Mpox virus Clade IIb infected Cynomolgus macaques via mimic natural infection routes closely resembled human mpox infection

ABSTRACT

Generating an infectious non-human primate (NHP) model using a prevalent monkeypox virus (MPXV) strain has emerged as a crucial strategy for assessing the efficacy of vaccines and antiviral drugs against human MPXV infection. Here, we established an animal model by infecting cynomolgus macaques with the prevalent MPXV strain, WIBP-MPXV-001, and simulating its natural routes of infection. A comprehensive analysis and evaluation were conducted on three animals, including monitoring clinical symptoms, collecting hematology data, measuring viral loads, evaluating cellular and humoral immune responses, and examining histopathology. Our findings revealed that initial skin lesions appeared at the inoculation sites and subsequently spread to the limbs and back, and all infected animals exhibited bilateral inguinal lymphadenopathy, eventually leading to a self-limiting disease course. Viral DNA was detected in post-infection blood, nasal, throat, rectal and blister fluid swabs. These observations indicate that the NHP model accurately reflects critical clinical features observed in human MPXV infection. Notably, the animals displayed clinical symptoms and disease progression similar to those of humans, rather than a lethal outcome as observed in previous studies. Historically, MPXV was utilized as a surrogate model for smallpox. However, our study contributes to a better understanding of the dynamics of current MPXV infections while providing a potential infectious NHP model for further evaluation of vaccines and antiviral drugs against mpox infection. Furthermore, the challenge model closely mimics the primary natural routes of transmission for human MPXV infections. This approach enhances our understanding of the precise mechanisms underlying the interhuman transmission of MPXV.

KEYWORDS:

• MPXV
• Clade IIb
• Cynomolgus macaques
• multiple routes
• typical symptoms

Introduction

Mpox is a viral disease caused by the monkeypox virus (MPXV), which is an enveloped, double-stranded DNA virus first discovered in monkeys at a laboratory in Denmark in 1958 [1]. The first reported human case was diagnosed in 1970 in a nine-month-old boy in the Democratic Republic of the Congo [2]. Symptoms of MPXV infection start with fever, generalized pain, swollen lymph nodes, and fatigue, followed by rashes, which often first appear on the face and then spread to the rest of the body. The distinctive lesions often appear macular, followed by papular, vesicular, and pustular [3]. The fatality rate ranges from 0–11%, with higher mortality rates among infants [4].

There are two distinct lineages of MPXV, Clade I and Clade II. Since 1970, mpox has occurred sporadically in Central and East Africa (Clade I) and West Africa (Clade II), with limited transmission to other regions [5,6]. However, beginning in early May 2022, mpox outbreaks emerged and rapidly spread to Europe, the Americas, and other parts of the world. As of October 2023, approximately 91,000 cases and 157 deaths have been reported in 115 countries [7].

All DNA sequences of viruses from the 2022 global mpox outbreak were associated with Clade IIb, and most of them belonged to the B.1 lineage of Clade IIb [7]. This outbreak was the most widespread and largest one reported in non-African regions [8]. While previous infections were primarily detected after direct contact with infected animals or travelling to affected areas, current transmission is predominantly through interhuman contact, particularly through sexual intercourse between males [9]. This unusual pattern of after spread indicates potential changes in viral genetic information and an increase in transmissibility, highlighting the need for further study of the pathogenesis, transmission, and medical strategies used to treat mpox.

Animal models provide a platform for identifying the disease pathogenesis of mpox, and they promote the development of vaccines and drugs [10]. Non-human primate (NHP) models are widely used due to their anatomical and immunological similarities to humans [11–16]. Studies have focused mainly on the lethal Zaire strain (Clade I). The epidemiology, transmission, and pathology of the strains in Clade IIb are significantly different from those of the Zaire strain [17]. Concerning pathogenicity in humans, the Zaire strain is considered to be slightly more fatal than the other strains [18]. Most NHP studies have developed models through intravenous, aerosol, and respiratory infection routes, but intravenous injection bypasses the early stages of disease development and leads to an accelerated fulminant disease course [19]. Additionally, the respiratory pathway was not the primary route of transmission during the outbreak of 2022 [7]. The most common mode of mpox transmission is through sexual intercourse, including contact with bodily fluids; lesions on the skin or internal mucosal surfaces, such as in the mouth or throat; respiratory droplets; and contaminated objects [4].

Reports on NHP models, specifically those involving the use of strains from the latest epidemic, are rare. In this study, we established a cynomolgus macaque model infected with WIBP-MPXV-001 isolated from a patient in Hong Kong during the 2022 outbreak [20]. The animals were exposed to the virus through multiple routes, including ocular, oral, rectal, and subcutaneous routes, mimicking interhuman transmission [4]. This approach led to the development of clinical features in macaques, closely resembling those observed in humans. To summarize, in this study, we established an NHP model for mpox that accurately reflects disease in humans, suggesting that this model is valuable for therapeutic and vaccine research.

Materials and methods

Biosafety and ethics statement

Infectious WIBP-MPXV-001 was handled in an animal biosafety level 3 (ABSL3) facility. The Institutional Animal Care and Use Committee of Wuhan Institute of Biological Products Co., Ltd. reviewed and approved all the experiments involving cynomolgus macaques. All experiments were also conducted in compliance with national guidelines and followed the Guide for the Care and Use of Laboratory Animals. Briefly, all the samples were collected after the animals were anesthetized with 5 mg/kg Zoletil 50 (Virbac, France).

Cells and viruses

Vero-E6 cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA), supplemented with 10% newborn calf serum (NBS) (Minhai Bio, China), 50 IU/mL penicillin, and 50 µg/mL streptomycin (Gibco, USA).

The MPXV strain WIBP-MPXV-001 was stored in our laboratory, and the isolation procedures have been described previously [20]. The titres of the virus inoculum were determined via plaque assay.

Animal experiments

In this study, three male cynomolgus macaques (5–6 years old) were used. The monkeys were free of illness or malformations and special pathogens, such as tuberculosis, simian immunodeficiency virus, and macacine herpesvirus 1. Three cynomolgus macaques were infected with WIBP-MPXV-001 using a consistent method of inoculation, involving simultaneous mucosal and skin infection. The animals were infected with 1.2 × 10⁸ PFU of WIBP-MPXV-001 via the mucosal plus skin route via the following routes: intraocular (0.1 mL per eye), intraoral (1.0 mL), intrarectal (1.3 mL), and subcutaneous (0.5 mL). Physical examination (PE), body temperature, weight, and lesion number were recorded on days 0, 1, 3, 5, 7, 9, 11, 14, and 16 post-infection (dpi) and on the day of euthanasia. Nasal swabs, throat swabs, anal swabs, and blood samples were collected and analyzed for viral load. Blister fluid swabs were also collected and assayed for viral load on days 7, 9, and 11. The swabs were collected in 2 mL of DMEM supplemented with 50 IU/mL penicillin and 50 µg/mL streptomycin. Blood samples were assayed for whole blood count and blood chemical activity, cytokine levels, and neutralizing antibody, and IgG antibody determination. Disease progression was mainly monitored through the number of skin lesions and the change in state from macular to scab for each monkey during PE. At the end of the study (18–20 dpi), the animals were euthanized and necropsied (Figure 1). Tissue samples from all major organ systems, such as the skin, lymphoid, endocrine, respiratory, genitourinary, gastrointestinal, heart, and brain systems, were collected to determine the viral load and perform histopathological analysis.

Figure 1. Experimental design and sample collection. Three adult male cynomolgus macaques (179327C, 186503C, and 186321C) were included in this study. At the onset of this experiment, the monkeys were challenged with 1.2 × 10⁸ PFU of WIBP-MPXV-001 via the mucosal plus skin route. All three monkeys were infected with WIBP-MPXV-001 using a consistent method of inoculation, involving simultaneous mucosal and skin infection. Body temperature, body weight, nasal/throat/anal/blister fluid swabs, biochemical changes, hematological changes, serum cytokines, and specific antibodies were measured throughout the observation period. Blood samples were not collected at some time points due to the poor condition of the infected monkeys. Histopathological examinations via H&E staining were conducted at 18 dpi (179327C), 19 dpi (186503C), and 20 dpi (186321C).

Detection of viral DNA by quantitative PCR

The swab samples were vortexed for 30 s and centrifuged at 3,000 rpm for 10 min at 4 °C. The supernatant was harvested for DNA detection. Tissue samples were homogenized with 1 mL of PBS using a tissue homogenizer (Xinzhi Biotechnology, China). Then, the samples were centrifuged, and the supernatant was collected. The swab or tissue sample supernatants (200 µL) were mixed with lysis buffer (500 µL) and proteinase K (20 µL). The mixtures were incubated at 70 °C for 15 min to inactivate the virus. The viral DNA was extracted using a nucleic acid extraction System (DAAN Gene, China), following the manufacturer’s instructions. The extracted DNA samples were analyzed for MPXV DNA using a MPXV nucleic acid testing kit (PCR-fluorescent probe method) (DAAN Gene, China) on an ABI QuantStudio 5 system.

MPXV plaque assay

The EDTA-treated blood, the supernatant of the swabs, and the tissue sample supernatants were stored at −20 °C. The titres of the infectious virus in the blood, swabs, and tissues were determined via plaque assay. The samples were incubated in 12-well plates (Corning, Germany) with Vero E6 cell monolayers in DMEM containing 1.5% carboxymethylcellulose, 2% NBS, 50 IU/mL penicillin and 50 µg/mL streptomycin (Gibco, USA). After incubating at 37 °C for 120 h, the plates were fixed with an equal volume of 8% paraformaldehyde for more than 1 h, the cell monolayers were stained with 0.5% crystal violet, and then the plaques were subsequently photographed and counted.

Clinical laboratory evaluation

Complete EDTA-pretreated blood samples were collected, and whole-blood counts were performed using a hematology analyzer (IDEXX, USA). The biochemical test was also conducted using a biochemical analyzer (IDEXX, USA).

Measurement of cytokines

Following the manufacturer’s instructions, the cytokine concentrations in the serum samples were determined using a Luminex® Non-Human Primate XL Cytokine Premixed Kit (R&D Systems, USA) with a Luminex 200 system (Bio-Rad Laboratories, China), and the data were analyzed using Bio-Plex Manager software (version 6.2.0). The concentration of each cytokine was calculated using a kit by comparison with the corresponding standard curve generated using purified cytokines.

Assessment of antibody levels via ELISA

The response of IgG antibodies against the MPXV-specific proteins A29, H3, M1R, I1L, B6R, and A35 was determined. The 96-well plates (Corning, Germany) were precoated with recombinant proteins A29, H3, M1R, I1L, B6R and A35 (0.5 µg/mL in 0.05 M carbonate buffer) at a volume of 100 µL per well, and then, incubated at 37 °C with blocking buffer (phosphate-buffered saline, 5% NBS, 0.5% Tween 20) for 1 h. Next, twofold serially diluted, heat-inactivated plasma samples (starting at 1:256) were added for 1 h at 37 °C, followed by incubation with horseradish peroxidase-labeled anti-monkey-IgG antibodies. After each incubation step, the plates were washed. The absorbance of the plates was at a wavelength of 450 nm using a microplate reader (Bio Tek, USA).

Neutralizing antibody assay

The plaque reduction neutralization test (PRNT) was performed to measure the levels of neutralizing antibodies in the serum samples. Vero E6 cells were seeded in 12-well plates (Corning, Germany) at a density of 5 × 10⁵ cells per well the day before the experiment. Heat-inactivated sera (56 °C for 40 min) were diluted (1:20), followed by a four-fold serial dilution in DMEM supplemented with 2% NBS, 50 IU/mL penicillin, and 50 µg/mL streptomycin. Each serum sample was incubated with 180 plaque-forming units of virus (PFU) for 1 h at 37 °C. The virus-serum mixture was added to the pre-formed Vero E6 cell monolayer and incubated for 1 h at 37 °C in a 5% CO₂ incubator. The supernatant was then removed and the cell monolayers were covered with methylcellulose (DMEM supplemented with 0.9% methylcellulose, 2% NBS, 50 IU/mL penicillin, and 50 µg/mL streptomycin). The next steps were performed as described in another study [20]. The neutralizing antibody titres were defined as the serum dilution, which resulted in a 50% reduction (PRNT₅₀) relative to the total number of plaques counted without antibodies. The PRNT₅₀ was given a value of 10 when no neutralization was observed.

Pathology examination

Post-mortem examinations were conducted on three cynomolgus macaques after they were euthanized. Tissue samples were collected from the lungs, trachea, heart, liver, kidneys, spleen, stomach, duodenum, colon, rectum, lymph nodes (submandibular, axillary, mesenteric, and inguinal), adrenal glands, testes, vas deferens, penis, skin (injection sites), and brain tissue. The tissue samples were fixed in 10% neutral formalin (Servicebio, China) for 14 days. After fixation, the tissues were embedded in paraffin and sectioned into approximately 5 µm thick slices. Hematoxylin and eosin (H&E) staining was performed, and images were acquired using an optical microscope for analysis.

Statistical analysis

All the statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, USA). The differences in viral loads among swabs were determined via one-way ANOVA. Differences were considered to be statistically significant at P < 0.05.

Results

Clinical manifestations

Clinical symptoms

The animals started to exhibit appetite loss and macules by day 3. The general condition of the animals deteriorated, and their activity decreased from days 5 to 9 post-infection. On day 5, signs of acute disease, including anorexia, weariness, and listlessness, were recorded. Papulovesicular rashes appeared in all three animals on day 5. Clinical symptoms, such as appetite loss, a decrease in activity, drowsiness, and depressed posture, became worse on days 7–9. By day 11, the animals started showing signs of recovery and increased activity, and they ate more. The lesions healed, and no new rashes were recorded. Lymphadenopathy of bilateral inguinal nodes was observed in all three animals from day 5 to day 11 (Figure 2(d)). In all animals, the lesions evolved to crustal stages by 14–16 days post-infection. All three animals recovered until they were euthanized on days 18–20 (Table 1).

Figure 2. Changes in skin lesions, body temperature, and body weight of cynomolgus macaques infected with WIBP-MPXV-001. (a) Skin lesions at the site of subcutaneous injection. The lesions progressed sequentially from papular to vesicular, pustular, scabs, and desquamating lesions. (b) Skin lesions at the site of mucosal infection. Symptoms in the eyes, mouth, and anus were most severe on day 7. (c) Progression of skin lesions. Rashes spread from the site of inoculation to the limbs and back. The lesions progressed to pustules (day 7), evolved to scabs (day 14), and ended as scarrings (day 20). (d) Manifestations of the lymph nodes. Swelling of the inguinal lymph nodes after infection on day 5 and day 7. (e) Change in body temperature. The rectal temperature was measured within 3 min after anesthesia. Elevation in body temperature of 2°C relative to the baseline temperature is defined as a high fever. The dotted line indicates the average baseline body temperature. The dashed line denotes a 2°C temperature increase above the average baseline. (f) Change in body weight. The percentage (%) change in body weight compared to baseline levels in animals infected with WIBP-MPXV-001 over time was determined. The results for each monkey are plotted.

Table 1. Clinical symptoms of mpox disease.

Clinical symptoms	Animal ID
	179327C	186503C	186321C
Temperature “spike” (fever > 2 °C)	Normal	Normal	Normal
Weight loss (> 5%)	Normal	Normal	Normal
Clinical signs ^a (day 7, day 9)	+ +	+ + +	+ + +
Appetite ^b (day 7, day 9)	+ +	+ +	+ + +
Days to duration of decreased activity	7	7	9
Onset of lesions	Day 3	Day 3	Day 5
Days to lesion resolution ^c	4–11	7–9	7–11
No. of lesions (day 11)	32	41	46
Outcome	Recovery	Recovery	Recovery
Euthanasia	Day 18	Day 19	Day 20

^a Clinical signs: –, none; +, mild; + +, substantial; + + +, intense; infected NHPs were monitored once daily for clinical signs of disease; ^bAppetite: –, normal; +, > 50% feed left; + +, only eat apples; + + +, loss of appetite; ^cLesion resolution: duration of lesion goes from macule to scar.

Lesions

Skin lesions, as a result of MPXV infection, first appeared on day 3 after infection. Subcutaneous injection with WIBP-MPXV-001 induced pustules, scabs, and scarring (Figure 2(a)), and rashes occurred on infected mucosal surfaces, such as the oral, ocular, and anorectal surfaces (Figure 2(b)). The peak number of lesions occurred on day 11. On day 3, the conjunctiva of 186321C began to show redness and swelling, and all three animals showed redness, swelling, and purulent secretion in the eyes by day 5. On day 7, the condition of the eyes worsened, and a large amount of secretion formed in both eyes, causing difficulty in keeping them open. In contrast to those observed in the eyes, the oral and anal symptoms were slightly less severe, with 1–2 rashes, which resolved within 5–7 days. The rashes on the face and trunk were later compared to those at the four infection sites; these rashes spread to the face and limbs beyond the site of infection by day 7 and then to the back by days 9–11. Table 2 and S1 present the dynamics of the skin lesions, including their number and severity. The papulovesicular lesions were morphologically similar in all three animals. The lesions progressed to pustular, evolved to scabs, and ended as scarring progressed (Figure 2(c)).

Table 2. Lesion count summary.

Animal ID	Route^a	Lesion count (face, oral, anorectal, ventral, limbs, (total))
		Day 1	Day 3	Day 5	Day 7	Day 9	Day 11	Day 14^b	Day 16
179327C	Intraocular (0.1 mL per eye) Intraoral (1.0 mL) Intrarectal (1.3 mL) Subcutaneous (0.5 mL)	0,0,0,0,0,(0)	0,1,0,3,0,(4)	2,1,0,5,13,(21)	2,2,0,9,17,(30)	2,2,0,9,17,(30)	2,2,0,9,19,(32)	2,0,0,4,20,(28)	1,0,0,0,7,(8)
186321C		0,0,0,0,0,(0)	0,0,0,1,0,(1)	0,2,0,1,0,(3)	2,2,1,1,18,(24)	4,2,2,1,20,(29)	4,2,2,1,37,(46)	5,0,0,2,30,(37)	2,0,0,0,28,(30)
186503C		0,0,0,0,0,(0)	1,0,0,1,0,(2)	2,1,3,2,0,(8)	6,2,3,5,5,(21)	6,2,3,7,8,(26)	6,2,3,6,24,(41)	2,0,0,4,14,(20)	0,0,0,1,7,(8)

^a Route: Three cynomolgus macaques were infected with WIBP-MPXV-001 using a consistent method of inoculation, involving simultaneous mucosal and skin infection; ^bDay 14: Most of the lesions evolved to crust stages by 14–16 days post-infection.

Body temperature and body weight

The body temperature increased mildly, starting on days 1–3 after the virus infection, and continued until days 7–9. The animals did not develop a high fever (an increase in body temperature of >2°C) [21] throughout the study despite becoming viremic and developing skin lesions (Figure 2(e)). Body weight slightly decreased starting three days after the animals were challenged due to the loss of appetite. The animals gained weight and were heavier at the end of the study than at the start of the study (Figure 2(f)).

Viral load in swabs, whole blood, and tissue samples

Real-time fluorescence quantitative PCR was performed to assess the viral load in nasal swabs, whole blood, and tissue samples after infection (Figure 3). After infection, all animals exhibited substantial viral shedding, with detectable viral DNA in nasal swabs (Figure 3(a)), throat swabs (Figure 3(b)), and rectal swabs (Figure 3(c)) as early as one day after infection; the DNA level reached its peak between day 5 and day 9. The shedding of the virus from the nose, throat, or rectum began to decrease after day 9. Except for the throat swabs of 186503C and 186321C, virus shedding was detected in all the other swab samples until the animals were euthanized. Although the timing of the infection varied slightly, the peak viral shedding did not significantly differ among the three monkeys through different shedding routes (nose, throat, or rectum) (Figure 3(f); P > 0.05). From day 7 to day 11, which corresponded to the peak period of blister formation, high viral loads were detected in the blister fluid swabs (Figure 3(d)). The blood viral loads of the monkeys are shown in Figure 3(e). Viral DNA remained undetected in the blood immediately after inoculation (day 1) but started to appear on day 3 and peaked on day 7. The viral load in the blood started decreasing after seven days and continued decreasing until the day of euthanasia. On day 7, when severe ocular symptoms were observed, we collected and tested ocular secretion swabs from the monkeys (Figure 3(g)). These swabs revealed higher viral loads, similar to those detected in the blister fluid. Notably, even on euthanasia day, viral DNA was detected in the ocular swabs, despite the recovery of the infected monkeys from ocular symptoms.

Figure 3. The viral loads in the swabs, whole blood, and tissue samples of cynomolgus macaques infected with WIBP-MPXV-001. Nasal swabs (a), throat swabs (b), and rectal swabs (c) were collected and placed in 2 mL of DMEM. The vertical axis represents the MPXV DNA load per millilitre of collected fluid. (d) Blister swabs were collected during the vesicular stage of the rash. Swabs with blister fluid were placed in 2 mL of DMEM. The vertical axis represents the MPXV DNA load per millilitre of collected fluid. (e) Quantitative PCR assays were performed using DNA extracted from EDTA whole blood, and the results are presented as the MPXV DNA load per millilitre of EDTA whole blood. (f) The peak viral loads recorded from different parts of the three monkeys were combined. The differences were determined via one-way ANOVA; however, no significant difference in the peak viral loads among the different swabs of the three infected monkeys was recorded (P > 0.05). (g) The amount of MPXV DNA per millilitre of collected fluid from ocular swabs. (h) Tissue samples were collected from euthanized monkeys 179327C, 186503C, and 186321C at 18, 19, and 20 dpi, respectively. The tissue samples were homogenized in 1 mL of PBS, and the supernatant was used for qPCR assays. The results showed the MPXV DNA load per gram of tissue. Blank spaces indicate that the DNA of the virus was not detected in the tissue (samples from mandibular lymph nodes and the colon were not collected for 186321C).

At 18, 19, and 20 dpi, the three monkeys (179327C, 186503C, and 186321C) were euthanized, and tissue samples were collected for qPCR analysis. As shown in Figure 3(h), the highest viral load was detected at the site of injection in all animals, and high viral loads were also found in the respiratory and lymphatic systems. Among the tissue samples tested, the viral load in 28 out of 29 tissues from the infected monkey 179327C exceeded the detection limit of the kit (200 copies/mL). For 186503C and 186321C, the ratios were 22/29 and 11/27, respectively (S2, Figure S2-1). In general, the viral loads in the corresponding tissues of 179327C were greater than those in the corresponding tissues of 186503C and 186321C, consistent with the chronological order of euthanasia for the three infected monkeys, indicating progressive recovery and clearance of the virus in the former.

Infectious MPXV was detected in some swabs collected from all three monkeys, including nasal swabs, throat swabs, rectal swabs, and blister fluid swabs (S3, Table S3-1). A blister fluid swab taken on day 7 from 179327C showed the highest viral titre (2.8 × 10⁶ PFU/mL). Additionally, relatively high viral loads were detected in rectal samples during the early stages of infection, which may be attributed to the rectum being one of the target sites for viral replication. Consistent with findings of the study by J. A. Tree et al [22], no lesions were observed in the cells of the inoculated blood samples (S3, Table S3-2). The viral titres in the skin from the injection sites and lung tissue homogenates of the three monkeys are shown in Table S3-3 (S3). Infectious viruses were detected in the skin of 179327C and 186503C. In addition, infectious viruses were also detected in the upper lung (right) and middle lung (right) of 179327C.

Biochemical analysis and hematology

Blood cell counts and biochemical parameters were measured in the infected monkeys. The results of the blood cell count tests showed the typical features of virus infection (Figure 4(a)). The absolute value of eosinophils started increasing on day 9, whereas the percentage of eosinophils started increasing on day 5. The lymphocyte count and the percentage of lymphocytes decreased from days 0 to 5 and then increased to normal levels. Monocyte counts and percentages exhibited similar trends, increasing from days 0 to 9 and then decreasing to near-initial levels. The absolute value of neutrophils in the three monkeys did not change considerably, but the percentage of neutrophils increased in the first 3–5 days after infection and then decreased by day 9. These variations may originate from changes in lymphocytes and monocytes. The platelet count in the infected monkeys decreased from days 0 to 5, and then increased, and the change in platelet volume showed a similar pattern.

Figure 4. Changes in the blood cell counts and biochemical parameters of cynomolgus macaques infected with WIBP-MPXV-001. (a) EDTA-treated whole blood was collected from the monkeys after exposure to WIBP-MPXV-001, and blood cell counts were performed using an IDEXX five-part blood cell counter. EO#: absolute value of eosinophils; EO%: percentage of eosinophils; LYMPH#: absolute value of lymphocytes; LYMPH%: percentage of lymphocytes; MONO#: absolute value of monocytes; MONO%: percentage of monocytes; NEUT#: absolute value of neutrophils; NEUT%: percentage of neutrophils; PCT: platelet count; PCT%: percentage of platelet count; PLT: platelet count. (b) Changes in the blood biochemical parameters of the monkeys after exposure to WIBP-MPXV-001 were assessed by collecting lithium heparin whole blood, centrifuging the blood to separate the plasma, and analyzing the results using an automated biochemical analyzer. ALB: albumin; LDH: lactate dehydrogenase. (c) Cytokine levels in the serum of infected monkeys. The concentration of cytokines in the samples was calculated by comparison with the corresponding standard curves generated using purified cytokines in the non-human primate Bio-Plex Kit.

The results of the blood biochemical tests showed that the parameters did not change significantly after infection, except for the levels of albumin and lactate dehydrogenase (Figure 4(b)). The blood ALB concentration decreased in the early-to-middle stage of infection (0–9 days), and then, gradually increased after nine days. The LDH concentration in the blood decreased from days 1 to 5, increased from days 5 to 11, and then fluctuated around the initial value.

Cytokine responses

The cytokine levels in the serum were detected using the Bio-Plex 200 System. The level of IL-6 and IFN-γ increased in all animals after infection (Figure 4(c)). IL-6 reached its peak level on days 3–5, and then decreased below the detection limit (considered to be 0). The change in IFN-γ was similar to the change in IL-6. The levels of IL-6 and IFN-γ in 179327C and 186503C reached their peak values at the same time, on day 3 and day 5, respectively. No significant changes in the levels of other cytokines (GM-CSF, IL-2, IL-5, IL-10, IL-13, IL-21, Granzyme B, IL-1β, IL-4, IL-12 p70, IL-17A, or TNF-α) were detected (S4).

Immune responses

The serum IgG antibody levels against six proteins (A29, H3, M1R, I1L, B6R, and A35) were measured via ELISA (Figure 5(a)). The time courses and levels of IgG response determined by ELISA were similar for two monkeys (179327C and 186321C), and IgG reactive to virus-specific proteins became detectable within 9 days after MPXV infection. The IgG antibody levels remained stable in all animals from days 14–16 until the day of euthanasia.

Figure 5. The humoral immune response in cynomolgus macaques after infection with WIBP-MPXV-001. (a) Development of IgG antibodies against the MPXV-specific proteins A29, H3, M1R, I1L, B6R, and A35 in blood samples collected on different days after infection, as measured by ELISA. The optical density at 450 nm (OD_{450 nm}) at a serum sample dilution of 1:256 is shown. The OD_{450 mn} values of blood samples at different dilution factors. (b) Neutralizing antibody titres against the MPXV in blood samples collected at different time points after the viral challenge were determined by the PRNT.A neutralizing antibody titre of 10 was assigned if no neutralizing reaction was detected.

The changes in the serum-neutralizing antibody titres at different time points after infection indicated changes in the humoral immune response in the animals (Figure 5(b)). The results showed that no neutralizing response was observed within the first five days after infection. From days 5 to 14, the neutralizing antibody titres slightly increased in all the animals. This increase in 179327C continued until the day of euthanasia, whereas the antibody titres decreased slightly in 186321C and 186503C on days 20 (euthanasia) and 16, respectively. All animals exhibited an increase in neutralizing antibodies after viral challenge, with the titres in the blood reaching a maximum of 730 PRNT₅₀.

Pathological and histopathological findings

Necropsy was performed on all animals. Gross lesions related to mpox were rarely found in organs, such as the heart, liver, kidneys, stomach, duodenum, colon, rectum, adrenal glands, and testes. During the acute phase of infection, the inguinal lymph nodes significantly increase in size. However, as the skin lesions and clinical symptoms of the animals relieved, the size of the lymph nodes returned to normal. No significant necrotic lesions were found in the axillary lymph nodes, mesenteric lymph nodes, or inguinal lymph nodes during the autopsy on the day of euthanasia.

Tissues with many copies of viral DNA, such as the lungs and skin, were subjected to pathological analysis, as they exhibited necrotic lesions. Interstitial pneumonia, characterized by widened alveolar septa, decreased alveolar spaces, abundant mononuclear cells and lymphocytes in the alveolar septa, and multifocal structural loss, was observed in all animals (Figure 6(a, b, and c)). In the skin of 186503C, skin ulceration, shedding of hair follicles and bulbs, granulation tissue formation, and inflammatory cell infiltration in the dermis were observed. These changes were accompanied by epithelial hyperplasia around the affected area (Figure 6(d, e, and f)). During the necropsy, splenomegaly (enlarged spleen) was found in all three animals, with 179327C showing more severe enlargement than the other two animals (Figure 6(g, h, and i)). Furthermore, individual hemorrhagic spots and ulcers were found in the colon and rectum of 179327C, but no lesions were observed in the digestive tracts of the other two animals (Figure 6(j and k)).

Figure 6. The following organ lesions and histopathological changes were observed in cynomolgus macaques infected with WIBP-MPXV-001. (a-c) Interstitial pneumonia was characterized by a decrease in alveolar space and the presence of a large number of inflammatory cells in the alveolar septa. The pathology of 186503C is shown in (b) at 5× magnification and in (c) at 20× magnification. The tissue sections were stained with hematoxylin and eosin (H&E) to visualize the cellular structures. (d-f) Lesions at the injection site, including skin damage, redness, and infiltration of a large number of mononuclear cells and lymphocytes in the dermis of 186503C, are shown in (e) at 2× magnification and in (f) at 10× magnification. H&E staining was performed to visualize the changes in tissues. (g-i) Splenomegaly. Animals had enlarged spleens without apparent lesions. From left to right, the images represent the spleens of 186503C, 179327C, and 186321C, respectively. (j-k) Hemorrhagic spots in the colon and ulcerative lesions in the rectum of 179327C.

Discussion and conclusion

An ideal animal model for human diseases should exhibit the natural route of pathogen transmission, reproduce the disease with an infectious dose equivalent to that causing disease in humans; and demonstrate a disease course, morbidity, and mortality similar to that recorded in human cases [23]. NHPs, including MPXV, are ideal animal models for treating emerging and re-emerging diseases [10]. However, reports of suitable NHP models for the mpox outbreak of 2022 are lacking. In this study, we established a NHP model of MPXV infection by simulating natural mpox infection using the prevalent strain from the 2022 outbreak.

The establishment of an animal model is linked to the question to which one wishes to answer. The aim of this study was to establish a cynomolgus macaque model of mpox that could accurately replicate the natural transmission route of contemporary strains and show typical manifestations of mpox infection in humans. Thus, the appropriate infectious dose and route of infection are crucial factors. Human, monkey and CAST/EiJ mouse disease virulence differs between Clade I strains and Clade II strains, the former being more virulent in NHPs [24,25]. In previous NHP studies focusing on MPXV (Clade I), the majority of inoculation doses ranged from 10⁴ to 5 × 10⁸ PFU [18,26,27]. The results of our study further demonstrated that, despite administering higher doses of WIBP-MPXV-001, a self-limiting disease phenotype was observed, which differed from the lethal or severe disease phenotypes associated with Zaire strain. These findings indirectly support the notion that Clade IIb exhibits lower virulence in the NHP than does Clade I. Additionally, MPXV can enter the body through broken skin, mucous membrane surfaces (such as the mouth, throat, eyes, genitalia, and rectum), or through the respiratory tract. In the current outbreak, the transmission of MPXV mainly occurred among men who have sex with men. In such cases, the aforementioned modes of viral entry into the body often occur simultaneously.

The animal model we established showed clinical symptoms, disease progression, and prognosis similar to those recorded in humans affected by mpox [28]. The cynomolgus macaques infected by the virus initially exhibited mild increases in temperature and skin symptoms, including skin rashes, perianal/genital rashes, and mucosal lesions; these symptoms after three days of infection and spread throughout the body from the site of infection. Fewer lesions were observed, consistent with the reported human symptoms [29]. Lymph node enlargement was also found in all three infected monkeys, which is a typical characteristic of mpox [28]. A broken mucosa may significantly contribute to the transmission of MPXV infection. Regarding mucosal damage to the rectum, the infected monkey 186503C showed the earliest and most severe symptoms in the anal region. The detection of viremia was consistent with the appearance of skin lesions. When viremia reaches its peak level, the most severe clinical manifestations and viral shedding are observed. Disease caused by WIBP-MPXV-001 was self-limiting, and symptoms began to resolve 14 days after infection. However, the infected monkeys exhibited several complications, including pulmonary inflammation and conjunctivitis [8], which might be related to our method of infection.

The detection of viral DNA is a preferred laboratory diagnostic test for MPXV. In this study, MPXV DNA was detected in nasal swabs, throat swabs, and rectal swabs, and the dynamics of viral shedding were similar to those reported in patients [9]. Specifically, MPXV DNA was detected in nasal swabs as early as one day post-infection, suggesting that it might be associated with the nasolacrimal system, through which the virus may enter the nasal cavity from the site of infection [30]. In addition to blister fluid, infectious MPXV has been detected in nasal, throat, and rectal swabs, suggesting the possibility of viral transmission through nasal, oral, or anal shedding. An autopsy revealed lesions in the lungs of the monkeys, with the highest content of viral DNA detected in respiratory tract locations, except for the injection site. Infectious virus was also detected in the lung tissue of 179327C. In the context of 2022 outbreak, some patients presented with respiratory symptoms, including sore throat and cough [31]. Infectious MPXV was also found in the saliva of patients [32], highlighting the susceptibility of the respiratory tract to the virus. In a UK-based sampling study, replicating MPXV was collected from hospital air samples [33]. While documented cases of human respiratory infection with mpox are currently lacking, animal models have reported instances of respiratory transmission of the virus [34]. Hence, further evaluation of the potential for respiratory transmission of MPXV is needed.

Several studies have shown that lymphoid tissues are the primary replication sites of MPXV. Due to the infection of lymphoid tissues, orthopoxviruses can affect distant organs via lymphohematogenous dissemination after the development of low-grade primary viremia [35,36]. In our study, during the early stages of infection, the monkeys exhibited low lymphocyte and platelet counts, probably due to virus-induced consumption or increased clearance [21]. On the fifth day after infection, an increase in lymphocytes was observed, indicating the adaptive immune response of the host to the virus. An increase in monocytes is a typical symptom of viral infection [37]. In reported cases of human mpox, increased white blood cell count and decreased platelet count are also common features of the disease [38]. An increase in the level of IL-6 and IFN-γ during infection further indicated that the immune system was activated. The levels of IgG and neutralizing antibodies in the blood started increasing on day 5 and reached higher levels by day 9. These immune responses contribute to a decrease in the viral load in the blood and the alleviation of symptoms of infection.

Our study has several limitations. A key uncertainty is the natural human infectious dose, which experimental studies have exceeded in many cases [18,23]. Additionally, distinguishing the role of individual transmission routes is challenging, as disease progression can vary depending on the method of exposure [35,39,40]. Invasive inoculation may induce symptoms different from those recorded after multi-route acquisition combined with mucosal exposure [40]. Experimental studies with animal models need to address questions regarding the frequency of different acquisition routes, particularly those associated with close contact, mucosal routes, and broken skin. Based on the available evidence, the likelihood of human-to-human respiratory transmission of MPXV appears to be low [7]. Although, in this study, we established a non-respiratory model, further research needs to assess the possibility of respiratory transmission.

To summarize, the cynomolgus macaque model established in this study accurately recapitulates the natural transmission route of MPXV and shows typical manifestations of mpox infection in humans. This model might be useful for evaluating vaccines and antiviral drugs for treating mpox infection. Additionally, as it is a non-respiratory challenge model, it provides a better understanding of the precise mechanisms underlying the interhuman transmission of mpox.

Acknowledgments

The authors thank Shanghai Institute of Biological Products Co., Ltd. for providing the reagents related to ELISA detection.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Key Research and Development Program of China (2021YFC2600204) and the Foundation Strengthening Project (2023–173ZD-124).