Experimental Evolution Studies in Φ6 Cystovirus

Abstract

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Full text

Citation: Singhal, S.; Balitactac, A.K.;
Nayagam, A.G.; Pour Bahrami, P.;
Nayeem, S.; Turner, P.E. Experimental
Evolution Studies in Φ6 Cystovirus.
Viruses 2024,16, 977. https://
doi.org/10.3390/v16060977
Academic Editors: Paul Gottlieb and
Aleksandra Alimova
Received: 27 April 2024
Revised: 5 June 2024
Accepted: 8 June 2024
Published: 18 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
viruses
Review
Experimental Evolution Studies in Φ6 Cystovirus
Sonia Singhal 1, *, Akiko K. Balitactac 1, † , Aruna G. Nayagam 1, †, Parnian Pour Bahrami 1,† , Sara Nayeem 1
and Paul E. Turner 2,3,4
1Department of Biological Sciences, San JoséState University, San José, CA 95192, USA;
akikokaitlin.balitactac@sjsu.edu (A.K.B.); aruna.gomathinayagam@sjsu.edu (A.G.N.);
parnian.pourbahrami@sjsu.edu (P.P.B.); sara.nayeem@sjsu.edu (S.N.)
2Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA;
paul.turner@yale.edu
3Program in Microbiology, Yale School of Medicine, New Haven, CT 06520, USA
4Center for Phage Biology and Therapy, Yale University, New Haven, CT 06511, USA
*Correspondence: sonia.singhal@sjsu.edu
†These authors contributed equally to this work.
Abstract: Experimental evolution studies, in which biological populations are evolved in a specific
environment over time, can address questions about the nature of spontaneous mutations, responses
to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years
of experimental evolution studies using the bacteriophage (phage)
Φ
6 cystovirus. Similar to many
lab-studied bacteriophages,
Φ
6 has a high mutation rate, large population size, fast generation time,
and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in
the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its
segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single
host cell make
Φ
6 a good non-pathogenic model for investigating the evolution of RNA viruses that
infect humans. We describe experiments that used
Φ
6 to address the fitness effects of spontaneous
mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges,
and mechanisms and consequences of the evolution of thermostability. We highlight open areas of
inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.
Keywords:
Φ
6; cystovirus; bacteriophage; experimental evolution; mutational effects; coinfection;
genetic exchange; host range; thermostability; genetic robustness
1. Introduction
Experimental evolution studies allow evolutionary processes to be examined in bi-
ological populations propagated over time in novel or control environments (Figure 1).
Such experiments have proven useful to address a wide variety of topics, including the
spontaneous nature of adaptive mutations, the role of population size in determining
responses to selection, and the origins and consequences of novel traits. Microbes, which
have relatively shorter generation times, larger population sizes, and (sometimes) higher
mutation rates than other organisms, have been particularly efficient for studying such
“evolution-in-action” [
1
]. In addition, microbes can be cryogenically frozen and later re-
vived, allowing for direct comparisons between ancestors and their evolved descendants,
and their small genome sizes foster affordable recombinant genetic experiments to explore
genotype-phenotype mapping. Evolutionary processes that could take years or decades to
examine in macro-organisms can be observed in real time in a matter of weeks or months
using microbial systems.
Viruses 2024,16, 977. https://doi.org/10.3390/v16060977 https://www.mdpi.com/journal/viruses

Viruses 2024,16, 977 2 of 17
Viruses 2024, 16, x FOR PEER REVIEW 2 of 18
Figure 1. Schematics of evolution experiments using serial transfer of phage populations in the la-
boratory. Typically, an ancestral clone or population of phages (circles) is allowed to infect bacterial
host cells under novel or control environmental conditions. At end of the passage (e.g., after 24 hours
of incubation), a sample of the phage population is transferred with dilution (e.g., 1:100, creating a
boleneck) into a fresh culture vessel with host cells, and a second sample is stored frozen to permit
later analyses. Over time, the phage population is expected to fix mutations (red circles) that increase
fitness (survival and/or reproduction) in the novel environment. (Top): Phages are serially trans-
ferred in liquid cultures containing bacterial host cells (rectangles). (Boom): Phages are grown on
lawns of bacterial hosts to form plaques (white circles, indicating zones of lysed bacterial cells). One
or more plaques are collected to obtain a cell-free lysate, followed by plating with dilution onto a
fresh host lawn.
In this review, we focus on the rich history of evolutionary studies using the bacteri-
ophage (phage) Φ6. The genome of Φ6 and other cystoviruses is divided into three dsRNA
segments (termed L, M, and S for their relative sizes, large, medium, and small) [2,3],
making it possible for different Φ6 genotypes to coinfect the same host cell and produce
hybrid progeny viruses via the process of reassortment [4]. As an RNA virus, phage Φ6
has an especially high mutation rate (2.7–5.0 × 10
−6
per nucleotide, [5]) and a short infection
cycle (a 70-minute [6] to 120-minute [7] lysis time). It shares similarities in structure and
assembly with eukaryotic Reoviruses [8,9]. These properties have made Φ6 an aractive
model for studying various evolutionary questions, such as the effects of mutation accu-
mulation on population fitness, the origins and adaptive maintenance of genetic exchange
(sex), the emergence of RNA viruses on novel hosts, and the evolution of virus thermo-
stability.
Figure 1. Schematics of evolution experiments using serial transfer of phage populations in the
laboratory. Typically, an ancestral clone or population of phages (circles) is allowed to infect bacterial
host cells under novel or control environmental conditions. At end of the passage (e.g., after 24 h
of incubation), a sample of the phage population is transferred with dilution (e.g., 1:100, creating
a bottleneck) into a fresh culture vessel with host cells, and a second sample is stored frozen to
permit later analyses. Over time, the phage population is expected to fix mutations (red circles) that
increase fitness (survival and/or reproduction) in the novel environment. (Top):Phages are serially
transferred in liquid cultures containing bacterial host cells (rectangles). (Bottom): Phages are grown
on lawns of bacterial hosts to form plaques (white circles, indicating zones of lysed bacterial cells).
One or more plaques are collected to obtain a cell-free lysate, followed by plating with dilution onto
a fresh host lawn.
In this review, we focus on the rich history of evolutionary studies using the bacterio-
phage (phage)
Φ
6. The genome of
Φ
6 and other cystoviruses is divided into three dsRNA
segments (termed L, M, and S for their relative sizes, large, medium, and small) [
2
,
3
],
making it possible for different
Φ
6 genotypes to coinfect the same host cell and produce
hybrid progeny viruses via the process of reassortment [
4
]. As an RNA virus, phage
Φ
6 has
an especially high mutation rate (2.7–5.0
×
10
−6
per nucleotide, [
5
]) and a short infection
cycle (a 70-min [
6
] to 120-min [
7
] lysis time). It shares similarities in structure and assembly
with eukaryotic Reoviruses [
8
,
9
]. These properties have made
Φ
6 an attractive model for
studying various evolutionary questions, such as the effects of mutation accumulation on
population fitness, the origins and adaptive maintenance of genetic exchange (sex), the
emergence of RNA viruses on novel hosts, and the evolution of virus thermostability.

Viruses 2024,16, 977 3 of 17
2. Exploring Fitness Effects of Mutations Using Φ6
2.1. Effects of Deleterious and Nearly Neutral Mutations
Mutations provide the raw material for evolution via natural selection, underlying
adaptive traits that affect relative fitness (survival and/or reproduction compared to other
genetic variants; Box 1). However, aside from mutations that provide such beneficial
effects, genetic changes can be deleterious or neutral for fitness. Thus, the frequency and
effect distribution of the mutations that occur in a population can heavily impact whether
evolutionary processes are adaptive, maladaptive, or neutral over time [10].
Box 1. Glossary of terms.
Coinfection: Occurs when two or more virus particles infect the same host cell.
Complementation: A decreased effect of a deleterious mutation, caused by a mutation at a
different position or, during viral coinfection, in a different genome.
Cost-free: A change in phenotype or genotype that is not associated with a detectable decrease
in fitness.
Epistasis: An interaction between two or more loci that causes a departure from a strictly
additive null expectation of their effects. When considering deleterious mutations, a combined
effect less deleterious than the null (higher fitness) indicates positive synergistic epistasis, and a
combined effect more deleterious than the null (lower fitness) indicates negative synergistic epistasis.
Evolvability: The capacity for a lineage to undergo greater evolutionary change in future
generations.
Fitness: The ability of a genotype to survive and produce offspring, relative to other genotypes
in the population.
Generalist: A biological entity that can use multiple different resources, such as a virus that is
capable of infecting different host genotypes or species.
Genetic drift: Changes in the allele frequencies in a population across generations, due to
random fluctuations.
Genetic robustness: The maintenance of a phenotype despite the introduction of mutations in
the genome.
Host range: The number of host genotypes or species that a virus is able to infect.
Muller’s ratchet: The theory proposed by H.J. Muller that predicts that the buildup of deleterious
mutations (and hence, fitness decline) is inevitable in an asexual population of a small size.
Multiplicity of infection (MOI): The ratio of virus particles to host cells. At an MOI < 1, cellular
infection by individual particles is likely, whereas at an MOI > 1, coinfection occurs with higher
probability.
Mutational load: A buildup of mutations in an evolving lineage which can reduce mean fitness
over time.
Mutation accumulation: A serial transfer experiment designed to assess the occurrence and
frequency of deleterious or nearly neutral mutations empirically, such as asexual populations that
experience bottlenecks of a single individual between each transfer.
Reassortment: In multi-partite (segmented) viruses, reassortment is the exchange of genome
segments during coinfection, which can result in the production of hybrid progeny.
Recombination: The formation of a new genotype through the breakage and joining of genetic
material from multiple (two or more) parental genotypes.
Sex: The exchange of genetic material between distinct parental genomes, resulting in hybrid
progeny.
Specialist: A biological entity that uses only a single resource, such as a virus that is confined to
infecting one host genotype or species.
Thermostability: The resistance to chemical or physical degradation when exposed to an elevated
temperature.
Trade-off : An increase in fitness or performance in one trait at the cost of decreased fitness or
performance in another trait.
Natural selection operates less efficiently in populations of small sizes, because random
fluctuations in allele frequencies across generations (genetic drift; Box 1) cause the inheritance
of mutations that are not necessarily beneficial for fitness. The geneticist H.J. Muller
posed an idea called ‘Muller’s ratchet’ [
11
,
12
] (Box 1), which predicted that this process of
evolution via genetic drift should be especially problematic in clonal (asexual) populations
of small sizes. Similar to a ratchet tool that only moves in one direction, these populations

Viruses 2024,16, 977 4 of 17
should inevitably accumulate deleterious mutations over time, creating a ‘mutational load’
(Box 1) that reduces average fitness. The theory assumes that the rate of reversions and
compensatory mutations must be less than that of deleterious mutations.
However, neutral and nearly neutral mutations often go undetected due to absent
or minimal fitness effects, and deleterious mutations can be eliminated by selection. This
makes it difficult to quantify their effects. To measure the distributions and impacts of
small-effect mutations, Chao and colleagues [
5
,
13
,
14
] performed mutation accumulation
(MA; Box 1) experiments using phage
Φ
6. During the serial transfer of the virus population
onto host lawns (Figure 1, bottom), an extreme bottleneck occurs if a single viral plaque (i.e.,
the progeny of a single founding genotype) is used to create the lysate that establishes the
next generation, because this plaque was formed by a single phage particle. If the plaque is
chosen at random, this process allows genetic drift to dominate over natural selection: the
chosen plaque may have spontaneous mutations that are either deleterious or have small
effects on fitness, and these mutations accumulate over subsequent transfers. Expected
decreases in fitness over succeeding generations can be observed by measuring changes in
fitness as the MA experiment proceeds (e.g., see Figure 2, bottleneck size = 1).
Viruses 2024, 16, x FOR PEER REVIEW 5 of 18
Figure 2. The relationship between boleneck size during transfers in an evolution experiment (see
Figure 1) and the average effect of mutations that reach high frequency in the population after 50–
100 transfers. The average mutational effect was calculated as the total difference in fitness between
the wild-type ancestor and the final population, divided by the estimated number of mutations.
Data are from Table 1 of Burch and Chao 1999 [15].
2.2. Effects of Adaptive Mutations
In contrast to MA experiments, studies that use very large populations sizes of virus
particles for bolenecks during serial transfers (Figure 1) should favor evolutionary
changes due to natural selection rather than genetic drift. Here, beneficial mutations can
more easily fix, and adaptation can proceed. Burch and Chao (1999) [15] used a Φ6 clone
from a previous MA experiment [13,16] as the founding ancestor of replicate populations
propagated under various boleneck sizes (10, 33, 100, 333, 1000, 2500, and 10,000 indi-
viduals). Under the assumption that the distribution of spontaneous beneficial mutations
should be dominated by those of small effect, it was predicted that adapting populations
must be sufficiently large in numerical size to fix beneficial mutations with strong fitness
effects. Results confirmed that populations that were evolved under smaller bolenecks
sequentially fixed mutations with minor fitness effects, while larger bolenecks permied
more rapid increases in fitness due to the fixation of large-effect mutations (Figure 2, [15]).
Thus, the experimental evolution of Φ6 lineages confirmed that the average population
size (i.e., the harmonic mean of the boleneck and the maximum population sizes) influ-
ences the opportunity for mutations of differing effect sizes to dictate adaptive change.
We note that selection experiments can also be used to examine the effects of epistasis
on evolution. Burch and Chao (2000) [14] looked at the trajectory of fitness changes in Φ6
populations over time when virus lineages were propagated using either of two different
ancestral phage genotypes (viral clones). Each lineage reached different fitness maxima,
suggesting that de novo mutations had variable epistatic effects that depended on their
occurrences in the distinct genome compositions of the founding ancestors [14].
3. Consequences of Coinfection
Evolutionary biologists have long considered the benefits and costs of sex (Box 1),
broadly defined as the exchange of genetic material between genomes, in populations. On
the one hand, a generalized benefit of sex is that it can reduce linkage disequilibrium: sex
can recombine multiple beneficial alleles into the same genetic background faster than
Figure 2. The relationship between bottleneck size during transfers in an evolution experiment
(see Figure 1) and the average effect of mutations that reach high frequency in the population after
50–100 transfers. The average mutational effect was calculated as the total difference in fitness between
the wild-type ancestor and the final population, divided by the estimated number of mutations. Data
are from Table 1 of Burch and Chao 1999 [15].
Chao (1990) [
13
] and Burch et al. (2007) [
10
] performed MA experiments to examine
how mutations of deleterious and small effect impacted the fitness of
Φ
6 populations. Fitness
was evaluated either by comparing the growth rate (average particle production) of phages
during or after MA relative to that of their founding ancestor [
13
], or through measurements
of plaque size [
5
,
10
], known in
Φ
6 to correlate with particle production measurements [
5
].
Both studies confirmed fitness decreases over time, consistent with predictions of Muller’s
ratchet. By comparing the empirical fitness data to simulations of fitness changes in evolving
populations, analyses confirmed that models based solely on the occurrence of deleterious
mutations provided a better fit than those based on the occurrence of beneficial mutations,
implying that MA lineages fixed harmful mutations [
5
,
10
]. Additionally, mutations of small
effect were more common than mutations of large effect [10].
Burch et al. (2007) [
10
] also conducted genetic sequencing of evolved phages to connect
decreases in fitness with specific underlying mutations. The majority of the mutations

Viruses 2024,16, 977 5 of 17
detected were single-nucleotide substitutions, located randomly among the three dsRNA
genome segments of
Φ
6. In some MA lines, there were more observed mutations than
discrete occurrences of fitness reductions [
10
], suggesting that some accumulated mutations
may have been neutral for fitness.
MA experiments can also be used to evaluate the presence and degree of epistasis
(interactions between mutations; Box 1). In the absence of epistasis, it is assumed that
each additional deleterious mutation will reduce fitness independently from the effects
of other genetic changes. However, if deleterious mutations exhibit positive synergistic
epistasis, then genotypes with additional mutations should experience lesser reductions in
fitness (causing higher than expected fitness) as mutations accrue. In contrast, if deleterious
mutations exhibit negative synergistic epistasis, then genotypes with additional mutations
should experience greater losses in fitness (faster declines in fitness than expected).
By tracking changes in fitness across genotypes with differing initial fitness values,
Burch and Chao (2004) [
5
] found that additional mutations in the
Φ
6 genome had a smaller
negative effect on fitness when viruses were already of low (but not high) fitness. This
finding suggested that epistasis among deleterious mutations is, on average, positively
synergistic in Φ6.
2.2. Effects of Adaptive Mutations
In contrast to MA experiments, studies that use very large populations sizes of virus
particles for bottlenecks during serial transfers (Figure 1) should favor evolutionary changes
due to natural selection rather than genetic drift. Here, beneficial mutations can more easily
fix, and adaptation can proceed. Burch and Chao (1999) [
15
] used a
Φ
6 clone from a previous
MA experiment [
13
,
16
] as the founding ancestor of replicate populations propagated under
various bottleneck sizes (10, 33, 100, 333, 1000, 2500, and 10,000 individuals). Under the
assumption that the distribution of spontaneous beneficial mutations should be dominated
by those of small effect, it was predicted that adapting populations must be sufficiently large
in numerical size to fix beneficial mutations with strong fitness effects. Results confirmed
that populations that were evolved under smaller bottlenecks sequentially fixed mutations
with minor fitness effects, while larger bottlenecks permitted more rapid increases in
fitness due to the fixation of large-effect mutations (Figure 2, [
15
]). Thus, the experimental
evolution of
Φ
6 lineages confirmed that the average population size (i.e., the harmonic
mean of the bottleneck and the maximum population sizes) influences the opportunity for
mutations of differing effect sizes to dictate adaptive change.
We note that selection experiments can also be used to examine the effects of epistasis
on evolution. Burch and Chao (2000) [
14
] looked at the trajectory of fitness changes in
Φ
6
populations over time when virus lineages were propagated using either of two different
ancestral phage genotypes (viral clones). Each lineage reached different fitness maxima,
suggesting that de novo mutations had variable epistatic effects that depended on their
occurrences in the distinct genome compositions of the founding ancestors [14].
3. Consequences of Coinfection
Evolutionary biologists have long considered the benefits and costs of sex (Box 1),
broadly defined as the exchange of genetic material between genomes, in populations. On
the one hand, a generalized benefit of sex is that it can reduce linkage disequilibrium: sex
can recombine multiple beneficial alleles into the same genetic background faster than
their expected occurrence via mutation alone, such that adaptation proceeds relatively
quickly. Similarly, sex can recombine harmful alleles into common backgrounds, so that
selection is more efficient at purging the resulting low-fitness genotypes from an evolving
population [
17
–
19
]. Conversely, sex may break apart beneficial combinations of alleles (e.g.,
coadapted gene complexes), illustrating a generalized cost of sex [20–22].
Viral sex cannot occur unless multiple, genetically distinct particles coinfect the same
host cell. Opportunities for viral sex should thus be determined by the probabilities for
coinfection (Box 1), which can be manipulated experimentally by controlling the multiplicity

Viruses 2024,16, 977 6 of 17
of infection (MOI), or the ratio of phage particles to bacterial cells. Assuming that a Poisson
process determines the extent of coinfection at any given MOI, at relatively low MOI (<<1.0),
it is expected that most infections will occur through a single phage entering a host cell on
its own, which favors asexuality (clonality). By contrast, at relatively high MOI (>>1.0), the
prediction is that the vast majority of infections will involve multiple (2 or more) phages
entering one bacterium, which fosters sexuality [
23
]. The tripartite genome of
Φ
6 (three
dsRNA segments per phage particle) can permit the formation of hybrid progeny viruses
containing segments from different coinfecting virus ‘parents’ [
24
] (Figure 3), a process
known as reassortment (Box 1). (Recombination, the breaking and joining of homologous
segments, is either nonexistent in natural
Φ
6 populations or occurs at rates too low to
measure [
25
–
27
].) Early experiments in
Φ
6 found that reassortment alone allowed mutated
segments to be combined into low-fitness hybrids that were then eliminated by selection,
reversing Muller’s ratchet and supporting an advantage of sex [13,16,19].
Viruses 2024, 16, x FOR PEER REVIEW 6 of 18
their expected occurrence via mutation alone, such that adaptation proceeds relatively
quickly. Similarly, sex can recombine harmful alleles into common backgrounds, so that
selection is more efficient at purging the resulting low-fitness genotypes from an evolving
population [17–19]. Conversely, sex may break apart beneficial combinations of alleles
(e.g., coadapted gene complexes), illustrating a generalized cost of sex [20–22].
Viral sex cannot occur unless multiple, genetically distinct particles coinfect the same
host cell. Opportunities for viral sex should thus be determined by the probabilities for
coinfection (Box 1), which can be manipulated experimentally by controlling the multiplic-
ity of infection (MOI), or the ratio of phage particles to bacterial cells. Assuming that a Pois-
son process determines the extent of coinfection at any given MOI, at relatively low MOI
(<<1.0), it is expected that most infections will occur through a single phage entering a
host cell on its own, which favors asexuality (clonality). By contrast, at relatively high MOI
(>>1.0), the prediction is that the vast majority of infections will involve multiple (2 or
more) phages entering one bacterium, which fosters sexuality [23]. The tripartite genome
of Φ6 (three dsRNA segments per phage particle) can permit the formation of hybrid prog-
eny viruses containing segments from different coinfecting virus parents [24] (Figure 3),
a process known as reassortment (Box 1). (Recombination, the breaking and joining of ho-
mologous segments, is either nonexistent in natural Φ6 populations or occurs at rates too
low to measure [25–27].) Early experiments in Φ6 found that reassortment alone allowed
mutated segments to be combined into low-fitness hybrids that were then eliminated by
selection, reversing Mullers ratchet and supporting an advantage of sex [13,16,19].
Figure 3. Possible consequences of coinfection in Φ6. Φ6 (the hexagons) has three genetic segments
(vertical bars), named Small, Medium, and Large for their relative lengths. When two or more Φ6
phage particles infect a bacterial cell (the rounded rectangle), they are able to exchange genetic in-
formation. In the case of recombination, crossover during replication results in segments that con-
tain genetic information from each phage parent. In the case of reassortment, the new phage pack-
ages an entire, genetically different segment. Phage particles may also cheat by preferentially pack-
aging one or more of their own segments into the capsid of a genetically distinct phage.
3.1. Constraints on Adaptation during Coinfection
The ability for sex to combine independent beneficial or deleterious mutations for
rapid improvement in population fitness suggested that sex may alter the rate at which
Φ6 lineages adaptively improve growth on their host bacteria. Turner and Chao (1998)
[23] leveraged the ability to manipulate the MOI of Φ6 populations to control the fre-
quency of coinfection, and hence whether sex is common versus infrequent during the
Figure 3. Possible consequences of coinfection in
Φ
6.
Φ
6 (the hexagons) has three genetic segments
(vertical bars), named Small, Medium, and Large for their relative lengths. When two or more
Φ
6 phage particles infect a bacterial cell (the rounded rectangle), they are able to exchange genetic
information. In the case of recombination, crossover during replication results in segments that
contain genetic information from each phage parent. In the case of reassortment, the new phage
packages an entire, genetically different segment. Phage particles may also ‘cheat’ by preferentially
packaging one or more of their own segments into the capsid of a genetically distinct phage.
3.1. Constraints on Adaptation during Coinfection
The ability for sex to combine independent beneficial or deleterious mutations for
rapid improvement in population fitness suggested that sex may alter the rate at which
Φ
6
lineages adaptively improve growth on their host bacteria. Turner and Chao (1998) [
23
]
leveraged the ability to manipulate the MOI of
Φ
6 populations to control the frequency
of coinfection, and hence whether sex is common versus infrequent during the viruses’
adaptive process. Three lineages of
Φ
6 were transferred for 250 virus generations at an
MOI of 0.002 (i.e., from 4
×
10
6
phages/mL to 2
×
10
9
bacteria/mL), whereas three other
lineages experienced an MOI of 5 (i.e., from 1
×
10
10
phages/mL to 2
×
10
9
bacteria/mL).
Phages were allowed to adsorb to their bacterial host in liquid at the treatment MOI (0.002
or 5), and were then plated on lawns of uninfected bacteria. To control phage population
sizes, different numbers of plaques were collected (Figure 1, bottom) for the next round of
adsorption: 500 plaques (each resulting from the progeny of one adsorbed phage) were
collected from the low-MOI populations, and 100 plaques (each resulting from the progeny

Viruses 2024,16, 977 7 of 17
of five adsorbed phages) were collected from the high-MOI populations. The fitness of
the evolved phages was measured at different time points in evolution by comparing the
growth rate (average particle production) of the test phages relative to that of the common
ancestor phage.
If genetic variation generated through sex (reassortment during coinfection) was
beneficial for adaptation, the high-MOI populations would show relatively greater fitness
than their clonal counterparts by the end of the experiment. However, this hypothesis
was not generally supported. In the low-MOI populations, the relative fitness in the
selected (MOI = 0.002) and unselected (MOI = 5) environments did increase monotonically
over evolutionary time. However, the high-MOI populations increased in fitness in their
selected (MOI = 5) environment, but performed poorly in the unselected (MOI = 0.002)
environment. This context-dependent result indicated that the
Φ
6 lineages evolved at a
high MOI suffered a performance trade-off. That is, the fitness of these virus populations
evolved under frequent coinfection relied on the continued ability to experience coinfection,
otherwise they showed poor performance in the low-MOI (clonal) environment [23].
This result was later explained by the realization that a high MOI permitted not
only the opportunity for reassortment, but also caused selection to differ between the
two treatments [
28
]. In the low-MOI populations, phages experienced selection to better
exploit the host cells to produce progeny, whereas in the high-MOI populations, frequent
coinfection caused phages to experience intracellular competition as well. Further study of
a representative phage genotype that had evolved at a high MOI,
Φ
H2, revealed that it had
higher fitness relative to the wild-type ancestor virus in the presence of coinfection, but
lower fitness when infecting cells on its own. This result was consistent with the famous
Prisoner’s Dilemma outcome in evolutionary game theory, which explains how cheating
can be positively selected in biological systems despite the evolution of lower fitness when
cheaters take over the population [
28
]. Measurements of viral traits for
Φ
H2 confirmed
that, when infecting cells on its own, the genotype was less productive (had a smaller burst
size) than the wild-type ancestor, indicating that when coinfecting host cells,
Φ
H2 ‘cheats’
by somehow gaining a productivity advantage over non-cheater viruses [29,30].
Turner and Chao (2003) [
30
] offered possible explanations for how cheating could
function mechanistically in
Φ
H2, based on observations of evolved cheaters in other virus
systems [
31
–
33
], but the details of the proposed mechanism have not been confirmed to date.
One possibility is that
Φ
H2 is relatively inefficient in making capsids when infecting cells
on its own [
29
], but is biased in favor of placing its own genome into all procapsids when
coinfecting a cell with the wild-type or other non-cheater viruses. If true, the underlying
basis for biased procapsid entry must be due to mutation(s) that distinguish the
Φ
H2
cheater from its wild-type ancestor. Support for this idea would be the observation that
one or more segments in the cheater virus are necessary and sufficient to reduce
Φ
H2
productivity during clonal infection at a low MOI, and to increase
Φ
H2 productivity in a
mixed, high-MOI infection. This can be examined by introducing each evolved segment of
the cheater virus into the wild-type genomic background and testing whether any of the
resulting reassortant hybrids shows a growth disadvantage in the low-MOI environment,
and a growth advantage at a high MOI (Figure 4). Interestingly, preliminary data are
consistent with the first hypothesis, and suggest that the evolved M segment of the
Φ
H2
cheater is alone capable of recapitulating the poor productivity of this virus at a low
MOI. The hypothesis of biased procapsid entry is also consistent with the earlier use of
a marker on the M segment to measure the fitness of the
Φ
H2 cheater relative to the
wild-type
Φ
6 in competition assays [
23
,
28
,
30
], suggesting that the allele(s) responsible
for cheating could reside solely on the M segment. Further confirmation, however, must
come from experiments demonstrating that the evolved M segment is also necessary and
sufficient to explain the fitness advantage of the
Φ
H2 cheater at a high MOI, and that one
or more mutations on this segment can drive the cheater’s preferential procapsid entry
during coinfection.

Viruses 2024,16, 977 8 of 17
Viruses 2024, 16, x FOR PEER REVIEW 8 of 18
marker on the M segment to measure the fitness of the ΦH2 cheater relative to the wild-
type Φ6 in competition assays [23,28,30], suggesting that the allele(s) responsible for cheat-
ing could reside solely on the M segment. Further confirmation, however, must come from
experiments demonstrating that the evolved M segment is also necessary and sufficient to
explain the fitness advantage of the ΦH2 cheater at a high MOI, and that one or more
mutations on this segment can drive the cheaters preferential procapsid entry during
coinfection.
Figure 4. Hypothesized fitnesses of wild-type Φ6, ΦH2 (cheater), and hybrid reassortants containing
one genomic segment of ΦH2 in the wild-type background when infecting host cells. ΦH2 performs
worse than the wild-type Φ6 at a low MOI, but beer than the wild-type Φ6 in a mixed, high-MOI
infection. Reassortant 1 shows no evidence that its effect on host bacterial growth differs from that
of the wild-type (i.e., the introduced genomic segment does not contain mutation(s) from ΦH2 for
poor productivity at a low MOI or high productivity at a high MOI). By contrast, Reassortant 2
recapitulates the poor productivity of ΦH2 on host cells at a low MOI and its high productivity at a
high MOI, suggesting that its introduced segment likely contains at least one mutation associated
with these phenotypes in ΦH2.
3.2. Constraints on Genetic Diversity due to Coinfection
Frequent versus infrequent coinfection should also be consequential for average ge-
netic diversity in phage populations. Because sex allows for the creation of genetic varia-
tion beyond typical changes (point mutations, insertions, deletions), virus populations
evolved at a high MOI should be more genetically diverse than lineages evolved under
clonality. After 300 generations of experimental evolution at a high or a low MOI, ten
clones (plaques) from treatment populations of Φ6 were chosen at random and partially
sequenced [34]. Contrary to expectations, the low-MOI (clonal) populations showed
higher average genetic diversity (13 mutations) compared to their high-MOI (sexual)
counterparts (4 mutations). This difference was largely due to greater numbers of muta-
tions in the low-MOI populations in the 5 untranslated regions of the S segment. Dennehy
et al. (2013) [34] also observed eight different mutations in the P5 lysis protein, five of
which were synonymous substitutions. Two of the synonymous mutations were beneficial
for fitness measured through competition assays. One of the beneficial mutations in P5 (a
change from g to u at nucleotide gene position 591) was found to occur in both the low-
MOI and high-MOI treatment populations, suggesting it provides a generalized ad-
vantage for virus growth, whereas the other beneficial P5 mutation (a change from c to u
at nucleotide gene position 507) was observed only in a low-MOI population [34].
Figure 4. Hypothesized fitnesses of wild-type
Φ
6,
Φ
H2 (cheater), and hybrid reassortants containing
one genomic segment of
Φ
H2 in the wild-type background when infecting host cells.
Φ
H2 performs
worse than the wild-type
Φ
6 at a low MOI, but better than the wild-type
Φ
6 in a mixed, high-MOI
infection. Reassortant 1 shows no evidence that its effect on host bacterial growth differs from that
of the wild-type (i.e., the introduced genomic segment does not contain mutation(s) from
Φ
H2 for
poor productivity at a low MOI or high productivity at a high MOI). By contrast, Reassortant 2
recapitulates the poor productivity of
Φ
H2 on host cells at a low MOI and its high productivity at a
high MOI, suggesting that its introduced segment likely contains at least one mutation associated
with these phenotypes in ΦH2.
3.2. Constraints on Genetic Diversity due to Coinfection
Frequent versus infrequent coinfection should also be consequential for average ge-
netic diversity in phage populations. Because sex allows for the creation of genetic variation
beyond typical changes (point mutations, insertions, deletions), virus populations evolved
at a high MOI should be more genetically diverse than lineages evolved under clonality.
After 300 generations of experimental evolution at a high or a low MOI, ten clones (plaques)
from treatment populations of
Φ
6 were chosen at random and partially sequenced [
34
].
Contrary to expectations, the low-MOI (clonal) populations showed higher average genetic
diversity (13 mutations) compared to their high-MOI (sexual) counterparts (4 mutations).
This difference was largely due to greater numbers of mutations in the low-MOI popu-
lations in the 5’ untranslated regions of the S segment. Dennehy et al. (2013) [
34
] also
observed eight different mutations in the P5 lysis protein, five of which were synonymous
substitutions. Two of the synonymous mutations were beneficial for fitness measured
through competition assays. One of the beneficial mutations in P5 (a change from gto u
at nucleotide gene position 591) was found to occur in both the low-MOI and high-MOI
treatment populations, suggesting it provides a generalized advantage for virus growth,
whereas the other beneficial P5 mutation (a change from cto uat nucleotide gene position
507) was observed only in a low-MOI population [34].
Based on these observations, the frequent opportunity for reassortment in phage
Φ
6
populations cultured at a high MOI seems to reduce the average genetic variation, despite
contrary expectations. The explanation could be that dsRNA segments that contain dele-
terious mutations are maintained in virus populations experiencing frequent coinfection,
because their deleterious effects can be masked (complemented) in trans by higher fitness
phages during intracellular coinfection [
23
,
28
,
35
] (Box 1). This effect would cause weaker
selection for removal of deleterious mutations from the virus genome [
35
]. Furthermore,

Viruses 2024,16, 977 9 of 17
frequent coinfection could foster exchange of entire segments (reassortment) at high enough
rates that variability is homogenized in a phage population. In contrast, a low-MOI en-
vironment can allow clonal interference, where independent viral mutants compete with
each other to fix in the phage population. Mutants of similar or equal fitnesses may persist
for prolonged periods, increasing genetic diversity in asexual populations [
34
]. Effects of
clonal interference may also explain why deleterious mutations in
Φ
6 were observed to be
purged faster in the absence of coinfection [35].
The rates of fitness improvement and levels of standing genetic variation observed in
Φ
6 experimental evolution studies tend not to support expectations for the evolutionary
benefits of sex. But these expectations assume that the evolutionary origin and/or adaptive
maintenance of viral sex in phage
Φ
6 is beneficial, and arose for the purpose of promoting
variation. The segmentation of the dsRNA genome in
Φ
6 and other viruses may have
evolved for other reasons, such as the efficient packaging of the RNA genome into capsids
during intracellular replication [4,36].
4. Host Range Evolution
The host range (Box 1) of a virus is the number of hosts (genotypes or species) that it
can infect to produce progeny particles. Although the host range should ideally include all
of the native host(s) that the wild-type virus typically infects, it is difficult or impossible to
know the true host range for a virus in its natural environment. Thus, the study of virus
host ranges in the laboratory often explores how mutational changes can either expand (to
include additional novel hosts) or reduce (eliminate the ability to infect one or more original
hosts) the number of susceptible hosts. Generalist viruses, which have a relatively broader
host range, seem to be more likely to emerge successfully on new hosts, whereas specialist
viruses with relatively narrower host ranges may be constrained in emergence [
37
–
41
]
(Box 1). Therefore, studies of host-range evolution in viruses are fundamentally important
for understanding the mechanisms that make viruses more or less likely to emerge on new
hosts in the future.
Phage
Φ
6 was originally isolated from bean straw infested with Pseudomonas syringae
pathovar phaseolicola bacteria (later reclassified as P. savastanoi pv. phaseolicola [
42
,
43
]), which
acts as the typical host in laboratory studies. Wild-type
Φ
6 also has the ability to infect
several related bacterial species: P. syringae pvs. persicae and tagetis, and P. savastanoi pv.
savastanoi [
44
]. Furthermore,
Φ
6 can easily gain single-nucleotide mutations that expand
its host range to infect novel hosts, including P. savastanoi pv. glycinea [
45
], P. syringae pv.
tomato [
44
,
46
,
47
] and pv. atrofaciens [
6
,
44
,
46
], and P. pseudoalcaligenes East River isolate A
(ERA) [
44
,
46
,
48
]. The ability for
Φ
6 to infect various hosts natively and to emerge on others
via single-step mutations has made it an attractive model for studying viral emergence and
the evolutionary genetics of virus specialism and generalism.
4.1. First-Step Mutations Fostering Emergence on Novel Hosts
Host-range mutants of
Φ
6 with broadened infectivity on novel host(s) often show
fitness decreases on the original host.
Φ
6 mutants that infect ERA [
49
,
50
], P. glycinea [
45
],
P. tomato [
49
], or P. atrofaciens [
46
,
49
] typically have lower fitnesses than wild-type
Φ
6 on
the typical lab host, P. phaseolicola. These studies suggest possible trade-offs (Box 1), where
expanded host-range genotypes that infect a novel host may pay a performance cost of
suboptimal or lack of growth on the native host, at least initially. However, these outcomes
also depend on the particular host-range mutations experienced by
Φ
6 [
45
,
50
]. For P.
glycinea host-range mutants, there is a positive correlation between the fitness on novel (P.
glycinea) and laboratory (P. phaseolicola) hosts [
45
], whereas host-range mutants isolated on
ERA bacteria do not show this general pattern [50].
The mutations that allow
Φ
6 to emerge on a novel host tend to be nonsynonymous
substitutions located in the gene that encodes the P3 spike protein, which interacts with
the host-cell receptor. Across four studies [
44
–
46
,
50
], 142 different amino acid substitutions
have been inferred to underlie changes in the host range of
Φ
6. In Figure 5, we show the

Viruses 2024,16, 977 10 of 17
frequency with which known host-range mutations in
Φ
6 have been isolated on novel
hosts, and indicate their positions within the p3 gene that encodes the spike protein. We
note that a few of these mutations, despite being isolated on one host, facilitate the infection
of additional novel hosts. For example, amino acid mutations D35A, initially isolated on P.
atrofaciens, and G515S, isolated on P. tomato, also allow for the infection of ERA; and S246T,
isolated on ERA, also allows for the infection of P. atrofaciens [44].
Viruses 2024, 16, x FOR PEER REVIEW 10 of 18
also depend on the particular host-range mutations experienced by Φ6 [45,50]. For P. gly-
cinea host-range mutants, there is a positive correlation between the fitness on novel (P.
glycinea) and laboratory (P. phaseolicola) hosts [45], whereas host-range mutants isolated
on ERA bacteria do not show this general paern [50].
The mutations that allow Φ6 to emerge on a novel host tend to be nonsynonymous
substitutions located in the gene that encodes the P3 spike protein, which interacts with
the host-cell receptor. Across four studies [44–46,50], 142 different amino acid substitu-
tions have been inferred to underlie changes in the host range of Φ6. In Figure 5, we show
the frequency with which known host-range mutations in Φ6 have been isolated on novel
hosts, and indicate their positions within the p3 gene that encodes the spike protein. We
note that a few of these mutations, despite being isolated on one host, facilitate the infec-
tion of additional novel hosts. For example, amino acid mutations D35A, initially isolated
on P. atrofaciens, and G515S, isolated on P. tomato, also allow for the infection of ERA; and
S246T, isolated on ERA, also allows for the infection of P. atrofaciens [44].
Figure 5 shows that changes at the p3 locus relate to differing abilities for these mu-
tants to infect certain host bacteria. Mutations at residue 8 (the square in Figure 5) occur
at a higher frequency when Φ6 is challenged to infect ERA and P. tomato bacteria, while
host shifts onto P. glycinea favor mutations at residue 554 (the triangle in Figure 5), and
host shifts associated with P. atrofaciens emergence are favored by mutations at residue
133 (the diamond in Figure 5). According to a study that examined these mutations in
light of the structure of the P3 protein, all three aforementioned amino acid changes were
predicted to be topologically close together [50]. This region of the protein may have a
particular functional role in the aachment of Φ6 to host cells, with different amino acid
replacements responsible for specific changes in the host range.
Figure 5. The frequency of host-range mutations across the Φ6 P3 spike protein. The mutants were
isolated on the specific host (colored circles), and the p3 gene was sequenced to identify mutations.
The number of mutations at each P3 residue isolated on a particular host was divided by the total
number of P3 mutants that were isolated from that host to obtain the frequency of mutations. Resi-
dues discussed in the text are marked with a square (residue 8), diamond (residue 133), and triangle
(residue 554). Data are from [44–46,50].
Interestingly, prior evolution of Φ6 on native P. phaseolicola bacteria also seems to
produce genetic and phenotypic changes that affect the potential for Φ6 to productively
infect other hosts. For example, Φ6 populations that were previously evolved on P.
Figure 5. The frequency of host-range mutations across the
Φ
6 P3 spike protein. The mutants were
isolated on the specific host (colored circles), and the p3 gene was sequenced to identify mutations.
The number of mutations at each P3 residue isolated on a particular host was divided by the total
number of P3 mutants that were isolated from that host to obtain the frequency of mutations.
Residues discussed in the text are marked with a square (residue 8), diamond (residue 133), and
triangle (residue 554). Data are from [44–46,50].
Figure 5shows that changes at the p3 locus relate to differing abilities for these mutants
to infect certain host bacteria. Mutations at residue 8 (the square in Figure 5) occur at a
higher frequency when
Φ
6 is challenged to infect ERA and P. tomato bacteria, while host
shifts onto P. glycinea favor mutations at residue 554 (the triangle in Figure 5), and host
shifts associated with P. atrofaciens emergence are favored by mutations at residue 133 (the
diamond in Figure 5). According to a study that examined these mutations in light of the
structure of the P3 protein, all three aforementioned amino acid changes were predicted
to be topologically close together [
50
]. This region of the protein may have a particular
functional role in the attachment of
Φ
6 to host cells, with different amino acid replacements
responsible for specific changes in the host range.
Interestingly, prior evolution of
Φ
6 on native P. phaseolicola bacteria also seems to pro-
duce genetic and phenotypic changes that affect the potential for
Φ
6 to productively infect
other hosts. For example,
Φ
6 populations that were previously evolved on P. phaseolicola at
a low MOI demonstrated higher rank order fitness when challenged to grow on P. tagetis
and P. savastanoi host bacteria, compared to their counterpart populations that experienced
evolution at a high MOI [
7
]. The relatively larger burst sizes of viruses evolved at a low
MOI [
29
] may have increased the likelihood that some progeny viruses would establish
infections on these novel hosts [
7
]. Differing selective pressures associated with strong
versus weak population growth of evolved viruses in one host environment may thus also
be consequential for the probability for successful viral emergence on alternative hosts.

Viruses 2024,16, 977 11 of 17
4.2. Evolutionary Adaptation on Novel Hosts
In nature, viruses are expected to shift onto novel hosts in steps. Initially, an ancestral
virus can only infect the host(s) constituting its original niche. The first stage of emergence
requires that one or more spontaneous mutations occur that broaden the ability of some
variants in the virus population to infect both the native and additional host(s). At the
next stage, selection to improve infection of the novel host can lead to virus adaptation
(increased fitness) [
45
]. Single-nucleotide host-shift mutations in the first stage must arise
in a limited time to permit the initial infection of a novel host, but the expectation is that
sustained transmission on the new host may not be possible. Thus, the second stage,
involving further adaptive evolution on the novel host, may result in viruses that become
specialists on the new host and can no longer infect the original (native) host(s). However,
this simple example does not include the many possibilities where emerging viruses might
encounter complex environments, especially ones that permit interactions with both native
and novel hosts through time. For example, virus evolution may occur on hosts that
vary across space (heterogeneous host environments) or through time (alternating host
encounters). Here, selection should favor the evolution of generalist viruses that can infect
multiple hosts. Several evolutionary studies on phage
Φ
6 have tested scenarios where
virus populations must maintain generalism over long periods of time on native and novel
hosts, or specialize on novel hosts.
When serially transferred in environments with a heterogenous mixture of native and
novel hosts,
Φ
6 populations were observed both to evolve generalism and to maintain
generalist traits over time. Evolved generalists grown with P. atrofaciens or ERA bacteria hosts
rarely demonstrated a fitness cost between performance on novel and native hosts [
6
,
51
,
52
],
and these lineages could even outcompete evolved specialist populations if the relative
proportions of novel hosts in the environment were sufficiently abundant [
6
,
51
]. Evolved
generalists also continued to bind (adsorb) more rapidly to the cells of the native host than to
those of the novel host [
51
]. These results indicate that evolved generalism in phage
Φ
6 can
be ‘cost-free’ (Box 1), in terms of avoiding strong fitness trade-offs across host environments as
emergence proceeds. Evolution in heterogeneous host environments may maintain selection
for the infection of both hosts, with the native host as a priority for selection in generalist
viruses and the novel host as an alternative resource when the native host cells are depleted.
Φ
6 selection experiments also demonstrate that generalism can be maintained in
alternating (temporally heterogeneous) host environments. Zhao and Duffy (2019) [
46
]
challenged generalist populations and specialist populations to evolve for 30 serial trans-
fers in experimental treatments containing either P. phaseolicola bacteria alone, or with
alternating infection of P. phaseolicola and novel host bacteria at each transfer. When a
specialist or a generalist population was transferred solely on P. phaseolicola, there was
no significant difference in observed fitness gains through time. However, when these
populations were serially transferred on alternating hosts, fitness on P. phaseolicola either
decreased (performance trade-off) or showed no significant difference from the ancestor’s
performance. Populations evolved on alternating hosts always improved in fitness on the
novel host used in selection, but fitness on another (unselected) novel species in the host
range either remained the same or decreased over time [46].
However, heterogeneous host conditions that facilitate generalism may reduce genetic
diversity, because mutations that are beneficial or neutral in one host environment may
be deleterious in another [
1
,
40
,
44
,
53
]. In support of this hypothesis, Zhao and Duffy
(2019) [
46
] found that, regardless of whether the founding phage was a specialist or a
generalist, populations transferred solely on P. phaseolicola exhibited a higher normalized
number of single-nucleotide polymorphisms (SNPs) than populations that experienced
infection of alternating host types. This suggests that a constant or native host environment
produces a greater diversity of genotypic variants than a fluctuating host environment.
Evolution in simpler environments consisting of a single novel host, on the other hand,
should be more likely to result in trade-offs suffered by viruses on the native host. In one
study,
Φ
6 generalists that were serially transferred solely on ERA host bacteria experienced

Viruses 2024,16, 977 12 of 17
reduced attachment rates on the original P. phaseolicola host [
52
], despite their continued
ability to infect both hosts. A separate study observed that adaptation to a single host led
to mutational changes which reduced the host range of
Φ
6, or even resulted in its complete
specialization on the new host [
49
]. Here, when a generalist genotype with an E8G amino
acid substitution in the P3 spike protein was evolved for 30 serial transfers on ERA host
bacteria, the phage populations accumulated amino acid changes in P3 that narrowed the
host range from six hosts (P. phaseolicola,P. persicae,P. savastanoi,P. tagetis,P. tomato, and
ERA) to either five hosts (amino acid substitution A31T caused an inability to infect P.
tomato) or just one host (mutants with amino acid substitution G247A could only infect
ERA bacteria) [
49
]. Individual point mutations can thus explain narrow host-range traits in
Φ
6, including extreme specialization for an evolved lineage that could no longer infect the
native host and could only infect ERA.
The ecological dynamics for emerging virus persistence and expected evolution poten-
tial on novel hosts can be examined in short-term studies that show whether viruses can
avoid extinction when challenged to thrive on native and novel hosts. Using phage
Φ
6 pop-
ulations, these experiments demonstrated that the probability of emergence should depend
on the size of the virus population. Dennehy et al. (2006) [
48
] cultured
Φ
6 populations
in environments that alternated daily between infection of novel ERA hosts and native P.
phaseolicola hosts. The authors found that lineages had higher fitnesses at large population
sizes, and lower fitnesses when population sizes were small. Across the population sizes
examined, the mean fitness was observed to be higher for viruses experiencing alternating
host treatments than for those propagated on the novel ERA bacteria alone. This study
showed that the persistence of emerging viruses on a novel host should be more likely
at relatively large population sizes or with a native source population, where the virus is
instead expected to go extinct on the novel host alone.
5. Thermostability Evolution
5.1. The Direct Evolution of Thermostability
The evolution of viruses on new hosts receives abundant attention in virology owing
to the importance of disease emergence. But viruses also experience abiotic changes in
their natural environments, and an inability to withstand such perturbations can prove
costly to infectivity. Several evolution studies in
Φ
6 have explored how viruses can resist
heat shock, or evolve greater thermostability (Box 1), in environments that manipulate the
frequency of an elevated-heat exposure [
54
–
56
].
Φ
6 is typically cultured in the laboratory at
a standard temperature of 25
◦
C. However, when exposed to 5-min heat shocks at extreme
temperatures such as 45–50
◦
C, the survival of the wild-type virus population decreases
substantially. Periodic exposure to heat shocks, in between serial transfers that permit
growth on bacteria at 25
◦
C (Figure 1, top), are shown to increase the average survival
of viruses at the elevated temperature. Point mutations that improve
Φ
6 survival in the
face of thermal stress commonly map to the P5 lysis protein [
55
–
57
] and the P8 outer shell
protein [
56
]. The P5 and P8 proteins are located on the outer surface of the virus particle’s
membrane, and are necessary for attachment to and initial infection of host cells [
58
,
59
],
making them vital structures for degradation avoidance under thermal stress. The V207F
amino acid substitution in P5 is especially commonly observed in these studies [
55
–
57
].
Crystal structure comparisons of the wild-type P5 protein and a P5 protein with the V207F
substitution revealed that phenylalanine fills a hydrophobic pocket in the P5 protein. This
mutation may thus enhance the thermostability of P5 by increasing the molecular van
der Waals interactions between amino acids, improving the survival of
Φ
6 under heat
stress [55].
However, a higher thermostability of proteins, particularly enzymes, may not be
generally beneficial for fitness. Many enzymes undergo conformational changes to catalyze
reactions, but increasing molecular forces within the protein for greater thermostability
may result in ‘stiffness’ that hinders catalytic function [
60
,
61
]. In
Φ
6, certain thermostable
mutations demonstrate this expected fitness trade-off. For example, wild-type-derived

Viruses 2024,16, 977 13 of 17
mutants with V207F exhibit higher thermostability, but a decreased viral growth rate at
25
◦
C [
55
], suggesting a trade-off between thermostability and growth. However, the effect
in
Φ
6 may also be genotype-dependent (V207F in a non-wild-type genetic background
increased both survival to heat shock and growth rates at 25
◦
C [
56
]). Comparisons of
the growth rate and thermostability of 10 single-nucleotide P5 and P8 mutants of
Φ
6 also
did not support a generalized trade-off between these traits [
56
], indicating that genetic
architecture matters for their interplay.
5.2. Thermostability and Genetic Robustness
Interestingly, selection in different biotic environments also influences the ability of
Φ
6 to survive under abiotic thermal stress. Clones from
Φ
6 populations evolved at a
low versus a high MOI for 300 generations (an extension of the coinfection-manipulated
experiments performed by Turner and Chao, 1998 [
23
]) showed both equivalent fitnesses at
a low MOI and equivalent survival to 45
◦
C heat-shock exposure. However, when these
populations were further evolved for 10 serial transfers in the presence of periodic heat
shocks, the increase in survival at 45
◦
C was greater for the viruses evolved previously
at a low MOI than for those evolved previously at a high MOI [
54
]. When permitting
these viruses to evolve under periodic heat shocks when the populations were initially
genetically diverse, the viruses previously evolved at a low MOI also adapted faster in
their thermotolerance to 45 ◦C than did their high-MOI counterparts [57].
These results suggest that viruses evolved at a low MOI may be advantaged in access-
ing beneficial thermostabilizing mutations. For example, if the low-MOI-evolved viruses
had a higher spontaneous mutation rate than the high-MOI-evolved viruses, they could be
capable of gaining adaptive thermostability more rapidly because of the higher input of mu-
tations. However, tests of the frequency of specific beneficial mutations, either a host-range
mutation or a mutation that confers resistance to a chemical (butylated hydroxytoluene)
that cleaves the P3 protein, revealed no measurable differences in the spontaneous mutation
rate between viruses evolved previously at a low versus high MOI [
54
,
62
]. Alternatively,
viruses evolved at a low MOI may have possessed one or more mutations that predisposed
them to the positive effects of input thermostabilizing mutations. In support of this hypoth-
esis, low-MOI-evolved viruses had greater mean survival than high-MOI-evolved viruses
at moderately high temperatures of 40–42
◦
C [
63
]. Sequencing of the resulting heat-shocked
populations revealed that the P5 mutation V207F, described above, was correlated with
these increases in survival [57].
A third hypothesis, perhaps more intriguing, is that viruses evolved at a low MOI are
genetically robust [
62
] (Box 1). Genetically robust genotypes are better able to maintain their
phenotypic traits in the face of spontaneous mutations, and thus experience a ‘buffer’ to
the effects of otherwise deleterious mutations. A genetically robust population is expected
to contain extant genotypes or those that are small (e.g., single) genetic steps away from
standing variants that harbor a greater diversity of neutral or nearly neutral mutational
options, which may be useful for adaptation when encountering other environments.
Because benefits of genetic robustness are offset by a generation (the offspring of genetically
robust parents will have higher fitness than the offspring of genetically non-robust parents,
but the parents themselves may have identical fitness) [
64
], genetic robustness may arise
as a correlated consequence of another trait, such as recombination [
22
,
65
] or, in cells,
well-connected metabolic networks [66].
In
Φ
6, complementation between different coinfecting variants (Box 1) can act as an
environmentally determined mechanism that beneficially masks the fitness effects of dele-
terious mutations in trans. During single infections, on the other hand, the harmful effects
of deleterious mutations are expressed. Frequent coinfection may thus relax selection for
the maintenance of genetic robustness, whereas mechanisms underlying robustness should
persist when single infection is more common. This hypothesis was supported by two lines
of evidence. First, although populations of
Φ
6 evolved at a low MOI had higher genetic
diversity than those evolved at a high MOI [
34
], the mean fitnesses of these populations

Viruses 2024,16, 977 14 of 17
were not significantly different [
62
], indicating that mutations in the low-MOI-evolved
populations were, on average, more neutral for fitness. The second line of evidence derived
from a mutation accumulation experiment (Box 1) using clones randomly isolated from the
high-MOI-evolved and low-MOI-evolved populations. Compared with high-MOI-evolved
clones, the low-MOI-evolved clones exhibited decreased fitness reductions and lesser vari-
ance in fitness changes following mutation accumulation. That low-MOI-evolved viruses
experienced fewer conditionally deleterious effects of random mutational inputs indicated
that prior evolution at a low MOI fostered the maintenance of genetic robustness [62].
The relationship between thermostability and genetic robustness further suggests that
genetic robustness in
Φ
6 may operate through protein stability. Stable proteins are more
likely to maintain their form and function in the face of both external thermal stress and
internally destabilizing or deleterious mutations [
67
–
69
]. Moreover, the greater stability
of these proteins should allow them to better tolerate mutations that are beneficial but
destabilizing [
67
,
69
,
70
]. Genetic robustness, mediated through protein stability, may thus
also improve protein evolvability [
68
–
70
] (Box 1). Further evolution of the low-MOI and high-
MOI
Φ
6 populations in other, non-thermal environments could help illuminate whether
their hypothesized genetic robustness also improves evolvability in environments beyond
those containing elevated temperatures.
6. Conclusions
Clearly, experimental evolution studies on
Φ
6 have contributed usefully to our un-
derstanding of various key topics in evolutionary biology, including the spontaneous
nature and fitness effects of randomly occurring and adaptive mutations; the evolutionary
consequences of coinfection, and its costs and benefits for viral adaptation; the mutations
responsible for viruses to infect a narrow versus broad range of original and/or novel
host species; and the roles of prior versus current environmental exposure in maintaining
performance under stressful conditions such as heat shock. Beyond elucidating details
of
Φ
6 and its cystovirus relatives, these studies show the use of
Φ
6 as a vital model for
understanding the evolution of vastly different virus systems due to its traits that remain
unique among known phages. The combination of a multi-partite dsRNA genome, the
ability to undergo segment reassortment, and the lipid outer membrane found in
Φ
6 makes
the virus an attractive non-pathogenic model for many viruses of disease importance, such
as the Reoviridae, influenza viruses, and SARS-CoV-2.
However, many unanswered questions still remain on the topics discussed in this
review. We conclude with a select list of questions for further study.
(1)
What specific mutations contribute to the evolution of cheating strategies and im-
proved fitness only during coinfection? It remains unclear whether the evolution of
cheating in
Φ
6 can be explained with as few as one mutation on a single RNA segment,
or with multiple mutations, such as those acting via epistasis across segments.
(2)
What is the frequency and degree of cost-free mutations for generalism (host-range
breadth) in
Φ
6? Although the balance of evidence suggests that cost-free generalism
in
Φ
6 persists in spatially heterogeneous and temporally alternating host environ-
ments, relatively few studies have measured the fitness of individual genotypes or
populations in both native and novel host environments. This makes it more difficult
to determine the presence and degree of fitness trade-offs. Correlated effects of muta-
tions that primarily improve fitness on the native host, for example, could explain the
tendency of Φ6 to evolve cost-free generalism.
(3)
How do genotype and phenotype (fitness) change when
Φ
6 evolves solely on a novel
host? The virology literature presents relatively few examples of point mutations
that lead either to a reduction in host range or to complete specialization on a new
host. There is also little evidence on how such substitutions might impact the fitness
effects of additional mutations during the emergence process. This would seem an
important next step in understanding the adaptation of RNA viruses after their initial
emergence on a new host, and models such as Φ6 would be valuable.

Viruses 2024,16, 977 15 of 17
(4)
How does evolution under frequent versus rare opportunities for coinfection alter
the broader genomic properties of
Φ
6, such as its ability to withstand deleterious
mutations (genetic robustness) and its survival in novel environments? Evidence
suggests that viruses evolved at a low MOI are putatively genetically robust and
experience advantages both when infecting novel hosts and under thermal stress.
However, the mechanisms that permit these advantages may not be universal. For
example, low-MOI-evolved viruses did not experience enhanced survival under UV
irradiation stress [
63
], suggesting that studies that compare low-MOI-evolved and
high-MOI-evolved
Φ
6 populations across a wider variety of stressful conditions
are warranted.
(5)
Does genetic robustness, hypothesized to occur in viruses previously evolved in low-
MOI conditions, improve evolvability in other, non-thermal environments? Given
the particular importance of RNA viruses in disease emergence, a relationship be-
tween genetic robustness and traits such as infectivity on novel hosts should leverage
tractable models such as Φ6.
Author Contributions: S.S.: Conceptualization, writing—original draft, writing—review and editing,
supervision. A.K.B., A.G.N., P.P.B. and S.N.: Writing—original draft, writing—review and editing.
P.E.T.: Writing—review and editing, supervision. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was funded by the National Institute of General Medical Sciences (Grant
#R16GM146706 to S.S.), and the Division of Research and Innovation at San JoséState University
(Award Number 23-SRF-08-051 to S.N.).
Acknowledgments: D. Goldhill and S. Arnold performed preliminary experiments with reassortants
of phages with evolved
Φ
H2 segments, described in the section “Coinfection can constrain adaptation
in Φ6”. Preliminary results of their experiments are included in this review with their permission.
Conflicts of Interest: The authors declare no conflicts of interest.
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