Although hepatotropic viruses are important causes of human disease, the intrahepatic immune response to hepatitis viruses is poorly understood due to a lack of tractable small animal models. Here we describe a murine model of hepatitis A virus (HAV) infection that recapitulates critical features of type A hepatitis in humans. We demonstrate that the capacity of HAV to evade MAVS-mediated type I interferon responses defines its host species range. HAV-induced liver injury was associated with interferon-independent intrinsic hepatocellular apoptosis and hepatic inflammation that unexpectedly results from MAVS and IRF3/7 signaling. This murine model thus reveals a previously undefined link between innate immune responses to virus infection and acute liver injury, providing a new paradigm for viral pathogenesis in the liver.

The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasienveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAVassociated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.exosome | multivesicular body | ESCRT | extracellular vesicle | picornavirus T he Picornaviridae are a large and diverse family of positivestrand RNA viruses that include numerous pathogens (1). Classically considered "nonenveloped," these viruses package their single-stranded genomes within stable icosahedral protein capsids that are released from cells following cell lysis. However, recent work has revealed that several picornaviruses gain egress from cells in a nonlytic fashion within the lumen of extracellular vesicles shed from the cell. Most notably, hepatitis A virus (HAV; genus Hepatovirus), an important cause of enterically transmitted hepatitis in humans, replicates without cytopathic effect and is released from cells as membrane-wrapped, "quasienveloped" (eHAV) virions similar in size and density to exosomes (2). eHAV virions have been identified in sera collected from persons with acute hepatitis A, as well as in supernatant fluids of infected cell cultures. These virions are completely cloaked in host-derived membranes that protect the capsid from neutralizing antibody until the quasi-envelope is degraded wi...

Current paradigms of cell intrinsic immunity to RNA viruses center on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors (RLRs) or Toll-like receptors (TLRs) that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons (IFNs) 1 . RNA viruses have evolved sophisticated strategies to disrupt these signaling pathways and evade elimination by cells, attesting to their importance 2 . Less attention has been paid how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RLR and TLR signaling in order to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show constitutively expressed IRF1 acts independently of MAVS, IRF3, and STAT1-dependent signaling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses (HAV and HCV), dengue virus (DENV) and Zika virus (ZIKV). Our findings reveal an unappreciated layer of hepatocyte intrinsic immunity to these positive-strand RNA viruses, and identify previously unrecognized antiviral effector genes.

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1−/− Ifnar1−/− and Tim4−/− Ifnar1−/− double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1−/− mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1−/−Ifnar1−/− mice compared to Ifnar1−/− mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.

Attention deficit is an early event in the cognitive impairment of patients with minimal hepatic encephalopathy (MHE). The underlying mechanisms remain unclear. Mismatch negativity (MMN) is an auditory event-related potential that reflects an attentional trigger. Patients with schizophrenia show impaired attention and cognitive function, which are reflected in altered MMN. We hypothesized that patients with MHE, similarly to those with schizophrenia, should show MMN alterations related with attention deficits. The aims of this work were to assess whether (1) MMN is altered in cirrhotic patients with MHE, compared to those without MHE, (2) MMN changes in parallel with performance in attention tests and/or MHE in a longitudinal study, and (3) MMN predicts performance in attention tests and/or in the Psychometric Hepatic Encephalopathy Score (PHES). We performed MMN analysis as well as attention and coordination tests in 34 control subjects and in 37 patients with liver cirrhosis without MHE and 23 with MHE. Patients with MHE show reduced performance in selective and sustained attention tests and in visuomotor and bimanual coordination tests. The MMN wave area was reduced in patients with MHE, but not in those without MHE. In the longitudinal study, MMN area improved in parallel with performance in attention tests and PHES in 4 patients and worsened in parallel in another 4. Logistic regression analyses showed that MMN area predicts performance in attention tests and in PHES, but not in other tests or critical flicker frequency. Receiver operating characteristic curve analyses showed that MMN area predicts attention deficits in the number connection tests A and B, Stroop tasks, and MHE, with sensitivities of 75%-90% and specificities of 76%-83%. Conclusion: MMN area is useful to diagnose attention deficits and MHE in patients with liver cirrhosis. (HEPATOLOGY 2012;55:530-539) A pproximately 33%-50% of patients with liver cirrhosis without clinical symptoms of encephalopathy show minimal hepatic encephalopathy (MHE), which can be unveiled using psychometric tests or neurophysiological analysis. [1][2][3][4] Patients with MHE show attention deficits and mild cognitive impairment. MHE reduces quality of life and is associated with increased risk of suffering with work, driving, and home accidents as well as clinical hepatic encephalopathy (HE) and reduced life span. [5][6][7][8][9][10] Attention deficits are an early manifestation of MHE. [11][12][13][14][15][16] Amodio et al. 16 reported that MHE affects primarily selective attention control. Weissenborn et al. 15 reported that patients with MHE show dysfunction in all attention subsystems. The brain areas involved in the attention system and the alterations