Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A. © 2018 Elsevier B.V

Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors

Background & Aims All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Results Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis

The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation

Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation ‘freezes’ IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir

Community-based Cluster Surveys on Treatment Preferences for Diarrhoea, Severe Diarrhoea, and Dysentery in Children Aged Less Than Five Years in Two Districts of Ghana

Hospital-based surveillance for severe diarrhoea has been recommended to assess the burden of disease due to rotavirus. However, information on healthcare-seeking patterns of residents in the hospital catchment area is needed first to obtain the burden of disease in the community using the hospital data. A community-based cluster survey was conducted in two districts of Ghana, each served by a single district hospital, to determine the prevalence of severe diarrhoea among and treatment preferences for children aged less than five years. Caretakers of 619 children in Tema, an urban district, and caretakers of 611 children in Akwapim South, a rural district, were interviewed. During the month preceding the survey, the prevalence of severe diarrhoea in children aged less than five years was similar in the two districts (13.6% urban and 12.9% rural), as was the proportion of mothers who sought care outside the home (69.0% urban and 70.9% rural). 48.8% of urban mothers of children with severe diarrhoea visited public/private clinics, 9.5% pharmacies, and 3.6% the district hospital. Whereas, 22.8% of rural mothers visited public/private clinics, 19.0% pharmacies, and 13.9% the district hospital. Results of the study suggest that rotavirus surveillance should be guided by community studies on healthcare-use patterns. Where hospital use is low for severe diarrhoea, rotavirus surveillance should include other health facilities

Community-based Cluster Surveys on Treatment Preferences for Diarrhoea, Severe Diarrhoea, and Dysentery in Children Aged Less Than Five Years in Two Districts of Ghana

ABSTRACT Hospital-based surveillance for severe diarrhoea has been recommended to assess the burden of disease due to rotavirus. However, information on healthcare-seeking patterns of residents in the hospital catchment area is needed first to obtain the burden of disease in the community using the hospital data. A community-based cluster survey was conducted in two districts of Ghana, each served by a single district hospital, to determine the prevalence of severe diarrhoea among and treatment preferences for children aged less than five years. Caretakers of 619 children in Tema, an urban district, and caretakers of 611 children in Akwapim South, a rural district, were interviewed. During the month preceding the survey, the prevalence of severe diarrhoea in children aged less than five years was similar in the two districts (13.6% urban and 12.9% rural), as was the proportion of mothers who sought care outside the home (69.0% urban and 70.9% rural). 48.8% of urban mothers of children with severe diarrhoea visited public/private clinics, 9.5% pharmacies, and 3.6% the district hospital. Whereas, 22.8% of rural mothers visited public/private clinics, 19.0% pharmacies, and 13.9% the district hospital. Results of the study suggest that rotavirus surveillance should be guided by community studies on healthcare-use patterns. Where hospital use is low for severe diarrhoea, rotavirus surveillance should include other health facilities

Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p

Social factors influencing child health in Ghana

Objectives Social factors have profound effects on health. Children are especially vulnerable to social influences, particularly in their early years. Adverse social exposures in childhood can lead to chronic disorders later in life. Here, we sought to identify and evaluate the impact of social factors on child health in Ghana. As Ghana is unlikely to achieve the Millennium Development Goals’ target of reducing child mortality by two-thirds between 1990 and 2015, we deemed it necessary to identify social determinants that might have contributed to the non-realisation of this goal. Methods ScienceDirect, PubMed, MEDLINE via EBSCO and Google Scholar were searched for published articles reporting on the influence of social factors on child health in Ghana. After screening the 98 articles identified, 34 of them that met our inclusion criteria were selected for qualitative review. Results Major social factors influencing child health in the country include maternal education, rural-urban disparities (place of residence), family income (wealth/poverty) and high dependency (multiparousity). These factors are associated with child mortality, nutritional status of children, completion of immunisation programmes, health-seeking behaviour and hygiene practices. Conclusions Several social factors influence child health outcomes in Ghana. Developing more effective responses to these social determinants would require sustainable efforts from all stakeholders including the Government, healthcare providers and families. We recommend the development of interventions that would support families through direct social support initiatives aimed at alleviating poverty and inequality, and indirect approaches targeted at eliminating the dependence of poor health outcomes on social factors. Importantly, the expansion of quality free education interventions to improve would-be-mother’s health knowledge is emphasised