Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps.

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

Satisfacción laboral, grado de burnout y calidad de vida profesional de los fisioterapeutas de atención hospitalaria de Castilla y León / Job satisfaction, level of burnout and quality of professional life of hospital care physiotherapists in Castilla y Leon

Antecedentes La satisfacción laboral, el burnout y la calidad de vida profesional de los fisioterapeutas es clave para mantener la calidad de los servicios prestados en los pacientes. Objetivos Describir la satisfacción laboral, el burnout y la calidad de vida profesional de los fisioterapeutas de Atención Hospitalaria de la Sanidad pública de Castilla y León (SACYL); identificar las diferencias existentes en la satisfacción laboral, el burnout y el nivel de calidad de vida profesional en los mismos; determinar la relación existente entre las tres variables. Materiales y métodos Se realizó un estudio descriptivo transversal, con muestreo de conveniencia, en fisioterapeutas de Castilla y León que trabajan en atención hospitalaria. Se recogieron datos de la satisfacción laboral (Font Roja), el burnout (Maslach Burnout Inventory) y la calidad de vida profesional (CVP-35) en su puesto de trabajo. Se llevó a cabo un análisis descriptivo y relacional de las variables analizadas. Resultados Participaron 72 fisioterapeutas, siendo 75% mujeres, con una media de edad de 42,8 ± 8,37 años. El nivel de burnout resultó medio-alto en las dimensiones de agotamiento emocional y despersonalización, bajo en realización personal y la satisfacción laboral baja (SMG = -0,04). Los valores de calidad de vida profesional alcanzan una puntuación de notable (7,15 ± 1,43). Conclusiones Los fisioterapeutas de atención hospitalaria del SACYL presentan una elevada motivación intrínseca, a pesar del escaso apoyo directivo que reciben. La mejora de ambas dimensiones conlleva un aumento de la satisfacción laboral y de la calidad de vida profesional, y un descenso de los niveles de burnout (especialmente en el agotamiento emocional y la despersonalización). (AU)

Background Job satisfaction, burnout and professional quality of life of physiotherapists is key to maintaining the quality of services provided to patients. Objectives To describe job satisfaction, burnout and professional quality of life of physiotherapists in hospital care in the public health system of Castilla y León (SACYL); to identify the existing differences in job satisfaction, burnout and the level of professional quality of life among them; to determine the relationship between the three variables. Materials and methods A cross-sectional descriptive study was carried out, with convenience sampling, in physiotherapists from Castilla y León working in hospital care. Data were collected on job satisfaction (Font Roja), burnout (Maslach Burnout Inventory) and professional quality of life (CVP-35) in the workplace. A descriptive and relational analysis of the variables analysed was carried out. Results Seventy-two physiotherapists participated, 75% of whom were women, with an average age of 42.8 ± 8.37 years. The level of burnout was medium-high in the dimensions of emotional exhaustion and depersonalisation, low in personal fulfilment and low in job satisfaction (SMG = −0.04). Professional quality of life values reach a score of remarkable (7.15 ± 1.43). Conclusions Hospital care physiotherapists of the SACYL present high intrinsic motivation, despite the low managerial support they receive. The improvement of both dimensions leads to an increase in job satisfaction and professional quality of life, and a decrease in burnout levels (especially in emotional exhaustion and depersonalisation). (AU)

Satisfacción laboral, grado de burnout y calidad de vida profesional de los fisioterapeutas de atención hospitalaria de Castilla y León / Job satisfaction, level of burnout and quality of professional life of hospital care physiotherapists in Castilla y Leon

Antecedentes La satisfacción laboral, el burnout y la calidad de vida profesional de los fisioterapeutas es clave para mantener la calidad de los servicios prestados en los pacientes. Objetivos Describir la satisfacción laboral, el burnout y la calidad de vida profesional de los fisioterapeutas de Atención Hospitalaria de la Sanidad pública de Castilla y León (SACYL); identificar las diferencias existentes en la satisfacción laboral, el burnout y el nivel de calidad de vida profesional en los mismos; determinar la relación existente entre las tres variables. Materiales y métodos Se realizó un estudio descriptivo transversal, con muestreo de conveniencia, en fisioterapeutas de Castilla y León que trabajan en atención hospitalaria. Se recogieron datos de la satisfacción laboral (Font Roja), el burnout (Maslach Burnout Inventory) y la calidad de vida profesional (CVP-35) en su puesto de trabajo. Se llevó a cabo un análisis descriptivo y relacional de las variables analizadas. Resultados Participaron 72 fisioterapeutas, siendo 75% mujeres, con una media de edad de 42,8 ± 8,37 años. El nivel de burnout resultó medio-alto en las dimensiones de agotamiento emocional y despersonalización, bajo en realización personal y la satisfacción laboral baja (SMG = -0,04). Los valores de calidad de vida profesional alcanzan una puntuación de notable (7,15 ± 1,43). Conclusiones Los fisioterapeutas de atención hospitalaria del SACYL presentan una elevada motivación intrínseca, a pesar del escaso apoyo directivo que reciben. La mejora de ambas dimensiones conlleva un aumento de la satisfacción laboral y de la calidad de vida profesional, y un descenso de los niveles de burnout (especialmente en el agotamiento emocional y la despersonalización). (AU)

Background Job satisfaction, burnout and professional quality of life of physiotherapists is key to maintaining the quality of services provided to patients. Objectives To describe job satisfaction, burnout and professional quality of life of physiotherapists in hospital care in the public health system of Castilla y León (SACYL); to identify the existing differences in job satisfaction, burnout and the level of professional quality of life among them; to determine the relationship between the three variables. Materials and methods A cross-sectional descriptive study was carried out, with convenience sampling, in physiotherapists from Castilla y León working in hospital care. Data were collected on job satisfaction (Font Roja), burnout (Maslach Burnout Inventory) and professional quality of life (CVP-35) in the workplace. A descriptive and relational analysis of the variables analysed was carried out. Results Seventy-two physiotherapists participated, 75% of whom were women, with an average age of 42.8 ± 8.37 years. The level of burnout was medium-high in the dimensions of emotional exhaustion and depersonalisation, low in personal fulfilment and low in job satisfaction (SMG = −0.04). Professional quality of life values reach a score of remarkable (7.15 ± 1.43). Conclusions Hospital care physiotherapists of the SACYL present high intrinsic motivation, despite the low managerial support they receive. The improvement of both dimensions leads to an increase in job satisfaction and professional quality of life, and a decrease in burnout levels (especially in emotional exhaustion and depersonalisation). (AU)

Quasispecies productivity.

The quasispecies theory is a helpful concept in the explanation of RNA virus evolution and behaviour, with a relevant impact on methods used to fight viral diseases. It has undergone some adaptations to integrate new empirical data, especially the non-deterministic nature of mutagenesis, and the variety of behavioural motifs in cooperation, competition, communication, innovation, integration, and exaptation. Also, the consortial structure of quasispecies with complementary roles of memory genomes of minority populations better fits the empirical data than did the original concept of a master sequence and its mutant spectra. The high productivity of quasispecies variants generates unique sequences that never existed before and will never exist again. In the present essay, we underline that such sequences represent really new ontological entities, not just error copies of previous ones. Their primary unique property, the incredible variant production, is suggested here as quasispecies productivity, which replaces the error-replication narrative to better fit into a new relationship between mankind and living nature in the twenty-first century.

Fluoride anodic films on stainless-steel fomites to reduce transmission infections.

The growing concern arising from viruses with pandemic potential and multi-resistant bacteria responsible for hospital-acquired infections and outbreaks of food poisoning has led to an increased awareness of indirect contact transmission. This has resulted in a renewed interest to confer antimicrobial properties to commonly used metallic materials. The present work provides a full characterization of optimized fluoride anodic films grown in stainless steel 304L as well as their antimicrobial properties. Antibacterial tests show that the anodic film, composed mainly of chromium and iron fluorides, reduces the count and the percentage of the area covered by 50% and 87.7% for Pseudomonas aeruginosa and Stenotrophomonas maltophilia, respectively. Virologic tests show that the same treatment reduces the infectivity of the coronavirus HCoV-229E-GFP, in comparison with the non-anodized stainless steel 304L.IMPORTANCEThe importance of environmental surfaces as a source of infection is a topic of particular interest today, as many microorganisms can survive on these surfaces and infect humans through direct contact. Modification of these surfaces by anodizing has been shown to be useful for some alloys of medical interest. This work evaluates the effect of anodizing on stainless steel, a metal widely used in a variety of applications. According to the study, the fluoride anodic layers reduce the colonization of the surfaces by both bacteria and viruses, thus reducing the risk of acquiring infections from these sources.

Puzzles, challenges, and information reservoir of SARS-CoV-2 quasispecies.

Upon the emergence of SARS-CoV-2 in the human population, it was conjectured that for this coronavirus the dynamic intra-host heterogeneity typical of RNA viruses would be toned down. Nothing of this sort is observed. Here we review the main observations on the complexity and diverse composition of SARS-CoV-2 mutant spectra sampled from infected patients, within the framework of quasispecies dynamics. The analyses suggest that the information provided by myriads of genomic sequences within infected individuals may have a predictive value of the genomic sequences that acquire epidemiological relevance. Possibilities to reconcile the presence of broad mutant spectra in the large RNA coronavirus genome with its encoding a 3' to 5' exonuclease proofreading-repair activity are considered. Indeterminations in the behavior of individual viral genomes provide a benefit for the survival of the ensemble. We propose that this concept falls in the domain of "stochastic thinking," a notion that applies also to cellular processes, as a means for biological systems to face unexpected needs.

Natural languages and RNA virus evolution.

Information concepts from physics, mathematics and computer science support many areas of research in biology. Their focus is on objective information, which provides correlations and patterns related to objects, processes, marks and signals. In these approaches only the quantitative aspects of the meaning of the information is relevant. In other areas of biology, 'meaningful information', which is subjective in nature, relies on the physiology of the organism's sensory organs and on the interpretation of the perceived signals, which is then translated into action, even if this is only mental (in brained animals). Information is involved, in terms of both amount and quality. Here we contextualize and review the main theories that deal with 'meaningful-information' at a molecular level from different areas of natural language research, namely biosemiotics, code-biology, biocommunication and biohermeneutics. As this information mediates between the organism and its environment, we emphasize how such theories compare with the neo-Darwinian treatment of genetic information, and how they project onto the rapid evolution of RNA viruses.

Viruses as archaeological tools for uncovering ancient molecular relationships.

The entry of a virus into the host cell always implies the alteration of certain intracellular molecular relationships, some of which may involve the recovery of ancient cellular activities. In this sense, viruses are archaeological tools for identifying unexpressed activities in noninfected cells. Among these, activities that hinder virus propagation may represent cellular defense mechanisms, for example, activities that mutagenize the viral genome such as ADAR-1 or APOBEC activities. Instead, those that facilitate virus propagation can be interpreted as the result of viral adaptation to-or mimicking-cellular structures, enabling the virus to perform anthropomorphic activities, including hijacking, manipulating, and reorganizing cellular factors for their own benefit. The alternative we consider here is that some of these second set of cellular activities were already in the uninfected cell but silenced, under the negative control of the cell or lineage, and that they represent a necessary precondition for viral infection. For example, specifically loading an amino acid at the 3'-end of the mRNA of some plant viruses by aminoacyl-tRNA synthetases has proved essential for virus infection despite this reaction not occurring with cellular mRNAs. Other activities of this type are discussed here, together with the biological context in which they acquire a coherent meaning, that is, genetic latency and molecular conflict.

Archaeological approaches to RNA virus evolution.

Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures.

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus.

The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC50) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.

Hepatitis C virus fitness can influence the extent of infection-mediated epigenetic modifications in the host cells.

Introduction: Cellular epigenetic modifications occur in the course of viral infections. We previously documented that hepatitis C virus (HCV) infection of human hepatoma Huh-7.5 cells results in a core protein-mediated decrease of Aurora kinase B (AURKB) activity and phosphorylation of Serine 10 in histone H3 (H3Ser10ph) levels, with an affectation of inflammatory pathways. The possible role of HCV fitness in infection-derived cellular epigenetic modifications is not known. Methods: Here we approach this question using HCV populations that display a 2.3-fold increase in general fitness (infectious progeny production), and up to 45-fold increase of the exponential phase of intracellular viral growth rate, relative to the parental HCV population. Results: We show that infection resulted in a HCV fitness-dependent, average decrease of the levels of H3Ser10ph, AURKB, and histone H4 tri-methylated at Lysine 20 (H4K20m3) in the infected cell population. Remarkably, the decrease of H4K20m3, which is a hallmark of cellular transformation, was significant upon infection with high fitness HCV but not upon infection with basal fitness virus. Discussion: Here we propose two mechanisms ─which are not mutually exclusive─ to explain the effect of high viral fitness: an early advance in the number of infected cells, or larger number of replicating RNA molecules per cell. The implications of introducing HCV fitness as an influence in virus-host interactions, and for the course of liver disease, are warranted. Emphasis is made in the possibility that HCV-mediated hepatocellular carcinoma may be favoured by prolonged HCV infection of a human liver, a situation in which viral fitness is likely to increase.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Viral Fitness, Population Complexity, Host Interactions, and Resistance to Antiviral Agents.

Fitness of viruses has become a standard parameter to quantify their adaptation to a biological environment. Fitness determinations for RNA viruses (and some highly variable DNA viruses) meet with several uncertainties. Of particular interest are those that arise from mutant spectrum complexity, absence of population equilibrium, and internal interactions among components of a mutant spectrum. Here, concepts, fitness measurements, limitations, and current views on experimental viral fitness landscapes are discussed. The effect of viral fitness on resistance to antiviral agents is covered in some detail since it constitutes a widespread problem in antiviral pharmacology, and a challenge for the design of effective antiviral treatments. Recent evidence with hepatitis C virus suggests the operation of mechanisms of antiviral resistance additional to the standard selection of drug-escape mutants. The possibility that high replicative fitness may be the driver of such alternative mechanisms is considered. New broad-spectrum antiviral designs that target viral fitness may curtail the impact of drug-escape mutants in treatment failures. We consider to what extent fitness-related concepts apply to coronaviruses and how they may affect strategies for COVID-19 prevention and treatment.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1−1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

Application of archaeological concepts to the interpretation of RNA virus quasi species evolution.

An enduring problem concerning the evolution of RNA viruses stems from the fact that their long-term rates of evolution (substitutions/ site/year) are lower than those calculated by comparing sequences of isolates collected over short time periods or within a single host (shortterm or intra-host evolution). This inconsistency has been attributed to several reasons, including deviations from the assumption of a molecularclock (constancy of mutational inputs as a function of time) and variations in viral multiplication rates, among others. We previously proposed a non-phylogenetic method for extracting information contained in mRNAs, that cannot be identified from examination of primary sequences alone, and that we called «archaeological¼ information. In this new approach, mRNAs are of interest as molecules, not for their primary sequence or encoded proteins but for encrypted information established in a remote past. In the present article, we propose that an archaeological approach may also contribute to explain higher short-term than long-term evolution rates in RNA viruses, in this case, by using the archaeological concept of palimpsest. The palimpsest is a record of historical changes, but it is not a successively ordered or a complete record, rather it is the product of two opposing activities, one of writing and rewriting and the other of erasing. In RNA virus quasispecies, the gain or loss of mutations is reflected in changes in the submolar frequency of myriads of variants in the population. The fact that mutation elimination is not always complete, turns viral quasispecies into complex palimpsests of viral variants or sub-populations thereof. Here we relate two main different temporalities of the quasispecies palimpsest (short- and long-term) to the stability of mutations in response to changes related to three components of the virus: the virions, the infected cell and the host cell lineage. Host cell lineage-related viral memory would be mostly irre versible as they are adaptive products to host cell changes. In contrast, memories related to the environment of the virion or responsive to the environment of the infected cell, which is shortterm mutational input, is less constrained provided the alteration in the ancestral information carried by the RNA is only transient. The two intermixed memory components result in two differently contributing mutation rates whose influence in the final result depends on whether the timescales used to take the sequences for comparison are short or long term.

Efficacy decrease of antiviral agents when administered to ongoing hepatitis C virus infections in cell culture.

We report a quantification of the decrease of effectiveness of antiviral agents directed to hepatitis C virus, when the agents are added during an ongoing infection in cell culture vs. when they are added at the beginning of the infection. Major determinants of the decrease of inhibitory activity are the time post-infection of inhibitor administration and viral replicative fitness. The efficacy decrease has been documented with antiviral assays involving the combination of the direct-acting antiviral agents, daclatasvir and sofosbuvir, and with the combination of the lethal mutagens, favipiravir and ribavirin. The results suggest that strict antiviral effectiveness assays in preclinical trials may involve the use of high fitness viral populations and the delayed administration of the agents, relative to infection onset.