Two plasmid constructs were made, one to incorporate CCP in the West African USA 2003 strain, and one to remove the gene from the Congo Basin ROC 2003 strain. The insertion of the CCP gene into USA 2003 clade was only begun after careful consideration and discussions with the institutional biosafety committee, the institutional animal care and use committee, and the select agent program. Approval was obtained since insertion of the gene encoding the CCP into the strain of virus that lacked its expression was an appropriate control to determine its function in virulence. Development of the ROCΔCCP and USA+CCP recombinant viruses required the incorporation of the selectable gpt gene marker from the pelP1-gpt vector. The gpt expression cassette was placed under the control of a vaccinia-derived synthetic early-late promoter (Figure 1). The complement control protein in ROC 2003 was knocked out by double homologous recombination at two flanking loci, deleting the gene and simultaneously conferring mycophenolic acid resistance to the recombinant. The USA+CCP recombinant was created by incorporating the Congo Basin strain CCP gene under the control of its predicted upstream promoter into the intergenic region between ORFs 176 & 177 (Figure 1).

Recombinants were analyzed for second site mutations by whole genome sequencing. Comparison of the USA+CCP genome (10,890 reads) to the published sequence showed one single nucleotide polymorphism (SNP) and one single nucleotide deletion, with neither located in coding or promoter regions. Mapping the sequencing reads from ROCΔCCP to the published ROC 2003 genome resulted in two contiguous sequences (contigs) (11,779 reads) separated by the deleted CCP region. Nucleotide comparisons revealed two SNPs. These SNPs affected a gene fragment of the A-type inclusion (ATI) protein causing an Asp to Val change at amino acid position 388 and a Val to Asp at position 386. These SNPs occurred in a repeat rich region of the truncated protein. It has been previously established that monkeypox viruses do not produce A-type inclusion bodies due to a frameshift mutation that causes premature truncation [4], [31]. Studies where the ati gene in cowpox virus was knocked out and compared to wild-type in mice have demonstrated limited effects on pathogenesis, adding to its classification as a non-essential protein product [32]. Since these two SNPs are within a truncated, non-essential gene, the alterations should not have a significant effect on the systemic disease pathogenesis of this recombinant.

Complement control protein expression in the USA+CCP recombinant and its absence in the ROCΔCCP knockout recombinant were evaluated by Western blot. Cell extracts and supernatants were prepared from infected BSC-40 cells at 24 hours post infection (hpi). The cell extracts and supernatants (concentrated ∼5X) were probed with rabbit-derived polyclonal antisera targeted against the vaccinia complement control protein (Figure 2A, 2B). Vaccinia-infected cell supernatants and cell extract samples were treated similarly to provide a positive control. In Western blot analysis, the expected size difference between the 35 kDa vaccinia CCP homologue and 24 kDa CCP in ROC 2003 and USA+CCP strains was readily apparent. CCP was not evident in the concentrated supernatants of ROCΔCCP or USA 2003-infected cells and the presence of CCP was equivalent in the cell extracts or concentrated supernatants from USA+CCP and ROC 2003-infected cells (Figure 2A and 2B). Quantification of the secreted monkeypox CCP (USA+CCP or ROC 2003), based on Western blot, was approximately five times lower than that detected from vaccinia WR infected cell supernatants (Figure 2A). There was similar accumulation of CCP within the ROC 2003 and USA+CCP cell extracts versus the vaccinia cell extract (Figure 2B). Importantly, there were no significant differences in CCP levels (supernatant or cell extract) between the ROC 2003 and USA+CCP recombinant at 24 hours post infection.

To determine at what stage of infection CCP was expressed, we used Ara-C, which results in the inhibition of late protein expression [33]. Cells were infected with either the ROC 2003 strain of monkeypox virus or vaccinia WR in the presence or absence of Ara-C, and supernatant was harvested and replaced with fresh media at the indicated times (10, 24, and 36 hpi, Figure 2C). The collected supernatants (∼5X concentrated) were analyzed by Western blot using a polyclonal anti-VCP antibody. Samples taken at earlier times (2, 4, and 6 hpi) were negative for CCP and VCP expression in the absence of Ara-C (data not shown). Growth of the virus from 6 to 10 hpi, allowed sufficient time for VCP to accumulate and be detected by Western blot, meanwhile monkeypox CCP expression was absent. In the sample collected at 24 hpi, the monkeypox homologue was first detected (Figure 2C). The addition of Ara-C eliminated the ability to detect VCP and monkeypox CCP by Western blot at any time point (data not shown). To evaluate the expression of monkeypox CCP in the USA+CCP recombinant, we chose a single time point (24 hpi) that based upon our time course experiment would have a sufficient amount of CCP expression. Western blots of supernatants (∼5X concentrated) from BSC-40 cells infected with USA+CCP MPXV, ROC 2003 MPXV, or vaccinia WR in the presence/absence of Ara-C were probed with anti-VCP rabbit polyclonal sera (Figure 2D). We found that USA+CCP and ROC 2003 accumulated similar levels of secreted CCP at 24 hours (Figure 2D). The CCP gene in USA+CCP is expressed at late times and likely under the control of the predicted upstream late promoter, derived from the ROC 2003 strain of monkeypox virus, contained within the construct.

We compared the biological activity of the secreted complement control protein from the USA+CCP strain and the ROC 2003 strain, using a C4b binding activity assay (Figure 3). This assay determined the ability of concentrated virus supernatants containing CCP to interact with human C4b coated on a microtiter plate. The monkeypox-infected cell supernatants, from ROC 2003 or USA+CCP infections, manifested equivalent levels of C4b binding (Figure 3). No C4b binding activity was seen in the supernatants from cells infected with either the ROCΔCCP or USA strains of MPXV.

To further evaluate whether the recombination manipulations affected the biologic properties of the viruses, relative to their growth kinetics, viral one step growth curves were compared. Over 72 hours, no significant differences in growth kinetics were observed between the recombinant and the parental viruses (Figure 4).

Previous work with black-tailed prairie dogs (Cynomys ludovicianus) has demonstrated that monkeypox virus challenge of this species provides an animal model that resembles many aspects of human systemic orthopoxvirus disease [5], [7], [8]. Four challenge groups, each containing four prairie dogs, were inoculated intranasally with 4×10⁵ pfu of ROC 2003, ROCΔCCP, USA 2003 or USA+CCP in a total volume of 10 microliters. This dose was ∼3 times greater than the LD₅₀ for the West African strain (1.29×10⁵ pfu) and ∼70 times greater than the LD₅₀ for the Congo Basin strain (5.9×10³ pfu) in prairie dogs [34]. All animals were observed daily for signs of illness, with weight measurements, swabs, and blood samples taken every other day for the duration of the study (30 days).

Similar to previous observations using the intranasal route of infection [8], the first observable presentation of illness was nasal swelling and lesions in the oral cavity (Figure 5). In animals challenged with the recombinant ROCΔCCP, there was a delay in illness onset. ROC 2003 challenged animals manifested nasal swelling at day 6 (2 of 4 animals) and primary lesions (all 4 animals) consisting of several flat, circular macules on the tongue and palate between days 8–10. In contrast, animals challenged with ROCΔCCP manifested nasal swelling later; only one animal showed any nasal swelling at day 8, and primary oropharyngeal lesions did not develop until day 10 (Figure 5, Table 1).

Viable virus and viral DNA was detected in the oral cavity as early as day 2 and remained for several days (Figures 6A, 7A), persisting after the development of orthopoxvirus-specific antibodies beginning on day 12 (Figure 8) in ROC 2003-infected animals. Similar to observation of delayed onset of oropharyngeal lesions, viable virus and viral DNA was detected only after day 4 in ROCΔCCP infected animals (Figures 6A, 7A).

Viral DNA was detected in whole blood of ROC 2003-infected animals at day 4, and not until day 6 in ROCΔCCP infected animals. The overall level of viral DNA in whole blood, on average, was lower in ROCΔCCP infected animals than in ROC 2003-infected animals. (Figure 7D). ROC 2003-infected animals developed a disseminated, vesiculo-pustular rash between days 10–12, with average lesion sizes measuring 5 mm in diameter appearing on the back, abdomen, armpit and hindleg regions of the prairie dogs (Table 1). The ROC 2003-infected prairie dogs developed secondary lesions on multiple sites, such as the back, armpits, and abdominal area; lesions were also observed on the footpads of all prairie dogs. In some animals, pustules developed on the lip and edges of the eyelids, and lesions were found on the genitals of two male animals. In most ROCΔCCP infected animals, secondary or generalized lesion onset was delayed with respect to the observations within the ROC-infected cohort, although in one ROCΔCCP infected animal (PD17) we observed the appearance of a secondary lesion on its neck on the same day as its first oral lesion (day 10). Secondary lesions were found in the other ROCΔCCP-infected prairie dogs between days 12–14 (Figure 5, Table 1), with a similar lesion distribution as seen in the ROC 2003-infected animals, but the lesion load was less than what was documented in the ROC 2003-infected animals (Table 1).

Twelve days post-infection it was evident that weight loss in the ROC 2003 group was occurring more rapidly and at a greater magnitude than any of the other challenge groups with 17% weight loss vs 8% for ROCΔCCP, 13% for USA 2003 and 11% for USA+CCP on this day (Figure 9). One animal was euthanized on this day (PD18) due to inappetence, lesion severity, lethargy, and labored breathing. The condition/health of the remaining animals in this group rapidly declined as an additional prairie dog was euthanized on day 14 (PD10) and the remaining two animals were euthanized by day 16, due to a combination of disease symptoms such as lethargy, labored breathing, and inappetence including >25% weight loss for PD9 (Figure 9, Table 1). Both viable virus and viral DNA were first detected on day 2 and persisted until the death of each animal (Figures 6 & 7, Table 1). Upon necropsy, viable virus was detected at varying levels in all sampled tissues with peak amounts found within the kidney for all four animals (Table 2).

Conversely, the ROCΔCCP-infected cohort demonstrated resolution of disease; weight gain reinitiated between days 18 and 20, by day 16 all lesions had scabbed over, and by day 22 most of the crusts had fallen off, leaving hypopigmented scars on the skin. Prior to euthanasia of the ROC-infected animals, there were no significant differences between the viable viral loads in oral, ocular or anal excreta of ROCΔCCP-infected and ROC 2003-infected animals on any day of the study, except for oral swabs taken on day 12 post-infection (p = 0.03) (Figure 6A, B, & C). On this particular sample day, the ROCΔCCP-infected cohort average viable virus load was lower than that of the wild-type ROC-infected cohort. The average oral and ocular shedding of viable ROCΔCCP virus peaked day 10, and consistently decreased until it was undetectable after day 22 (Figure 6A and 6B). Anal swabs detected viable ROCΔCCP through day 20 with peak average shedding occurring on day 16. The duration of viral shedding cannot be compared to the wild-type ROC 2003-infected animals since these succumbed to disease by day 16 post-infection, however the ROCΔCCP-infected animals demonstrated shedding of viable virus longer than seen for the USA-infected animals (Table 1 and Figure 6). The ROCΔCCP-infected cohort displayed slower weight loss than the wild-type ROC-infected cohort. Linear regression analysis of the average weight loss data from the highest to the lowest weight indicated that the slope of the ROC 2003-infected animals was significantly higher than in ROCΔCCP group (p = 0.03). The analysis also showed that the average weight loss in the ROCΔCCP group was not significantly different from the USA 2003 group (p = 0.69). These findings highlighted the fact that on average the ROC 2003-infected animals displayed signs of greater disease severity (such as rapid weight loss) than the ROCΔCCP-infected prairie dogs.

Although not statistically significant, all ROCΔCCP-infected prairie dogs seroconverted to the highest level of any of the four groups in the challenge study (Figure 8). At necropsy, no viable virus was found in any of the tissues from the ROCΔCCP-infected animals (Table 2) but weak positives for viral DNA were detected in the following tissues of at least two animals in this group: lungs, heart and kidney (data not shown).

Prairie dogs infected with USA 2003 did not show any signs of infection until day 10 when the first macules appeared on the tongues and palates of 2 animals (PD4 and PD16) (Figure 5). Only animals in the USA+CCP group exhibited the early symptom of nasal swelling on day 6 (PD12) and on day 8 (PD7) (Figure 5). Macular lesions appeared on the tongue or palate in all animals on day 10, similar to that observed with the USA 2003-infected prairie dogs.

Within the USA 2003 infected group, on day 10, one of the prairie dogs (PD2) in this group did not wake up post-anesthesia. PD2 did not demonstrate lesion development or other overt signs of disease on day 10, and did not have significant weight loss (<10%, Figure 9), and had very low viable virus levels in tissues upon necropsy (Table 2), suggesting its death most likely was due to complications with anesthesia and not related to monkeypox infection.

Unexpectedly, viable virus was detected relatively early (Day 2) in the oral swab of one USA 2003-infected animal (PD16), and in others by day 4. Viral excreta in ocular or anal swabs of animals infected with USA 2003 were not observed until day 6 and persisted in surviving animals out to day 14 for ocular swabs and day 18 for anal swabs (Figure 6). Viral DNA persisted longer than viable virus in oral, ocular and anal excreta samples (Table 1, Figures 6 and 7). Viral DNA was detected within the blood as early as 4 days post-infection and persisted until 22 days post-infection (Figure 7D). Animals challenged with the USA+CCP virus began to excrete virus in the oral, ocular and anal samplings on days 4, 6 and 8 respectively. After day 10, measured viable virus and DNA loads were consistently lower than that observed in the USA 2003-infected animals in the oral, ocular and anal swabs with the only exception being on a day 18 DNA oral swab (Figures 6 and 7). Viral DNA was detected in whole blood on day 6, persisted through day 16 and levels were less than that observed in USA 2003 infected animals throughout the study (Figure 7D). However, none of these differences in viable virus or viral DNA load were statistically significant.

Secondary or generalized lesions (1–3) appeared between days 12 and 14 on the anus of all USA 2003-infected animals and the genitals of two male prairie dogs. On day 14, PD4 was euthanized due to inappetence, lethargy, and labored breathing (Figure 5, Table 1). By day 16 all observed lesions became umbilicated and began to scab over; by day 24 most of the lesions had resolved leaving patches of hypopigmentated skin. As seen in the USA 2003-infected group, the USA+CCP-infected animals did not develop many secondary lesions, a few were found in the mouth or around the anus and these lesions were first identified on day 12 (Table 1). On day 18, two of the animals (PD12 and PD15) were euthanized due to lethargy, inappetence and labored breathing, along with weight loss near the euthanasia limit (Figure 9B). The rate of weight loss was similar for both the wild-type USA 2003-infected cohort and the USA+CCP-infected cohort. Linear regression analysis of the average weight loss data from the highest to the lowest weight showed no significant difference between the slopes of USA 2003 and USA+CCP-infected animals (p = 0.49).

Upon necropsy of the USA 2003-infected animals, viable virus was detected in the lungs, heart, kidney and spleen of PD4 who was euthanized on day 14, while in PD2, the animal who did not revive after anesthesia on day 10, viable virus was detected in the heart, lung, spleen and eyelid samples (Table 2). At necropsy, no viable virus was found in any of the tissues from the animals who survived the viral challenge, although low levels of viral DNA was present in the oral swabs of PD6 and PD16 (Figure 7).

Upon necropsy of the USA+CCP-infected animals that were euthanized on day 18 post-challenge (PD12 and PD15), viable virus was recovered from the lungs, kidney, liver and spleen. Viable virus was found in the liver of USA+CCP or ROC2003-infected animals who did not survive to day 30, but was not found in the liver of USA2003-infected PD4, which was euthanized on day 14 of the challenge study. At the day 30 necropsy of the survivors of the USA+CCP infection, no viable virus was found in any of the tissues, but there was a weak positive DNA signal detected in the eyelid sample of one animal (PD6).

The West African strain of monkeypox virus used for these experiments was MPXV-USA-2003-044 (USA 2003). This isolate was collected during the 2003 monkeypox outbreak in the USA from the lymph node of an infected prairie dog associated with the initial human case in Wisconsin [4]. The Congo Basin strain of monkeypox virus, MPXV-ROC-2003-358 (ROC 2003), was isolated from the rash of a 10-year-old girl infected in Impfondo, Republic of Congo, and admitted to the hospital on June 9, 2003 [39]. Both monkeypox strains were passaged three times prior to working stock production. Supernatant containing VCP produced by low-passage, Vaccinia virus Western Reserve (VACV WR) was utilized for comparisons to monkeypox CCP. BSC-40 cells [40], grown in RPMI 1640 containing 10% sterile-filtered, fetal bovine serum (FBS) were used for all cell culture infections and isolations of all parental and recombinant virus strains, unless otherwise specified.

All work was approved by the Institutional Biosafety Committee and the Select Agent office prior to the initiation of these experiments. The selectable genetic marker used for isolation was the Escherichia coli xanthine-guanine phosphoribosyltransferase or gpt gene, which confers resistance to growth inhibitor, mycophenolic acid [41], [42].

The CCP gene plus 123 bp of the upstream region was PCR amplified from ROC 2003 with primers CCP1, 5′-GTC GAC CTC ATA AGT C AC TGC CAT TGT TTT-3′ and CCP2, 5′-GGA TCC CAT TGT AGT TGT ATG AGT GT A TG-3′ using Platinum High Fidelity DNA Polymerase (Invitrogen, Carlsbad, CA), then ligated into the pZsGreen expression vector at the Sal I and Bam HI sites for the construction of pCDgpt. This insert was positioned into plasmid pCDgpt (Figure 1) adjacent to the gpt gene under the control of a vaccinia-derived, synthetic early-late promoter, 5′-GCA TAT GTA AAA GTT GAA AAT ATA TTT CTA TGC TAT AAA TA-3′ [43] and flanked by sequences homologous to the 765 bp non-coding intergenic region between ORFs 176 and 177 (annotated as type I membrane hemagglutinin and serine/threonine kinase, respectively) in the USA 2003 strain. Plasmid pABgpt (Figure 1) contained the synthetic early-late promoter controlling the gpt gene with upstream and downstream flanking regions homologous to ORFs 21 and 23 (annotated as unknown protein and kelch-like gene fragment, respectively) from the ROC 2003 genome.

The two plasmid constructs were integrated into their host genomes through an infection-transfection protocol. BSC-40 cells in RPMI+2% FBS are seeded in 6-well Costar plates (Corning Inc., Corning, NY) 24 hours prior to the experiment, with an average confluency of ∼80% per well. The cells are washed in Opti-MEM media, then either ROC 2003 or USA 2003 sucrose cushion-purified virus was added to each well at a MOI = 0.05. The viruses were incubated at 36°C, 6% CO₂ for 2 hours, and then replaced with Lipofectamine 2000 (Invitrogen) in Opti-MEM and 4 µg of the plasmid DNA of interest (pABgpt or pCDgpt) for 72 hours.

Virus plaques were purified using mycophenolic acid as the selection agent [41], [42]. The recombinant viruses successfully expressing xanthine-guanine phosphoribosyl-transferase were selected by growth in BSC-40 cells in 2X DMEM supplemented with 2% FBS, 25 µg/ml mycophenolic acid, 250 µg/ml xanthine, 10 µg/ml thymidine, 15 µg/ml hypoxanthine and 2 µg/ml aminopterin (Millipore, Phillipsburg, NJ). Each recombinant strain was plaque purified four times in a 25 µg/ml mycophenolic acid plus 2% agarose overlay containing 3% neutral red, and collected using a Pasteur pipet after incubation at 36°C, 6% CO₂ for 72 hours.

The West African recombinant strain of monkeypox virus, MPXV-USA-2003-044, in which CCP was incorporated into its genome through homologous recombination, was designated USA+CCP. The CCP knockout recombinant derived from the Central African strain, MPXV-ROC-2003-358, was designated ROCΔCCP. Each recombinant clone was sequenced using 454 sequencing in the CDC Biotechnology Core Facility to ensure the genomes were free of second site mutations.

BSC-40 cells were infected with VACV WR, ROC 2003, USA 2003, ROCΔCCP, or USA+CCP virus, at a MOI = 10 in Opti-MEM at 36°C, 6% CO₂ for 1 hour. The media was removed and replaced with Opti-MEM (Invitrogen, Carlsbad, CA) then incubated for 24 hours at 36°C and 6% CO₂. The supernatants were decanted into a centrifuge tube and then spun at 1000× g for 10 min to remove cell debris. After UV inactivation for 2 min. at 25°C, all harvested supernatants were subjected to ∼5-fold concentration (by volume) using the Ultracel YM-10 Centricon protein concentration columns (Millipore, Phillipsburg, NJ) with a 10 kDa molecular weight cut-off. Centricons were spun at 5000× g for 10 min at 25°C and filtrate discarded, retentate was collected at 1000× g for 2 min at 25°C. To harvest the cell lysates, the infected-cell monolayers were washed twice with Opti-MEM, and then RIPA buffer was added to the cells, which were then incubated at 4°C for 5 min (Thermo Scientific, Rockford, IL). The cells were collected and spun at 14,000× g for 15 min at 4°C.

For Western blot analysis, the above virus-infected cell supernatants and cell lysate samples were boiled for 5 min. in Laemmli loading buffer containing 2% sodium dodecyl sulfate and 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA). All supernatant and cell lysate samples (3 µg/ml per well) were loaded onto a 12% precast Tris-glycine polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and then run at 150 volts for 1 hour. Protein bands were transferred at 100 mA for 50 min to polyvinylidene fluoride (PVDF) membranes using a Mini-blot protean II chamber (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated in blocking buffer (1% non-fat milk, 0.1% Tween 20) for 2 hours. The blots were probed with polyclonal rabbit anti-VCP antibody (courtesy of Dr. John Lambris, University of Pennsylvania) at a 1∶1000 concentration for approximately 12 hours on a 10 rpm rocker at 4°C. The blots were also probed with polyclonal rabbit anti-transferrin at 1∶1000 to confirm to that wells received an equivalent load of protein. After the blot was washed with PBS containing 0.1% Tween 20, alkaline phosphatase conjugated goat anti-rabbit antibody (1∶3000) (Bio-Rad Laboratories, Hercules, CA) was added to the blot for 2 hours and the chemiluminescent signal detected using ChemiDoc XRS+ (Bio-Rad Laboratories, Hercules, CA).

BSC-40 cells were infected with VACV WR or ROC 2003 at MOI = 10 in Opti-MEM with or without 10 mg/ml cytosine arabinoside [Ara-C] (Sigma-Aldrich, St. Louis, MO) at 36°C, 6% CO₂ for 1 hour. The media was aspirated, cells washed twice with 5 ml of the appropriate media, and then 7 ml of the corresponding media was added to each flask and incubated at 36°C, 6% CO₂. Infected cell supernatants were collected at 2, 4, 6, 10, 24 and 36 hours post-infection (hpi) and replaced with fresh corresponding media after each harvest (Figure 2C). Ultracel YM-10 Centricon protein concentration columns were used to concentrate the harvested vaccinia and monkeypox supernatants. The samples were spun at 5000× g for 10 min at 25°C and filtrate discarded, retentate was collected at 1000× g for 2 min at 25°C. Samples were analyzed by Western blot as described above.

To compare the temporal expression of CCP between ROC 2003 and USA+CCP strains, we repeated the above experiment using those strains and VACV WR as a control in the presence/absence of 10 mg/ml Ara-C. Infected cell supernatants were harvested at 24 hours post-infection, then the vaccinia and monkeypox supernatants were ∼5X concentrated by Centricon, and all samples were analyzed by Western blot (Figure 2D).

Band intensities were measured using the Image Lab 3.0 software band density tool (Bio-Rad Laboratories, Hercules, CA).

C4b ELISAs were performed as described in the literature [9]. Briefly, Maxisorb ELISA plates (Nunc, Rochester, NY) were coated with human C4b (Complement Technologies, Tyler, Texas) at 5 µg/ml in PBS overnight at 4°C and then blocked for 2 hours at 37°C (5% BSA (Sigma A2934) and 0.1% Tween 20 in PBS). Concentrated supernatants (∼5X) from cells infected with ROC 2003, ROCΔCCP, USA 2003, or USA+CCP were serially diluted in low salt ELISA buffer (10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, 4% BSA, and 0.25% Nonidet-P40), added to wells in duplicate, and incubated for 1 hour at 37°C. Plates were then washed in low salt ELISA buffer (but not containing BSA), and rabbit anti-VCP (diluted 1∶5000 in low salt ELISA buffer) was added for 1 hour at 37°C. Following washing, a peroxidase-conjugated donkey anti-rabbit IgG (diluted 1∶8000 in low salt ELISA buffer) was added for 1 hour at 37°C. Plates were developed using TMB substrate (Invitrogen) and read at an OD of 450 nm. Data shown is the average of two experiments (Figure 3). A standard curve using rVCP (starting amount 2.5 ng) [9] (courtesy of Dr. John Atkinson, Washington University of School of Medicine) was also run in this assay to estimate the amount of MPXV CCP in the supernatant of infected cells.

Confluent monolayers (>95%) of BSC-40 cells in six-well plates were infected, in duplicate, with a MOI = 5 of ROC 2003, USA 2003, ROCΔCCP, or USA+CCP virus in RPMI 1640+2% FBS then incubated at 36°C and 6% CO₂ for 1 hour. The inoculum was removed and replaced with 1 mL fresh RPMI 1640+2% FBS media. Virus was harvested from the cells at 2, 6, 12, 18, 24, 36, 48, and 72 hours post-infection and stored at −70°C. On day of titration, the aliquots were freeze-thawed 3 times prior to serially diluting the samples and plating on six-well plates of BSC-40 cells. After 72 hour incubation, plates were stained with crystal violet+2% formalin and individual plaques were counted (Figure 4).

All animal work was conducted according to relevant national and international guidelines. The experimental designs and ethics of the study were discussed with the institutional biosafety committee, the institutional animal care and use committee, and the select agent program. Animals were cared for in accordance with the CDC Institutional Animal Care and Use Committee (IACUC) approved protocol 1431REGPRAC-A4. Wild-caught, juvenile black-tailed prairie dogs (Cynomys ludovicianus) were obtained from western Kansas. The animals were captured shortly after adolescence in 2004 and at the time of infection the prairie dogs had been in captivity for 4 years. During experimental infections, animals were housed individually in 12.13″×23.38″×9.00″ cages with aerosol filter tops (Thoren Caging Systems, Hazleton, PA). Cages were kept in a Duo-Flow biosafety cabinet (Lab Products, Inc. Seaford, DE) in the BSL-3 animal room. Animal handling was performed using BSL-3 personal protective equipment. Throughout the study, animals were maintained in a 12 hour light/12 hour dark cycle, and in addition to standard prairie dog chow (Exotic Nutrition Pet Co., Newport News, VA), the animals were provided with monkey biscuits (Exotic Nutrition Pet Co., Newport News, VA) for dietary enrichment.

Prairie dogs weighing between 875–1250 grams were infected intranasally with 4×10⁵ pfu of ROC 2003, USA 2003, ROCΔCCP, or USA+CCP while under inhaled anesthesia (5% isoflurane mixed with air). There were 4 animals in each of the monkeypox virus-challenged groups. The intranasal inoculum, titered at the time of challenge, was administered in a total volume of 10 µl 1× PBS, with each nostril receiving 5 µl of inoculum. Every 2 days swabs were taken from the mouth, anus and eye of each prairie dog under isoflurane anesthesia using sterile individual Dacron swabs (Epicentre Biotechnologies, Miami, FL) and stored at −70°C without diluent. In addition, 200 µl of whole blood was collected from the lateral saphenous or jugular vein in EDTA coated tubes (Vetlab Supply Inc., Miami, FL) for real-time PCR and viable virus assessment of total viral load; 100 µl was harvested in serum separating tubes (Becton, Dickinson and Co., Franklin Lakes, NJ) for serological analysis. On these sample days the weight and temperature of each prairie dog was recorded and tracked throughout the study. Any notable visual observations or occurrences were also documented on sampling days. For euthanasia criteria, we observed individual animals for the following signs of illness severity: inappetence (3 pts), labored breathing (5 pts), unsteady gate (3 pts), lack of responsiveness to touch (5 pts), and lethargy (3 pts). Points were given for each of these signs, any animal accruing a score indicative of severe illness (≥10 pts.) was humanely euthanized. In addition, any animal losing greater than 25% of their starting body weight was euthanized regardless of other clinical symptoms.

The planned length for this study was 30 days; if death did not occur prior to the end of the challenge experiment, animals were humanely euthanized and necropsied. Necropsies on all animals were performed according to IACUC standards in a BSL-3 laboratory and utilizing full BSL-3 personal protective equipment. Samples taken during necropsy, not including the swab and blood samples, were from the following sites: eyelid, heart, lung, liver, spleen, kidney, mesenteric lymph node, and gonad. Instruments (scalpel, scissors, forceps, etc.) were cleaned and decontaminated with 30% Lysol (Reckitt Benckiser Inc., Parsippany, NJ) and 10% bleach (Fisher Scientific, Pittsburgh, PA) between collections of each tissue. Tissues were frozen at −70°C prior to further processing.

All sample processing took place in the BSL-2 laboratory with BSL-3 practices. In order to analyze viral DNA in the blood, water was added as needed to bring the total volume to 200 µl, and the samples were incubated at 55°C for 1 hour. The EZ-1 DNA extraction robot (Qiagen, Valencia, CA) was used for viral DNA extraction of all collected blood samples using the tissue extraction protocol provided by the manufacturer. For all swabs collected during the challenge study, 400 µl PBS was added to the microfuge tube, incubated at room temperature for ∼5 minutes and then the swab extraction tube systems (SETS) (Roche, Indianapolis, IN) protocol was used to recover sample from the swab. A 100 µl aliquot of swab eluant was incubated in lysis buffer and proteinase K at 55°C for one hour prior to extraction of genomic DNA using the EZ-1 DNA extraction robot (Qiagen, Valencia, CA). For all virus strains, the remaining swab lysate was used for virus isolation. There was no viable virus isolation from the prairie dog whole blood samples due to technical difficulties.

Necropsy tissue samples were homogenized to slurry using a metal ball tissue grinder (SPEX SamplePrep LLC, Metuchen, NJ). Genomic DNA was prepared from a 100 µl slurry aliquot using the EZ-1 DNA extraction robot, after incubation in lysis buffer and proteinase K for 1 hour as described above. Remaining necropsy tissue sample slurry was used for virus isolation.

All swab, blood, and necropsy samples were analyzed in duplicate by real-time PCR using forward and reverse primers and probe complementary to the highly conserved orthopoxvirus DNA polymerase gene, E9L [8], [44].

Swab and necropsy samples that tested positive for the presence of orthopoxvirus DNA using the real-time PCR assay were evaluated for the presence of viable virus through growth in BSC-40 cells. These samples were titrated by tenfold dilution of the swab eluent/tissue slurry in RPMI+2% FBS media and incubated with the cells at 36°C and 6% CO₂ for 72 hours. Cells were stained with crystal violet+2% formalin and then the plaques were counted.

ELISA assays were used to analyze the collected blood for the presence of anti-orthopoxvirus-specific IgA and IgG. Ninety-six well microtiter plates were coated with 0.01 µg/well crude vaccinia virus-BSC40 cell lysate in carbonate buffer on one half of the plate and an equivalent volume of BSC-40 cell material diluted in carbonate buffer on the other half; plates were incubated overnight at 4°C. The plates were inactivated by incubating in 10% buffered formalin at room temperature for 10 min., after which they were blocked for 30 min at room temperature with assay diluent (PBS, 0.01 M, pH 7.4+0.05% Tween-20, 5% dried skim milk, 2% normal goat serum and 2% BSA) and washed in PBS plus 0.05% Tween-20. Experimental prairie dog sera (1∶100) was added to both halves of the plates and incubated for 1 hour at 37°C. Each plate was washed in PBST and ImmunoPure A/G conjugate (Pierce, Rockford, IL) was added at 1∶30,000 and then incubated for 1 hour at 37°C. Plates were then washed and a peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) added; image development took approximately 5–15 min. Stop solution was added and the absorbance was read on a spectrophotometer reading at 450 nm. All reported values represent the average of two wells of the same sample. Specimens were considered positive if the test sample absorbance was above the cut-off value (COV). The COV was determined averaging the cell lysate half of each plate and adding two standard deviations [8].

Sequencing of the ROCΔCCP and USA+CCP genomes was carried out using standard protocols of the Genome Sequencer FLX pyrosequencing platform (Roche Applied Science, Indianapolis, IN) using the shotgun sequencing protocol.

As data were not normally distributed, nonparametric statistical analyses were used [45]. Using the Wilcoxon rank-sum test, reported data averages were compared within sample day between the following groups, ROC 2003 vs. ROCΔCCP and USA 2003 vs. USA+CCP. All P-values lower than 0.05 were considered significantly different.