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PubMed2025

Use of Rift Valley Fever Virus Expressing NanoLuc Luciferase for the Assessment of Neutralizing Antibodies and Antivirals.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. In this chapter we describe the use of a replication-competent recombinant (r)RVFV expressing NanoLuc Luciferase (Nluc) for in vitro studies. The determination of parameters such as neutralizing antibodies in serum samples, or the antiviral activity of drugs is usually carried out using standard assays based on the assessment of cytopathic effect on cell cultures. The use of a virus encoding a traceable reporter protein allows to correlate the presence or absence of infection with the detection of the product in the infected cultures, thus tracking the level of RVFV infection in an objective, quantitative manner. In addition to this quantitative measurement of results, our protocol offers two other advantages, such as a shorter time to read, given that 48 h post-infection the production of the reporter protein is enough to give an accurate result, and the use of an attenuated virus, which reduces the risk of exposure.

Rift Valley fever virus↗

PubMed2025

Construction and In Vitro and In Vivo Analysis of Coxsackievirus B4 Reporter Viruses: Attenuated Virulence but Highly Efficient for Antiviral Drug Screening and Evaluation.

Coxsackievirus B4 (CVB4) is an enterovirus with one of the highest mortality rates following infection, yet research on it remains limited. To enhance the efficiency of CVB4 research, we developed the rCVB4-EGFP and rCVB4-NanoLuc reporter viruses. The replication kinetics of these reporter viruses in SH-SY5Y and HeLa cells were essentially consistent with those of the wild-type CVB4. A strong correlation was observed between the fluorescence and bioluminescence signals of rCVB4-EGFP and rCVB4-NanoLuc and viral titers at specific times postinfection. When evaluating the anti-CVB4 drug fluoxetine using these reporter viruses, the half-maximal effective concentrations derived from fluorescence signals, bioluminescence signal intensities, and viral genome copies were consistent. In In Vivo drug evaluations, because CVB4 can infect various tissues and organs, the bioluminescence signal of rCVB4-NanoLuc effectively demonstrated the antiviral effects of drugs, offering significant advantages over traditional tissue viral titer analysis. The reporter viruses exhibited reduced virulence compared with wild-type CVB4 both In Vitro, in SH-SY5Y and HeLa cells, and In Vivo, in ICR suckling mice. Although this reduced virulence may limit their application for studying pathogenic mechanisms, these reporter viruses can serve as highly efficient tools for high-throughput screening and evaluation of anti-CVB4 drugs, vaccines, and neutralizing antibodies.

Humans↗

PubMed2026

Redox-activated cholesterol-dependent cytolysin enables cytosolic release of liposomal cargo.

Precise intracellular delivery of biologic therapeutics remains a major challenge due to endosomal entrapment and inefficient delivery systems. Here, we develop a bioinspired platform that uses Streptolysin O (SLO), a member of the cholesterol-dependent cytolysin (CDC) family, for cytosolic cargo delivery. This delivery system incorporates an affibody for selective targeting and endocytosis and a redox-cleavable PEG-conjugated dithiol-ethyl carbonate linker (PEG-DEC) that reversibly inactivates SLO extracellularly. After endosomal uptake, the reductive intracellular environment removes the PEG layer, reactivating SLO to induce localized endosomal disruption and cargo release. This mechanism minimizes off-target toxicity while promoting efficient cytosolic delivery of diverse cargo, including doxorubicin (DOX), the fluorescent protein GFP and mApple, and the enzyme NanoLuciferase (NanoLuc) and lactate oxidase (LOX). PEGylated SLO exhibited significantly improved cytosolic release efficiency compared with conventional liposomal formulations, confirming the advantage as a controllable intracellular delivery module.

Liposomes↗

PubMed2026

Dual HBV cccDNA-linked HiBiT reporter hepatocyte models for screening of candidate cccDNA modulators.

Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, reporter readouts that facilitate early discovery of cccDNA-modulating agents. Here, we developed two complementary hepatocyte HiBiT reporter models: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16), in which a secreted split-NanoLuc HiBiT signal is linked to cccDNA-associated expression, and a Cre/Lox-based recombinant cccDNA (rcccDNA) reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA with a matched HiBiT readout. Screening of 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. Palovarotene, a retinoic acid receptor-γ agonist, was selected as an exemplar concordant hit and reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems without overt cytotoxicity at the tested concentrations. Together, this dual-reporter strategy supports efficient cross-model triage of candidate cccDNA modulators for subsequent orthogonal validation.

Humans↗

PubMed2026

Conserved innate immunity components limit transgene expression in adult planarians.

The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression still limits the elucidation of molecular mechanisms in in this system. Here, we establish a proof-of-principle system for plasmid-based expression of NanoLuciferase (NanoLuc) in S. mediterranea, employing commercially available transfection reagents and a panel of endogenous promoter sequences. Despite successful delivery, reporter expression remained low and transient. To identify biological barriers to robust transgene expression, we investigated the role of innate immune pathways. Candidate gene searches and biochemical pull-down of cytoplasmic DNA coupled to mass spectrometry identified several planarian homologs of conserved immune regulators and putative DNA sensors. Through RNA interference screening of conserved innate immune components, we uncover roles for S. mediterranea homologs of Tank-binding kinase 1 (TBK1) and macrophage mannose receptor 1 (MRC1) as potent repressors of transgene expression. Transcriptomic and functional analyses further implicate TBK1 in regulating broad innate immune and stress-response programs, akin to its vertebrate function. Together, our findings demonstrate that innate immune signaling limits transgene expression in S. mediterranea and suggest that modulating these pathways may be key to enabling stable and efficient genetic manipulation in planarians.

Animals↗

PubMed2025

Evaluation of Vaccinia Virus Infection in Mice Using Two-Reporter Recombinant Virus.

The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments. Recently, we described two reporter rVACV expressing luciferase (Nluc) and fluorescent (GFP or Scarlet) proteins to easily track viral infections in different systems, overcoming the limitations associated with the use of rVACV expressing a single luciferase or fluorescent reporter gene. Here, we describe the experimental procedures to carry out in vitro, in vivo and ex vivo studies using these novel bireporter-expressing rVACV, which also represent an excellent option to study the biology of VACV, including the use of these reporter viruses for testing new antivirals and vaccines, using cultured cells and/or well-characterized animal models of infection.

Animals↗

PubMed2025

Identification of glycogen synthase kinase 3alpha/beta as a host factor required for HBV transcription using high-throughput screening.

BACKGROUND AND AIMS: HBV leads to severe liver diseases, such as cirrhosis and HCC. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. APPROACH AND RESULTS: We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed HBeAg and HBsAg production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. CONCLUSIONS: Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.

Humans↗