{"paper_id":"60545621-8912-4e81-abe9-9b975d0f4b0f","body_text":"The genus  Orthopoxvirus  (OPXV) comprises several species including  Variola virus  (VARV),\n Monkeypox virus  (MPXV),  Cowpox virus  (CPXV) and  Vaccinia virus  (VACV) that\nare all, more or less, pathogenic for humans. Fortunately, the causative agent\nof smallpox, VARV, has successfully been eradicated by a worldwide\nvaccination campaign led by the World Health Organization (WHO) (Fenner,\n1988; World Health Organization, 1980). On the recommendation of the WHO,\nthe vaccination program was stopped in the 1980s because of severe side\neffects of the vaccine. The number of people lacking immunity against\nsmallpox and other zoonotic OPXV infections is therefore increasing\n(Shchelkunov, 2013). Consequently, a potential biowarfare attack with\nsmallpox would hit a nearly unprotected population. In addition, the\nzoonotic potential of orthopoxviruses, e.g., the increasing incidence of\nhuman monkeypox in the Democratic Republic of the Congo (Rimoin et al., 2010),\nis of a growing concern. The 2003 outbreak in the USA caused by prairie dogs\ninfected by imported exotic pets from Africa clearly showed that monkeypox\nis not restricted to Africa and can be transmitted at any time to any place\n(Centers for Disease, C. a. P., 2003; Reed et al., 2004). So\nfar, no licensed antiviral treatment for poxvirus infections is available.\nThere are two vaccines with irregular disposability. In the USA the\nsecond generation vaccine ACAM2000 ®  has been\nlicensed since 2007 ( http://www.fda.gov/ohrms/dockets/ac/07/briefing/2007-4292b2-02.pdf ), and\nImvanex ® , based on the modified vaccinia virus Ankara, received a\nmarketing authorization by the European Commission in 2013. Nevertheless, in\nlight of increasing numbers of zoonotic infections with different\northopoxviruses new vaccines and therapeutic agents are urgently needed.\nBoth have to be tested in adequate animal models. Since small animal models\nhave considerable limitations regarding disease pathology and\npharmacokinetics, animal models with nonhuman primates (NHPs), the closest\nrelatives to humans, are essential.\nOver the last decades several animal models for OPXV were developed in NHPs\n(Schmitt et al., 2014). Unfortunately, none of the animal models fulfills\nall the criteria needed and all have limitations (Hutson and Damon, 2010;\nSafronetz et al., 2013). Based on the Food and Drug Administration animal efficacy rule, new drugs and\nvaccines must be tested in more than one species and one of the species\nshould be a nonhuman primate animal model (Snoy, 2010).\nThe current nonhuman primate model for smallpox is the intravenous (i.v.)\ninoculation of MPXV, which causes a fulminant disease with many similarities\nto that of the human disease. A great disadvantage of the intravenous\ninfection route is that it does not mimic the natural route of smallpox\ntransmission, which occurs through close contact or inhaled aerosols. Key\nevents of a natural pox infection such as the alteration of the upper respiratory\ntract, a primary viremic phase and prodromal phases are skipped.\nNevertheless, these models cause a systemic disease with mortality rates of\nup to 100 % and can be used to evaluate the efficacy of anti-OPXV\ntherapeutics (Huggins et al., 2009) and vaccines (summarized in Schmitt et al., 2014).\nTo mimic the natural route of infection, respiratory models using MPXV,\nwhich often causes fibrinonecrotic bronchopneumonia that resembles the human\nMPXV and smallpox disease course, were developed (Goff et al., 2011; Johnson\net al., 2011a; Zaucha et al., 2001). However, the work with this animal\nmodel is complicated because of safety restrictions. The experiments must be\nperformed in a biosafety level (BSL)-3 containment facility. Furthermore, the\nVARV animal model, a suitable model for hemorrhagic smallpox used in some\ndrug efficacy studies (Huggins et al., 2009; Mucker et al., 2013) as well as\nfor pathogenesis studies (Wahl-Jensen et al., 2011), is restricted to two\nBSL-4 laboratories worldwide (Centers for Disease Control and Prevention, USA; and State Research Center of Virology\nand Biotechnology, Russia). Thus, alternative models are needed, and those\nbased on CPXV, which is classified as a BSL-2 pathogen, are increasing in interest.\nCPXV has the broadest host range of all OPXV. Compared to MPXV or VARV,\nresearch with CPXV can be done under median safety conditions. Cynomolgus\nmacaques have been infected intravenously and intrabronchially with CPXV to\nstudy pathogenesis (Johnson et al., 2011b; Smith et al., 2012). Small-particle aerosol inoculation in rhesus macaques resulted in a severe\nrespiratory disease (Johnson et al., 2015). Recently, we developed a\nnonhuman primate model based on marmosets experimentally infected with the\ncalpox virus that belongs to the  Cowpox virus  species (Kramski et al., 2010).\nCommon marmosets are highly susceptible to the calpox virus and they can be\ninfected experimentally via an intravenous or intranasal (i.n.) route\n(Mätz-Rensing et al., 2006, 2012). The intranasal route of infection\nresembles the natural infection route of smallpox and is therefore a\nsuitable model for the validation of therapeutics and vaccines. A\ndisadvantage of this model is that some species-specific reagents for\nanalyses are not available yet. The investigation of the immune\nand the inflammatory response is especially impeded as there is little\nknowledge on inflammatory markers in this species. In contrast, a wide range\nof commercial systems to investigate the inflammatory response and the\ncytokine and chemokine profile in the rhesus monkey model is available.\nWe therefore wanted to investigate whether the fulminant disease of\nmarmosets following infection with the calpox virus as described above can\nbe reproduced in rhesus monkeys. If successful this could represent an\nalternative model allowing for extended functional immunological studies on\northopoxvirus pathogenesis. Based on the assumption that older rhesus\nmonkeys might be more susceptible to calpox infection because of waning\nimmune competence, we inoculated aged monkeys with the calpox virus by the same\nroutes as described for marmosets (Kramski et al., 2010). Our results showed\nthat, compared to marmosets, this species was not only less susceptible to\ninfection but, depending on the route of inoculation, hardly developed\nsevere clinical disease.\nOverview of inoculation route and clinical, pathohistological and virological findings.*\n*  i.v., intravenous; i.n., intranasal; PFU, plaque forming units.\n\nSix healthy sexually mature female rhesus monkeys ( Macaca mulatta ) belonging to the age\ncategory “aged macaques” (Asquith et al., 2012) were obtained from the\nbreeding colony of the German Primate Center (see Table 1). The age was\nbetween 19 and 23 years. Older monkeys were chosen assuming that they were\nmore susceptible to orthopoxvirus infection than younger ones as reported\nfor humans (Fenner et al., 1989). During the experiments the animals were\nhoused in single cages with visual, olfactory and acoustic contact to one\nanother. They were allowed free access to food and water and provided\nstandard environmental enrichment. All animals were adequately fed and\ncared for in accordance with the German Animal Welfare act. The animal\nexperiments were approved by the responsible veterinary authorities\n(approval number: 33.14-42502-04-095/09) and performed in accordance with\nthe EU guidelines for the accommodation and care of animals used for\nexperimental and other scientific purposes. Animals were clinically checked\ntwice a day. Blood samples were collected on days 4, 7, 10, 14, 17, 21 and\nat necropsy. Animals 2–6 were also sampled on day 24 and 28. Animals that\nwere moribund or terminally ill were humanely euthanized using an overdose\nof barbiturates. Severe apathy, weight loss, and inflammation of the upper\nrespiratory tract in combination with pox-like alterations on skin and\nmucous membranes were the criteria used to define terminally ill animals. Only animal 1\nwas euthanized for animal welfare reasons at day 21 after infection. To\nconclude the study, all remaining animals were euthanized 10 weeks after\nviral exposure. Complete post-mortem examinations were performed on all\nanimals. A comprehensive organ spectrum was collected for histological,\nelectron microscopical and virological analyses at necropsy.\nThe calpox virus stock used for inoculation has been described before\n(Kramski et al., 2010). The viral titer was determined by plaque assay prior\nto its use in infection experiments. The virus stock contained 3.5  ×  10 6  PFU (plaque forming units) mL - 1 .\nThe rhesus monkeys were infected intravenously with two different infectious doses of\nthe calpox virus (Table 1), depending on\nthe virus titer, the volume per ampoule\n(200  µ L) and overall availability of the stock. The first i.v. dose\ncorresponded to 200  µ L of undiluted virus. In order to potentially\naugment clinical symptoms, the dose for the second i.v. inoculated animal was increased\nfivefold. Since whether intranasal inoculation would also lead to infection of rhesus monkeys like in marmosets was unpredictable, we chose\nundiluted virus for this route. For the purpose of virus inoculation, monkeys\nwere anesthetized by injecting 0.1 mL per 1 kg body weight of a mixture\ncontaining 5 % ketamine, 1 % xylazine and 0.01 % atropine into the hamstring muscles ( Musculus semimembranosus ,  Musculus semitendinosus ,\n Musculus biceps femoris ).\nA volume of 1 mL calpox virus was administered\nintravenously into the great saphenous vein. For intranasal inoculation,\nthree doses\nof 60  µ L\nof undiluted virus suspension were alternatingly applied into the nostrils.\nExtraction of total DNA from tissue was performed in a FastPrep apparatus\n(MP Biomedicals, Illkirch, France) with two intervals of 20 s at 6 m s - 1  for\ntissue homogenization as described by Kramski et al. (2010). Nucleic acid was\nextracted from lysate using the DNeasy Blood and Tissue Kit (Qiagen, Hilden,\nGermany). DNA from whole blood was extracted using the DNA Blood Mini Kit\n(Qiagen, Hilden, Germany). Extractions were performed according to the\nmanufacturer's instructions, and DNA was eluted in 100  µ L AE buffer\n(Qiagen). Quantification of viral genomes in purified DNA from tissue or\nblood was amplified with a calpox-virus-specific qPCR assay as reported\n(Kramski et al., 2010). Quantities of viral DNA were expressed as genome\nequivalents (GE) per mL of blood or for tissue samples per 10 6  copies\nof the c- myc  gene. To investigate oropharyngeal virus shedding, we analyzed\nsaliva for the presence of infectious virus by a plaque assay (Kramski et al., 2010).\nFor the detection of binding antibodies an indirect ELISA was performed\n(Miller et al., 2011), except that ELISA plates were coated with 400 ng virus\nlysate per well prepared from the calpox-virus-infected Hep2 cells by sonication\nand subsequent centrifugation (5 min at 200 g). Plasma samples were applied\nin a single 1 : 400 dilution, and for detection of binding a HRP-coupled goat\nanti-human IgG antibody (Invitrogen, Karlsruhe, Germany) was used.\nNeutralizing antibodies were determined by plaque reduction neutralization\ntest (PRNT) and the titer was calculated (Kramski et al., 2010).\nSamples for histopathology were immersion fixed in 10 % neutral buffered\nformalin. Tissues underwent routine histological processing and\nimmunohistological investigation as previously published (Mätz-Rensing\net al., 2012). Additionally, selected sections of skin samples were\nprocessed for transmission electron microscopy (TEM) after glutaraldehyde\nfixation (2.5 %) and embedding in Epon.\nDermal lesions at different time points after i.v. calpox infection,\n Macaca mulatta , animal no. 2.  (a)  Focal umbilicated pustular\nlesion beneath the nose, 17 days p.i. (arrows).  (b)  Severe focal papular\ndermatitis of the left trigger finger, 17 days p.i. (arrows).  (c)  Severe\nfocal ulcerative dermatitis of the right arm induced by confluent pustules,\n17 days p.i. (arrows).  (d)  Severe focal ulcerative dermatitis of the\nright arm covered by a crust, 21 days p.i. (arrows).\n\nTwo animals were inoculated by the intravenous route. Numerous classical pox\nlesions started to develop on day 7 (animal 1) and 10 (animal 2)\npost-inoculation. The first clinical symptoms in both animals were exanthemas of\nthe face and the upper parts of the extremities as well as small single\nmacular lesions that appeared on the face, trunk and legs. Macular lesions\nspread over the whole body and began to develop into small papular and\nvesicular lesions. On day 10 and 12, pustules were spread over the entire\nbody and began to umbilicate. In addition, lesions occurred on oral and\ngenital mucous membranes and the mucocutaneous junctions. The lesions were\ncompletely umbilicated between day 14 and 17 (Fig. 1a). Moreover, the palms\nof hands and soles of feet were affected. Here, umbilication of the lesions\nwas not observed (Fig. 1b). Lesions on the extremities tended to coalesce\n(Fig. 1c). The maximal number and size of lesions was observed at day 21\n(approximately 50 for animal 1 and 140 for animal 2). In animal 1 the lesions\nreached diameters of up to 3 cm, were highly confluent and became necrotic,\nleading to a massive phlegmon of the right leg. This animal was immediately\neuthanized for animal welfare reasons at day 21 post-infection (p.i.). In animal 2\nthe pox lesions reached a maximal size of 1 cm in diameter, started to\ncrust,\nand became dry and flattened (Fig. 1d). Scabs developed and fell off after\nabout 4 weeks. After 5 weeks all lesions were completely healed.\nDetection of calpox DNA in blood and tissues of infected animals.\n (a)  At different time points post-infection (see materials and\nmethods),\nblood samples were taken, DNA was prepared from whole blood and viral DNA was\ndetermined with a specific qPCR assay. Genome equivalents (GE) for viral DNA\nwere calculated per mL of blood.  (b)  Viral load in tissue. Post-mortem\nsamples from various tissues were collected and DNA was isolated. Viral genome\nequivalents were determined by q-PCR and correlated to 10 6  copies of the c- myc  gene.\nAfter intravenous infection, calpox virus DNA was first detectable in blood\nbetween day 4 (animal 1) and 7 (animal 2). In animal 1 viral DNA\ncontinuously increased until day 14 and reached a plateau with a maximum of\n1.7  ×  10 7  GE per mL blood on day 21, just prior to its euthanization for\nanimal welfare reasons. In animal 2 the viral load peaked at day 17 with\n7.7  ×  10 6  GE per mL blood and subsequently gradually decreased by almost\n2 orders of magnitude until the analysis was stopped (Fig. 2a).\nQuantification of viral DNA in various tissues on the day of necropsy\nrevealed that in animal 1 (day 21 p.i.) calpox virus was present in most of\nthe investigated tissues (Fig. 2b). The highest viral load of 1.4  ×  10 8  GE\nper 1  ×  10 6  copies of c- myc  was observed in the skin of animal 1. Lower\nviral loads in the range of 1.4  ×  10 2  to 9.3  ×  10 4  GE were measured\nin lymphatic, intestinal, mucosal, muscular and nervous tissues; in the liver; and in the\nrespiratory and reproductive organs. No viral DNA was found in\nthe heart, kidneys, adrenal glands, bladder, small intestine, parotid gland\nor ovaries. In animal 2 (day 70 p.i. and after recovery) calpox virus PCR was\nclearly positive in skin only (1.5  ×  10 2  GE), with low-level detection in\nthe parotid gland, bone marrow and spleen. The detection of high copy numbers\nof viral DNA in many tissues of animal 1 at the time of sacrifice as well as\nresidual viral DNA in some of the tissues in animal 2 after recovery\nindicated a systemic infection of the rhesus monkeys infected by the\ni.v. route (Fig. 2b). Saliva for the analysis of infectious virus was available\nfrom animals 2–6. In animal 2 all samples collected between day 4 and 24\nwere positive in the plaque assay, indicating the presence of infectious\nvirus in saliva (Table 2).\nOPXV-specific binding antibodies were detectable by ELISA in plasma of both\nanimals (Fig. 3a). Animal 1 and 2 seroconverted between day 7 and 10, and\nantibody levels increased until 3 weeks post-infection. As analyzed by\nPRNT, both intravenously infected animals started to develop neutralizing\nantibodies against OPXV between day 7 and 10. Maximum titers of 1 : 200 were\nobserved between day 21 and 28 post-infection.\nPathohistological investigation revealed typical poxvirus-induced skin\nlesions in animal 1. The lesions were characterized by superficial\nulceration of the epidermis. The ulcerated parts were covered by a\nserocellular crust (Fig. 4a). Adjacent parts of the epidermis showed\nirregular epidermal hyperplasia and signs of acantholysis, acanthosis and\nsyncytia formation of the basal keratinocytes. Numerous large Guarnieri\nbodies were found in altered epithelial cells. In deeper parts of the dermis,\nfollicular and sebaceous epithelia were also affected, leading to severe\nchronic granulomatous infection. The inflammatory infiltrate was\npredominated by lymphocytes and histiocytes and extended deeply into dermis\nand subcutis. Characteristic intracytoplasmic inclusion bodies were also found in\nenlarged vacuolated or degenerated cells of sebaceous glands (Fig. 4b).\nThe Guarnieri bodies measured 2–8  µ m and were distributed randomly\nwithin the altered epithelium. Animal 2 did not show pox-specific alterations.\nDetection of infectious particles in saliva.\nNo plaques ( - ), 1 plaque ( + ), 2–10 plaques ( + + ),  >  10 plaques ( + + + ),\nnot done (n.d.); i.v., intravenous; i.n., intranasal, p.i., post-infection.\nDetection of calpox-virus-specific antibodies in plasma of infected\nanimals. At different points in time post-infection (see Sect. 2.1),\nwhole blood was collected and plasma was prepared.  (a)  Antibodies\nspecifically binding calpox viral proteins were measured by ELISA and are shown\nas optical densities at 450 nm using plasma diluted 1 : 400.\n (b)  Neutralizing antibodies were determined with the plaque reduction\nneutralization assay (PRNT). The data are expressed as 50 % plaque reduction\ntiters. Animal numbers and the different inoculation routes (i.v., intravenous;\ni.n., intranasal) are given.\nDermal lesions at time of death (day 21 p.i. after i.v. calpox\ninfection),  Macaca mulatta , animal no. 1.  (a)  Severe subacute\ndermatitis with epidermal ulceration covered with serocellular crusts and severe\ngranulomatous inflammation in deeper parts of the dermis (HE stain).\n (b)  Inflammation of a sebaceous gland with typical intracytoplasmic\nGuarnieri bodies (arrow) positive for calpox virus antigen in immunohistochemistry.\n (c)  Transmission electron microscopy (TEM) showing multiple intracytoplasmic\ninclusions in infected epithelial cells which contain mature viral particles\n(arrows).  (d)  Enveloped viral particles were ovoid to brick shaped,\nhave a pale central zone and a size of 140  ×  260 nm (arrows; TEM).\nImmunohistochemistry and electron microscopy confirmed the presence of\ncalpox-virus-infected cells in the skin of animal 1. In dermal lesions, virus\nwas present in degenerated epithelial cells, dermal macrophages and altered\nsebaceous glands (Fig. 4b). The virus was not\ndetected in any of the other organs tested.\nElectron microscopy revealed virus particles with OPXV-like\nmorphology in intracytoplasmic inclusions in epithelial cells.\nUltrastructurally, numerous intracytoplasmic inclusion bodies were visible\n(Fig. 4c), presenting mature viral particles with a size of 140  ×  260 nm. The\nenveloped viral particles were ovoid to brick shaped with a pale central\nzone, presenting characteristic OPXV-like ultrastructural features (Fig. 4d).\nOnly one of the animals infected by the i.n. route showed mild clinical\nsymptoms. This animal (3) developed an enanthema of the pharynx mucosa\nat day 14 p.i., where three single papules occurred at day 17 and one at the\ncorner of the right eye. All lesions were completely healed by day 21. The\nother three intranasally infected animals showed neither clinical symptoms\nnor pathomorphologic alterations related to the calpox virus infection. No\nviral DNA was detected in blood or tissues of the animals infected intranasally in\ncontrast to the intravenously infected ones (Fig. 2a and b). Nevertheless,\ninfectious virus was sporadically isolated in saliva of animals 5 and 6.\nWith the exception of animal 3, OPXV-specific binding antibodies were\ndetectable by ELISA in plasma of all i.n. inoculated animals (Fig. 3a). All\nthree animals seroconverted between day 7 and 10, and antibody levels\nincreased until 3 weeks post-infection. They also developed neutralizing\nantibodies that appeared between day 14 and 21 after inoculation. Titers\nwere generally lower in these animals compared to the i.v. infected ones.\nInterestingly, in one of these three animals (animal 6), neutralizing titers\nincreased continuously until the end of the follow-up (Fig. 3b).\n\nWe previously showed that experimental low-dose intranasal infection of\ncommon marmosets with the calpox virus results in fatal disease. The calpox\nvirus belongs to the  Cowpox virus  species and was isolated from  Callithrix jacchus  during a\nnatural outbreak of the disease in a private New World monkey husbandry\n(Mätz-Rensing et al., 2006). High doses of the virus experimentally\napplied to marmosets reproducibly led to death within 4 to 7 days. Even the\nintranasal application of as low as 50 PFU of calpox virus to the New World\nmonkeys was infectious in 40 % of the animals and resulted in viremia and\nfatal outcome (Kramski et al., 2010). The route and dose used for viral\ninoculation of this monkey species mimics the natural transmission of\nsmallpox, thus representing a suitable model to study pathogenesis and to\nevaluate new vaccines and therapeutics against orthopoxvirus infection\n(Kramski et al., 2010; Mätz-Rensing et al., 2012). However, this animal\nmodel has some limitations due to the lack of species-specific or\ncross-reactive reagents, particularly for the analysis of innate immune\nresponses which hamper certain experimental approaches. The aim of the present\nstudy was to test whether the results from the marmoset studies can be\nrecapitulated in rhesus macaques, a nonhuman primate species that has been used in many\ndifferent research fields for decades.\nThe results of the study suggest that New World monkeys, i.e., common\nmarmosets, are more susceptible to the calpox virus than rhesus monkeys. High levels of viral\nreplication were observed in blood accompanied by the appearance of\nclassical pox lesions of varying degree only\nwhen this Old World monkey species was given high doses in the range of\n10 6  PFU of infectious virus intravenously. A comparable dose applied to\nmarmosets reproducibly led to death within 4 to 7 days (Kramski et al.,\n2010). A dose of 8.3  ×  10 3  PFU reliably led to infection and 100 %\nmortality in marmosets (Mätz-Rensing et al., 2012), while an\napproximately 100-fold higher dose of calpox virus inoculated\nintranasally in rhesus monkeys compared to that used in marmosets was not\nsufficient to induce viremia, let alone conspicuous clinical alterations.\nNonetheless, seroconversion in three out of four rhesus monkeys inoculated\nintranasally and the detection of infectious virus in saliva of two of the\nthree seroconverted animals suggest local low-level replication, presumably\nat the portal of virus entry, which could be the nasal epithelium. Those\nfindings can be defined as subclinical infection.\nIn contrast to marmosets, a very high dose of calpox virus is needed to\ninfect rhesus macaques. Nevertheless, the clinical symptoms observed in the\nrhesus macaques infected intravenously were typical of an orthopoxvirus infection.\nBoth animals developed viremia followed by characteristic skin lesions.\nWhereas the lesions in animal 1 showed similarities with the confluent\nordinary smallpox type with a higher fatality rate, lesions in animal 2\ncould be compared with a mild discrete ordinary type of smallpox. In both\nanimals, the pustules on the face, arms and extremities were numerous and\nrather sparse on the trunk. Lesions were all at the same stage; those that\nappeared earliest on face and upper extremities were more mature than those\nthat appeared later on other parts of the body. Lesions were also present on\nsoles and palms. Except on soles and palms, umbilication was a common\nfeature of the skin lesions similar to smallpox disease. Histological\nanalyses\ndemonstrated that cells of the sebaceous glands were highly susceptible,\nwhich is typical of the infection with other cowpox viruses or smallpox.\nFurthermore, the older age of the monkeys in this study should be\nconsidered. Upon necropsy, all animals presented with minor chronic diseases,\nsuch as pulmonary acariasis induced by  Pneumonyssus semicola  or endometriosis (see Table 1),\nwhich often represent clinically\ninapparent lesions in older animals. This could lead to the assumption that\nthese clinically inapparent diseases as well as the old age of the monkeys\nmight have affected their ability to resist the viruses,\nmaking them more susceptible\nto the administered calpox virus. In humans, children and elderly people\nseem to be more vulnerable to orthopoxvirus infections than middle-aged\npeople (Fenner et al., 1989). However, this does not seem to be the case with\nthe calpox virus in rhesus macaques. Apparently, old age does not seem to increase\nthe susceptibility to this virus. Intranasally infected rhesus monkeys\nremained healthy, and animals infected by the i.v. route developed relatively\nmoderate symptoms in relation to the high viral dose that was inoculated.\nThis confirms that the calpox virus is less pathogenic to macaques compared to\nmarmosets and leads to the question of species-specific pathogenicity that\nseems to be a phenomenon of CPXV.\nDespite high genetic homology, OPXV shows great differences in host range. On\nthe one hand, VARV, which is restricted to humans, has no known natural\nreservoir and is less pathogenic to nonhuman primates. On the other hand,\nCPXV with a known reservoir in rodents has a broad host range and is responsible\nfor lethal CPXV infections in different animal species and even humans\n(Essbauer et al., 2010; Eis-Hübinger et al., 1990). The combination of\nthe CPXV strain and host seems to play an important role in pathogenesis and\ndisease outcome. A CPXV infection in humans or cats usually leads to a\nself-limiting local infection, while the same virus strain induces death in\nsusceptible animal species as described for banded mongooses and jaguarundis\n(Kurth et al., 2009). Similar observations were made for other CPXV–host\ncombinations (Kurth et al., 2008). It could be shown that the calpox virus\nalso belonging to the CPXV species led to highly reproducible lethal disease\nin marmosets but was less pathogenic in mice (unpublished data). The\nresults of the present study demonstrated a lower pathogenicity of the same\nvirus for Old World monkeys and underline the great influence of virus–host\ninterrelationships for disease outcome. The molecular factors which\ndetermine the host range and host-specific pathogenicity are not very well\nunderstood yet. Taking all these facts into account, it could be assumed that the\ncowpox virus, which represents an Old World virus, has a higher virulence\nfor New World species such as marmosets, jaguarundis or prairie dogs.\nThe observation that the calpox virus is less pathogenic to rhesus macaques led\nto the question whether Old World monkeys are susceptible to CPXV in\ngeneral. During a natural cowpox outbreak in a sanctuary for exotic animals\nin the Netherlands, several animals of different macaque species developed\nneutralizing serum antibody titers, indicating an exposure to the virus, but\nonly three of them developed mild clinical symptoms. Experience of this\noutbreak suggests that macaques are susceptible to cowpox virus but that the\nclinical outcome is less severe than in New World monkeys (Martina et al.,\n2006; Mätz-Rensing et al., 2006). There is evidence that cynomolgus and\nrhesus macaques are susceptible to CPXV Brighton Red (CPXV-BR). Recently, 14\ncynomolgus macaques were inoculated intravenously with different doses of\nCPXV Brighton Red (5  ×  10 4   -  5  ×  10 7   -  PFU;\nJohnson et al., 2011b). A total of 9 out of 14 animals developed typical\npox-like skin lesions. Further findings included hemorrhages in a variety of\norgans, indicating a hemorrhagic course of disease. Infection was uniformly\nlethal within 12 days post-inoculation. The researchers concluded that this\nanimal model may serve as a model for hemorrhagic smallpox, which is more\nfeasible than the VARV model (Jahrling et al., 2004). The same researchers\nadapted a method of the intrabronchial and intra-alveolar infection with\nCPXV-BR to mimic the natural route of infection more closely. Intrabronchial\ninoculation and small-particle aerosol inoculation of cowpox BR led to a\nsevere respiratory disease in both macaque species (Smith et al., 2012;\nJohnson et al., 2015). Macaques inoculated by aerosol developed severe\nbronchointerstitial necrotizing pneumonia, but skin lesions were not\nobserved and the viral dissemination was limited. This shows that the route\nof infection strongly influences disease progression.\n\nRhesus macaques are less susceptible to calpox virus exposure. Symptomatic\ninfection requires high virus doses and depends on the route of application.\nTherefore, the rhesus monkey calpox model is not suited for calpox virus\nresearch and is of limited use in further intervention studies against OPXV.","source_license":"CC-BY-4.0","license_restricted":false}