Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Phase 3 Randomized Clinical Trial of the Safety and Efficacy of Travoprost Intraocular Implant in Patients with Open-Angle Glaucoma or Ocular Hypertension.

PURPOSE: To evaluate the safety and intraocular pressure (IOP)-lowering efficacy of 2 models of the travoprost intraocular implant (fast-eluting [FE] and slow-eluting [SE] types) from 1 of 2 phase 3 trials (the GC-010 trial). DESIGN: Multicenter, randomized, double-masked, sham-controlled, noninferiority trial. PARTICIPANTS: Patients with open-angle glaucoma or ocular hypertension having an unmedicated baseline mean diurnal IOP (average of 8 am, 10 am, and 4 pm time points) of ≥ 21 mmHg, and IOP of ≤ 36 mmHg at each of the 8 am, 10 am, and 4 pm timepoints at baseline. METHODS: Study eyes were randomized to the travoprost intraocular implant (FE implant [n = 200] or SE implant [n = 197] model) or to timolol ophthalmic solution 0.5% twice daily (n = 193). MAIN OUTCOME MEASURES: The primary outcome was mean change from baseline IOP in the study eye at 8 am and 10 am, at each of day 10, week 6, and month 3. Safety outcomes included adverse events (AEs) and ophthalmic assessments. RESULTS: Mean IOP reduction from baseline over the 6 time points ranged from 6.6 to 8.4 mmHg for the FE implant group, from 6.6 to 8.5 mmHg for the SE implant group, and from 6.5 to 7.7 mmHg for the timolol group. The primary efficacy end point was met; the upper limit of the 95% confidence interval of the difference between the implant groups and the timolol group was < 1 mmHg at all 6 time points. Study eye AEs, most of mild or moderate severity, were reported in 21.5%, 27.2%, and 10.8% of patients in the FE implant, SE implant, and timolol groups, respectively. The most common AEs included iritis (FE implant, 0.5%; SE implant, 5.1%), ocular hyperemia (FE implant, 3.0%; SE implant, 2.6%), reduced visual acuity (FE implant, 1.0%; SE implant, 4.1%; timolol, 0.5%), and IOP increased (FE implant, 3.5%; SE implant, 2.6%; timolol, 2.1%). One serious study eye AE occurred (endophthalmitis). CONCLUSIONS: The travoprost intraocular implant demonstrated robust IOP reduction over the 3-month primary efficacy evaluation period after a single administration. The IOP-lowering efficacy in both implant groups was statistically and clinically noninferior to that in the timolol group, with a favorable safety profile. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

How do people with chronic low back pain perceive specific and general exercise? A mixed methods survey.

PURPOSE: Exercise prescriptions for chronic low back pain (CLBP) often utilize reductionistic, trunk-focused exercise aimed at addressing proposed pain mechanisms. It is unknown if the use of these trunk-focused exercises imply beliefs to people with CLBP about the rationale for their use (e.g., etiology), even without concurrent biomedical narratives. This study aimed to explore people's perceptions of specific and general exercise without an accompanying narrative when experiencing CLBP. METHODS: An anonymous online survey was distributed. Mixed methods were utilized for analysis. Six-point Likert scales categorized people's beliefs about individual exercises. Open-ended questions were used to gather further beliefs which were then coded into themes. RESULTS: People with CLBP perceived specific exercise as more beneficial than general exercise. Eight themes and five subthemes were defined. A high volume of positive beliefs were centered around strengthening the low back and abdominal musculature, emphasizing the importance of correct technique. Negative beliefs were held against spinal flexion and external load. Both positive and negative beliefs were underpinned by spinal/pelvic stability being important as well as certain exercises being achievable or not. CONCLUSION: This study demonstrated that people with CLBP consider specific exercises to be more beneficial than general exercises for CLBP. Specific exercises irrespective of an accompanying narrative can imply meaning about the intent of an exercise. Understanding this requires practitioners to be mindful when prescribing and communicating exercise.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4.

OBJECTIVE: HCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy. DESIGN: HCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics. RESULT: The in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes. CONCLUSION: Rapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation.

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Synthesis of (±)-Emtricitabine and (±)-Lamivudine by Chlorotrimethylsilane-Sodium Iodide-Promoted Vorbrüggen Glycosylation.

By simple combination of water and sodium iodide (NaI) with chlorotrimethylsilane (TMSCl), promotion of a Vorbrüggen glycosylation en route to essential HIV drugs emtricitabine (FTC) and lamivudine (3TC) is achieved. TMSCl-NaI in wet solvent (0.1 M water) activates a 1,3-oxathiolanyl acetate donor for N-glycosylation of silylated cytosine derivatives, leading to cis-oxathiolane products with up to 95% yield and >20:1 dr. This telescoped sequence is followed by recrystallization and borohydride reduction, resulting in rapid synthesis of (±)-FTC/3TC from a tartrate diester.

Decoding 'Maximum Entropy' Deconvolution.

For over five decades, the mathematical procedure termed "maximum entropy" (M-E) has been used to deconvolve structure in spectra, optical and otherwise, although quantitative measures of performance remain unknown. Here, we examine this procedure analytically for the lowest two orders for a Lorentzian feature, obtaining expressions for the amount of sharpening and identifying how spurious structures appear. Illustrative examples are provided. These results enhance the utility of this widely used deconvolution approach to spectral analysis.

Epithelial-specific isoforms of protein 4.1R promote adherens junction assembly in maturing epithelia.

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of ß-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC50) of ∼40 µM. Among the macrocyclic PIs, simeprevir had the highest (EC50, 15 µM) and glecaprevir the lowest (EC50, >178 µM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC50s of 72 and 23 µM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC50 of 46 µM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC50 PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC50 boceprevir or 1-fold EC50 simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC50 remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication.

Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

Interpreting a recurrent neural network's predictions of ICU mortality risk.

Deep learning has demonstrated success in many applications; however, their use in healthcare has been limited due to the lack of transparency into how they generate predictions. Algorithms such as Recurrent Neural Networks (RNNs) when applied to Electronic Medical Records (EMR) introduce additional barriers to transparency because of the sequential processing of the RNN and the multi-modal nature of EMR data. This work seeks to improve transparency by: 1) introducing Learned Binary Masks (LBM) as a method for identifying which EMR variables contributed to an RNN model's risk of mortality (ROM) predictions for critically ill children; and 2) applying KernelSHAP for the same purpose. Given an individual patient, LBM and KernelSHAP both generate an attribution matrix that shows the contribution of each input feature to the RNN's sequence of predictions for that patient. Attribution matrices can be aggregated in many ways to facilitate different levels of analysis of the RNN model and its predictions. Presented are three methods of aggregations and analyses: 1) over volatile time periods within individual patient predictions, 2) over populations of ICU patients sharing specific diagnoses, and 3) across the general population of critically ill children.

Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells.

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

Continuous Prediction of Mortality in the PICU: A Recurrent Neural Network Model in a Single-Center Dataset.

OBJECTIVES: Develop, as a proof of concept, a recurrent neural network model using electronic medical records data capable of continuously assessing an individual child's risk of mortality throughout their ICU stay as a proxy measure of severity of illness. DESIGN: Retrospective cohort study. SETTING: PICU in a tertiary care academic children's hospital. PATIENTS/SUBJECTS: Twelve thousand five hundred sixteen episodes (9,070 children) admitted to the PICU between January 2010 and February 2019, partitioned into training (50%), validation (25%), and test (25%) sets. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: On 2,475 test set episodes lasting greater than or equal to 24 hours in the PICU, the area under the receiver operating characteristic curve of the recurrent neural network's 12th hour predictions was 0.94 (CI, 0.93-0.95), higher than those of Pediatric Index of Mortality 2 (0.88; CI, [0.85-0.91]; p < 0.02), Pediatric Risk of Mortality III (12th hr) (0.89; CI, [0.86-0.92]; p < 0.05), and Pediatric Logistic Organ Dysfunction day 1 (0.85; [0.81-0.89]; p < 0.002). The recurrent neural network's discrimination increased with more acquired data and smaller lead time, achieving a 0.99 area under the receiver operating characteristic curve 24 hours prior to discharge. Despite not having diagnostic information, the recurrent neural network performed well across different primary diagnostic categories, generally achieving higher area under the receiver operating characteristic curve for these groups than the other three scores. On 692 test set episodes lasting greater than or equal to 5 days in the PICU, the recurrent neural network area under the receiver operating characteristic curves significantly outperformed their daily Pediatric Logistic Organ Dysfunction counterparts (p < 0.005). CONCLUSIONS: The recurrent neural network model can process hundreds of input variables contained in a patient's electronic medical record and integrate them dynamically as measurements become available. Its high discrimination suggests the recurrent neural network's potential to provide an accurate, continuous, and real-time assessment of a child in the ICU.

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.

Quantitative assessment of linear noise-reduction filters for spectroscopy.

Linear noise-reduction filters used in spectroscopy must strike a balance between reducing noise and preserving lineshapes, the two conflicting requirements of interest. Here, we quantify this tradeoff by capitalizing on Parseval's Theorem to cast two measures of performance, mean-square error (MSE) and noise, into reciprocal- (Fourier-) space (RS). The resulting expressions are simpler and more informative than those based in direct- (spectral-) space (DS). These results provide quantitative insight not only into the effectiveness of different linear filters, but also information as to how they can be improved. Surprisingly, the rectangular ("ideal" or "brick wall") filter is found to be nearly optimal, a consequence of eliminating distortion in low-order Fourier coefficients where the major fraction of spectral information is contained. Using the information provided by the RS version of MSE, we develop a version that is demonstrably superior to the brick-wall and also the Gauss-Hermite filter, its former nearest competitor.

Integrated fundal assessment pathway for patients with suspected shunt malfunction via digital retinal images.

Introduction: Papilloedema is an important clinical sign in the assessment of neurosurgical patients presenting with suspected shunt malfunction.Methods: We describe our 18-month experience using a newly acquired nonmydriatic digital retinal camera to acquire fundal photographs for this purpose.Results: It was used to take digital photographs in 67 patients presenting with suspected shunt malfunction and 54% of these images were discussed with the on-call ophthalmology team for advice. Of these 81% had instant input provided by ophthalmology and the remainder were sent to the ophthalmology department for formal assessment. Of all shunt related regional referrals in the same time period, we found that only 13% of the referred patients were discussed with the on-call ophthalmology team. The rest had decisions made by the on-call neurosurgical team.Conclusions: We believe this new integrated pathway is clinically and cost-effective and recommend it as a useful tool in other neurosurgical units.

HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape.

BACKGROUND & AIMS: Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K. METHODS: The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis. RESULTS: Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions. CONCLUSIONS: Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays. LAY SUMMARY: Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models.

BACKGROUND & AIMS: Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. RESULTS: Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1-7 and Escape Variants.

BACKGROUND & AIMS: Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1-7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment. METHODS: We measured the efficacy of different concentrations of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1-7 NS5A proteins with or without RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or that contained RAS previously reported from patients. RESULTS: NS5A inhibitors had varying levels of efficacy against original and resistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had high levels of fitness. Pibrentasvir had the highest level of efficacy against variants; viruses with RAS at amino acids 28, 30, or 31 had no apparent resistance to pibrentasvir, and HCV with RAS at amino acid 93 had a low level of resistance to this drug. However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. For the remaining NS5A inhibitors tested, RAS at amino acids 28 and 93 led to high levels of resistance. Among these inhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants with RAS at amino acid 31 than the other agents. Variants with the pre-existing RAS T/Y93H acquired additional NS5A changes during escape experiments, resulting in HCV variants with specific combinations of RAS, showing high fitness and high resistance. CONCLUSIONS: We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent. These findings could improve treatment of patients with HCV infection.