Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery.

Massive efforts on both vaccine development and antiviral research were launched to combat the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We contributed, amongst others, by the development of a high-throughput screening (HTS) antiviral assay against SARS-CoV-2 using a fully automated, high-containment robot system. Here, we describe the development of this novel, convenient and phenotypic dual-reporter virus-cell-based high-content imaging assay using the A549+hACE2+TMPRSS2_mCherry reporter lung carcinoma cell line and an ancestral SARS-CoV-2_Wuhan_mNeonGreen reporter virus. Briefly, by means of clonal selection, a host cell subclone was selected that (i) efficiently supports replication of the reporter virus with high expression, upon infection, of the NeonGreen fluorescent reporter protein, (ii) that is not affected by virus-induced cytopathogenic effects and, (iii) that expresses a strong fluorescent mCherry signal in the nucleus. The selected clone matched these criteria with an infection rate on average of 75% with limited cell death. The average (R)Z'-factors of the assay plates were all >0.8, which indicates a robust assay suitable for HTS purposes. A selection of reference compounds that inhibits SARS-CoV-2 replication in vitro were used to validate this novel dual-reporter assay and confirms the data reported in the literature. This assay is a convenient and powerful tool for HTS of large compound libraries against SARS-CoV-2.

Cytopathic SARS-CoV-2 screening on VERO-E6 cells in a large-scale repurposing effort.

Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.

Metabolically Improved Stem Cell Derived Hepatocyte-Like Cells Support HBV Life Cycle and Are a Promising Tool for HBV Studies and Antiviral Drug Screenings.

More than 300 million people worldwide are diagnosed with a chronic hepatitis B virus (HBV) infection. Nucleos(t)ide viral polymerase inhibitors are available on the market and can efficiently treat patients with chronic HBV. However, life-long treatment is needed as covalently closed circular DNA (cccDNA) persists in the hepatocyte nucleus. Hence, there is a high demand for novel therapeutics that can eliminate cccDNA from the hepatocyte nucleus and cure chronically infected HBV patients. The gold standard for in vitro HBV studies is primary human hepatocytes (PHHs). However, alternatives are needed due to donor organ shortage and high batch-to-batch variability. Therefore, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are being explored as an in vitro HBV infection model. We recently generated hPSC lines that overexpress three transcription factors (HC3x) and that, upon differentiation in a high amino-acid supplemented maturation medium, generate a more mature hepatocyte progeny (HC3x-AA-HLCs). Here, we demonstrate that HBV can efficiently infect these HC3x-AA-HLCs, as was shown by the presence of HBV core (HBc) and surface antigens. A clear increasing release of HBV surface and e antigens was detected, indicating the formation of functional cccDNA. Moreover, back-titration of culture supernatant of HBV-infected HC3x-AA-HLCs on HepG2-NTCP cells revealed the production of novel infectious HBV particles. Additionally, an increasing number of HBc-positive HC3x-AA-HLCs over time suggests viral spreading is occurring. Finally, the HC3x-AA-HLC model was validated for use in antiviral drug studies using the nucleoside reverse-transcriptase inhibitor, lamivudine, and the HBV entry inhibitor, Myrcludex B.

CCL20, a direct-acting pro-angiogenic chemokine induced by hepatitis C virus (HCV): Potential role in HCV-related liver cancer.

The CCL20/CCR6 chemokine/receptor axis has previously been shown to contribute to the initiation and progression of hepatocellular carcinoma (HCC) through the recruitment of CCR6-positive leukocytes to the tumor microenvironment. In particular, high serum levels of CCL20 are reported in patients with HCC induced by the hepatitis C virus (HCV). A potential non-immune role for the CCL20/CCR6 axis in HCC development has not yet been investigated. Microarray analysis (Benkheil et al., paper submitted for publication), revealed that CCL20 is highly upregulated in hepatoma cells infected with HCV compared with non-infected hepatoma cells. To determine the role of the CCL20/CCR6 axis in HCV-related HCC, we first explored which cell populations express CCR6 in human liver tissue with chronic disease or HCC. Immunohistochemical (IHC) analysis revealed that CCR6 is present on endothelial cells (ECs) of portal blood vessels in livers with chronic HCV infection and in HCV- and alcoholic-HCC tissue. In addition, we found CCR6 to be expressed on primary macrovascular (HUVECs) and microvascular ECs (HMVEC-ds) where it co-expressed with the endothelial marker CD31. In vitro angiogenesis experiments revealed that CCL20 is a direct pro-angiogenic molecule that induces EC invasion, sprouting and migration through CCR6. Moreover, using the angiogenesis matrigel plug assay in immunodeficient NMRI-nu mice, we clearly showed that CCL20 induces blood vessel formation, by attracting CCR6-positive ECs. Finally, we demonstrated that HCV-induced CCL20 protein expression and secretion in hepatoma cells could be abolished by antiviral treatment, indicating that CCL20 expression is dependent on HCV replication. In contrast to HCV, HBV-infection resulted in a decreased expression of CCL20, implying a virus-specific effect. Taken together, we identified HCV-induced CCL20 as a direct pro-angiogenic factor that acts on endothelial CCR6. These results suggest that the CCL20/CCR6 axis contributes to hepatic angiogenesis, promoting the hypervascular state of HCV-HCC.

Time course of infarct healing and left ventricular remodelling in patients with reperfused ST segment elevation myocardial infarction using comprehensive magnetic resonance imaging.

OBJECTIVE: To describe the time course of myocardial infarct (MI) healing and left ventricular (LV) remodelling and to assess factors predicting LV remodelling using cardiac MRI. METHODS: In 58 successfully reperfused MI patients, MRI was performed at baseline, 4 months (4M), and 1 year (1Y) post MI RESULTS: Infarct size decreased between baseline and 4M (p < 0.001), but not at 1Y; i.e. 18 ± 11%, 12 ± 8%, 11 ± 6% of LV mass respectively; this was associated with LV mass reduction. Infarct and adjacent wall thinning was found at 4M, whereas significant remote wall thinning was measured at 1Y. LV end-diastolic and end-systolic volumes significantly increased at 1Y, p < 0.05 at 1Y vs. baseline and vs. 4M; this was associated with increased LV sphericity index. No regional or global LV functional improvement was found at follow-up. Baseline infarct size was the strongest predictor of adverse LV remodelling. CONCLUSIONS: Infarct healing, with shrinkage of infarcted myocardium and wall thinning, occurs early post-MI as reflected by loss in LV mass and adjacent myocardial remodelling. Longer follow-up demonstrates ongoing remote myocardial and ventricular remodelling. Infarct size at baseline predicts long-term LV remodelling and represents an important parameter for tailoring future post-MI pharmacological therapies designed to prevent heart failure.

Gingival microleakage of Class II resin composite restorations with fiber inserts.

PURPOSE: This investigation evaluated the effect of glass and polyethylene fiber inserts on the microleakage of Class II composite restorations with gingival margins on root surfaces. METHODS: Fifty-four intact molars were sterilized with Gamma irradiation and mounted in acrylic bases. Class II slot cavities were made on both proximal sides of each tooth (3 mm wide, 1.5 mm deep) with the gingival margin on the root surface. The teeth were divided into nine groups, according to the technique of restoration and type of bonding agent. Filtek P-60 (3M/ESPE) was used to restore all cavities. Two types of fiber inserts were used: glass fiber (Ever Stick, StickTech) and polyethylene (Ribbond-THM), with three bonding agents being employed: Scotch Bond Multipurpose (3M/ESPE), Clearfil SE Bond (Kuraray) and Xeno IV (Dentsply). In the experimental groups, 3 mm long fiber inserts were inserted into restorations at the gingival seat. The control groups had no fiber inserts. The restorations were made incrementally and cured with LED light (UltraLume5, Ultradent). The restored teeth were stored in water for two weeks, then thermocycled for 3000 cycles (5 degrees C and 55 degrees C). The tooth surfaces were sealed with nail polish, except at the restoration margins. The teeth were immersed in 2% procion red dye solution, sectioned and dye penetration was assessed to determine the extent of microleakage according to a six-point scale. RESULTS: The fiber groups generally showed reduced microleakage scores compared to the control groups. The Clearfil SE Bond (Kuraray)/Filtek P-60 (3M/ESPE) combination produced the lowest degree of microleakage, irrespective of fiber type. However, the glass fiber groups were more consistent in reducing microleakage than the polyethylene groups. CONCLUSIONS: The use of fiber inserts significantly reduced gingival microleakage in Class II composite restorations with gingival margins in dentin, irrespective of the adhesive used. Clearfil SE Bond (Kuraray)/Filtek P60 (3M/ESPE) produced the lowest microleakage scores.