Efficacy of Tecovirimat and Cidofovir Against MPXV-Induced Pneumonia, Skin Lesion, and Arthritis in the High-Risk Population-Relevant SCID Mouse Model

Efficacy of Tecovirimat and Cidofovir Against MPXV-Induced Pneumonia, Skin Lesion, and Arthritis in the High-Risk Population-Relevant SCID Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Efficacy of Tecovirimat and Cidofovir Against MPXV-Induced Pneumonia, Skin Lesion, and Arthritis in the High-Risk Population-Relevant SCID Mouse Model Hui-Jun Lu, Xinyu Cao, ning Shi, Xiangshu Qiu, Jiaxin Tian, Peng Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6483910/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract On February 27, 2025, WHO maintained monkeypox as a PHEIC following its third round of assessment. Human monkeypox virus infection primarily manifests with fever, lymphadenopathy, and rash. Severe cases may develop pneumonia, encephalitis, myocarditis and arthritis. After evaluation of three murine models (ICR, IFNAR1 −/− , and SCID), we identified SCID mice as stable hosts for clade IIb, modeling high-risk populations like patients with HIV. Three models, characterized by rash, pneumonia, and arthritis, were established for the pharmacodynamic evaluation of tecovirimat and cidofovir. Both drugs decreased virus titer in target organs during early infection and ensured 100% survival. As a limitation, cidofovir failed to inhibit viral DNA load in skin lesions, and monotherapy of cidofovir or tecovirimat was ineffective in prolonged intradermal infections. These data indicate that the therapeutic efficacy of tecovirimat and cidofovir is contingent upon distinct disease phenotypes and progression stages, underscoring the necessity for novel therapeutic interventions against monkeypox. Biological sciences/Microbiology/Virology/Antivirals Biological sciences/Microbiology/Virology/Pox virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As a significant member of the genus Orthopoxvirus from the family Poxviridae ( 1 ), monkeypox virus (MPXV) is taxonomically related to variola virus (smallpox virus). Although the lethality of MPXV is less than that of smallpox, MPXV has greater human transmissibility. Epidemiological data suggests the accelerated adaptive evolution of MPXV ( 2 , 3 ). Phylogenetic studies classify MPXV into four clades, including Ia, Ib, IIa, and IIb ( 4 ). Clade IIb has circulated among humans since 2016, leading to the 2022 pandemic ( 5 , 6 ). On July 21, 2022, the World Health Organization (WHO) first declared the MPXV outbreak a Public Health Emergency of International Concern (PHEIC). Due to the emergence of the newly identified clade Ib, which spread in the Democratic Republic of the Congo (DRC) and neighboring countries and regions beyond Africa, WHO announced the second PHEIC designation for monkeypox (mpox) on August 14, 2024 ( 7 ). Following a third round of assessment on February 27, 2025, WHO decided to maintain the PHEIC status of the outbreak due to the persistent risks of international spread and public health issues. The MPXV clade IIb predominantly affects men who have sex with men (MSM), accounting for 87% of the reported cases, of whom approximately 50% are HIV-positive. These patients with co-infection, particularly those with advanced HIV, exhibit severe complications and suffer from an increased risk of mortality ( 8 ). Among patients with MPXV and HIV co-infection, 29% develop pulmonary involvement ( 9 ). The first documented case of MPXV-induced pneumonia was observed in a male patient with advanced AIDS who presented with several complications and finally succumbed to sepsis ( 10 ). Cutaneous rash occurs in 95% of MPXV-infected patients ( 11 ). The rash is typically self-limiting in immunocompetent individuals, spontaneously resolving within 2–4 weeks ( 11 ). However, immunocompromised patients often develop more extensive and persistent skin lesions, have higher viral loads, suffer from prolonged disease, and are at a significantly increased risk of scarring ( 12 ). HIV-associated MPXV infection frequently manifests with arthritis, characterized by knee joint pain, abnormal synovial fluid cell counts, and MRI findings of synovitis and osteomyelitis ( 13 – 15 ). Notably, viral osteomyelitis is a classic complication of smallpox, affecting 2%-5% of infected children ( 16 ). Furthermore, symptoms of arthritis have been reported in patients with vaccinia virus infection, cowpox virus infection, and even among patients with HIV receiving the JYNNEOS vaccine (MPXV vaccine) ( 17 – 20 ). To date, no antiviral treatment has been definitively demonstrated to be effective against MPXV. However, several antiviral agents have been authorized for emergency use in certain countries and are under evaluation in clinical trials. WHO and several national guidelines recommended​tecovirimat as the first-line antiviral drug for mpox, showing efficacy in several clinical cases ( 21 – 23 ). However, recent studies reported that, during the mpox outbreak in the United States, severely immunocompromised patients who received multiple courses of tecovirimat exhibited poor outcomes (35.3%; 18 out of 51 patients) ( 24 , 25 ). Furthermore, tecovirimat resistance is a major clinical challenge, particularly in subjects with severe immune dysfunction. Resistance-associated mutations, such as F13L gene mutation, may lead to treatment failure and human-to-human transmission, emphasizing the need for cautious use of tecovirimat until obtaining more experimental data ( 26 – 28 ). Cidofovir, an emergency antiviral drug recommended by WHO, exhibits broad-spectrum activity against DNA viruses, such as MPXV. A patient with HIV and uncontrolled viremia exhibited marked improvement after switching to cidofovir after failure of treatment with tecovirimat and antibiotics ( 29 ). However, cidofovir exhibits nephrotoxicity, particularly in patients with HIV, with an incidence rate of nearly 25% ( 30 ). Additionally, the topical application of cidofovir may lead to adverse effects, such as skin erosions ( 31 ). Due to the risk of such adverse events, cidofovir is often reserved for critical cases or tecovirimat-resistant infections; therefore, there are limited experimental data on its monotherapy. The prodrug brincidofovir demonstrates reduced nephrotoxicity and exhibits anti-mpox activity, though its clinical efficacy, like that of the two aforementioned drugs, still needs more preclinical and clinical studies ( 32 ). In conclusion, the establishment of animal models that can accurately replicate the typical clinical manifestations of MPXV is a prerequisite for assessing the therapeutic efficacy of anti-viral drugs. Tecovirimat and cidofovir (and its derivative) are currently the main therapeutic options for mpox. However, the use of both drugs faces limitations due to insufficient clinical data regarding their efficacy and safety across different viral clades and in immunocompromised populations. Results Screening and Establishment of the Mouse Model of MPXV-Induced Pneumonia Intranasal (I.N.) inoculation is commonly employed for modeling pneumonia in small rodents, but the pathogenicity of MPXV varies across different mouse models. In this study, male ICR mice, IFNAR1 −/− mice, and SCID mice were intranasally inoculated with 10⁵ PFU MPXV or sterile saline (control group). After MPXV infection, ICR and IFNAR1 −/− mice exhibited a steady increase in body weight, which was comparable to the control group. SCID mice exhibited a significant decrease in body weight at 12 days post infection (dpi), which was recovered by 14 dpi (Fig. 1 A). After inoculation, no clinical symptoms, such as changes in behavior, appetite, fur, or appearance, were observed in any of the three mouse models. Gross examination of lung tissues collected at 3 dpi, 7 dpi, and 14 dpi exhibited no significant differences compared to the control group, except for mild hemorrhage and atrophy in the lung tissue of SCID mice at 14 dpi (Fig. 1 B). Additionally, qPCR and detection of virus titer were conducted to measure the viral DNA load and infectious virus titer in the lung tissues of the three mouse models at 3 dpi, 7 dpi, and 14 dpi. The results indicated that ICR mice were not susceptible to MPXV. Viral DNA and virus titer were at low levels at 3dpi, decreasing to the detection limit at 7 dpi and 14 dpi. IFNAR1 −/− mice exhibited peak lungs infection on 7 dpi, with a median viral DNA load of 10 4.48 copies/mL, and a low median virus titer of 10 2.43 TCID 50 /mL. Both viral load and titer decreased at 14 dpi. After intranasal inoculation, mice with SCID revealed a steady increase in lungs viral load and virus titer over time. This model exhibited the greatest increase in virus titer compared to the other two mouse models, reaching a median of 10 4.75 TCID 50 /mL at 14 dpi (Fig. 1 C, D). These findings suggest that SCID mice are the most stable and susceptible model for the intranasal infection study. Subsequently, SCID mice were intranasally inoculated with MPXV at the same virus titer (n = 8), and their clinical disease scores and survival rates were monitored for up to 28 dpi to observe the typical clinical symptoms. At 16 dpi, weight loss, lethargy, disheveled fur, swollen footpads, and the typical symptoms of MPXV infection, such as skin lesions of the tail were observed in SCID mice. The severity of clinical symptoms was measured, revealing a median disease score of 7 at 28 dpi (Fig. 1 E, Table 1 ), with a relatively low mortality rate of 25% (Fig. 1 F). Additionally, cytokine activation in the lungs was analyzed at the end of the experiment. The results showed significantly higher mRNA levels of pro-inflammatory factors (TNF-α and IL-6) and antiviral interferons (IFN-β and IFN-α1), while the mRNA levels of NAP3 were significantly downregulated (Supplementary Fig. 1A). These cytokines, particularly pro-inflammatory factors, may contribute to pulmonary pathology. Notably, compared to other mouse models of intranasal infection, footpad edema and cutaneous lesions were observed in this study. These symptoms indicate that intranasal MPXV infection in SCID mice can induce viremia during the late stage of infection. In summary, intranasal MPXV infection of ICR and IFNAR1 −/− mice resulted in self-limited resolution within a short timeframe, without exhibiting characteristic symptoms of pneumonia, making them unsuitable for drug evaluation. SCID mice exhibited the highest sensitivity to MPXV, with measurable changes in body weight, virus titer, tissue damage, and pro-inflammatory factors within 14 dpi. Extending the length of clinical monitoring to 28 dpi revealed the clinical symptoms of MPXV and led to a mortality rate of 25%. Screening and Establishment of the Mouse Model of MPXV-Induced Skin Lesion Skin scratches were created in the tails to simulate the MPXV-associated cutaneous rash. Male ICR, IFNAR1 −/− , and SCID mice were intradermally inoculated with 10⁵ PFU MPXV or sterile saline (control group). After virus inoculation, the body weights of ICR and IFNAR1 −/− mice increased steadily, with no significant difference between the model group and the control group. SCID mice exhibited a significant decrease in body weight from 10 dpi (Fig. 2 A). All three models exhibited certain scabs (width ≥ 1 mm), with ICR mice exhibiting the lowest probability of forming infectious scabs and recovery by 7 dpi. IFNAR1 −/− mice had the highest scab formation rate at 7–9 dpi, reaching 75% and beginning to recover from 10 dpi. SCID mice exhibited the highest scab formation rate at 6 dpi, reaching 100%, and the symptoms worsened over time (Fig. 2 B, C). Additionally, we measured viral DNA load and virus titer in the skin of the three mouse models at 3 dpi, 7 dpi, and 14 dpi using qPCR and virus titer detection. We found that the virus titer in ICR mice remained around the detection limit, with viral DNA detectable only at 3dpi and 7 dpi. IFNAR1 −/− mice were more sensitive to MPXV, but the virus titer decreased at 14 dpi, similar to the intranasal infection model. Consistent with the clinical symptoms, SCID mice exhibited the highest viral load and virus titer among the three models, which steadily increased over time (Fig. 2 D, E). The most susceptible SCID mice (n = 8) were re-infected via the percutaneous route to observe more typical clinical symptoms. The monitoring period was extended to 28 dpi. At 20 dpi, clinical symptoms, such as lethargy, disheveled fur, and rashes in non-lesional areas, were observed in SCID mice. The disease score at the end of the infectious period was 5 (Fig. 2 F, Table 1 ), with no deaths occurred (Fig. 2 G). The changes in the levels of pro-inflammatory factors (TNF-α and IL-6), chemokines NAP3 and antiviral interferons (IFN-β and IFN-α1) in the skin were consistent with those observed in the pneumonia model, with a further increase in GM-CSF levels (Supplementary Fig. 1B). In conclusion, SCID mice were the most sensitive to MPXV after intradermal infection, exhibiting body weight loss and characteristic scabs at 14 dpi. Systemic symptoms became evident at 20 dpi. Although no deaths were observed, spontaneous recovery did not occur. Screening and Establishment of the Mouse Model of MPXV-Induced Arthritis Male ICR, IFNAR1 −/− , and SCID mice were subcutaneously inoculated at footpads with 10⁵ PFU MPXV or sterile saline (control group) to simulate the joint lesions of MPXV infection. Following viral inoculation, the increase in the body weight of ICR mice was comparable to that of the control group. IFNAR1 −/− mice exhibited a significant decrease in body weight from 6 dpi, which normalized at 14 dpi. In contrast, SCID mice began to show a sustained decrease in body weight from 8 dpi (Fig. 3 A). Between 0–14 dpi, only ICR mice did not exhibit ankle joint swelling (width ≥ 2 mm). IFNAR1 −/− mice developed ankle joint swelling from 3 dpi, which resolved within three days. SCID mice gradually developed ankle joint swelling since 4 dpi. The incidence of ankle joint swelling reached 100% by 6 dpi, and the symptoms progressively worsened over time (Fig. 3 B, C). Consistent with clinical observations, the virus titer in the ankle joint of ICR mice remained near the detection limit, with only trace amounts of viral DNA detected at 3 dpi and 7 dpi. IFNAR1 −/− mice revealed moderate sensitivity to MPXV, with detectable viral loads and titer observed only at 3 dpi and 7 dpi. SCID mice exhibited a continuous increase in both viral load and titer throughout the disease course (Fig. 3 D, E). Both clinical symptoms and virus titer indicated that SCID mice were more susceptible to MPXV. Besides, HE staining of the ankle joints in SCID mice revealed inflammatory cell infiltration in the synovial tissue, accompanied by significant synovial cell hyperplasia and disarray. Interstitial fibrous tissue proliferation and collagen deposition were observed, with local pannus formation. The tidemark of the cartilage was blurred, and chondrocytes exhibited atrophy and uneven distribution. We also monitored the clinical disease scores and survival rates of SCID mice up to 28 days. SCID mice exhibited progressive ankle joint swelling, weight loss, lethargy, ruffled fur, papules, and skin lesions. The clinical symptom score peaked at the end of the infectious period (Table 1 ), reaching a score of 8 (Fig. 3 F), with a 100% mortality rate at 25 dpi (Fig. 3 G). The cytokine profiles in the ankle joint were generally consistent with those observed in the pneumonia model, although no increase was detected in IFN-α1 levels (Supplementary Fig. 1C). Notably, this study represents the first animal model of MPXV-induced arthritis and the first lethal model using Clade IIb strain in SCID mice. This model can help monitor weight changes, ankle joint swelling, and virus titer within 14 dpi. Skin lesions appeared from 16 dpi, and a 100% mortality rate was observed at 26 dpi, with no evidence of self-limiting recovery. This model is therefore ideal for short-term and long-term drug evaluations. Table 1 Clinical scoring system used for MPXV-infected SCID mice. Ategory Score Criteria Body Weight 0 No weight loss or weight loss < 10% 1 Weight loss between 10–19% 2 Weight loss ≥ 20% Appearance 0 Smooth fur and normal posture 1 Ruffled fur and hunched posture Responsiveness 0 Normal response to external stimuli (e.g., touch, sound) 1 Reduced to moderate movement 2 No response to external stimuli; possible paralysis Skin Lesions 0 No skin lesions (macules, papules, scabs) on the tail 1 Macules (1–4 mm in diameter) 2 Papules (2–5 mm in diameter) 3 Scabs (2–5 mm in diameter) Scab widths 0 No scabs or secretions at the tail root 1 Scab width between 1 mm and 3 mm 2 Scab width > 3 mm Ankle Joint Swelling 0 No visible ankle joint swelling 1 Ankle joint width between 2 mm and 5 mm 2 Ankle joint width > 5 mm Validation of Tecovirimat and Cidofovir in In Vitro Models Tecovirimat and cidofovir have demonstrated antiviral activity against MPXV in both clinical and preclinical studies. However, recent genomic alterations in the currently circulating clade IIb strains observed over the past two years may affect their susceptibility to these drugs. Therefore, the antiviral activity of tecovirimat and cidofovir against MPXV clade IIb was investigated. Based on the three disease models established previously, A549, HaCaT, and MC3T3 cell lines corresponding to the infected tissues, were selected with the commonly used Vero-E6 cells. These in vitro models were employed to measure the drug concentrations with 50% cytotoxicity and 50% viral inhibition (CC 50 , EC 50 ). The MC3T3 cell line, not previously reported in studies on MPXV, was confirmed to be susceptible to the MPXV Clade IIb strain. The CC 50 values for both tecovirimat and cidofovir were more than 200 µM in all four cell lines, suggesting low cytotoxicity. The EC 50 values for tecovirimat against MPXV ranged from 0.0029 µM to 0.0104 µM, which were lower than those for cidofovir (0.4414 µM to 2.03 µM) (Supplementary Fig. 3A-D). These results are consistent with recent findings on other clade IIb isolates ( 33 , 34 ). Overall, tecovirimat and cidofovir demonstrated potent inhibitory activity against MPXV clade IIb in several in vitro models. Tecovirimat and Cidofovir Exhibited Significant Therapeutic Efficacy in the Early Stages of MPXV Infection Given the broad and potent in vitro inhibitory effects of tecovirimat and cidofovir on MPXV, their in vivo efficacy was investigated using the three animal models established in this study. While the recommended treatment duration for tecovirimat and cidofovir is 14 days, clinical guidelines suggest extending the treatment length for severely immunocompromised patients (up to 16 weeks). Since the SCID model infected with the clade IIb strain exhibited characteristic rash in the late stages of the disease (14–28 dpi), both short-term (14 days) and long-term (28 days) treatment groups were established to evaluate the therapeutic effects of tecovirimat and cidofovir in the early and late phases of MPXV infection. The SCID intranasal, intradermal, and subcutaneous infection models were employed in this study (Fig. 4 A). Two hours after infection, each model group received tecovirimat (P.O., n = 8) or cidofovir (S.C., n = 8), and the control group received normal saline(P.O., n = 4; S.C., n = 4). The therapeutic efficacy of tecovirimat and cidofovir in the early stages of MPXV infection varied based on the route of infection. Robust efficacy was demonstrated by both drugs in the intranasal model, effectively reducing viral DNA load (Fig. 4 B) and virus titer (Fig. 4 C) in the lung tissues. H&E staining revealed severe pathological damage in the control group, including alveolar wall thickening, alveolar collapse, inflammatory cell infiltration, hemorrhage, and epithelial cell necrosis. In contrast, minimal pathological damage was observed in the lungs of the tecovirimat and cidofovir treatment groups. Significant differences in pathological scores were observed compared to the control group (Fig. 4 D, E, Supplementary Table 1). In the intradermal infection model, cidofovir failed to effectively control the viral DNA load (Fig. 4 C), and three mice developed scabs with a width of more than 1 mm. The tecovirimat treatment group showed near-complete recovery, with no scabs. H&E staining revealed severe fibrosis, inflammatory cell infiltration, cell necrosis, epidermal acanthosis, keratinocyte ballooning degeneration, and scab lesions in the challenge control group, with a median pathological score of 9 (Supplementary Table 2). Compared to the control group, significant improvement was observed in both drug treatment groups (Fig. 4 D, E). In the arthritis model, both tecovirimat and cidofovir effectively decreased the viral DNA load (Fig. 4 B) and virus titer (Fig. 4 C) in the ankle joint. The pathological scores of both drug treatment groups were significantly decreased compared to the control group (Fig. 4 D, E, Supplementary Table 3). However, tecovirimat and cidofovir did not completely suppress ankle joint swelling, and the incidence of unilateral mild swelling was 37.5% and 50% in the two groups, respectively. Severe pathological findings were observed in the control group, including fibrous tissue proliferation, inflammatory cell infiltration, chondrocytes necrosis, cartilage damage, and pannus formation. In contrast, mildly swollen ankle joint in the treatment groups exhibited only fibrosis and inflammatory cell infiltration. Ankle joint without swelling showed no pathological damage (Fig. 4 E). Except for cidofovir, which failed to suppress GM-CSF expression at ankle joint, the expression levels of the TNF-α and GM-CSF in the lung, skin, and ankle joint were significantly reduced by pharmacological treatment (Supplementary Fig. 3A). Overall, tecovirimat and cidofovir effectively suppressed infectious virus titer and histopathological changes in the early stages of pneumonia, skin lesions, and arthritis. However, cidofovir could not decrease viral loads in the skin lesion model. The Therapeutic Efficacy of Tecovirimat and Cidofovir in the SCID Models of Non-self-limiting Advanced-stage MPXV Infection SCID mice were subjected to an advanced-stage model via intranasal, intradermal, and subcutaneous routes and treated with tecovirimat and cidofovir for 28 days (Fig. 5 A). Both treatments effectively mitigated viral infection-induced weight loss. However, compared to the tecovirimat group, the cidofovir-treated group experienced weight loss across all three models (Fig. 5 B - D), likely due to the nephrotoxicity of cidofovir. Nevertheless, the long-term use of these drugs did not affect the survival of mice. Both tecovirimat and cidofovir increased survival rates to 100% in the intranasal infection model (62.5% survival in the control group) and the subcutaneous infection model (0% survival in the control group) (Supplementary Fig. 4A - C). In the later stages of the disease, both drugs alleviated a range of clinical symptoms caused by viremia (Supplementary Fig. 4D - F). Unfortunately, neither drug successfully inhibited scab formation at the scratch sites of the skin lesion model. In addition, the drugs did not reduce ankle joint swelling of the arthritis model (Fig. 5 E). Further studies on the viral DNA load and virus titer in the target organs of the three models revealed that tecovirimat and cidofovir effectively decreased virus titer (Fig. 5 F) and viral load (Fig. 5 G) of the lung tissues of the pneumonia model. The drugs also decreased the infectious virus titer in the ankle joint of the arthritis model (Fig. 5 G). However, none of these drugs suppressed the virus titer (Fig. 5 F) or load in the skin lesion (Fig. 5 G) of the skin lesion model. Furthermore, cidofovir could not inhibit the viral DNA load in the ankle joint of the arthritis model (Fig. 5 G). The persistent presence of the virus in the target organs may have led to a significant increase in histopathological scores of the target organs in the treatment groups across all three models. Histopathological scores for the lung, skin, and ankle joint were determined based on (Supplementary Tables 1–3). Notably, except for cidofovir, which significantly decreased the histopathological scores in the lung tissues of the pneumonia model, no other treatment groups exhibited significant differences compared to the virus challenge groups (Fig. 5 H). Consistent with these histopathological findings, significant differences in TNF-α and GM-CSF levels were observed between the treatment groups and the virus challenge group in the lung tissues of intranasal infection model. However, no changes were observed between the groups in the skin lesion model and the ankle joint model (Supplementary Fig. 3B). In summary, in the late stage of infection in SCID mice, treatment with tecovirimat and cidofovir effectively decreased the mortality rate, maintained body weight, and successfully reduced the risk of complications caused by viremia in all three models. However, none of the drugs could prevent the formation of scabs in the skin lesion model or the swelling of the ankle joint in the arthritis model. In the later stages of the disease, both drugs failed to control the pathological damage in the target organs. Only cidofovir effectively reduced the pathological scores of lung tissues (median = 6.5). Regarding the decrease in virus titer in the target tissues, both tecovirimat and cidofovir exhibited infection route-dependent efficacy, showing inhibitory effects only in the intranasal and subcutaneous models. Discussion Epidemiological data indicated that nearly half of patients with mpox suffer from HIV co-infection, which is associated with more severe clinical manifestations and higher mortality rates. A study conducted in the United States enrolled 395 patients with mpox, of whom 324 (82.0%) were diagnosed with HIV infection. In this cohort, severe mpox was observed in 19.5% of patients, and HIV coinfection was confirmed in 79.2% of patients ( 35 ). However, clinical research on this population has been limited due to low healthcare-seeking rates and low availability of samples due to social stigma. While traditional non-human primate models (such as SIV-coinfected macaques) can simulate immunodeficiency phenotypes, their prohibitive maintenance costs and stringent ethical restrictions impede high-throughput research demands ( 36 ). In this study, SCID mice were selected as a more available rodent model, representing the first model of SCID established using the Clade IIb strain responsible for the 2022 mpox outbreak. Although CAST/EiJ mouse models of skin lesion and pulmonary infection models in BALB/c mice and dormice for Clade IIb have been reported in recent years, these models only partly replicate localized symptoms or immune responses of MPXV-susceptible populations ( 34 , 37 – 39 ). In contrast, SCID mice, characterized by congenital T/B lymphocyte developmental blockade due to Prkdc gene mutation, more accurately mimic the depletion of CD4 + T cells observed in HIV-infected individuals ( 40 ). Their persistent viral replication and non-self-limiting disease progression closely align with the clinical manifestations of high-risk patients. Tecovirimat, brincidofovir, and cidofovir have not been specifically approved for mpox; however, these agents have been employed for some patients with MPXV. Brincidofovir was excluded from this study due to procurement limitations. The recommended in vivo treatment length for tecovirimat and cidofovir is 14 days, and the current drug evaluation studies in mouse models have administered these drugs for 14 days or less (7 days) ( 33 , 34 ). However, several studies have indicated that for severely immunocompromised patients with mpox, extending the treatment course of tecovirimat/cidofovir may improve clinical outcomes ( 25 , 41 – 44 ). Therefore, this study included an additional long-term treatment group to ensure complete viral clearance and prevent disease recurrence or secondary infections. In the short-term treatment group, both tecovirimat and cidofovir significantly reduced infectious virus titer and pathological scores of the target organs (lung, skin and ankle joint) across the three infection models, which is consistent with the results of recent reports ( 33 , 34 ). Unfortunately, tecovirimat and cidofovir were not very effective in the late stages of the disease. Neither tecovirimat nor cidofovir effectively reduced the histopathological progression and viral loads in the skin lesion or arthritis models. The limited therapeutic efficacy in patients with advanced-stage disease is consistent with a report from New York, where 6 out of 12 patients with HIV and advanced disease who received extended courses of tecovirimat combined with vaccinia immune globulin (VIG), cidofovir, and/or brincidofovir finally died ( 45 ). Notably, tecovirimat resistance-associated F13L gene mutations were detected in 44% of these patients ( 26 ). These findings highlight the limitations of long-term monotherapy with tecovirimat and cidofovir in immunocompromised individuals. Future studies should prioritize enhanced resistance monitoring for tecovirimat and toxicity surveillance for cidofovir, while exploring novel combination regimens to overcome current therapeutic shortcomings. Clinically, pneumonia caused by mpox infection is predominantly observed among patients with HIV co-infection ( 9 ). In patients with pneumonia, CT or X-ray examinations predominantly reveal pulmonary infiltration, ground-glass opacity, or consolidation, which are pathologically consistent with inflammatory cell infiltration, thickened alveolar walls, and alveolar collapse observed in our pneumonia model ( 10 , 46 ). Among all pathogenic models established in this study, the pneumonia model demonstrated the most favorable therapeutic outcomes, which can be attributed to the high blood flow in the lungs and the high permeability of alveolar epithelium facilitating drug accumulation in the lung tissues ( 47 , 48 ). With the exception of a patient with acute respiratory distress syndrome (ARDS) where tecovirimat was successfully administered clinically, current management of patients with MPXV and confirmed pneumonia remains symptomatic treatment and supportive therapy, with no antiviral agents ( 49 ). Given the favorable outcomes of both short-term and long-term treatments in this pneumonia model, tecovirimat and cidofovir can be administered to prevent severe adverse outcomes. In immunocompromised patients, the combination of antiretroviral therapy (ART) and adjunctive VIG should also be evaluated. Future development of nebulized (NEB) antiviral formulations (e.g., liposomal cidofovir) for direct delivery to the lungs is also warranted. In the skin lesion model, cidofovir could not effectively reduce viral DNA load, and in the late stage, neither tecovirimat nor cidofovir effectively inhibited the progression of skin lesion. This observation aligns with clinical findings in patients with advanced HIV, where no significant alteration in viral load was observed after administering a 33-day tecovirimat regimen (600 mg orally, twice daily) followed by topical (3%) and injected (5 mg/kg) cidofovir ( 29 ). Although cidofovir did not reduce the viral load in the skin of the intradermal infection model, it unexpectedly lowered the viral load in the lung tissue of SCID mice. This finding is consistent with the therapeutic effects of cidofovir on skin infections caused by cowpox virus ( 50 ). Viral inhibition is pharmacologically effective, though dermal bioavailability may be limited by the skin's low pH, high keratinization, and locally immunosuppressive microenvironment ( 51 , 52 ). This suggests that localized transdermal drug delivery systems (TDDS), such as nanoliposomal gels or microneedle patches, combined with systemic treatment, can achieve synergistic drug accumulation in the skin. Such an approach can enhance dual suppression of viral replication at the epidermal-dermal junction, accelerating pathological repair of skin lesion and viral clearance. Notably, recovery after infection with poxvirus frequently results in cutaneous scarring. A 1540 nm radiofrequency laser-based therapy, Secret Duo, has been approved by the U.S. FDA for treating scars. Clinically reported cases of arthritis are primarily present with generalized arthralgia, knee joint swelling, and MRI findings of synovitis and abnormal synovial fluid cell counts ( 13 – 15 ). These manifestations are highly consistent with ankle joint swelling, synovial damage, and inflammatory cell infiltration observed in our arthritis model. Our model represents the first preclinical model for mpox-associated arthritis, with its corresponding MC3T3 in vitro model being the first application for research on mpox infection. However, the main management for previous cases of arthritis remains symptomatic treatment, without administering any antiviral agents. Based on the favorable therapeutic outcomes observed in our arthritis model, tecovirimat and cidofovir can be considered for treating future cases of mpox-associated arthritis. When necessary, adjunctive therapies such as glucocorticoid, platelet-rich plasma (PRP) and low-intensity pulsed ultrasound (LIPUS) can be employed to promote cartilage repair, suppress the inflammatory responses, and alleviate pain. In conclusion, SCID mice were successfully employed to establish pneumonia, skin lesion, and arthritis models through various routes of MPXV infection. The models replicated the clinical manifestations observed in high-risk populations for mpox, including patients with HIV and organ transplant recipients. Utilizing these three models, comprehensive pharmacodynamic evaluations of tecovirimat and cidofovir were conducted across short-term and long-term treatment regimens, and potential strategies for combination therapy were proposed (Fig. 6 ), thereby providing robust preclinical data to guide the clinical management of patients with mpox. Materials and Methods Ethics Statement All animal experiments were conducted in AAALAC International-accredited facilities and adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Animal Care and Use Committee of the Changchun Veterinary Research Institute (IACUC approval no. AMMS-11-2023-041). All samples generated in the biosafety level 3 laboratory were inactivated according to IBC-approved standard operating procedures upon removal from the high-containment area. Cells and Virus MPXV (clade IIb, GenBank accession number: PP778666.1) was isolated from a patient in Guangzhou, China. The virus was cultured and amplified in Vero-E6 cells using DMEM medium (Sigma-Aldrich) containing 2% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 µg/mL streptomycin. Vero-E6 cells were cultured in DMEM containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 µg/mL streptomycin. No mycoplasma or contaminants were detected. All experiments involving infectious MPXV were conducted in a biosafety level 3 laboratory. Animal Experiment Design 4-5-week-old male ICR mice and SCID mice were purchased from Beijing HFK Biotechnology Co., Ltd., and 4-5-week-old male IFNAR1 −/− mice were purchased from Beijing Cyagen Biosciences Inc. Part 1: Mice from all three models were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n = 24) or normal saline (n = 8). The clinical symptoms were continuously monitored. ICR, IFNAR1 ⁻/⁻ , and SCID mice were euthanized at 3, 7, and 14 dpi, respectively. Lung, skin, and ankle joint were collected, minced, weighed to 0.1 g (wet weight), and then homogenized in 500 µL of PBS for subsequent analyses. Part 2: SCID mice were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n = 24) or normal saline (n = 8) to evaluate disease scores and survival rates. Disease scores and survival rates were measured from 0 dpi to 28 dpi. Part 3: SCID mice were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n = 48). Mice in each group received tecovirimat (oral, 100 mg/kg, once daily, n = 16), cidofovir (intravenous, 100 mg/kg, twice a week, n = 16), or normal saline (oral, n = 8; intravenous, n = 8). Mice were euthanized at 14 dpi and 28 dpi (n = 8). Lung, skin, and ankle joint were collected, minced, weighed to 0.1 g (wet weight), and homogenized in 500 µL of PBS for subsequent analyses. In Vitro Antiviral Efficacy Assays CC 50 Determination: Vero-E6 cell monolayers in 96-well plates were cultured to approximately 50% confluence and treated with different concentrations of tecovirimat (200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 µM) or cidofovir (200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 µM) in a total volume of 100 µL per well. Each drug concentration was prepared by five-fold serial dilution in the maintenance medium. After 72 hours of incubation, the medium was discarded, and the cells were washed twice with PBS. Subsequently, 100 µL of 10% CCK-8 reagent was added to each well, followed by incubation in a 5% CO 2 incubator at 37°C for 0.5-1 hour. The absorbance (OD) was measured at 450 nm using a microplate reader. EC 50 Determination: Vero-E6 cells were cultured in 24-well plates to near confluence and infected with MPXV at an MOI of 0.1. The infection was incubated for 1 hour at 37°C with 5% CO 2 , shaking every 15 minutes. After removing the inoculum, different concentrations of tecovirimat or cidofovir were added based on CC 50 . After a 72-hour incubation period, the supernatants were collected, and MPXV DNA was extracted using the MAGEN DNA extraction kit. The viral DNA levels were quantified by qPCR. The OD values and corresponding DNA copy numbers were entered into GraphPad Prism software, normalized to the control group (cells treated with no drug), and expressed as percentages. The concentrations (log-transformed) were plotted on the X-axis, and the percentage of cell viability or MPXV DNA content was plotted on the Y-axis. A four-parameter logistic curve was fitted, and nonlinear regression analysis was conducted to calculate the CC₅₀ and EC₅₀ values for each treatment group. Viral DNA Quantification (qRT-PCR) Tissue homogenates were centrifuged at 12,000 × g and 4°C for 15 minutes, and the supernatant (200 µL) was collected to detect and quantify the expression levels of MPXV. Viral nucleic acid was extracted using the Viral Nucleic Acid Quick Extraction Kit (MAGEN) following the manufacturer’s instructions, with an elution volume of 50 µL. Real-time fluorescence quantitative detection of the MPXV F3L gene was conducted using the TaqMan probe method (F: 5′-CTCATTGATTTTTCGCGGGATA-3′, R: 5′-GACGATACTCCTCCTCGTTGGT-3′, probe: FAM-CATCAGAATCTGTAGGCCGT-BHQ). The experiment was conducted using the Takara Premix Ex Taq™ (Probe qPCR) kit (TAKARA) for Real-Time PCR. The experiment was conducted using the Takara Premix Ex Taq™ (Probe qPCR) kit (TAKARA) for Real-Time PCR. The reactions were conducted on the Bio-Rad CFX96 Real-Time PCR Detection System. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Infectious Virus titer Quantification (TCID₅₀) The supernatant of tissue homogenate was serially diluted 10-fold. Then, 100 µL of each dilution was added to a 96-well plate containing a monolayer of Vero-E6 cells. The plate was incubated in a 5% CO 2 at 37°C. After 96 hours of incubation, the cytopathic effect (CPE) was observed. The virus titer was calculated using the Reed-Muench method. Histopathological Assessment Necropsy and tissue sampling were conducted following the protocols approved by the Institutional Biosafety Committee (IBC). Hematoxylin and eosin (H&E) staining of tissue samples was conducted using standard paraffin embedding methods. Tissue samples were fixed in 10% neutral-buffered formalin for at least 7 days, followed by dehydration, clearing, and embedding in paraffin. The tissues were sectioned into 4–5µm thick slices and mounted onto pre-coated APES slides. The tissue slides were deparaffinized in xylene, rehydrated through ethanol, stained with hematoxylin and eosin, and dehydrated. After clearing in xylene, the tissue sections were mounted with neutral balsam. The tissue slides were evaluated by a certified, double-blind pathologist. Measurement of Cytokine Levels RNA was extracted from 200 µL of homogenized tissue using an RNA Extraction Kit (Gene Script Biotech). The extracted mRNA was amplified in a CFX96 Real-Time PCR System (Bio-Rad) using the commercially available 2× Q1 SYBR qPCR Master Mix (Universal). For cytokine profiling, the following primer pairs were used: TNF-α (F: 5′-AGCCAGGAGGGAGAACAGA-3′, R: 5′-CAGTGAGTGAAAGGGACAGAAC-3′), IL-6 (F: 5′-CGGAGAGGAGACTTCACAGAG-3′, R: 5′-CATTTCCACGATTTCCCAGA-3′), NAP3 (F: 5′-TCCAGAGCTTGAAGGTGTTGCC-3′, R: 5′-AACCAAGGGAGCTTCAGGGTCA-3′), IFN-α1 (F: 5′-TAATTCCTACGTCTTTTCTTT-3′, R: 5′-TATGCCTGATCCCTGAACAGT-3′), IFN-β (F: 5′-AACCTCCTGGATGACATGCCTG-3′, R: 5′-AAATTGCCCCGTAGACCCTGCT-3′), GM-CSF (F: 5′-AACCTCCTGGATGACATGCCTG-3′, R: 5′-AAATTGCCCCGTAGACCCTGCT-3′) Expression levels were normalized to ​β-actin (F: 5′-GTGGGCCGCTCTAGGCACCAA-3′, R: 5′-CTCTTTGATGTCACGCACGATTTC-3′). The 2× Q1 SYBR qPCR Master Mix (Universal) was employed for qPCR. Cytokine levels were measured using the TNF-α ELISA Kit and GM-CSF ELISA Kit (Cloud-Clone Corp.). The homogenate was centrifugated and lysed with RIPA buffer to prepare for inflammatory factor detection. The experiments were conducted following the manufacturer’s instructions, and the absorbance was read at 450 nm using a spectrophotometer. Statistical Analysis and Data Visualization Data collection and analysis were conducted in a double-blind manner. Statistical analyses were conducted using GraphPad Prism 8.0. Data are presented as ​mean ± standard error of the mean (SEM). Inter-group differences were determined using t -tests or one-way analysis of variance (ANOVA) followed by two-tailed t -tests, unless otherwise specified. Statistical significance levels were defined as follows: not significant ( ns ), P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. The type of test used is indicated where appropriate. All data generated in this study are provided in the Source Data file. Declarations Acknowledgements This work was supported by the National Key Research and Development Program of China (2023YFD1800405) and CAMS Innovation Fund for Medical Sciences (2020-12M-5-001). Thanks to the Eighth Affiliated Hospital of Guangzhou Medical University for providing the clinical mpox samples.The authors would like to express their gratitude to EditSprings ( https://www.editsprings.cn ) for the expert linguistic services provided. References Jezek Z, Szczeniowski M, Paluku KM, Mutombo M (1987) Human monkeypox: clinical features of 282 patients. J Infect Dis 156(2):293–298 Isidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A et al (2022) Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6483910","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":446311360,"identity":"9b13481d-a878-4466-802f-f096747a2acd","order_by":0,"name":"Hui-Jun Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACAwbGBgMgLcPA3tj44AMpWngYeA43G84gTgsE8DBIpLdJcxCjxVwiuaGYdwcDj8HNhw3SDAx2croNBLRYzkhsMOY9A9RyG8goYEg2NjtAyGE3QFraIFqSZzAcSNxGvJabBxsO85Cm5QZjYzNxWs48bDCcC9QieSaxmXGGATF+OZ7+zOBtG4Mc3/Hjz398qLCTI6gFCNiAcfMfZgJh5SDA/IA4daNgFIyCUTBiAQAvCEGYTcvNfQAAAABJRU5ErkJggg==","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China. 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Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences,","correspondingAuthor":false,"prefix":"","firstName":"Xiangshu","middleName":"","lastName":"Qiu","suffix":""},{"id":446311364,"identity":"0fde060f-9ae8-4960-b8ba-9c140f628988","order_by":4,"name":"Jiaxin Tian","email":"","orcid":"","institution":"College of Animal Sciences, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou Zhejiang. Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, C","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Tian","suffix":""},{"id":446311365,"identity":"f1908bcf-8364-4695-83a2-8f124ae8b177","order_by":5,"name":"Peng Wang","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Wang","suffix":""},{"id":446311366,"identity":"a3443974-1dba-4f6e-9b1d-230c52b91316","order_by":6,"name":"Bocheng Liu","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China.","correspondingAuthor":false,"prefix":"","firstName":"Bocheng","middleName":"","lastName":"Liu","suffix":""},{"id":446311367,"identity":"7229bfb9-e66c-4b83-80eb-5809943734e3","order_by":7,"name":"Zhuo Ha","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China.","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Ha","suffix":""},{"id":446311368,"identity":"bfe2f1cf-23af-421e-8918-65ff9bba2d3f","order_by":8,"name":"He Zhang","email":"","orcid":"","institution":"Changchun Institute of veterinary medicine, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Zhang","suffix":""},{"id":446311369,"identity":"58ac33fa-3d0d-4494-a468-d18d7f834466","order_by":9,"name":"Chao Shang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Shang","suffix":""},{"id":446311370,"identity":"8f8ea8f9-3a21-4d34-8c37-ce0cc985e58d","order_by":10,"name":"Xiao Li","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China.","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Li","suffix":""},{"id":446311371,"identity":"165696bf-a7f0-409e-bc19-84ebaa77fe3b","order_by":11,"name":"Yubiao Xie","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China.","correspondingAuthor":false,"prefix":"","firstName":"Yubiao","middleName":"","lastName":"Xie","suffix":""},{"id":446311372,"identity":"5f9d001d-7fb3-4a15-999a-9136927f46a9","order_by":12,"name":"Yue-Long Shu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yue-Long","middleName":"","lastName":"Shu","suffix":""}],"badges":[],"createdAt":"2025-04-19 09:45:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6483910/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6483910/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67548-0","type":"published","date":"2025-12-18T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81197837,"identity":"c1835af4-ab03-4f67-8961-9e31883e0f14","added_by":"auto","created_at":"2025-04-23 10:41:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":595555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and viral dynamics analysis after intranasal infection of MPXV clade IIb in different mouse models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICR (blue), IFNAR1\u003csup\u003e-/-\u003c/sup\u003e (yellow), and SCID (red) mice were anesthetized with isoflurane and intranasally inoculated with MPXV (10\u003csup\u003e5\u003c/sup\u003e PFU/100 μL, clade IIb, n = 24) or sterile saline (control group, n = 8). (A) The body weight changes were monitored from 0 dpi to 14 dpi. At 3 dpi, 7 dpi, and 14 dpi, the ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mice were euthanized (n = 8), and the following were recorded for each group: (B) pulmonary tissue appearance, (C) lung viral DNA load (qPCR), and (D) infectious viral titer in the lungs (TCID\u003csub\u003e50\u003c/sub\u003e). After identifying the most susceptible mice, SCID mice were re-inoculated with MPXV (red, 10\u003csup\u003e5\u003c/sup\u003e PFU/100 μL, n = 8) or sterile saline (gray, 100 μL, n = 8) via intranasal inoculation. (E) Disease score and (F) survival rate of SCID mice were monitored from 0 dpi to 28 dpi. In (B), the experiments were repeated independently eight times, and a representative result is shown for each. In (C, D), TCID\u003csub\u003e50\u003c/sub\u003e and qPCR analyses were performed once, with 8 replicates per sample for TCID\u003csub\u003e50\u003c/sub\u003e. Dashed line: detection limit. Error bars represent mean ± standard error of the mean (SEM). Statistical differences between groups were determined by \u003cem\u003et\u003c/em\u003e-tests. Statistical significance levels are indicated as: *\u003cem\u003ep \u003c/em\u003e≤ 0.05\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/fdb486fa0a7db04e32374f67.png"},{"id":81199311,"identity":"f402ae4f-2b80-4e7c-a2cf-378a561d977a","added_by":"auto","created_at":"2025-04-23 10:57:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":356262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and viral dynamics analysis after intradermal infection of MPXV clade IIb in different mouse models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICR (blue), IFNAR1\u003csup\u003e-/-\u003c/sup\u003e (yellow), and SCID (red) mice were anesthetized with isoflurane and intradermally inoculated with MPXV (10\u003csup\u003e5\u003c/sup\u003e PFU/50 μL, clade IIb, n = 24) or sterile saline (control group, n = 8). (A) Body weight changes were monitored from 0 dpi to 14 dpi. (B) Incidence rate was continuously observed from 0 dpi to 14 dpi in ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mice. The scab width of ≥ 1mm at the tail was considered positive (blue, yellow, red), while scab width \u0026lt; 1mm was considered negative (white). The pie chart illustrates the number of diseased individuals at each time point in ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mouse samples (n = 8). (C) The typical scabs at the tail of ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mice were recorded and euthanized to measure (D) viral DNA load (qPCR) and (E) infectious viral titer (TCID\u003csub\u003e50\u003c/sub\u003e) in skin at 3, 7, and 14 dpi (n = 8). After identifying the most susceptible mice, SCID mice were re-inoculated with MPXV (red, 10\u003csup\u003e5\u003c/sup\u003e PFU/100 μL, n = 8) or sterile saline (gray, 100 μL, n = 8) via intradermal inoculation. (F) Disease scores and (G) survival rates of SCID mice were monitored from 0 dpi to 28 dpi. In (C), the experiments were repeated independently eight times, and a representative result is shown for each. Dashed line: detection limit. Error bars represent the mean ± standard error of the mean (SEM). Statistical differences between groups were determined by \u003cem\u003et\u003c/em\u003e-tests. followed by two-tailed \u003cem\u003et\u003c/em\u003e-tests. Statistical significance levels are indicated as follows: *\u003cem\u003eP\u003c/em\u003e ≤ 0.05; ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/274c1baa112f8363d02d58d4.png"},{"id":81197845,"identity":"bdee1c4f-b218-43c7-bf4f-3cfdb381a3d9","added_by":"auto","created_at":"2025-04-23 10:41:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and viral dynamics analysis after subcutaneous infection of MPXV clade IIb in different mouse models.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICR (blue), IFNAR1\u003csup\u003e-/-\u003c/sup\u003e (yellow), and SCID (red) mice were anesthetized with isoflurane and subcutaneously inoculated in the footpad with MPXV (10\u003csup\u003e5\u003c/sup\u003e PFU/100 μL, clade IIb, n= = 24) or sterile saline (control group, n = 8). (A) Body weight changes were monitored from 0 dpi to 14 dpi. (B) Incidence rate was continuously observed from 0 dpi to 14 dpi in ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mice. The width of ≥ 1mm at the ankle joint was considered positive (blue, yellow, red), while ankle joint width \u0026lt; 1mm was considered negative (white). The pie chart illustrates the number of diseased individuals at each time point in ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mouse samples (n = 8). (C) The typical swelling at the pootpad of ICR, IFNAR1\u003csup\u003e-/-\u003c/sup\u003e and SCID mice were recorded and euthanized to measure (D) viral DNA load (qPCR) and (E) infectious viral titer (TCID\u003csub\u003e50\u003c/sub\u003e) in footpad at 3, 7, and 14 dpi (n = 8). After identifying the most susceptible mice, SCID mice were re-inoculated with MPXV (red, 10\u003csup\u003e5\u003c/sup\u003e PFU/100 μL, n = 8) or sterile saline (gray, 100 μL, n = 8) via intradermal inoculation. (F) Disease scores and (G) survival rates of SCID mice were monitored from 0 dpi to 28 dpi. In (C), the experiments were repeated independently eight times, and a representative result is shown for each. Dashed line: detection limit. Error bars represent the mean ± standard error of the mean (SEM). Statistical differences between groups were determined by \u003cem\u003et\u003c/em\u003e-tests or one-way analysis of variance (ANOVA) followed by two-tailed \u003cem\u003et\u003c/em\u003e-tests. Statistical significance levels are indicated as follows: *\u003cem\u003eP\u003c/em\u003e ≤ 0.05; ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/33491383c39212b7238514e5.png"},{"id":81197853,"identity":"d3d8353b-c27d-4e18-b252-51cbebcf394e","added_by":"auto","created_at":"2025-04-23 10:41:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":910587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant Therapeutic Effects of Tecovirimat and Cidofovir in the Early Stages of MPXV Infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy schematic of \u003cem\u003ein vivo \u003c/em\u003eevaluation of tecovirimat and cidofovir was evaluated using SCID mice with intranasal (green, n = 24), intradermal (pink, n = 24), and subcutaneous (purple, n = 24) infection routes is shown. Tecovirimat (blue, P.O., n = 8), cidofovir (yellow, S.C., n = 8), or sterile saline (P.O. n = 4, S.C. n = 4) were administered for a 14-day treatment period, starting at two hours after infection. Mice were euthanized (red) at 14 dpi, and collecte lung, skin, and ankle joint. (B) Infectious viral titers and (C) DNA loads were measured using qPCR and TCID\u003csub\u003e50\u003c/sub\u003e assays. (D) Histopathological damage scores of the tissues were evaluated by a certified, double-blind pathologist. (E) The clinical symptoms and histopathology sections of the lung, skin, and joint are combined and presented. The black scale bar in H\u0026amp;E figure represents 100 μm. In (E), the experiments were repeated independently eight times, and a representative result is shown for each. Dashed line: detection limit. Error bars represent the mean ± standard error of the mean (SEM). Statistical differences between groups were determined by\u003cem\u003e t\u003c/em\u003e-tests or one-way analysis of variance (ANOVA) followed by two-tailed \u003cem\u003et\u003c/em\u003e-tests. Statistical significance levels are indicated as follows: \u003cem\u003ens\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; *\u003cem\u003eP\u003c/em\u003e ≤ 0.05; **\u003cem\u003eP\u003c/em\u003e ≤ 0.01; ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/2f026da522218d52cfb3266c.png"},{"id":81199680,"identity":"2aa1a8c8-078f-4cd6-b8c2-0741f9042c58","added_by":"auto","created_at":"2025-04-23 11:05:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":994166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic effects of tecovirimat and cidofovir in the prolonged disease model of MPXV are dependent on the route of infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy schematic of the \u003cem\u003ein vivo \u003c/em\u003eevaluation of tecovirimat and cidofovir, using SCID mice with intranasal (green, n = 24), intradermal (pink, n = 24), and subcutaneous (purple, n = 24) infection \u003cstrong\u003eroutesv is shown\u003c/strong\u003e. Tecovirimat (blue, P.O., n = 8), cidofovir (yellow, S.C., n = 8), or sterile saline (P.O. n = 4, S.C. n = 4) were administered for a 28-day treatment period, starting at two hours after infection. Surviving mice were euthanized (red) at 28 dpi, and those with a body weight loss exceeding 20% were euthanized earlier. (B - D) Body weight of tecovirimat and cidofovir treatment groups and the virus challenge control group was continuously monitored in all three infection models. (E) Clinical symptom assessments and histopathological observations were performed on the lung, skin, and joint of the three disease models, \u003cstrong\u003erespectively\u003c/strong\u003e. \u003cstrong\u003eThe \u003c/strong\u003eblack scale bar in H\u0026amp;E figure represents 100 μm. (F) Infectious viral titers and (G) DNA loads were measured using qPCR and TCID50 assays. (H) Histopathological damage scores of the tissues were evaluated by a certified, double-blind pathologist. In (E), the experiments were repeated independently eight times, and a representative result is shown for each. Dashed line: \u003cstrong\u003edetection limit. \u003c/strong\u003eError bars represent the mean ± standard error of the mean (SEM). Statistical differences between groups were determined by \u003cem\u003et\u003c/em\u003e-tests or one-way analysis of variance (ANOVA) followed by two-tailed \u003cem\u003et\u003c/em\u003e-tests. Statistical significance levels are indicated as follows: \u003cem\u003ens\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; *\u003cem\u003eP\u003c/em\u003e ≤ 0.05; **\u003cem\u003eP\u003c/em\u003e ≤ 0.01; ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/8aad93ea33aa63115c6ac522.png"},{"id":81197854,"identity":"ac6a45b7-5b29-4d34-915e-b86aa268a65e","added_by":"auto","created_at":"2025-04-23 10:41:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":640859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment of MPXV-Induced pneumonia, rash, and synovitis in immunocompromised patients.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/7c5bb0b5a0b0e0a88a9eba7b.png"},{"id":100958650,"identity":"90c82455-15aa-4aeb-b29f-c4dde81654ac","added_by":"auto","created_at":"2026-01-23 08:07:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5054982,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/fbbd896a-ea8e-4178-a7a1-1ad78e816b22.pdf"},{"id":81197840,"identity":"3163a60c-3b8d-4eb5-b8e8-d01cc151dfe0","added_by":"auto","created_at":"2025-04-23 10:41:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":991468,"visible":true,"origin":"","legend":"Supplementary figures and tables","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6483910/v1/65e942dfaff5617dfa0889e9.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Efficacy of Tecovirimat and Cidofovir Against MPXV-Induced Pneumonia, Skin Lesion, and Arthritis in the High-Risk Population-Relevant SCID Mouse Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a significant member of the genus \u003cem\u003eOrthopoxvirus\u003c/em\u003e from the family \u003cem\u003ePoxviridae\u003c/em\u003e (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), monkeypox virus (MPXV) is taxonomically related to variola virus (smallpox virus). Although the lethality of MPXV is less than that of smallpox, MPXV has greater human transmissibility. Epidemiological data suggests the accelerated adaptive evolution of MPXV (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Phylogenetic studies classify MPXV into four clades, including Ia, Ib, IIa, and IIb (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Clade IIb has circulated among humans since 2016, leading to the 2022 pandemic (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). On July 21, 2022, the World Health Organization (WHO) first declared the MPXV outbreak a Public Health Emergency of International Concern (PHEIC). Due to the emergence of the newly identified clade Ib, which spread in the Democratic Republic of the Congo (DRC) and neighboring countries and regions beyond Africa, WHO announced the second PHEIC designation for monkeypox (mpox) on August 14, 2024 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Following a third round of assessment on February 27, 2025, WHO decided to maintain the PHEIC status of the outbreak due to the persistent risks of international spread and public health issues.\u003c/p\u003e \u003cp\u003eThe MPXV clade IIb predominantly affects men who have sex with men (MSM), accounting for 87% of the reported cases, of whom approximately 50% are HIV-positive. These patients with co-infection, particularly those with advanced HIV, exhibit severe complications and suffer from an increased risk of mortality (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Among patients with MPXV and HIV co-infection, 29% develop pulmonary involvement (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The first documented case of MPXV-induced pneumonia was observed in a male patient with advanced AIDS who presented with several complications and finally succumbed to sepsis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Cutaneous rash occurs in 95% of MPXV-infected patients (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The rash is typically self-limiting in immunocompetent individuals, spontaneously resolving within 2\u0026ndash;4 weeks (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, immunocompromised patients often develop more extensive and persistent skin lesions, have higher viral loads, suffer from prolonged disease, and are at a significantly increased risk of scarring (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). HIV-associated MPXV infection frequently manifests with arthritis, characterized by knee joint pain, abnormal synovial fluid cell counts, and MRI findings of synovitis and osteomyelitis (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Notably, viral osteomyelitis is a classic complication of smallpox, affecting 2%-5% of infected children (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Furthermore, symptoms of arthritis have been reported in patients with vaccinia virus infection, cowpox virus infection, and even among patients with HIV receiving the JYNNEOS vaccine (MPXV vaccine) (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, no antiviral treatment has been definitively demonstrated to be effective against MPXV. However, several antiviral agents have been authorized for emergency use in certain countries and are under evaluation in clinical trials. WHO and several national guidelines recommended​tecovirimat as the first-line antiviral drug for mpox, showing efficacy in several clinical cases (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, recent studies reported that, during the mpox outbreak in the United States, severely immunocompromised patients who received multiple courses of tecovirimat exhibited poor outcomes (35.3%; 18 out of 51 patients) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Furthermore, tecovirimat resistance is a major clinical challenge, particularly in subjects with severe immune dysfunction. Resistance-associated mutations, such as F13L gene mutation, may lead to treatment failure and human-to-human transmission, emphasizing the need for cautious use of tecovirimat until obtaining more experimental data (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Cidofovir, an emergency antiviral drug recommended by WHO, exhibits broad-spectrum activity against DNA viruses, such as MPXV. A patient with HIV and uncontrolled viremia exhibited marked improvement after switching to cidofovir after failure of treatment with tecovirimat and antibiotics (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, cidofovir exhibits nephrotoxicity, particularly in patients with HIV, with an incidence rate of nearly 25% (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Additionally, the topical application of cidofovir may lead to adverse effects, such as skin erosions (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Due to the risk of such adverse events, cidofovir is often reserved for critical cases or tecovirimat-resistant infections; therefore, there are limited experimental data on its monotherapy. The prodrug brincidofovir demonstrates reduced nephrotoxicity and exhibits anti-mpox activity, though its clinical efficacy, like that of the two aforementioned drugs, still needs more preclinical and clinical studies (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, the establishment of animal models that can accurately replicate the typical clinical manifestations of MPXV is a prerequisite for assessing the therapeutic efficacy of anti-viral drugs. Tecovirimat and cidofovir (and its derivative) are currently the main therapeutic options for mpox. However, the use of both drugs faces limitations due to insufficient clinical data regarding their efficacy and safety across different viral clades and in immunocompromised populations.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eScreening and Establishment of the Mouse Model of MPXV-Induced Pneumonia\u003c/h2\u003e\n \u003cp\u003eIntranasal (I.N.) inoculation is commonly employed for modeling pneumonia in small rodents, but the pathogenicity of MPXV varies across different mouse models. In this study, male ICR mice, IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, and SCID mice were intranasally inoculated with 10⁵ PFU MPXV or sterile saline (control group). After MPXV infection, ICR and IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited a steady increase in body weight, which was comparable to the control group. SCID mice exhibited a significant decrease in body weight at 12 days post infection (dpi), which was recovered by 14 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). After inoculation, no clinical symptoms, such as changes in behavior, appetite, fur, or appearance, were observed in any of the three mouse models. Gross examination of lung tissues collected at 3 dpi, 7 dpi, and 14 dpi exhibited no significant differences compared to the control group, except for mild hemorrhage and atrophy in the lung tissue of SCID mice at 14 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, qPCR and detection of virus titer were conducted to measure the viral DNA load and infectious virus titer in the lung tissues of the three mouse models at 3 dpi, 7 dpi, and 14 dpi. The results indicated that ICR mice were not susceptible to MPXV. Viral DNA and virus titer were at low levels at 3dpi, decreasing to the detection limit at 7 dpi and 14 dpi. IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited peak lungs infection on 7 dpi, with a median viral DNA load of 10\u003csup\u003e4.48\u003c/sup\u003e copies/mL, and a low median virus titer of 10\u003csup\u003e2.43\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL. Both viral load and titer decreased at 14 dpi. After intranasal inoculation, mice with SCID revealed a steady increase in lungs viral load and virus titer over time. This model exhibited the greatest increase in virus titer compared to the other two mouse models, reaching a median of 10\u003csup\u003e4.75\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL at 14 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). These findings suggest that SCID mice are the most stable and susceptible model for the intranasal infection study.\u003c/p\u003e\n \u003cp\u003eSubsequently, SCID mice were intranasally inoculated with MPXV at the same virus titer (n\u0026thinsp;=\u0026thinsp;8), and their clinical disease scores and survival rates were monitored for up to 28 dpi to observe the typical clinical symptoms. At 16 dpi, weight loss, lethargy, disheveled fur, swollen footpads, and the typical symptoms of MPXV infection, such as skin lesions of the tail were observed in SCID mice. The severity of clinical symptoms was measured, revealing a median disease score of 7 at 28 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), with a relatively low mortality rate of 25% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Additionally, cytokine activation in the lungs was analyzed at the end of the experiment. The results showed significantly higher mRNA levels of pro-inflammatory factors (TNF-\u0026alpha; and IL-6) and antiviral interferons (IFN-\u0026beta; and IFN-\u0026alpha;1), while the mRNA levels of NAP3 were significantly downregulated (Supplementary Fig.\u0026nbsp;1A). These cytokines, particularly pro-inflammatory factors, may contribute to pulmonary pathology. Notably, compared to other mouse models of intranasal infection, footpad edema and cutaneous lesions were observed in this study. These symptoms indicate that intranasal MPXV infection in SCID mice can induce viremia during the late stage of infection.\u003c/p\u003e\n \u003cp\u003eIn summary, intranasal MPXV infection of ICR and IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice resulted in self-limited resolution within a short timeframe, without exhibiting characteristic symptoms of pneumonia, making them unsuitable for drug evaluation. SCID mice exhibited the highest sensitivity to MPXV, with measurable changes in body weight, virus titer, tissue damage, and pro-inflammatory factors within 14 dpi. Extending the length of clinical monitoring to 28 dpi revealed the clinical symptoms of MPXV and led to a mortality rate of 25%.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eScreening and Establishment of the Mouse Model of MPXV-Induced Skin Lesion\u003c/h3\u003e\n\u003cp\u003eSkin scratches were created in the tails to simulate the MPXV-associated cutaneous rash. Male ICR, IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, and SCID mice were intradermally inoculated with 10⁵ PFU MPXV or sterile saline (control group). After virus inoculation, the body weights of ICR and IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice increased steadily, with no significant difference between the model group and the control group. SCID mice exhibited a significant decrease in body weight from 10 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). All three models exhibited certain scabs (width\u0026thinsp;\u0026ge;\u0026thinsp;1 mm), with ICR mice exhibiting the lowest probability of forming infectious scabs and recovery by 7 dpi. IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had the highest scab formation rate at 7\u0026ndash;9 dpi, reaching 75% and beginning to recover from 10 dpi. SCID mice exhibited the highest scab formation rate at 6 dpi, reaching 100%, and the symptoms worsened over time (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Additionally, we measured viral DNA load and virus titer in the skin of the three mouse models at 3 dpi, 7 dpi, and 14 dpi using qPCR and virus titer detection. We found that the virus titer in ICR mice remained around the detection limit, with viral DNA detectable only at 3dpi and 7 dpi. IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were more sensitive to MPXV, but the virus titer decreased at 14 dpi, similar to the intranasal infection model. Consistent with the clinical symptoms, SCID mice exhibited the highest viral load and virus titer among the three models, which steadily increased over time (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, E).\u003c/p\u003e\n\u003cp\u003eThe most susceptible SCID mice (n\u0026thinsp;=\u0026thinsp;8) were re-infected via the percutaneous route to observe more typical clinical symptoms. The monitoring period was extended to 28 dpi. At 20 dpi, clinical symptoms, such as lethargy, disheveled fur, and rashes in non-lesional areas, were observed in SCID mice. The disease score at the end of the infectious period was 5 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), with no deaths occurred (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). The changes in the levels of pro-inflammatory factors (TNF-\u0026alpha; and IL-6), chemokines NAP3 and antiviral interferons (IFN-\u0026beta; and IFN-\u0026alpha;1) in the skin were consistent with those observed in the pneumonia model, with a further increase in GM-CSF levels (Supplementary Fig.\u0026nbsp;1B).\u003c/p\u003e\n\u003cp\u003eIn conclusion, SCID mice were the most sensitive to MPXV after intradermal infection, exhibiting body weight loss and characteristic scabs at 14 dpi. Systemic symptoms became evident at 20 dpi. Although no deaths were observed, spontaneous recovery did not occur.\u003c/p\u003e\n\u003ch3\u003eScreening and Establishment of the Mouse Model of MPXV-Induced Arthritis\u003c/h3\u003e\n\u003cp\u003eMale ICR, IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, and SCID mice were subcutaneously inoculated at footpads with 10⁵ PFU MPXV or sterile saline (control group) to simulate the joint lesions of MPXV infection. Following viral inoculation, the increase in the body weight of ICR mice was comparable to that of the control group. IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited a significant decrease in body weight from 6 dpi, which normalized at 14 dpi. In contrast, SCID mice began to show a sustained decrease in body weight from 8 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Between 0\u0026ndash;14 dpi, only ICR mice did not exhibit ankle joint swelling (width\u0026thinsp;\u0026ge;\u0026thinsp;2 mm). IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice developed ankle joint swelling from 3 dpi, which resolved within three days. SCID mice gradually developed ankle joint swelling since 4 dpi. The incidence of ankle joint swelling reached 100% by 6 dpi, and the symptoms progressively worsened over time (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Consistent with clinical observations, the virus titer in the ankle joint of ICR mice remained near the detection limit, with only trace amounts of viral DNA detected at 3 dpi and 7 dpi. IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice revealed moderate sensitivity to MPXV, with detectable viral loads and titer observed only at 3 dpi and 7 dpi. SCID mice exhibited a continuous increase in both viral load and titer throughout the disease course (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Both clinical symptoms and virus titer indicated that SCID mice were more susceptible to MPXV. Besides, HE staining of the ankle joints in SCID mice revealed inflammatory cell infiltration in the synovial tissue, accompanied by significant synovial cell hyperplasia and disarray. Interstitial fibrous tissue proliferation and collagen deposition were observed, with local pannus formation. The tidemark of the cartilage was blurred, and chondrocytes exhibited atrophy and uneven distribution.\u003c/p\u003e\n\u003cp\u003eWe also monitored the clinical disease scores and survival rates of SCID mice up to 28 days. SCID mice exhibited progressive ankle joint swelling, weight loss, lethargy, ruffled fur, papules, and skin lesions. The clinical symptom score peaked at the end of the infectious period (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), reaching a score of 8 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF), with a 100% mortality rate at 25 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). The cytokine profiles in the ankle joint were generally consistent with those observed in the pneumonia model, although no increase was detected in IFN-\u0026alpha;1 levels (Supplementary Fig.\u0026nbsp;1C).\u003c/p\u003e\n\u003cp\u003eNotably, this study represents the first animal model of MPXV-induced arthritis and the first lethal model using Clade IIb strain in SCID mice. This model can help monitor weight changes, ankle joint swelling, and virus titer within 14 dpi. Skin lesions appeared from 16 dpi, and a 100% mortality rate was observed at 26 dpi, with no evidence of self-limiting recovery. This model is therefore ideal for short-term and long-term drug evaluations.\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical scoring system used for MPXV-infected SCID mice.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAtegory\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCriteria\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eBody Weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo weight loss or weight loss\u0026thinsp;\u0026lt;\u0026thinsp;10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight loss between 10\u0026ndash;19%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight loss\u0026thinsp;\u0026ge;\u0026thinsp;20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAppearance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth fur and normal posture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRuffled fur and hunched posture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eResponsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal response to external stimuli (e.g., touch, sound)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReduced to moderate movement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo response to external stimuli; possible paralysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eSkin Lesions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo skin lesions (macules, papules, scabs) on the tail\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMacules (1\u0026ndash;4 mm in diameter)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePapules (2\u0026ndash;5 mm in diameter)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScabs (2\u0026ndash;5 mm in diameter)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eScab widths\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo scabs or secretions at the tail root\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScab width between 1 mm and 3 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScab width\u0026thinsp;\u0026gt;\u0026thinsp;3 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eAnkle Joint Swelling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo visible ankle joint swelling\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnkle joint width between 2 mm and 5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnkle joint width\u0026thinsp;\u0026gt;\u0026thinsp;5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of Tecovirimat and Cidofovir in\u003c/strong\u003e \u003cstrong\u003eIn Vitro\u003c/strong\u003e \u003cstrong\u003eModels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTecovirimat and cidofovir have demonstrated antiviral activity against MPXV in both clinical and preclinical studies. However, recent genomic alterations in the currently circulating clade IIb strains observed over the past two years may affect their susceptibility to these drugs. Therefore, the antiviral activity of tecovirimat and cidofovir against MPXV clade IIb was investigated. Based on the three disease models established previously, A549, HaCaT, and MC3T3 cell lines corresponding to the infected tissues, were selected with the commonly used Vero-E6 cells. These in vitro models were employed to measure the drug concentrations with 50% cytotoxicity and 50% viral inhibition (CC\u003csub\u003e50\u003c/sub\u003e, EC\u003csub\u003e50\u003c/sub\u003e). The MC3T3 cell line, not previously reported in studies on MPXV, was confirmed to be susceptible to the MPXV Clade IIb strain. The CC\u003csub\u003e50\u003c/sub\u003e values for both tecovirimat and cidofovir were more than 200 \u0026micro;M in all four cell lines, suggesting low cytotoxicity. The EC\u003csub\u003e50\u003c/sub\u003e values for tecovirimat against MPXV ranged from 0.0029 \u0026micro;M to 0.0104 \u0026micro;M, which were lower than those for cidofovir (0.4414 \u0026micro;M to 2.03 \u0026micro;M) (Supplementary Fig. 3A-D). These results are consistent with recent findings on other clade IIb isolates (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e). Overall, tecovirimat and cidofovir demonstrated potent inhibitory activity against MPXV clade IIb in several in vitro models.\u003c/p\u003e\n\u003ch3\u003eTecovirimat and Cidofovir Exhibited Significant Therapeutic Efficacy in the Early Stages of MPXV Infection\u003c/h3\u003e\n\u003cp\u003eGiven the broad and potent in vitro inhibitory effects of tecovirimat and cidofovir on MPXV, their \u003cem\u003ein vivo\u003c/em\u003e efficacy was investigated using the three animal models established in this study. While the recommended treatment duration for tecovirimat and cidofovir is 14 days, clinical guidelines suggest extending the treatment length for severely immunocompromised patients (up to 16 weeks). Since the SCID model infected with the clade IIb strain exhibited characteristic rash in the late stages of the disease (14\u0026ndash;28 dpi), both short-term (14 days) and long-term (28 days) treatment groups were established to evaluate the therapeutic effects of tecovirimat and cidofovir in the early and late phases of MPXV infection. The SCID intranasal, intradermal, and subcutaneous infection models were employed in this study (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Two hours after infection, each model group received tecovirimat (P.O., n\u0026thinsp;=\u0026thinsp;8) or cidofovir (S.C., n\u0026thinsp;=\u0026thinsp;8), and the control group received normal saline(P.O., n\u0026thinsp;=\u0026thinsp;4; S.C., n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n\u003cp\u003eThe therapeutic efficacy of tecovirimat and cidofovir in the early stages of MPXV infection varied based on the route of infection. Robust efficacy was demonstrated by both drugs in the intranasal model, effectively reducing viral DNA load (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) and virus titer (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) in the lung tissues. H\u0026amp;E staining revealed severe pathological damage in the control group, including alveolar wall thickening, alveolar collapse, inflammatory cell infiltration, hemorrhage, and epithelial cell necrosis. In contrast, minimal pathological damage was observed in the lungs of the tecovirimat and cidofovir treatment groups. Significant differences in pathological scores were observed compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, Supplementary Table 1). In the intradermal infection model, cidofovir failed to effectively control the viral DNA load (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), and three mice developed scabs with a width of more than 1 mm. The tecovirimat treatment group showed near-complete recovery, with no scabs. H\u0026amp;E staining revealed severe fibrosis, inflammatory cell infiltration, cell necrosis, epidermal acanthosis, keratinocyte ballooning degeneration, and scab lesions in the challenge control group, with a median pathological score of 9 (Supplementary Table 2). Compared to the control group, significant improvement was observed in both drug treatment groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). In the arthritis model, both tecovirimat and cidofovir effectively decreased the viral DNA load (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) and virus titer (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) in the ankle joint. The pathological scores of both drug treatment groups were significantly decreased compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, Supplementary Table 3). However, tecovirimat and cidofovir did not completely suppress ankle joint swelling, and the incidence of unilateral mild swelling was 37.5% and 50% in the two groups, respectively. Severe pathological findings were observed in the control group, including fibrous tissue proliferation, inflammatory cell infiltration, chondrocytes necrosis, cartilage damage, and pannus formation. In contrast, mildly swollen ankle joint in the treatment groups exhibited only fibrosis and inflammatory cell infiltration. Ankle joint without swelling showed no pathological damage (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Except for cidofovir, which failed to suppress GM-CSF expression at ankle joint, the expression levels of the TNF-\u0026alpha; and GM-CSF in the lung, skin, and ankle joint were significantly reduced by pharmacological treatment (Supplementary Fig.\u0026nbsp;3A).\u003c/p\u003e\n\u003cp\u003eOverall, tecovirimat and cidofovir effectively suppressed infectious virus titer and histopathological changes in the early stages of pneumonia, skin lesions, and arthritis. However, cidofovir could not decrease viral loads in the skin lesion model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Therapeutic Efficacy of Tecovirimat and Cidofovir in the SCID Models of Non-self-limiting Advanced-stage MPXV Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSCID mice were subjected to an advanced-stage model via intranasal, intradermal, and subcutaneous routes and treated with tecovirimat and cidofovir for 28 days (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Both treatments effectively mitigated viral infection-induced weight loss. However, compared to the tecovirimat group, the cidofovir-treated group experienced weight loss across all three models (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB - D), likely due to the nephrotoxicity of cidofovir. Nevertheless, the long-term use of these drugs did not affect the survival of mice. Both tecovirimat and cidofovir increased survival rates to 100% in the intranasal infection model (62.5% survival in the control group) and the subcutaneous infection model (0% survival in the control group) (Supplementary Fig.\u0026nbsp;4A - C). In the later stages of the disease, both drugs alleviated a range of clinical symptoms caused by viremia (Supplementary Fig.\u0026nbsp;4D - F).\u003c/p\u003e\n\u003cp\u003eUnfortunately, neither drug successfully inhibited scab formation at the scratch sites of the skin lesion model. In addition, the drugs did not reduce ankle joint swelling of the arthritis model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). Further studies on the viral DNA load and virus titer in the target organs of the three models revealed that tecovirimat and cidofovir effectively decreased virus titer (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF) and viral load (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG) of the lung tissues of the pneumonia model. The drugs also decreased the infectious virus titer in the ankle joint of the arthritis model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). However, none of these drugs suppressed the virus titer (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF) or load in the skin lesion (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG) of the skin lesion model. Furthermore, cidofovir could not inhibit the viral DNA load in the ankle joint of the arthritis model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). The persistent presence of the virus in the target organs may have led to a significant increase in histopathological scores of the target organs in the treatment groups across all three models. Histopathological scores for the lung, skin, and ankle joint were determined based on (Supplementary Tables 1\u0026ndash;3). Notably, except for cidofovir, which significantly decreased the histopathological scores in the lung tissues of the pneumonia model, no other treatment groups exhibited significant differences compared to the virus challenge groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Consistent with these histopathological findings, significant differences in TNF-\u0026alpha; and GM-CSF levels were observed between the treatment groups and the virus challenge group in the lung tissues of intranasal infection model. However, no changes were observed between the groups in the skin lesion model and the ankle joint model (Supplementary Fig.\u0026nbsp;3B).\u003c/p\u003e\n\u003cp\u003eIn summary, in the late stage of infection in SCID mice, treatment with tecovirimat and cidofovir effectively decreased the mortality rate, maintained body weight, and successfully reduced the risk of complications caused by viremia in all three models. However, none of the drugs could prevent the formation of scabs in the skin lesion model or the swelling of the ankle joint in the arthritis model. In the later stages of the disease, both drugs failed to control the pathological damage in the target organs. Only cidofovir effectively reduced the pathological scores of lung tissues (median\u0026thinsp;=\u0026thinsp;6.5). Regarding the decrease in virus titer in the target tissues, both tecovirimat and cidofovir exhibited infection route-dependent efficacy, showing inhibitory effects only in the intranasal and subcutaneous models.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEpidemiological data indicated that nearly half of patients with mpox suffer from HIV co-infection, which is associated with more severe clinical manifestations and higher mortality rates. A study conducted in the United States enrolled 395 patients with mpox, of whom 324 (82.0%) were diagnosed with HIV infection. In this cohort, severe mpox was observed in 19.5% of patients, and HIV coinfection was confirmed in 79.2% of patients (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). However, clinical research on this population has been limited due to low healthcare-seeking rates and low availability of samples due to social stigma. While traditional non-human primate models (such as SIV-coinfected macaques) can simulate immunodeficiency phenotypes, their prohibitive maintenance costs and stringent ethical restrictions impede high-throughput research demands (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In this study, SCID mice were selected as a more available rodent model, representing the first model of SCID established using the Clade IIb strain responsible for the 2022 mpox outbreak. Although CAST/EiJ mouse models of skin lesion and pulmonary infection models in BALB/c mice and dormice for Clade IIb have been reported in recent years, these models only partly replicate localized symptoms or immune responses of MPXV-susceptible populations (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In contrast, SCID mice, characterized by congenital T/B lymphocyte developmental blockade due to Prkdc gene mutation, more accurately mimic the depletion of CD4\u003csup\u003e+\u003c/sup\u003e T cells observed in HIV-infected individuals (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Their persistent viral replication and non-self-limiting disease progression closely align with the clinical manifestations of high-risk patients.\u003c/p\u003e \u003cp\u003eTecovirimat, brincidofovir, and cidofovir have not been specifically approved for mpox; however, these agents have been employed for some patients with MPXV. Brincidofovir was excluded from this study due to procurement limitations. The recommended \u003cem\u003ein vivo\u003c/em\u003e treatment length for tecovirimat and cidofovir is 14 days, and the current drug evaluation studies in mouse models have administered these drugs for 14 days or less (7 days) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). However, several studies have indicated that for severely immunocompromised patients with mpox, extending the treatment course of tecovirimat/cidofovir may improve clinical outcomes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Therefore, this study included an additional long-term treatment group to ensure complete viral clearance and prevent disease recurrence or secondary infections. In the short-term treatment group, both tecovirimat and cidofovir significantly reduced infectious virus titer and pathological scores of the target organs (lung, skin and ankle joint) across the three infection models, which is consistent with the results of recent reports (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Unfortunately, tecovirimat and cidofovir were not very effective in the late stages of the disease. Neither tecovirimat nor cidofovir effectively reduced the histopathological progression and viral loads in the skin lesion or arthritis models. The limited therapeutic efficacy in patients with advanced-stage disease is consistent with a report from New York, where 6 out of 12 patients with HIV and advanced disease who received extended courses of tecovirimat combined with vaccinia immune globulin (VIG), cidofovir, and/or brincidofovir finally died (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Notably, tecovirimat resistance-associated F13L gene mutations were detected in 44% of these patients (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). These findings highlight the limitations of long-term monotherapy with tecovirimat and cidofovir in immunocompromised individuals. Future studies should prioritize enhanced resistance monitoring for tecovirimat and toxicity surveillance for cidofovir, while exploring novel combination regimens to overcome current therapeutic shortcomings.\u003c/p\u003e \u003cp\u003eClinically, pneumonia caused by mpox infection is predominantly observed among patients with HIV co-infection (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In patients with pneumonia, CT or X-ray examinations predominantly reveal pulmonary infiltration, ground-glass opacity, or consolidation, which are pathologically consistent with inflammatory cell infiltration, thickened alveolar walls, and alveolar collapse observed in our pneumonia model (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Among all pathogenic models established in this study, the pneumonia model demonstrated the most favorable therapeutic outcomes, which can be attributed to the high blood flow in the lungs and the high permeability of alveolar epithelium facilitating drug accumulation in the lung tissues (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). With the exception of a patient with acute respiratory distress syndrome (ARDS) where tecovirimat was successfully administered clinically, current management of patients with MPXV and confirmed pneumonia remains symptomatic treatment and supportive therapy, with no antiviral agents (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Given the favorable outcomes of both short-term and long-term treatments in this pneumonia model, tecovirimat and cidofovir can be administered to prevent severe adverse outcomes. In immunocompromised patients, the combination of antiretroviral therapy (ART) and adjunctive VIG should also be evaluated. Future development of nebulized (NEB) antiviral formulations (e.g., liposomal cidofovir) for direct delivery to the lungs is also warranted.\u003c/p\u003e \u003cp\u003eIn the skin lesion model, cidofovir could not effectively reduce viral DNA load, and in the late stage, neither tecovirimat nor cidofovir effectively inhibited the progression of skin lesion. This observation aligns with clinical findings in patients with advanced HIV, where no significant alteration in viral load was observed after administering a 33-day tecovirimat regimen (600 mg orally, twice daily) followed by topical (3%) and injected (5 mg/kg) cidofovir (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Although cidofovir did not reduce the viral load in the skin of the intradermal infection model, it unexpectedly lowered the viral load in the lung tissue of SCID mice. This finding is consistent with the therapeutic effects of cidofovir on skin infections caused by cowpox virus (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Viral inhibition is pharmacologically effective, though dermal bioavailability may be limited by the skin's low pH, high keratinization, and locally immunosuppressive microenvironment (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). This suggests that localized transdermal drug delivery systems (TDDS), such as nanoliposomal gels or microneedle patches, combined with systemic treatment, can achieve synergistic drug accumulation in the skin. Such an approach can enhance dual suppression of viral replication at the epidermal-dermal junction, accelerating pathological repair of skin lesion and viral clearance. Notably, recovery after infection with poxvirus frequently results in cutaneous scarring. A 1540 nm radiofrequency laser-based therapy, Secret Duo, has been approved by the U.S. FDA for treating scars.\u003c/p\u003e \u003cp\u003eClinically reported cases of arthritis are primarily present with generalized arthralgia, knee joint swelling, and MRI findings of synovitis and abnormal synovial fluid cell counts (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These manifestations are highly consistent with ankle joint swelling, synovial damage, and inflammatory cell infiltration observed in our arthritis model. Our model represents the first preclinical model for mpox-associated arthritis, with its corresponding MC3T3 \u003cem\u003ein vitro\u003c/em\u003e model being the first application for research on mpox infection. However, the main management for previous cases of arthritis remains symptomatic treatment, without administering any antiviral agents. Based on the favorable therapeutic outcomes observed in our arthritis model, tecovirimat and cidofovir can be considered for treating future cases of mpox-associated arthritis. When necessary, adjunctive therapies such as glucocorticoid, platelet-rich plasma (PRP) and low-intensity pulsed ultrasound (LIPUS) can be employed to promote cartilage repair, suppress the inflammatory responses, and alleviate pain.\u003c/p\u003e \u003cp\u003eIn conclusion, SCID mice were successfully employed to establish pneumonia, skin lesion, and arthritis models through various routes of MPXV infection. The models replicated the clinical manifestations observed in high-risk populations for mpox, including patients with HIV and organ transplant recipients. Utilizing these three models, comprehensive pharmacodynamic evaluations of tecovirimat and cidofovir were conducted across short-term and long-term treatment regimens, and potential strategies for combination therapy were proposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e), thereby providing robust preclinical data to guide the clinical management of patients with mpox.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in AAALAC International-accredited facilities and adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Animal Care and Use Committee of the Changchun Veterinary Research Institute (IACUC approval no. AMMS-11-2023-041). All samples generated in the biosafety level 3 laboratory were inactivated according to IBC-approved standard operating procedures upon removal from the high-containment area.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCells and Virus\u003c/h3\u003e\n\u003cp\u003eMPXV (clade IIb, GenBank accession number: PP778666.1) was isolated from a patient in Guangzhou, China. The virus was cultured and amplified in Vero-E6 cells using DMEM medium (Sigma-Aldrich) containing 2% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin. Vero-E6 cells were cultured in DMEM containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin. No mycoplasma or contaminants were detected. All experiments involving infectious MPXV were conducted in a biosafety level 3 laboratory.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiment Design\u003c/h2\u003e \u003cp\u003e4-5-week-old male ICR mice and SCID mice were purchased from Beijing HFK Biotechnology Co., Ltd., and 4-5-week-old male IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were purchased from Beijing Cyagen Biosciences Inc. Part 1: Mice from all three models were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n\u0026thinsp;=\u0026thinsp;24) or normal saline (n\u0026thinsp;=\u0026thinsp;8). The clinical symptoms were continuously monitored. ICR, IFNAR1\u003csup\u003e⁻/⁻\u003c/sup\u003e, and SCID mice were euthanized at 3, 7, and 14 dpi, respectively. Lung, skin, and ankle joint were collected, minced, weighed to 0.1 g (wet weight), and then homogenized in 500 \u0026micro;L of PBS for subsequent analyses. Part 2: SCID mice were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n\u0026thinsp;=\u0026thinsp;24) or normal saline (n\u0026thinsp;=\u0026thinsp;8) to evaluate disease scores and survival rates. Disease scores and survival rates were measured from 0 dpi to 28 dpi. Part 3: SCID mice were anesthetized with isoflurane and inoculated intranasally, intradermally (in the tail), and subcutaneously (in the footpad) with MPXV (10⁵ PFU, n\u0026thinsp;=\u0026thinsp;48). Mice in each group received tecovirimat (oral, 100 mg/kg, once daily, n\u0026thinsp;=\u0026thinsp;16), cidofovir (intravenous, 100 mg/kg, twice a week, n\u0026thinsp;=\u0026thinsp;16), or normal saline (oral, n\u0026thinsp;=\u0026thinsp;8; intravenous, n\u0026thinsp;=\u0026thinsp;8). Mice were euthanized at 14 dpi and 28 dpi (n\u0026thinsp;=\u0026thinsp;8). Lung, skin, and ankle joint were collected, minced, weighed to 0.1 g (wet weight), and homogenized in 500 \u0026micro;L of PBS for subsequent analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eAntiviral Efficacy Assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e Determination: Vero-E6 cell monolayers in 96-well plates were cultured to approximately 50% confluence and treated with different concentrations of tecovirimat (200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 \u0026micro;M) or cidofovir (200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 \u0026micro;M) in a total volume of 100 \u0026micro;L per well. Each drug concentration was prepared by five-fold serial dilution in the maintenance medium. After 72 hours of incubation, the medium was discarded, and the cells were washed twice with PBS. Subsequently, 100 \u0026micro;L of 10% CCK-8 reagent was added to each well, followed by incubation in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C for 0.5-1 hour. The absorbance (OD) was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e Determination: Vero-E6 cells were cultured in 24-well plates to near confluence and infected with MPXV at an MOI of 0.1. The infection was incubated for 1 hour at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, shaking every 15 minutes. After removing the inoculum, different concentrations of tecovirimat or cidofovir were added based on CC\u003csub\u003e50\u003c/sub\u003e. After a 72-hour incubation period, the supernatants were collected, and MPXV DNA was extracted using the MAGEN DNA extraction kit. The viral DNA levels were quantified by qPCR. The OD values and corresponding DNA copy numbers were entered into GraphPad Prism software, normalized to the control group (cells treated with no drug), and expressed as percentages. The concentrations (log-transformed) were plotted on the X-axis, and the percentage of cell viability or MPXV DNA content was plotted on the Y-axis. A four-parameter logistic curve was fitted, and nonlinear regression analysis was conducted to calculate the CC₅₀ and EC₅₀ values for each treatment group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eViral DNA Quantification (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTissue homogenates were centrifuged at 12,000 \u0026times; g and 4\u0026deg;C for 15 minutes, and the supernatant (200 \u0026micro;L) was collected to detect and quantify the expression levels of MPXV. Viral nucleic acid was extracted using the Viral Nucleic Acid Quick Extraction Kit (MAGEN) following the manufacturer\u0026rsquo;s instructions, with an elution volume of 50 \u0026micro;L. Real-time fluorescence quantitative detection of the MPXV F3L gene was conducted using the TaqMan probe method (F: 5\u0026prime;-CTCATTGATTTTTCGCGGGATA-3\u0026prime;, R: 5\u0026prime;-GACGATACTCCTCCTCGTTGGT-3\u0026prime;, probe: FAM-CATCAGAATCTGTAGGCCGT-BHQ). The experiment was conducted using the Takara Premix Ex Taq\u0026trade; (Probe qPCR) kit (TAKARA) for Real-Time PCR. The experiment was conducted using the Takara Premix Ex Taq\u0026trade; (Probe qPCR) kit (TAKARA) for Real-Time PCR. The reactions were conducted on the Bio-Rad CFX96 Real-Time PCR Detection System. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInfectious Virus titer Quantification (TCID₅₀)\u003c/h2\u003e \u003cp\u003eThe supernatant of tissue homogenate was serially diluted 10-fold. Then, 100 \u0026micro;L of each dilution was added to a 96-well plate containing a monolayer of Vero-E6 cells. The plate was incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. After 96 hours of incubation, the cytopathic effect (CPE) was observed. The virus titer was calculated using the Reed-Muench method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Assessment\u003c/h2\u003e \u003cp\u003eNecropsy and tissue sampling were conducted following the protocols approved by the Institutional Biosafety Committee (IBC). Hematoxylin and eosin (H\u0026amp;E) staining of tissue samples was conducted using standard paraffin embedding methods. Tissue samples were fixed in 10% neutral-buffered formalin for at least 7 days, followed by dehydration, clearing, and embedding in paraffin. The tissues were sectioned into 4\u0026ndash;5\u0026micro;m thick slices and mounted onto pre-coated APES slides. The tissue slides were deparaffinized in xylene, rehydrated through ethanol, stained with hematoxylin and eosin, and dehydrated. After clearing in xylene, the tissue sections were mounted with neutral balsam. The tissue slides were evaluated by a certified, double-blind pathologist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Cytokine Levels\u003c/h2\u003e \u003cp\u003eRNA was extracted from 200 \u0026micro;L of homogenized tissue using an RNA Extraction Kit (Gene Script Biotech). The extracted mRNA was amplified in a CFX96 Real-Time PCR System (Bio-Rad) using the commercially available 2\u0026times; Q1 SYBR qPCR Master Mix (Universal). For cytokine profiling, the following primer pairs were used: TNF-α (F: 5\u0026prime;-AGCCAGGAGGGAGAACAGA-3\u0026prime;, R: 5\u0026prime;-CAGTGAGTGAAAGGGACAGAAC-3\u0026prime;), IL-6 (F: 5\u0026prime;-CGGAGAGGAGACTTCACAGAG-3\u0026prime;, R: 5\u0026prime;-CATTTCCACGATTTCCCAGA-3\u0026prime;), NAP3 (F: 5\u0026prime;-TCCAGAGCTTGAAGGTGTTGCC-3\u0026prime;, R: 5\u0026prime;-AACCAAGGGAGCTTCAGGGTCA-3\u0026prime;), IFN-α1 (F: 5\u0026prime;-TAATTCCTACGTCTTTTCTTT-3\u0026prime;, R: 5\u0026prime;-TATGCCTGATCCCTGAACAGT-3\u0026prime;), IFN-β (F: 5\u0026prime;-AACCTCCTGGATGACATGCCTG-3\u0026prime;, R: 5\u0026prime;-AAATTGCCCCGTAGACCCTGCT-3\u0026prime;), GM-CSF (F: 5\u0026prime;-AACCTCCTGGATGACATGCCTG-3\u0026prime;, R: 5\u0026prime;-AAATTGCCCCGTAGACCCTGCT-3\u0026prime;) Expression levels were normalized to ​β-actin (F: 5\u0026prime;-GTGGGCCGCTCTAGGCACCAA-3\u0026prime;, R: 5\u0026prime;-CTCTTTGATGTCACGCACGATTTC-3\u0026prime;). The 2\u0026times; Q1 SYBR qPCR Master Mix (Universal) was employed for qPCR.\u003c/p\u003e \u003cp\u003eCytokine levels were measured using the TNF-α ELISA Kit and GM-CSF ELISA Kit (Cloud-Clone Corp.). The homogenate was centrifugated and lysed with RIPA buffer to prepare for inflammatory factor detection. The experiments were conducted following the manufacturer\u0026rsquo;s instructions, and the absorbance was read at 450 nm using a spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis and Data Visualization\u003c/h2\u003e \u003cp\u003eData collection and analysis were conducted in a double-blind manner. Statistical analyses were conducted using GraphPad Prism 8.0. Data are presented as ​mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Inter-group differences were determined using \u003cem\u003et\u003c/em\u003e-tests or one-way analysis of variance (ANOVA) followed by two-tailed \u003cem\u003et\u003c/em\u003e-tests, unless otherwise specified. Statistical significance levels were defined as follows: not significant (\u003cem\u003ens\u003c/em\u003e), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001. The type of test used is indicated where appropriate. All data generated in this study are provided in the Source Data file.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2023YFD1800405) and CAMS Innovation Fund for Medical Sciences (2020-12M-5-001). Thanks to the Eighth Affiliated Hospital of Guangzhou Medical University for providing the clinical mpox samples.The authors would like to express their gratitude to EditSprings (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.editsprings.cn\u003c/span\u003e\u003cspan address=\"https://www.editsprings.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the expert linguistic services provided.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJezek Z, Szczeniowski M, Paluku KM, Mutombo M (1987) Human monkeypox: clinical features of 282 patients. J Infect Dis 156(2):293\u0026ndash;298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A et al (2022) Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med 28(8):1569\u0026ndash;1572\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGigante CM, Korber B, Seabolt MH, Wilkins K, Davidson W, Rao AK et al (2022) Multiple lineages of monkeypox virus detected in the United States, 2021\u0026ndash;2022, vol 378. Science, New York, NY, pp 560\u0026ndash;565. 6619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePati\u0026ntilde;o LH, Guerra S, Mu\u0026ntilde;oz M, Luna N, Farrugia K, van de Guchte A et al (2023) Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022. Emerg Microbes Infect 12(1):e2192830\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBunge EM, Hoet B, Chen L, Lienert F, Weidenthaler H, Baer LR et al (2022) The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl Trop Dis 16(2):e0010141\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesingu PA, Rubeni TP, Sundaresan NR (2022) Evolution of monkeypox virus from 2017 to 2022: In the light of point mutations. Front Microbiol 13:1037598\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVakaniaki EH, Kacita C, Kinganda-Lusamaki E, O'Toole \u0026Aacute;, Wawina-Bokalanga T, Mukadi-Bamuleka D et al (2024) Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo. Nat Med 30(10):2791\u0026ndash;2795\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaurenson-Schafer H, Sklenovsk\u0026aacute; N, Hoxha A, Kerr SM, Ndumbi P, Fitzner J et al (2023) Description of the first global outbreak of mpox: an analysis of global surveillance data. Lancet Global health 11(7):e1012\u0026ndash;e23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitj\u0026agrave; O, Alemany A, Marks M, Lezama Mora JI, Rodr\u0026iacute;guez-Aldama JC, Torres Silva MS et al (2023) Mpox in people with advanced HIV infection: a global case series. Lancet (London England) 401(10380):939\u0026ndash;949\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun G, TEJA KOLLI S, Asuzu C, Nihalani S, Zhu M, Stoeckel JE et al (2023) THE FIRST HUMAN TO HAVE NECROTIZING PNEUMONIA SECONDARY TO MONKEYPOX INFECTION. CHEST\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThornhill JP, Barkati S, Walmsley S, Rockstroh J, Antinori A, Harrison LB et al (2022) Monkeypox Virus Infection in Humans across 16 Countries - April-June 2022. N Engl J Med 387(8):679\u0026ndash;691\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Shea J, Zucker J, Stampfer S, Cash-Goldwasser S, Minhaj FS, Dretler A et al (2024) Prolonged Mpox Disease in People With Advanced HIV: Characterization of Mpox Skin Lesions. J Infect Dis 229(Supplement2):S243\u0026ndash;s8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLomb\u0026egrave;s A, Zmerli M, Nerozzi-Banfi E, Gozlan JM, Sellam J, Valin N (2023) Arthritis due to monkeypox virus: A case report. Joint bone spine 90(2):105492\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonti M, Mader T, Burmester-Kiang J, Aberle SW, Horvath-Mechtler B, Traugott M et al (2022) Monkeypox associated acute arthritis. Lancet Rheumatol 4(11):e804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMungmunpuntipantip R, Wiwanitkit V et al (2023) Comment on Arthritis due to monkeypox virus: A case report by Lomb\u0026egrave;s A. Joint Bone Spine. ;90:105492. 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Nature 458(7241):1034\u0026ndash;1038\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong G, Cheng L, Liu J, Zhou Y, Zhang C, Zong Y (2025) Establishment of an animal model for monkeypox virus infection in dormice. Sci Rep. ;15(1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarner BM, Klassen L, Sloan A, Deschambault Y, Soule G, Banadyga L et al (2022) In vitro and in vivo efficacy of tecovirimat against a recently emerged 2022 monkeypox virus isolate. Sci Transl Med 14(673):eade7646\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer Zu Natrup C, Clever S, Sch\u0026uuml;nemann LM, Tuchel T, Ohrnberger S, Volz A (2025) Strong and early monkeypox virus-specific immunity associated with mild disease after intradermal clade-IIb-infection in CAST/EiJ-mice. Nat Commun 16(1):1729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNonoyama S, Ochs HD (1996) Immune deficiency in SCID mice. Int Rev Immunol 13(4):289\u0026ndash;300\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuong MT, Tebas P, Ancha B, Baron J, Chary P, Isaacs SN et al (2024) Combination of Extended Antivirals With Antiretrovirals for Severe Mpox in Advanced Human Immunodeficiency Virus Infection: Case Series of 4 Patients. Open forum Infect Dis 11(3):ofae110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegrist EA, Sassine J (2023) Antivirals With Activity Against Mpox: A Clinically Oriented Review. Clin Infect diseases: official publication Infect Dis Soc Am 76(1):155\u0026ndash;164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Clercq E (2002) Cidofovir in the treatment of poxvirus infections. Antiviral Res 55(1):1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourner J, Redji Mbrenga FD, Malaka CN, Dunning J, Rojek A, Fandema E et al (2024) Expanded Access Programme for the use of tecovirimat for the treatment of monkeypox infection: A study protocol for an Expanded Access Programme. PLoS ONE 19(5):e0278957\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia EA, Foote MMK, McPherson TD, Lash MK, Bosompem AN, Bouscaren A et al (2024) Severe Mpox Among People With Advanced Human Immunodeficiency Virus Receiving Prolonged Tecovirimat in New York City. Open forum Infect Dis 11(6):ofae294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiepłucha HD, Bożejko M, Piesiak P, Serafińska S, Szetela B (2023) Bacterial Pneumonia and Cryptogenic Pleuritis after Probable Monkeypox Virus Infection: A Case Report. Infect disease Rep 15(6):795\u0026ndash;805\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlrashedi MG, Ali AS, Ahmed OA, Ibrahim IM (2022) Local Delivery of Azithromycin Nanoformulation Attenuated Acute Lung Injury in Mice. Molecules. ;27(23)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmola M, Vandamme T, Sokolowski A (2008) Nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases. Int J Nanomed 3(1):1\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTchoubou T, El-Hosni R, Dollat M, Jaquet P, Tournus C, Tandjaoui-Lambiotte Y et al (2023) Acute Respiratory Distress Syndrome due to Monkeypox Virus. Eur J case Rep Intern Med 10(11):004126\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarbet EB, Larson D, Anderson BJ, Bailey KW, Wong MH, Smee DF (2011) Evaluation of imiquimod for topical treatment of vaccinia virus cutaneous infections in immunosuppressed hairless mice. Antiviral Res 90(3):126\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnight FC, Gilchuk P, Kumar A, Becker KW, Sevimli S, Jacobson ME et al (2019) Mucosal Immunization with a pH-Responsive Nanoparticle Vaccine Induces Protective CD8(+) Lung-Resident Memory T Cells. ACS Nano 13(10):10939\u0026ndash;10960\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Park S, Kim HJ, Lee SJ, Jung WH (2023) The Interkingdom Interaction with Staphylococcus Influences the Antifungal Susceptibility of the Cutaneous Fungus Malassezia. J Microbiol Biotechnol 33(2):180\u0026ndash;187\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6483910/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6483910/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOn February 27, 2025, WHO maintained monkeypox as a PHEIC following its third round of assessment. Human monkeypox virus infection primarily manifests with fever, lymphadenopathy, and rash. Severe cases may develop pneumonia, encephalitis, myocarditis and arthritis. After evaluation of three murine models (ICR, IFNAR1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, and SCID), we identified SCID mice as stable hosts for clade IIb, modeling high-risk populations like patients with HIV. Three models, characterized by rash, pneumonia, and arthritis, were established for the pharmacodynamic evaluation of tecovirimat and cidofovir. Both drugs decreased virus titer in target organs during early infection and ensured 100% survival. As a limitation, cidofovir failed to inhibit viral DNA load in skin lesions, and monotherapy of cidofovir or tecovirimat was ineffective in prolonged intradermal infections. These data indicate that the therapeutic efficacy of tecovirimat and cidofovir is contingent upon distinct disease phenotypes and progression stages, underscoring the necessity for novel therapeutic interventions against monkeypox.\u003c/p\u003e","manuscriptTitle":"Efficacy of Tecovirimat and Cidofovir Against MPXV-Induced Pneumonia, Skin Lesion, and Arthritis in the High-Risk Population-Relevant SCID Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 10:41:26","doi":"10.21203/rs.3.rs-6483910/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d8d6f9e4-a442-4cd7-b8ee-e5ed1615bd0b","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47505756,"name":"Biological sciences/Microbiology/Virology/Antivirals"},{"id":47505757,"name":"Biological sciences/Microbiology/Virology/Pox virus"}],"tags":[],"updatedAt":"2026-01-23T08:07:42+00:00","versionOfRecord":{"articleIdentity":"rs-6483910","link":"https://doi.org/10.1038/s41467-025-67548-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-18 05:00:00","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-04-23 10:41:26","video":"","vorDoi":"10.1038/s41467-025-67548-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67548-0","workflowStages":[]},"version":"v1","identity":"rs-6483910","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6483910","identity":"rs-6483910","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}