The goal of this study was to determine if the proposed immunomodulatory effect of the monkeypox CCP was a major contributor to the documented disease differences between the West African and Congo Basin clades of monkeypox virus. To investigate the role of CCP in monkeypox virulence, we deleted it from the more virulent ROC 2003, a Congo Basin strain, and incorporated it into the less virulent USA 2003, belonging to the West African clade. The selectable marker, gpt
, has been used in the creation of recombinants for in vivo
studies successfully but its presence in the recombinants has the potential to be a limitation in this study, hence we conducted a C4b assay to confirm normal CCP activity and growth curve to demonstrate normal growth kinetics of the recombinants. The experimental design was discussed with multiple institutional ethics oversight committees prior to its execution. It was important to investigate the addition of CCP to the USA 2003 strain to better frame the scientific question regarding disease manifestations influenced by the presence or absence of CCP within these strains of monkeypox viruses. Due to several gene truncations/deletions in the West African strain, we sought to determine if the addition of a single virulence-associated gene was sufficient to impact its pathogenesis. We confirmed by single-step growth curves that the mutations introduced in the recombinant viruses had no effect on the in vitro
infectivity and growth of the virus. Using a C4b-binding ELISA, we confirmed that the CCP produced by the USA+CCP had the same activity as the CCP naturally expressed by ROC 2003. Liszewski, et al. reported that recombinant forms of VCP (rVCP) and the MPXV CCP had approximately the same C4b binding activity 
. Using the rVCP as a standard and the concentrated supernatants from MPXV-infected cells, we estimated that the concentration of CCP in the media of infected cells to be ~0.12 µg/ml. This estimation assumes that the activity or the protein in the supernatant of infected cells is similar to purified recombinant proteins. Through the Ara-C experiment, we also provided evidence that VCP and CCP were proteins expressed at the late stages of infection. VACV-infected cells produced more VCP than monkeypox-infected cells produce of its homologue ().
The parental and recombinant strains of virus were used in the prairie dog animal model, which has proven to be a reproducible model capable of distinguishing disease differences between the two clades of monkeypox virus 
. The intranasal route of infection mimics an upper respiratory exposure, which is an important route of transmission 
. Similar to intradermal infections of mice, guinea pigs, and rabbits with VCP-knockout virus 
, the ROCΔCCP group of infected animals displayed a marked reduction in disease severity, and in this study they produced fewer disseminated lesions with no mortality. The linear regression analysis of the weight loss data further highlights the reduced disease severity seen in the ROCΔCCP-infected group when compared to the wild-type ROC 2003-infected group. The loss of CCP significantly reduced the rate of weight loss compared to ROC 2003-infected group (p
0.03) and made the weight loss kinetics more closely resemble that observed in USA 2003-infected animals (p
0.69). The only other difference in the rate of weight loss that neared 95% statistical significance was between wild-type ROC 2003-infected and wild-type USA 2003-infected groups (p
0.057). The ROCΔCCP-infected group also appeared to have delayed signs of disease, with the onset of nasal swelling and primary lesions beginning later than the ROC 2003-infected group (). ROCΔCCP-infected animals excreted similar quantities of virus () at “peak” times post-infection when compared to ROC 2003 despite less severe disease with lower rash burden (). Detection of viral DNA in the blood of ROCΔCCP-infected animals was delayed and reduced with respect to parental ROC 2003-infected animals (), perhaps indicating that the loss of CCP reduced the ability to suppress elements of the innate response, enabling the hosts to localize the infection, thus increasing time until systemic viral disease.
The ROCΔCCP recombinant-infected prairie dogs elicited the strongest humoral immune response of all monkeypox virus strains tested (). Since all of the ROC 2003 animals succumbed to disease by day 16, we were unable to compare the magnitude of humoral immune response, persistence of viable virus and possibility for shedding/transmission between the ROC 2003 and ROCΔCCP groups. However, consistent with previous studies 
, the duration of viable virus excretion/viremia for the ROCΔCCP-infected animals was greater than that seen for USA 2003-infected animals, indicating other factors besides CCP affect the overall potential for shedding (and thus potentially transmission) of the virus. Overall, the ROCΔCCP-infected prairie dogs were able to avoid the severe morbidity, increased mortality and increased weight loss associated with the Congo Basin infection and instead manifested a disease progression more similar to the USA 2003-infected animals (, & ). The data presented here demonstrates that the monkeypox CCP homologue is a significant immunomodulatory protein for Congo Basin clade virulence. Our data also suggests that deletion of CCP from ROC 2003 renders the virus susceptible to clearance by the innate and adaptive immune system, and permits a robust adaptive immune response to virus infection.
Notably, the inclusion of CCP in the West African clade monkeypox virus did not enhance its virulence to levels comparable to that of the Congo Basin clade viruses, but appeared to accelerate development of some signs of the disease course. Overall, the mortality and morbidity associated with the USA 2003+CCP recombinant infections were within expected ranges estimated by the LD50
for the USA 2003-infected animals 
. When compared to the USA 2003-infected animals, there did appear to be some earlier signs of localized, “primary” disease, such as the observation of nasal swelling in PD12 (day 6, 8) and PD7 (day 8), the progression of disease in this regard is similar to the ROC 2003-infected group (). Other than earlier nasal involvement, all other later, generalized signs of disease remained unchanged from the USA 2003 group; for instance there was not an appreciable increase in the onset of, or number of, lesions (). Furthermore, the duration of viable virus shedding in oral swabs was almost indistinguishable (), providing greater evidence that the presence of CCP does not influence the potential for shedding/transmission of the virus. The presence of CCP may affect the tissue tropism and pathogenesis, the virus was found in the liver only in euthanized animals infected with either USA+CCP or ROC 2003 (). This remains a preliminary observation as none of the ROCΔCCP infected animals were euthanized during the disease course.
The prairie dog model system is useful because of the similarity to human systemic orthopoxvirus disease, and potential extrapolations to understanding human disease. However, the use of outbred animals does make experimental designs to achieve statistical significance, if outcome differences are subtle, challenging. For instance, in order to detect a significant difference (p<0.05) in the linear regression slopes of the weight loss in the USA 2003 vs. USA+CCP groups, we would have to reach a statistical power of 80% which would require 67 animals per group.
Our data indicates other factors besides CCP are necessary to significantly increase disease virulence. Although there are differences, this is a conclusion also reached by another group when testing a CCP knockout monkeypox virus recombinant in the non-human primate model 
. Homologues to several other virulence factors such as the myxoma virus M-T4, anti-apoptosis protein and B14R, interleukin-1 binding protein are also missing from the West African clade genomes and are inferred to be involved in determining disease pathogenesis and virulence 
. However, our data with the West African clade parental and recombinant viruses additionally suggests that the presence of CCP may have an effect on the host adaptive immune response as antibody levels were lower in the two surviving USA+CCP-infected animals versus the two surviving USA 2003-infected animals ().
These results also highlight CCP's importance as a potential target for immunotherapy. A strain of vaccinia virus lacking VCP, yet possessing all the other major antigenic epitopes, may be a promising vaccine due to its reduced virulence and potential to elicit a stronger immune response. Others have described the use of monoclonal antibodies (mAbs) to block the activity of the CCP homolog, SPICE, found in variola virus 
. Monoclonal antibodies directed against CCP 
and possibly several other established virulence-related proteins 
may prove to be effective in treating infected individuals.
Finally, an observation of this study which may impact the use of viral DNA measurements to track disease course and the effect of disease interventions was that all sampling group measurements of viral DNA had similar kinetics to that of viable virus. However, viral DNA persisted much longer than viable virus within the samples ( and ). The RT-PCR assay is likely to detect DNA fragments as well as genomic DNA belonging to intact viral particles, thereby viral DNA observed later during infection may be due to the clearing of the infection. A better understanding of what the presence of viral DNA signifies will be critical to determining its utility as a biomarker for studies evaluating vaccine or therapeutic efficacy.
This study demonstrates CCP expressed by ROC 2003, an example of the Congo Basin clade of monkeypox virus, is a virulence factor that significantly affects clinical disease progression, weight loss and mortality in the prairie dog animal model system. However, its expression in the West African clade virus, represented by USA 2003, is insufficient to elicit equivalent levels of disease comparable to the Congo Basin clade in this model system. These viruses have divergent evolutions 
, possibly due to different reservoir or susceptible host species, and thus may have developed different virulence mechanisms. Regardless, removal of CCP from the Congo Basin virus clade significantly reduced the morbidity and mortality associated with disease.