CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission

Free Will & Empirical Arguments for Epiphenomenalism

While philosophers have worried about mental causation for centuries, worries about the causal relevance of conscious phenomena are also increasingly featuring in neuroscientific literature. Neuroscientists have regarded the threat of epiphenomenalism as interesting primarily because they have supposed that it entails free will scepticism. However, the steps that get us from a premise about the causal irrelevance of conscious phenomena to a conclusion about free will are not entirely clear. In fact, if we examine popular philosophical accounts of free will, we find, for the most part, nothing to suggest that free will is inconsistent with the presence of unconscious neural precursors to choices. It is only if we adopt highly non-naturalistic assumptions about the mind (e.g. if we embrace Cartesian dualism and locate free choice in the non-physical realm) that it seems plausible to suppose that the neuroscientific data generates a threat to free will

Chikungunya Virus Neutralization Antigens and Direct Cell-to-Cell Transmission Are Revealed by Human Antibody-Escape Mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop “groove” as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Retaining Participants in Community-Based Health Research: A Case Example on Standardized Planning and Reporting

Background Effective strategies for participant retention are critical in health research to ensure validity, generalizability and efficient use of resources. Yet standardized guidelines for planning and reporting on retention efforts have been lacking. As with randomized controlled trial (RCT) and systematic review (SR) protocols, retention protocols are an opportunity to improve transparency and rigor. An RCT being conducted in British Columbia (BC), Canada provides a case example for developing a priori retention frameworks for use in protocol planning and reporting. Methods The BC Healthy Connections Project RCT is examining the effectiveness of a nurse home-visiting program in improving child and maternal outcomes compared with existing services. Participants (N = 739) were girls and young women preparing to parent for the first time and experiencing socioeconomic disadvantage. Quantitative data were collected upon trial entry during pregnancy and during five follow-up interviews until participants’ children reached age 2 years. A framework was developed to guide retention of this study population throughout the RCT. We reviewed relevant literature and mapped essential retention activities across the study planning, recruitment and maintenance phases. Interview completion rates were tracked. Results Results from 3302 follow-up interviews (in-person/telephone) conducted over 4 years indicate high completion rates: 90% (n = 667) at 34 weeks gestation; and 91% (n = 676), 85% (n = 626), 80% (n = 594) and 83% (n = 613) at 2, 10, 18 and 24 months postpartum, respectively. Almost all participants (99%, n = 732) provided ongoing consent to access administrative health data. These results provide preliminary data on the success of the framework. Conclusions Our retention results are encouraging given that participants were experiencing considerable socioeconomic disadvantage. Standardized retention planning and reporting may therefore be feasible for health research in general, using the framework we have developed. Use of standardized retention protocols should be encouraged in research to promote consistency across diverse studies, as now happens with RCT and SR protocols. Beyond this, successful retention approaches may help inform health policy-makers and practitioners who also need to better reach, engage and retain underserved populations