Mark Zwart

Dr. Mark Zwart

Senior Researcher


Droevendaalsesteeg 10
6708 PB Wageningen

+31 (0) 317 47 34 00

The Netherlands



From zero to hero? Whereas a few viruses cause major problems for human health, most don't infect people and are integral parts of nature. So what viruses do we find in the wild, and how do they impact ecosystems? That's what my team and I are exploring.


During my undergraduate degree, one of my mentors discouraged me from working on viruses because "we know everything about them". After all, we had more complete genome sequences than for any other organism and many insights in molecular biology are closely linked to viruses. What more could there be to learn about these simple life forms with relatively few genes? It turns out science was just beginning to scratch the surface! Nowadays we know that viruses are abundant in all environments, that they can have complex interactions with their host organisms despite their small sizes, and that they can impact whole ecosystems in many different ways. I have worked on the evolutionary biology, genetics and ecology of a number of different viruses, focusing on the plant viruses in recent years. I was initially interested in understanding the kinetics of virus infection and the evolution of major changes in their genomes. Now the focus of my work is on understanding how virus genome structure is linked to ecology and evolution, describing virus biodiversity in plant communities from natural ecosystems, and understanding how virus communities affect the composition and functioning of ecosystems. My dream is that we can predict patterns of virus spread and evolution in natural systems, and in doing so leverage these intriguing life forms.


Plant viruses Viruses


Peer-reviewed publicaties

  • Viruses

    Robust Approaches to the Quantitative Analysis of Genome Formula Variation in Multipartite and Segmented Viruses

    Marcelle Johnson, Mark Zwart

    When viruses have segmented genomes, the set of frequencies describing the abundance of segments is called the genome formula. The genome formula is often unbalanced and highly variable for both segmented and multipartite viruses. A growing number of studies are quantifying the genome formula to measure its effects on infection and to consider its ecological and evolutionary implications. Different approaches have been reported for analyzing genome formula data, including qualitative description, applying standard statistical tests such as ANOVA, and customized analyses. However, these approaches have different shortcomings, and test assumptions are often unmet, potentially leading to erroneous conclusions. Here, we address these challenges, leading to a threefold contribution. First, we propose a simple metric for analyzing genome formula variation: the genome formula distance. We describe the properties of this metric and provide a framework for understanding metric values. Second, we explain how this metric can be applied for different purposes, including testing for genome-formula differences and comparing observations to a reference genome formula value. Third, we re-analyze published data to illustrate the applications and weigh the evidence for previous conclusions. Our re-analysis of published datasets confirms many previous results but also provides evidence that the genome formula can be carried over from the inoculum to the virus population in a host. The simple procedures we propose contribute to the robust and accessible analysis of genome-formula data.
  • Frontiers in Virology

    Mixed viral infection constrains the genome formula of multipartite cucumber mosaic virus

    Dieke Boezen, Maritta Vermeulen, Marcelle Johnson, René A. A. van der Vlugt, Carolyn M. Malmstrom, Mark Zwart
    Many plant viruses have a multipartite organization, with multiple genome segments packaged into separate virus particles. The genome formula describes the relative frequencies of all viral genome segments, and previous work suggests rapid changes in these frequencies facilitate virus adaptation. Many studies have reported mixed viral infections in plants, often resulting in strong virus–virus interactions. Here, we tested whether mixed infections with tripartite alfalfa mosaic virus (AMV) and monopartite potato virus Y (PVY) affected the genome formula of the tripartite cucumber mosaic virus (CMV), our experimental model. We found that the CMV titer was reduced in mixed infections with its tripartite Bromoviridae relative AMV and in triple infections with both AMV and PVY, indicating notable virus–virus interactions. The variability of the CMV genome formula was significantly lower in mixed infections (CMV and AMV, CMV and PVY, and CMV and AMV and PVY) than in single infections (CMV only). These observations led to the surprising conclusion that mixed infections with two distinct viruses constrain the CMV genome formula. It remains unclear how common these effects are for different combinations of virus species and strains and what the underlying mechanisms are. We, therefore, extended a simulation model to consider three putative scenarios in which a second virus affected the genome formula. The simulation results also suggested that shifts in the genome formula occur, but may not be widespread due to the required conditions. One scenario modeled—co-infection exclusion through niche differentiation—was congruent with the experimental data, as this scenario led to reductions in genome formula variability and titer of the multipartite virus. Whereas previous studies highlighted host–species effects, our results indicate that the genome formula is also affected by mixed infections, suggesting that there is a broader set of environmental cues that affect the genome formula.
  • Frontiers in Virology

    Editorial: Predicting virus evolution

  • mBio

    The Fitness of Beta-Lactamase Mutants Depends Nonlinearly on Resistance Level at Sublethal Antibiotic Concentrations

    Andrew D. Farr, Diego Pesce, Suman G. Das, Mark Zwart, J. Arjan G. M. de Visser
    Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit and together make up the fitness landscape, which maps genotype space onto fitness under specified conditions. Experimentally derived fitness landscapes have demonstrated a predictability to evolution by identifying limited “mutational routes” that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilize indirect measures to determine fitness. We estimated the competitive fitness of mutants relative to all single-mutation neighbors to describe the fitness landscape of three mutations in a β-lactamase enzyme. Fitness assays were performed at sublethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. In the unstructured environment, the antibiotic selected for higher-resistance types—but with an equivalent fitness for a subset of mutants, despite substantial variation in resistance—resulting in a stratified fitness landscape. In contrast, in a structured environment with a low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with subinhibitory concentrations of antibiotics and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes.

    The evolution of antibiotic-resistant bacterial populations underpins the ongoing antibiotic resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained, and whether it can be predictable. To this end, competition experiments were performed with a combinatorially complete set of mutants of a β-lactamase gene subject to subinhibitory concentrations of the antibiotic cefotaxime. While some mutations confer on their hosts high resistance to cefotaxime, in competition these mutations do not always confer a selective advantage. Specifically, high-resistance mutants had equivalent fitnesses despite different resistance levels and even had selective disadvantages under conditions involving spatial structure. Together, our findings suggest that the relationship between resistance level and fitness at subinhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.
  • Virus Research

    Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula

    Dieke Boezen, Marcelle Johnson, Alexey Grum-Grzimaylo, René A. A. van der Vlugt, Mark Zwart

    Viruses show great diversity in their genome organization. Multipartite viruses package their genome segments into separate particles, most or all of which are required to initiate infection in the host cell. The benefits of such seemingly inefficient genome organization are not well understood. One hypothesised benefit of multipartition is that it allows for flexible changes in gene expression by altering the frequency of each genome segment in different environments, such as encountering different host species. The ratio of the frequency of segments is termed the genome formula (GF). Thus far, formal studies quantifying the GF have been performed for well-characterised virus-host systems in experimental settings using RT-qPCR. However, to understand GF variation in natural populations or novel virus-host systems, a comparison of several methods for GF estimation including high-throughput sequencing (HTS) based methods is needed. Currently, it is unclear how HTS-methods compare a golden standard, such as RT-qPCR. Here we show a comparison of multiple GF quantification methods (RT-qPCR, RT-digital PCR, Illumina RNAseq and Nanopore direct RNA sequencing) using three host plants (Nicotiana tabacum, Nicotiana benthamiana, and Chenopodium quinoa) infected with cucumber mosaic virus (CMV), a tripartite RNA virus. Our results show that all methods give roughly similar results, though there is a significant method effect on genome formula estimates. While the RT-qPCR and RT-dPCR GF estimates are congruent, the GF estimates from HTS methods deviate from those found with PCR. Our findings emphasize the need to tailor the GF quantification method to the experimental aim, and highlight that it may not be possible to compare HTS and PCR-based methods directly. The difference in results between PCR-based methods and HTS highlights that the choice of quantification technique is not trivial.
  • Ecology

    Environmental refuges from disease in host‐parasite interactions under global change

    Alena Gsell, Arjen Biere, Wietse de Boer, Irene de Bruijn, Götz Eichhorn, Thijs Frenken, Stefan Geisen, Henk P. van der Jeugd, Kyle Mason-Jones, Annelein Meisner, Maddy Thakur, Ellen Van Donk, Mark Zwart, Dedmer Van de Waal
    The physiological performance of organisms depends on their environmental context, resulting in performance–response curves along environmental gradients. Parasite performance–response curves are generally expected to be broader than those of their hosts due to shorter generation times and hence faster adaptation. However, certain environmental conditions may limit parasite performance more than that of the host, thereby providing an environmental refuge from disease. Thermal disease refuges have been extensively studied in response to climate warming, but other environmental factors may also provide environmental disease refuges which, in turn, respond to global change. Here, we (1) showcase laboratory and natural examples of refuges from parasites along various environmental gradients, and (2) provide hypotheses on how global environmental change may affect these refuges. We strive to synthesize knowledge on potential environmental disease refuges along different environmental gradients including salinity and nutrients, in both natural and food-production systems. Although scaling up from single host–parasite relationships along one environmental gradient to their interaction outcome in the full complexity of natural environments remains difficult, integrating host and parasite performance–response can serve to formulate testable hypotheses about the variability in parasitism outcomes and the occurrence of environmental disease refuges under current and future environmental conditions.
  • PLoS Genetics

    Empirical estimates of the mutation rate for an alphabaculovirus

    Dieke Boezen, Ghulam Ali, Manli Wang, Xi Wang, Wopke van der Werf, Just M. Vlak, Mark Zwart
    Mutation rates are of key importance for understanding evolutionary processes and predicting their outcomes. Empirical mutation rate estimates are available for a number of RNA viruses, but few are available for DNA viruses, which tend to have larger genomes. Whilst some viruses have very high mutation rates, lower mutation rates are expected for viruses with large genomes to ensure genome integrity. Alphabaculoviruses are insect viruses with large genomes and often have high levels of polymorphism, suggesting high mutation rates despite evidence of proofreading activity by the replication machinery. Here, we report an empirical estimate of the mutation rate per base per strand copying (s/n/r) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To avoid biases due to selection, we analyzed mutations that occurred in a stable, non-functional genomic insert after five serial passages in Spodoptera exigua larvae. Our results highlight that viral demography and the stringency of mutation calling affect mutation rate estimates, and that using a population genetic simulation model to make inferences can mitigate the impact of these processes on estimates of mutation rate. We estimated a mutation rate of μ = 1×10−7 s/n/r when applying the most stringent criteria for mutation calling, and estimates of up to μ = 5×10−7 s/n/r when relaxing these criteria. The rates at which different classes of mutations accumulate provide good evidence for neutrality of mutations occurring within the inserted region. We therefore present a robust approach for mutation rate estimation for viruses with stable genomes, and strong evidence of a much lower alphabaculovirus mutation rate than supposed based on the high levels of polymorphism observed.
  • Viruses

    Defective RNA Particles of Plant Viruses—Origin, Structure and Role in Pathogenesis

    Daria Budzyńska, Mark Zwart, Beata Hasiów-Jaroszewska
    The genomes of RNA viruses may be monopartite or multipartite, and sub-genomic particles such as defective RNAs (D RNAs) or satellite RNAs (satRNAs) can be associated with some of them. D RNAs are small, deletion mutants of a virus that have lost essential functions for independent replication, encapsidation and/or movement. D RNAs are common elements associated with human and animal viruses, and they have been described for numerous plant viruses so far. Over 30 years of studies on D RNAs allow for some general conclusions to be drawn. First, the essential condition for D RNA formation is prolonged passaging of the virus at a high cellular multiplicity of infection (MOI) in one host. Second, recombination plays crucial roles in D RNA formation. Moreover, during virus propagation, D RNAs evolve, and the composition of the particle depends on, e.g., host plant, virus isolate or number of passages. Defective RNAs are often engaged in transient interactions with full-length viruses—they can modulate accumulation, infection dynamics and virulence, and are widely used, i.e., as a tool for research on cis-acting elements crucial for viral replication. Nevertheless, many questions regarding the generation and role of D RNAs in pathogenesis remain open. In this review, we summarise the knowledge about D RNAs of plant viruses obtained so far.
  • New Phytologist

    Plant neighbours can make or break the disease transmission chain of a fungal root pathogen

    Eline Ampt, Jasper van Ruijven, Mark Zwart, Jos M. Raaijmakers, Aad J Termorshuizen, Liesje Mommer
    Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission.
    Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development.
    Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host–neighbour interactions changed which mechanisms steered disease transmission rate.
    Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.
  • Nature Ecology and Evolution

    Population size mediates the contribution of high-rate and large-benefit mutations to parallel evolution

    Martijn F. Schenk, Mark Zwart, Sungmin Hwang, Philip Ruelens, Edouard Severing, Joachim Krug, J. Arjan G. M. de Visser
    Mutations with large fitness benefits and mutations occurring at high rates may both cause parallel evolution, but their contribution is predicted to depend on population size. Moreover, high-rate and large-benefit mutations may have different long-term adaptive consequences. We show that small and 100-fold larger bacterial populations evolve resistance to a β-lactam antibiotic by using similar numbers, but different types of mutations. Small populations frequently substitute similar high-rate structural variants and loss-of-function point mutations, including the deletion of a low-activity β-lactamase, and evolve modest resistance levels. Large populations more often use low-rate, large-benefit point mutations affecting the same targets, including mutations activating the β-lactamase and other gain-of-function mutations, leading to much higher resistance levels. Our results demonstrate the separation by clonal interference of mutation classes with divergent adaptive consequences, causing a shift from high-rate to large-benefit mutations with increases in population size.
  • Journal of Theoretical Biology

    Second compartment widens plasmid invasion conditions

    Jesse Alderliesten, Mark Zwart, J. Arjan G. M. de Visser, Arjan Stegeman, Egil A. J. Fischer

    Understanding under which conditions conjugative plasmids encoding antibiotic resistance can invade bacterial communities in the gut is of particular interest to combat the spread of antibiotic resistance within and between animals and humans. We extended a one-compartment model of conjugation to a two-compartment model, to analyse how differences in plasmid dynamics in the gut lumen and at the gut wall affect the invasion of plasmids. We compared scenarios with one and two compartments, different migration rates between the lumen and wall compartments, and different population dynamics. We focused on the effect of attachment and detachment rates on plasmid dynamics, explicitly describing pair formation followed by plasmid transfer in the pairs. The parameter space allowing plasmid invasion in the one-compartment model is affected by plasmid costs and intrinsic conjugation rates of the transconjugant, but not by these characteristics of the donor. The parameter space allowing plasmid invasion in the two-compartment model is affected by attachment and detachment rates in the lumen and wall compartment, and by the bacterial density at the wall. The one- and two-compartment models predict the same parameter space for plasmid invasion if the conditions in both compartments are equal to the conditions in the one-compartment model. In contrast, the addition of the wall compartment widens the parameter space allowing invasion compared with the one-compartment model, if the density at the wall is higher than in the lumen, or if the attachment rate at the wall is high and the detachment rate at the wall is low. We also compared the pair-formation models with bulk-conjugation models that describe conjugation by instantaneous transfer of the plasmid at contact between cells, without explicitly describing pair formation. Our results show that pair-formation and bulk-conjugation models predict the same parameter space for plasmid invasion. From our simulations, we conclude that conditions at the gut wall should be taken into account to describe plasmid dynamics in the gut and that transconjugant characteristics rather than donor characteristics should be used to parameterize the models.
  • PLoS Biology

    Incomplete bunyavirus particles can cooperatively support virus infection and spread

    Erick Bermúdez-Méndez, Kirsten F. Bronsvoort, Mark Zwart, Sandra van de Water, Ingrid Cárdenas-Rey, Rianka P. M. Vloet, Constantianus J.M. Koenraadt, Gorben P. Pijlman, Jeroen Kortekaas, Paul J. Wichgers Schreur
  • Frontiers in Virology

    Five Challenges in the Field of Viral Diversity and Evolution

    Rafael Sanjuán, Christopher J.R. Illingworth, Jemma L. Geoghegan, Jaime Iranzo, Mark Zwart, Alexander T. Ciota, Gonzalo Moratorio, Selma Gago-Zachert, Siobain Duffy, Dhanasekaran Vijaykrishna
    Viral diversity and evolution play a central role in processes such as disease emergence, vaccine failure, drug resistance, and virulence. However, significant challenges remain to better understand and manage these processes. Here, we discuss five of these challenges. These include improving our ability to predict viral evolution, developing more relevant experimental evolutionary systems, integrating viral dynamics and evolution at different scales, systematic appraisal of the virosphere, and deepening our understanding of virus-virus interactions. Intensifying future research on these areas should improve our ability to combat viral diseases, as well as to exploit viral diversity and evolution for biotechnological purposes.
  • Viruses

    Metagenomic studies of viruses in weeds and wild plants

    Beata Hasiów-Jaroszewska, Dieke Boezen, Mark Zwart

    High throughput sequencing (HTS) has revolutionised virus detection and discovery, al-lowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection—often in the absence of disease symptoms—and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limita-tions of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel vi-ruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of met-agenomics as an approach.
  • Virus Evolution

    Unresolved advantages of multipartitism in spatially structured environments

    Mark Zwart, Stéphane Blanc, Marcelle Johnson, Susanna Manrubia, Yannis Michalakis, Mircea T. Sofonea

    Multipartite viruses have segmented genomes and package each of their genome segments individually into distinct virus particles. Multipartitism is common among plant viruses, but why this apparently costly genome organization and packaging has evolved remains unclear. Recently Zhang and colleagues developed network epidemiology models to study the epidemic spread of multipartite viruses and their distribution over plant and animal hosts (Phys. Rev. Lett. 2019, 123, 138101). In this short commentary, we call into question the relevance of these results because of key model assumptions. First, the model of plant hosts assumes virus transmission only occurs between adjacent plants. This assumption overlooks the basic but imperative fact that most multipartite viruses are transmitted over variable distances by mobile animal vectors, rendering the model results irrelevant to differences between plant and animal hosts. Second, when not all genome segments of a multipartite virus are transmitted to a host, the model assumes an incessant latent infection occurs. This is a bold assumption for which there is no evidence to date, making the relevance of these results to understanding multipartitism questionable.
  • Proceedings of the Royal Society B: Biological Sciences

    Chicken gut microbiome members limit the spread of an antimicrobial resistance plasmid in Escherichia coli

    Sarah J. N. Duxbury, Jesse Alderliesten, Mark Zwart, Arjan Stegeman, Egil A. J. Fischer, J. Arjan G. M. de Visser
    Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro, the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying blaCTX-M-1, in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.
  • BMC Microbiology

    Effect of donor-recipient relatedness on the plasmid conjugation frequency: a meta-analysis

    Jesse Alderliesten, Sarah J. N. Duxbury, Mark Zwart, J. Arjan G. M. de Visser, Arjan Stegeman, Egil A. J. Fischer
    Conjugation plays a major role in the transmission of plasmids encoding antibiotic resistance genes in both clinical and general settings. The conjugation efficiency is influenced by many biotic and abiotic factors, one of which is the taxonomic relatedness between donor and recipient bacteria. A comprehensive overview of the influence of donor-recipient relatedness on conjugation is still lacking, but such an overview is important to quantitatively assess the risk of plasmid transfer and the effect of interventions which limit the spread of antibiotic resistance, and to obtain parameter values for conjugation in mathematical models. Therefore, we performed a meta-analysis on reported conjugation frequencies from Escherichia coli donors to various recipient species.
  • Viruses

    Identification of loci associated with enhanced virulence in Spodoptera litura nucleopolyhedrovirus isolates using a deep sequencing approach

    Mark Zwart, Ghulam Ali, Elisabeth van Strien, Elio G.W.M. Schijlen, Wopke van der Werf, Just M. Vlak, Manli Wang
    Two genetically distinct Spodoptera litura nucleopolyhedrovirus (SpltNPVs) isolates from Pakistan were investigated by deep sequencing of their full genomes. Previously we reported that isolate SpltNPV-Pak-TAX1 kills S. litura (leafworm) larvae significantly faster than SpltNPV-Pak-BNG. Here we find that the consensus sequences of these two virus isolates shared 99% identity, suggesting they are closely related. The major difference between these two isolates is the absence of hr17 (putative enhancer of transcription and origin of replication) in SpltNPV-Pak-TAX1 and the absence of ORF125 with unknown function in SpltNPV-Pak-BNG. Analysis of the rates of nonsynonymous and synonymous single-nucleotide substitutions showed that strong purifying selection predominates, although for a small number of genes there was neutral or positive selection. The most striking case is ORF122, which encodes a putative viral fibroblast growth factor (FGF), known to be involved in the passage of virus from the midgut to the interior of the larva and linked to virus virulence in other baculoviruses. We found very little polymorphism within both virus isolates, a result at odds with observations for other baculoviruses, possibly suggesting recent dispersal of the virus in Pakistan. We have therefore identified two loci possibly linked to the enhanced virulence of the SpltNPV-Pak-TAX1. This information could help to understand the enhanced activity of SpltNPV-Pak-TAX1 and to select better SpltNPV isolates for the control of S. litura in Pakistan and elsewhere.
  • Virus Evolution

    On the stability of sequences inserted into viral genomes

    Anouk Willemsen, Mark Zwart
    Viruses are widely used as vectors for heterologous gene expression in cultured cells or natural hosts, and therefore a large number of viruses with exogenous sequences inserted into their genomes have been engineered. Many of these engineered viruses are viable and express heterologous proteins at high levels, but the inserted sequences often prove to be unstable over time and are rapidly lost, limiting heterologous protein expression. Although virologists are aware that inserted sequences can be unstable, processes leading to insert instability are rarely considered from an evolutionary perspective. Here, we review experimental work on the stability of inserted sequences over a broad range of viruses, and we present some theoretical considerations concerning insert stability. Different virus genome organizations strongly impact insert stability, and factors such as the position of insertion can have a strong effect. In addition, we argue that insert stability not only depends on the characteristics of a particular genome, but that it will also depend on the host environment and the demography of a virus population. The interplay between all factors affecting stability is complex, which makes it challenging to develop a general model to predict the stability of genomic insertions. We highlight key questions and future directions, finding that insert stability is a surprisingly complex problem and that there is need for mechanism-based, predictive models. Combining theoretical models with experimental tests for stability under varying conditions can lead to improved engineering of viral modified genomes, which is a valuable tool for understanding genome evolution as well as for biotechnological applications, such as gene therapy.
  • Viruses

    Bioinformatics Meets Virology

    Bashar Ibrahim, Ksenia Arkhipova, Arno C Andeweg, Susana Posada-Céspedes, François Enault, Arthur Gruber, Eugene V Koonin, Anne Kupczok, Philippe Lemey, Alice C McHardy, Dino P McMahon, Brett E Pickett, David L Robertson, Richard H Scheuermann, Alexandra Zhernakova, Mark Zwart, Alexander Schönhuth, Bas E Dutilh, Manja Marz

    The Second Annual Meeting of the European Virus Bioinformatics Center (EVBC), held in Utrecht, Netherlands, focused on computational approaches in virology, with topics including (but not limited to) virus discovery, diagnostics, (meta-)genomics, modeling, epidemiology, molecular structure, evolution, and viral ecology. The goals of the Second Annual Meeting were threefold: (i) to bring together virologists and bioinformaticians from across the academic, industrial, professional, and training sectors to share best practice; (ii) to provide a meaningful and interactive scientific environment to promote discussion and collaboration between students, postdoctoral fellows, and both new and established investigators; (iii) to inspire and suggest new research directions and questions. Approximately 120 researchers from around the world attended the Second Annual Meeting of the EVBC this year, including 15 renowned international speakers. This report presents an overview of new developments and novel research findings that emerged during the meeting.
  • Heredity

    Going, going, gone: predicting the fate of genomic insertions in plant RNA viruses

    Anouk Willemsen, José L. Carrasco, Santiago F. Elena, Mark Zwart
    Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate—the rate at which deletions removing the insertion occur—and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify
    the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.
  • Current Opinion in Virology

    Population bottlenecks in multicomponent viruses: First forays into the uncharted territory of genome-formula drift

    Serafin Gutiérrez, Mark Zwart
    Multicomponent viral systems face specific challenges when enduring population bottlenecks. These systems can lose coding information due to the lack of co-encapsidation of all the genetic information, at least in a proportion of the capsids in a population. Moreover, bottlenecks can also impact one of the main potential advantages of multicomponent systems: the regulation of gene expression through changes in gene copy frequencies at the population level. How these systems cope with population bottlenecks is far from being clear. Here, two non-exclusive scenarios are described. In the first scenario, population bottlenecks during host infection allow for the isolation of within-host populations with different gene frequencies, leaving the door opened for the selection of populations with adaptive gene frequencies. The second scenario postulates that viruses could influence bottleneck size, at least at certain steps of their life cycle, to limit random changes in gene frequencies. Examples of viral mechanism impacting bottleneck size at cell infection are available and, intriguingly, they can lead to either increases or reductions in bottleneck size. This situation opens the way for putative trade-offs on both gene frequencies and bottleneck sizes that could differ among multicomponent systems.
  • Heredity

    Unraveling the causes of adaptive benefits of synonymous mutations in TEM-1 β-lactamase

    Mark Zwart, Martijn F. Schenk, Sungmin Hwang, A Bertha Koopmanschap, Niek de Lange, Lion van de Pol, Tran Thi Thuy Nga, Ivan G. Szendro, Joachim Krug, J. Arjan G. M. de Visser
    While synonymous mutations were long thought to be without phenotypic consequences, there is growing evidence they can affect gene expression, protein folding, and ultimately the fitness of an organism. In only a few cases have the mechanisms by which synonymous mutations affect the phenotype been elucidated. We previously identified 48 mutations in TEM-1 β-lactamase that increased resistance of Escherichia coli to cefotaxime, 10 of which were synonymous. To better understand the molecular mechanisms underlying the beneficial effect of these synonymous mutations, we made a series of measurements for a panel containing the 10 synonymous together with 10 non-synonymous mutations as a reference. Whereas messenger levels were unaffected, we found that total and functional TEM protein levels were higher for 5 out of 10 synonymous mutations. These observations suggest that some of these mutations act on translation or a downstream process. Similar effects were observed for some small-benefit non-synonymous mutations, suggesting a similar causal mechanism. For the synonymous mutations, we found that the cost of resistance scales with TEM protein levels. A resistance landscape for four synonymous mutations revealed strong epistasis: none of the combinations of mutations exceeded the resistance of the largest-effect mutation and there were synthetically neutral combinations. By considering combined effects of these mutations, we could infer that functional TEM protein level is a multi-dimensional phenotype. These results suggest that synonymous mutations may have beneficial effects by increasing the expression of an enzyme with low substrate activity, which may be realized via multiple, yet unknown, post-transcriptional mechanisms.
  • Journal of Fish Diseases

    Quantitative analysis of the dose–response of white spot syndrome virus in shrimp

    Thuy T.N. Ngo, Alistair M. Senior, Antica Culina, Eduardo S.A. Santos, Just M. Vlak, Mark Zwart
    White spot syndrome virus (WSSV) is an important cause of mortality and economiclosses in shrimp farming. Although WSSV‐induced mortality is virus dose dependentand WSSV infection does not necessarily lead to mortality, the relationshipsbetween virus‐particle dose, infection and mortality have not been analysed quanti-tatively. Here, we explored WSSV dose–response by a combination of experiments,modelling and meta‐analysis. We performed dose–response experiments inPenaeusvannameipostlarvae, recorded host mortality and detected WSSV infection. Whenwe fitted infection models to these data, two models—differing in whether theyincorporated heterogeneous host susceptibility to the virus or not—were supportedfor two independent experiments. To determine the generality of these results, wereanalysed published data sets and then performed a meta‐analysis. We found thatWSSV dose–response kinetics is indeed variable over experiments. We could notclearly identify which specific infection model has the most support by meta‐analy-sis, but we argue that these results also are most concordant with a model incorpo-rating varying levels of heterogeneous host susceptibility to WSSV. We haveidentified suitable models for analysing WSSV dose–response, which can elucidatethe most basic virus–host interactions and help to avoid underestimating WSSVinfection at low virus doses.

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