Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A).

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.

The hepatitis E virus (HEV) causes large outbreaks and sporadic cases of acute viral hepatitis in developing countries. In the developed world, HEV occurrence has increased as a result of zoonotic transmission from swine. The cellular aspects of HEV infection, especially the determinants of entry, are poorly understood. In the absence of a robust in vitro culture system for HEV, it is not possible to produce high titre infectious virus that can be labeled for tracking its internalization. We have therefore used an Escherichia coli expressed HEV-like particle (HEV-LP) to study HEV entry. Following internalization, the HEV-LP initially trafficks to Rab5-positive compartments en route to acidic lysosomal compartments where it is degraded. Using pharmacological inhibitors, dominant negative and constitutively active mutants, and siRNA-mediated perturbations, we show that HEV entry requires dynamin-2, clathrin, membrane cholesterol and actin, but is independent of factors associated with macropinocytosis. The HEV-LP results were further validated through infection of liver cells with virus from the stool of an infected patient. The comparative analysis also showed involvement of the phosphatidylinositol-3-kinase/Akt pathway in an early post-entry step of viral replication. This report provides a detailed description of endocytic processes associated with HEV infection.

Hepatitis E virus in bottlenose dolphins Tursiops truncatus.

Hepatitis E virus (HEV) infects several animal species that act as zoonotic reservoirs for viral transmission. Solid and liquid residues from infected animals could lead to HEV contamination of food and surface waters. Evidence of human HEV infection through ingestion of seafood (shellfish, mussels) has been reported. Dolphins generally feed on fish and squid but are able to adapt to an environment and consume whatever prey is available. Clinical signs of infected dolphins include lethargy, inappetence, behavioral aberrations and increased serum alanine aminotransferase (ALT). The dolphins examined in this study were maintained at the National Aquarium, Havana, Cuba. A total of 31 dolphins were evaluated for HEV markers. Sera were collected and screened for total immunoglobin (Ig) anti-HEV. Sera and liver homogenate were tested for HEV RNA by nested RT-PCR using primers targeting the open reading frame 1. Phylogenetic analysis was performed using partial nucleotide sequences at the amplified RNA-dependent RNA polymerase gene. Total anti-HEV Ig was detected in 32.2% (10 of 31), and 16.1% (5 of 31) of these dolphins were positive by both serology and HEV RNA testing. Nucleotide sequence analyses revealed that HEV strains identified in dolphins were genotype 3. This virus may represent an environmental contamination of food or wastewater as a source of HEV exposure and infection. Our findings provide evidence that HEV is associated with liver disorders in cetaceans and that it is advisable to screen for exposure of this virus in captive dolphins, particularly animals with elevated serum ALT or compromised liver function test results of undetermined cause.

Measuring glutathione redox potential of HIV-1-infected macrophages.

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Proposed reference sequences for hepatitis E virus subtypes.

The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website (http://talk.ictvonline.org/r.ashx?C). The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

The microRNA miR-29a is associated with human immunodeficiency virus latency.

BACKGROUND: Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3'LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency. FINDINGS: We used two established cellular models of HIV-1 latency - the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication. CONCLUSIONS: The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

Consensus proposals for classification of the family Hepeviridae.

The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.

Molecular virology of hepatitis E virus.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. It is a nonenveloped virus with a ∼7.2 kilobases positive-stranded RNA genome. The molecular virology of HEV is getting better understood with the development of replicons and in vitro infection systems, and the discovery of related viruses that infect animal species other than humans. This review focuses on the virology of HEV and updates the current knowledge on the HEV genome and its constituent proteins--ORF1, ORF2, and ORF3, and the viral life cycle.

Halal cultivated meat: an untapped opportunity.

The global Halal food market is forecast to reach US$1.67 trillion by 2025, growing to meet the dietary demands of a rapidly increasing Muslim population, set to comprise 30% of the global population by mid-century. Meat consumption levels are increasing in many Muslim countries, with important implications for health and environmental sustainability. Alt protein products are currently being manufactured and positioned as one possible solution to reduce the environmental impact of meat consumption, yet, little is currently known about the Halal status of these products, nor the extent to which they appeal to Muslim consumers in emerging markets in Asia and Africa. Here, we explore key considerations regarding the acceptability of alt protein products for Muslim consumers, explore Halal certification requirements in the context of cultivated meat, and examine some unique beliefs within the Islamic faith that may support, as well as impede, widespread adoption of alt protein among the 2.8 billion Muslims of the future.

Hepatitis E.

Hepatitis E refers to liver disease caused by the hepatitis E virus (HEV), a small, nonenveloped virus with a single-stranded RNA genome. The virus has four genotypes, but only one serotype. Genotypes 1 and 2 exclusively infect humans, whereas genotypes 3 and 4 also infect pigs and several other mammalian species. Though HEV does not grow well in cell culture, several aspects of its biology and pathogenesis have been worked out using animal models and cell transfection studies, and by analogy with other related viruses. HEV itself appears noncytopathic, and the liver injury during hepatitis E may be mediated by the host immune response. In areas with poor sanitation, HEV infection is common and presents as outbreaks and also as sporadic cases with acute self-limited hepatitis. The transmission is feco-oral, usually through contaminated drinking water. The disease often affects young adults and is particularly severe among pregnant women and persons with preexisting liver cirrhosis. In the developed world, the disease is being increasingly recognized. It occurs as occasional sporadic cases, most often among elderly men with coexisting illnesses. These appear to be related to zoonotic transmission. Chronic infection is known among immunosuppressed persons in these regions and may progress to liver cirrhosis. Serological tests for diagnosis of HEV exposure and recent infection, namely immunoglobulin (Ig)G and IgM anti-HEV, respectively, need further improvement in sensitivity and specificity, particularly when used in developed countries. Two recombinant protein vaccines have undergone successful human trials, but are not yet commercially available. Recent development of cell-culture methods for HEV should allow a better understanding of this enigmatic agent.

The ORF3 protein of hepatitis E virus delays degradation of activated growth factor receptors by interacting with CIN85 and blocking formation of the Cbl-CIN85 complex.

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection in vivo and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis.

Plasma peptidome profiling of acute hepatitis E patients by MALDI-TOF/TOF.

BACKGROUND: Hepatitis E is endemic to resource-poor regions, where it manifests as sporadic cases and large waterborne outbreaks. The disease severity ranges from acute self-limited hepatitis with low mortality to fulminant hepatic failure with high mortality. It is believed that the host response plays an important role in determining the progression and outcome of this disease. We profiled the plasma peptidome from hepatitis E patients to discover suitable biomarkers and understand disease pathogenesis. RESULTS: The peptidome (< 10 kDa) fraction of plasma was enriched and analyzed by mass spectrometry. A comparative analysis of the peptide pattern of hepatitis E patients versus healthy controls was performed using ClinPro Tools. We generated a peptide profile that could be used for selective identification of hepatitis E cases. We have identified five potential biomarker peaks with m/z values of 9288.6, 7763.6, 4961.5, 1060.572 and 2365.139 that can be used to reliably differentiate between hepatitis E patients and controls with areas under the receiver operating characteristic curve (AUROC) values of 1.00, 0.954, 0.989, 0.960 and 0.829 respectively. A number of proteins involved in innate immunity were identified to be differentially present in the plasma of patients compared to healthy controls. CONCLUSIONS: Besides the utility of this approach for biomarker discovery, identification of changes in endogenous peptides in hepatitis E patient plasma has increased our understanding of disease pathogenesis. We have identified peptides in plasma that can reliably distinguish hepatitis E patients from healthy controls. Results from this and an earlier proteomics study are discussed.

Virus entry paradigms.

Viruses, despite being relatively simple in structure and composition, have evolved to exploit complex cellular processes for their replication in the host cell. After binding to their specific receptor on the cell surface, viruses (or viral genomes) have to enter cells to initiate a productive infection. Though the entry processes of many enveloped viruses is well understood, that of most non-enveloped viruses still remains unresolved. Recent studies have shown that compared to direct fusion at the plasma membrane, endocytosis is more often the preferred means of entry into the target cell. Receptor-mediated endocytic pathways such as the dynamin-dependent clathrin and caveolar pathways are well characterized as viral entry portals. However, many viruses are able to utilize multiple uptake pathways. Fluid phase uptake, though relatively non-specific in terms of its cargo, potentially aids viral infection by its ability to intersect with the endocytic pathway. In fact, many viruses despite using specialized pathways for entry are still able to generate productive infection via fluid phase uptake. Macropinocytosis, a major fluid uptake pathway found in epithelial cells and fibroblasts, is stimulated by growth factor receptors. Many viruses can induce these signaling cascades in cells leading to macropinocytosis. Though endocytic trafficking is utilized by both enveloped and non-enveloped viruses, key differences lie in the way membranes are traversed to deposit the viral genome at its site of replication. This review will discuss recent developments in the rapidly evolving field of viral entry.

Evidence of hepatitis E virus exposure among seronegative healthy residents of an endemic area.

OBJECTIVE: Hepatitis E virus (HEV) infection is endemic in the Indian subcontinent. Detection of serum anti-HEV IgG has traditionally been used to determine prior exposure to this virus. We studied HEV-specific recall immune responses in healthy subjects with and without detectable anti-HEV IgG. METHODS: Memory B and T cells specific for HEV recombinant proteins pORF2 and pORF3 were estimated among healthy subjects residing in an HEV-endemic region using enzyme-linked immunospot (ELISPOT) assays. RESULTS: Anti-HEV IgG-negative and anti-HEV IgG-positive healthy subjects had a similar median (range) number of IgG-secreting memory B cells specific for HEV pORF2 [percent of total IgG-producing cells: 0.39 (0-13.63) vs. 0.83 (0-12.78)] and HEV pORF3 [0.33 (0.05-12.35) vs. 1.01 (0.08-9.48)], and of IFN-γ-secreting memory T cells specific for HEV pORF2 [per one million PBMCs: 16 (0-220) vs. 36.5 (0-474)] and HEV pORF3 [166 (0-957) vs. 70.5 (0-533)]. Eight healthy volunteers residing in the USA and studied as controls lacked detectable T cells specific for HEV pORF2. CONCLUSION: ELISPOT assays may detect evidence of prior HEV infection in persons residing in areas endemic for this infection and lacking detectable anti-HEV IgG. Seroepidemiological studies that use the serum anti-HEV IgG test may underestimate the frequency of exposure to HEV.

HIV-1 Nef promotes endocytosis of cell surface MHC class II molecules via a constitutive pathway.

HIV-1 Nef has been reported to disrupt MHC class II (MHCII)-mediated Ag presentation by a dual strategy that comprises a reduction in cell surface levels of peptide-loaded mature MHCII molecules and a up-regulation of immature MHCII molecules. We show that Nef achieves relocation of MHCII away from the cell surface in monocytic cells by both delaying its transport to the cell surface and by accelerating endocytic removal of cell surface MHCII to a lysosomal compartment. Nef-induced MHCII endocytosis is cholesterol-sensitive but clathrin- and dynamin-independent. Internalized MHCII molecules traverse the early endosomal system and colocalize with pinocytic cargo before reaching lysosomes. Nef-triggered MHCII endocytosis requires Rab5 activity and lyst function, whereas lysosomal trafficking of internalized MHCII molecules requires Rab7 activity. We further show that a similar pathway can remove peptide-MHCII complexes from the surface of monocytic cells not expressing Nef. Our data suggest that Nef uses mechanisms involved in normal MHCII recycling and turnover to mediate the delivery of cell surface MHCII to a lysosomal destination. Thus, Nef-mediated endocytosis of MHCII provides a novel perspective on the regulation of normal MHCII trafficking.

Adaptive immune responses during acute uncomplicated and fulminant hepatitis E.

BACKGROUND AND AIM: Hepatitis E virus (HEV) infection is endemic in several developing countries. Clinical manifestations of this infection vary widely from asymptomatic infection to uncomplicated acute viral hepatitis and fulminant hepatic failure. The pathogenesis of this disease and the reason of varying disease severity remain unknown. In viral infections, tissue injury can be caused either by virus itself or by host immune responses directed against infected cells. We therefore studied adaptive immune responses to HEV antigens in patients with hepatitis E of varying disease severity and healthy controls. METHODS: Cytokine secreting CD4+ T cells and antibody-producing B cells specific for HEV were enumerated through intracellular cytokine staining and enzyme-linked immunosorbent spot assay, respectively. RESULTS: Patients with fulminant hepatitis E had a less marked expansion of HEV-specific interferon-γ or tumor necrosis factor-a secreting CD4+ T cells than patients with uncomplicated hepatitis E and healthy controls. These patients also had fewer CD4+ T cells that produce γ-interferon or tumor necrosis factor-a upon in vitro polyclonal stimulation. In addition, patients with fulminant disease had a more marked expansion of B cells that can secrete immunoglobulin G anti-HEV than patients with uncomplicated infection and control patients. CONCLUSION: These findings suggest that less-marked antiviral cellular immune responses and heightened antiviral humoral responses are associated with a more severe disease during HEV infection.

HIV-1 Nef induces a Rab11-dependent routing of endocytosed immune costimulatory proteins CD80 and CD86 to the Golgi.

The Nef protein of HIV-1 removes the immune costimulatory proteins CD80 and CD86 from the cell surface by a unique clathrin- and dynamin-independent, actin-based endocytic pathway that deploys coupled activation of c-src and Rac. In this study, we show that, similar to major histocompatibility complex class I (MHCI), Nef subsequently reroutes CD80 and CD86 to the Golgi region. However, not only are CD80/CD86 internalized by a different mechanism from MHCI but also the vesicular pathway of Golgi delivery for CD80/CD86 is distinct from that employed for MHCI. While MHCI passes through early endosomal and sorting compartments marked by Rab5/early embryonic antigen 1 and ADP ribosylation factor 6, respectively, CD80 and CD86 enter endocytic vesicles that do not acquire conventional early endosomal markers but remain accessible to fluid probes. Rather than being delivered to preexisting Rab11-positive recycling compartments, these vesicles recruit Rab11 de novo. Rab11 activity is also necessary for the delivery of CD80/CD86 in these transitional vesicles to the Golgi region. These data reveal an unusual pathway of endocytic vesicular traffic to the Golgi and its recruitment in a viral immune evasion strategy.