Introduction

Monkeypox is a zoonotic viral disease caused by infection with the Monkeypox virus (MPXV), which was initially identified in crab-eating macaques1 and subsequently detected in other non-human primates and rodents2. For a long time, monkeypox outbreaks were primarily confined to the rainforest regions of Central and West Africa, but in recent years, its prevalence has significantly expanded, spreading to Europe and North America. This trend has sparked considerable concern in the global public health ___domain3,4 MPXV is a linear, enveloped, double-stranded DNA (dsDNA) virus belonging to the Orthopoxvirus genus5. MPXV is classified into two major branches: the Congo Basin clade (Clade I) and the West African clade (Clade II). The fatality rate of infection with the Clade II strain can reach up to 10.6%, while that of the Clade II strain is about 3.6%. The Clade II strain can be subdivided into sub-branches IIa and IIb6. The clinical manifestations of MPXV infection are diverse and complex, including but not limited to fever, pneumonia, lymphadenopathy, and characteristic skin lesions. Severe cases can even be life-threatening7. Although the transmission range of MPXV is relatively limited, and it is often self-limiting, its genetic similarity and familial connection to the variola virus indicate that the potential public health risks of MPXV cannot be neglected8.

Currently, research on MPXV primarily focuses on its molecular biological characteristics, epidemiology, vaccine development, and treatment strategies8. However, there is a notable lack of research on the construction of animal models, particularly for small mammal models. The existing animal model systems primarily consist of non-human primates, such as crab-eating macaques9 and common marmosets10. These models can well simulate human infection symptoms but are limited by factors such as large size, high cost, and operational complexity. On the other hand, certain rodents like ground squirrels11 and tree squirrels12, although sensitive to MPXV, have unstable supplies and are difficult to domesticate, limiting their further application. Commonly used laboratory mouse models, such as the C57BL/6 strain, exhibit low susceptibility to MPXV13, while the CAST/EiJ strain, though sensitive to some MPXV strains14,15, is expensive and in short supply. In this context, dormice, as small and easily reared rodents, have gradually gained the attention of researchers. Previously, Schultz DA et al.4 and Earl PL et al.16have taken the lead in conducting preliminary investigations on the infection characteristics using African dormice and Clade I strain of MPXV, laying a certain foundation for subsequent research. However, it must be recognized that the virus has a strong mutation ability. It can adapt to the environment better through mutation, and this mutation may not only change the virus’s transmissibility and pathogenicity but also may give rise to new epidemic strains, thereby triggering widespread epidemics or even large-scale outbreaks of related diseases17,18,19. Unfortunately, research on dormice as an infection model for Clade II b is still scarce. In view of this research status, this study aims to fill this critical gap. This study employs the currently prevalent Clade II b strain of MPXV and inoculates it into dormice through the intranasal route at three different titers. Multiple methods, including clinical symptom observation, gross anatomical analysis, virus load measurement, and histopathological examination, are comprehensively utilized to evaluate the feasibility and effectiveness of dormice as an MPXV infection model. It provides reliable and economical animal model support for subsequent vaccine development, drug screening, and pathogenic mechanism research, thereby promoting the in-depth development of MPXV-related research.

Materials and methods

Virus, reagents, and instruments

In the present study, the monkeypoxvirus (MPXV) strain utilized is hMPXV/China/GZ8H-01/2023 (Genebank: PP778666.1), which was isolated from a patient in Guangzhou, China. Following genotyping analysis, this virus was identified as belonging to the Clade IIb subtype. Subsequently, the MPXV strain (hMpxV/China/GZ8H-01/2023) was propagated in Vero E6 cells. These cells were cultured in DMEM medium (Sigma - Aldrich) supplemented with 10% fetal bovine serum (Invitrogen), 50 U/mL penicillin, and 50 µg/mL streptomycin. Reagents included the Hematoxylin-Eosin (H&E) high-definition permanent staining kit (product code: G1076-500ML) from Wuhan Servicebio Technology Co., Ltd. Instruments used were a rotary microtome (model: RM2245) and a fluorescence microscope (model: DM2500) from Shanghai Leica Microsystem Co., Ltd.

Animals and grouping

Experimental subjects were Spanish dormice (Dryomys nitedula, Rodentia, Myoxidae, Dryomys), which were bred and maintained in the laboratory of Jiujiang University. All selected dormice were male, 4–5 months old, with a body weight of (20 ± 2) g. The animals were housed in an environment maintained at (23 ± 2) °C, with a relative humidity of 40 − 70%, a 12 - hour light - dark cycle, and ad libitum access to food and water. The use of experimental animals was approved by the Animal Welfare and Ethics Committee of Jiujiang University (approval number: JJU20240203), and the license number for experimental animal use is SYXK (Gan) 2022–0003. All methods were performed in accordance with the relevant guidelines and regulations. The experiment was conducted following ARRIVE guidelines (https://arriveguidelines.org).

The experimental animals were randomly assigned to four groups, comprising three infection groups (MPXV groups) and one control group. The dormice in the MPXV groups were intranasally inoculated with MPXV virus suspensions at titers of 103.5 PFU, 104.5 PFU, and 105.5 PFU, respectively (100 µL per animal), and were designated as the MPXV_103.5 PFU, MPXV_104.5 PFU, and MPXV_105.5 PFU infection groups. In contrast, the dormice in the control group (Mock group) received only an equal volume of PBS solution via intranasal inoculation and were completely separated from the infection groups. To comprehensively assess the impact of MPXV infection on the clinical status of the mice, daily observations of their clinical status were conducted during the experimental observation period after MPXV infection, and scores were assigned based on preset criteria (0: healthy, no abnormal manifestations; 1: lethargy, sleepiness; 2: any signs of chills, piloerection, hunching, or ataxia; 3: negative weight growth trend with significant differences compared to the Mock group; 4: paralysis of limbs, reduced activity; 5: moribund or deceased state).

Within 0 to 14 days post-MPXV infection, key clinical indicators such as body weight changes, feeding behavior, survival rate, and mental status of the mice were continuously monitored and recorded. On the 5th and 10th days post-infection, three dormice were randomly selected from both the Mock group and each of the MPXV groups. Tissue samples from the lungs, liver, spleen, and trachea were collected under isoflurane anesthesia. These samples were used for subsequent virus titer detection and pathological analysis. Meanwhile, the lung tissue samples from the 5th and 10th days of infection were photographed for the intuitive observation of pathological changes.

TCID50 assay for MPXV titer

The collected lung tissue samples were first subjected to homogenization and then underwent three freeze-thaw cycles to fully release MPXV virus particles. The virus in the supernatant was collected by centrifugation at 12,000 r/min for 10 min. A 96-well plate was used to set up virus dilutions ranging from 1 × 10− 1 to 1 × 10− 6, with three replicate wells for each dilution. The TCID50 values for each group were calculated using the Reed-Muench method to accurately assess the infection titer and replication efficiency of MPXV in the mice.

Observation of histopathological changes in dormouse tissue via H&E staining and quantitative assessment

The histopathological examination in this study followed the methods described previously in detail20. The specific steps were as follows: the collected lung tissue samples from dormice were first washed with cold PBS, then fixed in 10% neutral formalin for 48 h, followed by paraffin embedding and sectioning into 4 μm slices. After baking, H&E double staining, and neutral gum sealing, the slices were observed under an optical microscope, and the characteristics of histopathological changes were recorded in detail.

The histopathological scoring of lung tissue followed the established methods20. After MPXV infection in dormice, the observed histopathological changes in the lungs mainly included degeneration, necrosis, and desquamation of bronchial epithelial cells, inflammatory cell infiltration in the peribronchial and alveolar spaces, and the presence of exudate in bronchial and alveolar lumens. Based on the extent of the lesions, a detailed score was assigned to the degree of lung tissue pathology in each group: 0 indicated no pathology in the lung tissue; 1 indicated that the pathology accounted for less than 10% of the affected area; 2 indicated that the pathology accounted for 10-50% of the affected area; 3 indicated that the pathology accounted for 50–90% of the affected area; and 4 indicated that the pathology accounted for more than 90% of the affected area.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0.2 software. All data were expressed as mean ± standard deviation, and differences between multiple groups were compared using ANOVA. A P-value less than 0.05 was considered statistically significant.

Results

Clinical symptoms of animals

The experimental procedure is outlined in Fig. 1A. From the second day after MPXV challenge, compared to the Mock group, the dormice in the various MPXV-infected groups (MPXV_103.5 PFU, MPXV_104.5 PFU, and MPXV_105.5 PFU) exhibited a gradual decrease in body weight (Fig. 1B). By the fifth day post-challenge, particularly notable was the MPXV_105.5 PFU group, where some dormice entered a pre-mortem state or even died, leading to a sharp decline in survival rate in this group (Fig. 1C). The dormice infected with MPXV generally exhibited symptoms such as lethargy, significantly reduced activity, decreased appetite, and respiratory distress, accompanied by clinical pathological manifestations including sluggish movement, disheveled fur, chilliness and shivering, dull coat, and ataxia. It is noteworthy that the infection titer was positively correlated with the severity of clinical symptoms (Fig. 1D). Conversely, in the Mock group, no clinical abnormalities were observed throughout the experiment, with stable mental state, respiration, appetite, and body weight, and no deaths occurred (Fig. 1B,C).

Fig. 1
figure 1

Clinical manifestations of dormice infected with three different doses of MPXV. (A) Experimental flow chart; (B) Relative body weight; (C) Survival rate; (D) Clinical mean score. Compared with the Mock group (n = 10), * p < 0.05, ** p < 0.01, *** p < 0.001.

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Virus titer detection

After administering different titers of MPXV to dormice via the nasal route, tissue samples from their lungs, liver, kidneys, and trachea were collected on the 5th and 10th days for virus titer measurement. The results indicated a clear time- and titer-dependent increase in virus titer in various tissues as the infection time progressed and the inoculation titer increased (Fig. 2). Notably, the virus titer in lung tissue was significantly higher than that in other tissues at the same time points (Fig. 2). Furthermore, in the MPXV_103.5 PFU group, the MPXV titers in the liver, kidneys, and trachea were relatively low on the 5th and 10th days, with some even below the detection level, as shown in Fig. 2B,C, and 2D. Throughout the experiment, no MPXV titers were detected in any tissue samples from the Mock group.

Fig. 2
figure 2

Changes in viral titer in tissues and organs of dormice at different time points after infection with varying doses of MPXV. (A–D) represent lung, liver, spleen, and trachea, respectively. Compared with the Mock group (n = 3), *** p < 0.001.

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Impact of MPXV on lung histopathology

Upon infection of dormice with different titers of MPXV, significant pathological changes in the lungs were observable by gross examination. The lungs of the infected groups exhibited a color change to dark red to purplish red, with softened texture and edema, and loss of elasticity. In severe cases of infection, the lungs showed slight enlargement due to congestion, edema, and accumulation of inflammatory exudates. In contrast, the lungs of the Mock group were light pink, soft, and had good elasticity (Figs. 3A and 4A). Further microscopic examination after H&E staining revealed that infection with the three inoculation titers of MPXV resulted in varying degrees of damage to the lung tissue (Figs. 3B and 4B), with the MPXV_105.5 PFU group showing the most severe effects, manifested as widened lung interstitium (indicated by green arrows), filling of alveolar spaces with red blood cells (indicated by red arrows), inflammatory cell infiltration (indicated by black arrows), and desquamation of bronchiole epithelial cells with formation of exudates (indicated by asterisks). The lung tissue structure of the Mock group remained intact, with no signs of pathology (Figs. 3B and 4B). Additionally, compared to the Mock group, the pathological scores of the dormice lungs significantly increased after virus infection, and the scores were positively correlated with the infection titer (Figs. 3C and 4C). This indicates that high-titer MPXV infection causes more severe damage to the lung tissue of dormice.

Fig. 3
figure 3

The pathological changes in the lungs of dormice infected with different titers of MPXV on the 5th day. (A) Lung dissection; (B) H&E staining results of lung tissue from different groups, bar = 100 μm; (C) Histopathologic scoring of lung. Compared with the Mock group (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001.

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Fig. 4
figure 4

The pathological changes in the lungs of dormice infected with different titers of MPXV on the 10th day. (A) Lung dissection; (B) H&E staining results of lung tissue from different groups, bar = 100 μm; (C) Histopathologic scoring of lung. Compared with the Mock group (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001.

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Discussion

MPXV initially emerged in Central and West Africa and rapidly spread globally through inter-animal, animal-to-human, and human-to-human transmission21,22. Given the urgent situation of MPXV transmission, research on vaccine efficacy evaluation, drug screening, and virological, immunological, and pathological mechanisms is of paramount importance, necessitating the support of reliable animal models. Although previously established animal models have partially revealed the pathogenic mechanisms of MPXV, they still have limitations in fully simulating human infection symptoms8. Therefore, it is imperative to explore animal models that are closer to human physiological characteristics, convenient for laboratory breeding and management, and suitable for large-scale experimental applications.

The choice of inoculation route has a significant impact on the outcome of MPXV infection, which has been widely validated in numerous studies. Inoculation routes include intranasal, oral, intragastric, intradermal, intraperitoneal, and intravenous injection, each affecting the infection efficiency of the virus and the pathological response of the host to varying degrees. Studies have shown that adult common squirrels infected via the intranasal and oral routes exhibit significantly faster disease progression than those infected through skin scratching, presenting a series of typical symptoms including fever, reduced activity, anorexia, and respiratory distress. Although these animals have a mortality rate of up to 100%, they do not show obvious skin lesions12. Another study reported that thirteen-lined ground squirrels infected with the USA-03 strain at the same dose via intraperitoneal injection or intranasal route died within 6 to 9 days post-infection. Notably, the intraperitoneal injection route resulted in a shorter survival time compared to intranasal infection23, directly demonstrating the regulatory effect of the inoculation route on the severity of infection. Further analysis of experimental data from different animal models and virus strains reveals that differences in infection routes not only affect mortality rates but also alter the manifestation of clinical symptoms and pathological processes. For example, after intraperitoneal injection of the West African strain (USA-2003) into black-tailed prairie dogs, all animals died within 11 days, accompanied by focal necrosis of the liver and spleen and mild inflammatory changes in the lungs. In contrast, intranasal infection, while causing death in some animals, primarily resulted in pathological changes concentrated in the lungs and pleural cavity, accompanied by ulcerative changes in the lips, tongue, and buccal mucosa24. However, Gambian pouched rats did not show obvious symptoms after intranasal infection, while intradermal injection induced a series of significant manifestations such as lethargy, anorexia, weight loss, and skin lesions25. These findings collectively emphasize that the site of infection and the method of inoculation can determine the outcome of infection. This study shows that Spanish dormice have an 80% mortality rate after being infected with 105.5 PFU of MPXV. In contrast, African dormice infected with 200 PFU of the MPXV-ZAI-79 strain die within 10 days, with a 100% mortality rate4. Another study found that African dormice infected with 400 or 500 PFU of the MPXV-z06 strain succumb to the disease on the 13th and 16th days, respectively16.These findings highlight the complex and variable interactions between different virus strains and host species, revealing significant differences in their sensitivity to infection doses and lethality rates13. Specifically, Spanish dormice infected with 105.5 PFU of MPXV show markedly higher viral titers in lung tissues compared to other tissues at certain time points, which closely correlates with the severity of disease symptoms. Pathologically, the lung tissue exhibited changes in color and texture, interstitial widening, and red blood cell engorgement in the alveolar spaces. The pathological score was positively correlated with the viral titer. Additionally, a monkeypox virus infection model was established using African dormice, revealing histopathological manifestations such as rhinitis, lymph node necrosis, hepatocyte necrosis, multi-organ hemorrhage, and the presence of viral inclusion bodies in the cytoplasm of multiple tissues4. Despite being members of the same dormouse family, Spanish and African dormice belong to distinct genera. When African dormice are infected with the Clade Ib strain of MPXV, the highest viral titer is found in the liver, followed by the spleen and lungs4,16. In contrast, Spanish dormice infected with the Clade IIb strain show the highest viral titer in the lungs, followed by the trachea, liver, and spleen. These differences in viral loads and pathological characteristics between the two species may be attributed to variations in MPXV susceptibility, transmission routes, immune responses, or inherent characteristics of the virus strains. This fully demonstrates that the interactions between different virus strains and hosts not only affect the mortality rate but also have an important impact on virus replication and the pathological process. It can be seen that the interactions between different virus strains and hosts vary among different species and different strains of the same species, which reminds us that when selecting an animal model, we must comprehensively consider the host species, the characteristics of the virus strain, and their interactions.

During the exploration of the MPXV infection model, the impact of the infection route on the model’s clinical symptoms and virus distribution patterns cannot be overlooked. Previous studies have indicated that CAST/EiJ mice infected with MPXV via the intranasal route exhibit significantly higher viral titers in the lungs compared to other organs, such as the liver and spleen15,26. In this study, we infected dormice via the intranasal route and found that virus replication was more active in lung tissue, which is consistent with literature reports24,27. Specifically, MPXV titers in lung and liver tissues were significantly higher than those in other tissues during the infection process, indicating that the intranasal route can effectively simulate the natural replication process of the virus in the host. It is noteworthy that, apart from increased MPXV titers, our study also observed notable pathological changes in the lung tissue, such as congestion and widened lung interstitium, further confirming the specific damage caused by MPXV to lung tissue through the intranasal route. However, the liver did not exhibit significant pathological changes upon visual inspection, which may be related to differences in tissue sensitivity to MPXV and viral replication strategies8. In the process of establishing the MPXV-infected dormice animal model, we used three different MPXV titers and observed their effects on dormice. The dormice model infected with 105.5 PFU MPXV not only exhibited obvious clinical symptoms within 14 days post-infection but also showed significant changes in viral titer detection and histopathological analysis.

Conclusions

In summary, this study successfully established an MPXV-infected dormice animal model and initially explored the effects of different MPXV titers on dormice. Among them, the 105.5 PFU MPXV titer induced the most significant clinical symptoms and histopathological changes, and the virus had strong replication ability in various tissues, which could more comprehensively simulate the pathological process of MPXV infection. This provides an experimental platform that is closer to the natural infection process for further research on the pathogenic mechanism of MPXV and the evaluation of vaccine and drug efficacy.