In the present study, we compared the infection produced by West African and Congo Basin MPXV clades in inbred BALB/c and C57BL/6 mice. Subtle differences in pathogenicity between the two strains of MPXV were observed in this study. As seen in previous animal studies
, the Congo Basin strain of MPXV was more virulent than the West African strain as evidenced by the symptoms we observed. We have determined that laboratory mice may be a suitable animal model to study MPXV pathogenesis and future studies to further characterize the mouse innate and adaptive immune responses to monkeypox infection will help in understanding the host-virus interactions that lead to more attenuated disease presentations compared to people and other animals such as prairie dogs.
Localized signs and symptoms were more noticeable in the FP model, whereas generalized signs and symptoms were more evident in the IN challenge models. On the whole, for both mouse strains and both inoculation routes, the West African MPXV disease was less severe than that observed in the Congo Basin challenged groups. Although FP edema was observed in all mice that were FP inoculated, the edema observed in the West African infected group was comparatively less (no/slight limp) than that observed in the Congo Basin FP mice (severe limp with animal putting little/no weight on inoculated leg). Furthermore, the edema resolved in a shorter time period in the West African MPXV infected mice compared to the Congo Basin MPXV infected mice. Additionally, one of the Congo Basin FP animals lost weight, whereas none of the West African FP animals lost weight. When we compare the IN route of infection, unlike the Congo Basin IN animals which displayed some ruffled coats and weight loss, the animals in the West African IN group did not develop any noticeable symptoms. Both mouse strains that were challenged IN with Congo Basin MPXV had an observed trend of increased weight loss compared to West African MPXV challenged mice. However the weight loss was not considered statistically significant (C57BL/6 mice: mean % weight loss
0.56% West African vs. 8.16% Congo Basin, p
0.076; BALB/c mice: mean % weight loss
1.08% West African vs. 9.14% Congo Basin, p
0.099). However, utilizing the blood collection methods in this study, the immune response observed in the Congo Basin IN challenged mice was less than that observed in the West African IN challenged mice. Future studies on tracking virus during the course of infection may help to clarify this apparent difference in viral clearance by or virus presentation to the immune system.
The edema that occurred in the FP groups with both strains of MPXV suggests an acute inflammation. The observation that only mild ruffled coats and weight loss was seen in the Congo Basin IN animals and no observable symptoms in the West African IN animals could imply more of a generalized inflammatory response to infection in the Congo Basin IN infected animals. Alternatively, the weight loss that was seen in the Congo Basin IN animals may have been caused by localized inflammation in the nasal and oral cavities of these Congo Basin IN challenged animals resulting in decreased food consumption. However, the findings that all MPXV challenged groups had seroconverted at time of necropsy suggests that the animals did develop infection. Not surprisingly and as previously seen in the authors' studies with prairie dogs
, no viable virus was found in necropsy samples at 30 days p.i. However, additional evidence of systemic infection is provided by the positive PCR results in several tissue types including lungs, blood, lymph node, skin, liver and kidney samples at 30 days p.i. Although PCR positive tissue samplings were not found at day 30 in the Congo Basin IN challenged animals, in the West African MPXV IN challenged animals in which no symptoms were observed, at 30 days p.i. animals had several positive PCR samples suggestive of systemic infection.
Although all mice in the present study were considered OPXV antibody positive, it is noteworthy that the antibody levels seen in these animals were less than what we have observed when challenging prairie dogs with a comparable inoculums of MPXV
. Although these ELISA plates were run on different days, because our negative and positive controls are similar throughout ELISA runs it is possible to compare the data. The low levels of antibodies detected in the mouse sera could reflect the degree of infectious virus that is presented to the immune system in these inbred mouse strains. Future studies may better address the mouse innate and adaptive immune responses to monkeypox infection, and will help in understanding the host-virus interactions that lead to more attenuated disease presentations.
Inbred mice are as genetically alike as possible, being homozygous at virtually all of their loci. Such genetic homogenous models are ideal for studying human pathogens as symptoms of disease are very similar or identical between animals. Results therefore are often more easily interpreted than in a wild or out bred animal model in which genetic diversity can sometimes lead to conflicting or confusing results. Inbred mice, including BALB/c and C57BL/6 strains, are a very controlled and well understood animal model and specific commercial reagents have been created for these animals. It would also be possible to create knockout mice to further understand and characterize the relationship between the immune system and the virus. Having such an inbred mouse model for the study of MPXV could greatly increase our wealth of knowledge about this serious human pathogen.
A recent paper by co-authors of this manuscript 
used in vivo
imaging to study IP (intraperitoneal) inoculation of monkeypox in immunocompetent and immuno-compromised BALB/c mice. Although the IP route of infection does not mimic the natural transmission of this disease, the previous results were similar to our study in that the innate immune system in the immunocompetent animals kept the virus localized to the site of infection while the immuno-compromised animals developed systemic infections. Also, in both groups of animals in the previous study, the Congo Basin strain of monkeypox was more virulent which is also congruent with our results. Future in vivo
imaging studies with an IN route of infection would better mimic natural infection routes, and would provide a better understanding of “natural” patterns viral trafficking and the immune response(s) that protect against MPX infection in immunocompetent animals.
We have demonstrated that these mouse strains would not likely suffice as a model for human MPX disease due to the more subtle disease presentation compared to the disease course seen in humans, but may prove informative in the understanding of immune response which makes certain animals more capable of efficiently clearing MPXV. Both mice strains had similar clinical presentations, with the exception of more BALB/c mice developing ruffled coats when challenged with Congo Basin MPXV. Therefore, the BALB/c mouse strain should probably be used in future studies. However, based on symptoms of morbidity, the Congo Basin MPXV clade was more virulent in both strains of mice. The FP inoculation route provided the visual symptom of edema with both MPXV strains. However, the animals challenged IN with Congo Basin MPXV lost weight and 4/5 BALB/c mice developed ruffled coats unlike West African IN challenged animals, suggesting that the IN route might be better for clade comparison. Additionally, as previously mentioned, the IN route is the more probable natural route of infection, and therefore will be used in future studies. In particular, this mouse model may be used for non-invasive in vivo imaging in order to unravel many unknown aspects of MPX pathogenesis, including virulence factors, disease progression in rodent hosts, and viral shedding from infected animals. Furthermore, an in vivo inbred mouse model could be used to test antivirals and the next generation of OPXV vaccines for their ability to alter the course of monkeypox disease.