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Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment. ispartof: JOURNAL OF MEDICAL VIROLOGY vol:94 issue:7 pages:3101-3111 ispartof: location:United States status: published

PurposeThis paper aims to discuss the importance of having several entry points into the field, via gatekeepers who do not belong to law enforcement agencies, in contexts where the police cannot be defined as trustworthy.Design/methodology/approachThe argumentation of this paper is based on qualitative research on women and gangs in Honduras. An ethnographical methodology was implemented, which included over a year of observations, 65 interviews and two focus groups in gang-controlled communities and detention centers in Central America (with a focus on Honduras), between 2017 and 2020. The paper implements a feminist reflexive approach, focusing on patriarchy, positionality and silence.FindingsCollaborating with the police as gatekeepers in gang research needs to be reevaluated. In countries such as Honduras, the police are riddled with corruption and impunity, which eventually leads to mistrust among gang members and other citizens. Hence, it is recommended to approach other, non-law enforcement, gatekeepers, who often stand much closer to the gangs and have a less conflicted or biased position toward them and toward other people living in gang areas.Research limitations/implicationsA feminist reflexive approach is recommended for researching women and gangs, and thus also for choosing the right gatekeepers in the field, taking into account researchers’ and gatekeepers’ positionality.Originality/valuePolice corruption in relation to gangs and gang-related crimes often goes unreported and silences people living in gang-controlled areas. This paper exposes these conflicted roles, not only regarding police abusevis-à-visgangs and people living in gang areas but also in relation to gang researchers in the field.

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10 , as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15). ispartof: JOURNAL OF MEDICAL VIROLOGY vol:93 issue:7 pages:4454-4460 ispartof: location:United States status: published

BackgroundAs long as there is no vaccine available, having access to inhibitors of SARS-CoV-2 will be of utmost importance. Antivirals against coronaviruses do not exist, hence global drug re-purposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have potential activity against animal coronaviruses.MethodsUsing cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with SARS-CoV-2.ResultsItraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 μM; MTT assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 μM). Remdesivir inhibited viral replication with an EC50 = 0.4 μM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3 log10 drop and >4 log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively.DiscussionItraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).HighlightsItraconazole exerted in vitro low micromolar activity against SARS-CoV-2 (EC50 = 2.3 μM)Remdesivir demonstrated potent antiviral activity, confirming validity of the assayItraconazole has since shown no efficacy in a clinical study in hospitalized COVID-19 patients

The phenomenon of gangs in El Salvador and the Democratic Republic of Congo (DRC) has already been researched, but a comparative study of gangs and gang policies is lacking. In this paper we discuss several gang violence prevention initiatives regarding the Mara Salvatrucha and Barrio 18 in El Salvador, and the kuluna and bashege in Kinshasa, DRC. In order to analyze the different gang interventions, we implement the typology of first and second gang violence prevention initiatives (Rodgers et al. 2009), and propose the evolution towards a third generation of gang violence prevention interventions. While first generation initiatives are security and law-enforcement driven, and second generation initiatives socio-preventive driven, third generation initiatives are more politically driven. The latter indicates a shift towards a vision of dialogue and negotiations to deal with gang violence. However, the different generations are not predefined within time and third generation initiatives can also be followed up by first generation initiatives, which was for example the case in the gang truce in El Salvador. Also, comparative gang research includes challenges, especially when the gang phenomenon in one country is better researched and documented than in other countries. As such, we were unable to identify politically-driven initiatives in the DRC to compare with the ones in El Salvador. Further research is thus required. With this paper we not only aim to contribute to the literature on gang violence prevention and reduction initiatives, we also want to push researchers, practitioners and policymakers to look beyond the borders when setting up gang (violence) prevention and rehabilitation projects, and to learn from other regions where similar initiatives have been implemented to deal with comparable issues of gang violence.

Research on women in Central American gangs over the past two decades has focused on their being sexual objects or servants to the male gang members (Baird, A. (2018). Becoming the ‘Baddest’: Masculine trajectories of gang violence in Medellin. Journal of Latin American Studies, 50(1), 183–210. https://doi.org/10.1017/S0022216X17000761; Resendiz Rivera, N. E. (2017). Mujeres, pandillas y violencia en Guatemala. Cuadernos Inter.c.a.mbio sobre Centroamerica y el Caribe, 14(1), 50–75. https://doi.org/10.15517/c.a..v14i1.28614; Resendiz Rivera, N. E. (2017). Mujeres, pandillas y violencia en Guatemala. Cuadernos Inter.c.a.mbio sobre Centroamerica y el Caribe, 14(1), 50–75. https://doi.org/10.15517/c.a..v14i1.28614). Fieldwork conducted by the authors in El Salvador and Honduras over the past years suggests the position and role of women in gangs may have changed, whereby women have become more involved with gang tasks such as collecting extortion money or assassination. Academic research is lacking in this area, however, and the media maintain a rather paternalistic discourse, focusing on sensationalist stories of gang rape and femicides. In this article, we look at the way women are covered, or not, in popular media in relation to gangs. Focusing on digital newspapers, we make a comparison of media coverage in El Salvador and Honduras, the two Central American countries which are most affected by the widespread gang presence in the region Farah, D., & Babineau, K. (2017). The evolution of MS 13 in El Salvador and Honduras. Prism: A Journal of the Center for Complex Operations, 7(1), 58–73. While media coverage of women in gangs in Honduras has increased, with sensationalist stories focusing on the “cruelty” of female gang members, women remain in the shadow of male gang members in El Salvador. In these patriarchal societies, media coverage is gendered, with men in protagonist positions of gang leaders and women in inferior positions of mere partners of male gang members, with roles pertaining more to the household than the street.

Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R- group.

Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dose-dependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

Food insecurity is still remarkably high in the poorest areas of the KwaZulu-Natal province of South Africa. Many rural households struggle to have sufficient access to the food they need or prefer. This article explores the extent of food access insecurity and assesses the perceived impact on food security of an Empowerment for Food Security Programme that was launched in the Province in 2007. One of the programme aims was to improve agricultural practices in community gardens, home gardens and broiler production. Data were collected among 390 beneficiary households involved in these agricultural projects in 2010. Findings confirmed that experience-based food insecurity levels were still high, despite the agricultural support programme and the government income transfers. Nevertheless, respondents attribute an improved dietary diversity and better access to resources to the programme.

The focus of this chapter is on the reintegration of youth gang members in Honduras. The aim of this qualitative research is to broaden the knowledge of reintegration processes and programs in one specific country in Central America, namely, Honduras. A sample of 14 interviews with stakeholders in the field of maras and pandillas is being investigated and the results revealed some similarities between the different methods of reintegration. Our data suggest that a comprehensive process including the family, school, community, and governmental support via social politics is necessary to give the youngster a platform where he can reintegrate himself into society, and refrain from committing any criminal behavior. However, most respondents agreed upon the fact that leaving a gang is very difficult and includes high risks, the most infringing being assassination. Most stakeholders call for more investment in prevention at an early stage, i.e., before a youngster gets related to a gang.

Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naive neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential of HCMV to adapt to or influence different cellular environments to promote its own survival.

Human cytomegalovirus, a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' UTR of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of immediate early (IE) gene expression that is detectable by IE-specific cytotoxic T-cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral immediate early (IE) gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.

Human cytomegalovirus is an opportunistic double-stranded DNA virus with one of the largest viral genomes known. The 235 kB genome is divided in a unique long (UL) and a unique short (US) region which are flanked by terminal and internal repeats. The expression of HCMV genes is highly complex and involves the production of protein coding transcripts, polyadenylated long non-coding RNAs, polyadenylated anti-sense transcripts and a variety of non-polyadenylated RNAs such as microRNAs. Although the function of many of these transcripts is unknown, they are suggested to play a direct or regulatory role in the delicately orchestrated processes that ensure HCMV replication and life-long persistence. This review focuses on annotating the complete viral genome based on three sources of information. First, previous reviews were used as a template for the functional keywords to ensure continuity; second, the Uniprot database was used to further enrich the functional database; and finally, the literature was manually curated for novel functions of HCMV gene products. Novel discoveries were discussed in light of the viral life cycle. This functional annotation highlights still poorly understood regions of the genome but more importantly it can give insight in functional clusters and/or may be helpful in the analysis of future transcriptomics and proteomics studies.

Human cytomegalovirus (HCMV) is a ubiquitous virus that can cause serious sequelae in immunocompromised patients and in the developing fetus. The coding capacity of the 235 kbp genome is still incompletely understood, and there is a pressing need to characterize genomic contents in clinical isolates. In this study, a procedure for the high-throughput generation of full genome consensus sequences from clinical HCMV isolates is presented. This method relies on low number passaging of clinical isolates on human fibroblasts, followed by digestion of cellular DNA and purification of viral DNA. After multiple displacement amplification, highly pure viral DNA is generated. These extracts are suitable for high-throughput next-generation sequencing and assembly of consensus sequences. Throughout a series of validation experiments, we showed that the workflow reproducibly generated consensus sequences representative for the virus population present in the original clinical material. Additionally, the performance of 454 GS FLX and/or Illumina Genome Analyzer datasets in consensus sequence deduction was evaluated. Based on assembly performance data, the Illumina Genome Analyzer was the platform of choice in the presented workflow. Analysis of the consensus sequences derived in this study confirmed the presence of gene-disrupting mutations in clinical HCMV isolates independent from in vitro passaging. These mutations were identified in genes RL5A, UL1, UL9, UL111A and UL150. In conclusion, the presented workflow provides opportunities for high-throughput characterization of complete HCMV genomes that could deliver new insights into HCMV coding capacity and genetic determinants of viral tropism and pathogenicity.

Human cytomegalovirus (HCMV) is a common opportunistic pathogen that can cause devastating -morbidity and mortality amongst neonates and immune-compromised patients. The current standard of care for HCMV infection is limited to four antiviral compounds that have major limitations in terms of long--term use, toxicity, and use during pregnancy. To provide patients with alternative treatment options to decrease HCMV-related morbidity and mortality, new drugs with novel modes of action are warranted. Here, we describe a validated high-throughput fluorescence antiviral screening assay based on infection of fibroblast cells with a fluorescently tagged reference strain of HCMV (AD169-GFP) to screen and profile HCMV inhibitors.

Human cytomegalovirus (HCMV) is a common opportunistic pathogen that can cause devastating -morbidity and mortality amongst neonates and immune-compromised patients. The current standard of care for HCMV infection is limited to four antiviral compounds that have major limitations in terms of long--term use, toxicity, and use during pregnancy. To provide patients with alternative treatment options to decrease HCMV-related morbidity and mortality, new drugs with novel modes of action are warranted. Here, we describe a validated high-throughput fluorescence antiviral screening assay based on infection of fibroblast cells with a fluorescently tagged reference strain of HCMV (AD169-GFP) to screen and profile HCMV inhibitors.

Promyelocytic Leukaemia Protein nuclear bodies (PML-NBs) are dynamic nuclear protein aggregates. To gain insight in PML-NB function, reductionist and high throughput techniques have been employed to identify PML-NB proteins. Here we present a manually curated network of the PML-NB interactome based on extensive literature review including database information. By compiling the PML-ome, we highlighted the presence of interactors in the Small Ubiquitin Like Modifier (SUMO) conjugation pathway. Additionally, we show an enrichment of SUMOylatable proteins in the PML-NBs through an in-house prediction algorithm. Therefore, based on the PML network, we hypothesize that PML-NBs may function as a nuclear SUMOylation hotspot.