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What takes decades, centuries or millennia to happen with a natural ecosystem, it takes only days, weeks or months with a replicating viral quasispecies in a host, especially when under treatment. Some methods to quantify the evolution of a quasispecies are introduced and discussed, along with simple simulated examples to help in the interpretation and understanding of the results. The proposed methods treat the molecules in a quasispecies as individuals of competing species in an ecosystem, where the haplotypes are the competing species, and the ecosystem is the quasispecies in a host, and the evolution of the system is quantified by monitoring changes in haplotype frequencies. The correlation between the proposed indices is also discussed, and the R code used to generate the simulations, the data and the plots is provided. The virtues of the proposed indices are finally shown on a clinical case.

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.

RNA viruses are known to replicate by low fidelity polymerases and have high mutation rates whereby the resulting virus population tends to exist as a distribution of mutants. In this review, we aim to explore how genetic events such as spontaneous mutations could alter the genomic organization of RNA viruses in such a way that they impact virus replications and plaque morphology. The phenomenon of quasispecies within a viral population is also discussed to reflect virulence and its implications for RNA viruses. An understanding of how such events occur will provide further evidence about whether there are molecular determinants for plaque morphology of RNA viruses or whether different plaque phenotypes arise due to the presence of quasispecies within a population. Ultimately this review gives an insight into whether the intrinsically high error rates due to the low fidelity of RNA polymerases is responsible for the variation in plaque morphology and diversity in virulence. This can be a useful tool in characterizing mechanisms that facilitate virus adaptation and evolution.

Background Viruses can inhabit their hosts in the form of an ensemble of various mutant strains. Reconstructing a robust consensus representation for these diverse mutant strains is essential for recognizing the genetic variations among strains and delving into aspects like virulence, pathogenesis, and selecting therapies. Virus genomes are typically small, often composed of only a few thousand to several hundred thousand nucleotides. While constructing a high-quality consensus of virus strains might seem feasible, most current assemblers only generated fragmented contigs. It’s important to emphasize the significance of assembling a single full-length consensus contig, as it’s vital for identifying genetic diversity and estimating strain abundance accurately. Results In this paper, we developed FC-Virus, a de novo genome assembly strategy specifically targeting highly diverse viral populations. FC-Virus first identifies the k -mers that are common across most viral strains, and then uses these k -mers as a backbone to build a full-length consensus sequence covering the entire genome. We benchmark FC-Virus against state-of-the-art genome assemblers. Conclusion Experimental results confirm that FC-Virus can construct a single, accurate full-length consensus, whereas other assemblers only manage to produce fragmented contigs. FC-Virus is freely available at https://github.com/qdu-bioinfo/FC-Virus.git .

Influenza viruses exhibit high mutation rates and extensive genetic diversity, which hinder effective vaccine development and facilitate immune evasion (Taubenberger and Morens, 2006; Barr et al., 2010). These mutations arise from the error-prone viral RNA-dependent RNA polymerase, generating highly heterogeneous viral populations within individual hosts that conform to the quasi-species model of a cloud of related genomes evolving under selection (Domingo et al., 2012). Accurate characterization of this intra-host diversity is crucial for understanding viral evolution and improving vaccine design, yet conventional RNA sequencing often fails to detect low-frequency variants because of technical errors during sample preparation and sequencing. Here, we implement a single unique molecular identifier strategy that reduces sequencing artifacts and achieves an error rate of ~10⁻⁵, enabling single-particle–level quantification of quasi-species diversity. Mutation frequencies greatly exceeding background error confirm their biological origin, while information-theoretic metrics such as Shannon entropy and Jensen–Shannon divergence reveal non-random mutation distributions under selective constraints. This framework supports detailed studies of intra-host viral evolution and may inform artificial intelligence-driven prediction of mutational trajectories and more effective influenza vaccine strategies.

Measles virus (MeV) is dual-tropic: it replicates first in lymphatic tissues and then in epithelial cells. This switch in tropism raises the question of whether, and how, intra-host evolution occurs. Towards addressing this question, we adapted MeV either to lymphocytic (Granta-519) or epithelial (H358) cells. We also passaged it consecutively in both human cell lines. Since passaged MeV had different replication kinetics, we sought to investigate the underlying genetic mechanisms of growth differences by performing deep-sequencing analyses. Lymphocytic adaptation reproducibly resulted in accumulation of variants mapping within an 11-nucleotide sequence located in the middle of the phosphoprotein (P) gene. This sequence mediates polymerase slippage and addition of a pseudo-templated guanosine to the P mRNA. This form of co-transcriptional RNA editing results in expression of an interferon antagonist, named V, in place of a polymerase co-factor, named P. We show that lymphocytic-adapted MeV indeed produce minimal amounts of edited transcripts and V protein. In contrast, parental and epithelial-adapted MeV produce similar levels of edited and non-edited transcripts, and of V and P proteins. Raji, another lymphocytic cell line, also positively selects V-deficient MeV genomes. On the other hand, in epithelial cells V-competent MeV genomes rapidly out-compete the V-deficient variants. To characterize the mechanisms of genome re-equilibration we rescued four recombinant MeV carrying individual editing site-proximal mutations. Three mutations interfered with RNA editing, resulting in almost exclusive P protein expression. The fourth preserved RNA editing and a standard P-to-V protein expression ratio. However, it altered a histidine involved in Zn2+ binding, inactivating V function. Thus, the lymphocytic environment favors replication of V-deficient MeV, while the epithelial environment has the opposite effect, resulting in rapid and thorough cyclical quasispecies re-equilibration. Analogous processes may occur in natural infections with other dual-tropic RNA viruses.

We study periodic dynamics and error-threshold behavior in a delayed quasispecies model consisting of a master sequence and two mutant populations . The system, formulated as delay differential equations with time-periodic replication rates, yields new conditions for the existence and absence of -periodic solutions. Using topological degree arguments, we show that when mutation probabilities lie strictly between 0 and 1 and at least one fitness function is periodic, the system supports nontrivial positive periodic orbits, with or without backward mutations. This shows that fluctuating environments, such as circadian or treatment-induced cycles, can sustain oscillatory genotype distributions. Conversely, if mutations are strictly unidirectional and the master sequence is consistently dominated in fitness, no positive -periodic orbit arises. In this regime, the master sequence decays monotonically to extinction without time delays, while time delays induce non-monotonic decay, recovering the classical error-threshold phenomenon and linking it to cancer-related quasispecies dynamics.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

Quasispecies theory has been instrumental in the understanding of RNA virus population dynamics because it considered for the first time mutation as an integral part of the replication process. The key influences of quasispecies theory on experimental virology have been: (1) to disclose the mutant spectrum nature of viral populations and to evaluate its consequences; (2) to unveil collective properties of genome ensembles that can render a mutant spectrum a unit of selection; and (3) to identify new vulnerability points of pathogenic RNA viruses on three fronts: the need to apply multiple selective constraints (in the form of drug combinations) to minimize selection of treatment-escape variants, to translate the error threshold concept into antiviral designs, and to construct attenuated vaccine viruses through alterations of viral polymerase copying fidelity or through displacements of viral genomes towards unfavorable regions of sequence space. These three major influences on the understanding of viral pathogens preceded extensions of quasispecies to non-viral systems such as bacterial and tumor cell collectivities and prions. These developments are summarized here.

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