Alphaviruses, including Mayaro virus (MAYV) and Chikungunya virus (CHIKV), pose a growing public health threat, with increasing reports worldwide and no licensed antiviral treatments available. This study evaluated the antiviral activity of pyridine-3-carbohydrazide (P-3-C) against MAYV (strains BeAr and TRVL) and CHIKV (strain La Reunion, LR). Vero E6 or U-2OS cells were infected and treated with P-3-C for 24 h, and cell viability and replication levels were assessed. The 50% cytotoxic concentration (CC50), 50% effective concentration (EC50), and selective index (SI) were calculated. P-3-C showed concentration-dependent activity, strongly inhibiting post-entry stages of MAYV BeAr replication. Treatment reduced intracellular viral RNA levels by 2 log10 compared with controls and effectively decreased viral RNA release. P-3-C also inhibited MAYV TRVL (SI > 29.4) and CHIKV LR (SI > 48.8) in Vero E6 cells and retained selectivity in U-2OS cells (SI = 3.9 and 6.4, respectively). In a CHIKV U-2OS subgenomic replicon, an SI of 4.1 was found. Additionally, Fluorometric FRET-Based and MicroScale Thermophoresis (MST) assays indicated that P-3-C inhibited the nsP2 protease activity (IC50 = 12 µM ± 2) and exhibited binding affinity for nsP4 polymerase (Kd = 54 µM ± 1) from CHIKV, thereby suggesting that these non-structural proteins are potential molecular targets. Molecular docking positioned P-3-C within the catalytic pockets of nsP2 and nsP4 of both viruses. Overall, the data suggest that P-3-C exhibits a potential multitarget mechanism of action and highlight its antiviral activity against different alphavirus strains; however, this efficacy could be dependent on the cell line used.
N. Contreras, N. Cassani, Mikaela dos Santos Marinho et al.· Archives of Virology· 0 citations
Reverse genetics systems are crucial for facilitating the precise manipulation of viruses across a wide spectrum of translational and fundamental research pipelines. Here, we compared Circular polymerase extension reaction (CPER), Gibson assembly, and infectious subgenomic amplicons (ISA) for bacteria-free recovery of a positive sense RNA virus. Through optimisation of CPER, we demonstrated accelerated virus recovery and enhanced viral yields. We further investigated strategies to improve rescue efficiency across diverse positive-sense RNA virus families through incorporation of alternative promoters and non-coding elements. To evaluate the performance of the Aedes aegypti polyubiquitin promoter (AePUb) in tandem with a hammerhead ribozyme (HH Rbz) and a polymerase pause site for virus recovery in insect cells, we constructed a new fluorescent reporter genome using a 20 kb insect-specific mesonivirus. In vitro recovery by CPER of the mesonivirus was achievable in 1 day when using AePUb with HH Rbz, in comparison to a four-day recovery when using the minimal OpIE2-CA promoter. These elements were additionally assessed for rescue of the orthoflaviviruses, Binjari virus (BinJV) and dengue virus 2 (DENV-2), in insect cells (using AePUb); or in mammalian cells (using the CMV promoter) and for launch of DENV2 and SARS-CoV-2. Both BinJV and DENV-2 demonstrated improved rescue with the AePUb promoter and HH Rbz. However, the addition of the HH Rbz and the polymerase pause site to the CMV linker fragment showed no substantial differences to the standard CMV promoter systems for both DENV-2 and SARS-CoV-2, highlighting the context-specific benefits of their implementation. In summary, we demonstrated that a potent constitutive promoter system and a hammerhead ribozyme enhance the efficiency of positive-sense RNA virus rescue using CPER. Importance Reverse genetics systems are often limited by plasmid instability and variable efficiency of promoters across diverse cell lines. Extensive comparative approaches have yielded improvements across a variety of systems, however, there has been a paucity of publications that empirically compare novel advancements to established approaches. Here, we formalised and compared a series of reverse genetics advancements in the form of bacteria-free assembly methods, host promoters, pause sites, and ribozymes. These streamlined approaches expedite the existing methodologies and provide fundamental improvements to the field of synthetic virology. The advancements herein may support applications requiring efficient recovery of low fitness mutants and diverse mutational libraries and barcoded virus populations.
James R. Potter, Helen Mostafavi, A. Amarilla et al.· bioRxiv· 0 citations
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