A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

An Immunocompetent Mongolian Gerbil Model for Hepatitis E Virus Genotype 1 Infection.

BACKGROUND & AIMS: Hepatitis E virus (HEV), primarily genotype 1 (HEV-1), causes approximately 20.1 million infections, 44,000 deaths, and 3000 stillbirths annually. Current evidence indicates that HEV-1 is only transmitted in humans. Here, we evaluated whether Mongolian gerbils can serve as animal models for HEV-1 infection. METHODS: Mongolian gerbils were used for HEV-1 and hepatitis E virus genotype 3 infection experiments. HEV infection parameters, including detection of HEV RNA and HEV antigen, liver function assessment, and histopathology, were evaluated. RESULTS: We adapted a clinical isolate of HEV-1 for Mongolian gerbils by serial passaging in feces of aged male gerbils. The gerbil-adapted strain obtained at passage 3 induced a robust, acute HEV infection, characterized by stable fecal virus shedding, elevated liver enzymes, histopathologic changes in the liver, and seroconversion to anti-HEV. An infectious complementary DNA clone of the adapted virus was generated. HEV-1-infected pregnant gerbils showed a high rate of maternal mortality and vertical transmission. HEV RNA or antigens were detected in the liver, kidney, intestine, placenta, testis, and fetus liver. Liver and placental transcriptomic analyses indicated activation of host immunity. Tacrolimus prolonged HEV-1 infection, whereas ribavirin cleared infection. The protective efficacy of a licensed HEV vaccine was validated using this model. CONCLUSIONS: HEV-1 efficiently infected Mongolian gerbils. This HEV-1 infection model will be valuable for investigating hepatitis E immunopathogenesis and evaluating vaccines and antivirals against HEV.

Optimization of immunosuppression strategies for the establishment of chronic hepatitis E virus infection in rabbits.

Chronic hepatitis E mostly occurs in organ transplant recipients and can lead to rapid liver fibrosis and cirrhosis. Previous studies found that the development of chronic hepatitis E virus (HEV) infection is linked to the type of immunosuppressant used. Animal models are crucial for the study of pathogenesis of chronic hepatitis E. We previously established a stable chronic HEV infection rabbit model using cyclosporine A (CsA), a calcineurin inhibitor (CNI)-based immunosuppressant. However, the immunosuppression strategy and timing may be optimized, and how different types of immunosuppressants affect the establishment of chronic HEV infection in this model is still unknown. Here, we showed that chronic HEV infection can be established in 100% of rabbits when CsA treatment was started at HEV challenge or even 4 weeks after. Tacrolimus or prednisolone treatment alone also contributed to chronic HEV infection, resulting in 100% and 77.8% chronicity rates, respectively, while mycophenolate mofetil (MMF) only led to a 28.6% chronicity rate. Chronic HEV infection was accompanied with a persistent activation of innate immune response evidenced by transcriptome analysis. The suppressed adaptive immune response evidenced by low expression of genes related to cytotoxicity (like perforin and FasL) and low anti-HEV seroconversion rates may play important roles in causing chronic HEV infection. By analyzing HEV antigen concentrations with different infection outcomes, we also found that HEV antigen levels could indicate chronic HEV infection development. This study optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits and highlighted the potential association between the development of chronic HEV infection and immunosuppressants.IMPORTANCEOrgan transplant recipients are at high risk of chronic hepatitis E and generally receive a CNI-based immunosuppression regimen containing CNI (tacrolimus or CsA), MMF, and/or corticosteroids. Previously, we established stable chronic HEV infection in a rabbit model by using CsA before HEV challenge. In this study, we further optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits. Chronic HEV infection can also be established when CsA treatment was started at the same time or even 4 weeks after HEV challenge, clearly indicating the risk of progression to chronic infection under these circumstances and the necessity of HEV screening for both the recipient and the donor preoperatively. CsA, tacrolimus, or prednisolone instead of MMF significantly contributed to chronic HEV infection. HEV antigen in acute infection phase indicates the development of chronic infection. Our results have important implications for understanding the potential association between chronic HEV infection and immunosuppressants.

A novel multi-epitope peptide vaccine candidate targeting hepatitis E virus: An in silico approach.

Hepatitis E virus (HEV) is a foodborne virus transmitted through the faecal-oral route that causes viral hepatitis in humans worldwide. Ever since its discovery as a zoonotic agent, HEV was isolated from several species with an expanding range of hosts. HEV possesses several features of other RNA viruses but also has certain HEV-specific traits that make its viral-host interactions inimitable. HEV leads to severe morbidity and mortality in immunocompromised people and pregnant women across the world. The situation in underdeveloped countries is even more alarming. Even after creating a menace across the world, we still lack an effective vaccine against HEV. Till date, there is only one licensed vaccine for HEV available only in China. The development of an anti-HEV vaccine that can reduce HEV-induced morbidity and mortality is required. Live attenuated and killed vaccines against HEV are not accessible due to the lack of a tolerant cell culture system, slow viral replication kinetics and varying growth conditions. Thus, the main focus for anti-HEV vaccine development is now on the molecular approaches. In the current study, we have designed a multi-epitope vaccine against HEV through a reverse vaccinology approach. For the first time, we have used viral ORF3, capsid protein and polyprotein altogether for epitope prediction. These are crucial for viral replication and persistence and are major vaccine targets against HEV. The proposed in silico vaccine construct comprises of highly immunogenic and antigenic T-cell and B-cell epitopes of HEV proteins. The construct is capable of inducing an effective and long-lasting host immune response as evident from the simulation results. In addition, the construct is stable, non-allergic and antigenic for the host. Altogether, our findings suggest that the in silico vaccine construct may be useful as a vaccine candidate for preventing HEV infections.

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.

Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly.

Genotype 3 Hepatitis E virus (HEV-3) is an emerging threat for aging population. More than one third of older infected patients develops clinical symptoms with severe liver damage, while others remain asymptomatic. The origin of this discrepancy is still elusive although HEV-3 pathogenesis appears to be immune-mediated. Therefore, we investigated the role of CD8 T cells in the outcome of the infection in immunocompetent elderly subjects. We enrolled twenty two HEV-3-infected patients displaying similar viral determinants and fifteen healthy donors. Among the infected group, sixteen patients experienced clinical symptoms related to liver disease while six remained asymptomatic. Here we report that symptomatic infection is characterized by an expansion of highly activated effector memory CD8 T (EM) cells, regardless of antigen specificity. This robust activation is associated with key features of early T cell exhaustion including a loss in polyfunctional type-1 cytokine production and partial commitment to type-2 cells. In addition, we show that bystander activation of EM cells seems to be dependent on the inflammatory cytokines IL-15 and IL-18, and is supported by an upregulation of the activating receptor NKG2D and an exuberant expression of T-Bet and T-Bet-regulated genes including granzyme B and CXCR3. We also show that the inflammatory chemokines CXCL9-10 are increased in symptomatic patients thereby fostering the recruitment of highly cytotoxic EM cells into the liver in a CXCR3-dependent manner. Finally, we find that the EM-biased immune response returns to homeostasis following viral clearance and disease resolution, further linking the EM cells response to viral burden. Conversely, asymptomatic patients are endowed with low-to-moderate EM cell response. In summary, our findings define immune correlates that contribute to HEV-3 pathogenesis and emphasize the central role of EM cells in governing the outcome of the infection.

Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Mobilization of γδ T Cells and IL-10 Production at the Acute Phase of Hepatitis E Virus Infection in Cytomegalovirus Carriers.

Alterations in the γδ T cell compartment have been reported in immunocompromised individuals infected with hepatitis E virus (HEV)-g3. We now report the analysis of blood γδ T cells from acutely HEV-infected individuals in the absence of immunosuppression. In these patients, non-Vδ2 (ND2) γδ T cells outnumbered otherwise predominant Vδ2 cells selectively in human CMV (HCMV)-seropositive patients and were higher than in HCMVpos controls, mimicking HCMV reactivation, whereas their serum was PCR-negative for HCMV. Stimulation of their lymphocytes with HEV-infected hepatocarcinoma cells led to an HEV-specific response in γδ subsets of HCMVpos individuals. HEV infection was associated with a lowered expression of TIGIT, LAG-3, and CD160 immune checkpoint markers on ND2 effector memory cells in HCMVneg but not in HCMVpos HEV patients. γδ cell lines, predominantly ND2, were generated from patients after coculture with hepatocarcinoma cells permissive to HEV and IL-2/12/18. Upon restimulation with HEV-infected or uninfected cells and selected cytokines, these cell lines produced IFN-γ and IL-10, the latter being induced by IL-12 in IFN-γ-producing cells and upregulated by HEV and IL-18. They were also capable of suppressing the proliferation of CD3/CD28-activated CD4 cells in transwell experiments. Importantly, IL-10 was detected in the plasma of 10 of 10 HCMVpos HEV patients but rarely in controls or HCMVneg HEV patients, implying that γδ cells are probably involved in IL-10 production at the acute phase of infection. Our data indicate that HEV mobilizes a pool of ND2 memory cells in HCMV carriers, promoting the development of an immunoregulatory environment.

NLRP3 Inflammasome Activation is a Prognostic Marker of Recovery in HEV-Infected Patients.

Hepatitis E contributes to 3.3 million acute hepatitis cases worldwide with 30% mortality in pregnant women. Pathogenesis of Hepatitis E is complex; thus, the present study was aimed at inflammasomes and associated cytokines in the immunopathogenesis of viral hepatitis E. PBMCs were isolated from 45 HEV IgM/HEV RNA-positive AVH/ALF and 19 healthy individuals and processed for mRNA expressions of NLRs, RLRs, and cytokines. PBMCs were cultured and stimulated with HEV-pORF-2 peptide in vitro for mRNA expression by RT-PCR and cytokines levels in serum/culture supernatant by ELISA. siRNA transfection and post-silencing effect in AVH PBMCs were also assessed by NLRP3 gene expression and IL-1ß and IL-18 levels by ELISA. The results demonstrated high viral load in ALF than AVH cases. mRNA expression of NLRP3 in AVH patients was found to be positively correlated with IL-18 (r = 0.74) and IL-1ß (r = 0.68); P < 0.0001***. Significant levels of serum IL-1ß and IL-18 cytokines were observed in AVH as compared to ALF patients. The levels of IL-1ß in the culture supernatant in mock and stimulated conditions were significantly higher in AVH than in ALF patients. Significant downregulation in NLRP3 gene expression was correlated with the reduced levels of IL-1ß and IL-18 cytokines in NLRP3-siRNA-transfected PBMCs. This study highlighted the significance of upregulated NLRP3 inflammasome leading to increased production of IL-18 and IL-1ß cytokines in sera of AVH patients. Thus, it indicated the role of Th1 response acting through the NLRP3 pathway which might have been helpful in the recovery of AVH patients. These promising results open multiple treatment avenues where specific inhibitors can be designed to modulate the progress of disease and its pathogenicity.

Hepatitis E virus persists in the ejaculate of chronically infected men.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. METHODS: The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. RESULTS: In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. CONCLUSIONS: The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles. LAY SUMMARY: Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E.

The histologic presentation of hepatitis E reflects patients' immune status and pre-existing liver condition.

Infection with the hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Given that, the histopathology of hepatitis E is relatively poorly characterized, and it is unclear what exactly determines its remarkable variability. The aim of our study was a systematic analysis of hepatitis E histology, especially with regard to the clinical setting. Fifty-two liver samples (48 biopsies, 1 liver explant, 3 autopsy livers) from 41 patients with molecularly proven hepatitis E (28 HEV genotype (gt) 3, three gt 1, one gt 4 and 9 undetermined gt) were systematically evaluated for 33 histopathologic features. Following one approach, the biopsies were assigned to one of five generic histologic patterns. In another approach, they were subjected to hierarchical clustering. We found that 23/41 (56%) patients were immunocompromised, whereas 18 (44%) had no known immunosuppression. Five patients (12%) had pre-existing liver disease (LD). The histopathologic spectrum ranged from almost normal to acute, chronic, and steato-hepatitis to subtotal necrosis, and was thus distributed across all five generic patterns. Hierarchical clustering, however, identified three histopathologic clusters (C1-C3), which segregated along the immune status and pre-existing LD: C1 comprised mostly patients with pre-existing LD; histology mainly reflected the respective LD without pointing to the additional hepatitis E. C2 comprised mostly immunocompetent patients; histology mainly displayed florid hepatitis. C3 comprised mostly immunocompromised patients; histology mainly displayed smoldering hepatitis. Accordingly, C1-C3 differed markedly with respect to their clinical and histopathologic differential diagnoses. Hierarchical clustering suggests three groups with distinct histopathologies, indicating biologically different manifestations of hepatitis E. The association of histopathologic changes with the patient's immune status and pre-existing LD plausibly explains the diversity of hepatitis E histopathology, and suggests that these factors are the crucial underlying determinants. We expect our results to improve patient management by guiding the clinico-pathologic diagnosis of hepatitis E.

Prevalence of hepatitis E virus in children from Northeast of Argentina.

The aim of this study was to assess the prevalence of hepatitis E virus (HEV) in a young population from the Northeast region of Argentina. Four hundred and twelve patients under 18 years old, from rural areas of Chaco Province, were tested for anti-HEV immunoglobulin G (IgG) using enzyme-linked immunosorbent assay. Anti-HEV IgG antibodies were detected in 7 out of 412 patients, accounting for an overall 1.7% prevalence. HEV infection in developing countries is associated to lack of clean drinking water. Consequently, the seroprevalence observed in children in rural areas of Chaco, Argentina, where the access to tap water is less than 15%, was unexpectedly low.

An outbreak of hepatitis E in Yavatmal, India, 2019.

Hepatitis E, a public health concern in developing countries, frequently presents in epidemic, as well as in sporadic forms. This study investigated an outbreak of viral hepatitis at Yavatmal, Maharashtra, India in March 2019. Blood samples from 10 patients were received at Indian Council of Medical Research-National Institute of Virology, Pune to test for the presence of enterically transmitted hepatitis viruses. Subsequently, 49 suspected cases were screened for anti-hepatitis E virus (HEV)/hepatitis A virus (HAV) immunoglobulin M and immunoglobulin G (IgG) antibodies, alanine amino-transferase levels and HEV RNA. Water samples were screened for HEV and HAV RNA followed by phylogenetic analysis. Overall 32 of 49 (65.3%) suspected cases had recent acute HEV infection, while dual infection with HAV was noted in one case (2.04%). Forty-eight of 49 suspected cases were positive for anti-HAV IgG antibodies indicative of previously acquired immunity against HAV. Water samples had evidence of HEV contamination as detected by reverse transcription-polymerase chain reaction. Sequencing of HEV RNA from both patients (n = 2) and water samples (n = 5) indicated HEV genotype 1 to be the etiological agent of this outbreak. Serological and molecular evidence confirmed HEV as the etiology. Mixing of contaminated drain water with the domestic water supply may have triggered this outbreak. Subsequent changing of the defaulted water pipelines and its segregation from drain pipelines by the health authorities resulted in progressive decline of this outbreak. Despite the limitations, periodic surveillance of HEV exposure pattern and reporting of small outbreaks would supplement to the global disease burden data of hepatitis E.

First molecular characterization of the hepatitis E virus in humans in Cameroon: Confirmation of the HEV outbreak in Touboro, North-Cameroon.

Hepatitis E virus (HEV) is a major causative agent of acute viral hepatitis in many regions of the world including Africa. In Cameroon, there is no published molecular study on HEV in humans. However, based on serological assays, the first outbreak of HEV was detected in North-Cameroon. The objective of this study was to determine the molecular characterization of HEV that circulated during this period. A retrospective study design was used to select serum samples among those collected during the outbreak period. immunoglobulin M positive samples available in sufficient volumes to amplify HEV RNA were selected. RNA was extracted and then amplified by a real-time reverse transcription polymerase chain reaction (real time RT-PCR) assay, followed by a nested reverse transcription polymerase chain reaction (nested RT-PCR) assay for sequencing and phylogenetic analysis. Overall, 24 samples were selected and HEV RNA was amplified by real-time RT-PCR in 20 samples. Amongst these, 12 samples were positive for HEV RNA by nested RT-PCR and yielded good sequencing products. Phylogenetic analysis showed that 10 samples clustered with HEV genotype 1 (subtype 1e) and two samples clustered with HEV genotype 3 (subtype 3f). This study fills the gap of knowledge on the molecular epidemiology of HEV in Cameroon and confirms the first report of the hepatitis E outbreak in North-Cameroon.

Serological and molecular prevalence of hepatitis E virus among blood donors from Uruguay.

Hepatitis E virus (HEV) infection is considered a neglected disease of major concern in developed countries. Clinically, HEV occurs as an acute and self-limited disease, though chronic cases mostly associated to HEV-3 are now being commonly reported in immunocompromised individuals and solid organ transplant recipients. Transmission of HEV through blood and derivatives have been increasingly described in the last years, highlighting the importance of including this agent on the screening programs. Since 2010 both acute and chronic hepatitis E cases have been frequently reported in Uruguay. However, updated prevalence data among different population groups are lacking and HEV is not currently screened in blood banks. Herein, we report a seroprevalence and molecular survey of HEV in 400 plasma samples from blood donors. Overall, our results showed an HEV seroprevalence rate of 10% (40/400); almost 10-fold higher than 20 years ago. Total anti-HEV immunoglobulin antibodies were found to increase with age. Moreover, we reported an RNA detection rate of at least 0.75%, and two strains were sequenced. Phylogenetic analysis grouped them with human and swine HEV-3 strains from Uruguay. Data presented here should prompt public health policies of HEV screening in blood banks to minimize the risk of transfusion-transmitted hepatitis E.

Hepatitis E virus infection and waste pickers: A case-control seroprevalence study.

Whether waste pickers are a risk group for hepatitis E virus (HEV) infection is largely unknown. This study aimed to determine the association between HEV exposure and the occupation of waste pickers and the work characteristics of waste pickers. An age-and gender-matched case-control seroprevalence study of 86 waste pickers and 86 control subjects of the general population was performed. We determined anti-HEV IgG antibodies in sera of cases and controls using a commercially available enzyme-linked immunoassay. The McNemar's test was used to assess the association between HEV seropositivity and the occupation of waste picker. The association between HEV seropositivity and work characteristics of waste pickers was assessed by bivariate and logistic regression analyses. Anti-HEV IgG antibodies were detected in 14 (16.3%) of the 86 waste pickers and in 8 (9.3%) of the 86 control subjects (McNemar's pair test: odds ratio (OR) = 13.0; 95% confidence interval (CI): 0.73-230.77; p = .02). Bivariate analysis showed that HEV exposure was associated with an ill status (p = .01) and reflexes impairment (p = .009). Logistic regression analysis showed that HEV seropositivity was associated with increasing age (OR = 6.52; 95% CI: 1.95-21.78; p = .002) and raising pigs (OR = 12.01; 95% CI: 1.48-97.26; p = .02). This is the first age- and gender-matched case-control study on the association between HEV infection and the occupation of waste picker. Waste pickers represent a risk group for HEV infection. Factors associated with HEV seropositivity found in this study may help in the design of optimal planning to avoid HEV infection.

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.

High seroprevalence of viral hepatitis among animal handlers in Abeokuta, Ogun State, Nigeria.

Viral hepatitis is a deadly disease which can manifest as acute, chronic, hepatocellular carcinoma, and liver failure. Information about hepatitis is scarce among animal handlers. Due to Federal Government of Nigeria diversification programmes, many people are now involved in animal farming which can make them susceptible to viral hepatitis. This study aimed at determining the prevalence of Hepatitis B, C, and E viruses among animal handlers in Abeokuta, southwestern Nigerian. A total of 156 subjects were recruited for the study. Sociodemographic and risks factors data were fetched from subjects using interviewer-administered questionnaire. Blood samples were collected via venepuncture and tested for HCV, HBV, and HEV using ELISA technique. Results were analyzed using SPSS software version 21.0 and P value ≤ 0.05 was considered significant. The prevalence of HCV, HBV, and HEV were 46 (29.5%), 20 (12.8%), and 4 (2.6%) respectively while 6 (3.8%), 1 (0.6%), and 1 (0.6%) had co-infection of HBV-HCV, HBV-HEV, and HCV- HEV respectively. This study concludes that there is high prevalence of hepatitis C and B viruses among animal handlers in Abeokuta, Ogun state which is of significant public health problem, warranting further attention and research.

Interplay between Hepatitis E Virus and Host Cell Pattern Recognition Receptors.

Hepatitis E virus (HEV) usually causes self-limiting acute hepatitis, but the disease can become chronic in immunocompromised individuals. HEV infection in pregnant women is reported to cause up to 30% mortality, especially in the third trimester. Additionally, extrahepatic manifestations like neuronal and renal diseases and pancreatitis are also reported during the course of HEV infection. The mechanism of HEV pathogenesis remains poorly understood. Innate immunity is the first line of defense triggered within minutes to hours after the first pathogenic insult. Growing evidence based on reverse genetics systems, in vitro cell culture models, and representative studies in animal models including non-human primates, has implicated the role of the host's innate immune response during HEV infection. HEV persists in presence of interferons (IFNs) plausibly by evading cellular antiviral defense. This review summarizes our current understanding of recognizing HEV-associated molecular patterns by host cell Pattern Recognition Receptors (PRRs) in eliciting innate immune response during HEV infection as well as mechanisms of virus-mediated immune evasion.

The crystal structure of the emerging human-infecting hepatitis E virus E2s protein.

Hepatitis E virus (HEV) is a non-enveloped, globular particle that is responsible for acute hepatitis. HEV is classified into the Hepeviridae family and can be divided into four species (A-D). All HEV variants that infect humans are reported to belong to species A (HEV-A), except species C (HEV-C), which was reported to infect humans in December 2018. We determined the crystal structure of the HEV-C E2s domain at 1.8 Å resolution. It contains a classical 12-stranded ß-sandwich motif and forms dimers by hydrogen bonding, though the amino acid residues that form hydrogen bonds are quite different from the residues of HEV-A. The HEV-C E2s domain shares the common groove region with other structurally related viruses, and some subtle differences in this region may be related to host adoption or antibody binding. Antibody binding experiments and structural analysis revealed that HEV-C E2s is able to bind to the previously reported broad-spectrum antibody 8G12 but not bind to the antibody 8C11. Meanwhile, the structure analysis shows that HEV-C E2s does not have the key sites for binding to host cells as displayed by HEV-A (Genotype 1) E2s. These structural and biological findings present important implications for understanding the molecular mechanisms of host recognition and entry of HEV-C, as well as provide clues to the development of therapeutic antibodies and vaccines against HEV-C infection.