Emergence and selection of RNA virus variants: memory and extinction

Two features of viral quasispecies are reviewed: the presence of memory genomes as minority components of their mutant spectra, and viral extinction due to enhanced mutagenesis. Memory has been documented with several genetic markers of the important animal picornavirus foot-and-mouth disease virus (FMDV). The presence of memory genomes in viral quasispecies may accelerate their adaptive response whenever a selective constraint has already been experienced by a viral population during previous stages of its evolution. Enhanced mutagenesis has been shown to lead to losses of infectivity of a number of RNA viruses: poliovirus, vesicular stomatitis virus, human immunodeficiency virus type 1 and FMDV. These observations, based on the theoretical prediction of the existence of a copying error-threshold for maintenance of genetic information, may contribute to the development of a new antiviral strategy.     

Quasispecies dynamics and RNA virus extinction

The extinction of foot-and-mouth disease virus (FMDV) is strongly influenced by mutation rates, types of mutations, relative viral fitness and virus population regimens during infection. Here we review experimental results and theoretical models that describe a contrast between the effective extinction of FMDV subjected to increased mutagenesis, and the remarkable resistance to extinction of the same and related FMDV clones subjected to serial bottleneck events. The results suggest procedures to master key parameters to develop effective antiviral strategies based on virus entry into error catastrophe.     

Quasispecies and its impact on viral hepatitis

Quasispecies dynamics mediates adaptability of RNA viruses through a number of mechanisms reviewed in the present article, with emphasis on the medical implications for the hepatitis viruses. We discuss replicative and non-replicative molecular mechanisms of genome variation, modulating effects of mutant spectra, and several modes of viral evolution that can affect viral pathogenesis. Relevant evolutionary events include the generation of minority virus variants with altered functional properties, and alterations of mutant spectrum complexity that can affect disease progression or response to treatment. The widespread occurrence of resistance to antiviral drugs encourages new strategies to control hepatic viral disease such as combination therapies and lethal mutagenesis. In particular, ribavirin may be exerting in some cases its antiviral activity with participation of its mutagenic action. Despite many unanswered questions, here we document that quasispecies dynamics has provided an interpretation of the adaptability of the hepatitis viruses, with features conceptually similar to those observed with other RNA viruses, a reflection of the common underlying Darwinian principles.     

Minority report: hidden memory genomes in HIV-1 quasispecies and possible clinical implications

The RNA viruses replicate as complex distributions of closely related genomes termed viral quasispecies. The behavior of the evolving quasispecies and its response to selective pressures such as antiviral treatment is influenced by the ensemble of mutants that compose the viral population. One such influence is the presence of minority subpopulations in the mutant spectra of viral quasispecies. Biologically relevant mutants have long been known to be present as minority components of replicating viral populations. However, experiments designed with specific mutants of the animal pathogen foot-and-mouth disease virus in cell culture explained the presence of a class of minority genomes termed memory genomes. They descend from those variants that were dominant at an earlier phase of quasispecies evolution, and arise as a consequence of quasispecies dynamics, when viral populations are subjected to discontinuous selective pressures. The presence of memory genomes has also been documented during intrahost evolution of HIV-1 in vivo. The analysis of sequential viral samples of different HIV-1-infected patients showed that two distinct types of memory can operate in retroviruses: a replicative memory analogous to that observed in foot-and-mouth disease virus, as well as a reservoir memory derived from the integrative phase of the retroviral lifecycle. Despite being hidden as minority components of the HIV-1 viral population (ranging from about 0.1 to 20% of the total number of genomes in the quasispecies analyzed), memory genomes can drive the evolution of the virus during HIV-1 infections under antiviral therapy. The limited availability of current experimental data on minority HIV-1 subpopulations in vivo implies that further studies are required in order to define the cutoffs of clinically relevant minority genomes. Nevertheless, it is already evident that such low-abundance genomes remain undetectable by traditional genotyping methods such as consensus sequencing or conventional hybridization techniques. Several experimental systems are currently available for the detection and characterization of minority components of the mutant spectra of viral quasispecies including HIV, hepatitis C virus and hepatitis B virus. Some of these biotechnological approaches could, in the near future, be taken over and exploited in the clinical setting as useful biosensors with which to improve the management of HIV-infected patients.     

Characterization of minority subpopulations in the mutant spectrum of HIV-1 quasispecies by successive specific amplifications

RNA viruses do not replicate as defined genomic nucleotide sequences but rather as complex distributions of mutant genomes termed viral quasispecies. Quasispecies dynamics has a number of relevant biological consequences in ribo- and retroviruses, among these the possible presence of memory genomes as minority components of their mutant spectra. Minority memory genomes reflect those viral subpopulations that were dominant at an earlier phase of viral evolution, and can quickly re-emerge to react to certain selective pressures, as it was documented with HIV-1 in vivo. Therefore, an adequate clinical management of HIV-1 requires the development of experimental methods for the detection and quantification of minority viral subpopulations, even at levels of less than 1% of the total quasispecies. We describe a new approach based on successive, highly specific PCR amplifications, which allows the genetic characterization of minority genomes present in increasingly smaller proportion in viral populations. We have coined the term 'quasispecies diving' to reflect the progressive draw on minority or 'deeper' genomes in the mutant spectrum of the quasispecies. In the case of the multidrug-resistant HIV-1 strain analyzed here, quasispecies diving allowed the detection of mutant minority genomes at an unprecedented level of 0.0054% of the amplified viral population. This approach represents a general strategy for the genetic characterization of smaller minority genomes in complex molecular populations.     

RNA virus fitness

RNA viruses constitute the most abundant group of pathogens of man, animals and plants. They share high mutation rates which are in the range 10⁻³ to 10⁻⁵ misincorporations per nucleotide site and round of copying. This is due to the absence or low efficiency of proofreading-repair or postreplicative repair activities associated with replicating RNA. Populations of RNA viruses are extremely heterogeneous and form dynamic mutant swarms termed viral quasispecies. This genetic organisation implies that any individual mutant has only a fleeting existence; that is, RNA viral genomes are statistically defined but individually indeterminate. RNA viruses are able to accommodate their average nucleotide sequences to changes in environment. A parameter used to quantitate adaptation is fitness, or the relative ability of a virus to produce infectious progeny. Repeated transfers of one or a few particles (bottleneck events) generally lead to fitness losses. In contrast, large population passages allow competitive optimisation of mutant genomes and fitness gains. Of relevance to medical practice is the ability of viral quasispecies to overcome selective pressures imposed by vaccines and antiviral agents. Particularly dramatic have been the systematic isolations of HIV-1 mutants resistant to antiretroviral inhibitors in treated individuals. In addition to the ability of HIV-1 quasispecies to generate many mutant genomes in short times, calculations of mutation frequencies in the pol gene of HIV-1 populations have documented that mutations related to resistance to antiretroviral inhibitors preexist in the mutant swarms of HIV-1 quasispecies. It is not possible at present to anticipate whether a suitable drug cocktail may be capable of sustained inhibition of HIV-1 replication without selection of mutants resistant to the combination of antiviral agents. © 1997 John Wiley & Sons, Ltd.     

Quasispecies as a matter of fact: viruses and beyond

We review the origins of the quasispecies concept and its relevance for RNA virus evolution, viral pathogenesis and antiviral treatment strategies. We emphasize a critical point of quasispecies that refers to genome collectivities as the unit of selection, and establish parallels between RNA viruses and some cellular systems such as bacteria and tumor cells. We refer also to tantalizing new observations that suggest quasispecies behavior in prions, perhaps as a result of the same quantum-mechanical indeterminations that underlie protein conformation and error-prone replication in genetic systems. If substantiated, these observations with prions could lead to new research on the structure-function relationship of non-nucleic acid biological molecules.     

The error threshold

Basic principles underlying the population dynamics of bacteria and viruses are presented, with emphasis on RNA viruses. Concepts reviewed here include fitness, mutant generation, competition, selection, sequence space and the theoretical origins of quasispecies. A "wild-type" virus is no longer viewed as the fittest type, but as the center of gravity of a population landscape. A consequence of quasispecies is the existence of an error threshold for selective competence. The error threshold has a justification in quasispecies theory and lends itself to exploration of strategies to drive viral populations over the error threshold, the central theme of this volume.     

Genomic nucleotide sequence of a foot-and-mouth disease virus clone and its persistent derivatives. Implications for the evolution of viral quasispecies during a persistent infection.

The consensus nucleotide sequence of the entire genome of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) has been completed, and compared with that of two persistent derivatives R99 and R146, rescued after 99 and 146 passages of the carrier BHK-21 cells. Consensus sequences were determined directly from supernatants of persistently infected cells, without intervening cytolytic amplification of the viruses. These genomic sequences have also been compared with that of FMDV R100, a virus that was also rescued from persistently infected cells, but that was subjected to cytolytic amplification prior to sequencing. Mutation frequencies for R99 and R146 relative to C-S8c1 were in the range of 2.8x10(-3) to 7.7x10(-3) substitutions per nucleotide for the 5'-UTR and the L-, P1-, P2- and P3-coding regions. No mutations were fixed in the polymerase (3D)-coding region. Striking contrasts were noted regarding the distribution of mutation types along the persistent genomes, notably the complete absence of transversion mutations within the 5'-UTR, compared with 53% transversions in the L- and P1-coding regions. The sequencing results presented here, combined with previous sequences of FMDV C-S8c1 genomes at the onset of persistence, provide evidence of sequence fluctuations with a non-linear accumulation of mutations during prolonged persistence, a hallmark of quasispecies dynamics.     

Modification of foot-and-mouth disease virus after serial passages in the presence of antiviral polyclonal sera

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, this virus has a high rate of mutation. It has been proposed that selection exerted by the host's antibodies could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant populations (Nr), after serial passages of cloned FMDV A24 Cruzeiro strain on secondary monolayers of bovine fetal kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages under selective pressure, the virus population showed the following characteristics: (1) increased resistance to neutralization by APS; (2) altered electrophoretic mobility of structural viral proteins (VP1); (3) remarkable plaque size reduction, (4) a pronounced thermosensitivity (ts); and (5) decreased pathogenicity for mice, in both uncloned and cloned small plaque size populations. This indicates that FMDV populations under antibody pressure in vitro, have acquired, in addition to expected characteristics of natural FMDV variants (resistance to neutralization and altered viral structural proteins), phenotypic markers which correspond to attenuated, less virulent variants.     

Foot-and-mouth disease virus low-fidelity polymerase mutants are attenuated

Previous studies have shown that RNA viruses can be attenuated by either increased or decreased viral polymerase replication fidelity. Although foot-and-mouth disease virus (FMDV) high-fidelity RNA-dependent RNA polymerase (RdRp) variants with an attenuated phenotype have been isolated using mutagens, no FMDV mutant with a low-fidelity polymerase has been documented to date. Here, we describe the generation of several FMDV RdRp mutants using site-directed mutagenesis via a reverse genetic system. Mutation frequency assays confirmed that five rescued FMDV RdRp mutant populations had lower replication fidelity than the wild-type virus population, which allowed us to assess the effects of the change in replication fidelity on the virus phenotype. These low-fidelity FMDV RdRp mutants showed increased sensitivity to ribavirin or 5-fluorouracil (5-FU) treatment without a loss of growth capacity in cell cultures. In addition, decreased fitness and attenuated virulence were observed for the RdRp mutants with lower fidelity. Importantly, based on a quantitative analysis for fidelity and virulence, we concluded that lower replication fidelity is associated with a more attenuated virus phenotype. These results further contribute to our understanding of the replication fidelity of polymerases of RNA viruses and its relationship to virulence attenuation.     

Action of mutagenic agents and antiviral inhibitors on foot-and-mouth disease virus

Our current knowledge on foot-and-mouth disease virus (FMDV) entry into error catastrophe is reviewed. FMDV can establish cytolytic and persistent infections in the field and in cell culture. Both types of FMDV infection in cell culture can be treated with mutagens, with or without classical (non-mutagenic) antiviral inhibitors, to drive the virus to extinction. 5-Fluorouracil (FU) and 5-azacytidine (AZC) have been employed as mutagenic agents to treat cytolytic FMDV infections, and ribavirin (Rib) to treat persistent infections. Extinction is dependent on the relative fitness of the viral isolate, as well as on the viral load. In cytolytic infections, extinctions could be efficiently obtained with combinations of mutagens and inhibitors. High-fitness FMDV extinction could only be achieved with treatments that contained a mutagen, and not with combinations of inhibitors that exerted the same antiviral effect. Persistent infections could be cured with Rib treatment alone. The results presented here show entry into error catastrophe as a valid strategy for treatment of viral infections, although much work remains to be done before it can be implemented.     

Foot-and-mouth disease virus lacking the VP1 G-H loop: the mutant spectrum uncovers interactions among antigenic sites for fitness gain

The Arg-Gly-Asp (RGD) triplet found in the G-H loop of capsid protein VP1 of foot-and-mouth disease virus (FMDV) is critically involved in the interaction of FMDV with integrin receptors and with neutralizing antibodies. Multiplication of FMDV C-S8c1 in baby hamster kidney 21 (BHK-21) cells selected variant viruses exploiting alternative mechanisms of cell recognition that rendered the RGD integrin-binding triplet dispensable for infectivity. By constructing chimeric viruses, we show that dispensability of the RGD in these variant FMDVs can be extended to surrounding amino acid residues. Replacement of eight amino acid residues within the G-H loop of VP1 by an unrelated FLAG marker yielded infectious virus. Evolution of FLAG-containing viruses in BHK-21 cells generated complex quasispecies in which individual mutants included amino acid replacements at other antigenic sites of FMDV. Inclusion of such replacements in the parental FLAG clone resulted in an increase of relative fitness of the viruses. These results suggest structural or functional connections between antigenic sites of FMDV and underscore the value of mutant spectrum analysis for the identification of fitness-promoting genetic modifications in viral populations. The possibility of producing viable viruses lacking antigenic site A may find application in the design of new anti-FMD vaccines.     

Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications

Positive strand RNA virus populations are a collection of similar but genetically different viruses. They exist as viral quasispecies due to the high mutation rates of the low fidelity viral RNA-dependent RNA polymerase (RdRp). It is thought that this genomic heterogeneity is advantageous to the population, allowing for adaptation to rapidly changing environments that present varying types and degrees of selective pressure. However, one consequence of this extensive diversity is the susceptibility to mutagens that further increase sequence variation. Since RNA viruses live at the edge of maximal variability, an increase in the mutation rate is likely to force the virus beyond the tolerable mutation frequency into 'error catastrophe'. One such mutagen, ribavirin, is an antiviral nucleoside analog that is mutagenic to several RNA viruses. Ribavirin is incorporated into the viral genome causing lethal mutagenesis and a subsequent decrease in the specific infectivity. Even so, passaging poliovirus in the presence of low to intermediate concentrations of the drug leads to the emergence of a viral population resistant to the effects of ribavirin. These viruses have a point mutation in the RdRp that increases the overall polymerase fidelity. Interestingly, as predicted by the quasispecies theory, ribavirin resistant viruses are less adaptable, as they are more susceptible to other non-mutagenic antiviral drugs and are highly attenuated in vivo. Here, we review the mechanism of action of ribavirin on poliovirus and other RNA viruses, the possibility for escape via increased fidelity of the viral polymerase, the consequences of this response on viral population dynamics, and the biological implications for the therapeutic use of mutagenic antiviral agents.     

HCV准种多样性及其与临床关系的研究

目的     

Mutation pressure shapes codon usage in the GC-Rich genome of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. In this study, we report the results of a survey of codon usage bias of FMD virus (FMDV) representing all seven serotypes (A, O, C, Asia 1, SAT 1, SAT 2, and SAT 3). Correspondence analysis, a commonly used multivariate statistical approach, was carried out to analyze synonymous codon usage bias. The analysis showed that the overall extent of codon usage bias in FMDV is low. Furthermore, the good correlation between the frequency of G + C at the synonymous third position of sense codons (GC_3S) content at silent sites of each sequence and codon usage bias suggested that mutation pressure rather than natural (translational) selection is the most important determinant of the codon bias observed. In addition, other factors, such as the lengths of open reading frame (ORF) and the hydrophobicity of genes also influence the codon usage variation among the genomes of FMDV in a minor way. The result of phylogenetic analyses based on the relative synonymous codon usage (RSCU) values indicated a few obvious phylogenetic incongruities, which suggest that more FMDV genome diversity may exist in nature than is currently indicated. Our work might give some clues to the features of FMDV genome and some evolutionary information of this virus.     

Short hairpin RNA targeted to the highly conserved 2B nonstructural protein coding region inhibits replication of multiple serotypes of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is one of the most contagious agents of animals. Recent disease outbreaks in FMD-free countries have prompted the development of new control strategies that could improve the levels of protection against this virus. We have delivered a plasmid expressing a short hairpin RNA (shRNA) directed against a highly conserved sequence in the 2B nonstructural protein coding region of FMDV RNA to porcine cells. After virus infection, these cells showed a significant reduction in the synthesis of viral RNA and proteins, as well as a decrease in virus yield when compared to control cells. The antiviral effect was sequence specific and not attributable to induction of the interferon pathway. Since FMDV is an antigenically variable virus, we examined the effectiveness of this strategy against multiple serotypes and found that expressed 2B shRNA resulted in efficient silencing of at least 4 FMDV serotypes. Thus, RNA interference may be a potential alternative control strategy to limit the spread of this highly contagious virus in livestock.     

Hepatitis C virus genetic variability in patients undergoing antiviral therapy

Hepatitis C virus (HCV) has been the subject of intense research and clinical investigations due to its worldwide prevalence and major role in chronic liver disease. Like most RNA viruses, HCV circulates in vivo as a complex population of different but closely related viral variants, commonly referred to as a quasispecies. Recent studies suggest that ribavirin might exert an antiviral effect against HCV through both mutagenic effect and an impairment of RNA replication. The introduction of alpha interferon (IFN-alpha) plus ribavirin combination therapy was an important breakthrough in the treatment of chronic HCV infection. However, the rate of sustained virological response is still unsatisfactory, particularly in patients infected with HCV genotype 1. Viral persistence, a hallmark of HCV, may result from a dynamic control of the host response by the virus. In children with chronic HCV infection, the viral population is initially highly homogeneous, but diversifies during prolonged infection which seems to be a common event during chronic hepatitis C in childhood. Coinfection of human immunodeficiency virus 1 (HIV-1) patients by HCV can complicate the treatment of these patients with highly active antiretroviral therapy (HAART). HIV coinfection is associated with a decrease of HCV quasispecies variability, which appears to be reversed by effective HAART.