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Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium¿revealed by the changing composition of the mutant spectrum¿may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is 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 (MCIU), 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 (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 No. PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, Grant No. IDI-20200297. 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). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. 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 influence of quasispecies dynamics on long-term virus diversification in nature is a largely unexplored question. Specifically, whether intra-host nucleotide and amino acid variation in quasispecies fit the variation observed in consensus sequences or data bank alignments is unknown. Genome conservation and dynamics simulations are used for the computational design of universal vaccines, therapeutic antibodies and pan-genomic antiviral agents. The expectation is that selection of escape mutants will be limited when mutations at conserved residues are required. This strategy assumes long-term (epidemiologically relevant) conservation but, critically, does not consider short-term (quasispecies-dictated) residue conservation. We calculated mutant frequencies of individual loci from mutant spectra of hepatitis C virus (HCV) populations passaged in cell culture and from infected patients. Nucleotide or amino acid conservation in consensus sequences of the same populations, or in the Los Alamos HCV data bank did not match residue conservation in mutant spectra. The results relativize the concept of sequence conservation in viral genetics and suggest that residue invariance in data banks is an insufficient basis for the design of universal viral ligands for clinical purposes. Our calculations suggest relaxed mutational restrictions during quasispecies dynamics, which may contribute to higher calculated short-term than long-term viral evolutionary rates. The research at Centro de Biologia Molecular Severo Ochoa (CSIC-UAM) (CBMSO) was funded by Ministerio de Economia y Competitividad (MINECO), grant numbers SAF2014-52400-R and SAF2017-87846-R, and by Ministerio de Ciencia, Innovacion y Universidades (MCIU), grant number BFU2017-91384-EXP, and by Instituto de Salud Carlos III, grant number PI18/00210, and by PLATESA from Comunidad de Madrid/FEDER, grant numbers S2013/ABI-2906 and S2018/BAA-4370. 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 Investigacion en Red de Enfermedades Hepaticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundacion Ramon Areces and Banco Santander to the CBMSO are also acknowledged. The research at Vall d'Hebron Institut de Recerca (VHIR) was funded 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 Tecnologico Industrial (CDTI) from the MCIU, grant number IDI-20151125. The research at Centro de Astrobiologia (CAB) was supported by MINECO, grant numbers BIO2016-79618R and PID2019-104903RB-I00 (funded by EU under the FEDER program) and by the Spanish State research agency (AEI), grant number MDM-2017-0737 (Unidad de Excelencia "Maria de Maeztu"-Centro de Astrobiologia (CSIC-INTA). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. 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); With funding from the Spanish government through the "María de Maeztu Unit of Excellence" accreditation (MDM-2017-0737) Peer review