Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future

Prohibitin 1 Modulates Mitochondrial Stress-Related Autophagy in Human Colonic Epithelial Cells

Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1) or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease

Sources of Hepatitis E Virus Genotype 3 in the Netherlands

Four subtypes have been detected, and pathogenicity, zoonotic potential, or stability may vary between subtypes

The impact of inflammatory bowel disease on sexual health in men: A scoping review.

AIMS AND OBJECTIVES: To review the literature on the impact of inflammatory bowel disease on the sexual health of men, and make recommendations for nursing practice and research. BACKGROUND: Inflammatory bowel disease is a chronic condition of the gastrointestinal tract, causing symptoms that may impact upon sexual health. Specialist nurses are well positioned to assess and manage sexual health, but there is a lack of clinical guidance, especially in relation to men. DESIGN: A systematic scoping review following the Arksey and O'Malley (2005) framework reported in line with the PRISMA-ScR checklist (Tricco et al. 2018). METHODS: OVID MEDLINE ALL [R], OVID EMBASE [R], OVID PsychINFO, EBSCO CINAHL Complete, The Cochrane Library and ProQuest were searched. Inclusion and exclusion criteria were applied independently by two reviewers. Data was extracted, charted and summarised from eligible studies. RESULTS: Thirty-one studies met the inclusion criteria. These were synthesised under three categories: mediators, moderators, and descriptors of sexual health. Depression, disease activity and surgery were the most commonly cited disease-related factors to affect sexual health in men. The most commonly used assessment tool was The International Index of Erectile Function. Descriptors of function included; frequency of intercourse, libido and the ability to maintain a desired sexual role. CONCLUSIONS: The effect of inflammatory bowel disease on sexual health in men involves a complex interaction of physical and psychosocial factors. Researchers must explore areas outside of erectile function to understand how the disease impacts sexuality, sexual well-being and masculinity. This can be achieved through qualitative exploration of patient, partner and health professional experiences. Relevance to clinical practice A holistic nursing assessment of men with inflammatory bowel disease should include sexual health. Developing understanding of how the disease influences sexual interaction and expression will facilitate support that is relevant, accessible and of value to men living with the disease

Inactivation of infectious hepatitis E virus present in commercial pig livers sold in local grocery stores in the United States

Hepatitis E virus (HEV) is a zoonotic pathogen and pigs are a known reservoir. Recently we showed that approximately 11% of commercial pig livers sold in local U.S. grocery stores for food consumptions are contaminated by infectious HEV. In this study, a swine bioassay was used to determine if the infectious HEV in contaminated commercial pig livers could be inactivated by traditional cooking methods. Group 1 pigs (n=5) were each inoculated intravenously (I.V.) with a HEV-negative liver homogenate as negative controls, group 2 pigs (n=5) were each inoculated I.V. with a pool of two HEV-positive pig liver homogenates as positive controls, groups 3, 4 and 5 pigs (n=5, each group) were each inoculated I.V. with a pool of homogenates of two HEV-positive livers incubated at 56°C for 1 hr, stir-fried at 191°C for 5 min or boiled in water for 5 min, respectively. As expected, the group 2 positive control pigs all became infected whereas the group 1 negative control pigs remained negative. Four of the five pigs inoculated with HEV-positive liver homogenates incubated at 56°C for 1 hr also became infected. However, pigs in groups 4 and 5 did not become infected. The results indicated that HEV in contaminated commercial pig livers can be effectively inactivated if cooked properly, although incubation at 56°C for 1 hr cannot inactivate the virus. Thus, to reduce the risk of food-borne HEV transmission, pig livers must be thoroughly cooked

Genetic variability and the classification of hepatitis E virus

The classification of hepatitis E virus (HEV) variants is currently in transition without agreed definitions for genotypes and subtypes or for deeper taxonomic groupings into species and genera that could incorporate more recently characterized viruses assigned to the Hepeviridae family that infect birds, bats, rodents, and fish. These conflicts arise because of differences in the viruses and genomic regions compared and in the methodology used. We have reexamined published sequences and found that synonymous substitutions were saturated in comparisons between and within virus genotypes. Analysis of complete genome sequences or concatenated ORF1/ORF2 amino acid sequences indicated that HEV variants most closely related to those infecting humans can be consistently divided into six genotypes (types 1 to 4 and two additional genotypes from wild boar). Variants isolated from rabbits, closely related to genotype 3, occupy an intermediate position. No consistent criteria could be defined for the assignment of virus subtypes. Analysis of amino acid sequences from these viruses with the more divergent variants from chickens, bats, and rodents in three conserved subgenomic regions (residues 1 to 452 or 974 to 1534 of ORF1 or residues 105 to 458 of ORF2) provided consistent support for a division into 4 groups, corresponding to HEV variants infecting humans and pigs, those infecting rats and ferrets, those from bats, and those from chickens. This approach may form the basis for a future genetic classification of HEV into four species, with the more divergent HEV-like virus from fish (cutthroat trout virus) representing a second genus

Zoonotic hepatitis E: animal reservoirs and emerging risks

Hepatitis E virus (HEV) is responsible for enterically-transmitted acute hepatitis in humans with two distinct epidemiological patterns. In endemic regions, large waterborne epidemics with thousands of people affected have been observed, and, in contrast, in non-endemic regions, sporadic cases have been described. Although contaminated water has been well documented as the source of infection in endemic regions, the modes of transmission in non-endemic regions are much less known. HEV is a single-strand, positive-sense RNA virus which is classified in the Hepeviridae family with at least four known main genotypes (1–4) of mammalian HEV and one avian HEV. HEV is unique among the known hepatitis viruses, in which it has an animal reservoir. In contrast to humans, swine and other mammalian animal species infected by HEV generally remain asymptomatic, whereas chickens infected by avian HEV may develop a disease known as Hepatitis-Splenomegaly syndrome. HEV genotypes 1 and 2 are found exclusively in humans while genotypes 3 and 4 are found both in humans and other mammals. Several lines of evidence indicate that, in some cases involving HEV genotypes 3 and 4, animal to human transmissions occur. Furthermore, individuals with direct contact with animals are at higher risk of HEV infection. Cross-species infections with HEV genotypes 3 and 4 have been demonstrated experimentally. However, not all sources of human infections have been identified thus far and in many cases, the origin of HEV infection in humans remains unknown