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Frank SA. Immunology and Evolution of Infectious Disease. Princeton (NJ): Princeton University Press; 2002.

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Immunology and Evolution of Infectious Disease.

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Chapter 12Experimental Evolution: Foot-and-Mouth Disease Virus

Experimental evolution manipulates the environment of a population and observes the resulting pattern of evolutionary change. This allows one to study the selective forces that shape antigenic diversity. For example, one could manipulate immune selection by exposing parasites to different regimes of monoclonal antibodies. The parasites' evolutionary response reveals the adaptive potential and the constraints that shape patterns of antigenic variation.

In this chapter, I describe experimental evolution studies of foot-and-mouth disease virus (FMDV). I also use this virus as a case study to show how different methods combine to provide a deeper understanding of antigenic variation. These approaches include structural analysis of the virion, functional analysis of epitopes with regard to binding cellular receptors, sequence analysis of natural isolates, and experimental analysis of evolving populations.

The first section introduces the antigenicity and structure of FMDV. Structural studies provide the three-dimensional location of amino acids. This allows one to analyze how particular amino acid substitutions affect shape, charge, and interaction with antibodies. Structural information also aids functional analysis of substitutions with regard to binding cellular receptors or affecting other components of viral fitness.

The second section describes antibody escape mutants of FMDV. Most of these escape mutants were generated by application of monoclonal antibodies in controlled experimental studies. Several laboratory escape mutants occur in an exposed loop on the surface of the virion, which is also the site of a key antigenic region identified by sequencing natural isolates. This antigenic loop mediates binding to cellular receptors, an essential step for viral entry into host cells. The pattern of antibody escape mutants identifies varying and unvarying amino acid sites. The unvarying sites play an essential role in binding to host cells.

The third section continues discussion of binding to host cells and tropism for different host receptors. Experimental evolution studies show that in cell culture FMDV can evolve to use alternate cellular receptors. This switch in receptor tropism relieves the constraint on the previously unvarying amino acid sites in the key antigenic region. Consequently, escape mutants in that conserved region arise readily, demonstrating that the conserved sites play an important role in recognition by antibodies. This highlights the dual selective pressures by antibodies and receptor binding that may shape key antigenic sites.

The fourth section describes an experimental approach to analyze the fitness consequences of amino acid substitutions. Molecular studies can measure changes in binding affinity for antibodies and cellular receptors associated with changes in amino acid shape and charge. But substitutions ultimately spread or fail based on their consequences for the dynamics of growth and transmission. These aspects of fitness can be difficult to measure. I describe one study in which pigs were injected with a wild-type virus and various antibody escape mutants. The relative success of parental and mutant viruses provides clues about how particular amino acid substitutions may influence evolutionary dynamics.

The final section lists problems for future research.

General discussions and examples of experimental evolution can be found in Rose (1991), Bennett and Lenski (1999), Landweber (1999), Crill et al. (2000), and Stearns et al. (2000).

12.1. Overview of Antigenicity and Structure

FMDV is an RNA virus that frequently causes disease in domesticated cattle, swine, sheep, and goats (Sobrino et al. 2001). FMDV belongs to the Picornaviridae family of viruses, which includes poliovirus, human hepatitis A virus, and the human rhinoviruses (Racaniello 2001). FMDV populations maintain antigenic diversity in several rapidly evolving epitopes (Mateu et al. 1988; Feigelstock et al. 1996).

Seven major serotypes occur across the world (Sobrino et al. 2001). Phylogenetic distance between serotypes correlates reasonably well with antigenic distance measured by cross-reactivity to polyclonal antisera—in other words, phylogeny roughly matches serology at a broad scale of sequence divergence (Mateu 1995). By contrast, small-scale phylogenetic divergence does not correspond to patterns of antigenicity. One or a few amino acid substitutions within a serotype can greatly alter antibody recognition (Mateu et al. 1994).

Figure 12.1 illustrates the structure of the surface proteins of FMDV. Figure 12.2 shows how the three types of surface proteins combine to form the FMDV capsid. The most widely immunodominant epitopes occur on the GH loop of the VP1 surface protein (see fig. 12.3; Mateu et al. 1994; Mateu 1995; Sobrino et al. 2001). This loop has about 20 amino acids that contribute to several overlapping epitopes. These antibody binding sites appear to be determined mostly by the amino acids in the GH peptide (a continuous epitope). Antibodies that bind to an isolated GH peptide also neutralize intact viruses.

Figure 12.1. Schematic diagram of the foot-and-mouth disease virus surface proteins.

Figure 12.1

Schematic diagram of the foot-and-mouth disease virus surface proteins. Each of the three main surface proteins, VP1, VP2, and VP3, fills a trapezoidal space with eight β chains (arrows) labeled B-I and two α chains (cylinders). The loops (more...)

Figure 12.2. Structure of the foot-and-mouth disease virus.

Figure 12.2

Structure of the foot-and-mouth disease virus. There are three surface proteins, VP1, VP2, and VP3, labeled 1–3, respectively. Each protein presents an approximately trapezoidal shape on the surface. The three different proteins group into a structural (more...)

Figure 12.3. An enlarged view of the β loops and carboxyl termini shown in fig.

Figure 12.3

An enlarged view of the β loops and carboxyl termini shown in fig. 12.2.

Many antibody escape variants occur in the GH loop, leading to extensive genetic variation in this region. However, a conserved amino acid triplet, Arg-Gly-Asp (RGD), also binds to antibodies. This conserved triplet mediates binding to integrin host-cell receptors typically used in FMDV attachment and entry (Berinstein et al. 1995; Neff et al. 1998; Sobrino et al. 2001).

The GH loop of VP1 contains continuous epitopes that together define the hypervariable antigenic site A common to all serotypes. Discontinuous epitopes occur when amino acid residues from widely separated sequence locations come together conformationally to form a binding surface for antibodies. Two antigenic sites of serotypes A, O, and C have discontinuous epitopes that have received widespread attention (Mateu et al. 1994; Feigelstock et al. 1996).

The first discontinuous site occurs near the capsid's threefold axes of symmetry at the vertices of the pentagonal structural units (fig. 12.2). This site includes the BC loop of VP2 and the BB knob of VP3 in serotypes A, C, and O (fig. 12.3). In serotype C, the carboxyl terminus of VP1 also contributes to this site, and in serotype O, the EF loop of VP2 is sometimes involved. This region (antigenic site D) forms the second major immunodominant region of serotype C after antigenic site A in the GH loop of VP1 (Feigelstock et al. 1996).

A second discontinuous epitope occurs in serotype O. The GH loop and the carboxy-terminal (COOH) end of VP1 jointly form the binding region for some antibodies. However, in serotype C, the GH loop and the carboxy-terminal end of VP1 form independent, continuous epitopes. The high specificity of antibodies means that the sequence and conformational differences between serotypes change the detailed antigenic properties of particular regions. Studies focused on natural selection of particular amino acid residues must account for background differences of sequence and conformation among test strains.

12.2. Antibody Escape Mutants

Many antibody escape mutants have been sequenced (references in Martínez et al. 1997). One can develop a map of natural escape variants by comparing changes in sequence with differences in binding affinity to a panel of MAbs.

Two problems of interpreting selective pressures arise from an escape map based on natural variants. First, field isolates do not control the multitude of evolutionary pressures on variation. Mutants may spread either in direct response to antibody pressure, in response to other selective pressures, or by stochastic fluctuations independent of selective forces. Lack of variability may result either from lack of antibody pressure or from constraining selective pressures such as binding to host receptors.

The second problem for interpreting selective pressures from natural isolates concerns lack of control over genetic background. Whether a particular amino acid site affects antibody affinity may depend on conformation-changing variants at other sites.

Site-directed mutagenesis controls amino acid replacements in a fixed genetic background. One can alter sites that do not vary naturally to test for effects on antibody binding. Site-directed mutagenesis has provided useful information for FMDV (Mateu et al. 1998). But this method can only define changes in antibody binding; it does not show how viral populations actually respond to immune pressure.

Several studies have applied monoclonal or polyclonal antibodies to FMDV in laboratory culture (Mateu 1995; Sobrino et al. 2001). This allows direct control of selective pressure by comparing lines with and without exposure to antibodies. In addition, cultures can be started with genetically monomorphic viruses to control genetic background.

Martínez et al. (1997) began laboratory evolution studies from a single viral clone of serotype C. These viruses were grown on baby hamster kidney cells (BHK-21). All host cells were derived from a single precursor cell. Two separate viral lines were established. C-S8c1 developed through three successive plaque isolations. C-S8c1p100 began with C-S8c1 and developed through one hundred serial passages on a monolayer of BHK-21 cells. The host cells were refreshed from independent stock in each passage and therefore did not coevolve with the virus over the passage history.

In natural isolates, extensive sequence variability in the GH loop of VP1 correlates with escape from antibody neutralization. However, the Arg-Gly-Asp (RGD) sequence near the center of this GH loop is invariant in field isolates (Sobrino et al. 2001). Controlled studies of laboratory evolution provide some insight into the evolution of this region.

The monoclonal antibody SD6 binds to an epitope spanning residues 136–147 in the GH loop of VP1. Martínez et al. (1997) applied selective pressure by SD6 after establishment of the separate viral lines C-S8c1 and C-S8c1p100 by growing a cloned (genetically monomorphic) isolate in the presence of the antibody and sampling escape mutants. Nucleotide sequences of escape mutants were obtained. Each mutant (except one) escaped antibody neutralization by a single amino acid change.

Table 12.1 lists the changed amino acids in the escape mutants, excluding the one double mutant. The different locations of these mutations in the original (C-S8c1) line compared with the serially passaged (C-S8c1p100) line provide the most striking result of this study. The original line conserved the Arg-Gly-Asp (RGD) motif at positions 141–143. By contrast, the serially passaged line had numerous mutations within the RGD motif. Figure 12.4 contrasts the location of mutants for the two lines.

Table 12.1. Escape mutants of FMDV (from Martínez et al. 1997).

Table 12.1

Escape mutants of FMDV (from Martínez et al. 1997).

Figure 12.4. Amino acid sequence in the central region of the VP1 GH loop of foot-and-mouth disease virus.

Figure 12.4

Amino acid sequence in the central region of the VP1 GH loop of foot-and-mouth disease virus. The start and stop numbers label amino acid positions. The box shows the RGD motif at positions 141–143. The monoclonal antibody SD6 recognizes the continuous (more...)

Variants in the RGD motif had not previously been observed in spite of neutralizing antibodies' affinity for this region. The RGD motif was thought to be invariant because of its essential role in binding to the host cell. Yet the serially passaged line accumulated variants in this region. Those variants replicated with the same kinetics as the parental viruses of C-S8c1p100, with no loss in fitness. Baranowski et al. (2000) showed that lineages with an altered RGD motif use an alternative pathway of attachment and entry to host cells.

Martínez et al. (1997) sequenced the capsid genes from the original line, from the serially passaged line, and from an escape mutant of the serially passaged line. The escape mutant from the serially passaged line differed from the parental virus of this line only at a single site in the RGD region. Tolerance to replacements in the RGD region must follow from the differences accumulated by C-S8c1p100 during serial passage. Table 12.2 shows the 6 amino acids that differed between the original and serially passaged lines. Apparently those substitutions changed cell tropism properties of the virus and allowed variation in the previously invariant RGD motif.

Table 12.2. Capsid amino acids that differ between C-S8c1 and C-S8c1p100 (from Martínez et al. 1997).

Table 12.2

Capsid amino acids that differ between C-S8c1 and C-S8c1p100 (from Martínez et al. 1997).

12.3. Cell Binding and Tropism

Attachment and entry to host cells impose strong natural selection on some regions of the viral surface. Experimental evolution provides one approach to analyzing those selective forces, as described in the previous section. In this section, I briefly summarize further studies of amino acid variation in the FMDV capsid and the consequences for attachment and entry to host cells.

Natural isolates of FMDV use cellular integrin receptors for some of the steps in attachment and entry (Berinstein et al. 1995; Jackson, Sheppard, et al. 2000). Integrins are transmembrane glycoproteins composed of two different subunits, α and β. Various integrins mediate adhesion between cells, attachment of cells to the extracellular matrix, and signal transduction of pathways that affect cell proliferation, morphology, migration, and apoptosis (Springer 1990; Hynes 1992; Montgomery et al. 1994).

The integrin receptors rely on an RGD amino acid sequence of ligands in order to bind host proteins such as fibronectin, fibrinogen, and type I collagen (Fox et al. 1989). All field isolates of FMDV have the conserved RGD motif needed for interaction with the integrin receptors (Berinstein et al. 1995; Jackson, Sheppard, et al. 2000).

FMDV can evolve changes in receptor usage, as shown by the experimental evolution studies of Martínez et al. (1997) described above. In their study, certain FMDV lineages mutated in the RGD motif and lost the ability to use integrin receptors. The altered viruses had a high affinity for heparan sulfate (HS) (Jackson et al. 1996; Sa-Carvalho et al. 1997), a common carbohydrate component of surface proteoglycans found on many types of host cells (Salmivirta et al. 1996; Fry et al. 1999; Sasisekharan and Venkataraman 2000).

An affinity for HS has been reported for several viruses, including HIV-1, human cytomegalovirus, dengue virus, Sindbis virus, vaccinia virus, and adeno-associated virus type 2 (Schneider-Schaulies 2000). HS may also play a role in bacterial adhesion (Rostand and Esko 1997; Hackstadt 1999). Some of these cases of increased affinity for HS may be caused by adaptation of the pathogens to cell culture, as occurred in FMDV, Sindbis virus, and classical swine fever virus (Klimstra et al. 1998; Bernard et al. 2000; Hulst et al. 2000).

These various studies call attention to the complementary processes of attachment and entry (Haywood 1994). In some cases, viruses may first attach to host cells based on the kinetics of binding between viral and host attachment sites. Once viruses bind to host attachment sites, a second-phase kinetic process determines binding between viral and host receptors that initiate viral entry into host cells. For example, FMDV in cell culture may first bind to HS, a widespread component of the host cell surface. The viruses, attracted near the cell surface, may then encounter and bind to the relatively sparser host integrin receptors.

Viral kinetics may be modulated separately for preliminary attachment and secondary binding to the port of entry. Structural and kinetic studies of FMDV variants provide some clues about how modulation of attachment and binding may occur.

FMDV type O adaptation to cell culture favors a histidine to arginine substitution at position 56 of the surface protein VP3 (Fry et al. 1999). This amino acid change increases the positive charge of the viral surface at this site and strongly enhances binding to negatively charged HS. Structural studies show that HS binds near the point of contact between the three surface proteins, VP1, VP2, and VP3 (figs. 12.2 and 12.3), including contact with codon 56 of VP3 on the βB strand (fig. 12.1).

Serotype A12 does not acquire HS binding in cell culture, instead modifying amino acids near the RGD sequence that presumably allow tighter binding to integrin (Reider et al. 1994; Neff et al. 1998). Not surprisingly, genetic background affects the binding consequences of amino acid substitutions and the evolutionary changes that occur in different strains.

HS provides a relatively low-affinity receptor at high density on the surface of many cell types. FMDV variants with increased attraction to HS may interact with host cells in two different ways. First, viruses may enter host cells directly from attachment to HS without binding and entering through a second host receptor (Neff et al. 1998; Baranowski et al. 2000). Second, low-affinity and high-density HS may serve as an attractant that brings viruses into proximity of high-affinity and low-density integrins (Jackson et al. 1996; Fry et al. 1999; Baranowski et al. 2000).

Various host adhesion molecules such as vitronectin and fibronectin have affinity for both HS and integrin (Potts and Campbell 1994). HS and integrin are sufficiently close on the host cell surface to interact simultaneously with viral binding sites for HS and integrin (Fry et al. 1999). Studies of other pathogens have inferred a two-step process with low-affinity receptors serving as the first site of adsorption (reviewed in Jackson et al. 1996; Schneider-Schaulies 2000).

HS-binding variants of FMDV derived from cell culture cannot develop virulent infections in vivo (Sa-Carvalho et al. 1997; Neff et al. 1998). Similarly, equine encephalitis virus adapted for cell culture gained enhanced HS binding and subsequently produced relatively weak, rapidly cleared infections in mice when compared with the wild type (Bernard et al. 2000). HS-binding variants of Sindbis virus are also cleared rapidly from hosts (Byrnes and Griffin 2000). HS-binding variants of FMDV with arginine at codon 56 of VP3 reverted to histidine or cysteine in experimental in vivo infections, demonstrating strong selection and rapid evolution of reduced HS affinity (Sa-Carvalho et al. 1997).

Strong binding to HS impedes the spread of infection between host cells. Viral particles may adhere too strongly to cells that cannot be infected, or the rate of clearance may be raised by exposure on tissue surfaces. Fry et al. (1999) speculated that HS-binding variation provides different kinetics of infection and clearance in various tissues and also quantitative modulation of virulence. Thus, pathogens may adapt within the host to different tissues by altering HS affinity. In addition, reduced virulence may sometimes be favored when associated with enhanced persistence of infection, perhaps by sequestering viruses at low abundance in certain tissues. Surface stickiness may therefore influence several aspects of pathogen kinetics within the host and the consequences of infection on host morbidity and mortality.

No evidence presently suggests that HS binding plays an important role in natural isolates of FMDV. Rather, these analyses should be interpreted as a model for studying how particular amino acid substitutions can profoundly alter kinetics and cellular tropisms. In each case, the benefits for increased rates of entry to host cells balance against the costs of reduced spread and faster clearance from certain host compartments. Combined studies of experimental evolution in vitro and in vivo provide a useful tool for studying how selective forces shape parasite characters via particular amino acid substitutions.

Comparisons of HS versus integrin binding form a contrast between two very different types of host receptors interacting with two distinct regions of the FMDV capsid. Recent studies have turned to more subtle variations between FMDV isolates with regard to binding different integrin receptors.

Jackson, Blakemore, et al. (2000) compared the affinity of different viral genotypes for two integrin receptors, ανβ3 and α5β1. The standard RGD motif was required for both receptors. The following amino acid at the RGD+1 position influenced relative affinity for the two integrin types. For ανβ3, several different amino acids at RGD+1 allowed binding, consistent with this receptor's multifunctional role in binding several ligands. By contrast, α5β1 has narrower specificity, favoring a leucine at RGD+1.

Jackson, Blakemore, et al. (2000) compared two viruses that differed only at RGD+1, the first with an arginine and the second with a leucine. The first virus had relatively higher affinity for ανβ3 compared with α5β1. By contrast, the second virus had relatively higher affinity for α5β1 compared with ανβ3. For at least some antibodies that recognize RGDL, loss of leucine at RGD+1 abolishes recognition (see fig. 12.4; Martínez et al. 1997).

Thirty type O and eight type A field isolates had leucine at RGD+1. By contrast, five SAT-2 isolates had arginine, two Asia-1 isolates had methionine, and one Asia-1 isolate had leucine (Jackson, Blakemore, et al. 2000). These and other data suggest that most serotypes have leucine at RGD+1 and perhaps a higher affinity for α5β1. SAT-2 may either have greater affinity for ανβ3 or its binding may be conditioned by amino acid variants at other sites.

In another study, Jackson, Sheppard, et al. (2000) analyzed FMDV binding to a different integrin, ανβ6. This integrin binds relatively few host ligands and depends on an RGDLXXL motif with leucines at RGD+1 and RGD+4. Most FMDV isolates have leucines at those two positions. ανβ3 does not have stringent requirements at those sites, suggesting that ανβ6 may be an important natural receptor.

Overall, RGDLXXL binds to the widest array of integrins, at least over those studied so far, although relative affinities for different integrins may be modulated by substitutions at RGD+1 and perhaps other sites. It would be interesting to sample isolates from various host tissues that differ in the densities of the various integrin receptors and analyze whether any substitutions appear relative to isolates in other body compartments of the same host.

Viral success in different cell types or in different hosts may depend on variations in nonstructural genes that do not mediate binding and entry to host cells. For example, Núñez et al. (2001) serially passaged FMDV in guinea pigs. FMDV does not normally cause lesions in guinea pigs, but after serial passage, viral variants arose that caused disease. Among the several amino acid substitutions that arose during passage, a single change from glutamine to arginine at position 44 of gene 3A provided virulence. The function of 3A in FMDV is not known. In poliovirus, a distantly related picornavirus, 3A plays a role in virus-specific RNA synthesis.

These studies show the potential power of experimental evolution in studying evolutionary forces, particularly when combined with analysis of naturally occurring variation.

12.4. Fitness Consequences of Substitutions

Antibody escape mutants are typically isolated in one of two ways. First, pathogens may be grown in vitro with antibodies. This creates selective pressure for substitutions that escape antibody recognition. Second, naturally occurring variants from field isolates may be tested against a panel of antibodies. Certain sets of antibodies may bind most isolates, allowing identification of those variants that differ at commonly recognized epitopes.

Escape variants gain a fitness advantage by avoiding antibody recognition targeted to important epitopes. However, those pathogen epitopes may also play a role in binding to host cells, in release from infected cells, or in some other aspect of the pathogen's life cycle. Functional and structural studies of amino acid substitutions provide one method of analysis. That approach has the advantage of directly assessing the mechanisms by which amino acid variants affect multiple components of parasite fitness, such as escape from antibody recognition and altered host attachment characteristics.

Although functional and structural approaches can directly measure binding differences caused by amino acid substitutions in different genetic backgrounds, they cannot provide a good measure of all the fitness consequences associated with changes in genotype.

Carrillo et al. (1998) used an alternative approach to analyze the consequences of amino acid substitutions. They studied the relative fitnesses in vivo of a parental FMDV genotype and three mutant genotypes derived from the parental type. They measured relative fitness by competing pairs of strains within live pigs.

The parental type, C-S8c1, came from a C serotype isolated from a pig. The first monoclonal antibody–resistant mutant, MARM21, arose in a pig infected with C-S8c1. MARM21 differs from C-S8c1 by a single change from serine to arginine at VP1 139 (fig. 12.4), providing escape from the monoclonal antibody SD6.

The second mutant, S-3T1, came from a blood sample of a pig one day after experimental inoculation with C-S8c1. That isolate had a single change from threonine to alanine at VP1 135 (fig. 12.4). Only one of fifty-eight monoclonal antibodies differentiated between the parental type and S-3T1, and the difference in affinity was small. This supports the claim in figure 12.4 that position 135 is not strongly antigenic.

The third mutant, C-S15c1, derived from a field variant of type C1 isolated from a pig. This mutant type had eight amino acid differences in VP1 compared with C-S8c1. C-S15c1 did not react with monoclonal antibody SD6.

One of the three mutants was coinoculated with the parental type into each experimental pig. Two replicate pigs were used for each of the three pairs of mutant and parental types. Fever rose one day after infection and peaked two or three days postinfection. All animals developed vesicular lesions two to four days postinfection. For each animal, between two and seven samples were taken from lesions, and the relative proportions of the competing viruses were assayed by reactivity to monoclonal antibodies.

The small sample sizes do not allow strong conclusions to be drawn. Rather, the following two results hint at what might be learned from more extensive studies of this sort. First, the parental type strongly dominated MARM21 in all seven lesions sampled from the two experimental animals, comprising between 80 and 94% of the viruses in each lesion. The MARM21 mutation appears to confer lower fitness in vivo, at least in the two animals tested. The lower fitness may arise because the mutant was cleared more effectively by antibodies, bound less efficiently to host cells, or had reduced performance in some other fitness component.

Second, S-3T1 abundance relative to the parental type varied strongly between lesions. In the two lesions analyzed from one animal, the parental type comprised 67 ±3.4 and 3.2 ±1.5% (mean ± standard deviation). In the other animal, the three lesions analyzed had parental-type percentages of 75 ± 4.1, 25 ± 2.8, and 5.9 ± 1.2. Differences in dominance between lesions also occurred between C-S15c1 and the parental type. Variations in dominance may arise from stochastic sampling of viruses that form lesions, from differences in tissue tropism, or from some other cause.

Further studies of this sort may provide a more refined understanding of the multiple fitness consequences that follow from particular amino acid changes, their interactions with the genetic background of the virus, the role of different host genotypes, and the effect of prior exposure of hosts to different antigenic variants.

12.5. Problems for Future Research

1. Escape versus performance

Both antibodies and cellular binding impose strong natural selection on the GH loop of VP1. This leads to a general question: How much does immune pressure impede natural selection of functional performance?

Experimental evolution may provide some insight into this problem. Consider two experimental lineages, one passaged in immunodeficient hosts and the other passaged in immunocompetent hosts. If immune pressure constrains functional performance by improved cellular binding, then the immunodeficient line should respond with amino acid substitutions that improve binding function.

In this context, improved binding function means increased viral fitness rather than increased affinity of the virus for the host receptor. Changes in fitness can be measured by competing the original genotype against the genotype created by selection in immunodeficient hosts. It would be interesting to study how amino acid substitutions affect the kinetics of cellular binding and reproduction and how those kinetics arise from structural changes in shape and charge. One could also compete these same genotypes in the immunocompetent line to study how amino acid substitutions change response to antibodies.

2. Components of fitness

Serial passage experiments impose a complex set of selective pressures on different components of pathogen fitness. For example, collecting pathogens from hosts early after infection favors very rapid reproduction within the host, perhaps at the expense of survival over the entire course of infection. By contrast, collecting pathogens late after infection favors survival within the host rather than rapid growth.

In a naive host without prior exposure to the pathogen, early sampling may pick pathogens before strong antibody pressure develops. This may favor amino acid substitutions that promote improved cellular binding over avoidance of immune pressure. By contrast, late sampling may favor more strongly avoidance of antibody pressure. Early and late sampling in both immunocompetent and immunodeficient hosts would allow comparison of amino acid substitutions under varying selective pressures.

One could also examine evolutionary response in experiments to test the idea that heparan sulfate binding modulates the pathogen's stickiness to different tissues and consequently the dynamics of growth and clearance.

3. Adaptation to new hosts

The passage experiments in guinea pigs showed that small changes in FMDV genotype allow virulent infections to develop in novel hosts. Host adaptation forms the central problem in the study of emerging diseases. Experimental evolution provides a useful tool to identify the amino acid changes required to infect new hosts, to cause virulent infections in those hosts, to transmit between the new hosts, and to transmit back to the original host.

4. Genetic background

Pathogen genotypes that differ by many amino acids can have significantly altered protein shape and charge. It can be difficult to assess how those structural differences affect selection on particular amino acid sites. Experimental evolution studies could analyze a replicated design in which initial pathogen genotypes vary. This approach can identify how genetic background alters selective pressure at particular sites.

Different genotypes may be chosen from natural isolates to study the forces that shape particular variants in the field. Or special genotypes may be constructed to test hypotheses about how structure affects the fitness of amino acid substitutions at particular sites.

5. Experimental evolution of other pathogens

Most experimental evolution studies of pathogens have been conducted on RNA viruses. These viruses often grow easily in culture, grow to large population sizes, mutate frequently, and evolve quickly. RNA viruses also tend to have very small genomes, making it easy to identify and sequence evolving genes.

Experimental evolution will become an important tool for studying other kinds of pathogens. In section 5.5, I proposed an experiment to study programmed antigenic switching in organisms such as Borrelia hermsii.

The mechanistic issue concerned whether switch rates between different archival variants within a single genome could be modulated by natural selection, and if so, by what changes in DNA sequence or regulatory control. This highlights experimental evolution's role as a tool to study biochemical mechanism.

The evolutionary problem concerned the extent to which switch rates adapt to enhance bacterial fitness versus the extent to which mechanistic properties of switching constrain rates of switching between variants. This highlights experimental evolution's role in studying the constraints that govern evolutionary adaptation.

Copyright © 2002, Steven A Frank.
Bookshelf ID: NBK2393

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