An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Establishment of a robust rat hepatitis E virus fecal-oral infection model and validation for antiviral studies.

The hepatitis E virus (HEV) is a major cause of hepatitis, with an estimated 3.3 million symptomatic cases annually. There is no HEV-specific treatment besides the off-label use of ribavirin and a vaccine is only available in China and Pakistan. To aid the development of therapeutic and preventive strategies, there is a need for convenient HEV infection models in small laboratory animals. To this end, we make use of the rat hepatitis E virus. Human infections with this virus have been reported in recent years, making it a relevant pathogen for the establishment of a small animal infection model. We here report that oral gavage of a feces suspension, containing a pre-defined viral RNA load, results in a reproducible synchronized infection in athymic nude rats. This route of administration mimics fecal-oral transmission in a standardized fashion. The suitability of the model to study the effect of antiviral drugs was assessed by using ribavirin, which significantly reduced viral loads in the feces, liver, and other tissues.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

AAV9-mediated functional screening for cardioprotective cytokines in Coxsackievirus-B3-induced myocarditis.

Viral myocarditis (VM) is an important cause of heart failure (HF) in children and adults. However, the molecular determinants involved in cardiac inflammation and cardiomyocyte necrosis remain poorly characterized, and cardioprotective molecules are currently missing. Here, we applied an in vivo method based on the functional selection (FunSel) of cardioprotective factors using AAV vectors for the unbiased identification of novel immunomodulatory molecules in a Coxsackievirus B3 (CVB3)-induced myocarditis mouse model. Two consecutive rounds of in vivo FunSel using an expression library of 60 cytokines were sufficient to identify five cardioprotective factors (IL9, IL3, IL4, IL13, IL15). The screening also revealed three cytokines (IL18, IL17b, and CCL11) that were counter-selected and likely to exert a detrimental effect. The pooled overexpression of the five most enriched cytokines using AAV9 vectors decreased inflammation and reduced cardiac dilatation, persisting at 1 month after treatment. Individual overexpression of IL9, the top ranking in our functional selection, markedly reduced cardiac inflammation and injury, concomitant with an increase of anti-inflammatory Th2-cells and a reduction of pro-inflammatory Th17- and Th22-cells at 14 days post-infection. AAV9-mediated FunSel cardiac screening identified IL9 and other four cytokines (IL3, IL4, IL13, and IL15) as cardioprotective factors in CVB3-induced VM in mice.

Capsid Assembly Modulators as Antiviral Agents against HBV: Molecular Mechanisms and Clinical Perspectives.

Despite a preventive vaccine being available, more than 250 million people suffer from chronic hepatitis B virus (HBV) infection, a major cause of liver disease and HCC. HBV infects human hepatocytes where it establishes its genome, the cccDNA with chromosomal features. Therapies controlling HBV replication exist; however, they are not sufficient to eradicate HBV cccDNA, the main cause for HBV persistence in patients. Core protein is the building block of HBV nucleocapsid. This viral protein modulates almost every step of the HBV life cycle; hence, it represents an attractive target for the development of new antiviral therapies. Capsid assembly modulators (CAM) bind to core dimers and perturb the proper nucleocapsid assembly. The potent antiviral activity of CAM has been demonstrated in cell-based and in vivo models. Moreover, several CAMs have entered clinical development. The aim of this review is to summarize the mechanism of action (MoA) and the advancements in the clinical development of CAMs and in the characterization of their mod of action.

Genome Sequences and Phylogeny of Two Duck Hepatitis B Viruses.

Duck hepatitis B virus (DHBV) infection in Pekin ducks is a model for human hepatitis B. Sequence variations may contribute to host therapy responses against the virus. We provide full genome sequences of two DHBVs from France, their phylogenetic classification, and their sequence variability.

Hepatitis E virus replication and interferon responses in human placental cells.

Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV-infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental-derived cells (JEG-3). Following transfection of JEG-3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver-derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon-α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon-stimulated gene expression levels demonstrated an efficient HEV-dependent restriction in JEG-3. Conclusion: We showed differential tissue-specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell-culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated. (Hepatology Communications 2018;2:173-187).

Biological or pharmacological activation of protein kinase C alpha constrains hepatitis E virus replication.

Although hepatitis E has emerged as a global health issue, there is limited knowledge of its infection biology and no FDA-approved medication is available. Aiming to investigate the role of protein kinases in hepatitis E virus (HEV) infection and to identify potential antiviral targets, we screened a library of pharmacological kinase inhibitors in a cell culture model, a subgenomic HEV replicon containing luciferase reporter. We identified protein kinase C alpha (PKCα) as an essential cell host factor restricting HEV replication. Both specific inhibitor and shRNA-mediated knockdown of PKCα enhanced HEV replication. Conversely, over-expression of the activated form of PKCα or treatment with its pharmacological activator strongly inhibited HEV replication. Interestingly, upon the stimulation by its activator, PKCα efficiently activates its downstream Activator Protein 1 (AP-1) pathway, leading to the induction of antiviral interferon-stimulated genes (ISGs). This process is independent of the JAK-STAT machinery and interferon production. However, PKCα induced HEV inhibition appears independent of the AP1 cascade. The discovery that activated PKCα restricts HEV replication reveals new insight of HEV-host interactions and provides new target for antiviral drug development.

Study of hepatitis E virus infection of genotype 1 and 3 in mice with humanised liver.

OBJECTIVE: The hepatitis E virus (HEV) is responsible for approximately 20 million infections per year worldwide. Although most infected people can spontaneously clear an HEV infection, immune-compromised individuals may evolve towards chronicity. Chronic HEV infection can be cured using ribavirin, but viral isolates with low ribavirin sensitivity have recently been identified. Although some HEV isolates can be cultured in vitro, in vivo studies are essentially limited to primates and pigs. Since the use of these animals is hampered by financial, practical and/or ethical concerns, we evaluated if human liver chimeric mice could serve as an alternative. DESIGN: Humanised mice were inoculated with different HEV-containing preparations. RESULTS: Chronic HEV infection was observed after intrasplenic injection of cell culture-derived HEV, a filtered chimpanzee stool suspension and a patient-derived stool suspension. The viral load was significantly higher in the stool compared with the plasma. Overall, the viral titre in genotype 3-infected mice was lower than that in genotype 1-infected mice. Analysis of liver tissue of infected mice showed the presence of viral RNA and protein, and alterations in host gene expression. Intrasplenic injection of HEV-positive patient plasma and oral inoculation of filtered stool suspensions did not result in robust infection. Finally, we validated our model for the evaluation of novel antiviral compounds against HEV using ribavirin. CONCLUSIONS: Human liver chimeric mice can be infected with HEV of different genotypes. This small animal model will be a valuable tool for the in vivo study of HEV infection and the evaluation of novel antiviral molecules.

Relevance of cardiac parvovirus B19 in myocarditis and dilated cardiomyopathy: review of the literature.

Over the last decade, parvovirus B19 (B19V) has frequently been linked to the pathogenesis of myocarditis (MC) and its progression towards dilated cardiomyopathy (DCM). The exact role of the presence of B19V and its load remains controversial, as this virus is also found in the heart of healthy subjects. Moreover, the prognostic relevance of B19V prevalence in endomyocardial biopsies still remains unclear. As a result, it is unclear whether the presence of B19V should be treated. This review provides an overview of recent literature investigating the presence of B19V and its pathophysiological relevance in MC and DCM, as well as in normal hearts. In brief, no difference in B19V prevalence is observed between MC/DCM and healthy control hearts. Therefore, the question remains open whether and how cardiac B19V may be of pathogenetic importance. Findings suggest that B19V is aetiologically relevant either in the presence of other cardiotropic viruses, or when B19V load is high and/or actively replicating, which both may maintain myocardial (low-grade) inflammation. Therefore, future studies should focus on the prognostic relevance of the viral load, replicative status and virus co-infections. In addition, the immunogenetic background of MC/DCM patients that makes them susceptible to develop heart failure upon presence of B19V should be more thoroughly investigated.

A rat model for hepatitis E virus.

Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans). As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains). Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies.

Viral Macro Domains Reverse Protein ADP-Ribosylation.

UNLABELLED: ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE: Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified by macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the macro domain to PAR and simulates the demodification activity.

Hepatitis E virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity.

BACKGROUND & AIMS: Ribavirin monotherapy is the preferred treatment for chronic hepatitis E, although occasional treatment failure occurs. We present a patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype. We aimed to identify viral mutations associated with treatment failure and explore the underlying mechanisms. METHODS: Viral genomes were deep-sequenced at different time points and the role of identified mutations was assessed in vitro using mutant replicons, antiviral assays, cell culture of patient-derived virus and deep-sequencing. RESULTS: Ribavirin resistance was associated with Y1320H, K1383N and G1634R mutations in the viral polymerase, but also an insertion in the hypervariable region comprising a duplication and a polymerase-derived fragment. Analysis of these genome alterations in vitro revealed replication-increasing roles for Y1320H and G1634R mutations and the hypervariable region insertion. In contrast, the K1383N mutation in the polymerase F1-motif suppressed viral replication and increased the in vitro sensitivity to ribavirin, contrary to the clinical phenotype. Analysis of the replication of mutant full-length virus and in vitro culturing of patient-derived virus confirmed that sensitivity to ribavirin was retained. Finally, deep-sequencing of hepatitis E virus genomes revealed that ribavirin is mutagenic to viral replication in vitro and in vivo. CONCLUSIONS: Mutations Y1320H, G1634R and the hypervariable region insertion compensated for K1383N-associated replication defects. The specific role of the K1383N mutation remains enigmatic, but it appears to be of importance for the ribavirin resistant phenotype in this patient. LAY SUMMARY: Ribavirin is the most common treatment for chronic hepatitis E and is mostly effective, although some cases of ribavirin treatment failure have been described. Here, we report on a particular case of ribavirin resistance and investigate the underlying causes of treatment failure. Mutations in the viral polymerase, an essential enzyme for viral replication, appear to be responsible.

Update on hepatitis E virology: Implications for clinical practice.

Hepatitis E virus (HEV) is a positive-strand RNA virus transmitted by the fecal-oral route. The 7.2kb genome encodes three open reading frames (ORF) which are translated into (i) the ORF1 polyprotein, representing the viral replicase, (ii) the ORF2 protein, corresponding to the viral capsid, and (iii) the ORF3 protein, a small protein involved in particle secretion. Although HEV is a non-enveloped virus in bile and feces, it circulates in the bloodstream wrapped in cellular membranes. HEV genotypes 1 and 2 infect only humans and cause mainly waterborne outbreaks. HEV genotypes 3 and 4 are widely represented in the animal kingdom and are transmitted as a zoonosis mainly via contaminated meat. HEV infection is usually self-limited but may persist and cause chronic hepatitis in immunocompromised patients. Reduction of immunosuppressive treatment or antiviral therapy with ribavirin have proven effective in most patients with chronic hepatitis E but therapy failures have been reported. Alternative treatment options are needed, therefore. Infection with HEV may also cause a number of extrahepatic manifestations, especially neurologic complications. Progress in the understanding of the biology of HEV should contribute to improved control and treatment of HEV infection.

Stem cell-derived hepatocytes: A novel model for hepatitis E virus replication.

BACKGROUND & AIMS: Yearly, approximately 20million people become infected with the hepatitis E virus (HEV) resulting in over 3million cases of acute hepatitis. Although HEV-mediated hepatitis is usually self-limiting, severe cases of fulminant hepatitis as well as chronic infections have been reported, resulting annually in an estimated 60,000 deaths. We studied whether pluripotent stem cell (PSC)-derived hepatocytes, mesodermal and/or neuroprogenitor cells support HEV replication. METHODS: Human PSC were differentiated towards hepatocyte-like cells, mesodermal cells and neuroprogenitors and subsequently infected with HEV. Infection and replication of HEV was analyzed by qRT-PCR, RNA in situ hybridization, negative strand RT-PCR, production of infectious virions and transfection with a transient HEV reporter replicon. RESULTS: PSC-derived hepatocytes supported the complete replication cycle of HEV, as demonstrated by the intracellular presence of positive and negative strand HEV RNA and the production of infectious virions. The replication of the virus in these cells was inhibited by the antiviral drugs ribavirin and interferon-α2b. In contrast to PSC-derived hepatocytes, PSC-derived mesodermal cells and neuroprogenitors only supported HEV replication upon transfection with a HEV subgenomic replicon. CONCLUSION: We demonstrate that PSC can be used to study the hepatotropism of HEV infection. The complete replication cycle of HEV can be recapitulated in infected PSC-derived hepatocytes. By contrast other germ layer cells support intracellular replication but are not infectable with HEV. Thus the early steps in the viral cycle are the main determinant governing HEV tissue tropism. PSC-hepatocytes offer a physiological relevant tool to study the biology of HEV infection and replication and may aid in the design of therapeutic strategies.

Sofosbuvir Inhibits Hepatitis E Virus Replication In Vitro and Results in an Additive Effect When Combined With Ribavirin.

Infection with hepatitis E virus genotype 3 may result in chronic hepatitis in immunocompromised patients. Reduction of immunosuppression or treatment with ribavirin or pegylated interferon-α can result in viral clearance. However, safer and more effective treatment options are needed. Here, we show that sofosbuvir inhibits the replication of hepatitis E virus genotype 3 both in subgenomic replicon systems as well as a full-length infectious clone. Moreover, the combination of sofosbuvir and ribavirin results in an additive antiviral effect. Sofosbuvir may be considered as an add-on therapy to ribavirin for the treatment of chronic hepatitis E in immunocompromised patients.

The future of antivirals: broad-spectrum inhibitors.

PURPOSE OF REVIEW: Potent antivirals are successfully used for the treatment of infections with herpesviruses, hepatitis B and C viruses, HIV, and with some success for influenza viruses. However, no selective inhibitors are available for a multitude of medically important viruses, most of which are (re-)emerging RNA viruses. As it is impossible to develop drugs against each of these viruses, broad-spectrum antiviral agents (BSAA) are a prime strategy to cope with this challenge. RECENT FINDINGS: We propose four categories of antiviral molecules that hold promise as BSAA. Several nucleoside analogues with broad antiviral activity have been described and given the relatively conserved nature of viral polymerases, it may be possible to develop more broad-spectrum nucleoside analogues. A number of viral proteins are relatively conserved between families and may also be interesting targets. Host-targeting antiviral drugs such as modulators of lipid metabolism and cyclophilin inhibitors can be explored as well. Finally, the potent and broad antiviral function of the immune system can be exploited by the development of immune-modulating BSAA. SUMMARY: Despite the recent advances, the BSAA field is still in its infancy. Nevertheless, the discovery and development of such molecules will be a key aim of antiviral research in the coming decades.