We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine-5′-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC₅₀) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC₅₀ of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC₅₀ of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.

Background: Vancomycin-resistantEnterococci(VRE) infections are recurrently reported in different parts of India in the last two decades. However, an up-to-date, countrywide information concerning the prevalence and the rate of VRE in India is limited and hence this study aimed to estimate the pooled prevalence of VRE in India.Methods: A literature search was performed using various databases. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed throughout. Cross-sectional studies reporting the prevalence of VRE in India from human samples whereby at least twoEnterococciwere isolated between 1 January 2000 and 31 December 2022 were sought for inclusion. Data were extracted and analysed using Microsoft Excel and Comprehensive Meta-analysis version 4, respectively.Results: Nineteen studies were included in the analyses. A collective total of 3683Enterococciisolates were examined, of which 368 were VRE strains. The pooled prevalence of VRE in India was calculated at 12.4% (95% CI: 8.6–17.5;Q = 189.69;I² = 90.51%;p = < 0.001).E. faecaliswas the most frequently isolated species (1450 [39.37%]) followed byE. faecium(724 [19.66%]). Amongst the VRE strains,E. faeciumwas the most prevalent (214 [58.15%]) followed byE. faecalis(134 [36.41%]). An upsurge in the rate of VRE infections was observed in India over time: VRE prevalence was estimated at 4.8% between 2000 and 2010 and 14.1% between 2011 and 2020.Conclusion: This study presents the most up-to-date information on the rate of VRE infections in India. Though lower than the findings for some less developed countries, VRE prevalence in India is notable and on the rise.

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRβ, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells.