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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., Competing Interests: Authors KJ, DI, and LB are employed by the company Immudex ApS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Underwood, Sølund, Jacobsen, Binderup, Fernandez-Antunez, Mikkelsen, Inekci, Villadsen, Castruita, Pinholt, Fahnøe, Ramirez, Brix, Weis and Bukh.)

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., (Copyright © 2023 American Association for the Study of Liver Diseases.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 virus 1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer 3 . 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 life 4-8 . 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests Nina Weis has been clinical investigator, lecturer or on advisory boards for Abbvie, Gilead, Glaxo Smith Kline and Merck Sharp Dohme and has received unrestricted grants for research from Abbvie and Gilead without relation to the presented work and for the remaining authors there are no conflicts of interest. Christina Sølund has received support from MSD Denmark to attend the Nordic HIV and Virology Conference in Stockholm 2022. Anni Assing Winckelmann has received support from Gilead Denmark to attend the Nordic HIV and Virology Conference in Stockholm 2022., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Vaccines have relieved the public health burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and globally inactivated vaccines are most widely used. However, poor vaccination accessibility and waning immunity maintain the pandemic, driving emergence of variants. We developed an inactivated SARS-CoV-2 (I-SARS-CoV-2) vaccine based on a viral isolate with the Spike mutation D614G, produced in Vero cells in a scalable bioreactor, inactivated with β-propiolactone, purified by membrane-based steric exclusion chromatography, and adjuvanted with MF59-like adjuvant AddaVax. I-SARS-CoV-2 and a derived split vaccine induced persisting neutralizing antibodies in mice; moreover, lyophilized antigen was immunogenic. Following homologous challenge, I-SARS-CoV-2 immunized hamsters were protected against disease and lung pathology. In contrast with reports for widely used vaccines, hamster plasma similarly neutralized the homologous and the Delta (B.1.617.2) variant viruses, whereas the Omicron (B.1.1.529) variant was neutralized less efficiently. Applied bioprocessing approaches offer advantages regarding scalability and production, potentially benefitting worldwide vaccine coverage., Competing Interests: A.O., C.R.D.H., S.F., K.H., A.F.P., C.F-A., G.P.A., K.T.H., L.V.P., Y.Z., K.A.G., U.F., U.V.S., G.K.P., H.E.J., J.P.C., S.R., J.B., and J.M.G. declare no conflict of interests. P.M-G. is an inventor in pending patent applications related to the SXC purification method described in this work., (© 2023 The Authors.)

SARS-CoV-2 continues to pose a threat to human health as new variants emerge and thus a diverse vaccine pipeline is needed. We evaluated SARS-CoV-2 HexaPro spike protein formulated in Alhydrogel ® (aluminium oxyhydroxide) in Syrian hamsters, using an accelerated two dose regimen (given 10 days apart) and a standard regimen (two doses given 21 days apart). Both regimens elicited spike- and RBD-specific IgG antibody responses of similar magnitude, but in vitro virus neutralization was low or undetectable. Despite this, the accelerated two dose regimen offered reduction in viral load and protected against lung pathology upon challenge with homologous SARS-CoV-2 virus (Wuhan-Hu-1). This highlights that vaccine-induced protection against SARS-CoV-2 disease can be obtained despite low neutralizing antibody levels and suggests that accelerated vaccine schedules may be used to confer rapid protection against SARS-CoV-2 disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Christensen, Polacek, Sheward, Hanke, McInerney, Murrell, Hartmann, Jensen, Zimmermann, Jungersen, Illigen, Isling, Fernandez-Antunez, Ramirez, Bukh and Pedersen.)

The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

Background: Licensed vaccines against SARS-CoV-2 effectively protect against severe disease, but display incomplete protection against virus transmission. Mucosal vaccines providing immune responses in the upper airways are one strategy to protect against transmission., Methods: We administered Spike HexaPro trimer formulated in a cationic liposomal adjuvant as a parenteral (subcutaneous - s.c.) prime - intranasal boost regimen to elicit airway mucosal immune responses and evaluated this in a Syrian hamster model of virus transmission., Findings: Parenteral prime - intranasal boost elicited high-magnitude serum neutralizing antibody responses and IgA responses in the upper respiratory tract. The vaccine strategy protected against virus in the lower airways and lung pathology, but virus could still be detected in the upper airways. Despite this, the parenteral prime - intranasal booster vaccine effectively protected against onward SARS-CoV-2 transmission., Interpretation: This study suggests that parenteral-prime mucosal boost is an effective strategy for protecting against SARS-CoV-2 infection and highlights that protection against virus transmission may be obtained despite incomplete clearance of virus from the upper respiratory tract. It should be noted that protection against onward transmission was not compared to standard parenteral prime-boost, which should be a focus for future studies., Funding: This work was primarily supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653., Competing Interests: Declaration of interests D.C. is co-inventor on patents on the cationic adjuvant formulations (CAF). All rights have been turned over to Statens Serum Institut, which is a non-profit government research facility. The rest of the authors declare that there are no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.

With increasing numbers of vaccine-breakthrough infections worldwide, assessing the immunogenicity of vaccinated health-care workers that are frequently exposed to SARS-CoV-2-infected individuals is important. In this study, neutralization titers against SARS-CoV-2 were assessed one month after completed prime-boost vaccine regimens in health-care workers vaccinated with either mRNA-mRNA (Comirnaty ® , BioNTech-Pfzier, Mainz, Germany/New York, NY, USA, n = 98) or vector-based (Vaxzevria ® , Oxford-AstraZeneca, Cambridge, UK) followed by mRNA-based (Comirnaty ® or Spikevax ® , Moderna, Cambridge, MA, USA) vaccines ( n = 16). Vaccine-induced neutralization titers were compared to time-matched, unvaccinated individuals that were infected with SARS-CoV-2 and presented with mild symptoms ( n = 38). Significantly higher neutralizing titers were found in both the mRNA-mRNA (ID 50 : 2525, IQR: 1667-4313) and vector-mRNA (ID 50 : 4978, IQR: 3364-7508) prime-boost vaccine regimens when compared to SARS-CoV-2 infection (ID 50 : 401, IQR: 271-792) ( p < 0.0001). However, infection with SARS-CoV-2 induced higher titers when compared to a single dose of Vaxzevria ® ( p = 0.0072). Between mRNA-mRNA and vector-mRNA prime-boost regimens, the vector-mRNA vaccine regimen induced higher neutralization titers ( p = 0.0054). Demographically, both age and time between vaccination doses were associated with vaccine-induced neutralization titers ( p = 0.02 and p = 0.03, respectively). This warrants further investigation into the optimal time to administer booster vaccination for optimized induction of neutralizing responses. Although anecdotal ( n = 3), those with exposure to SARS-CoV-2, either before or after vaccination, demonstrated superior neutralizing titers, which is suggestive of further boosting through viral exposure.

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment., (© 2021. The Author(s).)

Background: Given the importance of neutralising antibodies in protection against SARS-CoV-2 infection, it is critical to assess neutralisation persistence long-term following recovery. This study investigated neutralisation titres against SARS-CoV-2 up to 6 months post-symptom onset in individuals with mild COVID-19., Methods: Plasma neutralisation titres in convalescent COVID-19 individuals were determined at baseline and 6 months post-symptom onset using a cell culture infectious SARS-CoV-2 assay. Total SARS-CoV-2 spike-specific IgG and IgA binding was measured using a lectin capture ELISA and compared between timepoints and correlated to neutralising titres., Findings: All 48 convalescent COVID-19 individuals were found to have detectable SARS-CoV-2 50% inhibitory dilution neutralisation titres (ID 50 ) at baseline and 6 months post-symptom onset with mean ID 50 of 1/943 and 1/411, respectively. SARS-CoV-2 neutralisation titres peaked within 1-2 months post-symptom onset. However, 50% of individuals showed comparable ID 50 at baseline and 6 months post-symptom onset. Both SARS-CoV-2 spike-specific IgG and IgA levels correlated well with neutralising titres. IgG binding was found to be sustained up to 6 months post-symptom onset, whereas IgA levels declined., Interpretation: This study demonstrates durability of SARS-CoV-2 spike-specific IgG and neutralisation responses following recovery from mild COVID-19. Thus, all subjects included in this study might potentially have protective levels of neutralising antibodies 6 months post-symptom onset. This study also demonstrates a relationship between spike-specific IgA and neutralisation decline, with implications for long-term protection against SARS-CoV-2 infection., Funding: Novo Nordisk Foundation, Independent Research Fund Denmark and Danish Agency for Science and Higher Education., Competing Interests: Declaration of Competing Interest Nina Weis has been clinical investigator, lecturer or on advisory boards for Abbvie, Gilead, Glaxo Smith Kline and Merck Sharp Dohme and has received unrestricted grants for research from Abbvie and Gilead without relation to the present work and for the remaining authors there are no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic., (Copyright © 2020 American Society for Microbiology.)