Critical Reviews in Microbiology, 28(3):149–185 (2002)

Immune Response to Poxvirus Infections in

Various Animals
Scott A. Smith and Girish J. Kotwal*
Department of Microbiology and Immunology, University of Louisville School of
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Medicine, Louisville, KY

* Address correspondence to: Girish J. Kotwal, Division of Medical Virology, Univ. of Capetown Medical
School, Cape Town, South Africa 7925. e-mail GJOTW01@yahoo.com

ABSTRACT: The study of infections of vertebrate animals by poxviruses has remained a dynamic
area of research for the last century. The host range of poxviruses vary from extremely narrow to
exceedingly broad, and they have been shown to enter their host by either the respiratory route or
through the skin. The severity of infection varies dramatically from one species to another, causing
anywhere from a local, self-limiting infection, to a devastating systemic disease, such as smallpox.
Although the immune response to poxvirus infections are very similar to that seen in other viral
infections, the poxviruses, unlike most other viruses (with the exception of Herpes viruses), are able
to defend themselves. They have been shown to carry a repertoire of proteins involved in immune
evasion and immune modulation. Poxviruses encode proteins involved in blocking many of the
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strategies employed by the host to combat viral infections; they encode for proteins that block
activity of many chemokines, cytokines, serine proteases, and even complement. Traditionally,
different animal models have been used to study the pathogenesis of poxvirus infections, and the
characterization of virulence genes using mutant poxviruses. Additionally, new animal models are
being developed to study the possible therapeutic uses many of these poxvirus immune modulating
proteins might have. This review discusses the host immune response against poxvirus infections
in various animals, the viral counter response to the host, and the animal models used to study
poxvirus infection and immune modulating proteins.

GLOSSARY:

ADCC antibody-dependent cell-mediated cytotoxicity

C4b-BP complement 4b binding protein
CAM chorioallantoic membrane
CCR CC-chemokine receptor
CPV cowpox virus
CR complement receptor
CrmA cowpox cytokine response modifier A
CTL cytotoxic T-lymphocyte
CXCR CXC-chemokine receptor
CX3CR CX3C-chemokine receptor
DAF decay-accelerating factor

1040-841X/02/$.50
© 2002 by CRC Press LLC

149
 EV ectromelia virus
fH factor H
FITC fluorescein-isothiocyanate
FPV fowlpox virus
GIF granulocyte-macrophage colony-stimulating factor-inhibitory factor
GM-CSF granulocyte-macrophage colony-stimulating factor
HIV human immunodeficiency virus
HS heparan sulfate
ICE interleukin-1β-converting enzyme
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IFN interferon
IFNR interferon receptor
IL interleukin
IL-1R interleukin 1 receptor
IL-18BP interleukin 18 binding protein
IMP inflammation modulatory protein
MAC membrane attack complex
MCCH molluscum contagiosum chemokine homolog
MCP membrane cofactor protein
MCV molluscum contagiosum virus
MGF myxoma virus growth factor
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MHC major histocompatibility complex

MIP macrophage inflammatory protein
MV myxoma virus
MVA modified vaccinia Ankara
NF nuclear factor
NK natural killer
ORF open reading frame
PAECs pig aortic endothelial cells
RCA regulators of complement activation
SCR short consensus repeat
serpin serine protease inhibitor
SFV Shope fibroma virus
sTNFR soluble tumor necrosis factor receptor
TGF transforming growth factor
TNF tumor necrosis factor
TNFR tumor necrosis factor receptor
tPA tissue-type plasminogen activator
uPA urokinase-type plasminogen activator
VCP vaccinia virus complement control protein
vIFNR viral interferon receptor
vIL viral interleukin

150
 vIL-1R viral interleukin 1 receptor
vIL-18BP viral interleukin 18 binding protein
vMIP viral macrophage inflammatory protein
vTNFR viral tumor necrosis factor receptor
VV vaccinia virus
VGF vaccinia virus growth factor
XCR C-chemokine receptor
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I. INTRODUCTION
The poxvirus family, Poxviridae, has the
largest virions of any animal virus family.
They appear oval or brick shaped, approxi-
mately 350 nm in length by 270 nm wide.
For this reason, vaccinia was the first animal
virus to ever be seen microscopically in 1886.
Poxviruses contain a large linear double-
stranded DNA genome of 130 to 280 kbp.
This large genome is responsible for encod-
ing all the genes necessary for their unique
cytoplasmic lifestyle. The members of the
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poxvirus family replicate almost entirely in-
dependent of the infected cell nucleus. There-
fore, being unable to utilize the host cell’s
enzymes to synthesize RNA or replicate
DNA, they must encode for these important
proteins in their own genome. In addition to
those proteins encoded by poxviruses re-
quired for transcription, replication, and
virion assembly, there are several nonessen-
tial proteins employed to establish a suitable
habitat. Several interesting nonessential pro-
teins encoded by poxviruses include: comple-
ment control protein (VCP), interleukin one
receptor homolog, tumor necrosis factor re-
ceptor homolog, and interferon receptor ho-
molog.1 These nonessential proteins, and
several others, play an important role in
modulating the host’s immune response to
the infection, thereby maintaining the viral
habitat.2
The family Poxviridae is divided into two
subfamilies, Chordopoxvirinae, which are
vertebrate poxviruses, and Entomopoxvirinae,
which are insect poxviruses — we only dis-
cuss the Chordopoxvirinae.3 There are eight
genera of vertebrate poxviruses (see Table 1).
Poxviruses have been found to infect verte-
brates ranging from birds to humans. Some of
these infections can be highly fatal; smallpox
virus (variola), which replicates only in hu-
mans, has killed more people than any other
infectious disease. In addition, myxoma vi-
rus, which infects rabbits, can have as high as
a 99% mortality rate. Much of the success of
poxviruses as pathogens is because of the
immune evasion strategies they employ.1
The poxvirus infection generally follows
one of two courses, a localized infection
resulting in benign skin lesions, or a sys-
temic infection, resulting in viral dissemina-
tion and usually death. Local pathogenesis
of molluscum contagiosum virus (MCV) and
Shope fibroma virus (SFV) infection has been
well documented. Although the pathogen-
esis and host response to infection is quite
different, both poxvirus diseases are self-
limiting and remain localized to the skin.
The generalized infections, however, are
characterized by several stages, ending in an
explosion of viral lesions. Ectromelia virus
infection of mice starts with an infection of
the skin, usually through the footpad. After
some replication, the virus spreads through
the local lymphatics to the bloodstream, re-
sulting in primary viremia. The virus then
replicates to high titers in the spleen and
liver before reentering the blood, resulting
in a secondary viremia. Finally, the virus
spreads to the skin, and a severe rash of
ulcerating lesions very rapidly overcomes
the mouse. The disease progression of vari-
151
 TABLE 1
Poxvirus Classification. The Family Poxviridae, Subfamily Chordopoxvirinae, is
Composed of Eight Genera. Listed Are the Most Studied Members of Each Genus
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ola virus infection in humans occurs simi-
larly, with high mortality rates.
The immune response to poxvirus infec-
tion is very complex. Each arm of the im-
mune system plays a vital role in the segre-
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gation and the elimination of a poxvirus
infection. Early in the infection, the innate
immune response attempts to keep the infec-
tion under control, while the acquired im-
mune response is mounted. Complement,
interferon, NK, and inflammatory cells help
control the early infection. Within a few days,
a very strong humoral immune response is
mounted, and poxvirus-specific antibodies
are produced. The antibodies can then help
control the infection through a variety of
mechanisms, including virus neutralization
and complement activation, obsonization, and
ADCC. In addition, a cell-mediated immune
response is mounted a few days into the
infection. Poxvirus-specific CTLs are gen-
erated and attack virus infected cells, ulti-
mately resulting in clearance of the poxvirus
infection.
Animal models have been used quite
extensively for the study of poxvirus disease
pathogenesis and exploring the therapeutic
potential of poxvirus immunomodulatory
proteins. To gain a better understanding of
152
the infection processes and genes involved
in interactions with the host, several animal
models have been developed to study vari-
ous poxvirus diseases and recombinant mu-
tant poxviruses. Animal infection models are
important for testing the efficacies of new
antiviral drugs or to explore new treatment
strategies. Models have been developed to
study the inflammatory response generated
by poxvirus infection, analyzing the degree
of inflammation and cellular infiltration both
quantitatively and qualitatively. This allows
for a better understanding of the genes asso-
ciated with poxvirus pathogenesis and re-
sults in better characterization of mutant
strains. Understanding pathogenesis assists
in the generation of more attenuated vaccine
strains and poxvirus vectors for use in live
vaccines in hopes of reducing complications
associated with traditional vaccines. Animal
models have also been instrumental in the
study of poxvirus immune modulating pro-
teins. Several poxvirus immunomodulatory
proteins have been investigated extensively
for their potential use as therapeutic agents.4
These immune blocking proteins have very
powerful antiinflammatory properties that
may be employed for treatment of many
injuries or diseases that involve excessive
 inflammation. Animal model systems have
been used to test the value of these proteins
for use in transplantation, vascular injury,
CNS injury, sepsis, arthritis, and restenosis.
The therapeutic effects of these proteins,
revealed by these model systems, are often
quite fascinating.
II. POXVIRUS INFECTIONS
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species whether a poxvirus remains localized
or disseminates. Myxoma virus, for example,
produces benign fibromas in wild rabbits, but
spreads explosively in domestic rabbits, result-
ing in a very high mortality. Several portals of
entry into the host have also been described
previously for the different poxvirus infections.5
Entry into the host via the skin has been re-
ported for myxoma virus and SFV. The respi-
ratory route, however, is best documented for

ectromelia virus, vaccinia virus, and variola

Poxviruses have been found to cause an
acute infection in a large variety of animal
species. The host range and lethality of the
poxvirus infection varies significantly. Some
poxviruses can infect a wide range of animals
(e.g., vaccinia), whereas others have a very
limited host range (e.g., variola) (see Figure 1).
Depending on the poxvirus and the host, some
infections remain local (e.g., MCV), while oth-
ers are disseminated throughout the organism
resulting in higher fatality rates (e.g., ectrome-
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virus. Of the huge number of poxvirus infec-
tions, only a few were chosen to represent the
various infection properties of the poxvirus
family. Here we describe briefly the pathogen-
esis and/or special features of several well-
studied poxvirus infections.
A. Ectromelia Virus Infection of Mice
Ectromelia virus (EV) infection and patho-

lia). In some cases, it depends on the host genesis has been studied extensively in mice.6

FIGURE 1. Pathology in various animals. (A) Characteristic smallpox (variola major) rash on the face and hand
(B) of a young boy several days into the illness, before lesions ulcerate. (World Health Organization, with
permission.) (C) Dome-shaped MCV papules on chest and around areola.127 (D) Human CPV infection.
Lesions are very painful and appear brownish with central vesicles.26 (E) Pig infected intravenously with SPV
14 days post-infection, and showing generalized ulcerating rash.33

153
 After infection through lesions of the footpad
with a virulent strain, the disease pathogen-
esis follows a well-documented disseminated
course. After the virus replicates rapidly at
the site of entry, it will pass through the lym-
phatic system to the blood. The primary vire-
mia then results in infection of the liver and
spleen, often within 5 days of the infection.
The virus then disseminates throughout the
mouse, resulting in a secondary viremia, and
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will grow to high titers in the epidermis and
hair follicles, resulting in the characteristic
rash. The mouse is most infectious between 6
and 21 days postinfection. In some mice,
BALB/c, for example, the infection is very
severe and death will usually result 6 to 7
days after infection because of sever liver
necrosis. Death occurs so quickly in these
mice that the rash does not even have time to
develop. In other strains of mice, most nota-
bly C57BL, the mortality is not as high.7 In
this case, the virus does not grow to as high a
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titer and disseminates more slowly. The mouse
will often survive with no appearance of a
rash.
B. Myxoma Virus Infection of
Rabbits
Myxoma virus (MV) causes a very le-
thal disease, myxomatosis, in the European
rabbit. Mosquitoes and fleas normally trans-
mit MV passively, causing a persistent be-
nign, nondisseminated disease in its natural
host, the North and South American rabbit.
However, when passed onto the European
rabbits, it causes a lethality of over 99%.
After infection of a European rabbit, the MV
replicates rapidly at the infection site and
then spreads systemically, resulting in infec-
tion of many tissues and development of
myxomas (secondary lesions).8 Extensive
lesions form throughout the areas of infec-
tion, both internally and externally, often
giving rise to secondary Gram-negative bac-
154
terial infections. In the 1950s, MV was de-
liberately released in southern Australia to
control overpopulations of feral European
rabbits. Initially it was a success, killing
masses of pest rabbits, but soon resistance
grew and the rabbits repopulated.
C. Shope Fibroma Virus Infection of
Rabbits
Shope fibroma virus (SFV), a close rela-
tive of myxoma, has a natural host range that
is limited only to eastern North American
cottontail rabbits. Transmitted by arthropod
vectors, SFV infection develops into a local-
ized (at the site of inoculation) benign tu-
mor; the tumorigenic effect it causes on its
host varies depending on the virus strain.9
Some strains exhibit disseminating effects
and can be found in the spleen, liver, and
kidneys, while others remain noninvasive
and show no signs of even locally spreading.
Independent of the infecting strain, the virus
is cleared by the immune system after ap-
proximately 2 weeks, and the rabbit recov-
ers.
D. Vaccinia Virus Infection
Vaccinia virus (VV) is the most studied
of the poxvirus family. Although its original
(natural) host remains to be found, it has a
broad host range, causing mild or occasion-
ally severe infection in a large number of
animals.10 Several feasible theories exist as
to the origin of vaccinia virus. VV is be-
lieved to have originated either from evolu-
tion of variola virus, evolution of cowpox
virus, or possibly a hybrid of variola and
cowpox viruses.5 Regardless of its origin,
vaccinia virus has played a central role in the
eradication of smallpox, conferring long-term
immunity to variola virus infection by cross-
reacting antibodies and cytotoxic T-lympho-
 cytes. VV is also employed for the expres-
sion of foreign genes used to vaccinate
against other disease-causing agents, such as
rabies. Although most human infections, or
immunizations, result in a very mild, self-
limiting local infection, occasionally dissemi-
nation occurred, and the development of a
very serious and sometimes fatal neurologi-
cal side effect ensues, known as post-vac-
cinial encephalitis.11 In most cases, however,
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VV infections remain localized to the infec-
tion site and usually ends in complete recov-
ery. The highly attenuated vaccinia strain,
modified vaccinia Ankara (MVA), is cur-
rently a potential vaccine candidate. The
sequence of MVA revealed 25 split and/or
mutated genes that result in truncated pro-
teins, and thereby diminished virulence.12
E. Variola Virus Infection of Humans
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Variola virus is the etiological agent for
the devastating disease smallpox. Two strains
of variola virus exist, variola major and vari-
ola minor. Variola major was associated with
high fatality rates of 5 to 40%, whereas in-
fection with variola minor resulted in less
than 1% fatality. Like ectromelia virus, vari-
ola virus has an extremely narrow host range,
infecting only humans. In addition, like ec-
tromelia virus, variola virus causes a sys-
temic infection. Infection occurs primarily
through the respiratory route (excretions of
the mouth and nose). The virus then repli-
cates and spreads into the bloodstream, caus-
ing a primary viremia. After infecting and
replicating in many organs of the body, high
levels of variola virus are again shed into the
blood, resulting in a secondary viremia. The
characteristic skin rash appears several days
into the illness and progresses to a severe
ulcerating rash that covers the body in about
10 days. Thanks to an aggressive vaccina-
tion campaign organized by the World Health
Organization (WHO), variola virus was eradi-
cated, the last reported case occurring in
Somalia in 1977.
F. Orf Virus Infection of Sheep,
Goats, and Humans
Orf virus infects the epithelium, forming
acute pustular lesions, primarily of sheep
and goats. Occasionally orf is zoonotic and
is transmitted to humans, often those that
frequently handle sheep. Human infections
are often on the hand or finger and rarely
require medical treatment. Antibiotics are
often given to prevent a possible secondary
bacterial infection from arising. Primary in-
fection in sheep is characterized by vesicle
formation, followed by pustulation, scabbing,
and finally resolution after about 4 weeks.
Although the immune response to this local
infection is reportedly vigorous, both hu-
moral and cell-mediated, reinfection often
occurs.13 Secondary infections, however, are
often resolved more rapidly, in 1 week, with
vesicle and scab formation occurring more
quickly.14 Recently, CD4+ T-lymphocytes
have been shown to play an important role in
the resolution of orf virus infection.15 Using
CD4+ and CD8+ T-lymphocyte depleted
lambs infected with orf, CD4+ T-lympho-
cyte depleted lambs were found to be unable
to clear the virus, whereas CD8+ T-cell de-
pleted lambs were able to resolve the infec-
tion. Specific antibody titers have also been
linked to the resolution of virus infection.
G. Molluscum Contagiosum Virus
Infection of Humans
Molluscum contagiosum virus (MCV),
like variola virus, exclusively infects humans.
Unlike variola virus, MCV causes very small
painless benign self-limiting lesions on
healthy individuals and only causes prob-
lems in children and immunocompromised
155
 adults (often disfiguring AIDS patients). In
addition to venereal transmission and direct
contact with lesions, MCV is also believed
to spread indirectly by tattooing, clothes,
and bath towels. MCV is found worldwide,
and studies done in Australia indicate that
23% of the overall population and 39% of
adults over 50 years of age are seropositive,
indicating that exposure is prevalent.16 Stud-
ies involving inoculation of volunteers dem-
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onstrate that MCV has an average incuba-
tion period of 2 to 7 weeks. Although lesions
usually regress within 3 months in healthy
individuals, lesions have persisted for up to
5 years in immunocompromised patients,
with reinfection occurring occasionally. The
treatment of MCV lesions is through a vari-
ety of destructive techniques applied locally,
such as cryotherapy, none of which seem to
work in patients with AIDS.17 MCV infection
is most noted for the lack of inflammation,
despite high virus titers, in the lesion. Lesions
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may persist for months without a sign of im-
mune cell infiltration. Studies have confirmed
the lack of T-lymphocytes and NK cells at the
erupted lesion site.18,19 Research into MCV is
difficult due to its inability to replicate in
tissue culture cells or animals. Recently, how-
ever, extended persistence of MCV DNA and
the expression of late proteins in an in vitro
cell culture has been demonstrated.20
H. Cowpox Virus Infection of Cattle,
Cats, and Humans
Cowpox virus (CPV), despite its name,
very rarely infects cattle causing a mild pus-
tular disease of the teats and udders. Cow-
pox has a very broad host range, in addition
to infecting cattle, cats, and humans, infec-
tions in rats, zoo animals (lions, elephants,
and anteaters), and gerbils have been re-
ported.21 The natural reservoir for the CPV
seems to be a small mammal; studies have
demonstrated its presence in several species
156
of wild voles and mice.22 Humans are ex-
posed to cowpox through contact with in-
fected udders, or more commonly through
contact with infected domestic cats. Domes-
tic cats, exposed while hunting rodents, have
been shown to be highly susceptible to in-
fection. In fact, a study where young cats
were infected with cowpox virus demon-
strated an explosive disseminating disease
with 100% fatality.23,24 Therefore, it is felt
that domestic cats act as intermediate hosts
in the circulation of CPV. The human dis-
ease is generally mild zoonotic disease, al-
though severe, sometimes fatal (usually in
immunocompromised patients) cases have
been reported.25 Lesions are often on the
face, hand, or neck, but occasionally gener-
alized possibly due to transfer from develop-
ing lesion(s) on the hand. Shortly after infec-
tion, the lesion(s) appears brownish and
becomes very painful after about a week.
Toward the end of the second week, the
lesion ulcerates and crusts over. Although
there is sometimes systemic involvement
(usually in immunocompromised patients),
the lesion usually heals within 3 to 8 weeks
without scaring.26
I. Lumpy Skin Disease Virus
Infection of Cattle
Lumpy skin disease virus (LSDV) ex-
hibits a narrow host range, only infecting
cattle, most exclusively in Africa, with only
one outside case being reported in the Middle
East.27 The disease causes hide damage, de-
creased milk yield, infertility, poor growth,
as well as cattle death, resulting in major
production loss and significant economic
problems. Lumpy skin disease is a subacute
to acute disease characterized by either gen-
eralized or localized nodular skin lesions
and lymphadenopathy.28 The generalization
of nodular skin lesions, due to systemic
spread of LSDV, occurs in about 50% of
 experimentally and naturally infected cattle
and correlates with high mortality. While
dermal inoculation often results in local in-
fection, intravenous inoculation of LSDV
was shown to result in a much greater like-
lihood that a severe disseminating infection
would take place.29 Vaccinations with at-
tenuated strains, which cause only a mild
local infection, have been shown effective in
inducing strong immunity and controlling
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the disease.30 Epidemiological, as well as
experimental, evidence has demonstrated that
LSDV is transmitted by arthropod vector,
either mosquitoes or sandflies.29,31 Therefore,
because of the increase in mosquito and bit-
ing fly abundance during the wet season,
disease incidence also rises.
J. Swinepox Virus Infection of Pigs
Swinepox virus (SPV) is found world-
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wide, often infecting swine in both devel-
oped and underdeveloped countries. SPV has
a narrow host range, infecting only porcine
species, and it causes a mild, benign, local
self-limiting infection. During infection, vi-
rus can only be isolated from lesion site and
draining lymph nodes, confirming the lack
of dissemination.32 When injected intrave-
nously, a uniform distribution of lesions was
observed after an incubation period of be-
tween 10 and 14 days.33 The ultrastructure of
SPV has been studied extensively in tissue
culture as well as the natural infection.34,35
K. Fowlpox Virus Infection of
Chickens
Fowlpox virus (FPV) is found to infect
primarily chickens and turkeys of all ages.
The disease stops egg production, stunts
growth of young birds, and causes blindness
and sometimes death, resulting in a major
production loss and significant economic
losses to the poultry industry. In recent years
vaccination by comb scarification, wing-web
stick, or feather follicle immunization with
attenuated vaccine strains confers immunity
to FPV infection.36 In addition, better man-
agement practices and improved hygienic
conditions helped prevent the spread of the
disease. FPV spreads by two separate routes,
each resulting in a different form of infec-
tion. Like SFV and LSDV, biting arthropod
vectors transmit FPV, resulting in the most
common form of the infection, the cutane-
ous form. This form of the disease is often
characterized by mild focal skin lesions,
generally on unfeathered areas, sometimes
leading to complete closure of the eyes, and
may result in secondary bacterial infections.
The second disease form, transmitted through
the mucous membranes of the mouth, is much
more severe and disseminating. In this form
of the disease, the diphtheritic form, the in-
fection of the upper respiratory tract often
results in suffocation due to closure of the
airway. Recently, recombinant FPV vaccines,
designed for the expression of foreign genes
in mammals (nonreplicating viral vector),
have been studied extensively. Recombinant
FPV vaccines, encoding common HIV-1 an-
tigens, have been studied for use as safe
HIV-1 vaccine candidates.37
III. HOST IMMUNE RESPONSE
The host immune response to poxvirus
infection is very powerful and multifaceted.
Additionally, the pathogenesis of localized
and systemic infections varies dramatically,
depending on the host and poxvirus species.
EV infection of mice, for example, is often
severe and disseminated, but does not in-
duce an inflammatory response. MCV infec-
tion in humans also does not invoke an in-
flammatory response, but remains localized.
Other poxvirus infections result in inflam-
mation and can be either systemic or local,
157
 for example, SFV is local, and FPV can be
systemic. The common immune response
mounted against the poxvirus infection cov-
ers all arms of the immune system. Early in
the immune response, the innate immune
system is called into battle. Interferon,
complement, NK, and inflammatory cells
rapidly try to keep the poxvirus under con-
trol until a more powerful acquired immune
response is mounted. Humoral and cell-me-
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diated immunity is very important for virus
clearance and the prevention of reinfection.
Poxvirus-specific antibodies play many roles,
including virus neutralization, opsonization,
and ADCC. The cell-mediated immune re-
sponse, poxvirus-specific CTLs, is thought
to play the most important role in controlling
poxvirus infection.
A. Role of Interferon
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Like other virus infections, interferons
play a critical role in the immune response
against poxvirus infection. All three types of
interferon, alpha, produced by fibroblasts,
beta, produced by leukocytes, and gamma,
produced by lymphocytes and NK cells, ef-
fectively control poxvirus growth. Fortu-
nately, poxviruses are strong inducers of
interferon production during animal infec-
tion. Interferon then has two roles in control-
ling poxvirus infection. First, it warns neigh-
boring cells that a virus is present, resulting
in induction of an antiviral state, consisting
of cell growth inhibition and the production
of MHC antigens. Secondly, it causes acti-
vation of macrophages, NK cells, and cyto-
toxic T-lymphocytes. The cellular response
that ensues thereby helps clear virus-infected
cells. Although IFN is critical for the clear-
ance of poxvirus infections, interferon alone
is not sufficient to protect the host from
severe disease. The role interferons play in
poxvirus infection and pathogenesis signifi-
cantly depends on the route of inoculation,
158
poxvirus species, and, most importantly, the
host.
To study the effect interferons have in
controlling poxvirus infection, knockout
mice, as well as anti-IFN antibody-treated
mice, have been infected with VV. Normal
mice have no problem clearing the infection;
however, anti-IFN-treated mice were unable
to clear the infection and died.38,39 Studies
using different IFN–/– and IFNR–/– knock-
out mice show similar results.40 However,
high levels of IFN in vivo do not always
correlate with the clearance of the infection.
During EV infection of mice, high levels of
IFN are present, and yet a lofty lethality
results.41 Interestingly, cell culture studies
show that the addition of exogenous IFN
results in abortive poxvirus infection. The
outcome seemed to be due in part to the
blocking of viral early mRNA translation.
The effect of interferon block on poxvirus
infection in culture clearly varied depending
on the strain of virus used.
B. Role of Complement
The role that complement plays in con-
trolling poxvirus infection has been studied
extensively. The complement system is com-
posed of over 30 serum proteins, some of
which are regulatory in nature. It is a very
important component of the innate immune
system and considered the first line of de-
fense against microbial infection. Although
three pathways of complement activation
have been found, only two have been widely
studied for their role in blocking poxvirus
infection: the classic pathway, initiated by
antibody fixation, and the alternative path-
way, initiated through the deposition of the
third component onto an activating surface.5
After the activation of either pathway,
proinflammatory chemotactic compounds are
released, resulting in leukocyte migration into
the area. The end result of complement acti-
 vation is the formation of the membrane
attack complex (MAC), creating holes in the
activating surface. As early as 1925, serum
neutralization of vaccinia virus was found to
be the result of heat-labile serum proteins,
later determined to be complement. How-
ever, no studies have been able to demon-
strate that complement alone can inactivate
purified poxvirus virions.
The classic pathway of complement ac-
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tivation is initiated through the binding of
antibodies and has been found to control
poxvirus infection by many mechanisms,
the first of which is virolysis. Once anti-
bodies are generated against the virus, se-
rum-induced lysis of the virus occurs
through the formation of the MAC.42 Al-
ternatively, in the absence of lysis, poxvi-
rus neutralization has been found to take
place through the build up of complement
components on the surface of the virus,
possibly blocking cell attachment. 43
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Complement activation results in leuko-
cyte migration and C3b fixation to the
poxvirus surface, which could lead to an
increased phagocytosis of extracellular
poxvirus through the binding of the C3b
receptor.44 Finally, poxvirus infection may
be controlled by complement through the
lysis of infected cells.45
The alternative pathway of complement
becomes activated through the deposition of
C3 to an activating surface in the absence of
antibody. Again, this pathway results in the
production of chemotactic compounds, as
well as the formation of the MAC. Cell cul-
ture studies of VV and FPV infection, dem-
onstrated a time-dependent deposition of C3
onto the surface of infected cells.46 This C3
deposition correlated with a decrease in vi-
rus yield, suggesting that the alternative path-
way of complement activation was control-
ling the infection. Additionally, the depletion
of C3 prior to infection of 3-day-old chick-
ens with FPV resulted in an increase in virus
yield, leading to 100% mortality.47 Addi-
tional studies using C5+/C5– congenic BALB/c
mice injected in the footpad with CPV
showed that C5 deficient mice exhibited a
significantly greater swelling response that
persisted longer and also showed signs of
hemorrhage, edema, and ulceration.48 Sev-
eral studies have suggested that the role of
complement in controlling poxvirus infec-
tion varies, depending on the virus strain and
host being studied.
C. Role of Inflammatory Cells
The inflammatory response to poxvirus
infection varies, depending on the species
and host. Lesions induced by VV, SFV, and
FPV infection, for example, are character-
ized by leukocyte infiltration, swelling, and
redness. Amazingly, however, no inflamma-
tory response is seen in EV or MCV infec-
tion. This is believed to be the result of viral
immune modulation. The role that inflam-
mation and inflammatory cells play in clear-
ing poxvirus infection has been studied for
many years. It is clear that inflammatory
cells play an important role in controlling
poxvirus infection through a number of dif-
ferent mechanisms. The first activity known
to contribute to poxvirus depletion is phago-
cytosis. Inflammatory cells are able to ph-
agocytize and destroy poxvirus virions that
are either antibody coated through binding
of the Fc receptor, or C3b coated, through
binding of the C3b receptor.49 Another way
inflammatory cells are able to limit a poxvi-
rus infection is through the release of oxy-
gen-dependent and -independent antimicro-
bial products.50 Finally, inflammatory cells
are able to release interferon and other
cytokines, resulting in induction an antiviral
state in local cells, as well as recruitment of
new leukocytes into the area. In addition to
the helpful effects contributed by leukocytes,
they have also been suspected of disseminat-
ing virus in infections like that caused by
159
 EV. This was evidenced by the recovery of
EV from leukocytes present in the blood and
thoracic lymphatic duct.
Experimental evidence shows that the
inflammatory response alone is not enough
to clear a poxvirus infection, without assis-
tance from an acquired immune response.51
CPV lacking the gene encoding an inflam-
mation modulating protein (serpin) showed
a stronger inflammatory response in the CAM
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model and did not lead to a reduction of
infection. This showed that the inflamma-
tory response could not single-handedly halt
infection. However, studies have shown that
an inflammatory response is required to slow
the progression of the poxvirus infection.
One study using macrophage-depleted mice
(using carrageenan) demonstrated that after
infection with a nonlethal dose of EV, an
increased level of virus was observed, re-
sulting in death of the mice.52 Interestingly,
if macrophages were depleted 3 days after
For personal use only.
EV infection, virus titers were still high,
even though normal levels of CTLs were
observed; this demonstrated the importance
of macrophages later in the infection pro-
cess. It is clear that the inflammatory re-
sponse, although essential for recovery, is
not enough to control poxvirus infection with-
out the help of other host defense mecha-
nisms.
D. Role of Antibody and ADCC
The effect that virus–specific antibodies
have on poxvirus infections have been in-
vestigated quite extensively. Specific anti-
bodies have been found to be important in a
number of mechanisms used to protect the
host. In addition to preventing virus
uncoating, antibodies can directly bind to
the poxvirus, causing aggregation and ob-
structing viral adsorption and internalization
into the cell. Furthermore, antibodies can
bind to the poxvirus, allowing for comple-
160
ment-mediated lysis or opsonization, result-
ing in phagacytosis via the Fc receptor.
Antibodies can also bind the infected host
cell, leading to antibody-dependent cell-
mediated cytotoxicity (ADCC). Despite the
numerous mechanisms created by specific
antibodies to destroy poxviruses, many stud-
ies have shown that antibody-mediated
mechanisms are unable to protect the host
during a primary infection.
Studies of children who received a pas-
sive immunization of gamma-globulin, fol-
lowing complications caused by vaccination
against smallpox, confirmed that antibody
had no effect in reducing virus titers. In ad-
dition, all patients with agammaglobuline-
mia were found to develop protective immu-
nity against smallpox following vaccination.
Even stronger evidence comes from the fact
that many patients who have succumbed to
complications from the smallpox vaccine
have had antivaccinia antibodies present in
their serum.53 This was confirmed with SFV
studies in rabbits; although neutralizing an-
tibody was detected in the serum of rabbits
infected with SFV, tumor growth continued.
Additionally, passive immunization of in-
fected immune compromised adult rabbits
with neutralizing antibodies to SFV also had
no effect on tumor growth. However, after
recovery from a primary poxvirus infection,
the host often develops antibodies respon-
sible for long-lasting immunity. The studies
looking at the ability of specific antibodies
generated during a primary infection to pre-
vent a secondary infection have come with
mixed results. Depending on the host, virus
species, infectious dose, and route of infec-
tion, the virus-specific antibodies may or
may not be protective. Overall, it seems that
poxvirus-specific antibodies are not as im-
portant for recovery from a primary infec-
tion as they are for the prevention of a sec-
ondary infection.
A subset of lymphocyte has been found
to possess the ability to lyse antibody bound
 virus-infected cells in a non-MHC-restricted
manner.54 Because this cellular activity is
dependent on cell-bound antibody, it is very
specific, but it is not present early in a pri-
mary infection. This process, known as
ADCC, has been demonstrated in many stud-
ies as being an important antibody and cell-
mediated mechanism used to combat sec-
ondary virus infection. The addition of
anti-VV immune serum to VV-infected cells
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has been shown to enhance this subset of
lymphocyte’s ability to selectively destroy
infected cells, without harming uninfected
cells.55 Like other immune mechanisms, the
effectiveness of ADDC in controlling pox-
virus infection depends on the poxvirus spe-
cies and host.5 It is clear, however, that gen-
erally ADCC is important for the repression
and elimination of poxvirus during reinfec-
tion.
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E. Role of NK Cells
The non-MHC restricted, nonspecific
cytolytic activity of NK cells can be seen long
before the development of virus-specific
CTLs. NK cells, which appear as large granu-
lar lymphocytes, are found at the area of virus
infection as part of the inflammatory response,
and part of the innate immune response. After
the release of interferon by infiltrating leuko-
cytes, NK cells become activated and begin
lysing virus-infected target cells by the re-
lease of perforin and granzymes.56 NK cells
are able to nonspecifically discriminate be-
tween normal cells and virus-infected cells,
which do not express adequate amounts of
major histocompatibility complex (MHC)
class I molecules on their surface. Studies
have consistently demonstrated the impor-
tance of NK cells early in the immune re-
sponse to poxvirus infections.
The early activity of NK cells during
poxvirus infection was shown in mice in-
fected intracranially with sublethal doses of
EV and VV.57 Long before CTL activity was
detected (day 6), NK cell-mediated cytolysis
was observed. Additional studies have sug-
gested that over 50% of VV infected cell
lysis observed in hamsters was due to NK
cells, not CTLs.58 Most surprisingly, how-
ever, are the studies showing that mice strains
resistant to EV infection have higher levels
of NK cell-mediated lysis.59 In addition, they
also showed that depletion of NK cells from
mice strains resistant to EV infection re-
sulted in high virus titers and severe disease.
Studies later suggested that it was not the
loss of NK cell-mediated cytolysis that gen-
erated these results, but the loss of IFN pro-
duced by the NK cells, reducing the stimu-
lation of other antiviral mechanisms.60 Thus
far, the studies have suggested that NK cells
play an important role early (first 3 days) in
the poxvirus infection, but cannot control
the spread and development of the infection
on their own.
F. Role of CTLs
While many components of the host
immune response are required for control-
ling poxvirus infections, it is generally felt
that the most important role is played by the
virus-specific CTL. The CTL (CD8+) is an
important part of the acquired cell-mediated
immune response. It is MHC class I restricted,
specifically recognizing virus-infected cells,
destroying them before virus maturation, and
thereby reduces the spread of infection. As
CTLs are part of the acquired immune re-
sponse, they are not present early in the pri-
mary infection. They are important for clear-
ance later in the infection, as well as the
rapid response and virus elimination after
reinfection.
Mouse studies of poxvirus-specific
CTLs, generated during primary infection,
vary slightly in their results, depending on
the strain used, route of inoculation, and
161
 virus dose.61 Primary CTL responses were
greatest with high doses of virus and intrave-
nous inoculation.62 Studies that measured the
CTL response in mice infected with EV
showed that virus-specific killing of target
cells peak around the sixth day of infec-
tion.63 After reinfection, the CTL response
was shown to be eight times greater and was
detected earlier, around the fourth day of
infection.64 These studies illustrate the clas-
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sic pattern for a learned CTL immune re-
sponse to primary virus infections, and the
faster, more powerful memory response to
reinfection. The importance of CTLs in vi-
rus clearance and recovery was demonstrated
many years ago in EV-infected mice. Due to
the clear correlation between the increase in
CTL activity and virus clearance and host
recovery, studies concluded that CTLs were
responsible for protection.61
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IV. VIRAL COUNTER RESPONSE
In order to defend themselves from the
many arms of the immune system, poxvi-
ruses must constantly evolve new, more
powerful methods of modulating the host
immune response. Being large DNA viruses,
poxviruses have enough room to encode
proteins not directly involved in replication.
Therefore, they often encode a repertoire of
proteins involved in habitat formation and
immune evasion (see Figure 2). These pro-
teins cover a wide range of activities, re-
flecting the scope and complexity of the
immune response that they counter in their
host. The survival of the virus depends on its
ability to create and perfect strategies to
conceal itself from this much larger and more
sophisticated opponent. The creativity and
potency of the resulting proteins are fasci-
nating and cover both intracellular and ex-
tracellular processes. Thus far, most of the
proteins that have been discovered are extra-
cellular. The term virokine was coined and
162
used to describe virally encoded proteins
that are secreted from the infected host cell.
Most virokines are powerful viral immune
modulating proteins created by capturing and
modifying genes responsible for regulating
the host immune response. Therefore, these
viral proteins share many structural and func-
tional features of that of the host proteins.
However, because there is limited room
within the viral genome, only those genes
very useful to the virus are maintained and
constantly adapted to make them smaller
and more potent. There have been virokines
discovered and shown to modulate many
important aspects of the immune response.
Many cytokine (IL-6, IL-10) and cytokine
receptor (TNFR, INFγR, IL-1βR) homologs
(also referred to as viroceptors or virokines
that mimic receptors) have been shown to
significantly disrupt cytokine-signaling path-
ways. This often results in the masking of
the host immune system from the viral infec-
tion, allowing the virus to grow to high ti-
ters. Chemokine homologs and chemokine
inhibitors (some of which are chemokine re-
ceptor homologs) were found to have a dra-
matic effect on leukocyte chemotaxis. These
virokines have been shown to either help the
virus evade immune detection, in some cases
by blocking the influx of leukocytes into the
area of infection, or in other cases, actually
cause the recruitment of leukocytes to in-
crease the pool of new host cells to allow for
better dissemination. Chemokine inhibitors
have been identified and shown to block cel-
lular influx into the areas of infection. There
are also virokines that block the most impor-
tant aspect of the innate immune system, the
complement system. These proteins are very
powerful; they not only prevent the formation
of the membrane attack complex (MAC), but
also block the complement cascade at mul-
tiple sites upstream to block the formation of
proinflammatory factors. Therefore, they are
also able to greatly reduce the migration of
leukocytes into the area of viral infection.
 In addition to those proteins encoded by
poxviruses that are secreted from the host
cell, there are also proteins that act intracel-
lularly. The most studied group of intracel-
lular poxvirus immune modulating proteins
are the serine proteinase inhibitors (serpins).65
Many poxviruses have been found to encode
serpins that modulate programmed cell death,
apoptosis. By blocking the cysteine protein-
ase interleukin-1β-converting enzyme (ICE),
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and the serine proteinase granzyme B, the
cowpox cytokine response modifier A
(CrmA), vaccinia virus SPI-2, and myxoma
virus Serp-2 all have been shown to inhibit
apoptosis.66 By blocking these intracellular
enzymes, these poxvirus serine protease in-
hibitor (serpins) proteins are able to prevent
TNF- and IFN-induced, as well as T-lym-
phocyte-mediated Fas-induced apoptosis.67
By protecting the infected cell from pro-
grammed cell death, the virus is able to rep-
licate to higher titers and continue host eva-
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sion.
A. Complement Regulatory Proteins
Many poxviruses encode proteins that
mimic components of the host complement
system in order to block the activation of
complement and thereby modulate the inflam-
matory response mounted by the host.68 The
vaccinia virus complement control protein
(VCP) was the first soluble microbial protein
shown to have complement binding activity
and postulated to have a role in evasion of
host complement. In the years since its dis-
covery, VCP has become one of the most
well-studied and best-characterized virokine.
Structurally, it is composed of four comple-
ment control protein (CCP) modules (also
called short consensus repeats), making it and
other homologous proteins members of the
regulators of complement activation (RCA)
superfamily. VCP exhibits functional simi-
larity to factor H, membrane cofactor protein
(MCP), decay-accelerating factor (DAF),
complement receptor one (CR1), and is struc-
turally most similar to human complement 4b
binding protein (C4b-BP). Purified bioactive
VCP has been shown to inhibit both the clas-
sic and alternative pathways of complement
activation through its ability to bind C3 and
C4 and act as a cofactor for factor I cleavage
of C3b and C4b.69 VCP, secreted from virally
infected cells, is believed to protect the cell
and the released viral progeny from host
complement attack. In addition, by blocking
complement activation at multiple sites, there
is a large reduction of C3a, C4a, and C5a
proinflammatory chemotactic factors, result-
ing in reduced cellular influx and inflamma-
tion.
VCP has also been shown to possess the
ability to bind heparin, as well as heparan
sulfate proteoglycans (heparin-like mol-
ecules) on the surface of human endothelial
cells. Because of this activity, VCP has been
shown to exhibit many properties that all
other complement control proteins are lack-
ing. In vivo studies have indicated that FITC
labeled VCP, when injected intravenously,
becomes localized in the endothelium. This
very interesting property of VCP has been
shown in vitro to reduce chemotactic migra-
tion of leukocytes in the presence and ab-
sence of the chemokine MIP-1α.70 In addi-
tion, VCP was able to inhibit antibody
binding to MHC class I molecules on human
endothelial cells.71 This suggested that VCP
could interfere with molecular interactions
with infected cells and possibly prevent an-
tibody-dependent cell-mediated cytotoxicity
(ADCC) as well as other cytotoxic cell inter-
actions with target cells. Amazingly, VCP
was shown to block interactions between pig
aortic endothelial cells (PAECs) and naïve
neutrophils and natural killer cells, prevent-
ing cell killing.72 In addition, cell culture
studies demonstrate that VCP blocks comple-
ment-mediated killing of PAECs by human
serum in a dose-dependent manner.
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164
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FIGURE 2. Poxvirus immune modulation. (1) Poxvirus binds the host cell, allowing for core entry and early gene transcription to initiate. At this time, many viral
proteins are produced to interfere with the host’s immune response in order to establish a suitable habitat for optimal growth. (2) Vaccinia virus complement control
protein (VCP) is produced and secreted from infected cells. VCP can block the activation of complement through its ability to bind C3b and C4b complement
components. VCP can also bind heparan sulfate proteoglycans on the cell surface, blocking many molecular and cellular interactions with the cell, like chemokine
and antibody binding. (3) Poxviruses also produce a soluble interleukin one receptor homolog (vIL-1βR) that is secreted from the cell, binding up soluble IL-1β
before it has a chance to reach the cell’s receptor. (4) Chemokine binding proteins, such as T1 and T7, inhibit chemokine binding to heparan sulfate proteoglycans
on the cell surface. (5) Soluble viral interleukin ten (vIL-10) is secreted from the cell, binds to IL-10 receptors on inflammatory cells, blocking cell activation. (6)
Soluble poxvirus interferon gamma receptor homolog (vIFN-γR) is also secreted from infected cells, binding and preventing host IFN-γ from binding the cellular
receptor. (7) Another poxvirus early protein that is responsible for immune modulation, soluble tumor necrosis factor receptor (vTNFR) homolog, binds host TNF
and prevents its interaction with the cellular receptor. (8) Poxvirus interleukin eighteen binding protein (vIL-18BP) is secreted form the infected host cell and binds
soluble IL-18, inhibiting its proinflammatory functions. (9) Orf virus encoded granulocyte-macrophage colony stimulating factor-inhibitory factor (GIF) is secreted
and binds to granulocyte-macrophage colony-stimulating factor (GM-CSF), blocking its ability to stimulate the production and secretion of IFNα. (10) Myxoma
virus SERP-1, which is the only secreted poxvirus serpin, blocks several serine proteases, leading to reduction of inflammation. (11) The intracellular poxvirus
serine protease inhibitor proteins, such as SPI-2, serp-2, and crmA, block the conversion of pre-caspase to active caspase one, thereby blocking its ability to
convert pro-IL-1β to IL-1β and the cell’s ability to undergo programmed cell death.

165
 B. Cytokine Homologs
The parapoxvirus Orf, which often infects
sheep and goats (sometimes humans), very
recently has been shown to encode a viral
homolog of IL-10 (vIL-10).73 This protein
shows the greatest degree of homology, 80%,
to sheep IL-10. Endogenous IL-10 plays many
important roles in down-regulating the immune
system. One very important function is to sup-
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press the activation of macrophages, thereby
blocking their ability to act as accessory cells
to activate T-cells or natural killer (NK) cells
and secrete cytokines. Thus, IL-10 acts as a
potent antiinflammatory agent and a powerful
immunosuppressor. Because of this activity, it
is believed that the Orf virus produces vIL-10
in order to inhibit the expression of various
cytokines that regulate the immune response
against viral infection, suppressing the activa-
tion of macrophages. The benefits of vIL-10
production for the virus are thereby to mask
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viral infection from the immune system long
enough to grow to high titers.
Very recently, a new virokine, having
homology to human interleukin 18 binding
protein (hIL-18BP), was uncovered in mol-
luscum contagiosum virus (MCV).74 Since
this time, it has also been found in other
poxviruses, including ectromelia, vaccinia,
and cowpox.75 IL-18 is a proinflammatory
cytokine important in activating natural killer
(NK) cells, inducing a T-helper 1 response,
and inducing the production of interferon
gamma (IFNγ). The MCV vIL-18BP has been
shown to bind to both human and murine IL-18
and inhibit the production of IFNγ in a dose-
dependent manner.74 The deletion of the gene
encoding vIL-18BP in vaccinia was shown
to dramatically attenuate the virus.75 In addi-
tion, the ectromelia virus vIL-18BP was
shown to block IL-18-induced NF-κB acti-
vation and induction of IFNγ. Therefore, this
strategy of viral evasion also acts to indi-
rectly block the strong antiviral effects of
IFNγ.
166
C. Cytokine Receptor Homologs
(Viroceptors)
Many members of the poxvirus family,
including myxoma, vaccinia, and cowpox,
encode homologs of the tumor necrosis fac-
tor receptor (TNFR). This viral tumor necro-
sis factor receptor (vTNFR) was originally
described in myxoma, where it is known as
T2, and Shope fibroma viruses (SFV), and
the term viroceptor was proposed to describe
them.76 TNF is a very important cytokine for
the early response to viral infection. It is a
very powerful proinflammatory agent, trig-
gering numerous immunological events, in-
cluding apoptosis of virally infected cells. In
order to block the effects of this powerful
cytokine, these poxviruses have captured and
modified the host gene for TNFR so that it
may be secreted in large amounts from the
infected cell. The vTNFR can bind soluble
TNF present in the nearby area, preventing
its binding to the cellular TNFR, thereby
blocking the action of this cytokine. Many
poxviruses encode several copies of this gene;
cowpox, for example, contains three sepa-
rate copies, showing its importance to viral
replication.77 In addition, when vTNFR (T2)
was deleted from myxoma virus the viru-
lence was reduced significantly. Interestingly,
vTNFRs show a great deal of host specific-
ity. Myxoma virus vTNFR, T2, will only
bind to rabbit TNF (not at all to human),
whereas the vTNFR encoded by vaccinia
and cowpox virus will bind human, mouse,
and rat TNF effectively.
Many poxviruses, including variola, ec-
tromelia, vaccinia, cowpox, and camelpox,
also encode an additional interesting cytokine
receptor homolog, viral interferon gamma
receptor (vINFγR). These proteins greatly
resemble the N-terminal, ligand binding (ex-
tracellular domain) region of their mamma-
lian counterparts. As with vTNFR, these
cytokine receptor homologs are also very
species specific. The first and most exten-
 sively studied of these proteins comes from
the myxoma virus and is known as T7.78 T7
is a 37-kDa N-linked glycoprotein that is the
most abundantly secreted protein of myxoma
virus. Exhibiting great species specificity,
T7 was shown to bind and block the effects
of rabbit INFγ, but had no effect in blocking
human or murine INFγ. In contrast, vaccinia
virus, which has a broad host range, can bind
and block a number of mammalian INFγs.
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The importance of this encoded protein to
myxoma virus virulence is evident, not only
because it is the most abundantly secreted
protein, but also because deletion of the gene
eliminates the 99% mortality rate exhibited
by the virus, and all infected rabbits recover.
Therefore, the T7 protein represents the major
virulence factor of myxoma virus. The im-
portance of this protein to viral virulence is
due to INFγ’s powerful antiviral effects. INFγ
serves numerous functions, including switch-
ing on many intracellular antiviral activities,
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and giving the signal to nearby cells that a
viral infection is occurring. Another cytokine
receptor homolog, produced most notably
by the orthopox members vaccinia and cow-
pox, is viral interleukin 1 receptor (vIL-1R).
This 33-kDa protein was first identified in
vaccinia virus and found to have the ability
to bind murine IL-1β in 1992.79 A similar
open reading frame was later identified in
cowpox virus and the protein product also
found to bind murine IL-1β. Interestingly,
supernatants taken from vaccinia virus in-
fected cells were found to inhibit IL-1-in-
duced murine lymphocyte proliferation. In
addition, recombinant vaccinia, unable to
express vIL-1R, showed remarkable attenu-
ation compared with the wild type in vivo.80
This demonstrates how the protein plays a
critical role in the pathology of vaccinia vi-
rus infection. The importance of IL-1 in
combating viral infection is large and was
demonstrated many years ago.81 The expres-
sion of IL-1 is induced early on in many
viral infections and results in a broad spec-
trum of inflammatory responses. NK cells
and B-cells proliferate in response to IL-1,
neutrophils degranulate, and T-cells produce
IL-2 and proliferate.
Another very recently identified virokine
was found encoded by the parapoxvirus, Orf.82
The Orf virus produces a secreted protein,
termed granulocyte-macrophage colony stimu-
lating factor-inhibitory factor (GIF), which was
shown to bind to and block the activity of GM-
CSF, as well as IL-2. Both of these cytokines
are very important in host antiviral immunity;
IL-2 is essential for its role in the activation of
T-cells, and GM-CSF for its ability to stimu-
late the production and secretion of IFNα. This
remarkable virokine plays a very novel role in
modulation of host defense as a means of pre-
serving viral habitat.
D. Chemokine Homologs
Many poxviruses encode proteins that
inhibit chemokines; the CC (β) chemokine
homolog encoded by the molluscipoxvirus
molluscum contagiosum (MCV), designated
as molluscum contagiosum chemokine ho-
molog (MCCH) or MC148, was discovered
only recently.83 This human disease causing
virus presumably utilizes this virokine to
block leukocyte trafficking to the site of in-
fection. MC148 is highly homologous to
members of the CC or β chemokine family;
however, it is truncated in the N-terminus of
the protein. The N-terminal portion of the
chemokine is required for proper binding
and activation of the cell.83 Therefore, the
MC148 chemokine homolog of MCV acts
by binding to the chemokine receptor, iden-
tified as being specifically CCR8, thereby
blocking endogenous chemokine binding.
The overall result of this protein is reduced
cellular influx and therefore reduced inflam-
mation. This protein seems to be geared to-
ward specifically blocking monocyte infil-
tration and dendritic cell function.
167
 E. Chemokine Inhibitors
Many poxviruses encode a protein that
blocks the effects of chemokines. Collectively,
these virokines have been termed the T1/35kDa
family, all of which possess eight conserved
cysteine residues and are shown to interact
with members of the CC and/or CXC classes
of chemokines.84 The myxoma virus T1 and
T7 proteins are the most widely studied mem-
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bers of this group. T1 shows no homology to
any host chemokine or chemokine receptor
and binds strongly (specifically) to members
of the CC class of chemokines, but only weakly
(nonspecifically) to CXC chemokines.65 Be-
cause of this ability, T1 has been shown to
block human monocyte migration in the pres-
ence of MIP-1α in vitro. The deletion of the
coding sequence for T1 resulted in a pronounced
increase in infiltrating monocytes and mac-
rophages into the site of infection in vivo.84
However, the loss of T1 had no significant
For personal use only.
effect on mortality, indicating that this gene
does not play a critical role in the pathogenesis
of the infection. T7, on the other hand, has
been shown to inhibit the activity of members
of C, CC, and CXC classes of chemokines in
a nonspecies-specific manner. This protein,
which is most noted for its anti-IFNγ activity,
has been shown to nonspecifically bind to the
heparan sulfate proteoglycan binding domain
of the chemokine, preventing gradient forma-
tion along the vascular wall, and inhibiting
chemotaxis.85 The addition of excess soluble
heparin completely prevents this activity of the
T7 protein, supporting this hypothesis. Because
the T7 protein also exhibits no homology to
any known chemokine, this strategy of
chemokine inhibition is quit novel.
F. Serine Protease Inhibitors
Several poxviruses have been shown to
encode proteins that are able to prevent
apoptosis, programmed cell death. The cow-
168
pox cytokine response modifier A (CrmA),
vaccinia virus SPI-2, and myxoma virus Serp-2
have all been shown to inhibit apoptosis
through the ability to block both the cysteine
proteinase interleukin-1β-converting enzyme
(ICE) and the serine proteinase granzyme
B.66,86,87 By blocking these intracellular en-
zymes, these poxvirus serine protease in-
hibitor (serpins) proteins are able to prevent
TNF and IFN-induced, as well as, T-lym-
phocyte-mediated (Ca+2-independent) Fas-
induced apoptosis.66,88 Therefore, these pro-
teins prevent destruction of infected cells,
even if they are recognized as being infected,
thus maintaining a habitat conducive to vi-
rus replication.
Many members of the poxvirus family
encode proteins that inhibit serine proteases
intracellularly, but only myxoma virus en-
codes one that acts as a serine protease inhibi-
tor (serpin) and secreted extracellularly. First
identified in 1990, this secreted glycoprotein,
Serp-1, has structural features that place it in
the serpin family of serine protease inhibi-
tors.89 Before its discovery, intracellular
serpins were found in vaccinia virus.90 Serpins
have many functions in maintaining homeo-
stasis of eukaryotic systems, from comple-
ment activation and blood clotting to the in-
flammatory response. In addition, mutations
in serpins have been linked to over 90 human
diseases. Given the importance of these pro-
teins, many pathogens have evolved to carry
their own serpins to disrupt host processes.
Myxoma virus Serp-1, for example, has been
shown in vivo to weaken the inflammatory
response to viral infection. When this gene is
deleted from the virus, a much greater inflam-
matory process is observed and the infection
is resolved much more rapidly. Purified re-
combinant Serp-1 has been shown to bind a
variety of proteases, including plasmin, throm-
bin, tissue-type plasminogen activator (tPA),
and urokinase-type plasminogen activator
(uPA), although the exact target has not been
found.91 This protein acts as a pseudosubstrate
 for these serine proteases, forming a stable
complex, preventing the protease from bind-
ing its true substrate. Due to the role of Serp-1
in modulating inflammation, recently it was
tested in a restenosis model system to evalu-
ate its therapeutic value.
V. ANIMAL MODELS FOR THE
STUDY OF POXVIRUS INFECTIONS
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the virus strains (see Figure 3). The degree of
inflammation and cellular infiltration can be
both quantitatively and qualitatively analyzed.
This allows for better characterization of mu-
tant strains and results in a better understand-
ing of genes associated with poxvirus viru-
lence (see Table 2). Understanding poxvirus
virulence aids in the production of better, more
attenuated, vaccine strains, in addition to pox-
virus vectors for use in new vaccines, in hopes

of reducing complications associated with tra-

Several animal models have been devel-
oped and used to study various poxvirus dis-
eases and poxvirus mutants to better our un-
derstanding of the infection processes and
virulence genes. Animal infection models are
important for exploring new treatment strate-
gies and are used to test the efficacies of anti-
viral drugs. Models have been developed to
study the inflammatory response generated by
poxvirus infection, as well as the virulence of
For personal use only.
ditional vaccines. Several important models
that have been used extensively in poxvirus
research are discussed in greater detail.
A. Chicken Embryo Chorioallantoic
Membrane (CAM) Model
The Chicken embryo chorioallantoic mem-
brane (CAM) model has been used extensively

FIGURE 3. Poxvirus infection models. (A) Footpad of normal mouse. (B) Swollen footpad of a mouse several
days after injection of CPV. The degree of inflammation can be measured. (C) Red pocks formed on CAM of
12-day-old chicken embryo 3 to 4 days after infection with CPV, showing no inflammatory response. (D) White
pocks formed on CAM of 12-day-old chicken embryo 3 to 4 days after infection with a mutant strain of CPV,
showing a massive inflammatory response.96 (E) Mouse intranasally infected with mutant VV, showing little
signs of infection. (F) Mouse intranasally infected with wild-type VV, showing significant signs (ruffled fur) of
pathology.

169
 TABLE 2
Poxvirus Animal Models for the Study of Poxvirus Infections. Many of the Experiments
Utilizing Various Animal Models to Study Poxvirus Infection Are Listed. Also Listed Are
the Poxvirus, or Mutant Poxvirus Tested and a Short Description of the Experimental
Model
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For personal use only.

170
 to study pathogenic poxvirus genes and for the
diagnosis of poxvirus infection. The CAM of an
11- to 12-day-old embryonated chicken egg is
inoculated with virus and incubated for 3 days.
The CAM is then removed and the color and
morphology of the pock lesions is examined.
This system is often used to study poxvirus
virulence genes and the presence or absence of
an inflammatory response. The CAM model is
limited, however, because chicken embryos lack
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a fully developed immune system. Therefore,
this model does not truly represent the infection
process of a poxvirus in vivo.
Experimentally, the CAM model has been
used to study poxvirus-encoded genes involved
in inflammation modulation, characterize new
poxvirus strains and mutants, and analyze ge-
netic traits of recombinant poxviruses. Recom-
binants between EV and VV were found to
possess pathogenicity markers from both parent
poxviruses using the CAM model.92 Recombi-
nant virus produced pocks that were large, hem-
For personal use only.
orrhagic, and ulcerating, far more severe than
that formed by the parents, indicating that patho-
genicity was enhanced in the recombinants. A
study of the cell proliferative response to VV
growth factor (VGF) was also examined in the
CAM model.93 VGF was found to produce le-
sions that contained more proliferating cells and
more infectious virus progeny. Similar studies
use the CAM to analyze the effect gene dele-
tions have on the pock morphology and cellular
infiltration.94–97 The deletion of genes encoding
inflammatory modulating proteins results in a
change of pock lesions from red to white, an
indication a massive cellular inflammatory re-
sponse. Finally, the CAM is used to characterize
new poxviruses and poxvirus strains, using varia-
tions in pock size, color, and morphology.98,99
B. Footpad Model for Quantitative
Analysis of Inflammation
The footpad injection model was devel-
oped to assess the inflammatory response
induced by poxvirus infection. Poxvirus is
injected into one of the hind footpads of an
adult mouse or rat. The primary inflamma-
tory response is then measured over several
days. In this model, unlike the CAM model,
a fully functional immune system is present,
and the inflammatory response can be calcu-
lated through direct measurement of the
swollen footpad. Like the CAM model, how-
ever, it is very quick and easy to perform.
Cellular infiltrate is often examined micro-
scopically from the footpad to better charac-
terize the specific cell types. This model is
used primarily to determine the role various
poxvirus genes have in regulating the in-
flammatory response.
Early experiments used the footpad
model to assess the cell-mediated immune
response to VV infection in rats.100 The ki-
netics of the cellular immune response was
found to be in accordance with the regular
time course. Footpad swelling started 3 days
after infection, peaked around day 6, and
disappeared after 2 weeks. The repose was
comparable to that reported previously in
mice. More recently, the footpad model was
employed to determine the role that a poxvi-
rus encoded complement regulating protein
might have on inflammation.48 To better
simulate an actual infection process, cow-
pox virus (CPV) was injected into the foot-
pads of mice, the natural host. Wild-type and
recombinant CPV without the gene encod-
ing the complement regulatory protein,
termed the inflammation modulatory protein
(IMP), was injected into the footpads of
mice.48,101 Additionally, C3 and MIP-1α
knockout mice were injected with CPV and
CPV lacking IMP, and the footpad swelling
measured. Significantly greater footpad
swelling was observed in the mice injected
with CPV lacking IMP. The results con-
firmed the importance that IMP has in re-
ducing cellular infiltration by inhibiting
complement activation, thereby reducing the
production of chemotactic factors.
171
 C. Air Pouch Model for Qualitative
Assessment of Inflammation
The subcutaneous air pouch injection
model was developed to assess the precise
cellular changes at the site of poxvirus infec-
tion. It has long been used to assess the
inflammatory response elicited by many
natural and synthetic agents, as well as the
ability of various drugs to block this re-
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sponse. Recently, it has been proven to be a
valuable tool for examining the cellular in-
filtrate at the site of poxvirus infection. The
model is simple; mice or rats are injected in
the back subcutaneously with 1 cc of air.
Virus or some other material of interest is
then injected into the air pouch. The air pouch
is then harvested at different time points; the
thin layer of connective tissue is spread across
a slide, stained (with May-Grunwald and
Giemsa), and the cellular infiltrate examined
microscopically. This model was used suc-
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cessfully to evaluate the importance IMP of
CPV has in limiting inflammatory cell
chemotaxis to the site of infection.101
D. Intracranial Injection Model
The virulence of poxvirus has often been
tested using the intracranial (LD50) injection
model. A direct correlation between
neurovirulence and virus propagation has
been shown previously with VV inoculated
intracranially, higher lethality resulted form
greater virus yield.102,103 Weanling mice
(3-to 4-week-old male BALB/c) are gener-
ally used for this model due to the greater
ease in which the softer skull can be pen-
etrated. Using a small-gauge needle, virus is
injected intracranially into one of the cere-
bral hemispheres of the mice. Long-term
mortality can be observed or the mice can be
sacrificed at various time points to deter-
mine the virus titer in the brain tissue. Ex-
perimentally, this model has been used to
172
define poxvirus virulence genes, most ex-
clusively in VV. The spontaneous deletion
mutant 6/2 of VV, which lacks 17 open read-
ing frames (ORF), was injected intracrani-
ally to demonstrate a loss in virulence.104 An
enormous loss in virulence was observed
compared to the wild-type VV strain. An
additional study using the intracranial injec-
tion model showed a substantial reduction of
VV virulence when a single 13.8-kDa ORF
was deleted.105 The single protein encoded
by this ORF contributed much of the viru-
lence demonstrated by wild-type strain of
VV. The overall goal of these studies are to
identify mutant strains of VV that may be
used for vaccination and gene therapy, with-
out the complication risks traditionally asso-
ciated with VV vaccine strains, such as
postvaccinial encephalitis.
E. Nude Mouse Model for
Evaluation of Virulence
Poxvirus virulence is also evaluated in
the nude (athymic) mouse model. The model
involves intraperitoneal injection of VV into
athymic mice, between 4 and 16 weeks of
age, and mortality is measured out to 60
days. A dose-dependent mortality is observed
with virulent strains of VV. This model has
been used quite extensively to investigate
the biological roles of various cytokines and
chemokines expressed by recombinant VV.
Nude mice are also used to study the viru-
lence of mutant vaccinia virus for possible
use as vaccine strains. Several deletion mu-
tants of VV have been shown to have re-
duced virulence, when compared with the
wild-type strain, when injected intraperito-
neally into nude mice.105 Other studies show
that expression of various cytokines, within
recombinant VV, reduces or prevents infec-
tion in nude mice. VV expressing human or
mouse IL-2 was rapidly eliminated, result-
ing in little or no mortality.106 A similar ef-
 fect, although not to the same degree, was
seen when VV expressing IL-15 was in-
jected into nude mice.107 The effect of IL-2
was not systemic, coinfecting with IL-2 ex-
pressing and virulent wild-type strains, re-
sulted in significant lethality.108 In addition,
the effects seen by IL-2 were abolished when
the mice were injected with monoclonal
antibody against IFNγ.109 This model has
also been used to evaluate the effects that
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VV expressed cytokines and chemokine have
on the host cellular response to infection.
The enhancement of NK cell activity was
attributed to many of the effects observed in
these studies.107,110
F. Intranasal Inoculation Model
Intranasal inoculation of mice with
CPV has been used as a model to study
orthopoxvirus respiratory disease. Mice, 4
For personal use only.
to 6 weeks of age, are inoculated intrana-
sally with aerosols containing CPV.111 Re-
cently, this model has been used to study the
efficiencies of antipoxvirus drugs in hopes
of developing better treatment strategies for
the highly fatal respiratory disease often as-
sociated with human monkeypox virus in-
fection.112 Severe disease, resulting in death,
starts to take effect by day 7 in untreated
mice. Mice develop pulmonary lesions, even-
tually involving most of the visceral pleura,
and often show ulcerating lesions on the tail,
indicating dissemination. Cidofovir, an ap-
proved antiviral drug, has been shown to
significantly reduce mortality using the in-
tranasal inoculation model.113,114 Addition-
ally, the intranasal inoculation model is used
to characterize mutant strains of VV. Intra-
nasal inoculation of mice with wild-type VV
resulted in a highly lethal disseminated in-
fection.115 However, mice intranasally in-
fected with VV lacking the thymidine kinase
gene showed no lethality and a lack of dis-
semination.116
VI. ANIMAL MODELS FOR THE
STUDY OF POXVIRUS IMMUNE
MODULATION
As discussed earlier, poxviruses are well
noted for their ability to produce a repertoire
of immune modulating proteins. Several of
these proteins have been investigated further
for their potential use as therapeutic agents
(see Table 3). These immune blocking pro-
teins have very powerful antiinflammatory
properties that may be employed for treat-
ment of many injuries or diseases that in-
volve excessive inflammation. Among other
things, poxvirus immunomodulatory proteins
have been explored for potential application
in transplantation, vascular injury, CNS in-
jury, sepsis, arthritis, and restenosis. Two
poxvirus-encoded proteins have been stud-
ied quite extensively, VCP and Serp-1; the
various animal models employed to evaluate
their therapeutic effectiveness is discussed
in greater detail.
A. Xenotransplantation Model
In order to test the ability of the vaccinia
virus complement control protein (VCP) to
block the rejection of grafted tissue trans-
planted between species, a suitable animal
model was developed. Guinea pig-to-rat het-
erotopic heart transplantation was performed
using young guinea pigs and large adult rats.
Guinea pig hearts were removed, prepared in
cold cardioplegic solution, and microsurgically
transplanted into the neck of the rat. With all
other vessels of the donor heart ligated, the
left jugular vein of the rat is sutured to the left
pulmonary artery of the heart and the right
carotid artery of the rat is sutured to the in-
nominate artery of the aorta. The clamps are
released, blood enters and warms the donor
heart, and the heart spontaneously begins to
beat. Steady perfusion of the donor heart tis-
sue with the rat’s blood, powered by the rat’s
173
 TABLE 3
Poxvirus Animal Models for the Study of Therapeutic Poxvirus Immune Modulation:
Many of the Experiments Utilizing Various Animal Models to Study Poxvirus Immune
Modulation Are Listed. Also Listed Are the Poxvirus Proteins Tested and a Short
Description of the Experimental Model
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heart, exposes the donor heart to the immune
system of the rat. Because this model repre-
sents a xenotransplant, the donor heart is
For personal use only.
quickly rejected, between 15 and 20 min after
blood flow is established. This rejection oc-
curs very rapidly due to the presence of anti-
bodies in the blood of the rat, which cross-
react to sugar moieties on the endothelium of
the guinea pig heart. This will then lead to the
activation of complement and thereby the de-
struction of the donor heart. Due to the comple-
ment inhibiting ability of VCP, as well as the
other properties contributed by its ability to
bind heparin, its capacity to block or delay
this rejection process was examined. Intrave-
nous administration of VCP was found to
delay xenograft rejection, by not only block-
ing complement activation, but also prevent-
ing antibody fixation to the donor endothe-
lium.117
B. Allograft and Restenosis Models:
To test the therapeutic ability of Serp-1,
the MV secreted antiinflammatory serine pro-
teinase inhibitor, to suppress artherosclerotic
174
plaque development, several animal models
were used.118 An aortic allograft model was
employed to investigate artheroscletotic plaque
formation following graft transplantation in
rats. A 1-cm section of lower abdominal aorta
was removed for the donor and sutured end to
end into the abdominal aorta of the recipient
rat. After blood flow was established, a single
dose of Serp-1 or saline was injected intrave-
nously. The tissue was then harvested for
histological examination to determine the
short- and long-term effects. The single dose
of Serp-1 was found to significantly reduce
inflammatory cell infiltration and later plaque
formation and aortic occlusion.119 This ability
to block artheroscletotic plaque formation was
also tested in a rabbit model for balloon
angioplasty. Cholesterol-fed rabbits were in-
fused with Serp-1 or saline shortly after being
subjected to balloon angioplasty. The injury
site tissue was removed at different time points
for histological examination. Again, Serp-1
was found to dramatically reduce cellular in-
vasion and plaque formation at the site of
injury.120
The M-T7 protein of MV, which is able
to inhibit IFN-γ and block chemokines, was
 also tested for its ability to suppress
artherosclerotic plaque development follow-
ing balloon angioplasty.121 Using both rat
and rabbit animal models, purified M-T7 or
saline was infused into the area after the
injury. In both models, histological analysis
after 1 month revealed a significant reduc-
tion in arterial hyperplasia. In addition, an
early inhibition of inflammatory cell inva-
sion into the area of the injury was observed.
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Another allograft model has been used re-
cently to study the ability of the MCV
chemokine antagonist MC148 to prolong
graft survival using gene transfer.122 A
heterotropic nonvascular heart transplant,
where a neonatal heart is implanted subcuta-
neously, was used in mice. Using plasmid-
mediated gene transfer, plasmid DNA en-
coding MC148 was transferred into the donor
heart before transplanting. The presence of
MC148 significantly decreased donor spe-
cific CTL infiltration into graft tissue and
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inhibited alloantibody production.
C. Head Injury Model
To evaluate the ability of VCP to modu-
late the inflammation and neurological dys-
function following traumatic brain injury,
the lateral fluid percussion brain injury model
was used. Rats receive a craniotomy to ex-
pose the dura before being attached to the
injury devise. The injury devise then sends a
bolus of saline that strikes the surface of the
brain, creating a pressure pulse that causes a
rapid but temporary displacement of brain
tissue. The severity of injury is predeter-
mined and very reproducible. Following the
injury, VCP or saline is microinjected into
the cortex underlying the site of injury and
the wound is closed. The effect that VCP
had on complement activation and inflam-
matory cell infiltration was then monitored
at various post-injury time points. It was
found that VCP, when present at the site of
injury, resulted in a significant reduction of
inflammatory cell infiltration and comple-
ment activation.123 However, these results,
which demonstrate a general reduction in
inflammation, do not necessarily imply that
neuronal sparing and/or cognitive recovery
will occur. Therefore, to assess whether ad-
ministration of VCP at the site of brain in-
jury can lead to a functional recovery, the
special memory of brain-injured rats was
evaluated using the Morris Water Maze.
VCP-treated rats were shown to perform sig-
nificantly better in the Morris Water Maze
than the control rats, indicating an increase
in cognitive recovery.124 The increased spa-
tial memory displayed by the VCP-treated
rats illustrates that its immune modulating
properties are, in fact, beneficial for recov-
ery following traumatic brain injury.
D. Spinal Cord Injury Model
To evaluate the ability of VCP to modu-
late the inflammation and neurological dys-
function following traumatic spinal cord in-
jury, the New York University impact
contusion model was used. Like with the
head injury model, adult rats receive a lami-
nectomy to expose the dura before being
fastened to the NYU weight-drop impactor
devise. The injury devise causes a very re-
producible contusion injury on the spinal
cord of the desired severity. Following the
injury, VCP or saline is microinjected into
the spinal cord parenchyma at multiple bilat-
eral sites underlying the injury and the wound
is closed. The effect that VCP had on in-
flammatory cell infiltration was then moni-
tored at various post-injury time points by
hematoxylin and eosin (H&E) staining and
the myeloperoxidase (MPO) assay. It was
found that VCP-treated spinal cords con-
tained fewer neutrophils present around the
site of the injury.125 To determine whether
the reduction of inflammatory cell infiltra-
175
 tion can coincide with functional recovery,
injured rats were evaluated using the Basso,
Beattie, and Bresnahan (BBB) locomotor
rating scale, and the grid walking test. VCP-
injected animals performed significantly
better in the grid walking test and scored
higher (although not significantly) on the
BBB locomoter scale for 6 weeks after in-
jury. This increase in motor function sug-
gests that rats receiving an injection of VCP
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at the site of injury exhibit greater neuronal
sparing and functional recovery.
E. Antigen-Induced Arthritis (AIA)
Model of Chronic Inflammation
In order to determine the effect that Serp-1
might have in alleviating chronic inflamma-
tion characteristic of arthritis, an antigen-
induced arthritis (AIA) model was used in
rabbits. Rabbits were first immunized with
For personal use only.
ovalbumin in complete Freund’s adjuvant to
generate a strong immune response. To in-
duce AIA in a single joint, rabbits received
an intraarticular (i.a.) injection of ovalbu-
min. Finally, after 2 weeks a second i.a.
injection of ovalbumin, followed by two daily
injections of transforming growth factor beta
(TGF-β) completed the induction of AIA.
Serp-1 was then injected i.a. 1 and 2 weeks
after the final ovalbumin injection, to test its
anti-arthritic effects. A histological analysis
of the synovium later revealed that Serp-1
injection had a dramatic effect on chronic
inflammation by reducing inflammatory cell
infiltration, cartilage erosion, and synovium
hyperplasia.126
VII. CONCLUSION
The study of poxvirus infection in vari-
ous animals is a very exciting and multifac-
eted topic for research. Poxviruses have been
found to infect a wide range of animal hosts,
176
causing a large diversity of disease patholo-
gies. The immune response to poxvirus infec-
tion mounted by the host is extremely power-
ful and of great complexity. However, the
poxvirus has become an ever more complex
and a worthy adversary, evolving new and
more powerful immune modulating agents.
The survival of the virus depends on its abil-
ity to create and perfect strategies to conceal
itself from this much larger and more sophis-
ticated opponent. Presently, the most heavily
studied aspect of poxvirus infection is the
immune modulating proteins. These amazing
proteins have tremendous potential for use as
therapeutic agents for the treatment of many
injuries and diseases, of an inflammatory na-
ture. The creativity and potency of the many
virokines is fascinating and may lend itself to
a new class of therapeutic agents. Someday
we may be able to make use of these powerful
proteins that have evolved to near perfection
for the treatment of diseases that cannot be
treated with conventional therapy. The use of
virokines as immunomodulatory agents is only
starting to be realized. In addition, new
virokines are constantly being uncovered,
often with new and novel features. Myxoma
virus, for example, secretes dozens of pro-
teins from the infected cell, only about five of
which have been isolated and characterized to
any extent.
Several animal models have been used
traditionally to study poxvirus infection, in-
cluding the CAM, nude mouse, and footpad
injection models. In addition, several models
have been developed and employed to evalu-
ate the therapeutic effectiveness of various
poxvirus immunomodulatory agents. These
models are designed to closely replicate hu-
man injury or disease and to assess the overall
usefulness of these poxviral agents. Several
of these proteins have shown great potential
in reducing inflammation, thus resulting in a
more favorable outcome. Although the study
of these poxvirus proteins has been quite ex-
tensive, one question that remains is whether
 they act as virulence factors that promote vi-
ral replication in host tissue, and if so what
are the mechanisms, and what do these pro-
teins do in terms of evading host defenses.
Until very recently, only a couple of investi-
gators have attempted to address the role of
virokines in whole animals. The data gath-
ered from these experiments could help to
answer many questions, such as why variola
virus is so deadly even though 80% of its
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ORFs possess 90% or greater sequence ho-
mology to that of vaccinia virus. An addi-
tional area that needs farther investigation,
one of the biggest mysteries in poxvirus re-
search is how the viruses acquired these im-
mune modulatory proteins. Many of these
poxvirus proteins possess high sequence ho-
mology to those immune regulatory proteins
found encoded by their natural host. There-
fore, the virus must have stolen them from the
host cell, modified them to be more compact
and more powerful, and turned them against
For personal use only.
their host. So how did a DNA virus, which
replicates in the cytoplasm, acquire the nucleic
acid sequence for such proteins? It is widely
believed that during coinfection with a
retrovirus, the RNA encoding these proteins
was converted to DNA and packaged by the
poxvirus — this hypothesis has yet to be
proven. Future experiments will help clear up
these remaining unanswered, often basic,
questions about poxvirus infection in animals.
ACKNOWLEDGMENTS
SAS is a recipient of the Center for Genetics
and Molecular Medicine graduate fellowship. GJK
is currently a senior International Wellcome Trust
Fellow in biomedical sciences in South Africa.
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