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4
Animal Models Using Variola
and Other Orthopoxviruses
I
n 1999, there were no suitable animal models for variola. This
l ed the IOM committee at that time to draw the following
c onclusion:
The existence of animal models would greatly assist the develop-
ment and testing of antiviral agents and vaccines, as well as studies
of variola pathogenesis. Such a program could be carried out only
with live variola virus.
Since 1999, some progress has been made in developing animal models.
However, it should be emphasized that there is still no animal model that
satisfactorily recapitulates all relevant aspects of human smallpox. Although
the nonhuman primate model described by Jahrling and colleagues (2004)
offers some features that are suggestive of later-stage, fulminant human small-
pox, the ability of this model system to mimic the wide spectrum of human
disease manifestations and pathophysiology remains uncertain. This chapter
describes efforts to use animal models to study variola infection and disease
in humans. Although such efforts have included use of the variola virus, the
inability to infect most animal species with variola has resulted in attempts to
use vaccinia, cowpox, and mousepox; monkeypox; and myxoma virus.
VARIOLA
As discussed in Chapter 3, there are no animal reservoirs for variola
virus in nature, and most animal species cannot be infected in the labora-
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0 LIVE VARIOLA VIRUS
tory. Attempts to develop nonhuman primate models for variola infec-
tion and disease in the 1960s met with only limited success (Hahon and
Wilson, 1960; Hahon, 1961; Lancaster et al., 1966; Westwood et al.,
1966). Although these attempts reportedly produced rash and systemic
illness in various nonhuman primate species, disease was inconsistent and
variable in severity with each of the routes of inoculation and host species
examined. Of note, the reports described primary and secondary viremias
and a mild, brief illness with fever and rash in Macaca irus and Macaca
mulatta exposed to variola virus via the aerosol route (Hahon and Wilson,
1960; Lancaster et al., 1966; Westwood et al., 1966).
Motivated by the potential utility of a realistic and consistent model with
which to test drugs and vaccines intended for possible use against smallpox
in humans, CDC revisited the possibility of developing a model of human
smallpox in nonhuman primates at the beginning of this decade (Jahrling et
al., 2004). After limited unsuccessful efforts to produce consistent, severe
disease with an aerosol device, the investigators turned to an intravenous
route of infection in cynomolgus macaques. Lethal disease with systemic
features reminiscent of late-stage severe human smallpox, including skin
vesicles and pustules, was achieved with doses of 109 plaque-forming units.
Lesser doses, as low as 106 plaque-forming units, produced less severe dis-
ease and fewer skin lesions in a dose-dependent but less consistent manner.
As described, this macaque model appears to truncate the natural course
of variola infection, bypassing the early respiratory tract replication of the
virus, primary viremia, and early clinical phases of human smallpox. On
the other hand, aspects of the systemic pathology resembled some of the
reported features of lethal, hemorrhagic smallpox in humans as described in
the historical record. But because the route of infection and the dose differed
substantially from those of the natural setting, there is reason to believe that
the mechanisms of pathogenesis in this model may vary from those that took
place during natural disease. For example, the initial instantaneous viremia
preempts a prodromal period and circumvents early local replication of virus
in the respiratory tract. An adequate assessment of these issues has not been
possible, in part because human smallpox was eradicated before modern
investigatory tools became available and also because relatively few studies
of the current nonhuman primate model have been published.
Nonetheless, this nonhuman primate model was subsequently used to
test therapeutics and vaccines (see Chapters 6 and 7, respectively), with the
understanding that it sets a stringent, perhaps overly demanding standard
for efficacy. Proposed improvements of this macaque model include using
an intratracheal route of inoculation to achieve a consistent and more real-
istic course of disease.
Elucidation of features of variola pathogenesis, a secondary goal in the
development of this macaque model, has been achieved using traditional
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ANIMAL MODELS USING VARIOLA
and more modern tools (Rubins et al., 2004). In addition to a variety of
histology-, hematology-, chemistry-, and immunology-based measurements,
genome-wide features of nonhuman primate gene expression were mea-
sured in serial peripheral blood specimens using DNA microarrays. The
latter revealed gene transcript abundance patterns indicative of prominent
interferon and cell proliferation responses, and notable for the absence of
responses associated with tumor necrosis factor alpha and transcription
factor NF-kappaB, which would otherwise be typical of many acute over-
whelming infections. These and other findings enhance understanding of the
mechanisms responsible for variola-associated morbidity and mortality, as
well as possible new targets for therapeutic intervention.
VACCINIA, COWPOX, AND MOUSEPOX
Vaccinia virus is perhaps the most widely used poxvirus in animal
models for studying variola virus infection because of its ready availability
and extensive knowledge base, and the susceptibility of laboratory rabbits
and mice to vaccinia infection. In rabbits in particular, infection with a
rabbit-adapted strain of vaccinia virus—rabbitpox virus—generates a dis-
ease that recapitulates some of the important features of smallpox, includ-
ing transmission between hosts by the aerosol route and a generalized rash
(Adams et al., 2007).
Laboratory mice can be lethally infected with several strains of vac-
cinia virus introduced intranasally or by aerosol. This mouse model yields
dose-dependent lethality, with up to 100 percent of animals dying, and
can be refined to result in a sublethal disease course in which disease
severity is quantified by weight loss followed by recovery over a period of
2–3 weeks. Although this model is characterized by a more rapid disease
onset—around 3 days—than is seen with smallpox and is not characterized
by a rash, it has been used to compare the efficacy of novel and traditional
vaccines and to conduct research on antiviral therapies (Bray et al., 2000;
Smee et al., 2001; Belyakov et al., 2003; Hooper et al., 2003; McCurdy et
al., 2004; Wyatt et al., 2004; Law et al., 2005; Phelps et al., 2005, 2007;
Abdalrhman et al., 2006; Ferrier-Rembert et al., 2007).
Inbred mice are also useful in models for cowpox (Bray et al., 2000;
Ferrier-Rembert et al., 2007) and mousepox (Fenner, 1949) infection, and
in both cases, a lethal challenge is obtainable. The mouse/cowpox model is
broadly similar to that of mouse/vaccinia, and has value in extending the
range of orthopoxviruses that can be used in a single host for the evaluation
of measures that may control orthopoxvirus infection. The mouse/mousepox
model is somewhat different in that it is lethal at very low doses and is
restricted to a single host, and its severity can be viewed as generating a
model that is more relevant to variola infection in humans. However, there
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LIVE VARIOLA VIRUS
are significant differences between the disease course of mousepox in mice
and most other acute orthopoxvirus infections. Mousepox is characterized by
large ulcerating lesions rather than the discrete maculo-papular rash charac-
teristic of smallpox, and extensive liver damage is seen as well (Jones et al.,
1997). Although the disease severity and host restriction make the mousepox
model attractive for testing control measures, this model must be treated with
caution given this differential pathology. However, the ability to perform
challenge experiments with vaccinia, cowpox, and mousepox viruses in a
single species when protection against all three viruses can be achieved with
traditional smallpox vaccine adds considerably to the confidence with which
extrapolations from these models to human smallpox can be made.
MONkEyPOX
Monkeypox is perhaps the most relevant orthopoxvirus with regard to
nonvariola animal models for smallpox. Monkeypox virus causes a some-
times fatal disease in humans whose clinical features and course are similar
to those of smallpox. The use of this virus in the laboratory requires bio-
safety level (BSL)-3 conditions in the United States, although the disease is
prevented in humans and animals with smallpox vaccine. The classification
of monkeypox virus as a select agent in the United States further compli-
cates and hinders work on this virus.
As its name implies, monkeypox virus causes disease in nonhuman
primates, and it has been used experimentally to cause disease in macaques
that is similar to smallpox and monkeypox in humans, and to evaluate pos-
sible countermeasures against smallpox (Earl et al., 2004; Stittelaar et al.,
2005). Monkeypox does not cause significant disease in laboratory mice;
however, the recent discovery that it causes disease in North American
prairie dogs has led to the examination of other, related ground-dwelling
squirrels, and there are now several rodent-based models for orthopoxvirus
disease using this virus (Tesh et al., 2004; Hutson et al., 2007).
The most important feature of monkeypox is not its similarity in
humans to smallpox. Rather, monkeypox is a public health problem in its
own right. At least 88 cases with 3 fatalities occur annually in endemic
regions of central Africa (Hutin et al., 2001, Levine et al., 2007; Parker
et al., 2007). Public health issues pertaining to monkeypox are beyond
the scope of this study. Nevertheless, the committee notes that human
monkeypox deserves attention because of its toll in endemic areas, and the
licensure of therapies for the disease would provide a tangible benefit for a
large at-risk population. Moreover, lessons learned from the development
of licensed medical countermeasures for human monkeypox might address
many of the uncertainties associated with extrapolation among different
orthopoxviruses in animal models.
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ANIMAL MODELS USING VARIOLA
MyXOMA VIRUS
Animal models with relevance to smallpox are generally restricted
to orthopoxviruses—members of the same genus as variola itself. One
nonorthopoxvirus is worthy of consideration, however, because of its
pathogenicity and the body of research based on its use. Myxoma virus,
a poxvirus of the leporipoxvirus genus, is a virus of New World rabbits
of the genus Sylvilagus, in which it causes an infection that is almost
asymptomatic and is nonlethal. When introduced to European Oryctolagus
rabbits, myxoma virus causes a fulminant ulcerating infection known as
myxomatosis, with a very high mortality rate (Stanford et al., 2007). The
severity of disease in myxoma virus-infected European rabbits invites par-
allels with smallpox, and although the two viruses differ significantly in
pathology, so, too, do smallpox and models using vaccinia, cowpox, and
mousepox.
Like orthopoxviruses, myxoma virus produces a number of proteins
that interact with elements of the immune system. Many of the lessons
learned from studies with myxoma virus directly inform and influence
understanding of orthopoxviruses and vice versa. However, myxoma virus
is sufficiently different from orthopoxviruses that smallpox vaccine does
not protect European rabbits from myxomatosis, and ST-246, a prom-
ising candidate antiviral drug for treatment of orthopoxvirus diseases,
including smallpox, has no activity against myxoma virus, which lacks the
specific target of the drug in orthopoxviruses. Consequently, data from
animal models using myxoma virus cannot be extrapolated to smallpox in
humans.
CHIMERIC VIRUSES
While the extreme host restriction of variola virus greatly facilitated the
smallpox eradication campaign, it also hampered research because no ani-
mal model using variola was available. In the 1960s, attempts were made
to address this gap by constructing chimeric viruses from variola and either
cowpox or rabbitpox (a rabbit-adapted strain of vaccinia virus) viruses
(Bedson and Dumbell, 1964a,b). These chimeric viruses were constructed
by coinfection of cell lines with the two viruses and plaque purification of
random recombinants between the two. At the time, the random nature of
the resulting recombinant viruses and the inability to fully sequence these
recombinants restricted their utility for research into the pathogenesis of
variola infection. Moreover, the advisability of adapting a human-only
virus to growth in animals that could thereby become potential reservoir
hosts was questioned. These chimeric viruses, which are stored under BSL-4
containment at CDC, were generated with methods that yield random
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LIVE VARIOLA VIRUS
TABLE 4-1 Usefulness of Animal Models and Human Infections for
Understanding the Pathogenesis of Variola and Other Orthopoxviruses
and for Developing Therapeutics and Vaccines
Small Animal Nonhuman Primate Human
Orthopoxviruses Contribution to Limited usefulness: Moderately useful
other than overall some potential to (especially vaccinia)
monkeypox and understanding of help in identifying for obtaining
variola pathogenesis of useful interventions information about
infections caused by against smallpox in antiviral activity of
humans,a but use of
poxviruses; candidate drugs and
vaccines.b
provides some nonhuman primates
evidence of antiviral for studies of
activity of drugs variola have
against poxviruses priority.
related to variola,
but not variola.
Monkeypox Limited usefulness: Moderately useful Most useful in
some potential to for obtaining suggesting likely
help in identifying information about benefits from
useful interventions antiviral activity of candidate therapeutics
against smallpox, candidate drugs and and vaccines against
but other vaccines. variola in the human
population.d
approaches are
more important.c
Variola Not an available Most useful in Not an available
option for suggesting likely option for developing
developing benefits from therapeutics or
therapeutics or candidate vaccines.
vaccines. therapeutics and
vaccines against
variola in the
human population.
aDual infection of nonhuman primates with simian immunodeficiency virus (SIV) and vaccinia
produces disease with features that mimic human smallpox.
bDisseminated vaccinia infection in humans produces disease with some features reminiscent
of smallpox.
cMonkeypox virus infection of ground squirrels has been used to assess monkeypox
vaccines.
dThis cell, monkeypox in humans, refers to naturally occurring disease. The study of this dis-
ease might provide an opportunity to assess diagnostics, therapeutics, and vaccines for their
utility in both monkeypox and variola.
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ANIMAL MODELS USING VARIOLA
recombinations of the parent virus genomes and have not been character-
ized by sequencing. Furthermore, while studies with these recombinants
might contribute to understanding of variola pathogenesis, such investiga-
tions could not substitute for those using variola virus and would have to
be carried out in BSL-4 facilities that should be used instead to support
experiments with variola virus that are essential for developing smallpox
therapeutics and vaccines.
USEFULNESS OF VARIOUS MODELS
Table 4-1 summarizes the usefulness of animal models and human
infection with monkeypox or vaccinia for understanding the pathogenesis
of variola and other orthopoxviruses and for developing therapeutics and
vaccines. Although some of these approaches are more useful than other,
none is ideal in recreating the equivalent of human smallpox.
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