JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in evaluation of virus neutralization.

BACKGROUND AIMS: HCV infection continues to be a major global health burden, despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human-liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. APPROACH RESULTS: We demonstrated infection of human-liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human-liver chimeric mice, with genetic stability. Furthermore, we showed that overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. CONCLUSIONS: These mouse-selected HCV recombinants enable triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human-liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.

Neutralizing antibody and CD8+ T cell responses following BA.4/5 bivalent COVID-19 booster vaccination in adults with and without prior exposure to SARS-CoV-2.

As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8+ T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8+ T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8+ T cells were the only responses detected after vaccination and non-spike-specific CD8+ T cells were only detected after infection. Both spike-specific and non-spike-specific CD8+ T cells were found at much lower frequencies than CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8+ T cell responses, which are maintained longitudinally.

B cell characteristics define HCV reinfection outcome.

BACKGROUND AND AIMS: In individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes. RESULTS: BnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of antigen imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development.

Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone.

IMPORTANCE: HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.

Hepatitis C virus alters the morphology and function of peroxisomes.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir.

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration-response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration-response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

The effect of smartphone-based monitoring and treatment including clinical feedback versus smartphone-based monitoring without clinical feedback in bipolar disorder: the SmartBipolar trial-a study protocol for a randomized controlled parallel-group trial.

INTRODUCTION: A substantial proportion of patients with bipolar disorder experience daily subsyndromal mood swings, and the term "mood instability" reflecting the variability in mood seems associated with poor prognostic factors, including impaired functioning, and increased risk of hospitalization and relapse. During the last decade, we have developed and tested a smartphone-based system for monitoring bipolar disorder. The present SmartBipolar randomized controlled trial (RCT) aims to investigate whether (1) daily smartphone-based outpatient monitoring and treatment including clinical feedback versus (2) daily smartphone-based monitoring without clinical feedback or (3) daily smartphone-based mood monitoring only improves mood instability and other clinically relevant patient-related outcomes in patients with bipolar disorder. METHODS AND ANALYSIS: The SmartBipolar trial is a pragmatic randomized controlled parallel-group trial. Patients with bipolar disorder are invited to participate as part of their specialized outpatient treatment for patients with bipolar disorder in Mental Health Services in the Capital Region of Denmark. The included patients will be randomized to (1) daily smartphone-based monitoring and treatment including a clinical feedback loop (intervention group) or (2) daily smartphone-based monitoring without a clinical feedback loop (control group) or (3) daily smartphone-based mood monitoring only (control group). All patients receive specialized outpatient treatment for bipolar disorder in the Mental Health Services in the Capital Region of Denmark. The trial started in March 2021 and has currently included 150 patients. The outcomes are (1) mood instability (primary), (2) quality of life, self-rated depressive symptoms, self-rated manic symptoms, perceived stress, satisfaction with care, cumulated number and duration of psychiatric hospitalizations, and medication (secondary), and (3) smartphone-based measures per month of stress, anxiety, irritability, activity, and sleep as well as the percentage of days with presence of mixed mood, days with adherence to medication and adherence to smartphone-based self-monitoring. A total of 201 patients with bipolar disorder will be included in the SmartBipolar trial. ETHICS AND DISSEMINATION: The SmartBipolar trial is funded by the Capital Region of Denmark and the Independent Research Fund Denmark. Ethical approval has been obtained from the Regional Ethical Committee in The Capital Region of Denmark (H-19067248) as well as data permission (journal number: P-2019-809). The results will be published in peer-reviewed academic journals, presented at scientific meetings, and disseminated to patients' organizations and media outlets. TRIAL REGISTRATION: Trial registration number: NCT04230421. Date March 1, 2021. Version 1.

Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae.

This review provides a summary of the recently ratified changes to genus and species nomenclature within the virus family Flaviviridae along with reasons for these changes. First, it was considered that the vernacular terms "flaviviral", "flavivirus", and "flaviviruses" could under certain circumstances be ambiguous due to the same word stem "flavi" in the taxon names Flaviviridae and Flavivirus; these terms could either have referred to all viruses classified in the family Flaviviridae or only to viruses classified in the included genus Flavivirus. To remove this ambiguity, the genus name Flavivirus was changed to Orthoflavivirus by the International Committee on Taxonomy of Viruses (ICTV). Second, all species names in the family were changed to adhere to a newly ICTV-mandated binomial format (e.g., Orthoflavivirus zikaense, Hepacivirus hominis) similar to nomenclature conventions used for species elsewhere in biology. It is important to note, however, that virus names remain unchanged. Here we outline the revised taxonomy of the family Flaviviridae as approved by the ICTV in April 2023.

Challenge Inoculum for Hepatitis C Virus Controlled Human Infection Model.

For any controlled human infection model (CHIM), a safe, standardized, and biologically relevant challenge inoculum is necessary. For hepatitis C virus (HCV) CHIM, we propose that human-derived high-titer inocula of several viral genotypes with extensive virologic, serologic, and molecular characterizations should be the most appropriate approach. These inocula should first be tested in human volunteers in a step-wise manner to ensure safety, reproducibility, and curability prior to using them for testing the efficacy of candidate vaccines.

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.

Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington's disease.

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

Pre-existing, treatment-specific resistance-associated substitutions in hepatitis C virus genotype 1 and 3 and viral RNA titers during treatment with direct-acting antivirals.

The introduction of direct-acting antiviral (DAA) treatment of hepatitis C virus (HCV) infected patients has greatly increased treatment success rates. However, viral response kinetics to DAA treatment may depend on pre-existing resistance-associated substitutions (RASs) in HCV. The aim of this study was to describe how pre-existing RASs affect DAA treatment-induced reduction in HCV RNA titers in HCV genotypes 1- and 3-infected individuals. Patients with HCV genotype 1 infection (N = 31) treated with either sofosbuvir/ledipasvir/ribavirin or paritaprevir/ombitasvir/ritonavir/dasabuvir/ribavirin and HCV genotype 3-infected patients (N = 16) treated with either sofosbuvir/daclatasvir/ribavirin or sofosbuvir/ribavirin were analyzed. HCV RNA levels were determined at baseline and frequently during treatment, and RAS profiles were obtained by deep sequencing at baseline. In total, 33/47 (70.2%) of the patients had baseline RASs. However, treatment-specific RASs were detected at baseline only in 12.9% and 18.8% of HCV genotypes 1- and 3-infected patients, respectively. In genotype 1-infected individuals, reduction in HCV RNA titer during the first week of treatment was not affected by evidence of either treatment-specific RASs or cirrhosis or treatment regimen. In genotype 3-infected individuals receiving sofosbuvir/daclatasvir/ribavirin, the presence of daclatasvir-specific NS5A RASs at baseline correlated with a reduced decline of HCV RNA in the first treatment week. For both genotypes 1- and 3-infected individuals, cirrhosis but not treatment-specific RAS were associated with the time of clearance of HCV RNA. It is, however, important to note that this study involves DAA regimens that were used only during the original introduction of interferon-free DAA-based treatments.

Differential activity of nucleotide analogs against tick-borne encephalitis and yellow fever viruses in human cell lines.

With no approved antiviral therapies, the continuous emergence and re-emergence of tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV) is a rising concern. We performed head-to-head comparisons of the antiviral activity of available nucleos(t)ide analogs (nucs) using relevant human cell lines. Eight existing nucs inhibited TBEV and/or YFV with differential activity between cell lines and viruses. Remdesivir, uprifosbuvir and sofosbuvir were the most potent drugs against TBEV and YFV in liver cells, but they had reduced activity in neural cells, whereas galidesivir retained uniform activity across cell lines and viruses. Ribavirin, valopicitabine, molnupiravir and GS-6620 exhibited only moderate antiviral activity. We found antiviral activity for drugs previously reported as inactive, demonstrating the importance of using human cell lines and comparative experimental assays when screening the activity of nucs. The relatively high antiviral activity of remdesivir, sofosbuvir and uprifosbuvir against TBEV and YFV merits further investigation in clinical studies.

The Role of CTLA-4 in T Cell Exhaustion in Chronic Hepatitis B Virus Infection.

Patients with chronic hepatitis B (CHB) gradually develop T cell exhaustion, and the inhibitory receptor molecule, cytotoxic T-lymphocyte antigen-4 (CTLA-4), may play a role in this phenomenon. This systematic review investigates the role of CTLA-4 in the development of T cell exhaustion in CHB. A systematic literature search was conducted on PubMed and Embase on 31 March 2023 to identify relevant studies. Fifteen studies were included in this review. A majority of the studies investigating CD8+ T cells demonstrated increased expression of CTLA-4 in CHB patients, though one study found this only in HBeAg-positive patients. Three out of four studies investigating the expression of CTLA-4 on CD4+ T cells found upregulation of CTLA-4. Several studies showed constitutive expression of CLTA-4 on CD4+ regulatory T cells. CTLA-4 blockade resulted in heterogeneous responses for all T cell types, as it resulted in increased T cell proliferation and/or cytokine production in some studies, while other studies found this only when combining blockade of CTLA-4 with other inhibitory receptors. Although mounting evidence supports a role of CTLA-4 in T cell exhaustion, there is still insufficient documentation to describe the expression and exact role of CTLA-4 in T cell exhaustion in CHB.

Toward a vaccine against hepatitis C virus.

Recent advances aid the development of vaccines to prevent chronic liver diseases.

Durability and breadth of neutralisation following multiple antigen exposures to SARS-CoV-2 infection and/or COVID-19 vaccination.

BACKGROUND: Given the importance of vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the prevention of severe coronavirus disease 2019 (COVID-19), detailed long-term analyses of neutralising antibody responses are required to inform immunisation strategies. METHODS: In this study, longitudinal neutralising antibody titres to an ancestral SARS-CoV-2 isolate and cross-neutralisation to delta and omicron isolates were analysed in individuals previously infected with SARS-CoV-2, vaccinated against COVID-19, or a complex mix thereof with up to two years of follow-up. FINDINGS: Both infection-induced and vaccine-induced neutralising responses against SARS-CoV-2 appeared to follow similar decay patterns. Following vaccination in previously infected individuals, neutralising antibody responses were more durable than prior to vaccination. Further, this study shows that vaccination after infection, as well as booster vaccination, increases the cross-neutralising potential to both delta and omicron SARS-CoV-2 variants. INTERPRETATION: Taken together, these results suggest that neither type of antigen exposure is superior for neutralising antibody durability. However, these results support vaccination to increase the durability and cross-neutralisation potential of neutralising responses, thereby enhancing protection against severe COVID-19. FUNDING: This work was supported by grants from The Capital Region of Denmark's Research Foundation, the Novo Nordisk Foundation, the Independent Research Fund Denmark, the Candys Foundation, and the Danish Agency for Science and Higher Education.

Molecular Determinants of Mouse Adaptation of Rat Hepacivirus.

The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.

Identification of the viral and cellular microRNA interactomes during SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a tremendous impact worldwide. Mapping virus-host interactions is critical to understand disease progression. MicroRNAs (miRNAs) are important RNA regulators, but their interaction with SARS-CoV-2 RNA was not experimentally investigated. Here, using Argonaute (AGO) cross-linking immunoprecipitation combined with RNA proximity ligation (CLEAR-CLIP), we provide unbiased mapping of SARS-CoV-2/miRNA interactions. We identified six main regions on the viral RNA bound primarily by one specific miRNA. Targeted mutagenesis and AGO1-3 knockdown demonstrated that these interactions are not critical for virus production. Moreover, we identified perturbed regulation of cellular miRNA interactions during infection, including non-compensated viral sequestration of the miR-15 family. Transcriptome analysis further showed that mRNAs targeted by this miRNA family are derepressed. This work delineates the interphase between miRNA regulation and SARS-CoV-2 infection and further contributes to deciphering the full molecular interactome of this virus.

Characterization of multi-DAA resistance using a novel hepatitis C virus genotype 3a infectious culture system.

BACKGROUND AND AIMS: The high HCV infection cure rates achieved with direct-acting antiviral (DAA) treatments could be compromised in the future by the emergence of antiviral resistance. Thus, it is essential to understand the viral determinants that influence DAA resistance, which is most prevalent in genotype 3. We aimed at studying how resistance to protease-, NS5A-, and NS5B-inhibitors influences the activities of glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, and sofosbuvir/velpatasvir/voxilaprevir in cell culture, and how the HCV genome adapts to selective pressure by successive rounds of treatment failure. APPROACH AND RESULTS: A previously developed in vivo infectious cDNA clone of strain S52 (genotype 3a) was adapted to efficiently replicate and propagate in human hepatoma cells (Huh7.5) using 31 adaptive substitutions. DAA escape experiments resulted in the selection of S52 variants with decreased drug susceptibility (resistance), which was linked to the emergence of known resistance-associated substitutions (RASs). NS5A-inhibitor resistance was sufficient to promote treatment failure with double-DAA but not triple-DAA regimens. Enhanced viral fitness associated with the selection of sofosbuvir resistance accelerated escape from DAAs. After serial DAA treatment failure, HCV genetic evolution led to a complex genome-wide network of substitutions, some of which coevolved with known RASs. CONCLUSIONS: Baseline NS5A-RAS can compromise the efficacy of double-DAA pangenotypic regimens for HCV genotype 3, and enhanced viral fitness can accelerate treatment failure. Persistence of RASs after successive treatment failure is facilitated by the remarkable evolutionary capacity and plasticity of the HCV genome. Proof-of-concept for the potential development of multi-DAA resistance is shown.