools, a vital step in providing safer blood for all recipients, especially those who are immunosuppressed.

Systematic review and meta-analysis of hepatitis E seroprevalence in Southeast Asia: a comprehensive assessment of epidemiological patterns.

The burden of hepatitis E in Southeast Asia is substantial, influenced by its distinct socio-economic and environmental factors, as well as variations in healthcare systems. The aim of this study was to assess the pooled seroprevalence of hepatitis E across countries within the Southeast Asian region by the UN division.The study analyzed 66 papers across PubMed, Web of Science, and Scopus databases, encompassing data from of 44,850 individuals focusing on anti-HEV seroprevalence. The investigation spanned nine countries, excluding Brunei and East Timor due to lack of data. The pooled prevalence of anti-HEV IgG was determined to be 21.03%, with the highest prevalence observed in Myanmar (33.46%) and the lowest in Malaysia (5.93%). IgM prevalence was highest in Indonesia (12.43%) and lowest in Malaysia (0.91%). The study stratified populations into high-risk (farm workers, chronic patients) and low-risk groups (general population, blood donors, pregnant women, hospital patients). It revealed a higher IgG-28.9%, IgM-4.42% prevalence in the former group, while the latter group exhibited figures of 17.86% and 3.15%, respectively, indicating occupational and health-related vulnerabilities to HEV.A temporal analysis (1987-2023), indicated an upward trend in both IgG and IgM prevalence, suggesting an escalating HEV burden.These findings contribute to a better understanding of HEV seroprevalence in Southeast Asia, shedding light on important public health implications and suggesting directions for further research and intervention strategies.Key pointsResearch QuestionInvestigate the seroprevalence of hepatitis E virus (HEV) in Southeast Asian countries focusing on different patterns, timelines, and population cohorts.FindingsSporadic Transmission of IgG and IgM Prevalence:• Pooled anti-HEV IgG prevalence: 21.03%• Pooled anti-HEV IgM prevalence: 3.49%Seroprevalence among specific groups:High-risk group (farm workers and chronic patients):• anti-HEV IgG: 28.9%• anti-HEV IgM: 4.42%Low-risk group (general population, blood donors, pregnant women, hospital patients):• anti-HEV IgG: 17.86%• anti-HEV IgM: 3.15%Temporal Seroprevalence of HEV:Anti-HEV IgG prevalence increased over decades (1987-1999; 2000-2010; 2011-2023): 12.47%, 18.43%, 29.17% as an anti-HEV IgM prevalence: 1.92%, 2.44%, 5.27%ImportanceProvides a comprehensive overview of HEV seroprevalence in Southeast Asia.Highlights variation in seroprevalence among different population groups.Reveals increasing trend in HEV seroprevalence over the years.Distinguishes between sporadic and epidemic cases for a better understanding of transmission dynamics.

Prevalence and molecular characterization of hepatitis E virus (HEV) from wild rodents in Hubei Province, China.

Hepatitis E, caused by the hepatitis E virus (HEV), is a global public health issue. Low similarity between the gene sequences of mouse and human HEV led to the belief that the risk of human infection was low. Recent reports of chronic and acute hepatitis E caused by murine HEV infection in humans in Hong Kong have raised global concerns. Therefore, it is crucial to investigate the epidemiology and prevalence of HEV in China. We comprehensively analyzed different rodent HEV strains to understand rocahepevirus occurrence in Hubei Province, China. The HEV positivity rate for was 6.43% (73/1136). We identified seven near-full-length rocahepevirus strains and detected rat HEV antigens in tissues from different mouse species. HEV has extensive tissue tropism and a high viral load in the liver. We highlight the genetic diversity of HEVs in rodents and underscore the importance of paying attention to their variation and evolution.

Multifunctional ORF3 protein of hepatitis E virus.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is transmitted primarily through the fecal-oral route and can cause acute hepatitis in humans. Since HEV was identified as a zoonotic pathogen, different species of HEV strains have been globally identified from various hosts, leading to an expanding range of hosts. The HEV genome consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. The ORF3 protein is the smallest but has many functions in HEV release and pathogenesis. In this review, we systematically summarize recent progress in understanding the functions of the HEV ORF3 protein in virion release, biogenesis of quasi-enveloped viruses, antigenicity, and host environmental regulation. This review will help us to understand HEV replication and pathogenesis mechanisms better.

Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein.

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Deregulation of immune response contributing to fulminant hepatitis in HEV infected pregnant women.

Hepatitis E virus (HEV) infection in pregnant women is associated with a wide spectrum of adverse consequences for both mother and fetus. The high mortality in this population appears to be associated with hormonal changes and consequent immunological changes. This study conducted an analysis of immune responses in pregnant women infected with HEV manifesting varying severity. Data mining analysis of the GSE79197 was utilized to examine differentially biological functions in pregnant women with HEV infection (P-HEV) versus without HEV infection (P-nHEV), P-HEV progressing to ALF (P-ALF) versus P-HEV, and P-HEV versus non-pregnant women with HEV infection (nP-HEV). We found cellular response to interleukin and immune response-regulating signalings were activated in P-HEV compared with P-nHEV. However, there was a significant decrease of immune responses, such as T cell activation, leukocyte cell-cell adhesion, regulation of lymphocyte activation, and immune response-regulating signaling pathway in P-ALF patient than P-HEV patient. Compared with nP-HEV, MHC protein complex binding function was inhibited in P-HEV. Further microRNA enrichment analysis showed that MAPK and T cell receptor signaling pathways were inhibited in P-HEV compared with nP-HEV. In summary, immune responses were activated during HEV infection while being suppressed when developing ALF during pregnancy, heightening the importance of immune mediation in the pathogenesis of severe outcome in HEV infected pregnant women.

Persistence of hepatitis E virus (HEV) subtypes 3c and 3e: Long-term cold storage and heat treatments.

Hepatitis E virus (HEV) is the causative agent of foodborne infections occurring in high income countries mainly by consumption of undercooked and raw pork products. The virus is zoonotic with pigs and wild boars as the main reservoirs. Several studies proved the presence of HEV-RNA in pork liver sausages, pâté and other pork by-products. However, the detection of HEV nucleic acids does not necessary correspond to infectious virus and information on the persistence of the virus in the food is still limited. To which extent and how long the virus can survive after conventional industrial and home-made conservation and cooking procedures is largely unknown. In the present study, we investigated the persistence of two subtypes of HEV-3, by measuring the viral RNA on cell supernatant of infected A549 cells, after long-term storage at +4 °C and -20 °C and after heating for short or long-time span. Results confirmed that either low temperature storage (+4 °C) or freezing (-20 °C) do not influence the survival of the virus, and only a moderate reduction of presence of its RNA after 12 weeks at +4 °C was observed. To the other side, heating at 56 °C for long time (1 h) or at higher temperatures (>65 °C) for shorter time inactivated the virus successfully.

Kinetics of Hepatitis E Virus Infections in Asymptomatic Persons.

To determine the kinetics of hepatitis E virus (HEV) in asymptomatic persons and to evaluate viral load doubling time and half-life, we retrospectively tested samples retained from 32 HEV RNA-positive asymptomatic blood donors in Germany. Close-meshed monitoring of viral load and seroconversion in intervals of ≈4 days provided more information about the kinetics of asymptomatic HEV infections. We determined that a typical median infection began with PCR-detectable viremia at 36 days and a maximum viral load of 2.0 × 104 IU/mL. Viremia doubled in 2.4 days and had a half-life of 1.6 days. HEV IgM started to rise on about day 33 and peaked on day 36; IgG started to rise on about day 32 and peaked on day 53. Although HEV IgG titers remained stable, IgM titers became undetectable in 40% of donors. Knowledge of the dynamics of HEV viremia is useful for assessing the risk for transfusion-transmitted hepatitis E.

An outbreak of hepatitis E virus genotype 4d caused by consuming undercooked pig liver in a nursing home in Zhejiang Province, China.

Hepatitis E infection is typically caused by contaminated water or food. In July and August 2022, an outbreak of hepatitis E was reported in a nursing home in Zhejiang Province, China. Local authorities and workers took immediate actions to confirm the outbreak, investigated the sources of infection and routes of transmission, took measures to terminate the outbreak, and summarized the lessons learned. An epidemiological investigation was conducted on all individuals in the nursing home, including demographic information, clinical symptoms, history of dietary, water intake and contact. Stool and blood samples were collected from these populations for laboratory examinations. The hygiene environment of the nursing home was also investigated. A case-control study was conducted to identify the risk factors for this outbreak. Of the 722 subjects in the nursing home, 77 were diagnosed with hepatitis E, for an attack rate of 10.66 %. Among them, 18 (23.38 %, 18/77) individuals had symptoms such as jaundice, fever, and loss of appetite and were defined as the population with hepatitis E. The average age of people infected with hepatitis E virus (HEV) was 59.96 years and the attack rate of hepatitis E among women (12.02 %, 59/491) was greater than that among men (7.79 %, 18/231). The rate was the highest among caregivers (22.22 %, 32/144) and lowest among logistics personnel (6.25 %, 2/32); however, these differences were not statistically significant (P > 0.05). Laboratory sequencing results indicated that the genotype of this hepatitis E outbreak was 4d. A case-control study showed that consuming pig liver (odds ratio (OR) = 7.50; 95 % confidence interval [CI]: 3.84-16.14, P < 0.001) and consuming raw fruits and vegetables (OR = 5.92; 95 % CI: 1.74-37.13, P = 0.017) were risk factors for this outbreak of Hepatitis E. Moreover, a monitoring video showed that the canteen personnel did not separate raw and cooked foods, and pig livers were cooked for only 2 min and 10 s. Approximately 1 month after the outbreak, an emergency vaccination for HEV was administered. No new cases were reported after two long incubation periods (approximately 4 months). The outbreak of HEV genotype 4d was likely caused by consuming undercooked pig liver, resulting in an attack rate of 10.66 %. This was related to the rapid stir-frying cooking method and the hygiene habit of not separating raw and cooked foods.

Hepatitis E Virus Infection in Voluntary Blood Donors in the Russian Federation.

Transfusion-transmitted hepatitis E virus (HEV) infection is an increasing concern in many countries. We investigated the detection rate of HEV viremia in blood donors in Russia. A total of 20,405 regular repetitive voluntary non-renumerated blood donors from two regions (Moscow and Belgorod) were screened for HEV RNA using the cobas® HEV test in mini-pools of six plasma samples. Samples from each reactive pool were tested individually. The average HEV RNA prevalence was 0.024% (95% CI: 0.01-0.05%), or 1 case per 4081 donations. No statistically significant differences in HEV RNA prevalence were observed between the two study regions. The PCR threshold cycle (Ct) values ranged from 25.0 to 40.5 in reactive pools, and from 20.9 to 41.4 in reactive plasma samples when tested individually. The HEV viremic donors had different antibody patterns. Two donor samples were reactive for both anti-HEV IgM and IgG antibodies, one sample was reactive for anti-HEV IgM and negative for anti-HEV IgG, and two samples were seronegative. At follow-up testing 6 months later, on average, four donors available for follow-up had become negative for HEV RNA and positive for anti-HEV IgG. The HEV ORF2 sequence belonging to HEV-3 sub-genotype 3a was obtained from one donor sample. The sequencing failed in the other four samples from viremic donors, presumably due to the low viral load. In conclusion, the HEV RNA detection rate in blood donors in Russia corresponds with data from other European countries, including those that implemented universal donor HEV screening. These data support the implementation of HEV RNA donor screening to reduce the risk of transfusion-transmitted HEV infection in Russia.

Hepatitis E Seroprevalence and Detection of Genotype 3 Strains in Domestic Pigs from Sierra Leone Collected in 2016 and 2017.

Hepatitis E virus (HEV) is the main cause of acute hepatitis in humans worldwide and is responsible for a large number of outbreaks especially in Africa. Human infections are mainly caused by genotypes 1 and 2 of the genus Paslahepevirus, which are exclusively associated with humans. In contrast, viruses of genotypes 3 and 4 are zoonotic and have their main reservoir in domestic and wild pigs, from which they can be transmitted to humans primarily through the consumption of meat products. Both genotypes 3 and 4 are widespread in Europe, Asia, and North America and lead to sporadic cases of hepatitis E. However, there is little information available on the prevalence of these genotypes and possible transmission routes from animal reservoirs to humans in African countries. We therefore analysed 1086 pig sera collected in 2016/2017 in four districts in Sierra Leone for antibodies against HEV using a newly designed in-house ELISA. In addition, the samples were also analysed for HEV RNA by quantitative real-time RT-PCR. The overall seroprevalence in Sierra Leone was low with only 44 positive sera and a prevalence of 4.0%. Two serum pools were RT-PCR-positive and recovered partial sequences clustered into the genotype 3 (HEV-3) of the order Paslahepevirus, species Paslahepevirus balayani. The results are the first evidence of HEV-3 infection in pigs from Sierra Leone and demonstrate a low circulation of the virus in these animals to date. Further studies should include an examination of humans, especially those with close contact with pigs and porcine products, as well as environmental sampling to evaluate public health effects within the framework of a One Health approach.

Evaluation of an automated platform for the detection of HEV RNA in plasma and stool.

INTRODUCTION: We evaluated the performance of the automated Altostar HEV RNA platform for detecting HEV RNA. METHODS AND RESULTS: Clinical performance was determined by testing 81 plasma samples and 10 fecal samples manually quantified previously with the Realstar RT-PCR assay using the Magnapure instrument for extraction. The assays were concordant for 79/81 plasma samples (97.5%) and 10/10 (100%) fecal samples. The two plasma samples that tested negative with the Altostar assay had a very low HEV RNA concentration (1.6 and 1.4 log10 IU/ml). Quantitative results obtained with the automated platform and the manual workflow were highly correlated (ρ= 0.98, p<0.01). The intra-run and inter-run standard deviation were 0.09 IU/ml and 0.13 IU/ml respectively. The assay was linear from 2 to 6 log IU/ml. The limit of detection determined by Probit analysis with the WHO HEV RNA standard was 7.6 [95% CI: 4.4-52.5] IU/ml. CONCLUSIONS: The Altostar platform enables highly accurate testing for the detection of HEV RNA in stool and the quantification of HEV RNA in plasma. This allowed us to shorten turnaround times and to save time for the technical staff.

Previous hepatitis E virus infection is associated with increased liver stiffness in patients with autoimmune hepatitis.

INTRODUCTION: Autoimmune hepatitis (AIH) is a chronic, progressive liver disease that, in most cases, may require lifelong immunosuppression. Hepatitis E virus (HEV) is a leading cause of acute, typically self­limited hepatitis worldwide, although immunocompromised patients may develop chronic hepatitis. OBJECTIVES: We aimed to evaluate the impact of HEV seropositivity on the clinical course of AIH. PATIENTS AND METHODS: The study involved a group of 374 adult patients with AIH (68% women; median [interquartile range] age, 34 [18-83] years; 38% with liver cirrhosis). Serum HEV immunoglobulin (Ig) G and IgM antibodies were measured by enzyme­linked immunosorbent assay, liver fibrosis was assessed by liver stiffness measurement (LSM), and liver cirrhosis was confirmed with liver histology or LSM. RESULTS: Fifty­five patients (15%) with AIH were HEV IgG­positive. These patients were older (P <0.001), had higher body mass index, and higher value of LSM (both P <0.05). In a multivariable model including the levels of alanine aminotransferase and IgG, the HEV seropositive status was associated with an increased risk of advanced liver fibrosis with odds ratio of 3.69 (95% CI, 1.26-10.77; P = 0.02), as reflected by liver stiffness equal to or above 10.5 kPa. HEV IgG seropositivity was, however, not linked with the type of treatment or worse AIH outcome. Seroprevalence of HEV in the patients with AIH was lower than in the general population of Polish blood donors (43%). CONCLUSIONS: Patients with AIH and HEV IgG­positive status seem to be at risk of more advanced liver fibrosis. However, the overall seroprevalence of HEV IgG is lower in patients with AIH than in blood donors in Poland.

Increased Prevalence of Antibodies to Hepatitis E Virus in Patients with Neurocysticercosis.

We explored the association between serological status for hepatitis E and neurocysticercosis (NCC) in neurologic patients attending a national neurological referral center in Lima, Perú, between the years 2008 and 2012. Anti-hepatitis E antibodies were evaluated in patients with and without NCC, and a control group of rural general population. Anti-hepatitis E IgG was found in 23.8% of patients with NCC, compared with 14.3% in subjects without NCC from a general rural population (P = 0.023) and 14.4% in subjects with neurological complaints without NCC (P = 0.027). Seropositive patients had a median age of 44 years compared with 30 years in seronegative patients (P <0.001). No significant differences in sex, region of residence, or liver enzyme values were found. Seropositivity to hepatitis E was frequent in this Peruvian population and higher in patients with NCC, suggesting shared common routes of infection.

Detection of Rat Hepatitis E Virus in Pigs, Spain, 2023.

We identified rat hepatitis E virus (HEV) RNA in farmed pigs from Spain. Our results indicate that pigs might be susceptible to rat HEV and could serve as viral intermediaries between rodents and humans. Europe should evaluate the prevalence of rat HEV in farmed pigs to assess the risk to public health.