Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

Novel hepatoviruses in synanthropic bats in the upper Midwestern United States.

A nearly complete genome sequence of hepatovirus G was isolated from an Eptesicus fuscus bat submitted for rabies virus testing due to human exposure in South Dakota. The predicted polyprotein sequence was 78.2% and 74.4% identical to genotypes G1 and G2, respectively, recovered from bats in Ghana. Quantitative PCR on 90 E. fuscus bats showed that eight (8.9%) were positive for hepatovirus G. Targeted sequencing of the VP2 region of the genome for five positive samples showed >99% identity to hepatovirus G strain Ef15893, demonstrating that hepatovirus G commonly circulates in E. fuscus bats in the upper Midwest.

Investigation of transfusion associated hepatitis C virus infection in Taiwan, 2015-2018.

Continuous strengthening of the safety of blood products to reduce the risk of transfusion-transmitted HCV in recipients is an important issue of Taiwanese government concern. Since 2013, highly sensitivity serology and NAT assays were simultaneously used for blood donation screening to shorten the window period of HIV, HBV and HCV infections. 15 cases of suspected transfusion-transmitted HCV infection were analyzed in 2015-2018. No HCV nucleic acid was detected among a total 91 bags of donated blood. Eleven cases among the 15 suspected recipients were positive for HCV nucleic acid, and 9 recipients had genotype results. Of these 9 recipients, five for genotype 1b (5/9, 55.6%), three for genotype 2a (3/9, 33.3%) and one for genotype 2b (1/9, 11.1%). We will continuously monitor the blood safety of recipients. There have been no confirmed cases of acute hepatitis C (AHC) infection due to transfusions of HCV contaminated blood product in 2015-2018 in Taiwan.

Need for recognizing atypical manifestations of childhood sporadic acute viral hepatitis warranting differences in management.

Various atypical manifestations have been described in acute viral hepatitis (AVH). We evaluated the prevalence, clinical features, response to treatment and outcome of various atypical manifestations of AVH in children. Consecutive children (≤ 18 years) with AVH due to hepatitis A, B, or E were studied while patients with acute or acute on chronic liver failure were excluded. Diagnosis of atypical manifestations was based on standard criteria. A total of 477 children with AVH (median age 7.0 (5-11) years, 74% boys) were seen; 22% (n = 106) had atypical manifestations. Prolonged cholestasis was the most common (11%), followed by ascites (7%), intravascular hemolysis (3%), relapsing hepatitis (2%), acute pancreatitis (1.3%), and thrombocytopenia (0.7%). Atypical manifestations were more common in HAV as compared to HBV (30% vs. 3%, p = 0.00) and HEV (30% vs. 15%, p = 0.07). Prolonged cholestasis was significantly more common in older children (20% in > 10 years vs. 9% in 6-10 years ; p = 0.009 and 5% in 0-5 years of age [p < 0.000]). Ascites was more common in younger children, although not significant. All patients recovered with supportive treatment.Conclusions: Twenty-two percent of children with AVH have atypical manifestations, more often with HAV infection, and prolonged cholestasis is most common. Recognition of these manifestations ensures correct diagnosis and treatment. What is Known: • Acute viral hepatitis is a major public health problem in developing countries. • There is limited information about atypical manifestations which may lead to unnecessary investigations, delayed diagnosis and morbidity. What is New: • Atypical manifestations are common in children, seen most often with HAV infection, and prolonged cholestasis is most common. • Prompt recognition of these manifestations helps in early diagnosis, appropriate management, and preventing unnecessary investigations. • Ensure follow-up until complete recovery and not to miss underlying chronic liver disease.

[Advances in liver immunology in 2018].

The mechanism of innate and adaptive immune responses to chronic infections with hepatotropic viruses (HBV, HCV) was studied in 2018. Its mechanism elucidated the dysregulation of natural killer (NK) cells, monocytes, B cells and T cells. In addition, a new target for immune regulation of HBV infection (TLR3/OX40L) was introduced. The discovery of new NK cell immune checkpoints, the involvement of mononuclear macrophages in liver failure and inflammation, sex hormone affecting intrahepatic-resistant bacterial infection through the regulation of humoral immunity, and the communication mechanism between liver and other immune organs have enriched people's understanding of liver immunology and its clinical significance.

Steroid therapy in children with fulminant hepatitis A.

Fulminant hepatic failure is a life-threatening disease. Hepatitis A virus (HAV) can cause fulminant hepatic failure and death in about 0.2% of cases. Extensive destruction of infected hepatocytes by immune-mediated lysis is thought to be the cause. We aimed to evaluate the use of steroid therapy in children with fulminant HAV. This study included 33 children with fulminant HAV in two groups. Steroid group: comprised of 18 children who received prednisolone (1 mg/kg/d) or its equivalent dose of methylprednisolone, and the nonsteroid group: comprised another 15 children who did not receive steroid therapy. Age and sex were matched for both groups (P > .05), and they were comparable regarding baseline clinical and laboratory characteristics. Of the steroid group, 15 patients survived and 3 died, while in the nonsteroid group, 4 patients survived and 11 died (P = .001). Of the living patients, 15 of 19 (78.9%) received steroids while only 3 of 14 (21.4%) of the dead patients received steroids (P = .001). Stepwise regression analysis showed that steroid therapy was the only independent variable associated with recovery (P = .001). Steroid therapy in children with fulminant HAV associated significantly with improved outcome and survival. Future studies on a larger population size are strongly recommended.

Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions.

Hypoxia inducible transcription factors (HIFs) activate diverse pathways that regulate cellular metabolism, angiogenesis, proliferation, and migration, enabling a cell to respond to a low oxygen or hypoxic environment. HIFs are regulated by oxygen-dependent and independent signals including: mitochondrial dysfunction, reactive oxygen species, endoplasmic reticular stress, and viral infection. HIFs have been reported to play a role in the pathogenesis of liver disease of diverse aetiologies. This review explores the impact of HIFs on hepatocellular biology and inflammatory responses, highlighting the therapeutic potential of targeting HIFs for an array of liver pathologies.

Foodborne hepatitis A outbreak associated with bakery products in northern Germany, 2012.

In October 2012, a hepatitis A (HA) outbreak with 83 laboratory-confirmed cases occurred in Lower Saxony. We defined primary outbreak cases as people with laboratory-confirmed HA and symptom onset between 8 October and 12 November 2012, residing in or visiting the affected districts. Secondary outbreak cases were persons with symptom onset after 12 November 2012 and close contact with primary cases. We identified 77 primary and six secondary cases. We enrolled 50 primary cases and 52 controls matched for age and sex, and found that 82% of cases and 60% of controls had consumed products from a particular bakery (OR=3.09; 95% CI: 1.15­8.68). Cases were more likely to have eaten sweet pastries (OR=5.74; 95% CI: 1.46­22.42). Viral isolates from five selected cases and three positively tested surfaces in the bakery had identical nucleotide sequences. One additional identical isolate derived from a salesperson of the bakery suffering from a chronic disease that required immunosuppressive treatment. Epidemiological and laboratory findings suggested that the salesperson contaminated products while packing and selling. Future risk assessment should determine whether food handlers with chronic diseases under immunosuppressive treatment could be more at risk of contaminating food and might benefit from HAV immunisation.

Genomic characterization of a novel Hepatovirus identified in Maranhão state, Brazil.

Bats are efficient reservoirs of a number of viruses with zoonotic potential, and are involved directly in the transmission cycle of many zoonoses. In the present study, which is part of a larger project that is documenting the viromes of the bat species found in the Mid-North states of Maranhão and Piauí, we analyzed 16 pooled samples obtained from four species of bat of the genus Artibeus-Artibeus obscurus, Artibeus cinereus, Artibeus lituratus and Artibeus planirostris. We describe and identify a Hepatovirus, denominated Hepatovirus H isolate sotense, which was found in a pool of internal organs (liver and lungs) extracted from a specimen of A. planirostris, a frugivorous bat, collected in the Cerrado biome of Maranhão state. This material was analyzed using new generation sequencing, which produced a contig of 7390 nucleotides and presented a degree of identity with a number of existing Hepatovirus sequences available for bats (amino acid identity of 61.5% with Bat hepatovirus C of Miniopterus cf. manavi, 66.6% with Bat hepatovirus G of Coleura afra, 67.4% with Hepatovirus G2 of Rhinolophus landeri, and 75.3% with Hepatovirus H2 of Rhinolophus landeri). The analysis of the functional domains of this contig confirmed a pattern consistent with the characteristics of the genus Hepatovirus (Picornaviridae). In the phylogenetic tree with several other Hepatovirus species, this genome also grouped in a monophyletic clade with Hepatovirus H (HepV-H1; HepV-H2, and HepV-H3) albeit on an external branch, which suggests that it may be a distinct genotype within this species. This is the first isolate of Hepatovirus H identified in bats from South America, and represents an important discovery, given that most studies of viruses associated with bats in the state of Maranhão have focused on the family Rhabdoviridae.

Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

[Experience of application of multiplex qPCR for differential diagnostics of intestinal viral infections].

AIM: Evaluate the effectiveness of multiplex reverse transcription (RT) and polymerase chain reaction with fluorescence detection in real time mode (qPCR) methods for differential detection of 11 groups of intestine viruses (adenoviruses, enteroviruses, polioviruses, hepatitis A and E viruses, group A and C rotaviruses, orthoreoviruses, noroviruses, sapoviruses and astroviruses) in various biological samples. MATERIALS AND METHODS: Panels of virus isolates and clinical samples characterized by reference methods were used to evaluate sensitivity of detection of various intestine viruses. Nucleic acids were isolated from study samples and multiplex RT and qPCR were carried out. RESULTS: Sensitivity of laboratory reagent kit (LRK) when compared with results obtained from reference methods was 100% for rotavirus A, adenovirus, enterovirus and norovirus, 88.9% for hepatitis E virus and 92.3% for hepatitis A virus, and diagnostic specificity - 99.4%. During analysis of 697 clinical samples from patients with acute intestine infection symptoms nucleic acids of various intestine viruses were isolated in 71.7%. CONCLUSION: Multiplex qRT-PCR was shown as an effective method of etiologic diagnostics of an intestine viral infection. Use of LRK was demonstrated to establish etiology of intestine diseases in 63 - 72% and in children with watery diarrhea - in approximately 90% of cases.

Internal initiation of translation.

Although the 5' cap-dependent scanning mechanism can account for the translational initiation of most mRNAs in eukaryotic cells, several viral and cellular mRNAs contain nucleotide sequences in their 5' non-coding regions that can mediate binding of ribosomes to the mRNA, regardless of the modification state of the 5' ends. During the past year, some nuclear proteins normally involved in RNA processing have been shown also to facilitate 'internal' ribosome binding. Unexpected dual functions have, therefore, been suggested for these RNA-binding proteins, in both RNA biogenesis in the nucleus and RNA translation in the cytoplasm.

Inactivation of Hepatovirus A in wastewater by 254 nm ultraviolet-C irradiation.

Hepatovirus A is known as a waterborne and foodborne virus that can be transmitted from one person to another through contaminated water and raw food. Therefore, it is necessary to survey the circulation of this type of enteric virus in the wastewater to prevent prospective outbreaks. Wastewater samples collected from WWTP El Menzeh I and Charguia I have been the subject for physicochemical, bacteriological (MPN) and virological analyses. Hepatovirus A (HAV) detection was carried out using the standard reverse transcription-polymerase chain reaction (RT-PCR). Hepatovirus A was detected respectively in 62% (63/102) and 66% (92/140) of the collected wastewater samples at El Menzeh I and Charguia I WWTPs. The treated effluent by natural oxidizing lagoon procedure was characterized by a poor physical-chemical and virological qualities but with excellent bacteriological quality. Consequently, this effluent is not suitable to be recycled and reused in agriculture or even dismissed in the environment. The treated sewage by activated sludge and rotating biodisk procedures turned out to be of a very good physical-chemical quality but with a poor bacteriological and virological quality. After tertiary UV-C254 nm irradiation, the faecal indicator bacteria concentration was mostly reduced and removed. These findings confirmed the need for improvement and upgrade of the treatment processes used in these two studied sewage purification plants and the necessity of implementation and establishment of a proper national virological standard to control the circulation rates of enteric viruses in Tunisian municipal wastewater.

Antiviral Properties of Alginate-Based Biomaterials: Promising Antiviral Agents against SARS-CoV-2.

The COVID-19 pandemic has made it essential to explore alternative antiviral materials. Alginate is a biodegradable, renewable, biocompatible, water-soluble and antiviral biopolymer with many potential biomedical applications. In this regard, this review shows 17 types of viruses that have been tested in contact with alginate and its related biomaterials. Most of these studies show that alginate-based materials possess little or no toxicity and are able to inhibit a wide variety of viruses affecting different organisms: in humans by the human immunodeficiency virus type 1, the hepatitis A, B, and C viruses, Sindbis virus, herpes simplex virus type 1 and 2, poliovirus type 1, rabies virus, rubella virus, and the influenza virus; in mice by the murine norovirus; in bacteria by the T4 coliphage, and in plants by the tobacco mosaic virus and the potato virus X. Many of these are enveloped positive-sense single-stranded RNA viruses, like SARS-CoV-2, which render alginate-based materials highly promising in the COVID-19 pandemic.

Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses.

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.

Cytokines and chronic liver disease.

From an immunological point of view, the healthy liver has been usually associated with the phenomenon of tolerance. A microenvironment of regulatory cytokines produced by liver Kuppfer cells and liver sinusoidal endothelial cells has contributed, together with resident dendritic cells, to generate a tolerogenic environment in this tissue. In this review we discussed the intrahepatic responses to different sorts of liver injury, such as hepatotrophic viruses, alcohol or putative self-antigens. In each case we analyzed the impact of different cytokines in the clinical outcome of the different pathological situations.

NK and NKT cells in liver injury and fibrosis.

The innate immune mechanisms of the liver represent an important first line of defense against bacterial products, toxins, and food antigens coming from the intestine. Natural Killer (NK) and Natural Killer T cells (NKT) are components of the innate immune system with increased presence in the liver compared to other organs and have been reported to participate in the inflammatory processes during hepatic diseases. However significant confusion has been noted in this field mainly due to changes in the characterization of these cells as new knowledge accumulates and due to differences in the approaches used for their study. Both cell types can mediate hepatic injury in several models but studies in human liver diseases have not managed to fully explain their functions. However accumulating evidence supports an antifibrotic role of NK cells mainly via an inhibitory effect on hepatic stellate cells by inducing apoptosis and via production of interferon-gamma. Therefore, downregulation of NK cells during most types of liver injury may facilitate liver fibrosis. Data about the role of NKT cells in liver fibrosis are limited. This review will summarize the studies about the role of NK and NKT cells in liver diseases with a special interest in hepatic injury and liver fibrosis.