Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.     

Host alternation is necessary to maintain the genome stability of rift valley fever virus

Background

Most arthropod-borne viruses (arboviruses) are RNA viruses, which are maintained in nature by replication cycles that alternate between arthropod and vertebrate hosts. Arboviruses appear to experience lower rates of evolution than RNA viruses that replicate in a single host. This genetic stability is assumed to result from a fitness trade-off imposed by host alternation, which constrains arbovirus genome evolution. To test this hypothesis, we used Rift Valley fever virus (RVFV), an arbovirus that can be transmitted either directly (between vertebrates during the manipulation of infected tissues, and between mosquitoes by vertical transmission) or indirectly (from one vertebrate to another by mosquito-borne transmission).

Methodology/Principal Findings

RVFV was serially passaged in BHK21 (hamster) or Aag2 (Aedes aegypti) cells, or in alternation between the two cell types. After 30 passages, these single host-passaged viruses lost their virulence and induced protective effects against a challenge with a virulent virus. Large deletions in the NSs gene that encodes the virulence factor were detectable from the 15^th serial passage onwards in BHK21 cells and from the 10^th passage in Aag2 cells. The phosphoprotein NSs is not essential to viral replication allowing clones carrying deletions in NSs to predominate as they replicate slightly more rapidly. No genetic changes were found in viruses that were passaged alternately between arthropod and vertebrate cells. Furthermore, alternating passaged viruses presenting complete NSs gene remained virulent after 30 passages.

Conclusions/Significance

Our results strongly support the view that alternating replication is necessary to maintain the virulence factor carried by the NSs phosphoprotein.     

West Nile virus experimental evolution in vivo and the trade-off hypothesis

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.     

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.     

Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.     

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.     

Insect-transmitted vertebrate viruses: Alphatogaviruses

Summary Alphatogaviruses, of which Sindbis virus (SV) is the prototype, replicate to high titer in the laboratory both in mosquito cells and in vertebrate cells. By studying the replication of SV in mosquito cells as well as in vertebrate cells, we were able to obtain several viral mutants which have novel phenotypes and have contributed to our basic knowledge of this virus family. These include three host range mutants: SV_AP15/21 which replicates normally in mosquito cells but is restricted in vertebrate cells and SV_CL35 and SV_CL58, which are restricted in mosquito cells but replicate normally in vertebrate cells. As well, two other mutants are described here: SV_LM21, which can replicate in methionine-starved mosquito cells and SV_MPA, which can replicate in mosquito cells treated with mycophenolic acid or ribavirin. The causal mutations of both SV_LM21 and SV_MPA are within the sequence encoding the nonstructural protein nsP1; these and other findings have enabled us to associate the capping and methylation of the viral mRNAs with the nsP1 protein. Our work serves to emphasize that it is both worthwhile and important to study the replication of arthropod-borne viruses in cells derived from the arthropod host as well as in cells derived from the vertebrate host.     

Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures

The mechanisms by which RNA arboviruses, including chikungunya virus (CHIKV), evolve and maintain the ability to infect vertebrate and invertebrate hosts are poorly understood. To understand how host specificity shapes arbovirus populations, we studied CHIKV populations passaged alternately between invertebrate and vertebrate cells (invertebrate ↔ vertebrate) to simulate natural alternation and contrasted the results with those for populations that were artificially released from cycling by passage in single cell types. These CHIKV populations were characterized by measuring genetic diversity, changes in fitness, and adaptability to novel selective pressures. The greatest fitness increases were observed in alternately passaged CHIKV, without drastic changes in population diversity. The greatest increases in genetic diversity were observed after serial passage and correlated with greater adaptability. These results suggest an evolutionary trade-off between maintaining fitness for invertebrate ↔ vertebrate cell cycling, where maximum adaptability is possible only via enhanced population diversity and extensive exploration of sequence space.Emergence of pathogenic RNA viruses is associated with their genomic variability and environmental changes that lead to novel host contacts. Many new and reemerging RNA viruses have been introduced into humans (10). Arthropod-borne viruses (arboviruses), including dengue virus (DENV), have caused recent epidemics by changing their host ranges to increase infection of humans (54). Adaptation to the urban mosquito Aedes albopictus may have expanded a 2005-2006 outbreak of Chikungunya virus (CHIKV) in Reunion Island, France (46), that subsequently circulated among humans in the absence of other amplifying hosts.Despite these emergence events, the evolutionary processes that mediate arbovirus host range changes are poorly understood, partly since arbovirus evolution is understudied. Arboviruses are transmitted horizontally between arthropod vectors and vertebrate reservoir hosts. They replicate rapidly and achieve large population sizes. Polymerases of RNA viruses lack proofreading to repair errors, leading to one substitution per ∼10⁻⁴ nucleotides (nt) copied (11, 36), corresponding to one error per 10-kb genome. This polymerase infidelity leads to diversification that produces closely related but nonidentical RNAs that together form a spectrum of mutants. Although arbovirus mutant spectra have been observed in nature (1, 8, 20, 55, 57), the diversity and divergence within the spectrum are not well described, and the phenotypic roles of minority RNAs are unknown. Understanding the mutation distribution in a heterogeneous arbovirus population is important, given that any variant can be favored by selection and ultimately affect fitness (12, 13). However, the relationships between fitness and RNA virus population diversity are poorly understood.Studies with other RNA viruses, including human immunodeficiency virus (HIV) (3, 58, 60), hepatitis C virus (14, 15, 25), and poliovirus (30, 52), indicate that intrahost population diversity is important for virus evolution, fitness, and pathogenesis. Unlike these vertebrate-only RNA viruses, arboviruses obligately cycle between vertebrates and arthropods, a process that imposes additional selective constraints on evolution and adaptation. Sequence comparisons of RNA arbovirus isolates show that they are relatively stable (18, 19), and genetic studies reveal that evolution is dominated by purifying selection (20-22, 57). This constancy of consensus sequence may derive from the need to infect disparate hosts that present conflicting demands for adaptation where sequence changes that improve fitness in one host may not be maintained in the alternate organism.Experimental evolution studies have been performed to study fitness trade-offs and the unique ability of arboviruses to simultaneously evolve to vertebrate and invertebrate hosts. In vitro evolution studies reveal three general patterns of arbovirus evolution: (i) fitness gains after serial passage in vertebrate or invertebrate cells (except in certain cases [7]) and losses in bypassed host cell types (DENV, Eastern equine encephalitis virus [EEEV], Sindbis virus [SINV], and vesicular stomatitis virus [VSV]) (17, 28, 51, 56); (ii) reduced fitness in new cells (VSV) (28), and (iii) fitness increases after alternating (invertebrate ↔ vertebrate) passage (DENV, EEEV, SINV, VSV) (17, 28, 51, 56). Together, these studies suggest that constraints on fitness differ in insect and vertebrate cells and can be virus specific but that arbovirus fitness is not limited by alternating between vertebrate and invertebrate hosts.An in vivo study revealed a similar pattern of arbovirus evolution: vertebrate-passaged Venezuelan equine encephalitis virus (VEEV) was five times more fit than its unpassaged parent, and mosquito-passaged VEEV was more infectious for vectors (6). Consensus sequences revealed that, despite becoming more fit, mutations in passaged populations were slight or absent. This suggests that fitness increases were mediated by minority genomes in the mutant spectrum. However, a major limitation of the in vivo experiments and other arbovirus evolution studies is that sequencing of individual RNAs from the mutant spectrum in passaged intrahost populations was not performed (although notable exceptions for flaviviruses exist [5, 22]). The identity of minority variants within intrahost arbovirus populations and their influences on phenotype have not been extensively examined.The goal of this study, therefore, is to understand how obligate host cycling shapes an intrahost arbovirus population. RNA viruses can tolerate increased niche breadth when they evolve in heterogeneous environments (48), a trait which may promote easier emergence and therefore help explain why arboviruses frequently emerge or reemerge to cause human and veterinary disease. One limitation of increasing breadth may be restricted genetic diversity, where only genomes accepted in both hosts can be maintained. To explore this possibility, we described arbovirus populations after invertebrate ↔ vertebrate cell cycling and compared them to populations that were artificially released from alternating passage via serial vertebrate or invertebrate cell transfer. Since previous studies indicate that intrahost RNA virus population diversity can determine phenotype (3, 37, 52) and given that increases in arbovirus fitness are not always associated with consensus genome changes (6, 27), we determined how arbovirus population diversity (mutation frequency) and distance (number of mutations by which each RNA differs from the consensus) relate to fitness. We predicted that genetic diversity and distance between RNAs in the mutant spectrum and the corresponding consensus are correlated with fitness and that diverse populations better escape varied selective pressures than genetically homogeneous populations since, by chance, they are more likely to contain an advantageous mutation(s) when the pressure is applied.Chikungunya virus (family Togaviridae, genus Alphavirus) was selected primarily because of its medical relevance. CHIKV has caused outbreaks of human disease characterized by arthralgia and myalgia since the 18th century and since 2004 in Africa, Indian Ocean islands, Southeast Asia, and Italy (32). Furthermore, no adaptation studies have been performed for CHIKV, and no intrahost population studies have been conducted for an alphavirus. An infectious clone was generated from the strain used for passages and was genetically marked to differentiate its RNAs from passaged CHIKV, so that after direct competition, the fitnesses of passaged populations could be compared to those for progenitors.     

Differential evolution of eastern equine encephalitis virus populations in response to host cell type

Arthropod-borne viruses (arboviruses) cycle between hosts in two widely separated taxonomic groups, vertebrate amplifying hosts and invertebrate vectors, both of which may separately or in concert shape the course of arbovirus evolution. To elucidate the selective pressures associated with virus replication within each portion of this two-host life cycle, the effects of host type on the growth characteristics of the New World alphavirus, eastern equine encephalitis (EEE) virus, were investigated. Multiple lineages of an ancestral EEE virus stock were repeatedly transferred through either mosquito or avian cells or in alternating passages between these two cell types. When assayed in both cell types, derived single host lineages exhibited significant differences in infectivity, growth pattern, plaque morphology, and total virus yield, demonstrating that this virus is capable of host-specific evolution. Virus lineages grown in alternation between the two cell types expressed intermediate phenotypes consistent with dual adaptation to both cellular environments. Both insect-adapted and alternated lineages greatly increased in their ability to infect insect cells. These results indicate that different selective pressures exist for virus replication within each portion of the two-host life cycle, and that alternation of hosts selects for virus populations well adapted for replication in both host systems.     

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.     

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.     

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.     

Vertical Transmission Selects for Reduced Virulence in a Plant Virus and for Increased Resistance in the Host

For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed (vertical) transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence.     

Sequential adaptive mutations enhance efficient vector switching by Chikungunya virus and its epidemic emergence

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics.     

Evolution of Sindbis Virus with a Low-Methionine-Resistant Phenotype Is Dependent Both on a Pre-Existing Mutation and on the Methionine Concentration in the Medium

SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium. Whether the passage was begun with SV319 or with SV321, the dominant virus population which emerged always acquired the second SVlm21 amino acid change. However, when the passage was begun with virus having neither the nt 319 or the nt321 mutation, even after many passages neither of these mutations was seen in the passaged virus population. Virus with the LMR phenotype emerged earlier when the virus encoded a wild-type RDRP (passage 4) rather than the mutant RDRP encoded by SVpzf (passage 7). When the methionine concentration in the medium of mosquito cells was increased to 250 µM, more than 20 passages were required until the LMR phenotype predominated. Competition experiments were carried out to compare the relative fitness of SVlm21, SVwt, SV319 and SV321 to each other. Our results indicated that SVlm21 was dominant to SVwt, as well as to both SV319 and SV321. However, SV319 and SV321 were able to co-exist with SVwt implying that in these mixed infection the presence of SVwt inhibited the emergence of SVlm21. Finally, our experiments highlight how a virus population by mutation and selection can adapt to the intracellular concentration of a simple metabolite, S-adenosylmethionine.     

A genetic background with low mutational robustness is associated with increased adaptability to a novel host in an RNA virus

Although mutational robustness is central to many evolutionary processes, its relationship to evolvability remains poorly understood and has been very rarely tested experimentally. Here, we measure the evolvability of Vesicular stomatitis virus in two genetic backgrounds with different levels of mutational robustness. We passaged the viruses into a novel cell type to model a host‐jump episode, quantified changes in infectivity and fitness in the new host, evaluated the cost of adaptation in the original host and analyzed the genetic basis of this adaptation. Lineages evolved from the less robust genetic background demonstrated increased adaptability, paid similar costs of adaptation to the new host and fixed approximately the same number of mutations as their more robust counterparts. Theory predicts that robustness can promote evolvability only in systems where large sets of genotypes are connected by effectively neutral mutations. We argue that this condition might not be fulfilled generally in RNA viruses.     

Spherical Influenza Viruses Have a Fitness Advantage in Embryonated Eggs,while Filament-Producing Strains Are Selected In Vivo

Influenza viruses can take on two distinct morphologies: filamentous or spherical. While the functional significance of each virion type is unclear, filaments are generally observed in low-passage-number isolates, while an exclusively spherical morphology is seen in strains grown extensively in laboratory substrates. Previous studies have shown that filamentous morphology is lost upon passage in eggs. The fact that the filamentous morphology is maintained in nature but not in the laboratory suggests that filaments provide an advantage in the host that is not necessary for growth in laboratory substrates. To test this hypothesis and identify naturally occurring mutations that alter morphology, we examined the effect of serial adaptation in eggs, MDCK cells, and guinea pigs. Two filamentous strains, A/Netherlands/602/2009 (H1N1) and A/Georgia/M5081/2012 (H1N1), were passaged in eggs and MDCK cells. Conversely, the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) was passaged in guinea pigs. We found that although passage in eggs and MDCK cells can lead to a loss of filaments, an exclusively spherical morphology is not required for highly efficient growth in either substrate. We did, however, identify two point mutations in the matrix of egg passage 10 isolates that confer spherical morphology and increased growth in eggs. In contrast, serial passage in guinea pigs resulted in the selection of filament-forming variants. Sequencing revealed point mutations to the PR8 matrix that, when introduced individually, yielded filaments. These findings suggest a functional role for filaments in the infected host and expand the breadth of mutations known to affect influenza virus shape.     

Repeated transfer of small RNA virus populations leading to balanced fitness with infrequent stochastic drift

The population dynamics of RNA viruses have an important influence on fitness variation and, in consequence, on the adaptative potential and virulence of this ubiquitous group of pathogens. Earlier work with vesicular stomatitis virus showed that large population transfers were reproducibly associated with fitness increases, whereas repeated transfers from plaque to plaque (genetic bottlenecks) lead to losses in fitness. We demonstrate here that repeated five-plaque to five-plaque passage series yield long-term fitness stability, except for occasional stochastic fitness jumps. Repeated five-plaque passages regularly alternating with two consecutive large population transmissions did not cause fitness losses, but did limit the size of fitness gains that would otherwise have occurred. These results underscore the profound effects of bottleneck transmissions in virus evolution.     

Delayed transmission selects for increased survival of vesicular stomatitis virus

Life-history theory predicts that traits for survival and reproduction cannot be simultaneously maximized in evolving populations. For this reason, in obligate parasites such as infectious viruses, selection for improved between-host survival during transmission may lead to evolution of decreased within-host reproduction. We tested this idea using experimental evolution of RNA virus populations, passaged under differing transmission times in the laboratory. A single ancestral genotype of vesicular stomatitis virus (VSV), a negative-sense RNA Rhabdovirus, was used to found multiple virus lineages evolved in either ordinary 24-h cell-culture passage, or in delayed passages of 48 h. After 30 passages (120 generations of viral evolution), we observed that delayed transmission selected for improved extracellular survival, which traded-off with lowered viral fecundity (slower exponential population growth and smaller mean plaque size). To further examine the confirmed evolutionary trade-off, we obtained consensus whole-genome sequences of evolved virus populations, to infer phenotype–genotype associations. Results implied that increased virus survival did not occur via convergence; rather, improved virion stability was gained via independent mutations in various VSV structural proteins. Our study suggests that RNA viruses can evolve different molecular solutions for enhanced survival despite their limited genetic architecture, but suffer generalized reproductive trade-offs that limit overall fitness gains.