Viral ecology and the maintenance of novel host use

Viruses can occasionally emerge by infecting new host species. However, the early phases of emergence can hinge upon ecological sustainability of the virus population, which is a product of both within-host population growth and between-host transmission. Insufficient growth or transmission can force virus extinction before the latter phases of emergence, where genetic adaptations that improve host use may occur. We examined the early phase of emergence by studying the population dynamics of RNA phages in replicated laboratory environments containing native and novel host bacteria. To predict the breadth of transmission rates allowing viral persistence on each species, we developed a simple model based on in vitro data for phage growth rate over a range of initial population densities on both hosts. Validation of these predictions using serial passage experiments revealed a range of transmission rates for which the native host was a source and the novel host was a sink. In this critical range of transmission rates, periodic exposure to the native host was sufficient for the maintenance of the viral population on the novel host. We argue that this effect should facilitate adaptation by the virus to utilize the novel host--often crucial in subsequent phases of emergence.     

Variable pleiotropic effects from mutations at the same locus hamper prediction of fitness from a fitness component

The relationship of genotype, fitness components, and fitness can be complicated by genetic effects such as pleiotropy and epistasis and by heterogeneous environments. However, because it is often difficult to measure genotype and fitness directly, fitness components are commonly used to estimate fitness without regard to genetic architecture. The small bacteriophage X174 enables direct evaluation of genetic and environmental effects on fitness components and fitness. We used 15 mutants to study mutation effects on attachment rate and fitness in six hosts. The mutants differed from our lab strain of X174 by only one or two amino acids in the major capsid protein (gpF, sites 101 and 102). The sites are variable in natural and experimentally evolved X174 populations and affect phage attachment rate. Within the limits of detection of our assays, all mutations were neutral or deleterious relative to the wild type; 11 mutants had decreased host range. While fitness was predictable from attachment rate in most cases, 3 mutants had rapid attachment but low fitness on most hosts. Thus, some mutations had a pleiotropic effect on a fitness component other than attachment rate. In addition, on one host most mutants had high attachment rate but decreased fitness, suggesting that pleiotropic effects also depended on host. The data highlight that even in this simple, well-characterized system, prediction of fitness from a fitness component depends on genetic architecture and environment.     

Characterization of Streptococcus thermophilus host range phage mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10(-6). Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.     

Superinfection of Laodelphax striatellus with Wolbachia from Drosophila simulans

Wolbachia are maternally inherited, intracellular alpha-proteobacteria that infect a wide range of arthropods. They manipulate the reproduction of hosts to facilitate their spread into host populations, through ways such as cytoplasmic incompatibility (CI), parthenogenesis, feminization and male killing. The influence of Wolbachia infection on host populations has attracted considerable interest in their possible role in speciation and as a potential agent of biological control. In this study, we used both microinjection and nested PCR to show that the Wolbachia naturally infecting Drosophila simulans can be transferred into a naturally Wolbachia-infected strain of the small brown planthopper Laodelphax striatellus, with up to 30% superinfection frequency in the F(12) generation. The superinfected males of L. striatellus showed unidirectional CI when mated with the original single-infected females, while superinfected females of L. striatellus were compatible with superinfected or single-infected males. These results are, to our knowledge, the first to establish a superinfected horizontal transfer route for Wolbachia between phylogenetically distant insects. The segregation of Wolbachia from superinfected L. striatellus was observed during the spreading process, which suggests that Wolbachia could adapt to a phylogenetically distant host with increased infection frequency in the new host population; however, it would take a long time to establish a high-frequency superinfection line. This study implies a novel way to generate insect lines capable of driving desired genes into Wolbachia-infected populations to start population replacement.     

Frequency and Fitness Consequences of Bacteriophage Φ6 Host Range Mutations

Viruses readily mutate and gain the ability to infect novel hosts, but few data are available regarding the number of possible host range-expanding mutations allowing infection of any given novel host, and the fitness consequences of these mutations on original and novel hosts. To gain insight into the process of host range expansion, we isolated and sequenced 69 independent mutants of the dsRNA bacteriophage Φ6 able to infect the novel host, Pseudomonas pseudoalcaligenes. In total, we found at least 17 unique suites of mutations among these 69 mutants. We assayed fitness for 13 of 17 mutant genotypes on P. pseudoalcaligenes and the standard laboratory host, P. phaseolicola. Mutants exhibited significantly lower fitnesses on P. pseudoalcaligenes compared to P. phaseolicola. Furthermore, 12 of the 13 assayed mutants showed reduced fitness on P. phaseolicola compared to wildtype Φ6, confirming the prevalence of antagonistic pleiotropy during host range expansion. Further experiments revealed that the mechanistic basis of these fitness differences was likely variation in host attachment ability. In addition, using computational protein modeling, we show that host-range expanding mutations occurred in hotspots on the surface of the phage''s host attachment protein opposite a putative hydrophobic anchoring domain.     

Characterization of the ELPhiS prophage from Salmonella enterica serovar Enteritidis strain LK5

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the Salmonella enterica serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of Salmonella were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting Salmonella serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.     

Ecological conditions that favor the evolution of intermediate-virulence in an environmentally transmitted parasite

In this paper we develop and analyze several populaion-dynamic models of an environmentally transmitted symbiotic parasite infecting an isolated population of susceptible hosts. In our most basic model infection acts only to decrease the average lifetime of the infected host, parasites are only transmitted to uninfected hosts, there is no recovery from infection, and the rate of parasite transmission is an increasing function of the level of parasite virulence. It is shown that invasion of the parasite-free equilibrium cannot occur for virulence levels that are either too high or too low. We then incorporate a number of modifications to the model, among them the possibility that host fertility is reduced by infection, and that transmission rate depends additionally on susceptible host density. It is shown that the essential nature of the conditions for invasion are preserved. Thus, natural selection for intermediate virulence is a generic property of a broad class of population models.     

Evolution of pathogen virulence: the role of variation in host phenotype

Selection on pathogens tends to favour the evolution of growth and reproductive rates and a concomitant level of virulence (damage done to the host) that maximizes pathogen fitness. Yet, because hosts often pose varying selective environments to pathogens, one level of virulence may not be appropriate for all host types. Indeed, if a level of virulence confers high fitness to the pathogen in one host phenotype but low fitness in another host phenotype, alternative virulence strategies may be maintained in the pathogen population. Such strategies can occur either as polymorphism, where different strains of pathogen evolve specialized virulence strategies in different host phenotypes or as polyphenism, where pathogens facultatively express alternative virulence strategies depending on host phenotype. Polymorphism potentially leads to specialist pathogens capable of infecting a limited range of host phenotypes, whereas polyphenism potentially leads to generalist pathogens capable of infecting a wider range of hosts. Evaluating how variation among hosts affects virulence evolution can provide insight into pathogen diversity and is critical in determining how host pathogen interactions affect the phenotypic evolution of both hosts and pathogens.     

The Vertical Distribution and Diversity of Marine Bacteriophage at a Station off Southern California

Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5-10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment.     

Pleiotropic costs of niche expansion in the RNA bacteriophage phi 6

Natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. When a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. Here we investigate the evolution of expanded host range (generalism) in the RNA virus phi6, an experimental model system allowing adaptive mutations to be readily identified. We isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous mutations in the phi6 gene P3, important in host attachment. Seven of these nine mutations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in phi6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses.     

Shifts in diversification rates and host jump frequencies shaped the diversity of host range among Sclerotiniaceae fungal plant pathogens

The range of hosts that a parasite can infect in nature is a trait determined by its own evolutionary history and that of its potential hosts. However, knowledge on host range diversity and evolution at the family level is often lacking. Here, we investigate host range variation and diversification trends within the Sclerotiniaceae, a family of Ascomycete fungi. Using a phylogenetic framework, we associate diversification rates, the frequency of host jump events and host range variation during the evolution of this family. Variations in diversification rate during the evolution of the Sclerotiniaceae define three major macro‐evolutionary regimes with contrasted proportions of species infecting a broad range of hosts. Host–parasite cophylogenetic analyses pointed towards parasite radiation on distant hosts long after host speciation (host jump or duplication events) as the dominant mode of association with plants in the Sclerotiniaceae. The intermediate macro‐evolutionary regime showed a low diversification rate, high frequency of duplication events and the highest proportion of broad host range species. Our findings suggest that the emergence of broad host range fungal pathogens results largely from host jumps, as previously reported for oomycete parasites, probably combined with low speciation rates. These results have important implications for our understanding of fungal parasites evolution and are of particular relevance for the durable management of disease epidemics.     

Genetic basis of infectivity evolution in a bacteriophage

Antagonistic coevolution between hosts and parasites is probably ubiquitous. However, very little is known of the genetic changes associated with parasite infectivity evolution during adaptation to a coevolving host. We followed the phenotypic and genetic changes in a lytic virus population (bacteriophage; phage Φ2) that coevolved with its bacterial host, Pseudomonas fluorescens SBW25. First, we show the rapid evolution of numerous unique phage infectivity phenotypes, and that both phage host range and bacterial resistance to individual phage increased over coevolutionary time. Second, each of the distinct phage phenotypes in our study had a unique genotype, and molecular evolution did not act uniformly across the phage genome during coevolution. In particular, we detected numerous substitutions on the tail fibre gene, which is involved in the first step of the host-parasite interaction: host adsorption. None of the observed mutations could be directly linked with infection against a particular host, suggesting that the phenotypic effects of infectivity mutations are probably epistatic. However, phage genotypes with the broadest host ranges had the largest number of nonsynonymous amino acid changes on genes implicated in infectivity evolution. An understanding of the molecular genetics of phage infectivity has helped to explain the complex phenotypic coevolutionary dynamics in this system.     

Parasite strain coexistence in a heterogeneous host population

Heterogeneity in host susceptibility and transmissibility to parasite attack allows a lower transmission rate to sustain an epidemic than is required in homogeneous host populations. However, this heterogeneity can leave some hosts with little susceptibility to disease, and at high transmission rates, epidemic size can be smaller than for diseases where the host population is homogeneous. In a heterogeneous host population, we model natural selection in a parasite population where host heterogeneity is exploited by different strains to varying degrees. This partitioning of the host population allows coexistence of competing parasite strains, with the heterogeneity-exploiting strains infecting the more susceptible hosts, in the absence of physiological tradeoffs and spatial heterogeneity, and even for markedly different transmission rates. In our model, intermediate-strategy parasites were selected against: should coexistence occur, an equilibrium is reached where strains occupied only the extreme ends of trait space, under appropriate conditions selecting for lower R₀.     

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.     

Dynamics of Baltic Sea phages driven by environmental changes

Phage predation constitutes a major mortality factor for bacteria in aquatic ecosystems, and thus, directly impacts nutrient cycling and microbial community dynamics. Yet, the population dynamics of specific phages across time scales from days to months remain largely unexplored, which limits our understanding of their influence on microbial succession. To investigate temporal changes in diversity and abundance of phages infecting particular host strains, we isolated 121 phage strains that infected three bacterial hosts during a Baltic Sea mesocosm experiment. Genome analysis revealed a novel Flavobacterium phage genus harboring gene sets putatively coding for synthesis of modified nucleotides and glycosylation of bacterial cell surface components. Another novel phage genus revealed a microdiversity of phage species that was largely maintained during the experiment and across mesocosms amended with different nutrients. In contrast to the newly described Flavobacterium phages, phages isolated from a Rheinheimera strain were highly similar to previously isolated genotypes, pointing to genomic consistency in this population. In the mesocosm experiment, the investigated phages were mainly detected after a phytoplankton bloom peak. This concurred with recurrent detection of the phages in the Baltic Proper during summer months, suggesting an influence on the succession of heterotrophic bacteria associated with phytoplankton blooms.     

Evolutionary rescue and the coexistence of generalist and specialist competitors: an experimental test

Competition for resources is thought to play a critical role in both the origins and maintenance of biodiversity. Although numerous laboratory evolution experiments have confirmed that competition can be a key driver of adaptive diversification, few have demonstrated its role in the maintenance of the resulting diversity. We investigate the conditions that favour the origin and maintenance of alternative generalist and specialist resource-use phenotypes within the same population. Previously, we confirmed that competition for hosts among φ6 bacteriophage in a mixed novel (non-permissive) and ancestral (permissive) host microcosm triggered the evolution of a generalist phenotype capable of infecting both hosts. However, because the newly evolved generalists tended to competitively exclude the ancestral specialists, coexistence between the two phenotypes was rare. Here, we show that reducing the relative abundance of the novel host slowed the increase in frequency of the generalist phenotype, allowing sufficient time for the specialist to further adapt to the ancestral host. This adaptation resulted in ‘evolutionary rescue’ of the specialists, preventing their competitive exclusion by the generalists. Thus, our results suggest that competition promotes both the origin and maintenance of biodiversity when it is strong enough to favour a novel resource-use phenotype, but weak enough to allow adaptation of both the novel and ancestral phenotypes to their respective niches.     

Host availability and the evolution of parasite life-history strategies

Parasites exploit an inherently patchy resource, their hosts, which are discrete entities that may only be available for infection within a relatively short time window. However, there has been little consideration of how heterogeneities in host availability may affect the phenotypic or genotypic composition of parasite populations or how parasites may evolve to cope with them. Here we conduct a selection experiment involving an entomopathogenic nematode (Steinernema feltiae) and show for the first time that the infection rate of a parasite can evolve rapidly to maximize the chances of infecting within an environment characterized by the rate of host availability. Furthermore, we show that the parasite's infection rate trades off with other fitness traits, such as fecundity and survival. Crucially, the outcome of competition between strains with different infection strategies depends on the rate of host availability; frequently available hosts favor "fast" infecting nematodes, whereas infrequently available hosts favor "slow" infecting nematodes. A simple evolutionarily stable strategy (ESS) analysis based on classic epidemiological models fails to capture this behavior, predicting instead that the fastest infecting phenotype should always dominate. However, a novel model incorporating more realistic, discrete bouts of host availability shows that strain coexistence is highly likely. Our results demonstrate that heterogeneities in host availability play a key role in the evolution of parasite life-history traits and in the maintenance of phenotypic variability. Parasite life-history strategies are likely to evolve rapidly in response to changes in host availability induced by disease management programs or by natural dynamics in host abundance. Incorporating parasite evolution in response to host availability would therefore enhance the predictive ability of current epidemiological models of infectious disease.     

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.     

Characterization of Streptococcus thermophilus Host Range Phage Mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10⁻⁶. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.