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Background & Aims Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods To analyze the evolution of the buoyant density of in vivo–derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet., Graphical abstract

New York City rats provide a gift to virologists Despite the development of curative drugs for hepatitis C virus (HCV) infection, global eradication of HCV will likely require a prophylactic vaccine. Progress toward a vaccine has been impeded by the absence of mouse models suitable for studying the immune response to HCV. Billerbeck et al. found that a HCV-related virus isolated from New York City rats produces an infection in laboratory mice that shares several immunological features with human infections (see the Perspective by Klenerman and Barnes). Their initial analyses of the infected mice revealed that acute clearance of the virus was dependent on T cells but not on natural killer cells. Science , this issue p. 204 ; see also p. 129

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in “humanized” mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

At least 170 million people are chronically infected with hepatitis C virus (HCV). Owing to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small nonhuman primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV-RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be owing to the diminished capacity of HCV to antagonize mitochondrial antiviral-signaling protein-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks.Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection, and vaccine development.

SummaryInterferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.

Differentiation of CD4(+) T cells into type 1 or type 2 subsets is mediated by the expression of the opposing lineage defining transcription factors T-bet and GATA-3. However, the existence of GATA-3(+) T-bet(+) CD4(+) T cells in mice suggests functional plasticity of these subsets. Little is known about type 1 and type 2 plasticity of human T-cell subsets in vivo. Here, we show that in the xenogeneic environment of humanized mice, which lacks a functional immune-regulatory network, human CD4(+) and, notably, CD8(+) T cells preferentially differentiate into interleukin (IL)-4(+) GATA-3(+) and IL-4(+) interferon-γ(+) GATA-3(+) T-bet(+) subsets. Treatment with recombinant human IL-12 or expansion of IL-12-producing human dendritic cells in vivo reverted this phenotype and led to the down-regulation of GATA-3 expression. These changes also correlated with improved antiviral immune responses in humanized mice. In conclusion, our study shows the capacity of human CD4(+) and CD8(+) T cells for stable co-expression of GATA-3 and T-bet in humanized mice and reveals a critical role for IL-12 in regulating this phenotype.

Li et al. describe a novel role for IRF2, previously known as a negative regulator of type I IFN signaling, in protection of mice from lethal viral neuroinvasion by facilitating the proper localization of B cells and antibodies to the central nervous system., The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2−/− mice after peripheral inoculation. Irf2−/− mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2−/− mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell–deficient μMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2−/− mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections.

Background & Aims Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34 + hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. Methods The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah −/− mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. Results Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah −/− mice by 5–100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. Conclusions Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. Lay summary Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.

In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. We demonstrate that three broadly nAbs-AR3A, AR3B, and AR4A-delivered with adeno-associated viral vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.