The development of direct-acting antivirals (DAAs) has revolutionized the state-of-the art treatment of HCV infections, with sustained virologic response rates above 90%. However, viral variants harboring substitutions referred to as resistance-associated substitutions (RASs) may be present in baseline levels and confer resistance to DAAs, thereby posing a major challenge for HCV treatment. HCV replicons have been the primary tools for discovering and evaluating the inhibitory activity of DAAs against viral replication. Interest in replicon systems has further grown as they have become indispensable for discovering genotype-specific and cross-genotype RASs. Here, we review functional replicon systems for HCV, how these replicon systems have contributed to the development of DAAs, and the characteristics and distribution of RASs for DAAs.

Herein, flower-shaped hydrophilic superparamagnetic iron oxide nanoclusters (IONCs) are synthesized via onepot thermolysis of iron acetylacetonate using triethanolamine (TEA) and diethylene glycol (DEG)/tetraethylene glycol (TTEG) mixtures at 9:1, 8:2 and 7:3 (v/v) ratios. The as-prepared 24-29 nm sized IONCs have displayed (i) saturation magnetization (Ms) values of~68-78 emu/g, (ii) hydrodynamic diameters of~95-192 nm and (iii) zeta potential values of +46 to +65 mV. Due to relatively high magnetization and water solubility, IONCs (prepared using 8:2 TEA:DEG, and 8:2 & 7:3 TEA:TTEG ratios) based aqueous ferrofluids i.e. NCAFF-1, NCAFF-2, and NCAFF-3 are investigated by calorimetric magnetic fluid hyperthermia (MFH) at 0.5-8 mg/ml concentrations by exposing them to the alternating magnetic fields (AMFs, H*f~2.4-9.9 GA m −1 s −1). The NCAFF-3 has demonstrated excellent time dependent temperature rise (42°C within 0.7-9.7 min) as compared to the NCAFF-1 and NCAFF-2. Moreover, the NCAFF-3 at 0.5 mg/ml concentration has exhibited enhanced heating efficacies with specific absorption rate (SAR) and intrinsic loss power (ILP) values of 142.4-909.4 W/g Fe and 4.2-14.7 nHm 2 /kg, respectively. Furthermore, the NCAFF-3 has presented better cytocompatibility, and substantially reduced proliferation capacity of HepG2 cancer cells in in vitro MFH studies. Thus, the IONCs based ferrofluids (NCAFF-3) are very promising candidates for MFH therapeutics applications.

To gain insight into the impact of mutations on the viability of the hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other, by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. The replication efficiencies of full-genome mutant libraries ranged between 71 and 329 focus-forming units (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capabilities, whereas those with high proportions of mutations had their replication capabilities restored. Hepatoma cells transfected with selected mutant libraries, with low (4 mutations per 10,000 bp copied), moderate (33 mutations), and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm, and Mutanth, respectively) were evaluated for changes in growth kinetics and particle-to-FFU unit ratio, virus protein expression, and modulation of host cell protein synthesis. Mutantm and Mutantl variants produced >3.0-log-higher extracellular progeny per ml than the parent, and Mutanth produced progeny at a rate 1.0-log lower. More than 80% of the mutations were in a nonstructural part of the mutant genomes, the majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that the HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate but highly selects for random, beneficial mutations. IMPORTANCE Hepatitis C virus (HCV) in vivo displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions in vitro because of limitations in inducing mutagenesis from the 5′ end to the 3′ end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of an HCV clone that can recapitulate all steps of the life cycle. Our study provides evidence of the capability of the HCV genome to overcome deleterious mutations and remain viable. Mutants that emerged from the libraries had diverse phenotype profiles compared to the parent, and putative adaptive mutations mapped to segments of the conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensures the survival and adaptation of HCV.