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4 Animal Models Using Variola and Other Orthopoxviruses I n 1999, there were no suitable animal models for variola. This l ed the IOM committee at that time to draw the following c onclusion: The existence of animal models would greatly assist the develop- ment and testing of antiviral agents and vaccines, as well as studies of variola pathogenesis. Such a program could be carried out only with live variola virus. Since 1999, some progress has been made in developing animal models. However, it should be emphasized that there is still no animal model that satisfactorily recapitulates all relevant aspects of human smallpox. Although the nonhuman primate model described by Jahrling and colleagues (2004) offers some features that are suggestive of later-stage, fulminant human small- pox, the ability of this model system to mimic the wide spectrum of human disease manifestations and pathophysiology remains uncertain. This chapter describes efforts to use animal models to study variola infection and disease in humans. Although such efforts have included use of the variola virus, the inability to infect most animal species with variola has resulted in attempts to use vaccinia, cowpox, and mousepox; monkeypox; and myxoma virus. VARIOLA As discussed in Chapter 3, there are no animal reservoirs for variola virus in nature, and most animal species cannot be infected in the labora- 

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0 LIVE VARIOLA VIRUS tory. Attempts to develop nonhuman primate models for variola infec- tion and disease in the 1960s met with only limited success (Hahon and Wilson, 1960; Hahon, 1961; Lancaster et al., 1966; Westwood et al., 1966). Although these attempts reportedly produced rash and systemic illness in various nonhuman primate species, disease was inconsistent and variable in severity with each of the routes of inoculation and host species examined. Of note, the reports described primary and secondary viremias and a mild, brief illness with fever and rash in Macaca irus and Macaca mulatta exposed to variola virus via the aerosol route (Hahon and Wilson, 1960; Lancaster et al., 1966; Westwood et al., 1966). Motivated by the potential utility of a realistic and consistent model with which to test drugs and vaccines intended for possible use against smallpox in humans, CDC revisited the possibility of developing a model of human smallpox in nonhuman primates at the beginning of this decade (Jahrling et al., 2004). After limited unsuccessful efforts to produce consistent, severe disease with an aerosol device, the investigators turned to an intravenous route of infection in cynomolgus macaques. Lethal disease with systemic features reminiscent of late-stage severe human smallpox, including skin vesicles and pustules, was achieved with doses of 109 plaque-forming units. Lesser doses, as low as 106 plaque-forming units, produced less severe dis- ease and fewer skin lesions in a dose-dependent but less consistent manner. As described, this macaque model appears to truncate the natural course of variola infection, bypassing the early respiratory tract replication of the virus, primary viremia, and early clinical phases of human smallpox. On the other hand, aspects of the systemic pathology resembled some of the reported features of lethal, hemorrhagic smallpox in humans as described in the historical record. But because the route of infection and the dose differed substantially from those of the natural setting, there is reason to believe that the mechanisms of pathogenesis in this model may vary from those that took place during natural disease. For example, the initial instantaneous viremia preempts a prodromal period and circumvents early local replication of virus in the respiratory tract. An adequate assessment of these issues has not been possible, in part because human smallpox was eradicated before modern investigatory tools became available and also because relatively few studies of the current nonhuman primate model have been published. Nonetheless, this nonhuman primate model was subsequently used to test therapeutics and vaccines (see Chapters 6 and 7, respectively), with the understanding that it sets a stringent, perhaps overly demanding standard for efficacy. Proposed improvements of this macaque model include using an intratracheal route of inoculation to achieve a consistent and more real- istic course of disease. Elucidation of features of variola pathogenesis, a secondary goal in the development of this macaque model, has been achieved using traditional

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 ANIMAL MODELS USING VARIOLA and more modern tools (Rubins et al., 2004). In addition to a variety of histology-, hematology-, chemistry-, and immunology-based measurements, genome-wide features of nonhuman primate gene expression were mea- sured in serial peripheral blood specimens using DNA microarrays. The latter revealed gene transcript abundance patterns indicative of prominent interferon and cell proliferation responses, and notable for the absence of responses associated with tumor necrosis factor alpha and transcription factor NF-kappaB, which would otherwise be typical of many acute over- whelming infections. These and other findings enhance understanding of the mechanisms responsible for variola-associated morbidity and mortality, as well as possible new targets for therapeutic intervention. VACCINIA, COWPOX, AND MOUSEPOX Vaccinia virus is perhaps the most widely used poxvirus in animal models for studying variola virus infection because of its ready availability and extensive knowledge base, and the susceptibility of laboratory rabbits and mice to vaccinia infection. In rabbits in particular, infection with a rabbit-adapted strain of vaccinia virus—rabbitpox virus—generates a dis- ease that recapitulates some of the important features of smallpox, includ- ing transmission between hosts by the aerosol route and a generalized rash (Adams et al., 2007). Laboratory mice can be lethally infected with several strains of vac- cinia virus introduced intranasally or by aerosol. This mouse model yields dose-dependent lethality, with up to 100 percent of animals dying, and can be refined to result in a sublethal disease course in which disease severity is quantified by weight loss followed by recovery over a period of 2–3 weeks. Although this model is characterized by a more rapid disease onset—around 3 days—than is seen with smallpox and is not characterized by a rash, it has been used to compare the efficacy of novel and traditional vaccines and to conduct research on antiviral therapies (Bray et al., 2000; Smee et al., 2001; Belyakov et al., 2003; Hooper et al., 2003; McCurdy et al., 2004; Wyatt et al., 2004; Law et al., 2005; Phelps et al., 2005, 2007; Abdalrhman et al., 2006; Ferrier-Rembert et al., 2007). Inbred mice are also useful in models for cowpox (Bray et al., 2000; Ferrier-Rembert et al., 2007) and mousepox (Fenner, 1949) infection, and in both cases, a lethal challenge is obtainable. The mouse/cowpox model is broadly similar to that of mouse/vaccinia, and has value in extending the range of orthopoxviruses that can be used in a single host for the evaluation of measures that may control orthopoxvirus infection. The mouse/mousepox model is somewhat different in that it is lethal at very low doses and is restricted to a single host, and its severity can be viewed as generating a model that is more relevant to variola infection in humans. However, there

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 LIVE VARIOLA VIRUS are significant differences between the disease course of mousepox in mice and most other acute orthopoxvirus infections. Mousepox is characterized by large ulcerating lesions rather than the discrete maculo-papular rash charac- teristic of smallpox, and extensive liver damage is seen as well (Jones et al., 1997). Although the disease severity and host restriction make the mousepox model attractive for testing control measures, this model must be treated with caution given this differential pathology. However, the ability to perform challenge experiments with vaccinia, cowpox, and mousepox viruses in a single species when protection against all three viruses can be achieved with traditional smallpox vaccine adds considerably to the confidence with which extrapolations from these models to human smallpox can be made. MONkEyPOX Monkeypox is perhaps the most relevant orthopoxvirus with regard to nonvariola animal models for smallpox. Monkeypox virus causes a some- times fatal disease in humans whose clinical features and course are similar to those of smallpox. The use of this virus in the laboratory requires bio- safety level (BSL)-3 conditions in the United States, although the disease is prevented in humans and animals with smallpox vaccine. The classification of monkeypox virus as a select agent in the United States further compli- cates and hinders work on this virus. As its name implies, monkeypox virus causes disease in nonhuman primates, and it has been used experimentally to cause disease in macaques that is similar to smallpox and monkeypox in humans, and to evaluate pos- sible countermeasures against smallpox (Earl et al., 2004; Stittelaar et al., 2005). Monkeypox does not cause significant disease in laboratory mice; however, the recent discovery that it causes disease in North American prairie dogs has led to the examination of other, related ground-dwelling squirrels, and there are now several rodent-based models for orthopoxvirus disease using this virus (Tesh et al., 2004; Hutson et al., 2007). The most important feature of monkeypox is not its similarity in humans to smallpox. Rather, monkeypox is a public health problem in its own right. At least 88 cases with 3 fatalities occur annually in endemic regions of central Africa (Hutin et al., 2001, Levine et al., 2007; Parker et al., 2007). Public health issues pertaining to monkeypox are beyond the scope of this study. Nevertheless, the committee notes that human monkeypox deserves attention because of its toll in endemic areas, and the licensure of therapies for the disease would provide a tangible benefit for a large at-risk population. Moreover, lessons learned from the development of licensed medical countermeasures for human monkeypox might address many of the uncertainties associated with extrapolation among different orthopoxviruses in animal models.

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 ANIMAL MODELS USING VARIOLA MyXOMA VIRUS Animal models with relevance to smallpox are generally restricted to orthopoxviruses—members of the same genus as variola itself. One nonorthopoxvirus is worthy of consideration, however, because of its pathogenicity and the body of research based on its use. Myxoma virus, a poxvirus of the leporipoxvirus genus, is a virus of New World rabbits of the genus Sylvilagus, in which it causes an infection that is almost asymptomatic and is nonlethal. When introduced to European Oryctolagus rabbits, myxoma virus causes a fulminant ulcerating infection known as myxomatosis, with a very high mortality rate (Stanford et al., 2007). The severity of disease in myxoma virus-infected European rabbits invites par- allels with smallpox, and although the two viruses differ significantly in pathology, so, too, do smallpox and models using vaccinia, cowpox, and mousepox. Like orthopoxviruses, myxoma virus produces a number of proteins that interact with elements of the immune system. Many of the lessons learned from studies with myxoma virus directly inform and influence understanding of orthopoxviruses and vice versa. However, myxoma virus is sufficiently different from orthopoxviruses that smallpox vaccine does not protect European rabbits from myxomatosis, and ST-246, a prom- ising candidate antiviral drug for treatment of orthopoxvirus diseases, including smallpox, has no activity against myxoma virus, which lacks the specific target of the drug in orthopoxviruses. Consequently, data from animal models using myxoma virus cannot be extrapolated to smallpox in humans. CHIMERIC VIRUSES While the extreme host restriction of variola virus greatly facilitated the smallpox eradication campaign, it also hampered research because no ani- mal model using variola was available. In the 1960s, attempts were made to address this gap by constructing chimeric viruses from variola and either cowpox or rabbitpox (a rabbit-adapted strain of vaccinia virus) viruses (Bedson and Dumbell, 1964a,b). These chimeric viruses were constructed by coinfection of cell lines with the two viruses and plaque purification of random recombinants between the two. At the time, the random nature of the resulting recombinant viruses and the inability to fully sequence these recombinants restricted their utility for research into the pathogenesis of variola infection. Moreover, the advisability of adapting a human-only virus to growth in animals that could thereby become potential reservoir hosts was questioned. These chimeric viruses, which are stored under BSL-4 containment at CDC, were generated with methods that yield random

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 LIVE VARIOLA VIRUS TABLE 4-1 Usefulness of Animal Models and Human Infections for Understanding the Pathogenesis of Variola and Other Orthopoxviruses and for Developing Therapeutics and Vaccines Small Animal Nonhuman Primate Human Orthopoxviruses Contribution to Limited usefulness: Moderately useful other than overall some potential to (especially vaccinia) monkeypox and understanding of help in identifying for obtaining variola pathogenesis of useful interventions information about infections caused by against smallpox in antiviral activity of humans,a but use of poxviruses; candidate drugs and vaccines.b provides some nonhuman primates evidence of antiviral for studies of activity of drugs variola have against poxviruses priority. related to variola, but not variola. Monkeypox Limited usefulness: Moderately useful Most useful in some potential to for obtaining suggesting likely help in identifying information about benefits from useful interventions antiviral activity of candidate therapeutics against smallpox, candidate drugs and and vaccines against but other vaccines. variola in the human population.d approaches are more important.c Variola Not an available Most useful in Not an available option for suggesting likely option for developing developing benefits from therapeutics or therapeutics or candidate vaccines. vaccines. therapeutics and vaccines against variola in the human population. aDual infection of nonhuman primates with simian immunodeficiency virus (SIV) and vaccinia produces disease with features that mimic human smallpox. bDisseminated vaccinia infection in humans produces disease with some features reminiscent of smallpox. cMonkeypox virus infection of ground squirrels has been used to assess monkeypox vaccines. dThis cell, monkeypox in humans, refers to naturally occurring disease. The study of this dis- ease might provide an opportunity to assess diagnostics, therapeutics, and vaccines for their utility in both monkeypox and variola.

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 ANIMAL MODELS USING VARIOLA recombinations of the parent virus genomes and have not been character- ized by sequencing. Furthermore, while studies with these recombinants might contribute to understanding of variola pathogenesis, such investiga- tions could not substitute for those using variola virus and would have to be carried out in BSL-4 facilities that should be used instead to support experiments with variola virus that are essential for developing smallpox therapeutics and vaccines. USEFULNESS OF VARIOUS MODELS Table 4-1 summarizes the usefulness of animal models and human infection with monkeypox or vaccinia for understanding the pathogenesis of variola and other orthopoxviruses and for developing therapeutics and vaccines. Although some of these approaches are more useful than other, none is ideal in recreating the equivalent of human smallpox. REFERENCES Abdalrhman, I., I. Gurt, and E. Katz. 2006. Protection induced in mice against a lethal orthopox virus by the Lister strain of vaccinia virus and modified vaccinia virus Ankara (MVA). Vaccine 24(19):4152–4160. Adams, M. M., A. D. Rice, and R. W. Moyer. 2007. Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox. Journal of Virology 81(20):11084–11095. Bedson, H. S., and K.R. Dumbell. 1964a. Hybrids Derived from the Viruses of Alastrim and Rabbit Pox. Journal of Hygiene (London) 62:141–146. Bedson, H. S., and K. R. Dumbell. 1964b. Hybrids derived from the viruses of variola major and cowpox. Journal of Hygiene (London) 62:147–158. Belyakov, I. M., P. Earl, A. Dzutsev, V. A. Kuznetsov, M. Lemon, L. S. Wyatt, J. T. Snyder, J. D. Ahlers, G. Franchini, B. Moss, and J. A. Berzofsky. 2003. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proceedings of the National Academy of Sciences of the United States of America 100(16):9458–9463. Bray, M., M. Martinez, D. F. Smee, D. Kefauver, E. Thompson, and J. W. Huggins. 2000. Cidofovir protects mice against lethal aerosol or intranasal cowpox virus challenge. Journal of Infectious Diseases 181:10–19. Earl, P. L., J. L. Americo, L. S. Wyatt, L. A. Eller, J. C. Whitbeck, G. H. Cohen, R. J. Eisenberg, C. J. Hartmann, D. L. Jackson, D. A. Kulesh, M. J. Martinez, D. M. Miller, E. M. Mucker, J. D. Shamblin, S. H. Zwiers, J. W. Huggins, P. B. Jahrling, and B. Moss. 2004. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature 428:182–185. Fenner, F., 1949. Mouse-pox (infectious ectromelia of mice): A review. The Journal of Immu- nology 63: 341–373. Ferrier-Rembert, A., R. Drillien, B. Meignier, D. Garin, and J.M. Crance. 2007. Safety, immunogenicity and protective efficacy in mice of a new cell-cultured Lister smallpox vaccine candidate. Vaccine 25(49):8290–8297. Hahon, N. 1961. Smallpox and related poxvirus infections in the simian host. Bacteriological Reviews 25:459–476.

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