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1 Centers for Disease Control and Prevention, MS G-43, 1600 Clifton Road NE, Atlanta, GA 30333, USA
2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
3 Centers for Disease Control and Prevention, MS G-12, 1600 Clifton Road NE, Atlanta, GA 30333, USA
Correspondence
Christina L. Hutson
zuu6{at}cdc.gov
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ABSTRACT |
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Supplementary figures are available with the online version of this paper.
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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INTRODUCTION |
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; Jezek et al., 1987) and has been used for the study of systemic orthopoxvirus infections. In humans, MPXV and variola virus have similar asymptomatic incubation periods ranging from 7–17 days followed by development of generalized rash. However, smallpox had a much higher secondary attack rate (25–96 %) than monkeypox (5–11 %) (Arita & Gromyko, 1982; Fine et al., 1988). Previous studies defined two distinct MPXV clades, West African and Congo Basin, with unique disease manifestations (Chen et al., 2005; Likos et al., 2005). Human disease associated with West African MPXV infection is less severe and associated with less human-to-human transmission compared to Congo Basin infection (Breman et al., 1980; Foster et al., 1972). In the Democratic Republic of Congo (Congo Basin), monkeypox has a reported case fatality rate of 
10–13 % (Arita et al., 1985; Breman et al., 1980; Meyer et al., 2002), compared with up to 40 % for smallpox (Breman & Henderson, 2002). Human monkeypox had never been reported outside of Africa until 2003, when an outbreak occurred in the USA resulting from human contact with infected prairie dogs (PDs) which were co-housed with imported African rodents (Hutson et al., 2007; Reed et al., 2004). This outbreak demonstrated the potential for MPXV importation and, together with observations of ongoing human monkeypox in Africa (Boumandouki et al., 2007; Hutin et al., 2001; Learned et al., 2005; Meyer et al., 2002; Mukinda et al., 1997; Mwanbal et al., 1997; Rimoin et al., 2007), further emphasized the importance of a more complete understanding of this serious human pathogen.
In vivo animal model comparisons of viruses from these two clades will provide valuable tools with which to evaluate host–pathogen factors responsible for pathogenic differences. Multiple animal species have been experimentally infected with MPXV (Marennikova & Seluhina, 1976; Shchelukhina & Marennikova, 1975; Tesh et al., 2004; Zaucha et al., 2001); however, most of these models have little in common with the human disease progression, presentation or mortality from either monkeypox or smallpox. Based on observations of PDs infected during the USA outbreak (Guarner et al., 2004; Hutson et al., 2007), and previous experimentally infected PDs (Xiao et al., 2005), we believe these animals will serve as a relevant model for human monkeypox. In the Xiao et al. study, only West African MPXV was used and it was administered via an intranasal and an intraperitoneal route. Here, we show that PDs are a valuable model for study of strain-dependent differences in MPXV virulence, something that has not been previously studied in these animals. Additionally, we infected with an intranasal as well as an intradermal route, both of which are plausible natural routes of infection. Our described animal model mimics human monkeypox disease progression and presentation more closely than previous models, and thus will serve as a valuable surrogate for future investigations of human systemic orthopoxvirus diseases, including smallpox.
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METHODS |
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Viruses.
The West African MPXV strain, MPXV-2003-044, was isolated during the 2003 USA outbreak, and has previously been fully sequenced (Likos et al., 2005; Reed et al., 2004). The Congo Basin MPXV strain, MPXV-2003-358, was collected from a 2003 outbreak of monkeypox in the Republic of Congo (ROC) and has been fully sequenced (Likos et al., 2005). Both viruses had undergone two passages in African green monkey kidney cells (BSC-40) prior to seed pool production. The titres for both virus inocula were retitrated after appropriate dilutions were made for PD inoculation.
Animal inoculation.
Infection of PDs with the two MPXV strains was done on separate occasions. The average starting weight for animals challenged with West African MPXV was 714.1 g (range 535–842 g), and the average for Congo Basin MPXV-challenged animals was 807.8 g (range 640–988 g). Animals were infected by either an intranasal (IN) or intradermal via scarification (ID-SC) route of inoculation while under anaesthesia. For both strains, groups of four animals were inoculated with 104.5 plaque-forming units (p.f.u.) in a total volume of 10 µl IN (5 µl in each nostril). Similarly, for both strains, four animals were inoculated with 104.5 p.f.u. in a total volume of 10 µl by ID-SC. For ID-SC animals, a small area on the right flank of the animals was shaved. Ten microlitres of virus solution was placed on this shaved area and a 28 gauge needle was used to gently probe to the superficial layers for ten replicates. Additionally for each strain, four animals were mock-infected with PBS.
Observations and sampling.
Daily visual observations of the animals were made and recorded throughout the study. Lesion count, temperature, weight and tissue/excreta samples were collected every third day post-infection (p.i.) without anaesthesia. For both MPXV strain challenge studies, the relatively less fur-covered areas of the face, inner hind legs and genitalia were used to count lesions. Blood was collected from the saphenous vein in EDTA-coated tubes. Sterile individual Dacron swabs were used to collect excreta and were stored frozen without diluent. Swabs, faeces and blood were processed and prepared for DNA analysis and virus isolation (see below).
Necropsy and tissue specimen collection.
If death did not occur as a result of infection, at 35 days (West African MPXV) and 49 days (Congo Basin MPXV) animals were humanely euthanized and necropsied. Euthanasia dates were determined based on estimated time to cessation of viable virus [RT-PCR values greater than cycle threshold (Ct)=34] in excreta. Necropsies on all animals were performed according to IACUC standards in a BSL-3 laboratory and utilizing full BSL-3 PPE. Instruments were cleaned and decontaminated with 3 % Amphyll and 10 % Clorox bleach between collections of each tissue. Tissues were frozen at –70 °C prior to further processing. Oral and ocular swabs were collected with sterile individual Dacron swabs and stored frozen without diluent. Serum was separated from whole blood and processed for serology (see below). Tissues and swabs were subsequently processed and further prepared for DNA analysis and virus isolation (see below).
Sample preparation.
Sample processing was performed under BSL-3 conditions. For DNA analysis of blood, water was added if necessary to bring total volume to 200 µl. The sample was incubated at 55 °C for 1 h to inactivate virus. The EZ-1 DNA extraction robot (Qiagen) was used for genomic DNA extraction of all blood samples. For swabs collected during the West African MPXV challenge, 400 µl PBS was added to the swab. The swab extraction tube systems (SETS) (Roche) protocol was used to recover sample from a swab. Genomic DNA was prepared using either the AquaPure DNA isolation kit (Bio-Rad), or the BioRobot MDx workstation (Qiagen). For swabs collected during the Congo Basin MPXV challenge, 1 ml PBS was added to the swab and vortexed. Genomic DNA was then prepared using the AquaPure DNA isolation kit. For both virus strains, remaining swab lysate was used for virus isolation (see below). Tissue and faecal samples were placed in disposable dounce homogenizers. PBS (1 ml) was added to each weighed tissue sample in the 50 ml sterile tissue grinder and ground thoroughly to create a slurry. Genomic DNA was prepared from a slurry aliquot (100 µl) with BioRobot MDx workstation or AquaPure DNA isolation kit and the remaining samples were used for virus isolation (see below).
Real-time PCR analysis.
Samples were tested by real-time PCR using forward and reverse primers and probe complementary to the conserved orthopoxvirus E9L (DNA polymerase) gene (Li et al., 2006). A representative sample from each animal was confirmed for MPXV DNA using forward and reverse primers and probe specific to the MPXV B6R gene (Li et al., 2006). MPXV DNA (10 fg–1 ng) was used as positive control for both tests.
Virus–tissue infectivity.
Previous analyses demonstrated that real-time PCR detection of MPXV DNA was significantly more sensitive than detection of p.f.u. (Hutson et al., 2007). Therefore, specimens were first tested for the presence of orthopoxvirus DNA by PCR and, if positive, were subsequently evaluated for viable virus by tissue culture propagation. Each swab, faeces or tissue sample was titrated using tenfold dilutions of swab eluent or tissue slurry on BSC-40 cell monolayers, incubated at 36 °C and 6 % CO2 for 72 h, and subsequently stained with crystal violet and formalin to reveal plaques.
Serological analysis.
A modified version of the ELISA was used for analysis of anti-orthopoxvirus immunoglobulin types A and G (Hutson et al., 2007). Microtitreplates (Immulon II; Dynatech) were coated with 0.01 µg per well crude vaccinia virus (Dryvax grown in BSC-40 cells) in carbonate buffer on one half of the plate and an equal volume of BSC-40 cell lysate diluted in carbonate buffer on the other half and incubated overnight at 4 °C. After inactivation with 10 % formalin (buffered) for 10 min at room temperature, plates were blocked for 30 min at room temperature with assay diluent [PBS, 0.01 M, pH 7.4 (Gibco)+0.05 % Tween-20, 5 % dried skim milk, 2 % normal goat serum and 2 % BSA] followed by three PBST (0.05 % Tween-20) washes. PD sera (diluted 1 : 100 in assay diluent) was added to both halves of the plates and incubated for 1 h at 37 °C. Plates were washed, and a 1 : 30 000 dilution (in assay diluent) of ImmunoPure A/G conjugate (Pierce) was added and incubated for 1 h at 37 °C. Plates were washed, and peroxidase substrate (Kirkegaard & Perry Laboratories) was added and allowed to develop for 5–15 min. After development, stop solution (Kirkegaard & Perry Laboratories) was added, and absorbance was read on a spectrophotometer at 450 nm. Values reported represent the average of duplicate wells for each sample. Both positive and negative human anti-vaccinia sera were used as assay controls. The BSC-40 cell lysate half of each plate was used to generate a cut-off value (COV) for each plate by averaging all the values of the BSC-40 lysate half and adding two SD. Specimens were considered positive if the test sample's value was above the COV.
Statistical analyses.
As data were not normally distributed, nonparametric statistical analyses were used (Lehmann, 1975). Levels of viral DNA, viable virus, anti-orthopoxvirus antibodies, and daily observations (weight, temperature, lesion counts) were compared between strains and routes of infection using the Wilcoxon rank-sum test. In order to evaluate differences in temperature, day zero temperature and the highest temperature recorded thereafter were used to determine the per cent increase in temperature for each PD. To evaluate weight differences, weights observed at day zero were used as the baseline and the lowest weight measured thereafter was used to determine per cent weight loss in each animal. Comparisons between the first and last day of DNA and viable virus were also evaluated between strains and routes using the Wilcoxon rank-sum test. ID-SC and IN groups were combined to increase numbers when comparing strains, and strains were combined to increase numbers when comparing route for DNA and viable virus. This was also done when comparing the number of lesions between strains. A P-value <0.05 was considered significant.
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RESULTS |
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Congo Basin MPXV challenge.
As was seen in the West African MPXV-infected animals, the animals inoculated with the Congo Basin strain of MPXV had similar disease presentations with generalized lesions appearing between days 9 and 12 (Supplementary Fig. S2, available in JGV Online). All four animals inoculated via the ID-SC route developed a SS lesion on day 6, also comparable to the West African ID-SC animals. Until the development of this primary lesion for the ID-SC group, or the disseminated lesions for the IN group, no observable symptoms were noted. Similarly to West African animals, lesion count was slightly less for the Congo Basin IN animals compared with the ID-SC animals, with the exception of one ID-SC animal which, unlike the West African animals, developed a SS lesion, but died on day 11 before disseminated lesions occurred. Another ID-SC animal was found dead on day 12 and one IN animal was found dead on day 13. Additionally, unlike the West African IN animals, all Congo Basin MPXV IN-infected animals developed lesions on the face and inner legs. Clinical symptoms were comparable between the two Congo Basin-inoculated groups. All of the surviving Congo Basin MPXV infected animals' secondary lesions began to resolve between days 18 and 28, similar to West African infected animals. One ID-SC animal developed an apparent secondary bacterial infection at the SS ( day 15), lasting until approximately day 35 and crusting over by day 42. The other animal that survived ID-SC inoculation never developed a bacterial infection at the site of scarification. For this animal, the primary lesion that developed began to resolve around day 15, with the scab completely falling off by day 30.
Molecular and virologic findings (Table 1
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West African MPXV challenge.
MPXV DNA was detectable in all samples (ocular, oral, nasal, faecal, blood) at days 9–15 from the West African MPXV ID-SC inoculated group as well as the IN-inoculated group (with the exception of faeces). Viral DNA was initially detected in blood, followed by oral and nasal excreta, then in ocular and faecal excreta (Fig. 1a) in the ID-SC-inoculated animals. Similar results were seen for IN-inoculated animals, with the exception of DNA initially being detected in oral secretions (Fig. 1b). Viable virologic analyses provided similar kinetics for each group, but virus isolation was not attempted from blood (Fig. 1c and d). Peak MPXV DNA was observed from day 6 to 15, with oral samples yielding the highest levels for both groups (Fig. 2a). For both inoculation routes, viable virus was detectable beginning on day 6 for oral and nasal swabs (with the exception of one IN animal which had detectable viable virus from the oral sample on day 3), day 9 for faecal and day 12 for ocular swabs. Viral shedding continued from these sites until days 15–21 (Fig. 1c and d). Peak MPXV viable virus was observed from day 9 to 18, with the oral sample having the highest load for both groups (Fig. 2b). Blood was collected from all West African MPXV-infected animals on day 35, and all had anti-orthopoxvirus antibodies (Fig. 3a). West African ID-SC-infected animals had significantly higher anti-orthopoxvirus antibodies than West African IN animals (P=0.03). At time of necropsy, all four animals in the ID-SC group had MPXV DNA detected in the lesion sample. In addition, three eyelid samples, one skin sample, one oral swab, one lymph node and one gonad were MPXV DNA-positive. The four IN-inoculated animals had two MPXV DNA-positive eyelid samples, as well as one skin and one lesion. None of these MPXV DNA-positive samples yielded viable virus.
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and 5). Congo Basin MPXV infected via ID-SC manifested DNA in blood earlier than other tissues, compared with those infected via the IN route (Fig. 4). However, all samples, with the exception of the ID-SC blood samples, yielded positive MPXV DNA beginning on days 6–9, comparable to what was seen in the West African MPXV samples. Also similar to the West African-inoculated animals, MPXV DNA levels peaked on days 9–21 (Fig. 6a). An interesting difference seen in the Congo Basin-challenged animals was that faecal samples yielded the highest MPXV DNA loads for both inoculation routes; however, comparable to the West African-infected group, the oral samples yielded the highest viable virus loads (Fig. 6). Also comparable to what was seen in the West African MPXV-inoculated animals, viable virus was detectable beginning on day 6 for samples from ID-SC and IN animals (Fig. 5). Viral shedding occurred for both groups until day 18–21 for all samples except the IN ocular sample, which had viable virus only until day 12 (Fig. 5). Peak MPXV viable virus was observed from days 12 to 18 (Fig. 6b), again similar to the peak observed during the West African MPXV infection. Blood was collected post-mortem on the three animals that died spontaneously, and on day 49 for the remaining animals. The blood from the animals that died during the study was negative for orthopoxvirus (OPXV) antibodies. The other five convalescent Congo Basin-inoculated animals had anti-OPXV antibodies detected at time of necropsy with similar levels as were observed in the serum collected from the West African-challenged animals (Fig. 3b). Unlike the West African-challenged animals, there was no significant difference seen between the antibody levels. At time of euthanasia, the five animals that survived had several MPXV DNA-positive samples which were negative for viable virus. This included two lesion samples and one skin sample from the ID-SC animals as well as one blood and one heart sample from the IN animals. The three animals that died during the course of the observations had high levels of both viral DNA and viable virus in the majority of samples tested (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Jezek et al., 1987; Reynolds et al., 2004), MPXV infection in PDs was asymptomatic during a 6–12 day incubation period, after which obvious skin lesions developed and subsequently resolved approximately 28 days p.i. for surviving animals. Prior to development of generalized lesions, viral shedding could be detected in oropharyngeal secretions, consistent with descriptions of the oral enanthem of human systemic orthopoxvirus disease. Generalized lesions developed and evolved at a similar rate in a centrifugal pattern, similar to disease progression for human monkeypox (Arita et al., 1985; Jezek et al., 1987).
In addition to disseminated vesiculo-pustular lesions, several other disease symptoms in the PD MPXV model were similar to those seen in human cases. One potentially painful symptom of human monkeypox cases is oral lesion development (Huhn et al., 2005; Jezek et al., 1987; Reynolds et al., 2006). Although we could not photograph lesions in the oral cavities of animals since animals were not anaesthetized during sampling, we were able to observe lesions on/inside the mouths of several animals. Furthermore, bloody oral swabs collected from some animals were probably caused by oral lesions. A complication in human disease is development of secondary bacterial skin infections at lesion sites (Jezek et al., 1987). Although no disseminated lesions developed secondary bacterial infections, several of the SSs did. Diarrhoea is seen in a small percentage of human cases (Jezek et al., 1987), and similarly, was observed in one infected animal. Bronchopneumonia is another rare, late sequela of human monkeypox. Although lower respiratory complications could not be confirmed, the gasping noise audible from one infected animal is suggestive of such complications, or could be indicative of upper airway oedema causing stridor. Crusty noses and nasal discharge from several of the animals suggests upper respiratory involvement; human disease in the USA and Africa has documented upper airway symptoms of sore throat and choryza (Huhn et al., 2004, 2005; Reynolds et al., 2006). Finally, inappetance and lethargy observed in PDs are commonly observed in humans (Jezek et al., 1987). A primary difference between the clinical presentations of human smallpox and monkeypox is the occurrence of lymphadenopathy in MPXV-infected individuals. Interestingly, we did not observe these sequelae in the PDs. However, we isolated viable virus from lymph node tissue in the three animals that succumbed to Congo Basin MPXV, indicating that the lymphatic system was involved during the course of infection, even though swelling was not noted. Either the condition did not occur, or was not substantial enough to be observed. Although disease manifestations are not identical in these two species, there are many similarities in disease progression, clinical manifestations and mortality rates between that seen in humans and in PDs infected with MPXV. Our model used a relatively low level of infectious dose as well as two plausible natural routes of infection, indicating that the PD is an ideal model for human MPXV infection.
Viral DNA was detected in the blood of most animals between days 6 and 15. Although we did not attempt viral isolation on blood samples, the presence of detectable MPXV DNA early in infection is evidence of viraemia during this time period. Viable virus was isolated from oral, ocular and nasal swabs, as well as faeces, during the study, demonstrating the shedding potential of MPXV via multiple excreta. Oral swabs were especially rich in viable virus, with average peak levels greater than 104 p.f.u. for the West African- and 105 p.f.u. for Congo Basin MPXV-infected animals. Interestingly, the faecal samples from the Congo Basin MPXV-infected animals had significantly more DNA (days 6–28) and viable virus (day 18) than the West African MPXV-infected animals (P-values between 3x10–4 and 0.046). This suggests, at least in this experimental animal model, there are potential strain-dependent differences in transmission. Viable virus levels peaked at similar times for both MPXV strains, days 9–18 for West African and 12–18 for Congo Basin. For both strains, infected animals continued shedding viable virus from certain samples until day 21 p.i., information lacking for 2003 USA monkeypox outbreak animals and which might be potentially useful for formulating future outbreak control measures. This period of viable MPXV shedding from PDs was similar to human monkeypox outbreaks, with isolation of viable virus from samples taken up to 18 days after the development of rash (Arita et al., 1985). The serology results confirmed that all animals, except those that died during the study, had developed immune responses to orthopoxviruses. This assay was designed to capture all Ig antibodies, but may not adequately capture IgM antibodies. The animals that perished most likely died before developing IgG responses which would have been detected with this ELISA. At time of necropsy, some animals that survived the challenge had low levels of viral DNA in a few tissues, but no viable virus. However, the three animals that died during the Congo Basin MPXV challenge had high levels of viral DNA and viable virus throughout the majority of tissues tested, suggesting that MPXV has a wide range of tissue tropism. In one or more of these three animals, samples including the oral swab, eyelid, tongue, lung, liver, spleen and scent glands had levels of viable virus at or above 107 p.f.u. ml–1.
Similar to human infection (Breman et al., 1980; Foster et al., 1972; Jezek et al., 1987; Likos et al., 2005; Reynolds et al., 2006), the Congo Basin MPXV strain was more pathogenic than the West African MPXV in the PD model. Morbidity was higher in Congo Basin MPXV-infected animals as measured by lesion count. A greater trend in weight loss and a significantly higher per cent increase in temperature in Congo Basin MPXV-challenged animals were also noted. Furthermore, mortality rates of 50 % (ID-SC) and 25 % (IN) were seen in the Congo Basin MPXV-inoculated animals, compared with 0 % in West African MPXV-inoculated animals. This is concordant with observations of human disease, where Congo Basin MPXV causes 10–13 % mortality compared with 0 % with West African MPXV. The ability to discern differences in virulence for MPXV from the two clades in this animal model should benefit future studies. Having such an animal model will hopefully allow better understanding/identification of MPXV virulence factors.
It is noteworthy that the observed mortality rate was lower than that observed in a previously published PD challenge study (Xiao et al., 2005) in which West African MPXV challenge administered IN caused 60 % mortality and administered intraperitoneally caused 100 % mortality. There are several possible explanations for this difference. Xiao et al. used a slightly higher dosage of virus (105 p.f.u.) and practices such as orbital bleeding and daily anaesthetizing for sampling, compared with our current study administering 104.5 p.f.u., bleeding from the saphenous vein and animal sampling every third day without anaesthesia. Also, throughout our study, animals were provided with highly palatable ‘monkey biscuits’, potentially a key dietary factor when anorexia occurred. Additionally, during the Xiao et al. study, the intraperitoneal animals did not develop disseminated lesions, suggesting that this inoculation does not mimic natural disease progression, unlike animals infected in our study.
The incubation period before onset of secondary lesions (12 days versus 9–12 days), as well as the time it took for lesions to resolve, were similar for IN and ID-SC West African MPXV-infected animals in our study. Interestingly, this observation is somewhat different than that seen in human cases during the USA outbreak, in which humans who received bites or scratches (the transmission route that the ID-SC model most closely mimics) had a shorter incubation period compared with infection via the respiratory route (Reynolds et al., 2006). Additionally, in the USA human monkeypox study, a significant difference in rash burden between the two routes of exposure was not observed. However, we did see significantly higher numbers of lesions in the ID-SC-infected animals compared with the IN-infected animals (P=0.02). We consistently observed the development of a primary lesion at the site of scarification (days 6–9) before the onset of disseminated lesions (days 9–12), which is similar to the disease progression of inoculation smallpox versus respiratory route-acquired smallpox (Fenner, 1948, 1990). Similarly, the incubation period of the probable respiratory route of human monkeypox was observed to be longer than inoculation/complex exposure to MPXV (Reynolds et al., 2006). The differences seen in these previous human studies, compared with our PD model, might be attributed to differences between a retrospective (USA investigation) versus a prospective (our study) study design, or different host–pathogen interactions in our experimental animal model compared with human infection. Also, the infection dose for our PD study may well have been higher than in naturally acquired human monkeypox cases.
There were several study limitations. Since we routinely collected swabs instead of whole tissue, samples cannot be directly compared to evidence of virus per gram of tissue, but instead must be related to internally consistent collection methods. For the faecal samples, not surprisingly, there were some bacterial and/or fungal contaminations that may have inhibited viral propagation in cells. Also, we had some sample preparation variation because the swabs from the Congo Basin MPXV challenge were extracted in a larger amount of PBS compared with West African MPXV challenge samples. Although this was accounted for when calculating MPXV yield, the slightly higher dilution may have lowered the amount of detectable virus in marginally positive samples. Also, because it is challenging to do large scale studies with large rodents, our relatively small sample size limited statistical analysis. Finally, because PDs are wild-caught animals, not inbred laboratory-acquired animals; there is individual animal variability which may affect the consistency of disease progression.
Our results are concordant with previous data (Xiao et al., 2005; Hutson et al., 2007; Guarner et al., 2004) that showed PDs are highly susceptible to MPXV. We found both inoculation routes gave similar infection results, and either route would be a good mode of inoculating animals in future experiments that may mimic natural transmission. Furthermore, because our model has a prolonged incubation period, disseminated lesions, as well as clear differences in virulence of the two MPXV strains, reminiscent of the differences in human diseases and MPXV strains, the PD MPXV model has the potential to be a valuable model for disseminated human orthopoxvirus disease, including both monkeypox as well as smallpox. The PD MPXV model will be valuable in the study of virulence factors within the two clades, orthopoxvirus therapeutics, transmission/shedding of MPXV, as well as the study of other human orthopoxviruses.
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Received 26 June 2008;
accepted 8 October 2008.
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