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Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes. IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO); SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU); and S2013/ABI-2906 (PLATESA from Comunidad de Madrid [CAM]/FEDER), S2018/BAA-4370 (PLATESA2 from CAM/FEDER), and PI18/00210 from Instituto de Salud Carlos III. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) (grant numbers PI16/00337) and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business (grant number IDI-20151125). Work at CAB was supported by MINECO grant BIO2016-79618-R (funded by the European Union under the FEDER program) and by the Spanish State Research Agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiologia (CSIC-INTA).

The European Association for the Study of the Liver (EASL) Recommendations on Treatment of Hepatitis C 2018, published in the Journal of Hepatology , specify that the recommendations are “primarily based on evidence from existing publications and presentations at international meetings” ([1][1

Lethal mutagenesis is an antiviral approach that consists of extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad-spectrum nature, which renders the strategy potentially effective against emergent RNA viral infections. Here we describe the synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method implemented in CompuSyn graphics software, with the average dose reduction index (DRI) being above 1 (68.02 101.6 for favipiravir and 5.83 6.07 for ribavirin) and the average combination indices (CI) being below 1 (0.52 0.28). Furthermore, analogue concentrations that individually did not extinguish high-fitness HCV in 10 serial infections extinguished high-fitness HCV in 1 to 2 passages when used in combination. Although both analogues displayed a preference for G ¡ A and C ¡ U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. The prospects for synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed, C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (grant CP14/00121), cofinanced by the European Regional Development Fund (ERDF). The Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III. The work in Madrid, Spain, was supported by grant SAF2014-52400-R from the Ministerio de Economía y Competitividad, grants SAF2017-87846-R, BFU2017-91384-EXP, and PI18/00210 from the Ministerio de Ciencia, Innovación y Universidades, grant S2013/ABI-2906 from PLATESA from the Comunidad de Madrid/FEDER, and grant P2018/BAA-4370 from PLATESA2 from the Comunidad de Madrid/FEDER. The work in Barcelona, Spain, was also funded by the Instituto de Salud Carlos III; by grant PI16/00337, cofinanced by the European Regional Development Fund (ERDF); and by the Centro para el Desarrollo Tecnológico Industrial (CDTI), Spanish Ministry of Economics and Competitiveness (MINECO) (grant IDI-20151125). Institutional grants from the Fundación Ramón Areces and Banco Santander to CBMSO are also acknowledged.

One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence including 38 (13%) synonymous mutations. WT and MAX viruses showed indistinguishable replication in MT-4 cells or PBMCs. Both viruses were subjected to serial passages in MT-4 cells with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (IC5014.6 ± 5.3 and 21.2 ± 9 nM for ATV, and 5. 9 ± 1.0 and 9.3 ± 1.9 for DRV, respectively). Ultra-deep sequence clonal analysis revealed that both viruses harbored previously described resistance mutations to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in WT, and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in MAX. Remarkably, G48L and L89I are rarely foundin vivoin PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P< 0.0001), suggesting higher selective pressure for change in this recoded virus. Our results indicate that HIV-1 protease position in sequence space delineates the evolution of its mutant spectra. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to the WT virus.IMPORTANCELarge-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus’s position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease impacts the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and the virus with the recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotype was indistinguishable between the recoded virus and the WT virus, suggesting that the synonymously recoded protease HIV-1 and the WT protease virus were equally robust and evolvable.

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment., Comunidad Autonoma de Madrid/FEDER) and by Fundacion Ramon Areces. The work in Barcelona was funded by Instituto de Salud Carlos III; by grants PI13/00456, PI15/00829, and PI16/00337 cofinanced by the European Regional Development Fund (ERDF); and by CDTI (Centro para el Desarrollo Tecnologico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20151125

Cell culture-produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of alpha interferon (IFN-α). Virus survival, genetic changes, fitness levels, and phenotypic traits have been examined. While high initial IFN-α doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus passaged in the presence of IFN-α acquired IFN-α resistance as evidenced by enhanced progeny production and viral protein expression in an IFN-α environment. A partial IFN-α resistance was also noted in populations passaged in the absence of IFN-α. All lineages acquired adaptative mutations, and multiple, nonsynonymous mutations scattered throughout the genome were present in IFN-α-selected populations. Comparison of consensus sequences indicates a dominance of synonymous versus nonsynonymous substitutions. IFN-α-resistant populations displayed decreased sensitivity to a combination of IFN-α and ribavirin. A phenotypic trait common to all assayed viral populations is the ability to increase shutoff host cell protein synthesis, accentuated in infections with IFN-α-selected populations carried out in the presence of IFN-α. The trait was associated with enhanced phosphorylation of protein kinase R (PKR) and eIF2α, although other contributing factors are likely. The results suggest that multiple, independent mutational pathways can confer IFN-α resistance to HCV and might explain why no unified picture has been obtained regarding IFN-α resistance in vivo © 2013 American Society for Microbiology., CDTI (Centro para el Desarrollo Tecnológico Industrial) from Ministerio de Ciencia e Innovación; Fundación Ramon Areces; Marie Curie International outgoing fellowship 219570; CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III; Consejo Superior de Investigaciones Científicas (CSIC); Greenberg Medical Research Institute; Starr Foundation

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvirspecific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvirspecific resistance mutations, linked to in vivo fitness of pretreatment viral populations., MINECO | Instituto de Salud Carlos III (ISCIII) (PI13/00456). This work, including the efforts of Rafael Esteban, was funded by MINECO | Instituto de Salud Carlos III (ISCIII) (PI15/00829). This work, including the efforts of Esteban Domingo, was funded by Ministerio de Economía y Competitividad (MINECO) (BFU-2011-23604); Comunidad Autónoma de Madrid (S2013/ABI-2906).

HepatitisCvirus (HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, incomparisonwith thoseof twocommercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1 and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing (69%1b,31%1a) in81 specimensandidentified amixed-subtype infection (1b/3a/1a) inone sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing inall but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in13 (16%)samples eachandwere unable to identify theHCVgenotype and/or subtype inmore thanhalfof the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methods andallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect toinforming treatment strategies withnewDAA-containing regimens across allHCVsubtypes

All Rights Reserved. HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypes., This study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5′ noncoding region (5′ NCR) of HCV. The probe contains a fluorescent reporter at the 5′ end and a fluorescent quencher at the 3′ end. The use of such a probe combined with the 5′-3′ nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5′ NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10 3 to 10 7 ). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.