Quasispecies Theory and the Behavior of RNA Viruses

A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy.     

Exploring the fitness landscape of an RNA virus by using a universal barcode microarray

Studies of viral pathogenesis have relied heavily on analyses of specific clones and their genetic determinants of virulence. It is sometimes difficult to apply this reductionist approach to the study of RNA viruses, which by virtue of their very high mutation rates, exist as a complex mixture of mutants. While quasispecies theory has provided an intellectual framework for exploring the relationship between the viral population structure and phenotype, experimental studies have been limited by the relatively poor resolution of traditional sequencing-based approaches. We have addressed this problem by developing a molecular barcoding strategy in which viral subpopulations are tagged with unique 20-nucleotide sequences. The behavior of these subpopulations can be monitored using a universal barcode microarray. We demonstrate the performance of our barcode microarray platform using poliovirus, a model RNA virus. Using this platform, we explored the fitness landscape occupied by an artificial quasispecies consisting of 48 randomly mutagenized clones. We were able to rapidly derive precise fitness measurements for a majority of these clones and identified a neutral space surrounding the wild type. The experimental paradigm presented here is readily adaptable to other viral systems and can potentially be used to track thousands of variants in a cost-effective manner.     

Unfinished Stories on Viral Quasispecies and Darwinian Views of Evolution

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis.     

Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake

RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro‐organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low‐latitude aquatic environments, this lake harbours an RNA virome dominated by positive single‐strand RNA viruses from the order Picornavirales probably infecting micro‐organisms. Antarctic picorna‐like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low‐complexity indexes. By contrast, APLV2‐APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run‐off‐mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity.     

Quasispecies made simple

Quasispecies are clouds of genotypes that appear in a population at mutation–selection balance. This concept has recently attracted the attention of virologists, because many RNA viruses appear to generate high levels of genetic variation that may enhance the evolution of drug resistance and immune escape. The literature on these important evolutionary processes is, however, quite challenging. Here we use simple models to link mutation–selection balance theory to the most novel property of quasispecies: the error threshold—a mutation rate below which populations equilibrate in a traditional mutation–selection balance and above which the population experiences an error catastrophe, that is, the loss of the favored genotype through frequent deleterious mutations. These models show that a single fitness landscape may contain multiple, hierarchically organized error thresholds and that an error threshold is affected by the extent of back mutation and redundancy in the genotype-to-phenotype map. Importantly, an error threshold is distinct from an extinction threshold, which is the complete loss of the population through lethal mutations. Based on this framework, we argue that the lethal mutagenesis of a viral infection by mutation-inducing drugs is not a true error catastophe, but is an extinction catastrophe.     

The Evolution of Virulence in RNA Viruses under a Competition–Colonization Trade-Off

RNA viruses exist in large intra-host populations which display great genotypic and phenotypic diversity. We analyze a model of viral competition between two viruses infecting a constantly replenished cell pool. We assume a trade-off between the ability of the virus to colonize new cells (cell killing rate or virulence) and its local competitiveness (replicative success within coinfected cells). We characterize the conditions that allow for viral spread by means of the basic reproductive number and show that a local coexistence equilibrium exists, which is asymptotically stable. At this equilibrium, the less virulent competitor has a reproductive advantage over the more virulent colonizer reflected by a larger equilibrium population size of the competitor. The equilibria at which one virus outcompetes the other one are unstable, i.e., a second virus is always able to permanently invade. We generalize the two-virus model to multiple viral strains, each displaying a different virulence. To account for the large phenotypic diversity in viral populations, we consider a continuous spectrum of virulences and present a continuum limit of this multiple viral strains model that describes the time evolution of an initial continuous distribution of virulence without mutations. We provide a proof of the existence of solutions of the model equations, analytically assess the properties of stationary solutions, and present numerical approximations of solutions for different initial distributions. Our simulations suggest that initial continuous distributions of virulence evolve toward a distribution that is extremely skewed in favor of competitors. At equilibrium, only the least virulent part of the population survives. The discrepancy of this finding in the continuum limit with the two-virus model is attributed to the skewed equilibrium subpopulation sizes and to the transition to a continuum. Consequently, in viral quasispecies with high virulence diversity, the model predicts collective virulence attenuation. This result may contribute to understanding virulence attenuation, which has been reported in several experimental studies.     

Mechanisms controlling virulence thresholds of mixed viral populations

The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold.     

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.     

Information catastrophe in RNA viruses through replication thresholds

RNA viruses are known to replicate at very high mutation rates. These rates are actually known to be close to their so-called error threshold. This threshold is in fact a critical point beyond which genetic information is lost through a so-called error catastrophe. However, the transition from a stable quasispecies to genetic drift and loss of information can also occur by crossing replication thresholds, below some replication rates, the viral population is suddenly unable to survive. Available data from hepatitis C virus population analysis [Mas, A., Ulloa, E., Bruguera, M., Furci?, I., Garriga, D., Fábregas, S., Andreu, D., Saiz, J.C., Díez, J., 2004. Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity. J. Gen. Virol. 85, 3619-3626] can be interpreted through this theoretical view, providing evidence for such a replication threshold. Here a simple model is used in order to provide evidence for such a phenomenon, consistent with available data.     

Investigation of bacteriophage MS2 viral dynamics using model discrimination analysis and the implications for phage therapy

Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population.     

Influence of the mutant spectrum in viral evolution: focused selection of antigenic variants in a reconstructed viral quasispecies

RNA viruses replicate as complex mutant distributions termed viral quasispecies. Despite this, studies on virus populations subjected to positive selection have generally been performed and analyzed as if the viral population consisted of a defined genomic nucleotide sequence; such a simplification may not reflect accurately the molecular events underlying the selection process. In the present study, we have reconstructed a foot-and-mouth disease virus quasispecies with multiple, low-frequency, genetically distinguishable mutants that can escape neutralization by a monoclonal antibody. Some of the mutants included an amino acid substitution that affected an integrin recognition motif that overlaps with the antibody-binding site, whereas other mutants included an amino acid substitution that affected antibody binding but not integrin recognition. We have monitored consensus and clonal nucleotide sequences of populations passaged either in the absence or the presence of the neutralizing antibody. In both cases, the populations focused toward a specific mutant that was surrounded by a cloud of mutants with different antigenic and cell recognition specificities. In the absence of antibody selection, an antigenic variant that maintained integrin recognition became dominant, but the mutant cloud included as one of its minority components a variant with altered integrin recognition. Conversely, in the presence of antibody selection, a variant with altered integrin recognition motif became dominant, but it was surrounded by a cloud of antigenic variants that maintained integrin recognition. The results have documented that a mutant spectrum can exert an influence on a viral population subjected to a sustained positive selection pressure and have unveiled a mechanism of antigenic flexibility in viral populations, consisting in the presence in the selected quasispecies of mutants with different antigenic and cell recognition specificities.     

Differences in accumulation and virulence determine the outcome of competition during Tobacco etch virus coinfection

Understanding the evolution of virulence for RNA viruses is essential for developing appropriate control strategies. Although it has been usually assumed that virulence is a consequence of within-host replication of the parasite, viral strains may be highly virulent without experiencing large accumulation as a consequence of immunopathological host responses. Using two strains of Tobacco etch potyvirus (TEV) that show a negative relationship between virulence and accumulation rate, we first explored the evolution of virulence and fitness traits during simple and mixed infections. Short-term evolution experiments initiated with each strain independently confirmed the genetic and evolutionary stability of virulence and viral load, although infectivity significantly increased for both strains. Second, competition experiments between hypo- and hypervirulent TEV strains have shown that the outcome of competition is driven by differences in replication rate. A simple mathematical model has been developed to analyze the dynamics of these two strains during coinfection. The model qualitatively reproduced the experimental results using biologically meaningful parameters. Further analyses of the model also revealed a wide parametric region in which a low-fitness but hypovirulent virus can still outcompete a high-fitness but hypervirulent one. These results provide additional support to the observation that virulence and within-host replication may not necessarily be strongly tied in plant RNA viruses.     

Genetic Diversity in RNA Virus Quasispecies Is Controlled by Host-Virus Interactions

Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work using Tobacco mosaic virus and Cucumber mosaic virus indicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.     

Quasispecies spatial models for RNA viruses with different replication modes and infection strategies

Empirical observations and theoretical studies suggest that viruses may use different replication strategies to amplify their genomes, which impact the dynamics of mutation accumulation in viral populations and therefore, their fitness and virulence. Similarly, during natural infections, viruses replicate and infect cells that are rarely in suspension but spatially organized. Surprisingly, most quasispecies models of virus replication have ignored these two phenomena. In order to study these two viral characteristics, we have developed stochastic cellular automata models that simulate two different modes of replication (geometric vs stamping machine) for quasispecies replicating and spreading on a two-dimensional space. Furthermore, we explored these two replication models considering epistatic fitness landscapes (antagonistic vs synergistic) and different scenarios for cell-to-cell spread, one with free superinfection and another with superinfection inhibition. We found that the master sequences for populations replicating geometrically and with antagonistic fitness effects vanished at low critical mutation rates. By contrast, the highest critical mutation rate was observed for populations replicating geometrically but with a synergistic fitness landscape. Our simulations also showed that for stamping machine replication and antagonistic epistasis, a combination that appears to be common among plant viruses, populations further increased their robustness by inhibiting superinfection. We have also shown that the mode of replication strongly influenced the linkage between viral loci, which rapidly reached linkage equilibrium at increasing mutations for geometric replication. We also found that the strategy that minimized the time required to spread over the whole space was the stamping machine with antagonistic epistasis among mutations. Finally, our simulations revealed that the multiplicity of infection fluctuated but generically increased along time.     

Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNA_pol) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNA_pol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNA_pol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.     

Functional inaccessibility of quiescent herpes simplex virus genomes

Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression.     

QSdpR: Viral quasispecies reconstruction via correlation clustering

RNA viruses are characterized by high mutation rates that give rise to populations of closely related genomes, known as viral quasispecies. Underlying heterogeneity enables the quasispecies to adapt to changing conditions and proliferate over the course of an infection. Determining genetic diversity of a virus (i.e., inferring haplotypes and their proportions in the population) is essential for understanding its mutation patterns, and for effective drug developments. Here, we present QSdpR, a method and software for the reconstruction of quasispecies from short sequencing reads. The reconstruction is achieved by solving a correlation clustering problem on a read-similarity graph and the results of the clustering are used to estimate frequencies of sub-species; the number of sub-species is determined using pseudo F index. Extensive tests on both synthetic datasets and experimental HIV-1 and Zika virus data demonstrate that QSdpR compares favorably to existing methods in terms of various performance metrics.     

Human immunodeficiency virus type 1 genetic evolution in children with different rates of development of disease.

The rate of development of disease varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected children. The reasons for these observed differences are not clearly understood but most probably depend on the dynamic interplay between the HIV-1 quasispecies virus population and the immune constraints imposed by the host. To study the relationship between disease progression and genetic diversity, we analyzed the evolution of viral sequences within six perinatally infected children by examining proviral sequences spanning the C2 through V5 regions of the viral envelope gene by PCR of blood samples obtained at sequential visits. PCR product DNAs from four sample time points per child were cloned, and 10 to 13 clones from each sample were sequenced. Greater genetic distances relative to the time of infection were found for children with low virion-associated RNA burdens and slow progression to disease relative to those found for children with high virion-associated RNA burdens and rapid progression to disease. The greater branch lengths observed in the phylogenetic reconstructions correlated with a higher accumulation rate of nonsynonymous base substitutions per potential nonsynonymous site, consistent with positive selection for change rather than a difference in replication kinetics. Viral sequences from children with slow progression to disease also showed a tendency to form clusters that associated with different sampling times. These progressive shifts in the viral population were not found in viral sequences from children with rapid progression to disease. Therefore, despite the HIV-1 quasispecies being a diverse, rapidly evolving, and competing population of genetic variants, different rates of genetic evolution could be found under different selective constraints. These data suggest that the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations are compatible with a Darwinian system evolving under the constraints of natural selection.     

Insights into RNA virus mutant spectrum and lethal mutagenesis events: replicative interference and complementation by multiple point mutants

RNA virus behavior can be influenced by interactions among viral genomes and their expression products within the mutant spectra of replicating viral quasispecies. Here, we report the extent of interference of specific capsid and polymerase mutants of foot-and-mouth disease virus (FMDV) on replication of wild-type (wt) RNA. The capsid and polymerase mutants chosen for this analysis had been characterized biochemically and structurally. Upon co-electroporation of BHK-21 cells with wt RNA and a tenfold excess of mutant RNA, some mutants displayed strong interference (<10% of progeny production by wt RNA alone), while other mutants did not show detectable interference. The capacity to interfere required an excess of mutant RNA and was associated with intracellular replication, irrespective of the formation of infectious particles by the mutant virus. The extent of interference did not correlate with the known types and number of interactions involving the amino acid residue affected in each mutant. Synergistic interference was observed upon co-electroporation of wt RNA and mixtures of capsid and polymerase mutants. Interference was specific, in that the mutants did not affect expression of encephalomyocarditis virus RNA, and that a two nucleotide insertion mutant of FMDV expressing a truncated polymerase did not exert any detectable interference. The results support the lethal defection model for viral extinction by enhanced mutagenesis, and provide further evidence that the population behavior of highly variable viruses can be influenced strongly by the composition of the quasispecies mutant spectrum as a whole.     

RNA virus evolution, population dynamics, and nutritional status

Trace elements exert a strong influence on immune function. Debilitated humoral and cellular immune responses may impair virus clearance in infected organisms, and favor the generation of virus variants with altered biological properties. The population size in evolving viral quasispecies, as well as increased mutagenesis trigered by oxidative stress, may contribute to altering the outcome of quasispecies evolution in infected hosts. The genetic plasticity of RNA viruses is one of the main obstacles for the control of the diseases they cause and probably a major force in the emergence of new viral pathogens. Recent results suggest links between nutritional deficiencies and the generation of variant viruses, a possibility that is addressed in the present article.