<p>Shiv Nadar University is a comprehensive, multidisciplinary, research-focused, and student-centric University offering a full range of academic programs at the undergraduate, postgraduate and doctoral level. With state-of-the-art infrastructure, the University comprises of academic wings, sophisticated labs, international standard sports facilities, amphitheaters, auditorium, conference rooms and smart classrooms. The University&rsquo;s goal is to become internationally recognized for the quality of its research and creative endeavors and their applicability to improving quality of life, generating new insights and expanding the boundaries of human knowledge creativity. Committed to excellence in teaching, research and service, the University aims to serve the higher education needs of India and the world beyond.</p>

Shiv Nadar University

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses. © 2022, Journal of Visualized Experiments. All rights reserved.

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Life Sciences

(SoNS) School of Natural Sciences

Journal of Visualized Experiments

Reverse Genetics to Engineer Positive-sense RNA Virus Variants

Drug resistance mutations of hepatitis C virus (HCV) negatively impact viral replicative fitness. RNA viruses are known to change their replication behaviour when subjected to suboptimal selection pressure. Here, we assess whether mutation supply in HCV is sufficiently large to allow the selection of its variants during dual or triple direct-acting antiviral (DAA) treatment associated with augmented virus fitness or impairment. We engineered randomly mutagenized full-genome libraries to create a highly diverse population of replication-competent HCV variants in cell culture. These variants exhibited escape when treated with NS5A/NS5B inhibitors (daclatasvir/sofosbuvir), and relapse on treatment with a combination of NS3/NS5A/NS5B inhibitors (simeprevir or paritaprevir/daclatasvir/sofosbuvir). Analysis of the relationship between virus fitness and drug resistance of JFH1-derived NS5A-5B variants showed a significant positive correlation (P=0.003). At the earliest time points, intracellular RNA levels remain unchanged in both the subgenomic replicon and infection assays, whereas extracellular RNA levels increased upto ten-fold compared to wild-type JFH1. Beneficial substitutions hyperstimulated phosphatidylinositol 4-phosphate during DAA treatment, and showed decreased dependence on cyclophilins during cyclosporine A treatment, indicating an interplay of virus-host molecular mechanisms in beneficial substitution selection that may necessitate infectious virus production. This comprehensive study demonstrates a possible role for HCV fitness of overcoming drug-mediated selection pressure. © 2022 Microbiology Society. All rights reserved.

Journal of General Virology

Enhanced fitness of hepatitis C virus increases resistance to direct-acting antivirals

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. © Copyright © 2020 Khan, Soni and Veerapu.

Fulltext

Frontiers in Cellular and Infection Microbiology

HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

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 &gt;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 errorprone 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. Copyright © 2020 American Society for Microbiology. All Rights Reserved.

Journal of Virology

Genome-wide mutagenesis of hepatitis c virus reveals ability of genome to overcome detrimental mutations

This work systematically describes one-step synthesis of hydrophilic functionalized superparamagnetic iron oxide nanoparticles (SPIONs) via thermolysis in presence of polyamines such as diethylene triamine (DETA), triethylene tetraamine (TETA), tetraethylene pentamine (TTEPA), or pentaethylene hexamine (PEHA) or mixture of a polyamine (TETA) and polyols such as diethylene glycol (DEG), triethylene glycol (TEG) or tetraethylene glycol (TTEG) while varying the polyamine: polyol (v/v) ratio, reaction temperature and reaction time. The saturation magnetization (Ms) values of the as-prepared polyamine (DETA/TETA/TTEPA/PEHA) coated SPIONs are determined in the range of 31.8–48.5 emu/g, which is altered in the range of 40.2–57.8 emu/g by the addition of a polyol (DEG/TEG/TTEG) to the polyamine (TETA) at different ratios. Moreover, the Ms. values are further improved to 64.6 and 66.8 emu/g at the optimized TETA:TEG (1:1) ratio by prolonging the reaction time up-to 2 h and the reaction temperature to 270 °C, respectively. In addition, the TETA-TEG coated SPIONs have displayed their average particle sizes, hydrodynamic sizes, and zeta potential (ζ) values in the range of 7–11 nm, 99–120 nm and +45 to +57 mV, respectively (indicating high water solubility). Finally, the TETA-TEG coated SPIONs with the highest Ms. and ζ values (i.e. 66.8 emu/g and +57 mV) are selected for the biological studies, where they have revealed excellent (i) cytocompatibility, and (ii) intracellular uptake in the cancer (HepG2 liver & MCF-7 breast) cells for the incubation periods of 24/48 h. Thus, the TETA-TEG coated SPIONs based aqueous ferrofluids have a great potential to be used in biomedical applications. © 2018 Elsevier B.V.

Journal of Molecular Liquids

One-step synthesis of hydrophilic functionalized and cytocompatible superparamagnetic iron oxide nanoparticles (SPIONs) based aqueous ferrofluids for biomedical applications

In this work, we have developed novel multifunctional magnetic-polymeric nanoparticles (MMPNs) based ferrofluids by encapsulating oleylamine (OM)-coated hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) inside the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) individually, and along with two drugs such as curcumin (Cur, a chemotherapeutic drug (CHD)), and/or verapamil (Ver, a calcium channel blocker (CCB)). Herein, several parameters such as weighed amount (wt\%) of PLGA polymer (i.e., Resomer), stabilizer (i.e., polyvinyl alcohol (PVA)), organic solvents, amount of the SPIONs (in liquid suspension and powder forms), and amount of the drugs (i.e., Cur or/and Ver) are varied during the encapsulation process to optimize the formulation of PLGA NPs. The resulting polymeric NPs including empty PLGA NPs (i.e., without SPIONs/drugs), and MMPNs such as SPIONs-loaded PLGA NPs, Cur-SPIONs-loaded PLGA NPs, Ver-SPIONs-loaded PLGA NPs, and Cur-Ver-SPIONs-loaded PLGA NPs have displayed (i) hydrodynamic diameters and zeta potentials in the range of 280.8–287.3 nm, and −21 to – 26 mV, respectively, and (ii) better encapsulation efficiency for the SPIONs/Cur/Ver. In addition, the MMPNs have exhibited (i) magnetization values in the range of 7.6–9.5 emu/g with superparamagnetic behaviour, (ii) concentration based time-dependent temperature raise up-to 42 °C (minimum therapeutic temperature in magnetic fluid hyperthermia (MFH)/thermotherapy) with heating efficacies i.e., specific absorption rate (SAR) and intrinsic loss power (ILP) values ranging from 7 to 36 W/gFe and 0.1–0.4 nHm2/kg, respectively and (iii) better cytocompatibility. Finally, the SPIONs and dual-drugs (Cur &amp;Ver) co-loaded PLGA NPs have shown enhanced therapeutic efficacy in HepG2 cancer cells via combined therapies (i.e., thermotherapy and chemotherapy), as compared to the individual therapy (i.e., thermotherapy or chemotherapy) using the SPIONs/Cur/Ver loaded PLGA NPs. Thus, the as-prepared SPIONs/dual-drugs co-loaded PLGA NPs (i.e., MMPNs based ferrofluids) are potential therapeutic candidates for multi-modal treatment of cancer in vitro using thermotherapy and chemotherapy. © 2019 Elsevier B.V.

Multifunctional magnetic-polymeric nanoparticles based ferrofluids for multi-modal in vitro cancer treatment using thermotherapy and chemotherapy

Herein, flower-shaped hydrophilic superparamagnetic iron oxide nanoclusters (IONCs) are synthesized via one-pot 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 &amp; 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/gFe and 4.2–14.7 nHm2/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. © 2018 Elsevier B.V.

One-pot synthesis of hydrophilic flower-shaped iron oxide nanoclusters (IONCs) based ferrofluids for magnetic fluid hyperthermia applications

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70\% of open reading frame 1 (ORF1) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10-3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. © 2018 American Society for Microbiology.

Novel synthesis and phenotypic analysis of mutant clouds for hepatitis E virus genotype 1

Successful hepatitis C virus (HCV) treatment is defined as the absence of viremia 6 months after therapy cessation. We previously reported that trace amounts of HCV RNA, below the sensitivity of the standard clinical assay, can reappear sporadically in treatment responders. Here, we assessed the infectivity of this RNA and infused 3 chimpanzees sequentially at 9-week intervals with plasma or PBMCs from patients who tested positive for trace amounts of HCV RNA more than 6 months after completing pegylated IFN-α/ribavirin therapy. A fourth chimpanzee received HCV RNA-negative plasma and PBMCs from healthy blood donors. The 3 experimental chimpanzees, but not the control chimpanzee, generated HCV-specific T cell responses against nonstructural and structural HCV sequences 6-10 weeks after the first infusion of patient plasma and during subsequent infusions. In 1 chimpanzee, T cell responses declined, and this animal developed high-level viremia at week 27. Deep sequencing of HCV demonstrated transmission of a minor HCV variant from the first infusion donor that persisted in the chimpanzee for more than 6 months despite undetectable systemic viremia. Collectively, these results demonstrate that trace amounts of HCV RNA, which appear sporadically in successfully treated patients, can be infectious; furthermore, transmission can be masked in the recipient by an extended eclipse phase prior to establishing high-level viremia.

Journal of Clinical Investigation

Trace amounts of sporadically reappearing HCV RNA can cause infection

Hepatitis C virus (HCV) is endemic in many countries due to its high propensity for establishing persistence. The presence of HCV-specific T cells in subjects repeatedly exposed to HCV who test negative for HCV RNA and antibodies and who do not have any history of HCV infection has been interpreted as T cell-mediated protection2-5. Here, we show in nonhuman primates that repeated exposure to human plasma with trace amounts of HCV induced HCV-specific T cells without seroconversion and systemic viremia but did not protect upon subsequent HCV challenge. Rather, HCV-specific recall and de novo T cell responses, as well as intrahepatic T cell recruitment and interferon-γ (IFN-γ) production, were suppressed upon HCV challenge, concomitant with quantitative and qualitative changes in regulatory T cells (Treg cells) that occurred after subinfectious HCV exposure and increased after HCV challenge. In vitro Treg cell depletion restored HCV-specific T cell responses. Thus, T cells primed by trace amounts of HCV do not generate effective recall responses upon subsequent HCV infection. Subinfectious HCV exposure predisposes to Treg cell expansion, which suppresses effector T cells during subsequent infection. Strategies to reverse this exposure-induced immune suppression should be examined to aid in the development of T cell-based vaccines against HCV and other endemic pathogens. © 2013 Nature America, Inc.

Nature Medicine

Subinfectious hepatitis C virus exposures suppress T cell responses against subsequent acute infection

Background &amp; Aims: Hepatitis C virus (HCV) infection is characterized by lack of immune-mediated liver injury despite a high level of HCV replication during the incubation phase, which lasts about 8 weeks. We investigated whether this results from delayed recruitment of HCV-specific T cells and whether it facilitates HCV persistence. Methods: Six chimpanzees were infected with HCV; blood and liver samples were collected for 28 weeks and analyzed for immune cells and chemokines. Results: Two chimpanzees developed self-limited infections, whereas the remaining 4 developed chronic infections. Levels of the chemokines CXCL10, CXCL11, CCL4, and CCL5 increased in blood and liver samples from all chimpanzees within 1 month of HCV infection. Chemokine induction correlated with intrahepatic type I interferon (IFN) responses in vivo and was blocked by neutralizing antibodies against IFN-β in vitro. Despite the early-stage induction of chemokines, the intrahepatic lymphocytic infiltrate started to increase no earlier than 8 weeks after HCV infection, when HCV-specific, tetramer-positive CD8+ T cells appeared in the circulation. The HCV-specific CD8+ T cells expressed chemokine receptors when they were initially detected in blood samples, so they could be recruited to the liver as soon as they entered the circulation. Conclusions: Chemokines are induced during early stages of HCV infection, which requires a type I IFNmediated response. The delayed onset of acute hepatitis does not result from delayed recruitment of HCV-specific T cells, but could instead be related to a primary delay in the induction of HCV-specific T cells. Divergent outcomes occur without evident differences in chemokine induction and T-cell recruitment. © 2011 AGA Institute.

Gastroenterology

Delayed induction, not impaired recruitment, of specific CD8+ T cells causes the late onset of acute hepatitis C

Background & Aims: Several studies have reported hepatitis C virus (HCV) RNA sequences in the circulation after treatment-induced or spontaneous recovery. We investigated whether the HCV RNA represents persistence of HCV infection or reinfection. Methods: We studied 117 patients who recovered from HCV infection (98 following therapy and 19 spontaneously). A reverse-transcription polymerase chain reaction assay was used to detect the 5′-untranslated region of HCV. T-cell responses were studied by enzyme-linked immunospot for interferon-γ. Results: Plasma samples from 15% of treatment-recovered patients and no spontaneously recovered patient tested positive for HCV RNA. Lymphocytes from 3 patients who responded to therapy and 1 who recovered spontaneously tested positive. The frequency of HCV RNA detection in plasma correlated inversely with the time after the end of treatment. Post-treatment HCV 5′-untranslated region sequences matched pretreatment sequences in 85% of cases. T-cell responses were significantly greater at time points with detectable trace amounts of HCV RNA than at time points without detectable HCV RNA (P = .035) and were primarily against nonstructural HCV antigens. The immune hierarchy was preserved over 5 years in patients whose post-treatment HCV RNA sequences matched pretreatment sequences, indicating HCV RNA persistence. An altered immune hierarchy with dominant immune responses, shifting from nonstructural to structural antigens, was observed in a single patient whose post-treatment HCV genotype differed from that of the pretreatment genotype, indicating HCV reinfection. Conclusions: Trace amounts of HCV RNA of pretreatment sequence persisted and reappeared sporadically in the circulation within 8 years after recovery from hepatitis C but not thereafter, indicating that patients are cured of HCV infection. Reappearance of HCV RNA induced HCV-specific T-cell responses. © 2011 AGA Institute.