Immunopathogenesis of poxvirus infections: forecasting the impending storm

REVIEW

Immunopathogenesis of poxvirus infections:forecasting the impending storm

Marianne M Stanford1,2, Grant McFadden1,2, Gunasegaran Karupiah3 and Geeta Chaudhri3

Variola virus, the causative agent of smallpox, is a member of the poxvirus family and one of the most virulent human pathogens

known. Although smallpox was eradicated almost 30 years ago, it is not understood why the mortality rates associated with the

disease were high, why some patients recovered, and what constitutes an effective host response against infection. As variola

virus infects only humans, our current understanding of poxvirus infections comes largely from historical clinical data from

smallpox patients and from animal studies using closely related viruses such as ectromelia, myxoma and monkeypox. The

outcome of an infection is determined by a complex interaction between the type of immune response mounted by the host

and by evasion mechanisms that the virus has evolved to subvert it. Disease pathogenesis is also a function of both host and

viral factors. Poxviruses are not only cytopathic, causing host tissue damage, but also encode an array of immunomodulatory

molecules that affect the severity of disease. The ability of the host to control virus replication is therefore critical in limiting

tissue damage. However, in addition to targeting virus, the immune response can inadvertently damage the host to such a degree

that it causes illness and even death. There is growing evidence that many of the symptoms associated with serious poxvirus

infections are a result of a ‘cytokine storm’ or sepsis and that this may be the underlying cause of pathology.

Immunology and Cell Biology advance online publication, 16 January 2007; doi:10.1038/sj.icb.7100033

Keywords: immune evasion; immunopathology; host response; poxvirus; smallpox

Variola virus, the causative agent of smallpox, is a virulent humanpathogen that has killed more people than all other infectious diseasescombined. It is not understood why the mortality rates were high,why some recovered or what constitutes an effective host responseagainst infection. Although smallpox was eradicated almost 30 yearsago, a renewed interest in poxvirus infections arise both fromthe potential threat of unintentional or intentional release of variolavirus and from the emergence of zoonotic poxvirus infections suchas monkeypox.

The poxvirus family is divided into two subfamilies: those thatinfect insects, Entomopoxvirinae, and those that infect vertebrates,Chordopoxvirinae. Viruses from a number of genera within Chordo-poxvirinae are able to infect humans. These include Orthopoxvirus,Parapoxvirus, Yatapoxvirus and Molluscipoxvirus.1 Of the genus Ortho-poxvirus, variola, vaccinia (the virus used for vaccination againstsmallpox) cowpox and monkeypox can infect humans and have thepotential of causing death.2 In addition, Molluscum contagiosumvirus, belonging to the genus Molluscipoxvirus, is a natural humanpathogen that causes benign disease in immunocompetent individualsbut can be a serious problem in immunodeficient patients.3

Poxviruses are large, linear double-stranded DNA viruses withgenome size ranging from 130 to 360 kbp.4 The central part of the

genome contains genes involved in key functions such as transcriptionand virus assembly, whereas those located at the termini are involvedin virus–host interactions, for example host range restriction andimmune evasion.5 Of more than 150 genes encoded by poxviruses, 49are common to all sequenced members of this family and 90 arecommon within the subfamily of chordopoxviruses.6 Not surprisingly,the most conserved genes are related to viral function and formthe central part of the genome. Detailed information on poxvirusbioinformatics has been recently collated and is available at www.poxvirus.org.

Viruses within a genus, such as Orthopoxvirus, are even more highlyconserved.6 This translates to a high degree of antigenic similarity,which has formed the basis of using one virus species to protectagainst infection with another.2 The first instance of modern immu-nization employed against any disease was by Edward Jenner who usedcowpox virus, which causes a benign infection in humans, to protectagainst smallpox. The eventual eradication of smallpox was achievedthrough the induction of protective immunity against the diseaseusing vaccine strains of vaccinia virus.7

Viral genes involved in interactions with the host are more variableand have been termed ‘virulence genes’.4,8 Poxviruses exhibit a range ofhost specificities from narrow, such as variola virus that only infects

Received 15 November 2006; accepted 16 November 2006

1Biotherapeutics Research Group, Robarts Research Institute, Ontario, Canada; 2Department of Microbiology and Immunology, University of Western Ontario, London, Ontario,Canada and 3Infection and Immunity Group, Division of Immunology and Genetics, The John Curtin School of Medical Research, Australian National University, Acton, AustralianCapital Territory, AustraliaCorrespondence: Dr G Chaudhri, Infection and Immunity Group, Division of Immunology and Genetics, The John Curtin School of Medical Research, Australian NationalUniversity, Acton, ACT 0200 Australia.E-mail: [email protected]

Immunology and Cell Biology (2007), 1–10& 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00

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humans, to broad, such as vaccinia and monkeypox viruses that areable to infect a large range of mammalian species. This attribute ofhost tropism is thought to be another function of the virulence genes.9

INTERACTION BETWEEN VIRUS AND HOST

Poxviruses infect their host via a number of different routes includingthrough the respiratory tract, skin abrasions and bites of arthropodvectors. The major route through which each specific poxvirus istransmitted varies and is related to the social behavior of its hostspecies. In addition, the route through which the virus enterscontributes to whether the resulting infection is localized or general-ized. Variola virus infection in humans was spread through inhalationof virus in aerosolized form via excretions from the mouth and nose ofinfected patients.10,11 This route of infection could result in severesystemic disease whereas cutaneous inoculation, also known asvariolation, produced milder disease with case fatality rates of 1–2%rather than 30–40% that were observed for naturally acquired small-pox.7 Indeed, the practice of cutaneous inoculation of materialobtained from pock lesions from infected patients into healthyindividuals was a technique historically used to immunize againstthe more severe form of the disease by the Chinese and Indians asearly as 1000 AD. The respiratory route has also been implicated in thespread of rabbitpox, ectromelia and vaccinia viruses to their respectivehosts.12,13

The primary means through which ectromelia, cowpox and vacciniaviruses enter their hosts is through skin abrasions, where they producelocal, primary skin lesions.13–16 Infection at this primary site can leadto systemic infection and then generalized skin lesions, depending onthe strain of virus and the immune status and genetic makeup of thehost. Another route of entry employed by some poxviruses is throughdeposition by arthropod vectors during a blood meal and this is themajor means of spread of myxoma virus in the wild.14 Poxviruses canalso be spread orally through bites and although it is thought to bea less significant mode, this route has been implicated in the spreadof ectromelia virus in mice and cowpox virus in cats.13,17

An infection is a race between replication and transmission of thevirus and mobilization of immune defenses by the host to eradicate it.For this reason, severity of disease and outcome of infection is afunction of both the infectious agent and the host response. The largesize and complex nature of poxviruses make them a significant targetfor the host’s immune response. Whereas smaller viruses may subvertthe host’s defenses by slipping through its gaps or by rapid replication,larger viruses require a more involved strategy to survive within thehost. In the face of a hostile environment, poxviruses have developedan array of molecules that are encoded by virulence genes anddesigned to directly subvert the defenses mounted by the host. Theplethora of viral molecules directed against components of theimmune response are thought to play modulatory roles and havebeen extensively reviewed elsewhere.4,8,9,18

These modulatory poxvirus-encoded proteins can be classed intotwo groups based on whether they act intracellularly or extracellularly(Figure 1).19–21 Virotransducer proteins act intracellularly and func-tion to interfere with the response to infection within the cell,including the induction of an antiviral state, the oxidative burst andapoptotic pathways. Virostealth proteins also act intracellularly andreduce the likelihood of detection by the immune system throughdownregulation of immune recognition molecules, such as majorhistocompatibility complex (MHC) class I and CD4.

Viral proteins that act extracellularly, classed as viromimics, func-tion to modulate the immune response and can be further classed intoviroreceptors and virokines. Viroreceptors are secreted or cell surface

glycoproteins that act to competitively bind host cytokines andchemokines thus interfering with their function. In contrast, virokinesare viral mimics of host cytokines, chemokines and growth factors thatact both to subvert the host responses, which are detrimental to virussurvival and to promote those responses that are favorable to viralreplication and spread. The nature of these viral immunomodulatorshas been extensively reviewed.4,8,15,16,18,19,22 Interestingly, there is noknown immunomodulatory protein that is shared by all poxviruses,and each virus species encodes its own unique combination ofproteins that allows it to be effective in evading the immune responseof its natural host. Furthermore, in some instances, viral immuno-modulatory proteins exhibit specificity of interaction that is limited tothe corresponding host molecule. The highly host-specific survivalstrategy employed by poxviruses may also contribute to the un-predictable outcome of infection when the virus jumps from itsevolutionary host species into a new host species.

The bulk of studies examining the relative importance of immuno-modulatory genes in poxviruses has been undertaken using targeteddeletion of these genes.20 This approach takes advantage of theknown genetic sequence of many poxviruses, as well as the ability toselectively ‘knock out’ genes using targeted disruption of specificsequences. Deletion of some of these genes has resulted in anattenuated or altered disease profile in the host, and proteinsencoded by them are involved in the expression of virulence,hence the term virulence genes. Other genes have been required forproductive infection in host cell lines in vitro, as well as maintenanceof a full infection in vivo and these have been termed ‘host range’genes.

Surprisingly, despite the large number of immunomodulatoryproteins encoded by each poxvirus, there does not appear to bemuch redundancy in the function of these molecules. Studies invaccinia, ectromelia, cowpox and myxoma viruses have shown thatthe deletion of a single gene can significantly decrease the pathogeni-city and replication capacity in vivo.20,23,24 In many cases, thisdecreased pathogenicity is correlated with a more robust earlyinflammatory response by the host, indicating that many poxvirusesparticularly target the innate and inflammatory portion of the hostresponse thus ensuring successful establishment of infection.

For its part, the host’s response to viral infection involves theactivation of a complex network in which numerous cell types andsoluble factors of the immune system participate. However, in addi-tion to targeting the virus, this response is thought to inadvertentlydamage the host to such a degree that it causes illness and even deathin certain poxvirus infections. In infections that are associated with

EXTRACELLULAR(Viromimicry)

Virokines Viroreceptors

Secreted

proteins

Intracellular

proteins

Intracellular serpinsAnti-apoptoticAntioxidantSteroid synthesisdsRNA binding proteinSignal transduction inhibitorsEIongation initiation factor-2α

homologues

IL-1β receptor homologueIFN receptor homologueTNF receptor homologueG-protein coupled receptor homologues

Extracellular serpinSemaphorin homologueCytokine homologuesChemokine homologuesIL-18 binding proteinChemokine binding proteinComplement binding proteinMulti-cytokine binding protein

VirostealthInterference with MHC Iand CD4 presentation

INTRACELLULAR

Virotransducers

Figure 1 Immunomodulatory proteins of poxviruses.

Immunopathogenesis of poxvirus infectionsMM Stanford et al

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Immunology and Cell Biology

benign disease, such as Molluscum contagiousum in humans, andboth Shope Fibroma virus and myxoma virus in Silvilagus species,there is little inflammatory response to the virus; thus, the virus causesonly superficial lesions in the skin and is eventually cleared. However,other poxviruses, such as variola, vaccinia, ectromelia, monkeypoxand myxoma viruses, cause generalized infection in their respectivehosts that can result in significant disease.

The first well-described model of generalized poxvirus infection wasectromelia virus in mice that results in a smallpox-like disease.25 Theoverall pattern of pathogenesis was confirmed in experiments withrabbitpox in rabbits and monkeypox in monkeys.2 The virus isdisseminated through the host via the lymph and blood primarilyby infected leukocytes, with a smaller contribution from free virions.A detailed temporal description of spread of virus through thehost, the resultant pathology and clinical symptoms is presentedelsewhere.2,7 A brief account is given below in the section onectromelia virus infection in mice.

A skin rash is the most characteristic feature of a generalizedpoxvirus infection and the individual lesion is referred to as a‘pock’. However, in highly susceptible hosts infected with virulentstrains of virus, uncontrolled virus replication leading to death occursbefore the skin lesions can develop. These severe forms of disease, forexample hemorrhagic-type smallpox in humans, can be seen in asubset of the population that is extremely susceptible, due to geneticfactors and immune status.

In addition to the cytopathic effects of poxviruses that cause tissuedamage, a contribution of the inflammatory response to pathogenesisis becoming increasingly appreciated. The overproduction of inflam-matory cytokines and other soluble mediators in response to infectioncan lead to sepsis and septic shock.26 Many of the symptomsassociated with serious poxvirus infections are similar to thoseobserved in other infectious diseases where a ‘cytokine storm’ resultingin sepsis is known to be the underlying cause of pathology. Thereis now accumulating evidence that uncontrolled inflammation andseptic shock may be involved in overwhelming and fatal poxvirusinfections.

SMALLPOX IMMUNOPATHOLOGY

The role of immunopathlogy in variola virus infection in humanshas been one of considerable debate. As a pathogen, variola virushas killed more members of the human population than allother infectious diseases combined, which makes understanding itspathology vital.9 Declaration of smallpox eradication by theWorld Health Organization and the subsequent restrictions forlaboratory investigation of variola virus has left a significant gapin the understanding of immunity and pathogenesis of thisinfection. The last known naturally occurring case of smallpoxoccurred in Somalia in 1977, which was long before the more currentadvances in the study of molecular virology and immunology.27,28

Thus, we have limited understanding of detailed virus hostinteractions.

To date, we do not understand why the two variants of variola,designated variola major, the main causative agent of smallpox, andvariola minor, or alastrim, differ so drastically in their pathogenicity.These strains differ by only 2% of their genome and share the majorityof key immunomodulatory proteins. However, variola major hada case fatality rate ranging from 30 to 40%, whereas alastrim’s wasapproximately 1%.29,30 It is therefore difficult to reconcile the minorgenetic differences between the virus strains with significant differ-ences in pathogenicity.31–34 Furthermore, although mortality ratesassociated with variola major infection were high, a significant subset

of the infected population recovered. The basis of this susceptibilityand resistance, and the host parameters associated with recovery, hasnot been elucidated.

It has not been possible to determine the mechanisms involved invariola major pathogenicity, given the strict species tropism of thevirus for humans. In addition, there is no known animal reservoir forthe virus. Animal models using non-human primates have beenrecently employed; however, these utilize high doses of virus that areadministered intravenously35 and their usefulness is limited as theyonly mimic post secondary viremia and end stages of disease. Despitethese drawbacks, the information gleaned from this model, in combi-nation with clinical data, provides us some understanding of theimmunopathology of smallpox.

Historical clinical data from patients provided information regard-ing disease progression and variations in the clinical manifestations.These have been described in detail elsewhere.7,36,37 The differentpatterns of illness observed in patients infected with variola majorranged from ordinary-type smallpox with raised pustular skin lesionsto very severe forms of disease that progressed too rapidly for thetypical rash to develop. These variations are thought to have beena function of genetic factors and immune status of the host. Aclassification system for the clinical types of variola major infection,proposed by Rao (1972),37 was adopted by the World Health Orga-nization scientific group on smallpox eradication. Four distinct clinicalvarieties ((1) hemorrhagic, (2) flat, (3) ordinary and (4) modified)were categorized based on the nature and evolution of the rash. Thesewere subdivided further into 12 categories derived from detailedclinical description.

Cases of modified-type smallpox, a mild form of the disease, usuallyoccurred in vaccinated persons and did not result in mortality. Up to90% of cases of the disease in unvaccinated individuals were of theordinary-type classification and had case fatality rates of about30%.7,37 A more severe form of the disease, referred to as flat-typesmallpox owing to characteristic flat, soft spreading skin lesions,accounted for about 5% of patients. This form was more commonin children and resulted in case fatality rates of 95% in unvaccinatedindividuals. Another severe form of the disease, or hemorrhagic-typesmallpox, seen in less than 3% of patients, was characterized bymucosal and skin hemorrhage. This was a rapidly progressing diseasewith the most common form being early hemorrhagic-type smallpox,which was associated with almost 100% mortality irrespective ofwhether the patient had been vaccinated or not. The late form ofhemorrhagic-type smallpox had case fatality rates of 90% in vacci-nated and 97% in unvaccinated individuals. Hemorrhagic-type small-pox was mostly seen in adults, especially pregnant women. Indeed,pregnant women with smallpox, of all classifications combined, hadthree to four times higher case fatality rates than non-pregnantwomen and men of the same age groups.7,37 One possible explanationfor this may be related to a systemic immune bias towards T-helpertype 2 responses associated with pregnancy.38 The importance ofT-helper type 1 cytokines in the generation of a successful hostresponse comes from animal studies of poxvirus infection39–42 andis underscored by the existence of poxvirus-encoded immuno-modulatory molecules that specifically target T-helper type 1, butnot T-helper type 2, cytokines.4,8,9,18

It is significant to note that whereas the pathophysiological basis ofvery severe illness and death is unknown, there was some suggestionthat there was a relationship between clinical severity and variola virusvirulence.43 However, this is unlikely to have been a major factor, asindividuals infected through contact with patients with severe formof disease usually developed ordinary-type smallpox.1 Thus, there is a


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significant contribution by the host response in determining themanifestation of disease severity and outcome of infection.

Very severe forms of smallpox that progressed rapidly and culmi-nated in death were associated with high levels of circulatingvirus.7,37,44,45 In these cases, the patient was unable to control earlyvirus replication and case fatality rates were very high even invaccinated individuals. A reevaluation of pathology records of patientswith smallpox has suggested that the cytopathic effect of variola viruswas a major cause of death from this disease.46

In most fatal cases of smallpox, the cause of death was difficultto pinpoint as organs such as the brain, lungs, heart, kidneys andliver were not severely damaged.7,36,37 Indeed, post-mortem examina-tion did not reveal significant lesions anywhere other than the skin ormucous membranes.7,47 Without the benefit of current clinical meth-ods used for evaluating pathology and disease, clinicians ascribed‘toxemia’ as a reason for death. Indeed, Dixon (1962)36 describes it asfollows: y‘I think one must recognize that the virus acts on a widerange of cells in many organs, interfering with function withoutnecessarily producing effects demonstrable by our current examinationtechniques. In our present ignorance of the cell changes, one can only saythat the patient dies from general toxemia’ (p. 88). This explanation isconsistent with our current understanding of damage caused by acytokine storm and sepsis.26 Furthermore, histopathological evalua-tion of lesions in both ordinary-type and hemorrhagic-type smallpoxrevealed signature hallmarks of inflammation such as dilation of bloodvessels, swelling of endothelial cells, adhesion of leukocytes, perivas-cular cuffing with lymphocytes, macrophages and polymorphonuclearcells, leukocyte infiltration into the tissue and fluid accumula-tion.7,47,48 Patients were reported to have depletion of intravascularfluid, leading to shock and renal failure and were described asresembling burn patients.49

Support for uncontrolled inflammation and sepsis being the causeof acute pathology and death in smallpox has come from a recentlyestablished non-human primate model of variola virus infection.35,50

Although this model does not represent the course of natural diseasein humans, it does mimic disease progression after secondary viremiaand infected animals show classical centrifugal lesions, coagulopathyand toxemia seen in end-stage hemorrhagic smallpox.35 Variola virusinfects monocytic cells and is disseminated via a cell-associatedviremia, resulting in high viral burdens and multi-organ failure.35

Using this model, the upregulation of inflammatory cytokines hasnow been demonstrated and a cytokine storm has been postulated asthe cause of pathology and lethality.35,50

LESSONS FROM ANIMAL MODELS OF POXVIRAL INFECTION

Our current understanding of immunity and immunopathology topoxvirus infections comes largely from studies on the response tovaccinia virus vaccination in humans and from animal studies usingrelated poxviruses, such as ectromelia, vaccinia, monkeypox andmyxoma viruses. As the outcome of a poxvirus infection is influenced

not only by the type of immune response mounted by the host butalso by the immune evasion mechanisms that the viruses have evolvedto subvert these responses, the most useful models are the ones thatstudy host response in the context of a natural infection (Table 1).

The mouse is an ideal model to study the interaction of poxviruseswith their host immune systems for several reasons. As the de factomodel of choice for many immunological studies, there are manyreagents available to elucidate host pathogen interactions. In addition,there are many well-characterized inbred strains as well as gene-deficient mice that allow for careful dissection of the key parametersinvolved in both immunity and immunopathology. The poxvirusesectromelia, cowpox and vaccinia virus are all regularly studied in thismodel. Vaccinia virus also infects mice and a broad range of otherspecies however, its natural host remains unknown.15,51 Similarly,cowpox virus, named for its ability to infect the teats of cows, hasrodents as a natural reservoir but can infect a broad range of otherspecies including cats, cows, zoo animals and humans.52–54 Theusefulness of vaccinia or cowpox virus infection in mice as a modelfor smallpox is limited as pathogenesis, disease progression andoutcome of infection are unlike that of variola virus. In contrast,ectromelia virus is only able to infect mice and, importantly, is anatural mouse pathogen.14,16,52 This model has been extensivelystudied and has provided useful insights into poxvirus pathogenesis.

The rabbit is another commonly used laboratory animal that issusceptible to poxvirus infection. Myxoma virus is a leporipoxviruswhose evolutionary and reservoir host are rabbits of the Sylvilagusgenus, where the virus causes a benign disease. Although the Europeanrabbit (Oryctolagus cuniculus) is not a reservoir host for any knownpoxviruses, myxoma virus causes a severe disease known as myxoma-tosis in these animals.21,22,24,55,56 The study of myxoma virus infectionin the rabbit allows for the comparisons of host virus interaction andoutcome of infection between its natural host and susceptible host.It further allows for identification and characterization of individualviral genes that result in the lethal phenotype observed in theEuropean rabbit.

The closest animal models for the study of human disease areprimate models. Many of the poxviruses that infect primates, such asmonkeypox, tanapox and yaba monkey tumor viruses, also have theability to infect humans.9 However, the clinically important humanpoxviruses, namely the variola virus and Molluscum contagiousumvirus do not naturally infect primates. The recently developed primatemodel of variola virus unfortunately requires unusually high virustitres, non-physiological inoculation routes and only mimics the endstages of disease.35,50 Similarly, monkeypox virus infection of non-human primate also requires high virus titres and non-physiologicalinoculation routes.57,58 Nevertheless, these studies have the advantageof being conducted in host systems that very closely resemble humans.

The ideal animal model of human poxvirus infections would be onewhereby infection occurs via a natural route with a low dose of viruscausing significant pathology. The host response in this model would

Table 1 Representative poxvirus infections

Virus Variola Ectromelia Myxoma Monkeypox

Genus Orthopoxvirus Orthopoxvirus Leporipoxvirus Orthopoxvirus

Reservoir host Humans Mice Rabbit (Brush) Rodents, squirrels

Zoonotic host None None Rabbits (European) Monkeys, humans

Pathology in reservoir host Mild to severe; smallpox Mild to severe; mousepox Benign cutaneous tumors Mild

Pathology in zoonotic host Not applicable Not applicable Severe; myxomatosis Mild to moderate severity; monkeypox


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closely resemble the response in humans. The pathology would closelymimic naturally occurring disease, and the model allows for manip-ulation of virus and host factors to determine the relative contributionof each to pathogenesis. As no one model fulfills all these criteria,a combination of models is required for an integrated approachto understanding poxvirus pathogenesis of clinically relevant diseases.In this review, we will focus on three animal models of poxvirusinfection that have been particularly important in informing ourcurrent understanding in this area. These are: ectromelia virus infec-tion of mice, myxoma virus infection of rabbits and monkeypox virusinfection in primates (Table 1).

Ectromelia virus infection of miceEctromelia and variola are closely related orthopoxviruses that arethought to have originated in rodents but over time each hascoevolved with its natural host, mouse and man, respectively. Bothviruses are infectious at very low doses and cause severe disease withhigh mortality rates.7,13,16,25 Although all orthopoxviruses are highlyconserved, sharing greater than 90% homology in the central 100 kbpregion of the genome,16 further specific similarities between mouse-pox, caused by ectromelia virus, and smallpox include virus replica-tion and transmission, cytokine responses39,50 aspects of pathologyand development of skin lesions in later stages of infection.7,59 Themousepox model25 is the most versatile in which the roles ofindividual components of innate and adaptive immunity have beeninvestigated. It has been extensively utilized to study virus–hostinteractions, genetic resistance to disease and viral immunol-ogy.39,59–71 Indeed, the mousepox model has been instrumental inestablishing critical roles of cell-mediated and antibody responses inthe control of poxvirus infections.

Host genetic factors are known to influence resistance or suscept-ibility to mousepox. Many inbred strains of laboratory mice have beencategorized as either resistant or susceptible to infection with ectro-melia virus. Both MHC and non-MHC genes determine resistance orsusceptibility. Strains such as A/J, BALB/c and DBA/2 exhibit highmortality to mousepox, whereas C57BL/6, C57BL/10 and 129 strainshave very low mortality, limited pathology and are classified resistant.Wild mice show variable susceptibility to mousepox.72

Studies crossing susceptible DBA/2 mice with resistant C57BL/6mice have identified at least four genetic loci that confer resistance tomousepox. The resistance to mousepox-1 (Rmp-1) locus maps to theNK gene complex;16,61 the Rmp-2 locus maps near the complement

component C5 gene;61 Rmp-3 locus is linked to the MHC and is alsogonad-dependent;60 and the Rmp-4 locus maps near the selectin genecomplex.61 Given the complexity of the host response and the immuneevasion strategies employed by the virus, we expect that there will bea number of other, as yet unidentified, host genes that are crucial forrecovery from poxvirus infection.

Similar to variola virus infection in humans, ectromelia virus causesan acute disease characterized by generalized viral spread and pathol-ogy in mice.25 Figure 2 outlines the progression of ectromelia virusinfection and development of disease. In mice infected with ectromeliavirus via the footpad, the virus replicates at the site of infection andreaches the draining lymph node as early as 8 h post-infection (p.i.).By 3–4 days p.i., free or cell-associated virus is transmitted via theefferent lymphatics and bloodstream to organs such as the bonemarrow, spleen and liver, where it replicates further. By about day 5p.i., virus from these organs is released into the blood, resulting insecondary viremia. At this stage, virus is detectable in most tissuesincluding the skin, where by day 9 p.i. it causes pock lesions thatulcerate within a few days. This is the most infectious stage and canlast up to about day 21 p.i.

In susceptible strains of mice, such as BALB/c, death occurs as earlyas day 7 p.i., owing to extensive necrosis of major organs, particularlythe liver and spleen, as a result of massive virus replication.These animals die before the development of any pock lesions.Resistant strains, such as C57BL/6, are able to control ectromeliavirus well and recover without the development of any lesions.Animals with intermediate susceptibility develop lesions as describedabove and a proportion of the animals recover from infection whereasothers succumb to disease.

Recovery of mice from ectromelia virus infection requires theeffector functions of NK cells, CD4 and CD8 T lymphocytes, Blymphocytes, macrophage subsets, nitric oxide and interferons(IFNs).39,59,63–66 The resistant strains of mice, such as C57BL/6,generate a strong T-helper 1 type cytokine response and potentantiviral cytotoxic T lymphocytes (CTL) and antibody responses,whereas these responses are either delayed and weak or lacking insusceptible strains of mice.39 These components of the host responsecombine to effectively control virus in resistant strains. Susceptiblestrains, such as A/J or BALB/c, have poor, delayed or absent IFN-g andantiviral CTL responses and succumb to infection.

The cause of pathology in mousepox is not well understood,although death in fatal cases is associated with high virus titers. The

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Skin: invasion, multiplicationRegional lymph node: multiplication

Blood stream: primary viremia

Spleen and liver: multiplication, necrosisBlood stream: secondary viremia

Skin: focal infection, multiplication

Foot swelling: primary lesion

Early rash: papules

Severe rash: ulceration

Day

Incu

batio

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a b

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Host-virus interaction

Figure 2 Mousepox: disease progression and outcome.


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cytopathic effects of virus are thought to be responsible for severenecrosis of liver and spleen, observed in susceptible animals.73,74

Although virus is detectable in these organs as early as a few daysp.i., notable necrosis in the liver and spleen does not occur untilwithin 24 h of death.73,74 Curiously, even significant leukocyte infiltra-tion into the liver is not observed till quite late in infection despite thepresence of substantial levels of viral antigen. It is likely that ectromeliavirus-encoded immune evasion molecules effectively delay the localrecruitment of leukocytes into the foci of infection. The contributionof immune mechanisms, such as a cytokine storm, to pathogenesisand death has not yet been investigated in mousepox.

Myxoma virus infection of European rabbitsStudy of myxoma virus infection in rabbits is a unique way to explorethe effect of viral immunomodulation on the outcome of infection inits natural evolutionary host (Silvilagus species) in which the viruscauses a relatively benign disease, compared with its susceptible host(Oryctolagus cuniculus, or the European rabbit) where it causes adevastating disease, known as myxomatosis, with nearly uniformmortality.75 Interestingly, myxoma virus infection produces a primarylesion in both types of lagomorphs; however, unlike in the susceptiblehost, in the European rabbit it progresses to secondary fulminatinglesions combined with immunosuppression. The severity of myxoma-tosis in laboratory-bred European rabbits was attributed to the lack ofcoevolution between pathogen and host. In Australia, where myxomavirus had been introduced over 50 years ago for biological control ofthe feral European rabbit population, the highly virulent myxomavirus first released is now attenuated and infection by field strains nolonger cause lethal myxomatosis.76 Contemporaneous to the evolutionof virus attenuation, feral rabbits have also evolved resistance so theyare no longer as susceptible to myxomatosis as laboratory-bred strains,indicating a coevolution of both host and virus towards dampeneddisease severity and pathology.77

Myxoma virus exists as two distinct types that have been isolatedfrom different geographic locations. Originally, myxoma virus wasdescribed in South America and was isolated from the tapeti or brushrabbit (S. brasiliensis); however, more recently myxoma virus has alsobeen described in the wild in California in the indigenous rabbitspecies (S. bachmani, S. nuttallii and S. audubonii).77 Strains of virusfrom each geographical region are well adapted to their natural orevolutionary hosts and cause a benign disease. Although the bulkof research into myxoma virus pathogenesis has concentrated onthe South American strains, there are some interesting differences inthe virulence between the South American and Californian strains, inboth evolutionary and susceptible rabbit hosts, which could shed lighton how the virus causes pathology.

In their evolutionary Silvilagus hosts, both South American andCalifornian strains of myxoma virus cause a local and benign diseasethat resolves within 10–40 days and results in a protective immunityagainst re-infection.77 Although detailed studies into this type ofmyxoma virus infection are lacking, it is reasonable to hypothesizethat the virus has coevolved sufficiently with the Silvilagus speciesimmune system to dampen the inflammatory response to initialinfection to a level that allows establishment of viral replication atthe primary site of infection. This would allow viral transmissionbetween hosts by arthropod vectors and the host immune response tocontrol virus replication and spread, thus allowing both virus and hostto survive. The degree of coevolution between virus and host withina geographic location is highlighted by the observable, but subtle,differences in the primary lesions when the South American myxomastrain is used to infect Californian leporids such as S. bachmani,

S. nuttallii and S. audubonii78 when compared with native SouthAmerican S. brasiliensis.

In European rabbits on the other hand, isolates of both SouthAmerican and Californian strains cause severe pathology and acutedisseminated disease. Most studies have been carried out using SouthAmerican isolates of myxoma virus, including Moses, SLS (standardlaboratory strain) and Lausanne strains. Infection is characterized byprominent primary and secondary cutaneous lesions, swollen eyelids,ears, nose and head, as well as mucopurulent discharge from the eyesand nose. In addition to the robust viral replication at the primary siteof infection, there is spread of virus to the draining lymph node within1 day of infection.78 Virus is then rapidly disseminated to other tissues,including distal skin sites, most likely by virus-infected leukocytes.There is extensive apoptotic death of cells of the immune systemand there is a resulting severe immunosuppression. Infected rabbitsoften contract supervening Gram-negative bacterial infections of therespiratory tract.78,79

California strains of myxoma virus cause an even more severeform of myxomatosis in European rabbits compared with the SouthAmerican isolates. Rabbits infected with these strains usually diebefore the appearance of secondary lesions (by days 7–8 comparedto days 10–12 for South American strains), show none of the classicsymptoms of myxomatosis described previously and exhibited addi-tional signs of central nervous system involvement, including muscletwitching, hypersensitivity to stimulation, depression and coma.Initial post-mortem analysis indicated presence of myxoma virus inthe brain, but recent studies have failed to find significant amountsof virus in any tissue.78,80,81 The severity of the response to Californianmyxoma virus suggests that serious immunopathology owing to acytokine storm may be a significant contributing factor to the death inthese animals, in a similar manner to severe variola virus infection.Although there have been extensive studies on the immunomodula-tory proteins of myxoma virus, a topic that is well reviewed else-where,21,24,55,56,76,82 little is known about the mechanisms involved inthe dramatic differences in pathology and disease outcome acrossleporid species. However, it is clear that both virus and host factorscontribute to the manifestation of disease caused by myxoma virus.

The deliberate release of the highly virulent SLS strain of myxomavirus from Brazil, in an effort to control the feral European rabbitpopulation in Australia in 1950, has provided a unique opportunity totrack the subsequent spread of the virus and its co-evolution with anew host. The introduction initially resulted in drastic reduction inrabbit numbers. However, investigations found that dominant fieldstrains of myxoma virus were significantly attenuated.14,76 Interest-ingly, the feral field rabbits also evolved resistance to infection so theywere no longer as susceptible to myxomatosis as the laboratory-bredstrains. Although infection of MHC-II-positive dendritic cells wasnoted at the site of infection for all myxoma virus strains, dependingon virus and host strain combinations, differences in the ensuinginflammatory response have been noted.83 In laboratory rabbits, infec-tion with the SLS strain resulted in lower inflammatory responses,dominated by polymorphonuclear leukocytes, as well as extensivedepletion and apoptosis of lymphocytes within lymph nodes andspleen.83 In contrast, infection of resistant field rabbits with either theSLS strain or the attenuated field strain Uriarra (Ur) resulted in anintense inflammatory response, dominated by mononuclear cells, andsignificantly less apoptosis and little lymphocyte depletion. This studyunderscores the importance of the initial inflammatory reaction andlater T-cell response in the successful resolution of myxoma virusinfection. A follow-up study examined the effect of the more virulentCalifornian strains of myxoma virus in both laboratory and resistant


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field rabbits.81 As expected, the field rabbits were also more resistant tosome of the pathological features of the Californian MSW strain, suchas massive lymphocyte apoptosis in lymphoid tissues; however, theystill succumbed to infection. Thus, although there is a spectrum ofvirulence observed in myxoma virus infections, ranging from mortal-ity to recovery and survival, the outcome clearly depends on acombination of virus and host factors.

The South American-derived Lausanne strain of myxoma virus hasbeen fully sequenced and preliminary mapping of the CalifornianMSW strain has also been completed.84,85 The major sequencedifference in the Californian strain, compared with the South Amer-ican strain, was the duplication within its terminal inverted repeats, offive complete (and one partial) open reading frames.85 The duplica-tion of several virulence genes in the Californian strain, which arepresent only in single copies in the South American strain, mayexplain the significant difference in the pathology and disease outcomeresulting from the infection of European rabbits with each of theseviruses. The identification of candidate genes provides a uniqueopportunity to elucidate specific parameters and mechanisms involvedin the pathogenesis of myxomatosis.

Monkeypox virus infection of primatesMonkeypox virus belongs to the genus Orthopoxvirus and has a broadhost range, producing lesions in most common laboratory animals,non-human primates and humans.2,86–88 Although the reservoir formonkeypox virus is not known, survey of wild animal species of thesecondary forest of Central Africa suggests that rodents or squirrels arethe natural hosts.89,90 Non-human primates and humans are thoughtto be incidental hosts and are zoonotically infected.91

Monkeypox in primates was initially described in 1958 as a diseaseof Java macaques held in captivity at the State Serum Institute inCopenhagen, Denmark. Similar outbreaks among captive primateswere subsequently reported at several other centers.2 In these out-breaks, infection resulted in a smallpox-like disease in monkeys andyet did not infect human handlers, despite close contact with sickanimals. Primates of different species exhibit a range of clinicalfeatures and differing susceptibilities to monkeypox with outcomesranging from subclinical infection in some species to severe illness andhigh mortality in others.2,92 Recently, a model using cynomolgusmonkeys infected with monkeypox virus has been developed to mimicsmallpox in humans57 and this model provides a system in which totest vaccination and antiviral therapies. Conventional vaccinia immu-nization protects monkeys from monkeypox infection,93 and new,more attenuated vaccine strains are similarly effective.94,95 In addition,antiviral compounds such as cidofovir have been tested in this model,showing superior effectiveness in preventing monkeypox to ‘ringvaccination’, which was the standard procedure given to humanpatients during smallpox outbreaks.96

The first human monkeypox infection was described in 1970.97–100

Until the recent outbreak of 2003 in USA,101 all human monkeypoxcases had occurred in tropical rain forests of West and Central Africawith the largest outbreak in 1996–1997 in two regions of KasaiOriental, Democratic Republic of Congo.102–106 The clinical diseaseitself is virtually indistinguishable from ordinary-type smallpox and,as for other members of the genus Orthopoxvirus, protection frommonkeypox is provided by immunization with another orthopoxvirussuch as vaccinia virus. For this reason, it is believed that the smallpoxeradication campaign, with mass vaccinations, had kept human casesof monkeypox in check. However, the emergence of a youngerunvaccinated population, born after the eradication of smallpox,may be an important factor in the reemergence of monkeypox in

humans. Fears of further threats from human monkeypox werecompounded by the outbreak of the disease in the USA in 2003.101

This represented the first cluster of human infection outside Africa,although it was traced back to a shipment of Gambian rats from WestAfrica. No patients died in the US outbreak101 and this contrasts withcase fatality rates of about 10% (mostly limited to children) reportedfor some African outbreaks.7

Human monkeypox may clinically resemble smallpox but itsepidemiology is very different. Monkeypox mostly affects childrenunder 10 years of age and it occurs sporadically or in small clusters,indicating that person-to-person spread is inefficient.2 However, thereis some suggestion that the secondary transmission rates, calculatedfrom outbreaks in the Democratic Republic of Congo, have increasedover the last three decades.107 Person-to-person transmission in mosthuman infections occur through close contact with infected animals.

Strains of monkeypox virus fall into two large clades, strainsisolated from Central Africa and strains isolated from WesternAfrica.108 Western African strains have been shown to be significantlyless virulent than the Central African strains, and in general all strainsare less virulent than variola virus. Interestingly, unlike the CentralAfrican strains, the less virulent Western African strains of monkeypoxdo not encode a functional inhibitor of complement enzymes.109

Indeed, the cofactor activity of the inhibitor of complement enzymesencoded by a central African strain is significantly less efficient thanthe activity of its counterpart encoded by variola virus.110 This may beone factor in determining differences in pathology and disease severitycaused by each of the above viruses. The main pathological differencebetween smallpox and monkeypox in humans is lymphadenopathy,seen in 90% of unvaccinated monkeypox patients, but not a commonfeature of smallpox.2,107 The failure to control the inflammatoryresponse through complement control proteins, as well as the abilityof the virus to stimulate robust immune responses in lymph nodes,could explain why monkeypox virus is generally less fatal than variolavirus infections.

CONCLUDING REMARKS

The elegant dance played between the immune system of the host andthe immunomodulatory proteins of poxviruses is an excellent exampleof the evolutionary co-dependence of pathogen and host. The host hasevolved complex defense mechanisms to keep the destructive nature ofinvading microorganisms at bay and to clear them effectively whileprotecting against further attack. As a counterpoint, pathogens areconstantly under pressure to evade the immune response of their hostand, poxviruses in particular, actively manipulate the host immunesystem to survive and replicate.111

For the time being, we have won the battle against smallpox inhumans, but with the potential threat of deliberate or accidentalrelease of variola virus, we cannot afford to be complacent. Indeed,there is also risk associated with the emergence of strains of mon-keypox virus that are more virulent or have higher person-to-persontransmission rates. Since the eradication of smallpox almost 30 yearsago, mass vaccinations have been discontinued, leaving the youngermembers of the human population unvaccinated and the older oneswith waning immunity to poxviral infections. Current vaccines haveunacceptable levels of adverse effects and there are increasing numbersof immunocompromised individuals affected with AIDS and otherconditions. Although safer vaccines and antivirals against orthopox-viruses are currently being developed,96,112,113 a better understandingof the pathogenesis of disease and virus host interaction remainscritical in the development of new strategies for effective interventionin established infection.


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Lessons learned from poxviruses have helped develop new andexciting therapies for human disease. As vaccinia virus has alreadybeen used extensively as a vaccine, with relatively good safety recordfor attenuated strains, it is a front-runner as a vector for use in newvaccination strategies and gene therapy.114–118 Another possible areathat shows potential is the use of poxviruses as therapeutic oncolyticagents.119,120 The ability of viral proteins to manipulate and dampeninflammatory responses is of interest in many human diseases whereinflammation is the major cause of the disorder, such as arthritis andarthrosclerosis.121–125 Poxvirus proteins as immunomodulators havetherefore been the target of new drug discovery.126,127 One exampleis the poxvirus-encoded serine-protease inhibitor, Serp-1, that hasanti-inflammatory properties and is currently undergoing phase IIclinical trials for treatment of vascular inflammation in patientswith acute coronary syndrome.128

Elucidation of the interactions between poxviruses and their host isallowing a better understanding of the delicate balance that is struck toensure the mutual survival of both. Knowing the mechanisms criticalfor virus control and resolution of infection, and those involved inimmunopathogenesis, may allow us to intervene in such a way thatdissociates the two processes to tip the balance in favor of the host.Moreover, we may be able to exploit the strategies that these pathogenshave evolved over many millennia, to minimize pathology and diseaseseverity, not only in unrelated infectious disease but also in otherconditions where immune and inflammatory responses need to becontrolled.

ACKNOWLEDGEMENTSM Stanford is supported by a Postdoctoral Fellowship provided by the Pamela

Greenaway Kohlmeier Translational Breast Cancer Research Unit of the London

Regional Cancer Program. G McFadden holds a Canada Research Chair in

Molecular Virology and is an International Scholar of The Howard Hughes

Medical Institute. This work was supported by grants from the National Cancer

Institute of Canada and Canadian Institutes of Health Research to G McFadden

and from the Howard Hughes Medical Institute to G Karupiah and National

Health and Medical Research Council of Australia to G Karupiah and

G Chaudhri. We thank John Barrett and Steven Nazarian for critical reading

of the manuscript and for their helpful comments.

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Immunopathogenesis of poxvirus infections: forecasting the impending storm

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