Experimental manipulation of population‐level MHC diversity controls pathogen virulence evolution in Mus musculus

The virulence levels attained by serial passage of pathogens through similar host genotypes are much higher than observed in natural systems; however, it is unknown what keeps natural virulence levels below these empirically demonstrated maximum levels. One hypothesis suggests that host diversity impedes pathogen virulence, because adaptation to one host genotype carries trade‐offs in the ability to replicate and cause disease in other host genotypes. To test this hypothesis, with the simplest level of population diversity within the loci of the major histocompatibility complex (MHC), we serially passaged Friend virus complex (FVC) through two rounds, in hosts with either the same MHC genotypes (pure passage) or hosts with different MHC genotypes (alternated passage). Alternated passages showed a significant overall reduction in viral titre (31%) and virulence (54%) when compared to pure passages. Furthermore, a resistant host genotype initially dominated any effects due to MHC diversity; however, when FVC was allowed to adapt to the resistant host genotype, predicted MHC effects emerged; that is, alternated lines show reduced virulence. These data indicate serial exposure to diverse MHC genotypes is an impediment to pathogen adaptation, suggesting genetic variation at MHC loci is important for limiting virulence in a rapidly evolving pathogen and supports negative frequency‐dependent selection as a force maintaining MHC diversity in host populations.     

Host sexual dimorphism affects the outcome of within‐host pathogen competition

Natural infections often consist of multiple pathogens of the same or different species. When coinfections occur, pathogens compete for access to host resources and fitness is determined by how well a pathogen can reproduce compared to its competitors. Yet not all hosts provide the same resource pool. Males and females, in particular, commonly vary in both their acquisition of resources and investment in immunity, but their ability to modify any competition between different pathogens remains unknown. Using the Daphnia magna–Pasteuria ramosa model system, we exposed male and female hosts to either a single genotype infection or coinfections consisting of two pathogen genotypes of varying levels of virulence. We found that coinfections within females favored the transmission of the more virulent pathogen genotype, whereas coinfections within male hosts resulted in equal transmission of competing pathogen genotypes. This contrast became less pronounced when the least virulent pathogen was able to establish an infection first, suggesting that the influence of host sex is shaped by priority effects. We suggest that sex is a form of host heterogeneity that may influence the evolution of virulence within coinfection contexts and that one sex may be a reservoir for pathogen genetic diversity in nature.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

Superinfection drives virulence evolution in experimental populations of bacteria and plasmids

A prominent hypothesis proposes that pathogen virulence evolves in large part due to a trade‐off between infectiousness and damage to hosts. Other explanations emphasize how virulence evolves in response to competition among pathogens within hosts. Given the proliferation of theoretical possibilities, what best predicts how virulence evolves in real biological systems? Here, I show that virulence evolution in experimental populations of bacteria and self‐transmissible plasmids is best explained by within‐host competition. Plasmids evolved to severely reduce the fitness of their hosts even in the absence of uninfected cells. This result is inconsistent with the trade‐off hypothesis, which predicts that under these conditions vertically transmitted pathogens would evolve to be less virulent. Plasmid virulence was strongly correlated with the ability to superinfect cells containing competing plasmid genotypes, suggesting a key role for within‐host competition. When virulent genotypes became common, hosts evolved resistance to plasmid infection. These results show that the trade‐off hypothesis can incorrectly predict virulence evolution when within‐host interactions are neglected. They also show that symbioses between bacteria and plasmids can evolve to be surprisingly antagonistic.     

In vivo fitness associated with high virulence in a vertebrate virus is a complex trait regulated by host entry, replication, and shedding

The relationship between pathogen fitness and virulence is typically examined by quantifying only one or two pathogen fitness traits. More specifically, it is regularly assumed that within-host replication, as a precursor to transmission, is the driving force behind virulence. In reality, many traits contribute to pathogen fitness, and each trait could drive the evolution of virulence in different ways. Here, we independently quantified four viral infection cycle traits, namely, host entry, within-host replication, within-host coinfection fitness, and shedding, in vivo, in the vertebrate virus Infectious hematopoietic necrosis virus (IHNV). We examined how each of these stages of the viral infection cycle contributes to the fitness of IHNV genotypes that differ in virulence in rainbow trout. This enabled us to determine how infection cycle fitness traits are independently associated with virulence. We found that viral fitness was independently regulated by each of the traits examined, with the largest impact on fitness being provided by within-host replication. Furthermore, the more virulent of the two genotypes of IHNV we used had advantages in all of the traits quantified. Our results are thus congruent with the assumption that virulence and within-host replication are correlated but suggest that infection cycle fitness is complex and that replication is not the only trait associated with virulence.     

VACCINATION AND REDUCED COHORT DURATION CAN DRIVE VIRULENCE EVOLUTION: MAREK’S DISEASE VIRUS AND INDUSTRIALIZED AGRICULTURE

Marek’s disease virus (MDV), a commercially important disease of poultry, has become substantially more virulent over the last 60 years. This evolution was presumably a consequence of changes in virus ecology associated with the intensification of the poultry industry. Here, we assess whether vaccination or reduced host life span could have generated natural selection, which favored more virulent strains. Using previously published experimental data, we estimated viral fitness under a range of cohort durations and vaccine treatments on broiler farms. We found that viral fitness maximized at intermediate virulence, as a result of a trade‐off between virulence and transmission previously reported. Our results suggest that vaccination, acting on this trade‐off, could have led to the evolution of increased virulence. By keeping the host alive, vaccination prolongs infectious periods of virulent strains. Improvements in host genetics and nutrition, which reduced broiler life spans below 50 days, could have also increased the virulence of the circulating MDV strains because shortened cohort duration reduces the impact of host death on viral fitness. These results illustrate the dramatic impact anthropogenic change can potentially have on pathogen virulence.     

IS HIV SHORT‐SIGHTED? INSIGHTS FROM A MULTISTRAIN NESTED MODEL

An important component of pathogen evolution at the population level is evolution within hosts. Unless evolution within hosts is very slow compared to the duration of infection, the composition of pathogen genotypes within a host is likely to change during the course of an infection, thus altering the composition of genotypes available for transmission as infection progresses. We develop a nested modeling approach that allows us to follow the evolution of pathogens at the epidemiological level by explicitly considering within‐host evolutionary dynamics of multiple competing strains and the timing of transmission. We use the framework to investigate the impact of short‐sighted within‐host evolution on the evolution of virulence of human immunodeficiency virus (HIV), and find that the topology of the within‐host adaptive landscape determines how virulence evolves at the epidemiological level. If viral reproduction rates increase significantly during the course of infection, the viral population will evolve a high level of virulence even though this will reduce the transmission potential of the virus. However, if reproduction rates increase more modestly, as data suggest, our model predicts that HIV virulence will be only marginally higher than the level that maximizes the transmission potential of the virus.     

Generalized selection to overcome innate immunity selects for host breadth in an RNA virus

Virus‐host coevolution has selected for generalized host defense against viruses, exemplified by interferon production/signaling and other innate immune function in eukaryotes such as humans. Although cell‐surface binding primarily limits virus infection success, generalized adaptation to counteract innate immunity across disparate hosts may contribute to RNA virus emergence potential. We examined this idea using vesicular stomatitis virus (VSV) populations previously evolved on strictly immune‐deficient (HeLa) cells, strictly immune competent (MDCK) cells, or on alternating deficient/competent cells. By measuring viral fitness in unselected human cancer cells of differing innate immunity, we confirmed that HeLa‐adapted populations were specialized for innate immune‐deficient hosts, whereas MDCK‐adapted populations were relatively more generalized for fitness on hosts of differing innate immune capacity and of different species origin. We also confirmed that HeLa‐evolved populations maintained fitness in immune‐deficient nonhuman primate cells. These results suggest that innate immunity is more prominent than host species in determining viral fitness at the host‐cell level. Finally, our prediction was inexact that selection on alternating deficient/competent hosts should produce innate viral generalists. Rather, fitness differences among alternating host‐evolved VSV populations indicated variable capacities to evade innate immunity. Our results suggest that the evolutionary history of innate immune selection can affect whether RNA viruses evolve greater host‐breadth.     

Genotype specificity among hosts,pathogens, and beneficial microbes influences the strength of symbiont‐mediated protection

The microbial symbionts of eukaryotes influence disease resistance in many host‐parasite systems. Symbionts show substantial variation in both genotype and phenotype, but it is unclear how natural selection maintains this variation. It is also unknown whether variable symbiont genotypes show specificity with the genotypes of hosts or parasites in natural populations. Genotype by genotype interactions are a necessary condition for coevolution between interacting species. Uncovering the patterns of genetic specificity among hosts, symbionts, and parasites is therefore critical for determining the role that symbionts play in host‐parasite coevolution. Here, we show that the strength of protection conferred against a fungal pathogen by a vertically transmitted symbiont of an aphid is influenced by both host‐symbiont and symbiont‐pathogen genotype by genotype interactions. Further, we show that certain symbiont phylogenetic clades have evolved to provide stronger protection against particular pathogen genotypes. However, we found no evidence of reciprocal adaptation of co‐occurring host and symbiont lineages. Our results suggest that genetic variation among symbiont strains may be maintained by antagonistic coevolution with their host and/or their host's parasites.     

Host genotype and genetic diversity shape the evolution of a novel bacterial infection

Pathogens continue to emerge from increased contact with novel host species. Whilst these hosts can represent distinct environments for pathogens, the impacts of host genetic background on how a pathogen evolves post-emergence are unclear. In a novel interaction, we experimentally evolved a pathogen (Staphylococcus aureus) in populations of wild nematodes (Caenorhabditis elegans) to test whether host genotype and genetic diversity affect pathogen evolution. After ten rounds of selection, we found that pathogen virulence evolved to vary across host genotypes, with differences in host metal ion acquisition detected as a possible driver of increased host exploitation. Diverse host populations selected for the highest levels of pathogen virulence, but infectivity was constrained, unlike in host monocultures. We hypothesise that population heterogeneity might pool together individuals that contribute disproportionately to the spread of infection or to enhanced virulence. The genomes of evolved populations were sequenced, and it was revealed that pathogens selected in distantly-related host genotypes diverged more than those in closely-related host genotypes. S. aureus nevertheless maintained a broad host range. Our study provides unique empirical insight into the evolutionary dynamics that could occur in other novel infections of wildlife and humans.Subject terms: Molecular evolution, Bacterial evolution, Bacterial genetics     

Bacteria-phage coevolution and the emergence of generalist pathogens

Understanding the genetic constraints on pathogen evolution will help to predict the emergence of generalist pathogens that can infect a range of different host genotypes. Here we show that generalist viral pathogens are more likely to emerge during coevolution between the bacterium Pseudomonas fluorescens and the lytic phage SBW25Φ2 than when the same pathogen is challenged to adapt to a nonevolving population of novel hosts. When phages were able to adapt to nonevolving novel hosts, the resulting phenotypes had relatively narrow host ranges compared with coevolved phages. Evolved (rather than coevolved) phages also had lower virulence, although they attained virulence similar to that of coevolved phages after continued adaptation to a nonevolving population of the same host. We explain these results by using sequence data showing that the evolution of broad host range is associated with several different amino acid substitutions and therefore occurs only through repeated rounds of selection for novel infectivity alleles. These findings suggest that generalist bacteriophages are more likely to emerge through long-term coevolution with their hosts than through spontaneous adaptation to a single novel host. These results are likely to be relevant to host-parasite systems where parasite generalism can evolve through the acquisition of multiple mutations or alleles, as appears to be the case for many plant-bacteria and bacteria-virus interactions.     

Two optimal mutation rates in obligate pathogens subject to deleterious mutation

Pathogen species with high mutation rates are likely to accumulate deleterious mutations that reduce their reproductive potential within the host. By altering the within-host growth rate of the pathogen, the deleterious mutation load has the potential to affect epidemiological properties such as prevalence, mean pathogen load, and the mean duration of infections. Here, I examine an epidemiological model that allows for multiple segregating mutations that affect within-host replication efficiency. The model demonstrates a complex range of outcomes depending on pathogen mutation rate, including two distinct, widely separated mutation rates associated with high pathogen prevalence. The low mutation rate prevalence peak is associated with small amounts of genetic diversity within the pathogen population, relatively stable prevalence and infection dynamics, and genetic variation partitioned between hosts. The high mutation rate peak is characterized by considerable genetic diversity both within and between hosts, relatively frequent invasions by more virulent types, and is qualitatively similar to an RNA virus quasispecies. The two prevalence peaks are separated by a valley where natural selection favors evolution toward the optimal within-host state, which is associated with high virulence and relatively rapid host mortality. Both chronic and acute infections are examined using stochastic forward simulations.     

Trade-off between virulence and vertical transmission and the maintenance of a virulent plant pathogen

The continuum hypothesis predicts that parasites should evolve reduced virulence if they have higher opportunity for vertical transmission. However, when there is a trade-off between virulence and vertical transmission, selection may favor horizontal transmission and higher virulence. Atkinsonella hypoxylon is a fungal pathogen that reduces Danthonia fitness by 50% or more when it completely castrates hosts' chasmogamous inflorescences, despite the high opportunity for vertical transmission through cleistogamous seeds. Sporadically, infected hosts with partially castrated inflorescences (which have higher fecundity than completely castrated hosts) are observed in natural populations. Why are partially castrated plants rare if selection favors reduced virulence? We investigated whether there was genetic diversity for virulence among A. hypoxylon genotypes and the relationship between virulence and vertical transmission. We found that the fungal genotype significantly affects the occurrence of partial castration in Danthonia compressa. The proportion of seedlings that were vertically infected by their maternal plant was lower for partially castrated than for completely castrated plants. Our results demonstrate a trade-off between virulence and vertical transmission, explaining the maintenance of more virulent, completely castrating fungal genotypes in natural populations, and suggest that vertical transmission in plants is more complex than what is considered in current models.     

The Integral Role of Genetic Variation in the Evolution of Outcrossing in the Caenorhabditis elegans-Serratia marcescens Host-Parasite System

Outcrossing is predicted to facilitate more rapid adaptation than self-fertilization as a result of genetic exchange between genetically variable individuals. Such genetic exchange may increase the efficacy of selection by breaking down Hill-Robertson interference, as well as promoting the maintenance of within-lineage genetic diversity. Experimental studies have demonstrated the selective advantage of outcrossing in novel environments. Here, we assess the specific role of genetic variation in the evolution of outcrossing. We experimentally evolved genetically variable and inbred populations of mixed mating (outcrossing and self-fertilizing) Caenorhabditis elegans nematodes under novel ecological conditions—specifically the presence of the virulent parasite Serratia marcescens. Outcrossing rates increased in genetically variable host populations evolved in the presence of the parasite, whereas parasite exposure in inbred populations resulted in reduced rates of host outcrossing. The host populations with genetic variation also exhibited increased fitness in the presence of the parasite over eight generations, whereas inbred populations did not. This increase in fitness was primarily the result of adaptation to the parasite, rather than recovery from initial inbreeding depression. Therefore, the benefits of outcrossing were only manifested in the presence of genetic variation, and outcrossing was favored over self-fertilization as a result. As predicted, the benefits of outcrossing under novel ecological conditions are a product of genetic exchange between genetically diverse lineages.     

The Relationship between Host Lifespan and Pathogen Reservoir Potential: An Analysis in the System Arabidopsis thaliana-Cucumber mosaic virus

Identification of the determinants of pathogen reservoir potential is central to understand disease emergence. It has been proposed that host lifespan is one such determinant: short-lived hosts will invest less in costly defenses against pathogens, so that they will be more susceptible to infection, more competent as sources of infection and/or will sustain larger vector populations, thus being effective reservoirs for the infection of long-lived hosts. This hypothesis is sustained by analyses of different hosts of multihost pathogens, but not of different genotypes of the same host species. Here we examined this hypothesis by comparing two genotypes of the plant Arabidopsis thaliana that differ largely both in life-span and in tolerance to its natural pathogen Cucumber mosaic virus (CMV). Experiments with the aphid vector Myzus persicae showed that both genotypes were similarly competent as sources for virus transmission, but the short-lived genotype was more susceptible to infection and was able to sustain larger vector populations. To explore how differences in defense against CMV and its vector relate to reservoir potential, we developed a model that was run for a set of experimentally-determined parameters, and for a realistic range of host plant and vector population densities. Model simulations showed that the less efficient defenses of the short-lived genotype resulted in higher reservoir potential, which in heterogeneous host populations may be balanced by the longer infectious period of the long-lived genotype. This balance was modulated by the demography of both host and vector populations, and by the genetic composition of the host population. Thus, within-species genetic diversity for lifespan and defenses against pathogens will result in polymorphisms for pathogen reservoir potential, which will condition within-population infection dynamics. These results are relevant for a better understanding of host-pathogen co-evolution, and of the dynamics of pathogen emergence.     

The evolution of parasite host range in heterogeneous host populations

Theory on the evolution of niche width argues that resource heterogeneity selects for niche breadth. For parasites, this theory predicts that parasite populations will evolve, or maintain, broader host ranges when selected in genetically diverse host populations relative to homogeneous host populations. To test this prediction, we selected the bacterial parasite Serratia marcescens to kill Caenorhabditis elegans in populations that were genetically heterogeneous (50% mix of two experimental genotypes) or homogeneous (100% of either genotype). After 20 rounds of selection, we compared the host range of selected parasites by measuring parasite fitness (i.e. virulence, the selected fitness trait) on the two focal host genotypes and on a novel host genotype. As predicted, heterogeneous host populations selected for parasites with a broader host range: these parasite populations gained or maintained virulence on all host genotypes. This result contrasted with selection in homogeneous populations of one host genotype. Here, host range contracted, with parasite populations gaining virulence on the focal host genotype and losing virulence on the novel host genotype. This pattern was not, however, repeated with selection in homogeneous populations of the second host genotype: these parasite populations did not gain virulence on the focal host genotype, nor did they lose virulence on the novel host genotype. Our results indicate that host heterogeneity can maintain broader host ranges in parasite populations. Individual host genotypes, however, vary in the degree to which they select for specialization in parasite populations.     

Temperature-dependent costs of parasitism and maintenance of polymorphism under genotype-by-environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.     

Multiple infection dynamics has pronounced effects on the fitness of RNA viruses

Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two‐fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka‐Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed.     

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens

Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek''s disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.     

Evolution of virulence in heterogeneous host communities under multiple trade-offs

Many pathogens and parasites are transmitted through hosts that differ in species, sex, genotype, or immune status. In addition, virulence (here defined as disease-induced mortality) and transmission can vary during the infectious period within hosts of different state. Most models of virulence evolution assume that transmission and virulence are constant over the infectious period and that the host population is homogenous. Here, we examine a multispecies susceptible-infected-recovered (SIR) model where transmission occurs within and between species, and transmission and virulence varied during the infectious period. This allows us to understand virulence evolution in a broader range of situations that characterize many emerging diseases. Because emerging pathogens are by definition new to their host populations, they should be expected to rapidly adapt after emergence. We illustrate these evolutionary effects using the framework of adaptive dynamics to examine how virulence evolves after emergence in response to the relative strength of selection on pathogen fitness and mutational variance for virulence. We illustrate the role of evolution by simulating adaptive walks to an evolutionarily stable virulence. We found that the magnitude of between-species transmission and the relative timing of transmission and mortality across species were of primary importance for determining the evolutionarily stable virulence.