Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.

Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in immunocompromised individuals, and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about the strain diversity of the 235-kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the amount of information available for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hot spots of diversity in the genome. Importantly, 75% of strains are not genetically intact but contain disruptive mutations in a diverse set of 26 genes, including the immunomodulatory genes UL40 and UL111A. These mutants are independent of culture passage artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. The recombination density was significantly higher than those of other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142, and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCEHuman cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased toward a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, these data have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity. Human cytomegalovirus (HCMV), the prototype member of the herpesvirus subfamily Betaherpesvirinae, is a widespread and important pathogen. Seroprevalence in the adult population ranges from 45% to 100% (1). After primary infection, HCMV establishes a lifelong, latent infection in myeloid progenitor cells (2). This virus causes mild to ...

Dengue virus (DENV) causes ~96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue 1,2 . There are no antivirals available to prevent or treat dengue. We describe a highly potent DENV inhibitor (JNJ-A07) that exerts nano-to picomolar activity against a panel of 21 clinical isolates, representing the natural genetic diversity of known geno-and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus unveiling an entirely novel mechanism of antiviral action. JNJ-A07 has an excellent pharmacokinetic profile that results in outstanding efficacy against DENV infection in mouse infection models. Delaying start of treatment until peak viremia results in a rapid and significant reduction in viral load. An analogue is currently in further development. MAIN TEXTDengue is currently considered one of the top10 global health threats 1 . Annually, an estimated 96 million develop dengue disease 2 , which is likely an underestimation [3][4][5] . The incidence has increased ~30-fold over the past 50 years. The virus is endemic in 128 countries in (sub-)tropical regions, with an estimated 3.9 billion people at risk of infection. A recent study predicts an increase to 6.1 billion people at risk by 2080 6 . The upsurge is driven by factors such as rapid urbanization and the sustained spread of the mosquito vectors [6][7][8] . DENV has four serotypes (further classified into genotypes), which are increasingly co-circulating in endemic regions. A second infection with a different serotype increases the risk of severe dengue 9,10 . The vaccine Dengvaxia ® , which is approved in a number of countries for those aged ≥9 years, is only recommended for those with previous dengue exposure 11,12,13 . There are no antivirals for the prevention or treatment of dengue; the development of pan-serotype DENV inhibitors has proven challenging 14,15 .

BackgroundTumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells’ genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC).MethodsThe efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization.ResultsThe overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified.ConclusionsCTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GαS, we have measured inhibition of ligand-dependant, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.