Multiple effects of silymarin on the hepatitis C virus lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell

IL-1β Production through the NLRP3 Inflammasome by Hepatic Macrophages Links Hepatitis C Virus Infection with Liver Inflammation and Disease

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease

Innate Immune Tolerance and the Role of Kupffer Cells in Differential Responses to Interferon Therapy Among Patients With HCV Genotype 1 Infection

In patients with hepatitis C virus (HCV) infection, interferon alfa (IFN-α) alters expression of IFN-stimulated genes (ISGs), but little is understood about factors that determine outcomes of therapy. We used a systems biology approach to evaluate the acute response of patients with chronic hepatitis C to IFN-α therapy

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Mechanism of hepatic inflammation during hepatitis C virus infection

Thesis (Ph.D.)--University of Washington, 2013Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer, but the processes that promote hepatic inflammation by HCV are not defined. We used a systems biology analysis together with multiple lines of evidence to demonstrate that interleukin-1&beta (IL-1&beta) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1&beta compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells- resident hepatic macrophages- are the primary cellular source of hepatic IL-1&beta during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages and Kupffer cells recovered from normal donor liver produce IL-1&beta after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1&beta processing and secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggered the MyD88-mediated TLR7 signaling pathway to induce IL-1&beta mRNA expression. HCV uptake concomitantly induced a potassium efflux that activated the NLRP3 inflammasome for IL-1&beta processing and secretion. RNA sequencing analysis comparing THP1 cells and liver samples from chronic hepatitis C virus-infected patients revealed that viral engagement of the NLRP3 inflammasome stimulates IL-1&beta production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identified intrahepatic IL-17beta production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1&beta activity could offer therapeutic approaches to reduce hepatic inflammation and mitigate disease

Functional characterization of core genes from patients with acute hepatitis C virus infection

Background. The hepatitis C virus (HCV) core protein is implicated in diverse aspects of HCV-induced pathogenesis. There is a paucity of information on core in acute hepatitis C infection.<p></p> Methods. We analyzed core gene sequences and protein functions from 13 patients acutely infected with HCV genotype 1.<p></p> Results. Although core isolates differed slightly between patients, core quasispecies were relatively homogeneous within each patient. In 2 of 4 patients studied temporally, core quasispecies did not change over time. Comparison with more than 2700 published core isolates indicated that amino acid changes from a prototype reference strain found in acute core isolates were present in chronically infected persons at low frequency (6.4%; range, 0%-32%). Core isolates associated with lipid droplets to similar degrees in Huh7 cells. Core diffusion in cells was not affected by nonconservative changes F130L and G161S in the lipid targeting domain of core. Core isolates inhibited interferon-stimulated response element- and nuclear factor κB-dependent transcription and tumor necrosis factor α-induced nuclear translocation of nuclear factor κB and were also secreted from Huh7 cells.<p></p> Conclusions. The data suggest that upon transmission, core quasispecies undergo genetic homogenization associated with amino acid changes that are rarely found in chronic infection and that, despite genetic variation, acute core isolates retain similar functions in vitro

HCV triggers the NLRP3 inflammasome and IL-1β maturation through induction of potassium efflux after macrophage uptake.

<p>(<b>A</b>) Secreted IL-β protein levels (upper panel) and immunoblot analysis of IL-1β (lower panel set) of THP-1 treated with transfection reagent or transfected with either with full length HCV RNA or polyU/UC RNA or exposed to HCV (moi = 0.01). (<b>B</b>) IL-β mRNA expression in THP1 stably expressing non-targeting control shRNA or shRNA specific to NLRP3 or caspase-1. (<b>C</b>) Immunoblot of caspase-1 and IL-1β in THP1 stably expressing non-targeting control the indicated shRNA. (<b>D</b>) THP-1 were pre-treated with DMSO (control) or with 6.25, 12.5, 25, 50, 100, 200 µM of potassium channel inhibitor glybenclamide (Glyben) for 2 hrs followed by mock treatment (M; control) or HCV (moi = 0.01) exposure in the presence of glyben for an additional 1 hr. (E) IL-1β p17 abundance in THP-1 cultured in normal media or in media containing NaCl (100 mM) or KCl (100 mM) for 1 hr followed by mock-treatment (-) or exposure to HCV (moi = 0.01) in the same media for an additional 1 hr.</p

IL-1β signaling and the NRLP3 inflammasome are required for immunity against WNV.

<p>6–10 wk old age matched WT (closed circles; n = 26) or <i>Il-1r^−/−</i> (open squares; n = 14) (<b>A</b>) or <i>Caspase-1^−/−</i> (open circles; n = 12) and <i>Nlrp3^−/−</i> (closed square; n = 12) (<b>D</b>) or WT (closed circles, n = 5) and <i>Nlrc4^−/−</i> (open circles; n = 4) (<b>E</b>) or WT (closed circles, n = 5) and <i>Myd88^−/−</i> (closed square; n = 5) (<b>F</b>) animals were infected with 100 PFU WNV-TX via s.c. foot-pad inoculation and monitored for survival over the course of 14–16 days (<b>A, D–F</b>). Infected WT (closed circles) or <i>Il-1r^−/−</i> (open squares) animals from panel A, were monitored daily for weight loss (<b>B</b>) or scored for hind limb paralysis and morbidity (<b>C</b>) to day 16 post infection. *p<0.05, ** p<0.005, *** p<0.0005.</p

HCV stimulates IL-1β production upon uptake by macrophages.

<p>(<b>A</b>) IL-1β mRNA expression (upper panel) and secreted IL-1β protein levels (lower panel) from primary monocyte-derived macrophages of healthy human donor blood. Cells were mock-treated or treated with infectious HCV supernatant (moi = 0.01 based on Huh7 focus forming units (ffu) or treated with 1 µg/ml of LPS and ATP 5 mM (LPS/ATP; positive control). (<b>B</b>) Intracellular cytokine staining of treated CD14+ cells recovered from saline washout of healthy donor liver. Cells were left untreated (unstim) or were cultured with conditioned media (mock, negative control), LPS (positive control) or treated with UV-inactivated HCV (moi = 0.01 based on Huh7 focus forming units (ffu)). Data are shown from one donor and are representative two experiments each of cells collected from two independent donors. In the analysis shown the frequency of IL-1β-expressing cells was: unstim, 2.7%; mock, 6.4%; LPS, 76.5%; UV-HCV. 67.6%. (<b>C</b>)–(<b>I</b>) Analysis of THP1 cells. (<b>C</b>) IL-1β mRNA expression post exposure to HCV. (<b>D</b>) IL-1β protein secretion after treatment with variable doses of HCV (moi = 0.001, 0.01 or 0.1 Huh7 ffu) or LPS/ATP at 1 µg/ml for 24 hr. (<b>E</b>) Immunoblot showing the kinetics of caspase-1 activation after HCV exposure. (<b>F</b>) Levels of secreted IL-1β over a time course after HCV exposure (moi = 0.01 based on Huh7 ffu). (<b>G</b>) IL-1β levels secreted 24 hr after exposure to (left to right) cell culture media, sucrose solution, sucrose-purified culture media, infectious HCV supernatant or sucrose-purified HCV virions. (<b>H</b>) IL-1β mRNA expression in pre-treated with DMSO (control), bafilomycin (2.5 uM) or cytochalasin D (10 µM) cells and exposed to media or infectious HCV supernatant (moi = 0.01 Huh7 ffu). (<b>I</b>) Levels of secreted IL-1β 24 hr post treatment with conditioned media alone (mock) or treatment with live infectious HCV (HCV, moi = 0.01) or UV-inactivated HCV (HCV-UV). <i>*P = 0.0175</i> and <i>***P = 0.0005</i>, by student <i>t-test</i>.</p