The interaction of hepatitis A virus (HAV) with soluble forms of its cellular receptor 1 (HAVCR1) share the physiological requirements of infectivity in cell culture

<p>Abstract</p> <p>Background</p> <p>Hepatitis A virus (HAV), an atypical <it>Picornaviridae </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

The transcriptome landscape of 3D-cultured placental trophoblasts reveals activation of TLR2 and TLR3/7 in response to low Trypanosoma cruzi parasite exposure

Vertical transmission of Trypanosoma cruzi (T. cruzi) become a globalized health problem accounting for 22% of new cases of Chagas disease (CD). Congenital infection is now considered the main route of CD spread in non-endemic countries where no routine disease testing of pregnant women is implemented. The main mechanisms that lead to fetal infection by T. cruzi remain poorly understood. Mother-to-child transmission may occur when bloodstream trypomastigotes interact with the syncytiotrophoblasts (SYNs) that cover the placenta chorionic villi. These highly specialized cells function as a physical barrier and modulate immune responses against pathogen infections. To model the human placenta environment, we have previously used a three-dimensional (3D) cell culture system of SYNs that exhibits differentiation characteristics comparable to placental trophoblasts. Further, we have shown that 3D-grown SYNs are highly resistant to T. cruzi infection. In this work, we used RNA sequencing and whole transcriptome analysis to explore the immunological signatures that drive SYNs’ infection control. We found that the largest category of differentially expressed genes (DEGs) are associated with inflammation and innate immunity functions. Quantitative RT-PCR evaluation of selected DEGs, together with detection of cytokines and chemokines in SYNs culture supernatants, confirmed the transcriptome data. Several genes implicated in the Toll-like receptors signaling pathways were upregulated in 3D-grown SYNs. In fact, TLR2 blockade and TLR3/7 knockdown stimulated T. cruzi growth, suggesting that these molecules play a significant role in the host cell response to infection. Ingenuity Pathway Analysis of DEGs predicted the activation of canonical pathways such as S100 protein family, pathogen induced cytokine storm, wound healing, HIF1α signaling and phagosome formation after T. cruzi exposure. Our findings indicate that SYNs resist infection by eliciting a constitutive pro-inflammatory response and modulating multiple defense mechanisms that interfere with the parasite’s intracellular life cycle, contributing to parasite killing and infection control

Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

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

Data_Sheet_1_The transcriptome landscape of 3D-cultured placental trophoblasts reveals activation of TLR2 and TLR3/7 in response to low Trypanosoma cruzi parasite exposure.PDF

Vertical transmission of Trypanosoma cruzi (T. cruzi) become a globalized health problem accounting for 22% of new cases of Chagas disease (CD). Congenital infection is now considered the main route of CD spread in non-endemic countries where no routine disease testing of pregnant women is implemented. The main mechanisms that lead to fetal infection by T. cruzi remain poorly understood. Mother-to-child transmission may occur when bloodstream trypomastigotes interact with the syncytiotrophoblasts (SYNs) that cover the placenta chorionic villi. These highly specialized cells function as a physical barrier and modulate immune responses against pathogen infections. To model the human placenta environment, we have previously used a three-dimensional (3D) cell culture system of SYNs that exhibits differentiation characteristics comparable to placental trophoblasts. Further, we have shown that 3D-grown SYNs are highly resistant to T. cruzi infection. In this work, we used RNA sequencing and whole transcriptome analysis to explore the immunological signatures that drive SYNs’ infection control. We found that the largest category of differentially expressed genes (DEGs) are associated with inflammation and innate immunity functions. Quantitative RT-PCR evaluation of selected DEGs, together with detection of cytokines and chemokines in SYNs culture supernatants, confirmed the transcriptome data. Several genes implicated in the Toll-like receptors signaling pathways were upregulated in 3D-grown SYNs. In fact, TLR2 blockade and TLR3/7 knockdown stimulated T. cruzi growth, suggesting that these molecules play a significant role in the host cell response to infection. Ingenuity Pathway Analysis of DEGs predicted the activation of canonical pathways such as S100 protein family, pathogen induced cytokine storm, wound healing, HIF1α signaling and phagosome formation after T. cruzi exposure. Our findings indicate that SYNs resist infection by eliciting a constitutive pro-inflammatory response and modulating multiple defense mechanisms that interfere with the parasite’s intracellular life cycle, contributing to parasite killing and infection control.</p

Data_Sheet_2_The transcriptome landscape of 3D-cultured placental trophoblasts reveals activation of TLR2 and TLR3/7 in response to low Trypanosoma cruzi parasite exposure.PDF

Vertical transmission of Trypanosoma cruzi (T. cruzi) become a globalized health problem accounting for 22% of new cases of Chagas disease (CD). Congenital infection is now considered the main route of CD spread in non-endemic countries where no routine disease testing of pregnant women is implemented. The main mechanisms that lead to fetal infection by T. cruzi remain poorly understood. Mother-to-child transmission may occur when bloodstream trypomastigotes interact with the syncytiotrophoblasts (SYNs) that cover the placenta chorionic villi. These highly specialized cells function as a physical barrier and modulate immune responses against pathogen infections. To model the human placenta environment, we have previously used a three-dimensional (3D) cell culture system of SYNs that exhibits differentiation characteristics comparable to placental trophoblasts. Further, we have shown that 3D-grown SYNs are highly resistant to T. cruzi infection. In this work, we used RNA sequencing and whole transcriptome analysis to explore the immunological signatures that drive SYNs’ infection control. We found that the largest category of differentially expressed genes (DEGs) are associated with inflammation and innate immunity functions. Quantitative RT-PCR evaluation of selected DEGs, together with detection of cytokines and chemokines in SYNs culture supernatants, confirmed the transcriptome data. Several genes implicated in the Toll-like receptors signaling pathways were upregulated in 3D-grown SYNs. In fact, TLR2 blockade and TLR3/7 knockdown stimulated T. cruzi growth, suggesting that these molecules play a significant role in the host cell response to infection. Ingenuity Pathway Analysis of DEGs predicted the activation of canonical pathways such as S100 protein family, pathogen induced cytokine storm, wound healing, HIF1α signaling and phagosome formation after T. cruzi exposure. Our findings indicate that SYNs resist infection by eliciting a constitutive pro-inflammatory response and modulating multiple defense mechanisms that interfere with the parasite’s intracellular life cycle, contributing to parasite killing and infection control.</p

A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9.

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

A novel nanoluciferase-based system to monitor <i>Trypanosoma cruzi</i> infection in mice by bioluminescence imaging

<div><p>Chagas disease, caused by the intracellular protozoan <i>Trypanosoma cruzi</i>, 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. <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal <i>in vivo</i> 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. <i>Ex vivo</i> 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.</p></div

HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis.

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

Monitoring the course of TcCOL-NLuc chronic infection in Swiss Webster mice by live bioluminescence imaging.

<p>(A) Representative ventral view images of mouse #1 infected with TcCOL-NLuc tissue culture-derived trypomastigotes (5 x10³ parasites) taken at the indicated time points. NI: non-infected control mouse. Log ₁₀ heat-map scale represents bioluminescence intensity (blue: low; red: high). (B) Total abdominal bioluminescence (total flux) for seven individual mice and their mean (red line) measured in the regions of interest indicated in (A) and over the course of infection. The grey line indicates the detection threshold determined as the mean (solid line) and mean ± 2 s.d. (dashed lines) of background luminescence of the control uninfected mouse. (C) Blood parasitemia (trypomastigotes/ml) determined by microscopy over time for each individual mouse and mean (blue line). Grey line indicates the limit of detection (1.58 x10⁴ parasites/ml blood).</p