Group Title: Virology Journal 2008, 5:104
Title: Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias
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Title: Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias
Series Title: Virology Journal 2008, 5:104
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Creator: van der Walt E
Martin DP
Varsani A
Polston JE
Rybicki EP
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Experimental observations of rapid Maize streak virus evolution
reveal a strand-specific nucleotide substitution bias
Eric van der Walt', Darren P Martin2, Arvind Varsanil,3, Jane E Polston4 and
Edward P Rybicki*1,2


Address: 'Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa, 2Institute of Infectious Disease and
Molecular Medicine, University of Cape Town, Cape Town, South Africa, 3Electron Microscope Unit, University of Cape Town, Cape Town, South
Africa and 4University of Florida, Interdisciplinary Centre for Biotechnology Research, Bradenton, USA
Email: Eric van der Walt eric.vanderwalt@kapabiosystems.com; Darren P Martin darrin.martin@uct.ac.za;
Arvind Varsani arvind.varsani@uct.ac.za; Jane E Polston jep@ufl.edu; Edward P Rybicki* ed.rybicki@uct.ac.za
* Corresponding author



Published: 24 September 2008 Received: 17 June 2008
Virology journal 2008, 5:104 doi: 10. 186/1743-422X-5-104 Accepted: 24 September 2008
This article is available from: http://www.virologyj.com/content/5/1/104
2008 Walt et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the
taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10-4
substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA
viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases
with fidelities approximately 10 000 times greater than those of error-prone viral RNA
polymerases. Although high ssDNA virus evolution rates were first suggested in evolution
experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has
never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive
value of high geminivirus mutation rates remain unanswered.
Results: We determined the short-term evolution rate of MSV using full genome analysis of virus
populations initiated from cloned genomes. Three wild type viruses and three defective artificial
chimaeric viruses were maintained in plant for up to five years and displayed evolution rates of
between 7.4 x 10-4 and 7.9 x 10-4 subs/site/year.
Conclusion: These MSV evolution rates are within the ranges observed for other ssDNA viruses
and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven
distribution of mutations within the defective virus genomes suggests that some of the changes may
have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are
consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA
viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.



Background have been shown to evolve at rates between 10-3 to 10-5
Most research on virus evolution has focused on RNA substitutions per site per year (subs/site/year) [1-4]. In
viruses, which are generally subject to relatively high rates contrast and consistent with the hypothesis that
of mutation due to their dependence on error-prone DNA polymerase fidelity influences evolution rates double
dependent RNA polymerases. Accordingly, RNA viruses stranded DNA (dsDNA) bacteriophages, papillomavi-

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ruses and polyomaviruses evolve at rates in the region of
10-9 subs/site/year [5,6]. Intriguingly, and possibly contra-
dicting the premise that polymerase fidelity is the major
universal determinant of evolution rates, figures closer to
those of RNA viruses (~10-4 subs/site/year) have been
reported for the small single stranded DNA (ssDNA)
anelloviruses [7-9] and parvoviruses [10-12]. Further-
more, direct estimates of the basal or biochemical rates at
which mutations occur during each replication cycle of
ssDNA bacteriophages have also indicated that these rates
approach those of RNA viruses [5,13] For a good general
review on the topic of virus mutation and evolution rates
see [14].

The ssDNA geminiviruses represent extremely important
threats to commercial agriculture and basic subsistence
farming throughout the tropical and temperate regions of
the world [15-18]. The geminiviruses are a highly diverse
group comprising more characterized species than any
other virus family [19]. Although interest in geminivirus
evolution has, until recently, been largely focused on the
undeniably important role of recombination in the gener-
ation of novel species and strains [20-25], it is the accu-
mulation of point mutations that is the ultimate source of
diversity within the family.

Very little is known about the timescales over which gem-
inivirus diversification has occurred. The apparent
absence of any members of the most divergent geminivi-
rus genus the mastreviruses in the New World strongly
suggests that the earliest geminiviruses only evolved after
the break-up of Gondwanaland ~100 million years ago
[26]. Additionally, all available phylogenetic evidence
indicates that the geminiviruses currently found in the
Americas were introduced there much more recently:
most extant New World geminiviruses probably evolved
from one or a few progenitor begomoviruses that were
possibly introduced as recently as 20 000 years ago along
with human colonists from Asia via the Bering land bridge
[27], and a few species originating in the middle East and
Asia have been accidentally released in the Americas in
modem times [28,29].

Importantly, indirect estimates of geminivirus evolution
rates and direct experimental measurement of geminivi-
rus mutation frequencies both indicate that, as is the case
for some other ssDNA virus groups, geminiviruses are
evolving at an unexpectedly rapid rate. Duffy & Holmes
[30], using Bayesian coalescent based analysis of gemini-
viruses causing Tomato yellow leaf curl disease (eight sep-
arate old world begomovirus species), reported that the
average genome-wide rate at which mutations have been
fixed in the genomes of these viruses over the past 20 years
has been approximately 2.88 x 10-4 subs/site/year. While
the credibility interval of this estimate is quite broad, it is


95% certain that the last common ancestor of the eight
species studied existed within the past 41 000 years. It is
noteworthy that the most probable date for the origin of
these viruses, which represent approximately the same
breadth of diversity as that currently observable amongst
new-world begomoviruses, is between 3000 and 9000
years ago a figure that fits well with the hypothesis that
humans and begomoviruses may have colonised the
Americas at approximately the same time.

Although only two direct experimental measurements of
geminivirus mutation frequencies appear in the literature,
both confirm that these viruses are capable of evolving at
rates of between 10-3 and 10-4 subs/site/year. The first,
using a "biologically cloned" MSV population maintained
for up to four years in both maize and in a Coix sp., esti-
mated a genome-wide evolution rate of between 2.6 x 10-
4 and 5.5 x 10-4 subs/site/year [31] within individual
infected plants. The second, using infectious cloned
tomato yellow leaf curl China virus (TYLCCV) isolates
maintained for between 60 and 120 days in Nicotiana
benthamiana and tomato plants, detected evolution rates
of between 1.4 x 10-3 and 2.2 x 10-3 subs/site/year in a
genome region that included the rep gene and the inter-
genic region [32].

Two reports of high-frequency reversions of specific non-
lethal deleterious mutations in the rep genes of MSV
[33,34] and isolates of various begomovirus species [35]
indicate that the basal rate at which mutations occur in
geminivirus genomes may be orders of magnitude higher
than the rate at which mutations become fixed within
these genomes. At a particular genomic site analysed in
one of these experiments, a highly adaptive reversion
mutation was detectable in 5/8 independent MSV infec-
tions within 10 days of inoculation [33] implying that the
virus is capable of adaptive evolution rates rivalling those
of even the most rapidly evolving RNA viruses.

Thus, the population wide evolution rates estimated for
geminiviruses by Duffy and Holmes [30] are slightly
lower than evolution rates directly observed within indi-
vidual infections [31,32], which are in turn lower than
mutation rates implied by mutation frequency studies
involving highly adaptive reversion mutations [33-35].
These differences in estimated evolution rates probably
reflect the effects of population size and selection pressure
on the rate at which mutations become fixed in a popula-
tion [13]. Selection operates more effectively on larger
populations, with advantageous mutations rising to fixa-
tion and deleterious mutations being purged quicker than
for small populations [36]. Furthermore, it has been
experimentally verified in various systems that, consistent
with the popular theoretical concept of scaling a fitness
peak, rates of evolutionary adaptation to new environ-


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ments are initially rapid but eventually slow down and
level off [37-42]. This is because as a sequence ascends a
fitness peak the fraction of possible advantageous muta-
tions permitting upward movement becomes progres-
sively smaller. The fraction reaches zero as the peak is
attained, at which point the evolution rate should match
the rate of selectively neutral genetic drift. As a result of
these factors, short-term evolution rates estimated from
small populations of a virus species, such as those meas-
ured within individual infected plants over a few years,
will be somewhere between the basal rate at which muta-
tions occur for that species and the long-term rate at
which the species is evolving over tens or hundreds of
years [13].

To accurately measure the rate at which MSV genomes
accumulate mutations over periods of a few years, and to
study the relationship between fitness and evolution rate,
we studied nucleotide substitutions arising in defective
mutant and wild-type MSV genomes during infections of
maize and sugarcane. Three of the genomes analysed were
unusual in that they were low-fitness laboratory con-
structed MSV chimaeric viruses comprising genome com-
ponents we knew to be specifically maladapted to survival
in maize [23,43]. In addition to estimating the short-term
MSV evolution rate within individual hosts, we present
evidence that MSV exhibits strand specific nucleotide sub-
stitution imbalances that are consistent with a recent pro-
posal by Duffy and Holmes [30] that high mutation rates
in ssDNA viruses are due to mutagenic processes that spe-
cifically affect ssDNA molecules.

Results and discussion
Mutations occur at high frequencies during MSV infections
With the intention of studying evolution rates and pat-
terns of nucleotide substitution in MSV, sweetcorn plants
were initially agroinoculated with clones of three wild-
type MSV strains MSV-Tas, MSV-Kom and MSV-Set -
and three defective laboratory constructed recombinant
viruses K-MP-S, K-MP-CP-S and S-CP-K (Figure 1). All
are described in detail by van der Walt et al. [43].

We used two approaches to avoid the severe population
bottlenecks that were likely to occur during insect trans-
mission in the course of our experiments. Our first
approach, used with all viruses other than MSV-Tas, uti-
lised three plants infected with each virus to initiate serial
transmissions via leafhopper, with each transmission last-
ing several days and involving tens of leafhoppers. Our
second approach, used with MSV-Tas, was to avoid serial
leafhopper transmissions altogether. To achieve this, a
single sugarcane plant (cultivar Uba) was infected with
the wild-type isolate MSV-Tas via leafhopper transmission
from an agroinoculated sweetcorn plant [44], and main-
tained in an infected state for five years. Although MSV-


Tas was originally isolated from wheat, it produces rela-
tively severe symptoms in sugarcane [44], indicating that
it was not particularly maladapted to this perennial host.

Following twelve passages through sweetcorn over a one-
year period, no obvious changes in symptomatology were
observed for any of the serially transmitted viruses (data
not shown). At the end of the one-year period, viral
genomes were cloned from one symptomatic plant
infected with each of the viruses. Full-length genomic
sequences were obtained for two individual MSV genomic
clones from each plant, except for K-MP-S, for which only
one genome was sequenced. Similarly, seventeen full-
length MSV-Tas genomes were cloned and sequenced
from the five year old infection of sugarcane.

Figure 1 and 2 respectively show the positions of all of the
mutations identified in the nine genome sequences from
maize and the 17 genome sequences from sugarcane,
while Additional files 1 and 2 respectively detail the nude-
otide and protein sequence context and the specific
sequence changes in each individual clone from maize
and sugarcane. All of the genomes sequenced contained at
least one mutation with respect to the original parental
viruses; the most mutations in any single genome was
four (El-01, MSV-Kom; E2-01, K-MP-S) for the maize
viruses and 18 (SC-E02) for the sugarcane viruses. Besides
three identical clone pairs (E5-01 and E5-02; E7-01 and
E7-02; E3 and F7) all 20 remaining genomes were unique.

A total of 66 different mutations were detected overall: 15
in the viruses from maize and 51 in the viruses from sug-
arcane. Two of these were deletion mutations (mutation
12 in El-02 and mutation 33 in SC-E-02 and F10; Figures
1 and 2 respectively) and one was an insertion mutation
(mutation 44 found in all clones from sugarcane).
Whereas the insertion mutation was at a site in the LIR
that seems to tolerate insertions and deletions in related
MSV isolates, both the deletion mutations are likely to be
lethal in that they cause rep frame shifts that should result
in the expression of seriously truncated and partially mis-
translated Rep proteins. For example, a 16 nt deletion in
SC-E-02 and F10 would be predicted to result in loss of
the rep intron acceptor site and premature termination of
repA some thirty codons before the normal stop site. It is
very unlikely that SC-E-02 and F10 could somehow
express a functional Rep despite this deletion in that both
also carry a substitution mutation (mutation 30 in Figure
2 and Additional file 2) that introduced a premature stop
codon at Rep position 257.

While these deletion mutations should disable the viruses
carrying them, many of the 63 nucleotide substitution
mutations are probably neutral in that the vast majority
did not alter any nucleotide or amino acid sequence


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repB


repA


WUIJJUI! I.U.U~JIUrnIJ.L ~U!IL.J ~LWU.


v-or


14 15





-41


".." "". "....... "".'"


Figure I
Mutations in MSV-Kom/-Set parental and chimaeric viruses. Short vertical lines above or below the centre line indi-
cate homology at informative sites to either MSV-Kom or MSV-Set, respectively. Long vertical lines above the centre line rep-
resent positions not homologous to either MSV-Kom or MSV-Set sequence (i.e. mutated sites). Mutations are numbered, and
refer to those listed in Additional file I. The positions of ORFs and the virion-strand replication origin stem-loop sequence are
indicated in shaded red (MSV-Kom) or green (MSV-Set). The diagrams are to scale.


motifs with either known or suspected functionality and,
based on their having PAM250 scores > 1 [45], most of the
predicted amino acid changes are probably relatively con-


servative. Notable exceptions were three independent
mutations that disrupted the most distal of three potential
C-sense TATA boxes in clones El-01 (mutation 14 in Fig-


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10 11


5


E1-01
KNco



E1-02
KNco



E2-01
K-MP-S



E4-01
K-MP-CP-S


6,7 9


1 3


E4-03
K-MP-CP-S



E5-01
E5-02
SNco


E7-01
E7-02
S-CP-K


200bp


3


aM


Virology Joumnal 2008, 5:104


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http://www.virologyj.com/content/5/1/104


arcane would have been 2.6 x 10-4 subs/site/year only
1.1-fold higher than the lower rate estimated by Isnard et
al. [31].


It is important to note that the MSV evolution rates we
have measured should be considered "short-term small-
population" evolution rate estimates, and they are almost
certainly an over-estimation of longer-term population-
wide rates [13]. Whereas an ideal evolution rate estimate
:- : -- - would be the rate at which mutations become fixed within
)')/'i'/' \ \I \the global MSV population, our short-term small-popula-
tion estimates more closely reflect the rate at which muta-
tions accumulate in MSV genomes during a single
infection. This rate provides an indication of the maxi-
repB repA v-ori
LIR mum rate at which MSV could evolve; however, it is the
slower rate at which such mutations become fixed,


through drift and positive selection, that determines how
rapidly large MSV populations evolve over tens or hun-
dreds of years.

Nevertheless, based on the evolution rate estimates
reported here and elsewhere [30-32], it is becoming
increasingly apparent that geminiviruses are probably
evolving as fast as some RNA viruses[3,4,46,47] and
orders of magnitude faster than dsDNA viruses [48,49].
This represents a significant departure from the natural
assumption that the synthesis of geminivirus genomes by
host DNA polymerases [50,51] implies relatively error-
free virus replication and therefore mutation rates similar
to those experienced by plant genomic DNA [52,53]. At
least two other diverse ssDNA viruses seem to have nucle-
otide substitution rates in the range of 10-4 subs/site/year
- parvoviruses [11,12] and anelloviruses [7] which
implies that high mutation rates may be a common, if not
universal, feature among ssDNA viruses.

Nucleotide substitution biases suggest a possible cause of
high MSV mutation rates
Because of our relatively scant understanding of plant
DNA replication in general, and more specifically of the
host factors involved in geminivirus replication [51,54],
the mechanisms underlying the surprisingly high muta-
tion rates seen in geminiviruses remain a topic of specula-
tion. There are, however, some clues about where to start
looking. As early as 1997, Roossinck [53] noted that since
replicating geminivirus DNA is apparently not methylated
[55] it is possible that normal host mechanisms for mis-
match repair may not operate during their replication
[56]. Both Ge et al. [32] and Duffy and Holmes [30] made
the same proposal. Duffy and Holmes [30] suggested two
additional possibilities: i) because geminivirus DNA is
only transiently double-stranded during rolling-circle rep-
lication, it may not be suitable for base-excision repair; ii)
the biased substitution patterns may be explained either
by spontaneous deamination potentially more likely to


Figure 2
Mutation frequencies in seventeen MSV-Tas derived
genomes isolated after five years of maintenance in
sugarcane. The histogram represents the proportions of
the 17 analysed genomes that carried the different mutations.
Beneath the histogram, the positions of ORFs and the virion-
strand replication origin are indicated in shaded grey. The
genomic locations of the 51 analysed mutations are indicated
by vertical black lines overlaying the genome map. Mutation
numbers correspond to those in Additional file 2. mp =
movement protein gene; cp = coat protein gene; RepA+RepB
= replication associated protein gene;repA = RepA gene.



ure 1 and see Additional file 1), SC-E02, SC-F01, C5, F10
and F5 (mutations 45 and 46 in Figure 2 and see Addi-
tional file 2).

MSV displays evolution rates similar to those of other
ssDNA viruses
Whereas the average evolution rate of the nine genome
sequences from maize was 7.4 x 10-4 subs/site/year (20
substitutions in 24183 nucleotides sequenced), the aver-
age rate for the seventeen sequences from sugarcane was
7.9 x 10-4 subs/site/year (180 substitutions in 45713
nucleotides sequenced). While these rates are approxi-
mately half those recently determined for the related
begomovirus, TYLCCV. (Ge et al., 2007), they are between
3- and 4-fold higher than a previous estimate of MSV evo-
lution rates [31].

It is not entirely surprising that our evolution rate estimate
is higher than that made by Isnard et al. [31] because
whereas our estimates are based on mutational distances
from known progenitor sequences, theirs are based on
distances from a population consensus sequence. Had we
used a consensus of the 17 MSV-Tas derived clones
instead of the MSV-Tas progenitor sequence itself, our
evolution rate estimate for the viruses maintained in sug-


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1 3 5 7 I IS : :t
24 l l


mp cp


Virology Joumnal 2008, 5:104








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occur in ssDNA [57-59] or by the action of deaminating
host enzymes [60].

One way to explore these alternative possibilities is to
examine substitution biases. Duffy and Holmes [30]
detected high rates of C-T and G->A transitions that were
possibly indicative of increased C and G deamination
rates. As deamination rates are probably higher for
ssDNA, this was taken to imply that high begomovirus
mutation rates might be at least partially attributable to
the considerable fraction of their life-cycles spent in
ssDNA form.

However, another way of using substitution biases as an
indicator of ssDNA specific mutagenic processes is to
compare the substitution rates of complementary substi-
tutions. If ssDNA is specifically prone to a mutagenic
process that, for example, results in an increased rate of
T->C transitions, then there should be evidence of signif-
icantly more T-C transitions on the virion strand (the
only strand that spends any appreciable time in a single


stranded state) than on the complementary strand. As the
two strands are complementary, one need only compare
rates of complementary T->C and A-G transitions on the
virion strand to determine whether the mutagenic mecha-
nism in question is more active on ssDNA.

We examined the 63 substitution mutations to determine
whether there was any evidence of substitution biases in
MSV. Table 1 lists the number of observed mutations of
each substitution type, as well as the expected frequencies
taking initial genome-wide nucleotide frequencies into
account. We found that G->T transversions were over-rep-
resented in both the maize and sugarcane evolution
experiments, and that this over-representation was highly
significant when either the MSV-Tas sequence dataset was
analysed alone (chi square p < 10-8) or when all the muta-
tion data from both experiments were considered collec-
tively (chi square p = 5.4 x 10-7; Table 1). Though not
statistically significant in our relatively small dataset, the
complementary C->A changes appeared to be consistently
under-represented. That there is such an obvious imbal-


Table I: Analysis of nucleotide substitution and mutation distribution biases in MSV genome sequences derived from evolution
experiments in maize (MSV-Kom, -Set and defective recombinant sequences) and sugarcane (MSV-Tas sequences).


MSV-Kom,-Set and defective recombinants


Substitution (V-sense)


MSV-Tas


All mutants analysed


X2 p-value Obs. Exp. X2 p-value Obs. Exp. X2 p-value


Transversions
A->C 2
A-T I
C->A 0
C-G I
G- C I
G->T 3
T->A 0
T ->G 0
Transitions
A-> G 3
C->T I
G->A 2
T- C 0
Ins 0
Del I
GC Content
A/T-G/C 5
G/C-A/T 6
Transitions (Ts) vs transversions (Tv)
Tv 8


Ts/Tv 0.75 0.5
Coding vs non-coding genome regions
Coding 13 12.4
Non-coding 2 2.6
Synonymous (S) vs non-synonymous (N) mutations
N 6 10.1
S 7 2.9
dN/dS 0.24 1.0


0.43
0.86
0.28
0.94
0.84
0.09
0.25
0.25

0.08
0.94
0.46
0.25



0.50
0.50

0.45
0.45


0.67
0.67

6x 10-3
6x 10-3


3 4.26
3 4.26
2 3.65
5 3.65
4 3.87
13 3.87
3 4.26
4 4.26

4 4.26
3 3.65
3 3.87
2 4.26
I


13 17
21 15

37 32.7
12 16.3
0.32 0.5


0.54
0.54
0.39
0.48
0.95
< 10-8
0.54
0.90

0.90
073
0.66
0.27



0.07
0.07

0.19
0.19


42 42.1 0.99
9 9.9 0.99


27 30.4
12 8.6
0.64 1.0


5 5.39
4 5.39
2 4.62
6 4.62
5 4.90
16 4.90
3 5.39
4 5.39

7 5.39
4 4.62
5 4.90
2 5.39
I
2

18 21.6
27 19.0

45 42
18 21
0.40 0.5


0.87
0.55
0.22
0.52
0.96
5.4 x 10-7
0.30
0.55

0.48
0.77
0.96
0.14



0.05
0.05

0.42
0.42


55 54.4 0.85
II 11.6 0.85


33 40.5
19 11.5
0.49 1.0


0.012
0.012


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ance in the complementary G->T and C->A transversions
strongly supports the hypothesis that a mutagenic process
causing G->T transversions on the virion DNA strand (the
strand predominantly found in single stranded form) is at
least partially responsible for higher than expected muta-
tion rates in MSV.

Probably as a consequence of the high rate of G->T muta-
tions, there was evidence of a significant trend towards
lower GC content over the course of the evolution experi-
ments when all mutations were collectively considered
(chi square p = 0.05). However, despite the high G->T
mutation bias, there was no significant trend in favour of
transversion mutations over transition mutations
(Table 1).

Whereas guanine and cytosine deamination of virion
sense ssDNA has been cited as a possible cause of the
increased frequencies of G->A and C->T transitions
observed in begomoviruses [30], the over representation
of G->T transversions we have observed in MSV is proba-
bly caused by some other form of damage to single
stranded MSV DNA. One possible mechanism is the oxi-
dation of guanine into 8-oxoguanine which then base-
pairs with adenine during replication and causes G->T
transversions. Formation of 8-oxoguanine is known to be
the most common cause of spontaneous G->T transver-
sions in many organisms [61-64]. That an increased rate
of G->T transversions has been associated with time spent
as ssDNA [65-67] fits very well with the notion that
increased rates of MSV mutation may be at least partially
attributable to either increased rates of 8-oxoguanine for-
mation or decreased rates of 8-oxoguanine lesion repair in
virion sense ssDNA.

Negative selection predominates but some mutations may
be adaptive
Mutations were distributed among coding and non-cod-
ing sites more or less as expected, given their relative num-
bers (Table 1). The ratio of non-synonymous to
synonymous substitutions (dN/dS) was significantly less
than one when either the maize experiment dataset (col-
lectively including sequences derived from wt MSV-Kom,
MSV-Set and the defective chimaeric viruses) was consid-
ered in isolation (chi square p = 6.0 x 10-3) or when all
data was collectively considered (chi square p = 1.2 x 10-
2; Table 1). This indicated that the sequences, particularly
those from maize, were most likely evolving under a pre-
dominance of negative (or purifying) rather than positive
(or diversifying) selection. Unfortunately our datasets
contained insufficient diversity and too few sequences for
the kinds of site-by-site selection analyses that enable
detection of individual sites evolving under positive selec-
tion against a background of negative selection [68,69].


We nevertheless thought it probable that evidence of
adaptive evolution might be detectable amongst the
mutations found in the defective chimaeric virus dataset.
Disruptions of specific interactions between CP and MP
and between CP and some other as yet unidentified viral
genome regions) are apparently responsible for the
reduced fitness of these chimaeric viruses [23,43]. We
hypothesised that fitness losses caused by transferring mp,
cp or mp-cp coding regions between MSV-Kom and MSV-
Set might have been partially recouped through compen-
satory mutations within the mp-cp cassette that restored
damaged interactions either within the mp-cp cassette, or
between the cassette and the remainder of the MSV
genome. It was anticipated that the most obvious sign of
such "repaired interactions" would be mutations within
the mp-cp cassettes of defective chimaeric viruses that
changed identity from that of one parental sequence to
the other.

However, only one mutation (13 in Figure 1 and see Addi-
tional file 1) out of eight detected in the defective chimae-
ric viruses represented a change from one wild-type
parental sequence to the other. This mutation was one of
four (mutations 6, 7 and 9 in Figure 1 were the others)
that occurred at sites that were polymorphic between
MSV-Kom and MSV-Set. This is close to the expected
number (4/3 = 1.3) of conversions between MSV-Kom
and MSV-Set polymorphisms if one assumes random
mutation. In the context of reports that some MSV
mutants either revert or experience compensatory muta-
tions at high rates to restore fitness [33-35] and that MSV
can adaptively overcome host resistance within a period
of about a year [31], we were surprised by this result.
Together with the fact that we observed no changes in the
symptomatology of any of our defective chimaeric viruses
after a year in maize, this lends support to the results of
our dN/dS analyses (Table 1) indicating that few, if any,
of the observed genetic changes were beneficial evolution-
ary adaptations.

The only indication of positive selection that we found in
the defective chimaeric virus dataset was a significantly
elevated number of substitutions in the mp-cp cassette of
these viruses. We compared the distribution of mutations
between the mp-cp and repA-repB coding regions in the
defective MSV-Kom/-Set chimaeras with the mutation dis-
tributions seen in the progeny genomes of wild type MSV-
Kom, -Set, and -Tas infections. In both the MSV-Kom/Set
and the MSV-Tas datasets, neither the mp-cp cassette nor
the repA-repB cassette contained disproportionately more
mutations than could be accounted for by chance. Simi-
larly, the number of mutations in the repA-repB cassette of
the defective chimaeric viruses was not significantly
higher than expected by chance. However, the mp-cp cas-
sette of these viruses contained eleven times more substi-


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tutions per site than did the rest of their genomes (chi
square p-value = 0.014). On the other hand, considering
that only two of these substitutions resulted in (relatively
conservative) non-synonymous changes (mutations 2
and 7, see Additional file 1) any positive selection that
may have occurred was likely to have been acting on non-
coding aspects of the DNA sequences such as those iden-
tified by Shepherd et al. [33].

Conclusion
We have presented evidence from controlled evolution
experiments lasting up to five years that indicates that
MSV experiences high rates of evolution close to those
recently approximated in shorter term experiments for
another geminivirus species [32]. Collectively these
results add credibility to reports that on a long term global
scale geminiviruses may be evolving at rates as high as
those reported for many RNA viruses [30]. For the first
time we show strand-specific substitution biases which
directly indicate that at least some of the mutational proc-
esses underlying high MSV evolution rates are acting pref-
erentially on ssDNA. While the increased mutability of
ssDNA may neatly account for disparities between the
evolution rates of ssDNA and dsDNA viruses, proof of this
may ultimately require a detailed comparative analysis of
the individual impacts of all mutagenic reactions and
repair pathways acting on single and double stranded
DNA molecules.

Methods
Virus isolates, plasmids, bacterial strains, plants and
leafhoppers
Agroinfectious clones of MSV-Kom, MSV-Set, K-MP-S, K-
MP-CP-S and S-CP-K [43,70] have been described previ-
ously. Agrobacterium tumefaciens C58C1 [pMP90] was
used to deliver viral DNA to maize cv. Jubilee (sweetcom)
seedlings by agroinoculation as described by Martin et al.
[71]. The MSV-Tas infected sugarcane plant (cultivar Uba)
used in this study was the same as that mentioned in a
previous publication [44]. A virus-free Cicadulina mbila
colony maintained at the University of Cape Town since
1990 was used as a source of leafhoppers during transmis-
sions [72].

Leafhopper transmission of viruses
C. mbila leafhoppers and infected plants were maintained
isolated in purpose-built cages (410 mm x 410 mm x 710
mm, w x d x h) at approximately 21 oC with indirect nat-
ural light augmented by Grolux"T fluorescent tubes for 12
hours per day. Each cage contained plants infected with a
single virus genotype. Initially three 25-day-old plants
infected by agroinoculation with each ofMSV-Kom, MSV-
Set, K-MP-S, K-MP-CP-S, and S-CP-K were placed in sepa-
rate isolation cages with c.a. 100 adult leafhoppers and
three uninfected 8-day-old maize seedlings per cage.


When symptoms became visible on new plants the older
plants were removed from the cage and replaced with
seedlings; this cycle was repeated approximately monthly.
The entire experiment lasted for 12 months, during which
the viruses were passage through 12 generations of
maize plants.

Initiation of a MSV-Tas infection in a single sugarcane
plant (cv. Uba) by leafhopper transmission from an
agroinoculated maize plant is described in [44]. This
infected sugarcane plant was maintained for five years at
25 C with 16 hours of light per day provided by Grolux
fluorescent tubes.

Isolation, cloning and sequencing of viral DNA
Replicative form, double-stranded virus DNA was
extracted from plants as described by Palmer et al. [73].
Isolated virus genomes were ligated either into the BamHI
site of pUC18 using standard techniques (all clones
labelled Ex-Oy and SC-Ex-Ox) [74] or using phi29 DNA
polymerase (TempliPhi'", GE Healthcare, USA) as
described previously [75,76] (all clones labelled Cx, Ex
and Fx where C, E, F indicate that clones were obtained
from different shoots). Briefly, the amplified concatamers
were digested with BamHI, to yield ~2.7-kb linearised
viral genomes which were ligated with linearised
pGEMZf+ (Promega Biotech). Individual genome
sequences were determined by the University of Cape
Town DNA Sequencing Service (Molecular and Cell Biol-
ogy Department, UCT), the University of Florida Interdis-
ciplinary Center for Biotechnology Research DNA
sequencing service, or commercially sequenced (Macro-
gen Inc., Korea) using the primer set described by Owor et
al. [75]. All mutations were verified by at least two
sequencing runs. All parental virus clones were re-
sequenced in both directions.

Sequence analysis
The expected frequency for a given substitution ofnt. X for
nt. Y (fEx>y) was calculated assuming all substitution
types were equally likely, as f EXy = (Px x M)/3 where Px
is the fractional proportion of nucleotide X (= A, G, T or
C) in the parental sequence, and M is the total number of
observed mutations. Significant deviation from the
expected number of mutations of a given type was tested
using a 2 x 2 chi square test (ie. observed and expected
substitutions numbers of a particular type x observed and
expected substitution numbers of all other types pooled).
Expected transition (Ts) and transversion (Tv) frequencies
were calculated by summing the expected frequencies of
the relevant substitutions. Significant deviation of
observed Tv and Ts values from those expected under the
null hypothesis ofTv/Ts = 2 (i.e. all mutations occur at the
same frequency irrespective of whether they are transi-



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Table 2: Distribution of mutations by genomic region.

Genomic region Mutations in region (region size)


Mutations in rest of genome
(region size)


Fold difference in sub/site X2 p-value


MSV-Kom/Set parental viruses
mp + cp
repA + repB
MSV-Kom/Set chimaeras
mp + cp
repA + repB
MSV-Tas
mp + cp
repA + repB


SYates' chi-squared p-values; p-value < 0.05.


tions or transversions) was calculated using a 2 x 2 chi
square test.

To calculate the proportions of nonsynonymous muta-
tions per nonsynonymous site (dN) and proportions of
synonymous mutations per synonymous site (dS), the
numbers of nonsynonymous and synonymous sites in
each coding region were obtained using the Datamonkey
web-server http://www. datamonkey.org/[61]. The num-
bers of synonymous and nonsynonymous mutations in
each coding region were determined manually. Deviation
of observed dN and dS values from those expected assum-
ing a dN/dS ratio of 1 (i.e. neutrality) was tested using a 2
x 2 chi square test.

List of abbreviations used
CP: Coat protein; cp: Coat protein gene; dsDNA: double
stranded DNA; LIR: Long intergenic region; MP: move-
ment protein; mp: movement protein gene; MSV: Maize
streak virus; NSP: Nuclear shuttle protein; ORF: Open
reading frame; PCR: Polymerase chain reaction; Rep: rep-
lication associated protein; rep : replication associate pro-
tein gene; SD: Standard deviation; SIR: Short intergenic
region; ssDNA: Single stranded DNA; TYLCV: Tomato yel-
low leaf curl virus.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
EvdW conceived the study, carried out the experiments,
analysed the data and prepared the manuscript. AV helped
carry out the experiments. DPM helped analyse the data
and prepare the manuscript. JP helped carry out the exper-
iments. EPR supervised the study, secured funding for its
execution and helped prepare the manuscript. All authors
read and approved the final manuscript.


Additional material


Acknowledgements
The authors wish to thank Siobain Duffy for her extremely insightful review
of this paper and for offering an excellent explanation of the oxidative proc-
ess that may be responsible for the mutation biases we observed. They also
thank the South African National Research Foundation (NRF) for funding
the research. EvdW was supported by the NRF, AV was supported by the
Carnegie Corporation of New York, DPM was supported by the NRF and
the Wellcome Trust.

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Mutations in MSV-Kom, MSV-Set and defective recombinants passage
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Click here for file
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