In the current study, transmission of the two MPXV clades was minimal via respiratory transmission. Transmission did not occur in any naive animals cage-housed at a fixed 4 inches across from West African MPXV cage-housed infected animals. Low rates of transmission occurred during Congo Basin MPXV transmission studies; 1/4 (25%) naive animals housed across from Congo Basin MPXV high-dose challenged animals developed an infection and neither of the 2 naive animals housed across from two low-dose Congo Basin MPXV challenged/infected symptomatically ill animals became infected. Compared to prior studies, based on these findings from a small sample set, respiratory transmission appears to be less efficient than contact as a mechanism of transmission within this model.
These findings are in contrast to a previous study we did with the prairie dog MPXV model to study transmissibility potential of the West African clade MXPV 
. In the previous study, when naive animals were housed in cages across from MPXV infected animals similar in methods as the current study; 100% of the naive animals were infected with MPXV. However, the cages were placed closer together (approximately one inch apart) and were not separated by a metal rod as in the current study; therefore the cages could have shifted and become in close enough proximity for animals to touch noses. Therefore, one hypothesis which warranted testing was whether the animals in the previous study were able to touch noses and exchange nasal discharge (previously shown to be virus-laden). Based on the findings from the current study in which none of the naive animals housed across from West African MPXV infected prairie dogs became infected, we can hypothesize that animals in the previous study were indeed able to exchange nasal discharge due to the close proximity of the cages. Based on the comparison of these two studies, we have demonstrated that transmission of West African MPXV is only likely to occur if animals are in very close proximity/direct contact with one another. However, one of our Congo Basin MPXV high-dose challenged animals in this study did transmit the virus to a naive/exposed animal which became infected and subsequently died. This difference in rate of transmission and increased disease burden in secondarily infected animals between the two MPXV clades could be attributed to the greater viral shedding in Congo Basin MPXV infected animals; these animals excreted 1–2 logs higher loads of virus than West African MXPV infected animals challenged with the same inoculation dose. Previous studies with this animal model also showed higher loads of viable virus from samples collected from Congo Basin MXPV infected prairie dogs compared to West African MXPV challenged animals 
Only three of the four animals challenged with 5×103
pfu of West African MPXV became infected and only two of the four challenged with low-dose Congo Basin MPXV became infected. There are several possible explanations for why these animals were not infected after experimental inoculation. Although the animals are completely anesthetized during inoculation, it is possible that the virus was not efficiently delivered via intranasal challenge. We have previously seen a steep dose response in this animal model, even half a log lower than the intended viral inoculation could result in little or no disease. This steep dose response was noted in a previous study in which 6×103
pfu of each MPXV clade resulted in overt MPXV disease in all four animals 
. However, in the same study, approximately one log lower dose of 6×102
pfu resulted in only one of the four challenged animals becoming infected for each clade. If the intended inoculation was efficiently given, another possibility is that this animal’s innate immune system was able to effectively clear the virus before the adaptive immune system became involved. Because these are wild/outbred animals, we do observe differences in disease response and progression.
One of the major limitations of the current study is the small number of animals utilized. Although we were able to show animal to animal transmission of MPXV within this and previous studies, additional respiratory transmission studies with MPXV would be worthwhile. The described prairie dog MPXV animal model will be useful to study anti-virals or therapeutics that may stop/decrease transmission of the virus. However, to truly understand the transmissibility potential of the two MPXV clades, studies that evaluate aerosol/particle numbers and size will be of benefit 
. Such controlled studies that are able to generate different particle sizes as well as different transmission scenarios (i.e. coughing and breathing) to assess the spread of MPXV aerosols would be very informative in understanding the transmission potential of MPXV amongst potential animal reservoirs as well as within human populations.
All four of the animals challenged with 5×103
pfu (high-dose) of Congo Basin MPXV developed signs and symptoms of characteristic MPXV illness including disseminated skin pustules 10–13 days p.i., upper airway disease, anti-OPXV antibodies and viral shedding. However, three of these animals were euthanized due to extreme morbidity on days 13 or 14 p.i. Of the four naive animals housed across from the Congo Basin MPXV challenged animals, only the naive animal housed across from the challenged animal that survived infection developed characteristic MPXV illness. Skin lesions on this secondarily infected animal were first observed 20 days after the start of the study; a maximum number of 15 lesions were counted at peak infection. This animal developed a measurable antibody titer and began shedding virus from oropharyngeal samples on day 20, and perished due to infection 34 days from study initiation. Because three of the four Congo Basin MXPV primary challenged animals were euthanized during the course of infection, we did a follow-up study with a lower challenge dosage of Congo Basin MPXV to see if the transmission rate would have been higher had the primary challenged animals survived infection. Only two of the animals challenged with 7×102
pfu of Congo Basin MPXV developed infection, similar to results we have seen previously with a comparable challenge dose 
; neither of the two naive animals housed across from the two infected animals showed evidence of MPXV infection. If we combine the animals successfully primarily infected with Congo Basin MPXV from each challenge dose (n
6), we observed evidence of exposure to, and infection with MPXV in 1/6 (16.6%) of the naive animals. Infection, resulting in illness, was transmitted if exposure was to a symptomatically ill animal that was shedding virus for a period greater than seven days.
In this and previous studies with the prairie dog MPXV model, we observe the development of disseminated lesions on animals between days 10–13 p.i. and the detection of OPXV antibodies between days 13–17 
. We can therefore use this data to hypothesize when the naive animal (PD8021) was likely exposed to MPXV. The secondarily infected animal that was infected by Congo Basin MPXV was probably exposed between days 11–14 p.i. based on lesion presentation (initial time of antibody production was not used as we were not able to collect serum from this animal on two consecutive days). Since peak viral shedding from the oral cavity occurs around day 13 from those animals experimentally challenged, the 11–14 day hypothesized timeframe of MPXV exposure would be conceivable.
Western Blot of serum samples from representative primary challenged animals () were screened for immunodominant bands. Three bands were consistently seen in all of these primary-challenged animals (). One immunodominant doublet was seen in the range of 30–39 kDa. This homologous doublet is consistent with two immune-dominant proteins characterized in vaccinia virus infections (a closely related OPXV): the 32 kDa D8 envelop protein, and the 39 kDa A4 core protein 
. The third immunodominant band measured 62 kDa in primary challenged animals and is consistent with the molecular weight of A10, a well-characterized vaccinia virus core protein 
. These three immunodominant bands were also observed in another study which utilized MPXV infected prairie dog sera as positive controls 
. Serum from all of the naive/uninfected prairie dogs produced no bands, supporting the conclusion that proteins detected in sera from primary challenged animals were due to reactions between anti-MPXV antibodies and viral antigens and that the naive animals without overt symptoms were not exposed to MPXV during the course of the study. The primary challenged animals without overt signs of disease were also negative for bands by Western blot, supporting the belief that these animals were not infected.
Animals that died due to MPXV infection had high loads of virus within all tested tissues taken at necropsy; with the exception of PD 9049 that was euthanized later in infection. This animal was observed to be recovering from infection, but euthanasia was mandated due to weight loss. However, the molecular data and gross pathology indicated that this animal was in the final stages of viral clearance. Unlike the other animals that died or were euthanized at peak infection, this animal did not have enlarged/necrotic submandibular lymph nodes, but did have enlarged blood-engorged mesentery lymph nodes. In a previous study we have seen that the submandibular lymph nodes are associated with early replication of the virus within this animal model, while the mesentery lymph nodes are involved later during infection, probably during a second replication of the virus (Hutson et al, manuscript in preparation). Within all of the animals that died, extensive gross pathological changes were observed in numerous organs including liver, spleen, lymph nodes, intestines and the uterus/ovaries. Pox lesions were also observed on several internal tissues, both by gross pathology as well as histological examination of tissues from one of these animals. The liver had pathological changes observed in all of the animals that died during this study, and these observations were supported by blood chemistry results. The trends of increased hepatic enzyme ALT and blood urea nitrogen (BUN) seen on day 13 within the animals that died during infection suggest decreased liver function. Infection with MPXV in a ground squirrel model also reported impaired blood chemistry levels; including ALP and ALT values 
. However in primate challenge studies with MPXV, these blood chemistry values were not reported to differ from uninfected animals 
. In our current study, the albumin levels were low in the three animals that died from infection compared to uninfected animals, also suggestive of liver disease. Hypoalbuminemia was also a common finding in humans infected with MPXV; occurring in 50% of patients 
Evidence has been given to support differences in transmissibility of the two MPXV clades within humans 
. The West African clade has been documented to rarely transmit human to human; during the U.S. outbreak with the West African clade, no human to human cases were seen 
. However, the Congo Basin clade of MPXV has had up to six sequential transmissions of virus from human to human documented 
. The increased transmissibility of Congo Basin MXPV within a human setting is one of the noted differences between the two viral strains 
. Although disease transmission was minimal during our study, apparent respiratory transmissibility of the Congo Basin clade virus was slightly greater than that of the West African MXPV clade virus (16.7% and 0% respectively) within the prairie dog MPXV model. In the current study, the increased transmissibility of the Congo Basin MPXV clade by respiratory route compared to the West African clade may be due to greater respiratory shedding of Congo Basin MXPV. Based on these findings, respiratory transmission of MPXV appears to be less efficient than close or direct contact within the prairie dog MPXV model.
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.