Group Title: Virology Journal 2008, 5:39
Title: Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N) methanocarbathymidine
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Title: Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N) methanocarbathymidine
Series Title: Virology Journal 2008, 5:39
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Quenelle DC
Keith KA
Moyer RW
Turner PC
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Malaria Journal BioIed Cen



Research

Clinically immune hosts as a refuge for drug-sensitive malaria
parasites
Eili Y Klein1,2, David L Smith3, Maciej F Bonil,2,4 and
Ramanan Laxminarayan* 1,4


Address: 'Resources for the Future, 1616 P St., NW, Washington, DC 20036, USA, 2Department of Ecology and Evolutionary Biology, Princeton
University, Princeton, NJ 08544, USA, 3Department of Zoology, University of Florida, Gainesville, FL 32611, USA and 4Princeton Environmental
Institute, Princeton University, Princeton, NJ 08544, USA
Email: Eili Y Klein klein@rff.org; David L Smith davesmith@ufl.edu; Maciej F Boni boni@rff.org;
Ramanan Laxminarayan* ramanan@rff.org
* Corresponding author



Published: 25 April 2008 Received: 28 January 2008
Malaria journal 2008, 7:67 doi: 10. 186/1475-2875-7-67 Accepted: 25 April 2008
This article is available from: http://www.malariajournal.com/content/7/l/67
2008 Klein 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: Mutations in Plasmodium falciparum that confer resistance to first-line antimalarial
drugs have spread throughout the world from a few independent foci, all located in areas that were
likely characterized by low or unstable malaria transmission. One of the striking differences
between areas of low or unstable malaria transmission and hyperendemic areas is the difference in
the size of the population of immune individuals. However, epidemiological models of malaria
transmission have generally ignored the role of immune individuals in transmission, assuming that
they do not affect the fitness of the parasite. This model reconsiders the role of immunity in the
dynamics of malaria transmission and its impact on the evolution of antimalarial drug resistance
under the assumption that immune individuals are infectious.
Methods: The model is constructed as a two-stage susceptible-infected-susceptible (SIS) model of
malaria transmission that assumes that individuals build up clinical immunity over a period of years.
This immunity reduces the frequency and severity of clinical symptoms, and thus their use of drugs.
It also reduces an individual's level of infectiousness, but does not impact the likelihood of becoming
infected.
Results: Simulations found that with the introduction of resistance into a population, clinical
immunity can significantly alter the fitness of the resistant parasite, and thereby impact the ability
of the resistant parasite to spread from an initial host by reducing the effective reproductive
number of the resistant parasite as transmission intensity increases. At high transmission levels,
despite a higher basic reproductive number, R0, the effective reproductive number of the resistant
parasite may fall below the reproductive number of the sensitive parasite.
Conclusion: These results suggest that high-levels of clinical immunity create a natural ecological
refuge for drug-sensitive parasites. This provides an epidemiological rationale for historical patterns
of resistance emergence and suggests that future outbreaks of resistance are more likely to occur
in low- or unstable-transmission settings. This finding has implications for the design of drug policies
and the formulation of malaria control strategies, especially those that lower malaria transmission
intensity.


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Background
Malaria is the leading cause of death in children under five
in sub-Saharan Africa [ 1]. Prompt treatment with effective
antimalarial drugs could prevent much of the morbidity
and mortality associated with clinical malaria, but the
evolution of resistance has diminished the therapeutic
efficacy of two previous first-line antimalarials, chloro-
quine (CQ) and sulphadoxine-pyrimethamine (SP). His-
torically, it has been suggested that resistance to both CQ
and SP emerged from a limited number of de novo selec-
tion events in areas of low or unstable transmission [2,3].
Genetic evidence suggests that most CQ-resistant parasites
in the world today are descended from one of four
founder events [4,5] that occurred in Southeast Asia,
South America and Papua New Guinea, and then spread
to other regions, including sub-Saharan Africa. Evidence
also suggests that resistance to SP originated from only a
few foci [6-8]. Several hypotheses about the de novo muta-
tion rate and the selection pressure resistant parasites face
and their relation to transmission intensity have been pro-
posed to explain why CQ resistance originated in what is
presumed to be low or unstable transmission areas out-
side of sub-Saharan Africa, including (a) a lower fre-
quency of resistant alleles in higher transmission areas
because of within-host competition [9]; (b) less drug
treatment (per parasite) in higher transmission areas
[2,9,10]; and (c) a lower frequency of selfing in higher
transmission areas, which increases the probability that
multilocus resistant genotypes will be broken up by the
action of Mendelian segregation [9,11,12]. An additional
explanation is that in high transmission areas, where
immunity is better developed, mutant parasites are less
likely to survive a host immune response [10,13].

Although the evolution of resistant parasites within a sin-
gle host is a significant risk factor for the emergence of
resistance within a population, the effectiveness of an
antimalarial drug is affected only if these resistant para-
sites spread, a process that is driven both by the overall
rate of transmission and by the relative fitness of drug-
resistant and drug-sensitive parasites. Parasite fitness is
related to the population treatment rate and to the biolog-
ical cost of resistance. Because of differences in vector
ecology and biting preferences, transmission intensities
vary from less than one infectious bite per decade to more
than 1,000 per year [14], and the basic reproductive
number (R0) can exceed 3,000 [15]. Higher transmission
intensity is associated with a higher level of clinical immu-
nity to malaria reduced frequency and severity of clinical
symptoms in older children and adults which results in
a reduction in the need for antimalarial drugs [16-19].
Immunity to malaria has consequences for transmission
as well; blood-stage immunity reduces asexual parasite
and gametocyte densities in older children and adults
[20], and transmission-blocking immunity can block


development of the parasite in the mosquito [21]. Impor-
tantly, despite significant reductions in clinical symptoms
and infectiousness, older individuals still become infected
and remain infectious to mosquitoes, albeit at relatively
lower levels, even in holoendemic areas [22-24].

Even though immunity affects both the transmission
dynamics and treatment rates, epidemiological models
have generally assumed that immune individuals are not
infectious to mosquitoes, and thus do not contribute to
parasite fitness [20,25-27]. Consequently, an epidemio-
logical model for the spread of resistance found no differ-
ence in the ability of resistant parasites to spread at
different transmission rates [27], or why resistance to CQ
emerged outside of sub-Saharan Africa.

This paper reconsiders the role of immunity in the trans-
mission dynamics of malaria and its effect on the evolu-
tion of antimalarial drug resistance under the assumption
that immune individuals are infectious. Using a novel epi-
demiological model with two immune stages, nonim-
mune and clinically immune, the role of immunity in the
emergence of resistance is reexamined based on quantita-
tive effects associated with clinical immunity. Individuals
in the clinically immune stage have lower transmission,
lower incidence of clinical disease, and consequently a
lower rate of drug treatment. Based on the defined rela-
tionship between transmission intensity, immunity, and
clinical malaria, the model is used to explore the relation-
ship between vector ecology, human epidemiology, and
the ability of a resistant parasite to spread.

Methods
Individuals living in areas of endemic P. falciparum trans-
mission develop immunity to malaria with age [28] and
exposure [29,30]; immunity is manifest as a decline in
parasite densities (both trophozoites and gametocytes) in
the blood [20], a lower probability of transmission from
humans to mosquitoes [22,23], and a decline in the fre-
quency and severity of clinical malaria [16-19], though lit-
tle decline in the probability of becoming infected
[24,31]. Thus, it is assumed that individuals develop a
form of clinical immunity over time in which they are less
likely to infect mosquitoes or manifest clinical symptoms
but are no less likely to become infected.

The model is based on earlier models developed for the
Garki Project [20], in which individuals acquire immunity
after being infected for a period of time. However, the
number of infected classes was simplified and the
assumption that individuals develop full transmission-
blocking immunity was relaxed. As with the Garki model,
clinical immunity is incorporated as a second immune
stage in a susceptible-infected-susceptible (SIS) model.
Though, in this model clinically immune individuals


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remain infectious to mosquitoes and infect mosquitoes
with lower probability; also, a smaller fraction of new
infections progress to clinical malaria. Clinical episodes of
malaria are important for the evolution of resistance
because individuals who develop clinical symptoms are
more likely to seek treatment; thus an increasing fre-
quency of clinical episodes is associated with increasing
drug pressure. Consequently, in this model clinically
immune individuals represent a refuge for drug-sensitive
pathogens because of lower treatment rates. The evolution
of resistance is incorporated by assuming that individuals
can be infected by either resistant or sensitive parasites.

Population dynamics
It is assumed that both nonimmune and clinically
immune individuals can be susceptible, infected with sen-
sitive parasites, or infected with resistant parasites. The
human population density in each state is denoted S, I,,i,
I,, where the subscripts w and x denote infections with
drug-sensitive wild-type and resistant phenotypes, respec-
tively, and the i subscript denotes the immune stage. The
population size is normalized to one, and the population
birthrate B is set equal to the per capital death rate of the
human population, p, so that the total population size
stays constant.

Entomology
Model notation follows Macdonald [32] and Smith and
McKenzie [33]; m denotes the number of mosquitoes per
human and a the human feeding rate (the number of bites
on humans per mosquito per day). The instantaneous
death rate is g (e is the probability of a mosquito surviv-
ing one day), and n is the number of days required for
sporogony. Vectorial capacity (V), the number of infec-
tious bites by a mosquito over its lifetime, is then given by
the formula V = ma2e-gl/g.

The fraction P of bites on humans that infect a mosquito
depends on the differing transmission intensities of the
two-stages, cI and c2, and the number of humans in each
stage; thus P = cl(Iwl + I,1) + c2(Iw2 + Ix2). The sporozoite
rate, or the fraction of infectious mosquitoes, is aPe-g /(g +
aP). The entomological inoculation rate (EIR), the
number of infectious bites per person per day, is calcu-
lated as the product of the human biting rate (ma) and the
sporozoite rate (aPe -'/(g + aP)). The force of infection, or
happenings rate (h), is bEIR, where b, the infectivity rate,
measures the fraction of bites in humans that produce a
patent infection. It follows that h=(bVP)/(1 + sP), where s
= a/g is called the stability index, the number of bites on a
human per vector per lifetime. The fraction of infections
that are drug sensitive is F, = (c1Ik1 + c2IJw)/P and the frac-
tion that are drug resistant is F = (cl4I + c2l2)/P. Happen-
ings rates for drug-sensitive and drug-resistant infections
are h, = Fwh and h = Fxh, respectively.


Immunity acquisition and parasite clearance
Clinical immunity is assumed to develop in infected indi-
viduals after ten years. Once individuals gain immunity,
protection is retained through biting and is lost at a faster
rate (y) than it is gained (6). The values are based on a sig-
nificant number of age-prevalence studies suggesting that
children acquire immunity after approximately five to 10
years [29,30,34], and additional studies that suggest a
strong role of biting in maintaining immunity, and a loss
of immunity that occurs after exposure to infection is
eliminated [35]. Because not all infections result in fever
and other associated symptoms, it is assumed that clinical
symptoms arise in infected individuals at a rate 7i and that
a fraction, are treated and cleared of parasites. Thus, exist-
ing infections are cleared by drugs at the rate pi = fi,. It is
further assumed that a fraction of new infections in sus-
ceptible individuals develop clinical symptoms and are
treated with drugs and cleared immediately prior to the
development of gametocytes, thus precluding the possi-
bility of transmission. In these individuals it is as if the
infection never occurred.

Mutations conferring resistance to antimalarial drugs are
likely to be disadvantageous to the parasite. For example,
resistance to CQ in P. falciparum has been shown to have
a fitness cost of approximately 25 percent in vitro [36] and
5 percent in vivo [37,38]. Although a fitness cost could
conceivably occur at any or all stages of the parasite life-
cycle, where it is implemented in this model is not partic-
ularly important for the transmission dynamics (though it
could be important in other models), so it is assumed that
resistant infections are cleared at a faster rate and therefore
transmit for a relatively shorter period. Thus, infections
clear naturally at rate r, when an individual is infected
with a drug-sensitive phenotype and r for drug-resistant
infections.

Equations
Based on the above assumptions, model dynamics are
described by a simple set of coupled ordinary differential
equations:

Si = B + YS2 + I1(p, + r.) + Ir( Sl(h(1 1,) + h, + )
(1)


Iiw = Slh(1 41) iwl(pi + rw + 0 + P)


ix Sbli _I'1(r +0+P)


S2 = Iw2(2 + r) + x2 S2(hw(1 2) + h + y + P)
(4)


iw2 = S2hw(1 2) + Iw1 w2(P2 + r + +)


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Figure I
SIS two-stage model. Susceptible individuals (S) become infected (I) with wild-type infections at the rate h,(I-i) and resist-
ant infections at the rate hx, where h, is the happenings rate (see text) and is the rate at which new infections result in clini-
cally manifested symptoms that are treated and resolved prior to the formation of gametocytes. Infected individuals naturally
clear resistant infections at rate rxi, and they clear wild-type infections at the rate p,+r,,, where p, is the rate of drug treatment.
Infected individuals acquire semi-immunity at rate 0and lose immunity at rate yif they are no longer infected. Individuals die
from all states at a background rate of gt and are born at rate B as nonimmune susceptibles (process not shown).


Ix2 = S '1 + IxO Ix2(r + i) (6)

A diagram of the model is found in Figure 1.

Results
The principal assumptions for the analysis were that
immune individuals are less infectious to mosquitoes (c2
treated (p2 < p1 and 2 < 1), and that parasites face a resist-
ance-related fitness cost (rx > rJ). While recent evidence
has suggested that individuals with clinical-immunity
may recover at faster rates [39], it was assumed that recov-
ery from infection was independent of immune status in
the base model. Based on these assumptions, the equilib-
rium of the system was determined without resistance and
then the resulting spread of resistance was simulated
across a range of transmission rates (from less than one to
more than 600 infectious bites per person per year) using
a set of baseline assumptions (Table 1).

At equilibrium without resistance, the results were con-
sistent with other models of transmission. As vectorial
capacity increases, the proportion of the population that
is nonimmune, and thus subject to frequent attacks,
increases sharply and then slowly decreases, while the


proportion of immune individuals increases steadily until
it plateaus at a fairly high level (Figure 2). As expected
[24,31], the infected proportion of the population nears
100 percent at high transmission levels, though a signifi-
cant portion of infected individuals are clinically
immune. In addition, the results suggest that clinical epi-
sodes of malaria a proxy for hospital admissions for
malaria as well as severe malaria plateau at intermediate
levels of transmission, which is consistent with other
research [30].

Because symptomatic individuals are more likely to use
antimalarial drugs, the number of individuals experienc-
ing a clinical episode affects the transmission dynamics by
changing the drug pressure facing the parasite. Individuals
that acquire clinical immunity, have reduced rates of clin-
ical episodes [16-19], which reduces the rate of drug use.
Though individuals will continue to receive antimalarial
treatments throughout their lives, these treatments occur
less frequently as immunity increases, and often can be
unconnected to the peaks of parasitemia [3]. This reduc-
tion in the treatment rate reduces the resistance selection
pressure, providing a refuge for drug-sensitive parasites. In
previous epidemiological models of transmission, these
immune individuals were not assumed to have a signifi-
cant qualitative effect on the dynamics of the system,


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Table I: Baseline Parameter Values


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Acquisition of clinical immunity
Loss of clinical immunity
Fraction of new infections that are treated and cleared

Rate clinical symptoms arise (o,) times Fraction treated (f) equals rate existing infections are cleared by drugs (p)

Disease induced death rate
Human feeding rate
Infectivity rate
Mosquito death rate
Number of days required for sporogony
Recovery rate


because young children were assumed to dominate trans-
mission events [25]. However, clinically immune individ-
uals are infectious and can significantly affect the
dynamics of transmission when the distribution between
sensitive and resistant infections is different in nonim-
mune and clinically immune individuals.

Distributional differences in the frequencies of infection
types are driven by drug treatment rates, and these differ-
ences can be calculated as differences in the relative fitness
of resistant parasites compared with sensitive parasites.
Parasite fitness was calculated as the basic reproductive
number (R0) of both the resistant and the sensitive para-
sites when the population is completely naive (sub-
scripted by 1) and fully immune (subscripted by 2). The
R0-values of the sensitive parasites are


w bV(1-41) [ + c2 (7a)
Rowl ---0---- 61 + -------- (7a)
8+pl+*W+lw I P2+Tw+I


_R bVc2(1-2) (7b)
R,w2 P2+T (7b)

and the R0-values of the resistant strains are


Ro,x 1 + C (8a)
O+rx+P rx+P


bVc2
RO,x2 = (8b)
rx+p f

Previous models suggested that the parasite with the high-
est R0 would be the most fit and should predominate
[27,40]. That conclusion always holds when rx= rw, c1 = c2,
and p, = p2. However, differences in the drug treatment
rates between clinically immune and nonimmune indi-
viduals can change the result, and it was found that resist-
ant parasites may not spread even when they have the


-1 10 years
Y- 2 years
,1 0.3
42 0.01
af, = P 0.025(0.2) = 1/200
2f2= P2 0.01(0.2) = 1/500
m 180/100000/year
a 0.3
b 0.8
g 1/10
n 10
rw 1/(165/b)
r. rw(fitness cost)


dominant Ro. This can be explained by calculating the
effective reproductive number of the resistant parasite, R,
where Rx = R,, .3 and d is the fraction of the population
that can be infected and will transmit the resistant infec-
tion. The effective reproductive number of the resistant
parasite is


Rx= (SS + S2)((1 -f) R,x +fR,x2)


where f is the fraction of the population that is clinically
immune, and S1 and S2 are the proportion of the popula-
tion that is susceptible and nonimmune, and susceptible
and clinically immune, respectively.

The results suggest that as the transmission rate increases,
the fraction of clinically immune individuals increases,
significantly reducing the effective reproductive number
of the resistant parasite. At certain parameter values it was
even possible for the effective reproductive number of the
resistant parasite to fall below 1 while the reproductive
number of the sensitive parasite remained above 1, effec-
tively abrogating the ability of resistant parasites to spread
(Figure 3). Thus, at higher transmission levels it may be
possible for the resistant parasite to have the dominant Ro
but be unable to spread because of the population-wide
level of immunity.

Though the ability of the resistant parasite to spread is not
always compromised, the generalizable result is that at all
levels of fitness cost (including none), it is easier for resist-
ant parasites to spread at lower levels of transmission (Fig-
ure 4). This reduction in the ability of individuals to
transmit the resistant parasite suggests an important
implication: it is relatively easier for resistance to spread in
an area of lower transmission than in an area of higher
transmission. This also helps explain, from an epidemio-
logical perspective, why resistance in general tends to
evolve faster in lower-transmission settings.





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E


r Co
E


o

t
o
oa
0.


3 -2 1 0 1 2 3 4
Log ofVectonal Capacity

Figure 2
Steady state infection level. The proportion of people
with P. falciparum infections increases extremely rapidly as
vectorial capacity increases, but there is a large shift between
those who are nonimmune and those who are immune.
Semi-immune individuals mirror the trajectory of total infec-
tions as vectorial capacity increases and quickly become the
large majority of infections. At low levels of vectorial capac-
ity, the proportion of the population that is clinically immune
remains extremely low. However, as vectorial capacity
increases, the proportion of individuals that are clinically
immune nonimmunee) increases (falls). The clinical incidence
rate increases sharply at first but then plateaus as the pro-
portion of the population that is clinically immune increases.
The clinical incidence rate is defined at equilibrium as the
sum of susceptible individuals who become infected with a
wild-type phenotype and develop an immediate clinical infec-
tion plus individuals already infected who become sympto-

matic h,S4, + li.s where i and j are the


phenotype of the infection and the immune stage, respec-
tively.



Discussion
Simulations of the model found a significant relationship
between malaria immunity and the ability of resistant par-
asites to spread from an initial locus. Previous epidemio-
logical models of malaria transmission assumed that
immune individuals were not infectious; however, the
best available evidence in malaria suggests that immune
individuals remain infectious, though they transmit less
efficiently to mosquitoes [22-24,31] and the frequency
and severity of clinical disease declines [16-19]. Because


-3 -2 -1 0 1 2 3 4 5 6
Log of Vectorial Capacity

Figure 3
Immunity reduces resistant parasite fitness. The clini-
cally immune class influences the ability of a resistant parasite
to invade. Since individuals in this class do not progress to
clinical malaria as often, they are treated less often; this cre-
ates a refuge for the wild-type parasites. As vectorial capacity
increases, the clinically immune class is maintained at higher
and higher levels until it becomes biologically impossible for
the resistant parasite to spread. This paradigm exists because
of the population of clinically immune individuals, without
whom the resistant parasite would be able to spread at any
vectorial capacity (as shown by the dashed line above).



they remain infectious but asymptomatically infected,
their relatively lower usage of antimalarials creates a natu-
ral refuge for sensitive parasites, similar to the way non-
transgenic crops act as refugia for Bt-sensitive insects in
agriculture [41]. Increases in the transmission rate result
in concomitant increases in the proportion of clinically
immune individuals, which increases the size of the ref-
uge and reduces the ability of the resistant parasite to
spread. This suggests an epidemiological rationale for the
more likely emergence of resistance in low- or unstable-
transmission settings.

The presence of a refuge of clinically immune individuals
alters the ecological landscape that resistant parasites face,
and introduces a mechanism for the coexistence of resist-
ant and sensitive parasites. Simple models of directly
transmitted infections [40], and a previous model of
malaria transmission [27], suggested there was a critical
threshold level of treatment above which resistance would
fix and below which it would not spread. However, in
those models, there was either no immunity, or immune
individuals were assumed to be noninfectious and were,
therefore, irrelevant for transmission and selection for



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; Ro
resistant parasite Proportion clinically immune



,. re




------------------ ----------------------------------
-- -----
< resistant parasite
*


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control project in the Garki region of Nigeria [35]. The
mathematical model of control developed for the Garki
Base project considered the clearance of infection in hosts that
have been infected by more than one parasite brood
20% [[20,45], and the development of blood-stage immunity
and transmission-blocking immunity in the human host
S33% [20]. Despite a model that assumed parasite infections
43% were complex and evidence that gametocytes remain
50% present throughout life, the Garki model made a simplify-
ing assumption that new infections remained infectious
for a short period of time. Thereafter, infections persisted
until the person either cleared the infection or developed
immunity. The Garki model called this a semi-immune
state. In this semi-immune state, humans could become
re-infected, but they were never infectious. Mathematical
epidemiologists later noted that individuals who were
.I. infected but not infectious were epidemiologically irrele-
-6 -4 -2 0 2 4 6 vant for transmission, and the Garki model was simplified
Vectorial Capacity into an SIRS compartment model in which the dynamics
4 of infection in the semi-immune population were
e reproductive rate and fitness cost of resist- replaced by a recovered and immune state with immune
*he higher the fitness cost of resistance (I rjr,), the boosting [25,26]. The SIRS model was a familiar model to
he absolute level of the effective reproductive mathematical epidemiologists, and it has been the basis
. Qualitatively, this suggests an important implication: for many subsequent papers on malaria epidemiology,
ays more difficult for the parasite to evolve (i.e., but the assumptions about immunity have been propa-
and spread) resistance in higher-transmission set- gated and revised without much critical thought.


tings.


resistance. Clinical immunity changes the criteria for
determining the critical threshold so that the fraction of
clinical episodes that are treated, as well as the level of
immunity in the community, which determines the frac-
tion of new infections that result in a clinical episode, are
both important. Therefore, in populations where immu-
nity has developed, the resistant parasite faces a different
fitness landscape; they may not be the most fit, despite
having the highest R0, which can prevent the parasite from
either fixing or spreading. This result also suggests that dif-
ferential treatment levels of host-groups can create differ-
ential ecological niches allowing the resistant and
sensitive parasites to coexist [42].

History of epidemiological models of malaria
There is a striking difference between the findings of this
paper and a previously published epidemiological model
of antimalarial resistance [27]. To explain the differences,
a brief history of malaria models is warranted. A quantita-
tive approach to malaria epidemiology organized around
the parasite life-cycle was first described mathematically
by Ross [43], and later revised by Macdonald to consider
mosquito mortality during sporogony [32,44]. In the
1970s several major innovations in malaria modeling
were first introduced and field-tested during a malaria


This paper is an attempt to demonstrate the importance of
immunity in modeling the transmission dynamics of
malaria with respect to the introduction of resistance.
While the implications of the results are significant for
drug policy, it is prudent to note that as with all models a
number of simplifying assumptions have been made. In
incorporating immunity, the process of immunity acqui-
sition has been simplified so that individuals acquire
immunity in a stepwise fashion after 10 years of continu-
ous infection, and they lose immunity after two years of
continuous non-infection. This is a simplification of the
complicated process that is immunity. It ignores age
effects, differences between immune individuals at older
ages and younger ages, and faster acquisition of immu-
nity, which may occur in areas of higher transmission [46]
or after fewer challenges in low transmission areas [47].
While this is a simplification, evidence suggests that it
gives a representation of reality that is close enough to
make qualitative observations [29,30,34,35]. Despite the
simplification of immunity, the basic model (without
resistance) accords well with both field data and other
models of transmission dynamics, and changes in these
values did not change the qualitative results. Thus, the
model, as formulated, likely captures the qualitative dif-
ferences that exist between areas with different transmis-
sion levels, and is, hopefully, only the first step in more





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U CT

co

UJ


Figure
Effect
ance. T
lower ti
number
it is alw
emerge


Malaria Journal 2008, 7:67








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detailed modeling of the spread of antimalarial resistance
which takes account of the importance of immunity.

Conclusion
The link between the transmission rate and the probabil-
ity that de novo mutants can both arise and spread is a crit-
ical issue for developing strategies to mitigate resistance
emergence. Though it is impossible to be certain, histori-
cal analysis of the evolution of resistance has suggested
that it originated in low or unstable transmission areas
[2,3]. Quantitative population genetic models have sup-
ported this assertion, suggesting that there may be genetic
reasons why resistance is less likely to evolve in high-
transmission settings [3,9,11,12], but individual models
may not always explain population-level phenomena.
Epidemiological models, which can take account of spe-
cific population characteristics, have been hampered by
the assumption that immune individuals play no qualita-
tive role in the dynamics of transmission. Consequently,
an epidemiological model could not offer an explanation
for why resistance emerged outside Africa [27]. The model
in this paper demonstrates that the existence of a refuge
for drug-sensitive parasites in high-transmission areas can
slow or prevent the evolution of resistance; this has enor-
mous implications for the design of effective malaria con-
trol strategies.

Efforts to control malaria have been undermined by the
emergence of resistance, which now exists to all known
antimalarials except the artemisinin compounds. To help
ensure a longer period of efficacy for artemisinin, the
World Health Organization has proposed a global sub-
sidy to comply with its mandate that all new artemisinin-
based therapies be deployed as combinations [48]. One
concern with this strategy is that increased use in high-
transmission areas may engender resistance at a faster rate.
The results as determined by this model, mitigates that
concern, showing that the existence of a refuge of immune
individuals makes this outcome highly unlikely. How-
ever, in areas with low transmission intensity, the relative
paucity of immune individuals increases the risk that
resistance will emerge. Because the emergence of resist-
ance to an antimalarial in any country threatens the via-
bility of the drug in all countries [49], areas of relatively
lower transmission should be a focus in controlling the
emergence of resistance. As a corollary, eliminating
malaria from areas of low transmission intensity may
have the global benefit of prolonging the effective lifetime
of antimalarial drugs.

Although areas of low transmission should implement
strategies to reduce the likelihood of resistance, high-
transmission areas are also a concern because transmis-
sion intensity is reduced through mass distribution of
insecticide-treated bed nets. The analysis in this paper sug-


gests that among-host selection for resistance increases as
functional immunity wanes and the refuge for drug-sensi-
tive parasites shrinks or disappears. Thus, implementation
of transmission reduction strategies should also include
improved surveillance for drug resistance with resources
to deploy appropriate containment strategies to prevent
the geographical dissemination of resistance.

The intention of this study was to evaluate the qualitative
importance of immunity in the transmission dynamics of
malaria and its role in the development of resistance. Epi-
demiological models of malaria transmission, which can
synthesize various factors about how emergence and
spread are linked to the composition of the population,
can be useful tools in determining optimal drug treatment
strategies to extend the effective life of newly introduced
therapies; however, they must take account of immune
individuals and the refuge they provide for sensitive para-
sites.

Authors' contributions
DLS and EYK developed the model. All authors contrib-
uted to the analysis and writing and have read and
approved the final version of the manuscript.

Acknowledgements
We are grateful to Peter Billingsley and Sunetra Gupta for their input on
the design of the model as well as Karen I Barnes, Anders Bjorkman, lan C
Boulton, Karen P Day, Abdoulaye Djimde, Arjen M Dondorp, Ogobara
Doumbo, Rose McGready, Malcolm Molyneux, Myaing M Nyunt, Christo-
pher V Plowe, Ric N Price, Allan Schapira and Nicholas J White for their
comments on the model at the Gordon Research Conference on Malaria.
This research was supported by a grantfrom the Bill & Melinda Gates Foun-
dation. Decisions concerning the study design; the collection, analysis, and
interpretation of data; the writing of the manuscript; and the decision to
submit the manuscript for publication, were made entirely by the authors
with no input from the Bill & Melinda Gates Foundation.

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Malaria Journal 2008, 7:67







Virology Journal BioMed



Research W

Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L)
replicates well in vitro and in vivo, but is hypersensitive to the
antiviral drug (N)-methanocarbathymidine
Mark N Prichard*l, Earl R Kern', Debra C Quenelle1, Kathy A Keithl,
Richard W Moyer2 and Peter C Turner2


Address: 'Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL 35233, USA and 2Department of Molecular
Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610, USA
Email: Mark N Prichard* mprichard@peds.uab.edu; Earl R Kern ekern@peds.uab.edu; Debra C Quenelle dquenelle@peds.uab.edu;
Kathy A Keith kkeith@peds.uab.edu; Richard W Moyer rmoyer@mgm.ufl.edu; Peter C Turner ptumer@mgm.ufl.edu
* Corresponding author


Published: 5 March 2008
Virology journal 2008, 5:39 doi:10. 1186/1743-422X-5-39
This article is available from: http://www.virologyj.com/content/5/1/39


Received: 24 January 2008
Accepted: 5 March 2008


2008 Prichard 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: The vaccinia virus (W) F2L gene encodes a functional deoxyuridine triphosphatase
(dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the
incorporation of deoxyuridine residues into the viral genome. Previous studies with with a
complex, multigene deletion in this virus suggested that the gene was not required for viral
replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo.
Although the crystal structure for this enzyme has been determined, its potential as a target for
antiviral therapy is unclear.
Results: The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral
replication. The resulting virus replicated well in cell culture and its replication kinetics were almost
indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to
be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with
the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be
expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir
and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus.
The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was
hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was
altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular
dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an
increased reliance on the salvage of thymidine by the viral thymidine kinase.
Conclusion: We confirmed that F2L was not required for replication in cell culture and
determined that it does not play a significant role on virulence of the virus in intranasally infected
mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect
metabolic differences in the mutant virus.


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Central


=es







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Background
All free-living organisms have mechanisms to minimize
the incorporation of uracil in their genomes. These resi-
dues in DNA can arise either through misincorporation of
dUTP by DNA polymerase or the spontaneous deamina-
tion of cytosine and can result in A:T transition mutations
in one of the nascent strands [1]. Minimizing the incorpo-
ration of these bases and excising those that arise prevents
the accumulation of deleterious mutations. The enzymes
uracil DNA glycosylase (UNG) and deoxyuridine triphos-
phatase (dUTPase) arose very early in evolutionary terms
and act in concert to protect organisms from uracil resi-
dues [2,3]. Enzymes with dUTPase activity catalyze the
dephosphorylation of dUTP to minimize its incorpora-
tion into genomic DNA, while UNG family members
repair uracil residues from DNA by base excision repair.

These protective enzymes are also present in many viruses
including retroviruses, herpesviruses and orthopoxviruses
[4]. Proteins with UNG activity are either encoded by
these viruses, or recruited by viral proteins and are
thought to be important in viral replication [1]. Similarly,
dUTPase homologs are encoded by many lentiviruses, as
well as all herpesviruses and orthopoxviruses and are pre-
sumed to minimize potential damage by the incorpora-
tion of uracil residues [3]. Both herpes simplex virus
(HSV) and vaccinia virus (W) encode homologs of dUT-
Pase and the viral enzymes hydrolyze dUTP to dUMP and
require divalent cations for their activity [5,6]. The dUT-
Pase encoded by the F2L gene of VV is a 16.5 kiloDalton
protein that forms homotrimers and enzymatic studies
determined that the Km for dUTP was 1 iM and that it was
competitively inhibited by 8-azido-ATP [7]. Recently, the
crystal structure of this trimeric enzyme was determined
and proved to be closely related to that of the human
homolog, although the central channel was somewhat


larger in the viral enzyme. These results suggested that the
development of specific inhibitors of this enzyme might
be possible [8].

If the dUTPase fulfills an essential role in viral replication
then inhibitors of this enzyme might have the potential to
be used in the treatment of orthopoxvirus infections. One
previous report described a recombinant virus with a large
deletion resulting in the elimination of 55 open reading
frames including F2L [9]. This recombinant was viable
suggesting that the dUTPase was not required for replica-
tion in cell culture, although it did not exclude the possi-
bility that it might be important for replication in vivo. In
HSV, deletion of the dUTPase homolog did not effect the
replication of the virus in vitro, however its virulence was
reduced by 1000-fold in mice following footpad inocula-
tion and reduced replication in the CNS was also observed
[10]. To assess the potential of the dUTPase as a target for
antiviral therapy, F2L was deleted in the WR strain of VV
and the replication of the mutant virus (W A F2L-gfp) was
evaluated in cell culture and in intranasally infected mice.
The deletion of F2L had a minimal impact on viral repli-
cation in vitro and did not appear to significantly reduce
the virulence in vivo suggesting that it did not contribute
appreciably to disease, and that it was not a good target for
the development of antiviral therapies.

Results
A recombinant virus lacking only the dUTPase gene was
constructed by homologous recombination in the WR
strain of VV. In this virus, F2L was replaced with the gfp
gene driven by the synthetic E/L promoter. Fluorescent
recombinant plaques were plaque purified three times to
eliminate any contaminating wild type (wt) parental
virus. The insertion of the gfp resulted in the deletion of
most of the F2L open reading frame from amino acids


F3L VACWR042


UTPase
VACWR041
F1L VACWR040


VV WR wt


PE/L gfp


N-terminal
10 aa of F2L


C-terminal
8 aa of F2L


= VV A F2L-gfp


Figure I
Genomic structure of the F2L region of VV A F2L-gfp. The F2L gene in VV strain WR (shaded arrow in the top line) was
replaced with the gfp gene driven by the synthetic E/L promoter (black arrow in the bottom line). The resulting virus was des-
ignated VV A F2L-gfp and contained a deletion in F2L corresponding to amino acids I 1-129 of the open reading frame.



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Virology Joumnal 2008, 5:39







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11-129 (Fig. 1). The engineered mutation did not appear
to affect plaque size and confirmed that F2L was not
required for replication in cell culture (data not shown).
The replication kinetics of this virus were assessed in
human foreskin fibroblast (HFF) cells at an MOI of 0.001
PFU/cell. Results from this experiment suggested that the
F2L mutant (VVAF2L-gfp) replicated well in these cells
and yielded titers that were reproducibly reduced com-
pared to those of the parent virus (Fig. 2). However, the
slight impairment in replication was so minor that it is
unlikely to be an important factor in cell culture. These
results suggested that the dUTPase is not required for the
replication of VV in cell culture and are consistent with the
previous report.

Although deletion of the dUTPase gene did not appear to
affect replication in cell culture, it is possible that it may
play a significant role in vivo as has been seen with the
dUTPase knockout virus in HSV. To test this hypothesis, 3
week old female, BALB/c mice were anesthetized with ket-
amine-xylazine and inoculated intranasally with 10-fold
dilutions of the viruses in a volume of 40 pl (20 pl per
nostril). Two isolates ofwt VV WR, and VV A F2L-gfp were
evaluated in this experiment to assess the virulence char-
acteristic of these strains. Mice were observed daily for 21
days and were evaluated for clinical signs of infection and
mortality. Infection with 1.6 x 104 PFU or greater resulted


7
oVV A F2L-gfp
--VV WRwt






2






0 12 24 36
Time (hours post infection)

Figure 2
Replication kinetics of VV A F2L-gfp in HFF cel
wells of 6-well plates were infected with the W
(square symbols) or VV A F2L-gfp (circular syn
from each well was harvested at 2, 8, 12, 24, 3
post infection. All samples including inocula we
duplicate and average titers are shown with er
resenting the standard deviations.


Table I: W A F2L-gfp exhibits virulence characteristics that are
similar to the parent virus.


Virus (PFU/mouse)a


W-WR, UAB
1.6 x I04
1.6 x 103
1.6 x 102
16
1.6

W-WR, Turner
Stock 1.6 x 108
1.6 x 107
1.6 x 106
1.6 x 105
1.6 x 104

W A F2L-gfp
Stock 6 x 107
6x 106
6x 105
6 x 104
6 x 103


Mortality
Number Percent


MDDb



6.8 0.8
8.3 0.6





3.2 0.4
3.9 0.3
5.1 0.3
6.2 0.4
7.9 1.0


4.2 0.4
4.7 0.5
6.1 0.6
7.1 0.3
8.4 0.5


a. Anesthetized mice were inoculated intranasally with 40 glI of virus
(20 gl/nostril).
b. Mean day of death (MDD) is shown with the standard deviation.

in 100% mortality, while some animals survived with 10-
fold less virus (Table 1). Thus, no significant differences
were observed in the virulence among the two isolates of
WR and the mutant virus. These results suggested that
dUTPase is not required for virulence in mice and its
removal does not appear to impact viral replication in ani-
mals.


Deletion of the dUTPase is predicted to effect pyrimidine
metabolism in infected cells and may alter the susceptibil-
ity of the mutant virus to some antiviral drugs. A set of
thymidine analogs was selected and a standard plaque
reduction assay was used to evaluate the susceptibility of
the dUTPase mutant and the parent virus. The mutant
remained fully sensitive to all of the drugs tested including
cidofovir (CDV), idoxuridine (IDU), and two thymidine
analogs reported to require phosphorylation by the VV
48 60 thymidine kinase (TK) [11]. The only significant differ-
ence observed in the mutant virus was the modest but
repeatable increase in the efficacy of N-methanocarbathy-
Is. Triplicate midine (N-MCT) (Table 2). This compound is a carbocy-
R strain of VV clic thymidine analog that inhibits the replication of VV
ibols). Virus both in vitro and in vivo [12,13], and also appears to
6, and 48 h require phosphorylation by the viral TK [12].
re titered in
ror bars rep- Discussion
Results presented here are consistent with a previous
report that showed that the dUTPase was not required for


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'1


Virology Joumnal 2008, 5:39







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Table 2: Susceptibility of VV A F2L-gfp to selected thymidine analogs and cidofovir.


Compound Name


WA F2L-gfp (EC50, M)a


IDU
N-MCT
PFT3
PFT4
CDV


2.5 0.9
6.2 3.5
2.0 0.6
2.3 1.1
II 4.2


W WR wt (EC50, gtM)

2.4 0.6
12 1.6
2.4 0.4
2.5 0.1
10 0.3


a. Concentration required to reduce plaque formation by 50%. Values shown are the average of duplicate determinations with the standard
deviations shown.


replication in cell culture using a virus containing a mul-
tigene deletion [9]. They also suggest that the function of
this protein is of modest importance in the replication of
VV in vivo. These results contrast with those reported pre-
viously for the dUTPase negative mutants of HSV where
virulence was severely affected in mice [10]. It is unclear if
the observed differences in replication in mice reflect real
differences in the biology of these viruses, or are related to
differences in the animal models including route of infec-
tion. In HSV, direct intracranial inoculation resulted in a
10-fold reduction in virulence of the mutant, whereas
footpad inoculation increased the LD50 from approxi-
mately 103 PFU to more than 106 PFU of the mutant virus.
Thus, the observed virulence of the HSV mutant was
dependent on the route of administration and it is unclear
if reduced virulence would be observed following intrana-
sal inoculation.

We show here that there is little if any attenuation when
mice are intranasally infected with VV in which the dUT-


dUTP



dUDP dUTPase



dUMP ,-
thymidylate
synthetase
dUrd


dTTP


I
dTDP


I
dTMP (N-MCT-MP)
Sthymidine
kinase
Thd (N-MCT)


Figure 3
Model of dUTPase function in VV and a potential explanation
of (N)-MCT hypersensitivity. Deletion of dUTPase is pre-
dicted to reduce intracellular pools of its dUMP product,
which is converted to dTMP by thymidylate synthetase. This
is significant since dTMP is predicted to compete with the
monophosphate metabolite of N-MCT (N-MCT-MP) for sub-
sequent anabolic reactions or as substrates for the target
enzyme.


Pase has been deleted. Additional experiments are
required to resolve this issue and the rabbit model of VV
infection and might be a more sensitive indicator of
reduced virulence associated with the mutant virus [14]. It
is also possible that reduced virulence might be observed
if infection was initiated through inoculation at periph-
eral sites.

Differences in pyrimidine metabolism were predicted to
occur in the absence of the dUTPase so a set of selected
thymidine analogs were used as potential indicators of
metabolic differences. The efficacy of the CDV control
virus was unchanged in the mutant, as was the activity of
IDU and two thymidine analogs reported previously [11].
However, the mutation in VV AF2L-gfp appeared to confer
some hypersensitivity to the drug (N)-MCT. The mecha-
nism of action of this compound is incompletely under-
stood, although it appears to require phosphorylation by
the viral TK to the monophosphate (N-MCT-MP) [12].
This is significant since intracellular pools of dUMP and
dTMP are predicted to be reduced in the absence of the
viral dUTPase. Thus, the increased ratios of N-MCT-
MP:dTMP and N-MCT-MP:dUMP should reduce competi-
tion for subsequent anabolic reactions or as substrates for
the target enzyme, perhaps thymidylate synthetase (Fig.
3). It is unclear why this was not observed with IDU and
the other compounds, but it likely reflects the specific
mechanisms of the individual drugs.

Conclusion
Data presented here suggest that VV dUTPase is not
required for viral replication in cell culture. The deletion
of F2L also does not appear to impact the virulence of the
virus in mice following intranasal infection. These studies
suggest that dUTPase is not a particularly good target for
the development of antiviral therapies, although it
remains possible that other animal models may identify
an important function of this enzyme. The mutation does
not affect the efficacy of most antiviral drugs including
CDV although it appears to confer a modest hypersensi-
tivity to N-MCT and may reflect metabolic differences in
the mutant virus.


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Methods
Cells, viruses, and drugs
Recombinant virus designated W AF2L-gfp and the
parental wt W strain WR (Turner) were received from Dr.
Pete Turner, University of Florida, Gainesville, FL. VV
strain WR (UAB) used in the animal studies was obtained
from the American Type Culture Collection (ATCC), Man-
assas, VA. Working stocks of both W-WR isolates and W
AF2L-gfp were propagated in Vero cells obtained from
ATCC. Human foreskin fibroblasts were prepared as pri-
mary cultures from freshly obtained newborn human
foreskins as soon as possible after circumcision. Culture
medium for both cell lines was minimum essential
medium (MEM) with Earle's salts containing 10% fetal
bovine serum and standard concentrations of L-
glutamine, penicillin and gentamicin.

The drugs tested included CDV, IDU, N-MCT, 5-(2-
amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-
yl)-1-(2-deoxypento-furanosyl)-pyrimidine-2,4(1H,3H)-
dione (PFT3), and 1-(2-deoxypentofuranosyl)-5- [(3-
methyl-5-oxo- -phenyl-4,5-dihydro-4H-pyrazol-4-yli-
dene)pyrimidine-2,4(1H,3H)-dione (PFT4). Compounds
designated PFT3 and PFT4 were synthesized by Paul Tor-
rence (Northern Arizona University) and were described
previously [11]; N-MCT was described previously [15];
CDV was a gift of Mick Hitchcock at Gilead sciences and
IDU was purchased from Sigma Aldrich (St. Louis, MO).

Construction of W AF2L-gfp
A recombinant was constructed from W strain WR with
most of F2L replaced with the gfp gene driven by the syn-
thetic E/L promoter by methods similar to those described
previously [16]. Primers IDT 715 (5'-ATGCTGCTTGGGT-
TAATATGCCG-3') against the F3L gene upstream from
F2L and IDT 716 (GCGAAGCTTAACTGGTGAGTTAATAT-
TCATGTTGAAC, HindIII site underlined) against the com-
plement of F2L residues 3-31 were used to PCR amplify a
487-bp fragment upstream from F2L. A 531-bp PCR prod-
uct consisting of the downstream flank from F2L was
made using primers IDT 717 (GCGCTCGAGAGGGTIT-
GGATCAACAGGAC, XhoI site underlined) against F2L
residues 417-436 and IDT 718 (CATACATCGTCTAC-
CCAATTCGG) against F1L. HindlII-digested, dephospho-
rylated IDT 715+716 PCR product and Xhol-digested,
dephosphorylated IDT 717+718 PCR product were
ligated to a HindlII-XhoI restriction fragment consisting of
the PE/L promoter linked to the gfp gene. The ligation mix
was PCR amplified with primers IDT 715 and IDT 718 to
generate a 1.8-kb product of gfp flanked by portions of
F3L and F1L. CV-1 cells were infected with wt W-WR,
transfected with the F3L-gfp-F1L DNA, and plaques
expressing gfp were isolated by fluorescence. The resulting
virus was designated W AF2L-gfp and contained a dele-
tion in F2L corresponding to amino acids 11-129 of the


open reading frame. The genomic structure and purity of
this virus was confirmed by PCR using primers IDT 715 +
718. No fragment of 1.4 kb corresponding to wt F2L plus
flanks was detected, but a 1.8 kb fragment of F3L-gfp-F1L
was present.

Growth curves
To determine the in vitro replication of the viruses, HFF
cells were incubated in 6 well plates for 24 h prior to infec-
tion at 370C with 5% CO2 and 90% humidity. Triplicate
wells were infected with wt W-WR or W A F2L-gfp at an
MOI of 0.001. Infected plates were frozen at-80 C at 2, 8,
12, 24, 36 and 48 h post infection. Duplicate titrations of
each of the triplicate wells were conducted in HFF cells in
6 well plates. Plaques were enumerated and titers were
determined for each time point and virus.

Determination of antiviral activity
Plaque reduction assays were performed using HFF cells
added to six well plates and incubated 48 h prior to infec-
tion. On the day of assay, drug at two times the final
desired concentration was diluted serially 1:5 in 2X MEM
with 10% FBS to provide six concentrations. Aspiration of
culture medium from triplicate wells for each drug con-
centration was followed by addition of 0.2 ml/well of
diluted virus which would give 20-30 plaques per well in
MEM containing 10% FBS or 0.2 ml medium for drug tox-
icity wells. The plates were incubated for one h with shak-
ing every 15 minutes. An equal amount of 1% agarose was
added to an equal volume of each drug dilution and this
mixture was added to each well in 2 ml volumes and the
plates incubated for three days. The cells were stained with
a 0.02% solution of neutral red (Sigma, St. Louis, MO) in
PBS and incubated for 5-6 h. The stain was aspirated, and
plaques counted using a stereomicroscope at 10x magni-
fication and 50% effective concentration (EC50) values
were calculated by standard methods.

Virulence
Three week old female, BALB/c mice were anesthetized
with ketamine-xylazine and inoculated intranasally with
10-fold dilutions of the viruses in a volume of 40 Al (20
Al per nostril). Two isolates of WR, and W A F2L-gfp were
evaluated in this experiment to assess the virulence char-
acteristic of these strains. Mice were observed daily for 21
days and were evaluated for clinical signs of infection and
mortality.

Abbreviations
Hour (h), wild type (wt), plaque forming unit (PFU), vac-
cinia virus (W), herpes simplex virus (HSV), (N)-meth-
anocarbathymidine (N-MCT), cidofovir (CDV),
idoxuridine (IDU), N-MCT monophosphate (N-MCT-
MP), deoxyuridine triphosphatase (dUTPase), deoxyurid-
ine triphosphate (dUTP), deoxyuridine diphosphate


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(dUDP), deoxyuridine monophosphate (dUMP), green
fluorescent protein (GFP), deoxythymidine monophos-
phated (TMP), uracil DNA glycosylase (UNG), human
foreskin fibroblast (HFF), lethal dose 50% (LD50), effec-
tive concentration (ECs5), American type culture collec-
tion (ATCC), 5-(2-amino-3-cyano-5-oxo-5,6,7,8-
tetrahydro-4H-chromen-4-yl)-1-(2-deoxypento-furano-
syl)-pyrimidine-2,4(1H,3H)-dione (PFT3), 1-(2-deox-
ypentofuranosyl)-5-[(3-methyl-5-oxo-l-phenyl-4,5-
dihydro-4H-pyrazol-4-ylidene)pyrimidine-2,4(1H,3H)-
dione (PFT4), minimum essential medium (MEM), cen-
tral nervous system (CNS).

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
MNP contributed to the design of experiments, analysis of
the data and the drafted the manuscript. ERK contributed
to the conception of the studies and the critical review of
the manuscript. DCQ contributed to the design of the
experiments and analysis of the data. KAK contributed to
the acquisition and interpretation of data. RWM contrib-
uted to the conception of the studies and the critical
review of the manuscript. PCT contributed to the design of
experiments, the acquisition and analysis of data and the
critical review of the manuscript.

Acknowledgements
These studies were supported by Public Health Service contracts NOI-AI-
30049 and NO I-Al-15439, grant 2R56A1015722-25 to RWM and grant I-
U54-AI-057157 from the NIAID, NIH.

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