Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain

AbstractInfections with hepatitis C virus (HCV) genotype 3 exhibit differences in clinical phenotype including an increase in response to interferon therapy and development of steatosis. To initiate studies on genotype 3, we created a chimeric genotype 1b replicon containing a genotype 3a NS5A domain. The chimera was capable of efficient colony formation after the selection of a novel dominant adaptive mutation. Thus, domains from highly different strains can interact to form a functional replicase. A new genotype 1a replicon was constructed as well. Genotype specific influence on interferon sensitivity was examined using genotype 1a, 1b and chimeric 1b–3a replicons. The genotype 3a NS5A domain did not increase the sensitivity of the chimeric replicon to IFNα. The results suggest that NS5A is not sufficient to convey the increased IFNα response by genotype 3 or the replicon model is not capable of mimicking the events involved in increased sustained viral response

The Chimpanzee Model of Viral Hepatitis: Advances in Understanding the Immune Response and Treatment of Viral Hepatitis

Chimpanzees (Pan troglodytes) have contributed to diverse fields of biomedical research due to their close genetic relationship to humans and in many instances due to the lack of any other animal model. This review focuses on the contributions of the chimpanzee model to research on hepatitis viruses where chimpanzees represented the only animal model (hepatitis B and C) or the most appropriate animal model (hepatitis A). Research with chimpanzees led to the development of vaccines for HAV and HBV that are used worldwide to protect hundreds of millions from these diseases and, where fully implemented, have provided immunity for entire generations. More recently, chimpanzee research was instrumental in the development of curative therapies for hepatitis C virus infections. Over a span of 40 years, this research would identify the causative agent of NonA,NonB hepatitis, validate the molecular tools for drug discovery, and provide safety and efficacy data on the therapies that now provide a rapid and complete cure of HCV chronic infections. Several cocktails of antivirals are FDA approved that eliminate the virus following 12 weeks of once-per-day oral therapy. This represents the first cure of a chronic viral disease and, once broadly implemented, will dramatically reduce the occurrence of cirrhosis and liver cancer. The recent contributions of chimpanzees to our current understanding of T cell immunity for HCV, development of novel therapeutics for HBV, and the biology of HAV are reviewed. Finally, a perspective is provided on the events leading to the cessation of the use of chimpanzees in research and the future of the chimpanzees previously used to bring about these amazing breakthroughs in human healthcare

Peeling Bifurcations of Toroidal Chaotic Attractors

Chaotic attractors with toroidal topology (van der Pol attractor) have counterparts with symmetry that exhibit unfamiliar phenomena. We investigate double covers of toroidal attractors, discuss changes in their morphology under correlated peeling bifurcations, describe their topological structures and the changes undergone as a symmetry axis crosses the original attractor, and indicate how the symbol name of a trajectory in the original lifts to one in the cover. Covering orbits are described using a powerful synthesis of kneading theory with refinements of the circle map. These methods are applied to a simple version of the van der Pol oscillator.Comment: 7 pages, 14 figures, accepted to Physical Review

Lack of susceptibility of baboons to infection with hepatitis B virus

Historically, hepatitis B virus (HBV) has been considered species specific and unable to infect baboons. Based on this premise, two patients with HBV end-stage liver disease underwent baboon liver xenotransplantation. To study whether baboons are susceptible to HBV infection, four baboons (two receiving immunosuppressive therapy) were inoculated with HBV. Animals were followed for 6 months: clinical examinations and biochemical studies were normal, hepatitis B surface antigen and hepatitis B core antigen staining of biopsies was negative, and HBV serology remained negative. HBV polymerase chain reaction was transiently positive in one animal, which most likely reflects the initial inoculation. This pilot study corroborates historical evidence and beliefs that baboons are resistant to HBV

Amplification of simian retroviral sequences from human recipients of baboon liver transplants

Investigations into the use of baboons as organ donors for human transplant recipients, a procedure called xenotransplantation, have raised the specter of transmitting baboon viruses to humans and possibly establishing new human infectious diseases. Retrospective analysis of tissues from two human transplant recipients with end-stage hepatic disease who died 70 and 27 days after the transplantation of baboon livers revealed the presence of two simian retroviruses of baboon origin, simian foamy virus (SFV) and baboon endogenous virus (BaEV), in multiple tissue compartments. The presence of baboon mitochondrial DNA was also detected in these same tissues, suggesting that xenogeneic 'passenger leukocytes' harboring latent or active viral infections had migrated from the xenografts to distant sites within the human recipients. The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation

Therapeutic silencing of microRNA-122 in primates with chronic hepatitis

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide with over 170 million infected individuals who are at greatly increased risk of developing liver failure and hepatocellular carcinoma. The current standard anti-HCV therapy, which combines pegylated interferon-α (IFN-α) with ribavirin provides sustained clearance of HCV in only about 50% of patients and is often associated with serious side effects (1, 2). Therapies that target essential host functions for HCV may provide a high barrier to resistance and, thus, could present an effective approach for the development of new HCV antiviral drugs. MicroRNA-122 (miR-122) is a highly abundant, liver-expressed microRNA, which binds to two closely spaced target sites in the 5' non-coding region (NCR) of the HCV genome, resulting in upregulation of viral RNA levels (3, 4). Interaction of miR-122 with the HCV genome is essential for accumulation of viral RNA in cultured liver cells and both target sites are required for modulation of HCV RNA abundance (3-5). Previously, we reported on potent and specific miR-122 silencing in vivo using a LNA-modified phosphorothioate oligonucleotide (SPC3649) complementary to the 5'-end of miR-122 leading to long-lasting decrease of serum cholesterol in mice and African green monkeys (6). Here, we investigated the potential of miR-122 antagonism by SPC3649 as a new anti-HCV therapy in chronically infected chimpanzees (genotype 1). Baseline measurements were obtained from four chimpanzees for four weeks, the last two of which included an intravenous (i.v.) placebo dose of saline. Two animals each were assigned to the high and low dose groups, 5 and 1 mg kg -1 , respectively, and were treated with i.v. injections of SPC3649 on a weekly basis for 12 week

Primate innate immune responses to bacterial and viral pathogens reveals an evolutionary trade-off between strength and specificity

Despite their close genetic relatedness, apes and African and Asian monkeys (AAMs) differ in their susceptibility to severe bacterial and viral infections that are important causes of human disease. Such differences between humans and other primates are thought to be a result, at least in part, of interspecies differences in immune response to infection. However, because of the lack of comparative functional data across species, it remains unclear in what ways the immune systems of humans and other primates differ. Here, we report the whole-genome transcriptomic responses of ape species (human and chimpanzee) and AAMs (rhesus macaque and baboon) to bacterial and viral stimulation. We find stark differences in the responsiveness of these groups, with apes mounting a markedly stronger early transcriptional response to both viral and bacterial stimulation, altering the transcription of ∼40% more genes than AAMs. Additionally, we find that genes involved in the regulation of inflammatory and interferon responses show the most divergent early transcriptional responses across primates and that this divergence is attenuated over time. Finally, we find that relative to AAMs, apes engage a much less specific immune response to different classes of pathogens during the early hours of infection, up-regulating genes typical of anti-viral and anti-bacterial responses regardless of the nature of the stimulus. Overall, these findings suggest apes exhibit increased sensitivity to bacterial and viral immune stimulation, activating a broader array of defense molecules that may be beneficial for early pathogen killing at the potential cost of increased energy expenditure and tissue damage

Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R

ABSTRACT Many DNA tumor viruses promote cellular transformation by inactivating the critically important tumor suppressor protein p53. In contrast, it is not known whether p53 function is disrupted by hepatitis C virus (HCV), a unique, oncogenic RNA virus that is the leading infectious cause of liver cancer in many regions of the world. Here we show that HCV-permissive, liver-derived HepG2 cells engineered to constitutively express microRNA-122 (HepG2/miR-122 cells) have normal p53-mediated responses to DNA damage and that HCV replication in these cells potently suppresses p53 responses to etoposide, an inducer of DNA damage, or nutlin-3, an inhibitor of p53 degradation pathways. Upregulation of p53-dependent targets is consequently repressed within HCV-infected cells, with potential consequences for cell survival. Despite this, p53 function is not disrupted by overexpression of the complete HCV polyprotein, suggesting that altered p53 function may result from the host response to viral RNA replication intermediates. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting HCV replication, showing that p53 inhibition results directly from viral activation of PKR. The hepatocellular abundance of phosphorylated PKR is elevated in HCV-infected chimpanzees, suggesting that PKR activation and consequent p53 inhibition accompany HCV infection in vivo . These findings reveal a feature of the host response to HCV infection that may contribute to hepatocellular carcinogenesis. IMPORTANCE Chronic infection with hepatitis C virus (HCV) is the leading cause of liver cancer in most developed nations. However, the mechanisms whereby HCV infection promotes carcinogenesis remain unclear. Here, we demonstrate that HCV infection inhibits the activation of p53 following DNA damage. Contrary to previous reports, HCV protein expression is insufficient to inhibit p53. Rather, p53 inhibition is mediated by cellular protein kinase R (PKR), which is activated by HCV RNA replication and subsequently suppresses global protein synthesis. These results redefine our understanding of how HCV infection influences p53 function. We speculate that persistent disruption of p53-mediated DNA damage responses may contribute to hepatocellular carcinogenesis in chronically infected individuals

Human pDCs preferentially sense enveloped hepatitis A virions

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-α production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-α induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs

microRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection

Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy