Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

Mutation analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: evidence for hotspots of genetic change.

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.     

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.     

Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.

Sequencing of multiple recombinant clones generated from polymerase chain reaction-amplified products demonstrated that the degree of heterogeneity of two well-conserved regions of the hepatitis C virus (HCV) genome within individual plasma samples from a single patient was consistent with a quasispecies structure of HCV genomic RNA. About half of circulating RNA molecules were identical, while the remaining consisted of a spectrum of mutants differing from each other in one to four nucleotides. Mutant sequence diversity ranged from silent mutations to appearance of in-frame stop codons and included both conservative and nonconservative amino acid substitutions. From the relative proportion of essentially defective sequences, we estimated that most circulating particles should contain defective genomes. These observations might have important implications in the physiopathology of HCV infection and underline the need for a population-based approach when one is analyzing HCV genomes.     

Monitoring sequence space as a test for the target of selection in viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.     

Synchronous loss of quasispecies memory in parallel viral lineages: a deterministic feature of viral quasispecies

Viral quasispecies are endowed with a memory of their past evolutionary history in the form of minority genomes of their mutant spectra. To determine the fate of memory genomes in evolving viral quasispecies, we have measured memory levels of antigenic variant of foot-and-mouth disease virus (FMDV) RED, which includes an Arg-Glu-Asp (RED) at a surface antigenic loop of the viral capsid. The RED reverted to the standard Arg-Gly-Asp (RGD), and the RED remained as memory in the evolving quasispecies. In four parallel evolutionary lineages, memory reduction followed a strikingly similar pattern, and at passage 60 memory levels were indistinguishable from those of control populations (devoid of memory). Nucleotide sequence analyses indicated that memory loss occurred synchronously despite its ultimate molecular basis being the stochastic occurrence of mutations in the evolving quasispecies. These results on the kinetics of memory levels have unveiled a deterministic feature of viral quasispecies. Molecular mechanisms that may underlie synchronous memory loss are the averaging of noise signals derived from mutational input, and constraints to genome diversification imposed by a nucleotide sequence context in the viral genome. Possible implications of the behaviour of complex, adaptive viral systems as experimental models to address primary mechanisms of neurological memory are discussed.     

Duration and fitness dependence of quasispecies memory.

The duration and fitness dependence of memory in viral quasispecies evolving in cell culture have been investigated using two genetic markers of foot-and-mouth disease virus (FMDV). In lineages of antigenic variant FMDV RED, which reverted to FMDV RGD, memory FMDV RED genomes were detected after 50 infectious cycles, and memory level was fitness dependent. In growth-competition experiments between a reference FMDV RGD and two different FMDV RED populations, a 7.6-fold higher fitness of the initial FMDV RED population resulted in 30 to 100-fold higher memory level. In lineages of low-fitness clones containing an elongated internal polyadenylate tract, revertants lacking excess adenylate residues became dominant by passage 20. However, genomes including a larger number of adenylate residues were detected as memory genomes after at least 150 infectious cycles. Thus, quasispecies memory can be durable and is fitness dependent, as predicted from the growth competition of two mutant forms of a genome. An understanding of factors influencing quasispecies memory levels and duration may have implications for the extended diagnosis of viruses based on the quantification of minority genomes.     

Genome Instability in Picornaviruses

Picornaviruses are small animal RNA viruses and include etiological agents of poliomyelitis, foot and mouth disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.     

Genetic structure and population variability of tomato yellow leaf curl China virus

Geminiviruses have circular single-stranded DNA genomes and are important pathogens in tropical and subtropical regions, but their population diversity and variability are poorly understood. Here, we have investigated variations accumulating in Tomato yellow leaf curl China virus (TYLCCNV), a geminivirus in the genus Begomovirus of the family Geminiviridae. The population variation was analyzed in a naturally infected tomato (Solanum lycopersicom) plant and in Nicotiana benthamiana and tomato plants experimentally infected with a swarm of TYLCCNV DNA clones to provide an identical sequence for initiation of infection. Our results demonstrate that the population of TYLCCNV in a naturally infected tomato plant was genetically heterogeneous and that rapid mutation occurred in the populations amplified from N. benthamiana and tomato plants that had been infected with cloned DNA. This feature of the population of TYLCCNV in these plants consisted of the consensus sequence and a pool of mutants that are not identical but are closely related to the consensus sequence, and it coincides with the quasispecies concept described for many RNA viruses. The mutation frequency was circa 10(-4) in N. benthamiana and tomato at 60 days postinoculation, a value comparable to that reported for plant RNA viruses. The quasispecies-like nature of the TYLCCNV populations suggested that TYLCCNV is capable of rapid evolution and adaptation in response to changing agricultural practices.     

Genomic instability in picornaviruses

Picornaviruses are small animal RNA viruses and include wtiological agents of poliomyelitis, foot and mouse disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.     

Mutagenesis-induced, large fitness variations with an invariant arenavirus consensus genomic nucleotide sequence

Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.     

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Background

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Results

We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.

Conclusions

Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.     

Bioinformatics prediction of siRNAs as potential antiviral agents against dengue viruses

Dengue virus (DENV 1-4) represents the major emerging arthropod-borne viral infection in the world. Currently, there is neither an available vaccine nor a specific treatment. Hence, there is a need of antiviral drugs for these viral infections; we describe the prediction of short interfering RNA (siRNA) as potential therapeutic agents against the four DENV serotypes. Our strategy was to carry out a series of multiple alignments using ClustalX program to find conserved sequences among the four DENV serotype genomes to obtain a consensus sequence for siRNAs design. A highly conserved sequence among the four DENV serotypes, located in the encoding sequence for NS4B and NS5 proteins was found. A total of 2,893 complete DENV genomes were downloaded from the NCBI, and after a depuration procedure to identify identical sequences, 220 complete DENV genomes were left. They were edited to select the NS4B and NS5 sequences, which were aligned to obtain a consensus sequence. Three different servers were used for siRNA design, and the resulting siRNAs were aligned to identify the most prevalent sequences. Three siRNAs were chosen, one targeted the genome region that codifies for NS4B protein and the other two; the region for NS5 protein. Predicted secondary structure for DENV genomes was used to demonstrate that the siRNAs were able to target the viral genome forming double stranded structures, necessary to activate the RNA silencing machinery.     

Frequent frameshift and point mutations in the SH gene of human metapneumovirus passaged in vitro

During the preparation of recombinant derivatives of the CAN97-83 clinical isolate of human metapneumovirus (HMPV), consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This heterogeneity was suggestive of sizable subpopulations containing mutations. An analysis of molecularly cloned cDNAs confirmed the presence of mixed populations. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequencing. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild-type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There were also two instances of nucleotide deletions and numerous instances of nucleotide substitution point mutations, mostly in the SH gene. The occurrence of mutant subpopulations was greatly reduced by the replacement of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. We suggest that we frequently detected subpopulations in which the expression of full-length SH protein was ablated because it provided a modest selective advantage to this clinical isolate in vitro. Adaptation involving the functional loss of a gene is unusual for an RNA virus.     

Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission

One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.     

Evaluation of intra-host variants of the entire hepatitis B virus genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.     

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.     

Contingent Neutrality in Competing Viral Populations

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.