Evolutionary genomics of host adaptation in vesicular stomatitis virus

Populations experiencing similar selection pressures can sometimes diverge in the genetic architectures underlying evolved complex traits. We used RNA virus populations of large size and high mutation rate to study the impact of historical environment on genome evolution, thus increasing our ability to detect repeatable patterns in the evolution of genetic architecture. Experimental vesicular stomatitis virus populations were evolved on HeLa cells, on MDCK cells, or on alternating hosts. Turner and Elena (2000. Cost of host radiation in an RNA virus. Genetics. 156:1465-1470.) previously showed that virus populations evolved in single-host environments achieved high fitness on their selected hosts but failed to increase in fitness relative to their ancestor on the unselected host and that alternating-host-evolved populations had high fitness on both hosts. Here we determined the complete consensus sequence for each evolved population after 95 generations to gauge whether the parallel phenotypic changes were associated with parallel genomic changes. We also analyzed the patterns of allele substitutions to discern whether differences in fitness across hosts arose through true pleiotropy or the presence of not only a mutation that is beneficial in both hosts but also 1 or more mutations at other loci that are costly in the unselected environment (mutation accumulation [MA]). We found that ecological history may influence to what extent pleiotropy and MA contribute to fitness asymmetries across environments. We discuss the degree to which current genetic architecture is expected to constrain future evolution of complex traits, such as host use by RNA viruses.     

Consequences of host adaptation for performance of vesicular stomatitis virus in novel thermal environments

Although it is widely assumed that the selective advantage of niche specialization drives species biodiversity, some theory suggests that generalists are favored over specialists when environments change unexpectedly. But this idea is rarely tested empirically, and its relevance is unknown for microparasites such as RNA viruses. Due to their small genome sizes pleiotropy is not uncommon in RNA viruses. Therefore, the genetic architectures underlying generalist traits may be indirectly molded by selection to better prepare generalist organisms for growth in new environments. Previously, vesicular stomatitis viruses were evolved to specialize on a single host, or to generalize on multiple hosts. Here we test whether virus generalists arising in the context of host adaptation also perform differently than specialists when viruses grow at novel temperatures. We compared thermal reaction norms of performance, within and among groups of viral specialists and generalists. Results showed that host adaptation was consequential for some fitness traits at novel temperatures due to modification of pleiotropic viral genes. Contrary to theoretical predictions, host generalists were selectively disadvantaged at extreme cool and warm environments. Multi-host adaptation may compromise the evolved thermostability of viral proteins, creating a cost of host generalization when viruses replicate at extreme temperatures.     

Evolutionary feedback mediated through population density, illustrated with viruses in chemostats

A cornerstone of evolutionary ecology is that population density affects adaptation: r and K selection is the obvious example. The reverse is also appreciated: adaptation impacts population density. Yet, empirically demonstrating a direct connection between population density and adaptation is challenging. Here, we address both evolution and ecology of population density in models of viral (bacteriophage) chemostats. Chemostats supply nutrients for host cell growth, and the hosts are prey for viral reproduction. Two different chemostat designs have profoundly different consequences for viral evolution. If host and virus are confined to the same chamber, as in a predator-prey system, viral regulation of hosts feeds back to maintain low viral density (measured as infections per cell). Viral adaptation impacts host density but has a small effect on equilibrium viral density. More interesting are chemostats that supply the viral population with hosts from a virus-free refuge. Here, a type of evolutionary succession operates: adaptation at low viral density leads to higher density, but high density then favors competitive ability. Experiments support these models with both phenotypic and molecular data. Parallels to these designs exist in many natural systems, so these experimental systems may yield insights to the evolution and regulation of natural populations.     

Dengue Virus RNA Structure Specialization Facilitates Host Adaptation

Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3’UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.     

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.     

Are RNA Viruses Adapting or Merely Changing?

RNA viruses and retroviruses fix substitutions approximately 1 million-fold faster than their hosts. This diversification could represent an inevitable drift under purifying selection, the majority of substitutions being phenotypically neutral. The alternative is to suppose that most fixed mutations are beneficial to the virus, allowing it to keep ahead of the host and/or host population. Here, relative sequence diversification of different proteins encoded by viral genomes is found to be linear. The examples encompass a wide variety of retroviruses and RNA viruses. The smoothness of relative divergence spans quasispeciation following clonal infection, to variation among different isolates of the same virus, to viruses from different species or those associated with different diseases, indicating that the majority of fixed mutations likely reflects drift. This held for both mammalian and plant viruses, indicating that adaptive immunity doesn't necessarily shape the relative accumulation of amino acid substitutions. When compared to their hosts RNA viruses evolution appears conservative. Received: 16 November 1999 / Accepted: 10 March 2000     

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.     

The molecular population genetics of the Tomato spotted wilt virus (TSWV) genome

RNA viruses are characterized by high genetic variability resulting in rapid adaptation to new or resistant hosts. Research for plant RNA virus genetic structure and its variability has been relatively scarce compared to abundant research done for human and animal RNA viruses. Here, we utilized a molecular population genetic framework to characterize the evolution of a highly pathogenic plant RNA virus [Tomato spotted wilt virus (TSWV), Tospovirus, Bunyaviridae]. Data from genes encoding five viral proteins were used for phylogenetic analysis, and for estimation of population parameters, subpopulation differentiation, recombination, divergence between Tospovirus species, and selective constraints on the TSWV genome. Our analysis has defined the geographical structure of TSWV, attributed possibly to founder effects. Also, we identify positive selection favouring divergence between Tospovirus species. At the species level, purifying selection has acted to preserve protein function, although certain amino acids appear to be under positive selection. This analysis provides demonstration of population structuring and species-wide population expansions in a multisegmented plant RNA virus, using sequence-based molecular population genetic analyses. It also identifies specific amino acid sites subject to selection within Bunyaviridae and estimates the level of genetic heterogeneity of a highly pathogenic plant RNA virus. The study of the variability of TSWV populations lays the foundation in the development of strategies for the control of other viral diseases in floral crops.     

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.     

Adaptation of Puumala hantavirus to cell culture is associated with point mutations in the coding region of the L segment and in the noncoding regions of the S segment

We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A-->G and U-->C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.     

Antagonistic coevolution limits population persistence of a virus in a thermally deteriorating environment

Understanding the conditions under which rapid evolutionary adaptation can prevent population extinction in deteriorating environments (i.e. evolutionary rescue) is a crucial aim in the face of global climate change. Despite a rapidly growing body of work in this area, little attention has been paid to the importance of interspecific coevolutionary interactions. Antagonistic coevolution commonly observed between hosts and parasites is likely to retard evolutionary rescue because it often reduces population sizes, and results in the evolution of costly host defence and parasite counter-defence. We used experimental populations of a bacterium Pseudomonas fluorescens SBW25 and a bacteriophage virus (SBW25Φ2), to study how host-parasite coevolution impacts viral population persistence in the face of gradually increasing temperature, an environmental stress for the virus but not the bacterium. The virus persisted much longer when it evolved in the presence of an evolutionarily constant host genotype (i.e. in the absence of coevolution) than when the bacterium and virus coevolved. Further experiments suggest that both a reduction in population size and costly infectivity strategies contributed to viral extinction as a result of coevolution. The results highlight the importance of interspecific evolutionary interactions for the evolutionary responses of populations to global climate change.     

Rate of novel host invasion affects adaptability of evolving RNA virus lineages

Although differing rates of environmental turnover should be consequential for the dynamics of adaptive change, this idea has been rarely examined outside of theory. In particular, the importance of RNA viruses in disease emergence warrants experiments testing how differing rates of novel host invasion may impact the ability of viruses to adaptively shift onto a novel host. To test whether the rate of environmental turnover influences adaptation, we experimentally evolved 144 Sindbis virus lineages in replicated tissue-culture environments, which transitioned from being dominated by a permissive host cell type to a novel host cell type. The rate at which the novel host ‘invaded’ the environment varied by treatment. The fitness (growth rate) of evolved virus populations was measured on each host type, and molecular substitutions were mapped via whole genome consensus sequencing. Results showed that virus populations more consistently reached high fitness levels on the novel host when the novel host ‘invaded’ the environment more gradually, and gradual invasion resulted in less variable genomic outcomes. Moreover, virus populations that experienced a rapid shift onto the novel host converged upon different genotypes than populations that experienced a gradual shift onto the novel host, suggesting a strong effect of historical contingency.     

The two faces of mutation: extinction and adaptation in RNA viruses

From a population standpoint, two main features characterize the replication of RNA viruses and viruses that use RNA as a replicative intermediate: high genetic variability, and enormous fluctuations in population size. Their genetic variability mainly reflects a lack of the proof-reading and post-replicative error correction mechanisms that operate during cellular DNA replication, but recombination and segment exchange can also play an important role. Viral population size can change tremendously as a consequence of transmission between hosts or between different tissues within an infected host. A new infection can be initiated with very few particles that subsequently expand many trillion-fold. Repeated bottleneck events can lead to drastic fitness losses or even to viral extinction, whereas continuously large population sizes result in fitness gains and adaptation. Here we review experimental evidence for the effects of mutation, selection, and genetic drift on the adaptation and extinction of RNA viruses.     

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.     

Cost of host radiation in an RNA virus

Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host. However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats.     

Insights into the selective pressures restricting Pelargonium flower break virus genome variability: Evidence for host adaptation

The molecular diversity of Pelargonium flower break virus (PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3' untranslated region (3' UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3' UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host Chenopodium quinoa and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to C. quinoa-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.     

Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989-12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.     

Faster Adaptation in Smaller Populations: Counterintuitive Evolution of HIV during Childhood Infection

Analysis of HIV-1 gene sequences sampled longitudinally from infected individuals can reveal the evolutionary dynamics that underlie associations between disease outcome and viral genetic diversity and divergence. Here we extend a statistical framework to estimate rates of viral molecular adaptation by considering sampling error when computing nucleotide site-frequencies. This is particularly beneficial when analyzing viral sequences from within-host viral infections if the number of sequences per time point is limited. To demonstrate the utility of this approach, we apply our method to a cohort of 24 patients infected with HIV-1 at birth. Our approach finds that viral adaptation arising from recurrent positive natural selection is associated with the rate of HIV-1 disease progression, in contrast to previous analyses of these data that found no significant association. Most surprisingly, we discover a strong negative correlation between viral population size and the rate of viral adaptation, the opposite of that predicted by standard molecular evolutionary theory. We argue that this observation is most likely due to the existence of a confounding third variable, namely variation in selective pressure among hosts. A conceptual non-linear model of virus adaptation that incorporates the two opposing effects of host immunity on the virus population can explain this counterintuitive result.     

STOCHASTIC TEMPERATURES IMPEDE RNA VIRUS ADAPTATION

Constant environments are often assumed to favor the evolution of specialization whereas exposure to changing environments may favor the evolution of generalists. Here we explored the phenotypic and molecular changes associated with evolving an RNA virus in constant versus fluctuating temperature environments. We used vesicular stomatitis virus (VSV) to determine whether selection at a constant temperature entails a performance trade‐off at an unselected temperature, whether virus populations evolve to be generalists when selected in deterministically changing temperature environments, and whether selection under stochastically changing temperatures prevents evolved generalization, such as by constraining the ability for viruses to adaptively improve. We observed that all VSV lineages evolved at constant temperatures showed fitness gains in their selected temperature with little evidence for trade‐offs in performance in the unselected environment. Evolution in deterministically and stochastically changing temperatures led to populations with the highest and lowest overall fitness gains, respectively. Sequence analysis revealed little evidence for convergent molecular evolution among lineages within the same treatment. Across all temperature treatments, the majority of genome substitutions occurred in the G (glycoprotein) gene, suggesting that this locus for the cell‐binding protein plays a key role in dictating VSV performance under changing temperature.     

Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

Background

Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.

Principal Findings

Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.

Significance

Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.