MONKEYPOX ZOONOTIC ASSOCIATIONS: INSIGHTS FROM LABORATORY EVALUATION OF ANIMALS ASSOCIATED WITH THE MULTI-STATE US OUTBREAK

Acquisition of potentially infected animals.

Animal specimens were submitted to CDC for MPXV evaluation by state departments of health and agriculture; these animals were sent as frozen carcasses or were transported as live animals to the CDC in compliance with the Institutional Animal Care and Use Committee (IACUC) standards. Animals involved directly in the Ghana shipment and animals associated with persons with monkeypox were among the first to be submitted for evaluation and analyses. Subsequently, the state of Illinois provided all animals from the exotic animal dealership in Illinois suspected to have been the site of MPXV transmission between African and North American species. Many arrived at CDC after the last reported case of human monkeypox in the United States.

Animal maintenance, necropsy, and tissue specimen collection.

Live animals were housed and observed for visible signs of orthopoxvirus infection in accordance with CDC IACUC (species-specific) protocols. Necropsies on animals were performed according to IACUC standards in a biosafety level (BSL)-3 laboratory with high efficiency particulate air (HEPA)-filtered down-draft necropsy table and using full BSL-3 personal protective equipment. When available, 18 samples were individually collected from each animal. The 18 samples included oral swab, ocular swab, eyelid, tongue, lesion (if apparent), blood, serum, heart, lung, liver, spleen, kidney, abdominal skin, lymph node, gonad, urine, feces, and brain. To avoid cross-contamination, instruments (scalpel, scissors, forceps, etc.) were cleaned with 20% Lysol (Lysol-Reckitt Benckiser Inc., Parsippany, NJ) and 10% Clorox bleach (Bleach-Fisher Scientific, Pittsburgh, PA) between collection of each tissue. Tissue snips of approximately equal size from each of the readily homogenized soft tissues including heart, lung, liver, spleen, kidney, lymph node, gonad, and brain tissue were combined in one tube to form the “pooled tissue” sample. Tissues were frozen and stored at −70°C before further processing. Oral and ocular swabs were collected on sterile individual Dacron swabs and stored frozen without diluent. Pooled tissue collections were used to screen for evidence of Orthopoxvirus DNA before the analysis of individual tissue samples. Serum was separated from whole blood collected by either cardiac puncture from fully anesthetized animals before death or from venous collections from live, anesthetized animals.

Tissue preparation.

All tissue processing work was performed under BSL-3 conditions. Pooled or individual tissues, along with 1.0 mL of phosphate-buffered saline (PBS), were placed in a sterile 50-mL disposable dounce homogenizer (Kendall Large Tissue Grinders, catalog 3500SA; Tyco Healthcare, Kendall Company, Mansfield, MA) and ground thoroughly to create a tissue slurry. Virus was isolated from the slurry, and virus DNA was prepared (catalog 732-6340; Bio-Rad, Hercules, CA) from an aliquot of the slurry (see below).

Real-time polymerase chain reaction analysis.

If any pooled specimens were positive in the initial Orthopoxvirus real-time polymerase chain reaction (PCR) test, subsequent real-time PCR testing of individual tissue specimens was done to detect and quantify viral DNA in the various individual organs. Forward and reverse PCR primers and probe, complimentary to regions of the E9L (DNA polymerase) gene, were designed to enable detection of all known Eurasian or-thopoxviruses except for variola.²³ Monkeypox DNA (10 fg to 1 ng) was used as a positive, quantified control, and six wells with water were used as negative controls. Reactions were placed in a 7700 or 7900 ABI SDS (Applied Biosystems, Foster City, CA) detector and subjected to the following thermal cycle parameters: 95°C for 10 minutes and then 95°C for 15 seconds and 63°C for 1 minute for 45 cycles.

Tissues that tested positive as a result of evaluation with the generic E9L Orthopoxvirus real-time PCR assay were further evaluated with a MPXV-specific real-time PCR assay to provide virus species specificity to the diagnosis and to further quantify the viral DNA present in the samples. Forward and reverse primers and probe for this assay were specific to the B6R gene of monkeypox.²³ Monkeypox DNA (50 fg to 5 pg) was used as a positive, quantified control, and six wells with water were used as negative controls. Reactions were placed in a Biorad iCycler (Bio-Rad) and subjected to the following thermal cycle parameters: 94°C for 10 seconds and then 94°C for 5 seconds, 57°C for 15 seconds, and 70°C for 20 seconds for 45 cycles.

Virus–tissue infectivity.

Tissue specimens that tested PCR positive for MPXV DNA were also evaluated for viable virus in cell culture. Plaque-forming virus was detected after making 10-fold dilutions of tissue slurry and adding the dilutions to BSC-40 cell monolayers (African green monkey kidney cell line), which were incubated at 36°C, 6% CO₂ for 72 hours in liquid medium and subsequently fixed and stained with formalin and crystal violet to reveal plaques.

Serologic analysis.

A modified enzyme-linked immunosorbent assay (ELISA) was used for analysis of anti-Orthopoxvirus immunoglobulin types A and G.²⁴ Microtiter plates (Immulon II; Dynatech, Chantilly, VA) were coated with purified vaccinia virus (DryVax) at 0.01μg/well in carbonate buffer overnight at 4°C. After inactivation with 10% formalin (buffered) for 10 minutes at room temperature, plates were blocked for 30 minutes at room temperature with assay diluent (PBS, 0.01 mol/L, pH 7.4 [cat. 93-0223 DK; GibcoBRL, Gaithersburg, MD] + 0.05% Tween-20, 5% dried skim milk, 2% normal goat serum, and 2% bovine serum albumin followed by washing three times in PBST [0.05% Tween-20]). Animal test sera were added to plates at dilutions of 1:100, 1:400, 1:1,600, and 1:6,400 in assay diluent, and plates were incubated for 1 hour at 37°C. Plates were washed, and a 1:30,000 dilution (in assay diluent) of Immunopure A/G conjugate (Pierce ImmunoPure Protein A/G/Peroxidase conjugated, 32490; Pierce, Rockford, IL) was added and incubated for 1 hour at 37°C. Plates were washed, and peroxidase substrate (50-76-05; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and allowed to develop for 5–15 minutes. After development, stop solution (50-85-05; Kirkegaard & Perry Laboratories) was added, and optical densities (ODs) were read on a spectrophotometer (Spectra-MAX 190; Molecular Devices, Sunnyvale, CA) 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. Negative controls were used to generate a cut-off value (COV) for each plate by averaging the negative controls and adding three times the SD. Specimens were considered positive if two consecutive serum dilutions were above the COV.

Aggregate animal test results.

Tissues from a total of 249 animals of 26 different species were tested with real-time PCR DNA detection methods. Of these, 33 individual animals tested positive for MPXV-specific DNA. Of these, virus isolation was attempted from 30 animals, and 22 of these animals were virus isolate positive. In addition, 28 of 172 animals (from 16 different species) tested positive for anti-Orthopoxvirus antibodies (Table 1).

Virus detected in African animals.

Among the imported African rodents that were examined, three species showed evidence of MPXV infection. These included three rope squirrels (Funisciuris sp.), two giant pouched rats (Cricetomys spp.), and nine dormice (Graphiuris sp.).

The rope squirrels (Funisciurus sp.) had been separated from the remainder of the shipment immediately after arrival in the United States (Texas) and had been trans-shipped to New Jersey. Many of these were reported to have died on-site in New Jersey and were frozen. A subset of these were later sent to the CDC for evaluation, and 3 of 11 had varying degrees of detectable MPXV DNA and/or infectious virus throughout tissue samples. (Figures 1 and 2). Because of the condition of the carcasses, we were unable to obtain serum from these animals for Orthopoxvirus antibody assays.

Tissues from two pouched rats (Cricetomys sp.) had MPXV DNA and/or infectious virus. One of these animals, identified as a juvenile, died shortly after arrival in Texas and was preserved as a frozen carcass. This animal (099) showed relatively high concentrations of both viral DNA and infectious virus throughout all tissues (e.g., the spleen from this animal had 2 billion viral genome equivalent/g tissue and 20 million infectious particles; Figure 3). The second MPXV DNA-positive pouched rat had been sent to a distributor in Illinois (IL1)¹⁷ and was purchased and taken to Indiana. From this apparently healthy animal, MPXV DNA was found in gonad tissue only (data not shown). No infectious virus was recovered from this animal, but a serum sample showed evidence of anti-Orthopoxvirus antibodies. Indeed, 12 of 18 pouched rats (Cricetomys spp.), sampled as apparently healthy specimens from the original shipment, tested positive for orthopoxvirus antibodies (Figure 4).

Nine of 40 African dormice (Graphiurus spp.) yielded positive results for MPXV by real-time PCR, and infectious virus was recovered from 8 of these animals (Figures 5 and 6). Only one of these nine dormice (320) had serum recovered for antibody testing, and this sample was positive (Figure 7). At least seven of the nine PCR- or isolate-positive dormice (originally from the Ghana shipment) were submitted to the CDC after having been maintained at the exotic pet distributor (IL1). The origin of two of nine of the virus-positive dormice could not be definitively identified (Table 1). Many of the MPXV-infected dormice (positive by real-time PCR or virus culture) died without obvious signs or external lesions. However, two of these dormice showed evidence of prolonged presence of MPXV DNA. Both of these animals arrived at the CDC alive, at which time PCR-positive oral and/or ocular swabs were obtained. The first dormouse, which was among those acquired from IL1 distributor, died spontaneously without obvious antemortem illness ~1 month after arrival at the CDC. Postmortem tests were positive for viral DNA and infectious virus. The second dormouse (320), derived from the same source, lived at the CDC for ~6 months before it was killed. Postmortem tests for this dormouse were positive for MPXV DNA but negative for infectious virus; however, this animal was strongly positive for Orthopoxvirus antibodies (Figure 7). Twenty-eight dormice, previously housed at a distributor in Iowa (IA distributor),¹⁷ showed no evidence of infection by real-time PCR. Twenty-four of the 28 dormice housed at distributor IA were tested for Orthopoxvirus antibodies, and 1 of these (294) had a weak antibody response to Orthopoxvirus antigen (Figure 7).

MPXV-negative animals from African shipment.

In addition to the three rodent genera discussed above, three additional species shipped on the April 2003 African shipment were evaluated for Orthopoxvirus DNA by real-time PCR and/or for evidence of Orthopoxvirus genus–specific antibodies. These included 2 cusimanse (Crossarchus obscurus), 1 genet (Genetta genetta), and 27 sun squirrels (Helosciurus gambianus). All 30 of these individual animals were found to be negative for MPXV DNA. Additionally, 10 of the sun squirrels were tested for anti-Orthopoxvirus antibodies, and all 10 were Orthopoxvirus sero-negative (Table 1).

North American species results.

Although at least three African species showed MPXV infection, all human cases of monkeypox during the 2003 US outbreak were associated with MPXV-infected black-tailed prairie dog (Cynomys ludovicianus) contact¹⁷ (unpublished data). These prairie dogs had been housed in the same Illinois establishment (IL1) as giant pouched rats that originated from Ghana. Fourteen of 20 prairie dogs submitted for testing were PCR positive for MPXV DNA, and infectious virus was recovered from 9 of 11 (82%). Of these, all 14 were associated with confirmed human monkeypox cases (unpublished data). Although the quantitative DNA viral loads and infectious virus titers varied between individual tissues and excreta, the prairie dogs generally had higher yields of virus or viral DNA than did the other animal species. Some of these tissues had virus in excess of 1 billion PFU/g tissue (Figures 8 and 9).

Evidence for MPXV in additional species.

Several additional mammals, which had been housed at distributor IL1 but which were clearly not part of the shipment of animals from Ghana, also showed evidence of MPXV infection. A southern opossum (Didelphis marsupialis, native to South America) had varying degrees of MPXV DNA and infectious virus throughout its tissues (data not shown). Monkeypox virus DNA also was detected in a hedgehog (Atelerix sp.), a gray short-tailed opossum (Monodelphis domestica), and a jerboa (Jaculus sp.), all originating from distributor IL1 (Table 1). The hedgehog and jerboa died without obvious antemortem illness after ~3 months at the CDC. The gray short-tailed opossum died during anesthesia after ~4 months at the CDC. All three of these animals had low amounts of viral DNA in several tissue types, although no infectious virus was recovered (data not shown).

There were also several animals associated with distributor IL1 that showed Orthopoxvirus antibodies while being both negative for viral DNA and infectious virus. These included 1 of 2 chinchillas (Chinchilla lanigera), 10 of 16 coatimundis (Nasua nasua), 2 of 43 hedge hogs (Atelerix spp.), 1 of 6 southern opossums (Didelphis marsupialis), and 11 of 19 giant pouched rats (Cricetomys spp.). All 19 giant pouched rats were subsequently traced to the original Ghana shipment (unpublished data; Table 1).

Many additional animals from the Illinois distributor were found to be negative for orthopoxvirus infection by real-time PCR, serology, and viral culture and showed no outward evidence of poxvirus infections (Table 1).

A young woodchuck (Marmota monax) that had been co-housed with a prairie dog suspected of being infected with MPXV had real-time PCR evidence of MPXV DNA in 13 of the tissues sampled, including gonad, spleen, and tongue (Table 1).

Overview.

These data help to further delineate the range of species infected with MPXV during the 2003 US outbreak, as well as provide a better understanding of appropriate testing algorithms for sensitive diagnoses. At least three African genera associated with the shipment of animals from Ghana were MPXV isolate positive. However, for these three genera, it was impossible to determine if infection occurred before, during, or shortly after importation. Genetic comparison of MPXV genomes isolated from a human, a prairie dog, a rope squirrel, a dormouse, and a giant pouched rat associated with the US outbreak revealed these isolates to be essentially identical (unpublished data).²⁵ Laboratory data showed that multiple prairie dogs, many of which were epidemiologically linked to human cases of disease, harbored large amounts of MPXV.

Collection of individual tissues from various MPXV-infected animals provided information regarding virus and viral DNA within the infected animals. However, kinetics of virus replication and distribution in various tissues could not be determined in the absence of longitudinal infection studies.

Preliminary studies, and direct comparisons with individual organs from animals, showed that the real-time PCR assays used in these studies were more sensitive for detection of MPXV infection than even viral culture (e.g., Figures 1, 2, 3, 5, 6, 8, and 9); however, the presence of readily detectable MPXV DNA was highly correlated (r = 0.81, P < 0.001; Spearman rank-correlation test) with the presence of infectious virus. Therefore femtograms of MPXV DNA per gram tissue and plaque-forming units per gram tissue have a significant strong positive correlation and add further support to the conclusion that the viral plaque-forming units were caused by MPXV. Real-time PCR, therefore, was considered an advantageous diagnostic tool, because it was also generally less labor intensive and less costly than tissue culture.

Distribution of virus within tissues: implications for diagnosis and modes of virus transmission.

We did not observe a single tissue type that was uniformly highest in its MPXV DNA for all species and hence always clearly optimal for testing for MPXV-specific DNA. We had the largest sample sizes of tissues from prairie dogs and dormice from which tissue comparisons could be made. Considering DNA results from 10 of 14 PCR-positive prairie dogs (Figure 8), the eyelid (10/10), skin (10/10), and lymph node (10/10) were uniformly positive for MPXV DNA. When pox-like lesions were noted on individual prairie dogs and sampled, five of five such samples were positive for MPXV by real-time PCR. Among nine MPXV DNA-positive dormice, the blood (6/6), eyelid (8/8), gonad (8/8), heart (9/9), kidney (9/9), liver (9/9), lung (8/8), lymph node (4/4), skin (9/9), and urine (4/4) yielded positive PCR results (Figure 5). In the future, a smaller collection of tissues for screening from each animal can now be collected without losing the ability to identify infected animals. The brain, eyelid, gonad, lymph node, spleen, skin, and lesion (when present), when comparing all MPXV PCR-positive animals, had an average detection rate of 85%. The liver, heart, lung, and tongue had a slightly lower probability of MPXV detection, with a rate of 81%, whereas blood, urine, kidney, and feces had a detection rate of 72%.

When comparing viable virus levels (PFU/g tissue) from infected dormice and prairie dogs, there were some noteworthy findings. For seven MPXV-positive prairie dogs, the skin and tongue tissues seemed to be relatively consistent sources for recovery of viable virus (five of seven) compared with the other tissues tested (Figure 9). In contrast, almost all dormice tissues were uniformly positive for viable virus, with the exception of the feces, which only yielded detectable virus in two of eight fecal samples tested (Figure 6). Without more information relating to the pathogenesis of MPXV infections at the time of sampling, it is not possible at this juncture to say if the apparent differences in viable virus distributions among tissues between these two species of animals was a reflection of virus–host tissue tropisms or simply the state of disease progression when the different sets of animals were sampled. Another difference observed occurred in at least three animals (pouched rat 99, Figure 3; dormouse 1, Figure 6; prairie dog 6, Figure 9), in which evidence of MPXV was detected in their kidneys without detectable virus in their urine at the time of urine collection.

The findings of MPXV and viral DNA in various tissues from a wide variety of animal species are concordant with previous observations that MPXV infects a wide range of potential experimental hosts.^8–11 The potential for infected prairie dogs to shed virus and potentially transmit virus to humans and other animals by a variety of routes seems plausible (i.e., biting, licking, scratching, direct contact). Whether any of these sources of virus could have resulted in air-borne infectious material remains speculative. We documented viable virus in the lesion, tongue, skin, lung, and eyelid samples from infected prairie dogs associated with the outbreak; observations that are consistent with the published pathology descriptions of prairie dogs associated with the outbreak and data that had previously suggested that both respiratory and mucocutaneous transmission of MPXV was possible.^26,27 The presence of MPXV in multiple tissues from these prairie dogs was also consistent with data from subsequent experimental infections of prairie dogs.²⁸ We observed a high median level of viable virus in prairie dog fecal samples and one prairie dog urine sample, suggesting potential for fomite transmission (Figure 9).

Why African species were not directly implicated in human disease, despite findings of generalized and occasionally high-titered MPXV infections, remains a mystery. In general, of the tissues we had the opportunity to study, prairie dog tissues reached higher virus titers than did African species, although examples of dormouse tissue (e.g., liver) had MPXV titers equivalent to those of high-titered prairie dog tissue. Behavioral features of prairie dog and human interactions (e.g., anecdotal reports suggested extensive human–prairie dog handling, as well as reports of prairie dog biting and scratching) may have also predisposed the prairie dogs to be especially effective vectors for human disease.

Specificity of animal ELISA test.

Of 138 animals that were tested with both the MPXV real-time PCR and Orthopoxvirus-generic ELISA, 3 were real-time PCR positive. Of these three MPXV real-time PCR–positive animals, one was Orthopoxvirus antibody negative (a gray short-tailed opossum). However, control tests showed that the serum of this opossum had a low avidity for the commercially available anti-serum conjugate, and hence, the true antibody status could not be accounted for with the methods used in this study; this observation emphasizes the requisite evaluation of serologic reagent specificity. Establishing precise cut-off values for determination of positive and negative antibody titers was not possible in the absence of known, control-negative sera from exotic species; identification of serologic COV was instead done using a statistical algorithm and by comparison with known negative human sera. As with previous studies that investigated the presence of Orthopoxvirus-generic antibodies, the cross-reactive nature of such antibodies precludes concluding with certainty that animals with evidence for anti-Orthopoxvirus antibodies had been exposed only to monkey-pox and no other orthopoxviruses. Certain animals showed no evidence for infectious MPXV or viral DNA but did test positive for anti-Orthopoxvirus antibodies. To the best of our knowledge, all of the Orthopoxvirus antibody–positive animals had been housed under conditions wherein there was clear evidence of MPXV transmission among species (as determined by PCR and virus isolation), and monkeypox infections would have been a likely source of antibody conversion. Absence of anti-Orthopoxvirus antibodies was relevant to interpretation of absence of possible MPXV infections.

Which animals were the imported disease reservoirs?

Our results strongly support the observation that at least two African genera were infected with MPXV at the time of arrival in North America (rope squirrel and giant pouched rat). Dormice also may have been infected before and during transit; however, no samples of dormice were available from immediately after the importation event. The absence of clear evidence for active dormice infection at distributor IA is consistent with the secondary infection of dormice MPXV infections at distributor IL1, subsequent to importation. Evidence derived after the importation event has suggested that some dormice and giant pouched rats survived infections but had evidence of prolonged persistence of MPXV or MPXV DNA in their tissues and/or evidence of Orthopoxvirus antibodies.

Importantly, none of the 27 sun squirrels recovered from the original African shipment had any evidence of real-time PCR monkeypox DNA in tissue and/or swab samples. Furthermore, 10 of 10 of these sun squirrels were found to be negative for Orthopoxvirus antibody. Representatives of this genus (Heliosciurus) have previously been shown to have anti-Orthopoxvirus antibodies in previous studies in Central Africa,^12,15,16 and subsequent to these observations, sun squirrels were included on the list of possible species involved with naturally occurring sylvan monkeypox. It is worth noting that none of the sun squirrels seemed to acquire infections during the collection and shipping process from Africa to the United States or during subsequent transshipment to New Jersey, unlike the rope squirrels that were part of the same Ghanaian shipment to New Jersey (through Texas). It is now well recognized that there are two distinct clades of MPXV,^25,29 and their distributions seem to correspond to the Congo River Basin compared with more coastal West Africa (origin of the 2003 rodents imported from Ghana). These observations provide evidence for speculation that the sun squirrel may serve as a potential reservoir for the Congo Basin MPXV clade but perhaps not for the West African clade of MPXV, consistent with the suggestion of separate reservoirs for each MPXV clade.²⁵

Laboratory evidence of transmission.

The events, or animal husbandry practices, that may have contributed to the seminal transmission(s) of MPXV from African species to prairie dogs and other species will be the subject of a more detailed analysis. Our results are consistent with several species having been infected within the environment of a specific distributor’s primary holding compound (a converted, stand-alone garage). It is, however, unclear from these laboratory findings whether all of these species were infected from one or more of the African species associated with the initial shipment or whether the amplification of virus in additional species (e.g., prairie dogs) may have played a role in subsequent rounds of infections. The infection of a captive ground hog (also a burrowing sciurid) in contact with a prairie dog does provides at least one reasonably clearly defined example for prairie dog MPXV transmission to another North America species (other than humans). Elucidation of modes of transmission between species, for example as by fomite, arthropod, or possible aerosolized infectious material, will be critical for understanding MPXV ecology and will be the subject of additional experimental studies.

Several species of animals associated with distributor IL1 were shown to be MPXV isolate–positive, MPXV PCR–positive, Orthopoxvirus antibody–positive, or a combination of all three. These included examples of marsupials (two species of opossums), insectivores (hedgehogs), and examples of multiple rodent species (Table 1). Hedgehog, pygmy opossum, and jerboa showed evidence for persistence of MPXV DNA in various tissues. It is unclear how much mortality was associated with MPXV infections with various animal species before recognition of the disease outbreak.

Among the live animals that were sent to the CDC in response to the outbreak, we observed no obvious signs of viral infection (e.g., lesions, ruffled fur), with the exception of fatalities among one cohort of dormice and with the exception of one dormouse that died (without visible lesions) 1 month after arrival at CDC. Although there was clear evidence of past infections (MPXV DNA and Orthopoxvirus-positive serology), we found no further obvious signs of poxvirus disease among the remaining animals.

The contrasting absence of obvious disease and only minimal antibody titers in dormice from the same African shipment that were trans-shipped to distributor IA suggests that either the majority of the dormice became infected at distributor IL1 or that environmental conditions there favored enhancing the course of monkeypox progression and transmission.

Potential for establishment of sylvatic MPXV.

The potential for MPXV to become an ongoing disease within native North American species or introduced, invasive species largely remains an open question. The recent recognition of a population of introduced African pouched rats in the Florida Keys suggests the possibility for potential African MPXV reservoir species to themselves become established in the Western Hemisphere.³⁰ To study possible transmission to wild animals after the 2003 MPXV outbreak, the US Department of Agriculture (USDA) Wildlife Services and the US Geologic Survey’s National Wildlife Health Center (NWHC) live-trapped animals in proximity to premises of six Wisconsin human MPXV cases (and associated captive prairie dogs). Trapped animals were tested at the NWHC for MPXV infection by serology (hemagglutination inhibition with vaccinia antigen) and standard PCR assay.³¹ Specimens from 201 animals, primarily rodents, were tested. Species included opossums, shrews, several squirrel species, chipmunks, voles, several mice species, a woodchuck, a skunk, a rat, and an eastern cottontail rabbit. By these methods, no evidence of infection with orthopoxvirus was detected (Christopher Brand, NWHC, and David Nelson, USDA, personal communication, 2004).

Further trapping studies were performed in Illinois by the Illinois Wildlife Services program and included three locations linked by trash disposal routes to distributor IL1. A total of 43 animals from these three sites were trapped during August 2003 including several species of mice, raccoons, a skunk, opossums, a prairie vole, and a rat. PCR and serology testing were the same as described above. All 43 animals were negative for evidence of MPXV infections (Kirk Gustad, personal communication, 2004). Any attempt at direct comparison of test results must recognize that standard PCR is typically considered to be at least 100 times less sensitive than real-time PCR.

Sobering precedents exist for accidental or intentional introductions of poxviruses into novel host species in novel environments including myxoma virus (a Leporipoxvirus) in Australia and Europe and vaccinia virus (another member of the genus Orthopoxvirus) in South America.^32–35

The emergence of MPXV in the United States has been another reminder of the potential impact of global commerce on both human and non-human infectious disease. Additionally, the outbreak of human monkeypox and the association with various non-human animal species will help to refocus attention on the natural history of monkeypox in Africa.