A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.

Identification of Z-Tyr-Ala-CHN2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.

Development and optimization of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: Evaluation of 5676 Phase 1 Passed Structures.

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.

The Hepatitis B Virus Interactome: A Comprehensive Overview.

Despite the availability of a prophylactic vaccine, chronic hepatitis B (CHB) caused by the hepatitis B virus (HBV) is a major health problem affecting an estimated 292 million people globally. Current therapeutic goals are to achieve functional cure characterized by HBsAg seroclearance and the absence of HBV-DNA after treatment cessation. However, at present, functional cure is thought to be complicated due to the presence of covalently closed circular DNA (cccDNA) and integrated HBV-DNA. Even if the episomal cccDNA is silenced or eliminated, it remains unclear how important the high level of HBsAg that is expressed from integrated HBV DNA is for the pathology. To identify therapies that could bring about high rates of functional cure, in-depth knowledge of the virus' biology is imperative to pinpoint mechanisms for novel therapeutic targets. The viral proteins and the episomal cccDNA are considered integral for the control and maintenance of the HBV life cycle and through direct interaction with the host proteome they help create the most optimal environment for the virus whilst avoiding immune detection. New HBV-host protein interactions are continuously being identified. Unfortunately, a compendium of the most recent information is lacking and an interactome is unavailable. This article provides a comprehensive review of the virus-host relationship from viral entry to release, as well as an interactome of cccDNA, HBc, and HBx.

In vitro activity of itraconazole against SARS-CoV-2.

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).

Lack of antiviral activity of darunavir against SARS-CoV-2.

OBJECTIVES: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. METHODS: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. RESULTS: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 µM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 µM). CONCLUSIONS: Overall, the data do not support the use of DRV for the treatment of COVID-19.

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus.

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.

HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types.

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-naïve 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 miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells.

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.

High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination.

Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCE: Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

Glucocorticosteroids trigger reactivation of human cytomegalovirus from latently infected myeloid cells and increase the risk for HCMV infection in D+R+ liver transplant patients.

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.

Functional annotation of human cytomegalovirus gene products: an update.

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.

A method enabling high-throughput sequencing of human cytomegalovirus complete genomes from clinical isolates.

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.

A fluorescence-based high-throughput screening assay for identifying human cytomegalovirus 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.

Crosstalk between viruses and PML nuclear bodies: a network-based approach.

Due to the recent advances in instrumental and scientific methods, cell biology data are generated with increasing speed and quantity. One of these fast developing fields is the crosstalk between promyelocytic leukemia protein nuclear bodies (PML-NBs) and viruses. PML-NBs are dynamic nuclear protein aggregates which are targeted by entire viral particles, viral proteins or viral nucleic acids. Their possible anti-viral properties motivated researchers to investigate the interaction between PML-NBs and viruses in depth. Based on extensive literature data mining, we created a comprehensive PML-NB/virus crosstalk Cytoscape network, which groups not only the most common relations but also less well described findings. The network is easy to navigate and provides a biologically relevant overview which can help finding interesting case studies.

A manually curated network of the PML nuclear body interactome reveals an important role for PML-NBs in SUMOylation dynamics.

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.

Colistin sulfate as a suitable substitute of thallium acetate in culture media intended for mycoplasma detection and culture.

Thallium acetate in concentrations of 500 to 1000 mg/l is tolerated in the culture by the most mollicutes of the orders Mycoplasmatales and Acholeplasmatales and by this reason it is added in the culture media as a selective element for the detection and propagation of mycoplasmas and acholeplasmas. Because of the high toxicity of thallium acetate and its accumulation in the environment, thallium acetate is not biodegradable, an alternative was searched. The results and analysis of tests with nine mollicute species are presented here. It is recommended to replace thallium acetate in the formulations where it is used and colistin sulfate is proposed as its substitute.