Search

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. [ABSTRACT FROM AUTHOR]

We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved. • The omicron SARS-CoV-2 variant remains susceptible for GS-441524, Remdesivir, EIDD-1931, Molnupiravir and Nirmatrelvir. • The target genes of these antivirals are highly conserved between SARS-CoV-2 variants. • Similarly, the alpha, beta, gamma and delta variants remain susceptible to these antivirals. [ABSTRACT FROM AUTHOR]

Comparable to the related Ebola virus, Marburg virus is an emerging zoonotic pathogen that causes hemorrhagic fever with a high mortality rate. Therefore, handling of Ebola virus and Marburg virus is limited to biosafety level 4 facilities, of which only a limited number exists worldwide. However, researchers have developed several virus alternatives that are safe to handle in lower biosafety settings. One particularly interesting approach is the engineering of biologically contained Ebola virus by removing an essential gene from the virus genome and providing this missing gene in trans in a specific cell line. Because the virus is confined to this specific cell line, this results in a system that is safe to handle. So far, Ebola virus is the only virus for which biological containment has been reported. Here, we describe the first successful rescue of biologically contained Marburg virus and demonstrate that biological containment is also feasible for other filoviruses. Specifically, we describe the development of containment cell lines for Marburg virus through lentiviral transduction and show the growth and safety characteristics of eGFP-expressing, biologically contained Marburg virus in these cell lines. Additionally, we exploited this newly established Marburg virus system to screen over 500 compounds from available libraries. Lastly, we also validated the applicability of our biologically contained Marburg virus system in a 384-well format, to further illustrate the usefulness of this novel system as an alternative for high-throughput MARV screening of compound libraries. [ABSTRACT FROM AUTHOR]

Despite recent advancements in the development of vaccines and monoclonal antibody therapies for Ebola virus disease, treatment options remain limited. Moreover, management and containment of Ebola virus outbreaks is often hindered by the remote nature of the locations in which the outbreaks originate. Small-molecule compounds offer the advantage of being relatively cheap and easy to produce, transport and store, making them an interesting modality for the development of novel therapeutics against Ebola virus disease. Furthermore, the repurposing of small-molecule compounds, previously developed for alternative applications, can aid in reducing the time needed to bring potential therapeutics from bench to bedside. For this purpose, the Medicines for Malaria Venture provides collections of previously developed small-molecule compounds for screening against other infectious diseases. In this study, we used biologically contained Ebola virus to screen over 4,200 small-molecule drugs and drug-like compounds provided by the Medicines for Malaria Venture (i.e., the Pandemic Response Box and the COVID Box) and the Centre for Drug Design and Discovery (CD3, KU Leuven, Belgium). In addition to confirming known Ebola virus inhibitors, illustrating the validity of our screening assays, we identified eight novel selective Ebola virus inhibitors. Although the inhibitory potential of these compounds remains to be validated in vivo , they represent interesting compounds for the study of potential interventions against Ebola virus disease and might serve as a basis for the development of new therapeutics. • We created a biologically contained Ebola virus assay. • This system allows working with infectious Ebola virus in a BSL-2/BSL-3 facility. • Screening of >4,000 compounds using this assay identified several novel Ebola virus inhibitors. [ABSTRACT FROM AUTHOR]