Multiple monkeypox virus (MPXV) animal models have been discussed in previous studies, but no small animal models, nor most non-human primate models, demonstrated the protracted asymptomatic incubation phase seen in systemic human orthopoxvirus illness. Herein, we characterize a black-tailed prairie dog (PD) (Cynomys ludovicianus) model of infection, via intranasal and intradermal exposures, with the two MPXV clades. Daily observations of the animals were made (food consumption, general symptoms, disease presentation), while weights and virus evaluations (ocular, nasal, oropharyngeal, faeces, blood) were obtained/made every third day. Generalized rash became apparent 9–12 days post-infection for all animals. Individual animals demonstrated a range of symptoms consistent with human monkeypox disease. Measurable viraemias and excretas were similar for both clade-representative strains and persisted until at least day 21. Greater morbidity was observed in Congo Basin strain-challenged animals and mortality was observed only in the Congo Basin strain-challenged animals. The PD model is valuable for the study of strain-dependent differences in MPXV. Additionally, the model closely mimics human systemic orthopoxvirus disease and may serve as a valuable non-human surrogate for investigations of antivirals and next generation orthopoxvirus vaccines.

Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 10⁵ plaque-forming units (PFU; 90% lethal dose [LD₉₀]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID–1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID–1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [C_max]) and the time of the last quantifiable concentration (AUC_last) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival. IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today9s current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

Monkeypox virus(MPXV) infection of the prairie dog is valuable to studying systemic orthopoxvirus disease. To further characterize differences in MPXV clade pathogenesis, groups of prairie dogs were intranasally infected (8×103 p.f.u.) with Congo Basin (CB) or West African (WA) MPXV, and 28 tissues were harvested on days 2, 4, 6, 9, 12, 17, and 24 postinfection. Samples were evaluated for the presence of virus and gross and microscopic lesions. Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen earlier in CB challenged animals (day 4) than WA challenged animals (day 6). For both groups, primary viremia (indicated by viral DNA) was seen on days 6–9 through day 17. CB MPXV spread more rapidly, accumulated to greater levels, and caused greater morbidity in animals compared to WA MPXV. Histopathology and immunohistochemistry (IHC) findings, however, were similar. Two animals that succumbed to disease demonstrated abundant viral antigen in all organs tested, except for brain. Dual-IHC staining of select liver and spleen sections showed that apoptotic cells (identified by TUNEL) tended to colocalize with poxvirus antigen. Interestingly splenocytes were labelled positive for apoptosis more often than hepatocytes in both MPXV groups. These findings allow for further characterization of differences between MPXV clade pathogenesis, including identifying sites that are important during early viral replication and cellular response to viral infection.

Monkeypox is a zoonotic disease endemic to central and western Africa, where it is a major public health concern. Although Monkeypox virus (MPXV) and monkeypox disease in humans have been well characterized, little is known about its natural history, or its maintenance in animal populations of sylvatic reservoir(s). In 2003, several species of rodents imported from Ghana were involved in a monkeypox outbreak in the United States with individuals of three African rodent genera (Cricetomys, Graphiurus, Funisciurus) shown to be infected with MPXV. Here, we examine the course of MPXV infection in Cricetomys gambianus (pouched Gambian rats) and this rodent species’ competence as a host for the virus. We obtained ten Gambian rats from an introduced colony in Grassy Key, Florida and infected eight of these via scarification with a challenge dose of 4X10⁴ plaque forming units (pfu) from either of the two primary clades of MPXV: Congo Basin (C-MPXV: n = 4) or West African (W-MPXV: n = 4); an additional 2 animals served as PBS controls. Viral shedding and the effect of infection on activity and physiological aspects of the animals were measured. MPXV challenged animals had significantly higher core body temperatures, reduced activity and increased weight loss than PBS controls. Viable virus was found in samples taken from animals in both experimental groups (C-MPXV and W-MPXV) between 3 and 27 days post infection (p.i.) (up to 1X10⁸ pfu/ml), with viral DNA found until day 56 p.i. The results from this work show that Cricetomys gambianus (and by inference, probably the closely related species, Cricetomys emini) can be infected with MPXV and shed viable virus particles; thus suggesting that these animals may be involved in the maintenance of MPXV in wildlife mammalian populations. More research is needed to elucidate the epidemiology of MPXV and the role of Gambian rats and other species. Post smallpox eradication, Monkeypox virus (MPXV) has emerged as the most important human health threat within the Orthopoxvirus genus. Sporadic outbreaks of monkeypox within Africa, concern over the potential of the virus to move outside of its natural range, as well as the increasing proportion of unvaccinated people now susceptible to MPXV (due to cessation of smallpox vaccination), makes it important to understand how the virus is transmitted to humans within Africa. Thus far the natural reservoir(s) of MXPV has eluded identification; however several rodent species including African pouched rats (Cricetomys spp.) have been implicated as possible reservoirs. Cricetomys are often found living in close proximity to humans (and invading homes) and additionally serve as a food source within Africa. Therefore, it is important to utilize laboratory methods to examine the course of MPXV infection in Cricetomys and thus determine this rodent species’ competence as a host for the virus. We challenged eight animals with MPXV (4X10⁴ pfu) and assessed clinical symptoms and molecular markers of disease. Our results show Cricetomys can be infected with MPXV and shed high loads of virus via multiple routes, supporting the hypothesis that they may be involved in the maintenance and transmission of the virus within Africa.

Although monkeypox virus (MPXV) studies in wild rodents and non-human primates have generated important knowledge regarding MPXV pathogenesis and inferences about disease transmission, it might be easier to dissect the importance of virulence factors and correlates of protection to MPXV in an inbred mouse model. Herein, we compared the two clades of MPXV via two routes of infection in the BALB/c and C57BL/6 inbred mice strains. Our studies show that similar to previous animal studies, the Congo Basin strain of MPXV was more virulent than West African MPXV in both mouse strains as evidenced by clinical signs. Although animals did not develop lesions as seen in human MPX infections, localized signs were apparent with the foot pad route of inoculation, primarily in the form of edema at the site of inoculation; while the Congo Basin intranasal route of infection led to generalized symptoms, primarily weight loss. We have determined that future studies with MPXV and laboratory mice would be very beneficial in understanding the pathogenesis of MPXV, in particular if used in in vivo imaging studies. Although this mouse model may not suffice as a model of human MPX disease, with an appropriate inbred mouse model, we can unravel many unknown aspects of MPX pathogenesis, including virulence factors, disease progression in rodent hosts, and viral shedding from infected animals. In addition, such a model can be utilized to test antivirals and the next generation of orthopoxvirus vaccines for their ability to alter the course of disease.