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Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, affects 8-10 million people worldwide and represents a major public health challenge. There is no effective treatment or vaccine to control the disease that is characterized by a mild acute phase followed by a chronic life-long infection. Approximately 30% of chronically infected individuals develop cardiac and/or digestive pathologies. T. cruzi can invade a wide variety of nucleated cells, but only persists at specific tissues in the host. However, the mechanisms that determine tissue tropism and the progression of the infection have not been fully described. Identification of infection niches in animal models has been difficult due to the limited quantity of parasite-infected cells and their focal distribution in tissues during the chronic phase. To better understand the course of chronic infections and parasite dissemination, we developed a bioluminescence imaging system based on the use of transgenic T. cruzi Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal in vivo imaging showed parasites predominantly distributed in the abdominal cavity and surrounding areas throughout the infection. Bioluminescence signal reached a peak between 14 to 21 days post infection (dpi) and decreased progressively over time. Total animal luminescence could still be measured 126 dpi while parasites remained undetectable in blood by microscopy in most animals. Ex vivo imaging of specific tissues and organs dissected post-mortem at 126 dpi revealed a widespread parasite distribution in the skeletal muscle, heart, intestines and mesenteric fat. Parasites were also detected in lungs and liver. This noninvasive imaging model represents a novel tool to study host-parasite interactions and to identify parasite reservoirs of chronic Chagas Disease.

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease, and an effective vaccine is not available. In this study, we compared the immunogenicity and protection efficacy of recombinant proteins corresponding to different domains of the SARS-coronavirus spike protein. Trimeric recombinant proteins were created by fusing the foldon domain derived from T4 bacteriophage to the carboxy-termini of individual domains of the spike protein. While the full-length ectodomain (S) of the spike protein, the full-length ectodomain fused to foldon (S-foldon), the S1 domain (S1), S1-foldon, and the S2 domain(S2) antigens all elicited comparable antibody titers as measured by ELISA, S-foldon induced a significantly higher titer of neutralizing antibody and S2 protein did not elicit virus neutralizing antibodies. When tested in a mouse virus replication model, all the mice vaccinated with the S1, S1-foldon, S, or S-foldon were completely protected.

Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets.

Hepatitis A virus (HAV) infects African green monkey kidney (AGMK) cells via the HAV cellular receptor-1 (havcr-1), a mucin-like type 1 integral-membrane glycoprotein of unknown natural function. The ectodomain of havcr-1 contains an N-terminal immunoglobulin-like cysteine-rich region (D1), which binds protective monoclonal antibody (MAb) 190/4, followed by an O-glycosylated mucin-like threonine-serine-proline-rich region that extends D1 well above the cell surface. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc portion of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVR-Fc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified through protein A-agarose columns. In a solid-phase assay, HAV bound to D1-Fc in a concentration-dependent manner whereas background levels of HAV bound to PVR-Fc. Binding of HAV to D1-Fc was blocked by treatment with MAb 190/4 but not with control MAb M2, which binds to a tag epitope introduced between the D1 and Fc portions of the immunoadhesin. D1-Fc neutralized approximately 1 log unit of the HAV infectivity in AGMK cells, whereas PVR-Fc had no effect in the HAV titers. A similarly poor reduction in HAV titers was observed after treating the same stock of HAV with murine neutralizing MAbs K2-4F2, K3-4C8, and VHA 813. Neutralization of poliovirus by PVR-Fc but not by D1-Fc indicated that the virus-receptor interactions were specific. These results show that D1 is sufficient for binding and neutralization of HAV and provide further evidence that havcr-1 is a functional cellular receptor for HAV.

This article addresses how low-income urban adolescents view the fairness of different aspects of American society, including how wealth is distributed, the nature of legal constraints, and overall social opportunities and legitimacy. This research emerged from efforts to understand the moral and emotional nature of some adolescents' aggressive tendencies. Recently it has become clearer that aggression can serve many purposes and that, for some adolescents, aggression is a coherent though problematic response to larger familial, neighborhood, and institutional forces. Consequently, the authors focus on the connections between low-income adolescents' perceptions of institutional and interpersonal fairness, certain aggressive tendencies, and related emotion judgments. At the same time, relatively little is known about how low-income adolescents as a group perceive the fairness of wealth distribution and other broad aspects of American society. Consequently, a second important goal is to examine these adolescents' normative beliefs about institutional fairness at a time of growing financial and educational inequalities in the United States.

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.

Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC50] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.

Background Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, usurps the HAV cellular receptor 1 (HAVCR1) to infect cells. HAVCR1 is a class 1 integral membrane glycoprotein that contains two extracellular domains: a virus-binding immunoglobulin-like (IgV) domain and a mucin-like domain that extends the IgV from the cell membrane. Soluble forms of HAVCR1 bind, alter, and neutralize cell culture-adapted HAV, which is attenuated for humans. However, the requirements of the HAV-HAVCR1 interaction have not been fully characterized, and it has not been determined whether HAVCR1 also serves as a receptor for wild-type (wt) HAV. Here, we used HAV soluble receptor neutralization and alteration assays to study the requirements of the HAV-HAVCR1 interaction and to determine whether HAVCR1 is also a receptor for wt HAV. Results Treatment of HAV with a soluble form of HAVCR1 that contained the IgV and two-thirds of the mucin domain fused to the Fc fragment of human IgG1 (D1 muc-Fc), altered particles at 37°C but left a residual level of unaltered particles at 4°C. The kinetics of neutralization of HAV by D1 muc-Fc was faster at 37°C than at 4°C. Alteration of HAV particles by D1 muc-Fc required Ca, which could not be replaced by Li, Na, Mg, Mn, or Zn. Neutralization of HAV by D1 muc-Fc occurred at pH 5 to 8 but was more efficient at pH 6 to 7. D1 muc-Fc neutralized wt HAV as determined by a cell culture system that allows the growth of wt HAV. Conclusion The interaction of HAV with soluble forms of HAVCR1 shares the temperature, Ca, and pH requirements for infectivity in cell culture and therefore mimics the cell entry process of HAV. Since soluble forms of HAVCR1 also neutralized wt HAV, this receptor may play a significant role in pathogenesis of HAV.

The hepatitis A virus cellular receptor 1 (HAVCR1/TIM1), a member of the T-cell immunoglobulin mucin (TIM) family, is an important atopy susceptibility gene in humans. The exact natural function of HAVCR1/TIM1 and the inverse association between HAV infection and prevention of atopy are not well understood. To identify natural ligands of human HAVCR1/TIM1, we used an expression cloning strategy based on the binding of dog cells transfected with a human lymph node cDNA library to a HAVCR1/TIM1 Fc fusion protein. The transfected cells that bound to the human HAVCR1/TIM1 Fc contained cDNA of human immunoglobulin alpha 1 heavy (Igα1) and lambda light (Igλ) chain and secreted human IgA1λ antibody that bound to the cell surface. Cotransfection of the isolated Igα1 and Igλ cDNAs to naïve dog cells resulted in the secretion of IgA1λ that bound to HAVCR1/TIM1 Fc but not to a poliovirus receptor Fc fusion protein in a capture enzyme-linked immunosorbent assay. The interaction of HAVCR1/TIM1 with IgA was inhibited by monoclonal antibodies (MAbs) against Igα1 and Igλ, excess IgA1λ, or anti-HAVCR1/TIM1 MAb. IgA did not inhibit HAV infection of African green monkey cells, suggesting that the IgA and the virus binding sites are in different epitopes on HAVCR1/TIM1. IgA enhanced significantly the neutralization of HAV by HAVCR1/TIM1 Fc. Our results indicate that IgA1λ is a specific ligand of HAVCR1/TIM1 and that their association has a synergistic effect in virus-receptor interactions.

Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics., Author Summary Hepatitis C virus (HCV) causes a persistent infection that can lead to hepatocellular carcinoma and liver cirrhosis. Interferon (IFN)-based treatments are ineffective for some HCV genotypes. HCV research has been hampered by the lack of suitable cell culture systems. With the discovery of a unique HCV genotype 2a isolate that can replicate in the human liver cell line Huh7, some obstacles were overcome. However, there remains the need of systems to grow IFN-resistant genotypes and serum-derived isolates. Here we show that the presence of adenovirus-associated RNAI (VA RNAI), a known IFN antagonist, permitted establishment of a persistent infection of genotype 1b in VeroE6 cells that were passaged weekly for more than 2 years. The persistent virus induces strong cytopathic effect (CPE), a feature that allowed the development of a CPE-based assay to test HCV-specific inhibitors, neutralization by anti-HCV immunoglobulins and by anti-CD81 antibody, and HCV-specific siRNA. Our system provides the first persistent culture of genotype 1b virus and a convenient assay that can be used for therapeutics development.

Interferon-alpha (IFN-alpha) mono-therapy is largely ineffective for most of the hepatitis C virus (HCV)-infected patients that receive it. The addition of ribavirin to IFN therapy has increased the response rate dramatically. While many factors are implicated in determining the success rate for IFN therapy, viral genotype seems to play a crucial role. Examining differences in viral gene sequences has and will continue to advance our understanding as to how HCV and other viruses circumvent the IFN response. Here we review the different ways that HCV evades the immune response elicited by IFN.