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.

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.

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.

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.

Broad and Dynamic Diversification of Infectious Hepatitis C Virus in a Cell Culture Environment.

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.

Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection.

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.

Synergistic lethal mutagenesis of hepatitis C virus.

Lethal mutagenesis is an antiviral approach that consists in 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 that renders the strategy potentially effective against emergent RNA viral infections. Here we describe 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 as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display 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. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

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.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy.

The 5' untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II-IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg(2+)-induced switch between two alternative conformations: from 'open', elongated morphologies at 0-2 mM Mg(2+) concentration to a 'closed', comma-shaped conformation at 4-6 mM Mg(2+). This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4-6 mM Mg(2+) in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I-II)+(V-VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.

Geneticin Stabilizes the Open Conformation of the 5' Region of Hepatitis C Virus RNA and Inhibits Viral Replication.

The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.

Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA.

The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200 nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself.

Health-related quality of life and nursing-sensitive outcomes in mechanically ventilated patients in an Intensive Care Unit: a study protocol.

BACKGROUND: Mechanical ventilation (MV) is one of the most utilised techniques in the intensive care unit (ICU), but it can cause sequelae that can negatively influence the patient's health-related quality of life (HRQL). Nursing-sensitive outcomes (NSOs) can also influence the HRQL. Assessing the HRQL of mechanically ventilated patients admitted to an ICU and its relation to nurse-sensitive outcomes will give healthcare professionals with valuable information to improve patient care. METHODS: Prospective longitudinal cohort study in which all patients admitted to the ICU at Hospital Universitari Vall d'Hebron who undergo MV for more than 48 h will be included. The study will last 12 consecutive months. HRQL will be assessed by the completion of the SF-36 and the Saint Georges Respiratory Questionnaire. Pre-admission HRQL assessment will be performed by the main caregiver, and after ICU discharge, the assessment will be performed by the patient him/herself. The same questionnaires will also be completed one year after ICU discharge. Other variables (sociodemographic and those related to reason for ICU admission, ICU length of stay, MV, ICU stressors and NSO) will be included in a multiple regression model to assess their relation to the patient's HRQL. DISCUSSION: This study will show the relationship between the HRQL perceived by patients and their main caregiver, what the HRQL is one year after discharge from ICU, and what the impact of MV, NSO and ICU stressors and other clinical outcomes on the patient's HRQL is. Determining mechanically ventilated patients' HRQL and its relation to NSO and ICU stressors as well as other clinical variables will enable early nursing interventions to try to minimise possible sequelae and improve the patient's welfare. TRIAL REGISTRATION: ClinicalTrials.gov ID:NCT02636660Registration Date: 17th December 2015.

High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods.

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

RNA self-cleavage activated by ultraviolet light-induced oxidation.

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C(79) and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U(78) and CSFV U(44) in others. Among other reaction products, the majority of biochemically active products detected contained 3'-phosphate and 5'-phosphate-end groups at the newly generated termini, along with a much lower amount of 3'-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (~33 nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures.

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.

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.

Clinician treatment choices for post-traumatic stress disorder: ambassadors survey of psychiatrists in 39 European countries.

BACKGROUND: Considering the recently growing number of potentially traumatic events in Europe, the European Psychiatric Association undertook a study to investigate clinicians' treatment choices for post-traumatic stress disorder (PTSD). METHODS: The case-based analysis included 611 participants, who correctly classified the vignette as a case of PTSD, from Central/ Eastern Europe (CEE) (n = 279), Southern Europe (SE) (n = 92), Northern Europe (NE) (n = 92), and Western Europe (WE) (N = 148). RESULTS: About 82% woulduse antidepressants (sertraline being the most preferred one). Benzodiazepines and antipsychotics were significantly more frequently recommended by participants from CEE (33 and 4%, respectively), compared to participants from NE (11 and 0%) and SE (9% and 3%). About 52% of clinicians recommended trauma-focused cognitive behavior therapy and 35% psychoeducation, irrespective of their origin. In the latent class analysis, we identified four distinct "profiles" of clinicians. In Class 1 (N = 367), psychiatrists would less often recommend any antidepressants. In Class 2 (N = 51), clinicians would recommend trazodone and prolonged exposure therapy. In Class 3 (N = 65), they propose mirtazapine and eye movement desensitization reprocessing therapy. In Class 4 (N = 128), clinicians propose different types of medications and cognitive processing therapy. About 50.1% of participants in each region stated they do not adhere to recognized treatment guidelines. CONCLUSIONS: Clinicians' decisions for PTSD are broadly similar among European psychiatrists, but regional differences suggest the need for more dialogue and education to harmonize practice across Europe and promote the use of guidelines.

Direct mapping of nanoscale compositional connectivity on intact cell membranes.

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains < 120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (< 150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

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.

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.