Comparative pathogenicity study of monkeypox virus Clade Ib and Clade IIb in a rabbit model

ABSTRACT

Mpox has emerged as a significant public health concern, spreading across continents with the emergence of new variants. Clade IIb, from the recent global outbreak, and the newly identified Clade Ib have shown changes in transmission dynamics and possibly in severity. We developed a New Zealand White rabbit model of Mpox by using a combination of intradermal and intravenous routes of administration. Following infection, the rabbits developed erythematous papular lesions on the skin, which progressed to crust and scab formation by the second week, ultimately leading to healing and seroconversion. Infection with the Clade Ib and IIb.A.2.1 lineages revealed that both Monkeypox virus (MPXV) variants caused skin and testicular lesions, as well as viral excretion from the nasal cavity, skin, and testicular tissue. However, there were no significant differences in disease severity between the two variants. These findings suggest that this rabbit model could be useful for evaluating countermeasures against MPXV.

Introduction

Mpox is a zoonotic disease caused by an Orthopoxvirus and has recently emerged as a public health concern of increasing importance across the globe [1]. The disease was first identified in the Democratic Republic of Congo in 1970 and remained endemic to Central and West Africa for decades [2,3]. Infections were primarily linked to contact with infected rodents in these regions [4–6]. However, in 2022, Mpox spread globally, marking the largest reported epidemic outside of Africa [7]. This prompted the World Health Organization (WHO) to declare it a Public Health Emergency of International Concern from 23 July 2022, to 10 May 2023 [8]. Unlike previous Mpox infections, which were typically associated with travel to endemic areas or contact with animals, the 2022 epidemic was linked to sexual contact between individuals [7]. In 2024, there was another increase in Mpox cases reported in Central and Eastern Africa, leading the WHO to declare a second Public Health Emergency of International Concern on 14 August 2024 [9,10].

Phylogenetically, the virus is divided into two distinct clades: Clade I (Central African or Congo Basin) and Clade II (West African) [11]. The average case fatality rates (CFR) reported from previous Mpox outbreaks (before 2022) are 10.6% for Clade I and less than 4% for Clade II [12]. The CFR estimates for Clade I were also influenced by limited access to healthcare, primarily due to outbreaks in rural areas. Following the global spread of the virus in 2022, an accelerated microevolution of the Monkeypox virus (MPXV) has been observed, leading to further sub-classification of the clades into subclades (Ia, Ib, IIa, IIb) and lineages (A, B, C) [13]. The global surge in Mpox cases from 2022 to 2023 was primarily associated with the Clade IIb MPXV, while Clade Ib was predominantly reported during the infection surge in Central and Eastern Africa in 2024 [14]. The Clade II lineage A and its subvariants were reported from 2017 to 2023 in Africa, the USA, the UK, Spain, Malaysia, Thailand and Japan [15]. The Clade IIb.B.1 variant was the predominant lineage reported during the 2022 epidemic [16]. The Clade IIb.C.1 variant also emerged as a major variant from mid-2023. Additionally, the Clade IIb.A.2 lineage was reported during this outbreak in the USA, UK, India, and Thailand [15]. These variants have exhibited multiple nucleotide substitutions and deletions in their genomes, but their effects on gene functions are still unknown. Distinct nucleotide changes have been observed in the A.2 variants, suggesting their independent emergence. Notably, a seven-nucleotide deletion in the OPG 174 gene, which influences virulence, has been identified in this variant [17]. OPG 174 is known to suppress host immune response; mutations in it could alter virulence and could serve as a possible target for therapeutic interventions [18].

With the evolution of the virus, it is important to understand the ecology and epidemiology of the disease as well as the virus properties like pathogenesis, virulence, transmissibility, etc. Animal models are important tools to study virus properties as well as for the evaluation of vaccines or therapeutics. Many animal models like laboratory mouse strains, guinea pigs, dormice, rabbits, prairie dogs, squirrels and non-human primate models have been studied for MPXV susceptibility [19]. These models have shown varying degrees of susceptibility to infection, but not all the clinical aspects of human Mpox disease could be reproduced. Among these, wild rodents like ground squirrels, prairie dogs, dormice and Cast/EiJ mice were highly susceptible but are difficult to obtain for research studies in all parts of the world [20–23]. The non-human primate models are relatively expensive and not readily available [24]. Rabbits are less expensive and are easier to handle and have been used as animal models for other pox viruses like rabbit pox and vaccinia virus, but their immunologic and anatomic differences from humans are a disadvantage [19]. Two earlier studies have demonstrated susceptibility of rabbits to MPXV using the Copenhagen strain of 1976 (Clade I) and the Clade IIb C.1.1 variant [5,25].

The refinement and application of the available models will help us to better understand the dynamics of the disease and assess the effectiveness of interventions aimed at preventing and managing Mpox. The majority of the animal model studies have used historic isolates [19]. The studies on recent isolates are limited to a few studies in Cast/EiJ mice, BALB/c mice, and the rabbit model [26–28]. In this study, we have compared the disease caused by the MPXV Clade Ib and Clade IIb.A.2.1 viruses isolated during the 2022–2023 epidemic using a rabbit model by combining intradermal and intravenous routes of infection.

Materials and methods

Virus

Monkeypox virus (Accession no: PP_004G599.1) belonging to the Clade IIb.A.2.1 lineage isolated from the blister fluid of a Mpox patient and Clade Ib lineage (Accession no: PP_004G68A.1) isolated from the skin lesion swab of a Mpox patient in Vero (ATCC® CCL-81™) cells (ATCC, USA) were used for the study [17,29]. Growth kinetics analysis was performed to characterize the isolates. The growth kinetics were estimated in Vero cells. For this, six well tissue culture plates with Vero cells were infected with 0.1 multiplicity of infection and were incubated for an hour at 37°C in a CO₂ incubator. The plates were washed with phosphate-buffered saline and incubated with maintenance medium (minimal essential media with 2% fetal bovine serum). The cell culture supernatant, along with the scraped cells were collected at 24-hour intervals and was titrated in Vero cells, and titers were determined by the Reed and Muench method (Figure S1). At 24-hour post-infection, a 1.75-fold increase in titer was observed for both isolates. This increase continued at subsequent time points, with peak virus titer occurring on day 4 post-infection for both virus isolates, followed by a decline (Figure S2). The isolates were sequence verified by whole genome sequencing as described earlier [17]. The virus isolates were passaged three times, and the titer of the Clade IIb.A.2.1 isolate was 2·5 × 10⁷ TCID₅₀/ml and the Clade Ib isolate was 3.16 × 10⁶ TCID50/ml on titration in Vero cells as per the Reed and Muench method [30].

Animal experiments

The Institutional Animal Ethics Committee of ICMR-National Institute of Virology (ICMR-NIV), Pune, approved the experiment under the approval number [ICMR-NIV/IAEC/2022/MCL/05]. The guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, were followed during the experiment. New Zealand white rabbits (3–4 months old, sub-adults) procured from the laboratory animal facilities licensed by CCSEA were used for the study. Rabbits were housed in conventional stainless-wired steel cages in the Maximum Containment facility (ABSL-4). The animals were provided with ad libitum food and water, and were randomly allocated to experimental groups after two weeks of acclimatization. The caretakers and technicians were blinded to the group allocation. Baseline body weights were measured before the experiment. The ARRIVE guidelines checklist is attached as a supplementary file.

An initial experiment was performed to understand the susceptibility of the New Zealand white rabbit model to MPXV infection by two different methods of virus inoculation. The female rabbits with weights ranging from 1.4 to 1.9 kg (3–4 months old) were used. Eight rabbits were infected intradermally (0.7 ml, with a dose of 2.5 × 10⁷ TCID50/ml) with MPXV Clade IIb A.2.1 variant, and four rabbits were infected with both intradermal (ID) and intravenous (IV) administration (0.5 ml ID and 0.2 ml IV, with a dose of 2.5 × 10⁷ TCID50/ml). Three rabbits were kept as healthy controls. The dorsum of the rabbits was shaved, and the animals were observed for 21 days for clinical signs. The skin lesions were checked daily, and the number of disseminated lesions was counted. Oral swab, rectal swab, and nasal swab samples were collected in 1 ml sterile cell culture media using nylon flocked swabs on alternate days for 21 days. The rabbits were euthanized on 21 days by overdose of isoflurane anesthesia. The serum samples collected on 21 days were tested for anti-MPXV IgG antibodies.

Based on the results of the first experiment, the combined intradermal intravenous method was chosen for the second study to further evaluate the model by two different MPXV isolates belonging to Clade Ib and IIb.A.2.1. In the second experiment, five adult male rabbits, each weighing between 1.4 and 1.9 kg, were infected through both ID and IV administration (0.5 ml ID and 0.2 ml IV, with a total dose of 3.16 × 10⁶ TCID50/ml) with either MPXV Clade Ib or Clade IIb.A.2.1 isolates. To ensure equal titers for infection in both groups, the Clade IIb.A.2.1 isolate was diluted with sterile medium to create a uniform titer before administration. Male rabbits were chosen for this experiment based on availability and previous literature stating that male rabbits exhibit testicular pathology [25]. The rabbits were observed for clinical signs for 14 days. Nasal and oral swabs were taken from the rabbits on alternate days until the 14th day post-inoculation. Additionally, skin crust samples were collected from rabbits that showed healing of the scab. On the 14th day, the rabbits were euthanized by overdose of isoflurane anesthesia, and organ samples, including skin, testes, lungs, liver, spleen, and lymph nodes (axillary/inguinal and mesenteric), were collected. Serum samples were also collected on the 14th day and checked for IgG antibodies.

Viral load estimation by real-time qPCR

Swab samples, skin crusts, scabs or tissue homogenate samples were used for the nucleic acid extraction. DNA extraction was performed using the MagMAX™ viral/pathogen nucleic acid isolation kit (Cat # A48310, Thermo Scientific, USA) as per the manufacturer’s instructions. Real-time qPCR was performed further using published primers for the envelope protein gene (B6R) for the viral DNA [31].

Virus isolation from infected rabbit samples

The virus isolation was performed in Vero (ATCC® CCL-81™) cells. Twenty-four well plates seeded with Vero cells were infected with the nasal swab, skin scabs and testes homogenate samples. After one hour of adsorption time, the maintenance media (minimal essential media with 2% fetal bovine serum) were added, and the plates were incubated for 5 days at 37°C in a CO₂ incubator. The plates were observed daily for the cytopathic effect. The observations were recorded on the 5th day. The samples showing cytopathic effect were further passaged and confirmed by real-time qPCR.

Anti-MPXV rabbit IgG ELISA

Gamma-inactivated MPXV antigen was prepared in a carbonate buffer (pH 9.2, 0.05 M). The 96-well ELISA plates were coated with 100 µL of diluted MPXV antigen in wells A-D and with normal Vero cell lysate in wells E-H. The coated plates were incubated at 4°C for 16–18 hours. After incubation, the plates were washed three times with PBST (pH 7.2–7.4). Then, 100 µL of blocking buffer was added, and the plates were incubated for 2 hours at 25–30°C. Following this, the blocking buffer was aspirated, and the plates were vacuum-dried, sealed, and stored at 4°C. Rabbit sera were diluted at a ratio of 1:1000, and a volume of 100 µL was added to the wells and incubated at 37°C for 1 hour. For titration, 2 fold dilutions of serum samples starting from 1:100 was used. The plates were washed three times. Next, 100 µL of anti-rabbit IgG-HRP (in a concentration of 1:15000 in 5% milk PBST) was added, and the plates were incubated for another hour. After washing three times, 100 µL of TMB substrate was added, and the plates were incubated for 10 minutes at 25–28°C. The reaction was halted by adding 2 M sulfuric acid, and the absorbance was measured at 450 nm. An optical density (OD) value equal to or greater than 0.2, along with a positive/negative (P/N) ratio of 1.5 or greater, was considered positive; otherwise, the sample was considered negative.

Histopathology and immunohistochemistry

Organ samples fixed in 10% buffered formalin were processed for histopathology by hematoxylin and eosin (H&E) staining [32]. Coded tissue samples were blindly scored by a pathologist. For immunohistochemical evaluation, skin and testis tissue sections were used. The tissue sections were rehydrated, and 0.3% hydrogen peroxide in methanol was used for antigen retrieval. Polyclonal anti-MPXV mouse serum (generated in-house using formalin-inactivated MPXV antigen in mice) was used as the primary antibody. The sections were incubated with primary antibody (1:200 dilution) overnight and washed four times with PBS. The mouse and rabbit-specific HRP/DAB (ABC) detection immunohistochemistry kit (Abcam, ab64264) was used for immunostaining.

Statistical analysis

For statistical analysis, GraphPad Prism, Version 9.1.0, was used. Data were presented as mean/geometric mean ± standard deviation. Statistical significance between groups was determined using a t-test or analysis of variance (ANOVA). A p-value of <0.05 was considered statistically significant.

Results

Comparison of disease by MPXV in adult rabbits by intradermal and combined intradermal-intravenous inoculation

We infected 8 rabbits by the intradermal route and 4 rabbits by the combined intradermal intravenous route with MPXV for this experiment (Figure 1(A)). Among the rabbits, 3 out of 8 that were infected via the intradermal route developed skin lesions, while all 4 rabbits infected through both the intradermal and intravenous exhibited cutaneous lesions. The skin lesions appeared in rabbits infected by either mode of infection approximately 4–5-day post-infection, initially as erythematous raised areas. These lesions progressed to dark red papules and gradually developed into crusts by days 9–10. The scabs typically fell off within 2–3 weeks, and the lesions completely resolved by the end of the 3 weeks (Figure 1(B-G)). The number of lesions varied among the rabbits (Table 1). The disseminated lesion count was recorded on the shaved backs of the rabbits as well as on their ear pinnae. The size of the lesions varied, ranging from pinpoint size to a maximum diameter of 0.7 cm. The site of intradermal infection also displayed a progression from a bleb to a firm, raised red area within 2 days. This lesion became dark red over the following days, eventually forming crusts before healing.

Figure 1.

Experimental design and the skin lesions developed in rabbits after experimental infection of the MPXV by intradermal plus intravenous infection. A) Experimental study design where two groups were infected with MPXV Clade IIb.A.2.1 through intradermal (n = 8) and combined intradermal and intravenous (n = 4) inoculation. B) the intradermal inoculation site (2 erythematous areas) on the dorsum of the rabbit on the 2nd day after MPXV inoculation, showing erythema. The dorsum of rabbits showing erythematous papules of varying sizes around the intradermal inoculation site C) on day 4, D) on day 5, E) on day 10 and F) on day 14 post MPXV infection. G) the shaved dorsum of the mock control rabbit showing normal skin.

Table 1.

The details of experimental MPXV inoculation in rabbits by different routes and the clinical signs observed.

Sl no.	Route of infection	Virus titer	Clinical signs	No. of skin lesions
1	Intradermal	2.5 × 107 TCID50/ml (MPXV Clade IIb.A.2.1)	Erythematous papules on skin (3/8)	Rabbit 1: ~40
				Rabbit 2: 20
				Rabbit 3: ~50
				Rabbit 4: 0
				Rabbit 5: 0
				Rabbit 6: 0
				Rabbit 7: 0
				Rabbit 8: 0
2	Intradermal + Intravenous	2.5 × 107 TCID50/ml (MPXV Clade IIb.A.2.1)	Erythematous papules on skin (4/4)	Rabbit 1: ~20
				Rabbit 2: ~50
				Rabbit 3: ~20
				Rabbit 4: ~50

The rabbits were monitored for 21 days after virus infection. During the first week of infection, there was a slight rise in average rectal temperature (non-significant) compared to baseline measurements in both groups (Figure 2(A)). The body weights of the rabbits infected through intradermal inoculation and through both intradermal and intravenous inoculation of MPXV showed an increasing trend over the 21-day study period (Figure 2(B)). MPXV DNA was detected in the oral and rectal swabs of 2 out of 8 rabbits, as well as in the nasal swabs of 3 out of 8 rabbits infected through the ID route alone. In rabbits infected by both intradermal and intravenous routes, MPXV DNA was found in the oral swabs of 2 rabbits, the rectal swabs of 1 rabbit, and the nasal swabs of all 4 rabbits tested (Figure 2(C,D)). By day 21 post-infection, all rabbits that were infected exhibited the presence of anti-MPXV IgG antibodies (Figure 2(E)). The titers of the ID group ranged from 6400 to 25,600 whereas of the ID + IV group ranged from 6400 to 12,800. Furthermore, skin scabs collected from rabbits with skin lesions in both groups (3 rabbits each from ID and ID + IV) showed the presence of MPXV DNA (Figure 2(F)).

Figure 2.

Comparison of disease in rabbits by intradermal inoculation and intradermal plus intravenous inoculation of MPXV. A) Line graph showing the average body weight per cent change (mean ± SD) of rabbits measured sequentially every 5 days after MPXV infection. B) Line graph showing the average rectal temperature (mean ± SD) of rabbits measured every day after infection for 14 days. Bar graph showing the MPXV DNA copies detected per ml (Geometric mean ± SD) in the C) oral swab and D) nasal swabs samples of the rabbits collected on alternate days post viral infection till 21 days (*represents a p value of 0.0182 by mann-whitney test on day 6). E) Scatter plot depicting the anti-MPXV IgG titers (mean ± SD) measured by anti-MPXV IgG ELISA in samples collected 21 days after MPXV infection. F) Bar graph showing the MPXV DNA copies detected per ml (Geometric mean ± SD) in the skin scab samples collected from rabbits. The dotted lines indicate the limit of detection of the assay.

Comparison of the disease by MPXV clade Ib and clade IIb.A.2.1 virus in rabbits by combined intradermal-intravenous inoculation

Rabbits infected with either Mpox clades Ib or IIb.A.2.1 (n = 5 each) showed normal appetite and activity during the 14-day study period (Figure 3(A)). No significant weight loss was observed in either group (Figure 3(B)). A slight increase (non-significant) in average rectal temperature, compared to baseline measurements, was noted in the rabbits during the first week of infection (Figure 3(C)). By day 14 post-infection, anti-MPXV IgG antibodies were detectable in both Clade Ib and Clade IIb.A.2.1 infected rabbits (Figure 3(D)). The endpoint titers ranged from 400 to 1600 for Clade Ib and from 6400 to 12,800 for Clade IIb.A.2.1, which was significantly higher from the former. Skin rashes began to develop approximately five days’ post-infection in both groups. These rashes presented as erythematous raised areas of varying sizes, some of which progressed to dark red papules and later formed crusts. Smaller lesions resolved without developing crusts. The number of lesions varied among the rabbits both within and between the Clade Ib and Clade IIb.A.2.1 infected groups. Specifically, Clade IIb.A.2.1-infected rabbits exhibited between 15 to more than 50 lesions, while Clade Ib-infected rabbits had 4 to 25 lesions (Table 2). The lesions were primarily observed in the shaved areas of the dorsum and the ear pinnae of a few rabbits. Additionally, in one Clade IIb.A.2.1-infected rabbit, we noted a diffuse cutaneous lesion in the scrotal sac that progressed to crust and scab formation, and this scab sample tested positive for MPXV DNA (1.01×10⁵ viral DNA copies/ml).

Figure 3.

Comparison of disease caused by MPXV Clade Ib and Clade IIb.A.2.1 in rabbits by intradermal plus intravenous inoculation. A) Study design. B) Line graph showing the average body weight (mean ± SD) of rabbits measured sequentially every 4 days after MPXV infection. C) Line graph showing the average rectal temperature (mean ± SD) of rabbits measured on alternate days post-MPXV infection. D) Scatter plot depicting the anti-MPXV IgG titers (mean ± SD) measured by anti-MPXV IgG ELISA in samples collected 14 days after MPXV infection (**represents a p value of 0.0079 by Mann-whitney test). E) Bar graph showing the MPXV DNA copies detected per ml (Geometric mean ± SD) in the nasal swab samples collected from rabbits till 14 days. F) Bar graph showing the MPXV DNA copies detected per ml (Geometric mean ± SD) in the testes, lungs, liver and spleen samples collected from rabbits on 14-day post-virus infection. The dotted lines indicate the limit of detection of the assay.

Table 2.

The details of the comparison of the MPXV Clade Ib and Clade IIb.A.2.1 experimental infection in rabbits and the clinical signs observed.

Sl no.	Groups (No. of animals, Virus dose)	Clinical signs	No. of skin lesions	MPXV DNA detection /Virus isolation
				Nasal swab	Skin scabs	Testes
1	MPXV Clade Ib (n = 5, 3.16 × 106 TCID50/ml)	Cutaneous lesions (4 out of 5 rabbits infected)	Rabbit 1: 0	+ /+	ND	-/-
			Rabbit 2: ~25	+/+	+/+	+/+
			Rabbit 3: ~10	+/+	ND	+/-
			Rabbit 4: ~4	+/+	+/+	+/-
			Rabbit 5: ~10	+/+	ND	+/+
2	MPXV Clade IIb (n = 5, 3.16 × 106 TCID50/ml)	Cutaneous lesions (4 out of 5 rabbits infected)	Rabbit 1: 0	+/+	ND	-/-
			Rabbit 2: ~20	+/+	+/+	+/+
			Rabbit 3: ~25	+/-	+/+	+/+
			Rabbit 4: ~50	+/+	+/+	+/-
			Rabbit 5: ~15	+/+	ND	+/+

“+” indicates DNA detected or virus isolated, ‘–‘indicates absence of MPXV DNA or virus isolation, “ND” indicates sample not collected.

MPXV DNA was detected in the oral swabs of only 2 out of 5 rabbits from both groups on days 8 and 10 post-infection. In contrast, viral DNA in nasal swabs was detectable as early as day 4 and remained so until day 14 post-infection (Figure 3(E)). By days 8 and 10 post-infection, MPXV DNA was found in the nasal swabs of all rabbits from both Clade Ib and Clade IIb.A.2.1. Virus isolation was successful from nasal swab samples [PCR cycle threshold (Ct) range: 21 to 34] collected on day 8, as well as from skin scab samples (Ct range: 17 to 36) from both Clade Ib and Clade IIb.A.2.1 infected rabbits, when cultured in Vero cells. The infected cell monolayer exhibited rounding and lysis of the cells (Figure 4(A,B), Table 2). Testes samples collected on day 14 from Clade Ib (4 out of 5 rabbits) and Clade IIb.A.2.1 (4 out of 5 rabbits) infected rabbits also tested positive for viral DNA (Figure 3(F)). Virus isolation attempts from these samples (Ct range: 21 to 34) resulted in a cytopathic effect in Vero cells (Figure 4(C,D), Table 2). In addition to the testes, samples from the lungs (one from each of the Clade Ib and IIb.A.2.1 infected groups), the liver (3 rabbits from the Clade Ib group), and the spleen (3 rabbits from the Clade Ib group) showed positive results for viral DNA; whereas virus isolation/titration from these samples were not successful. However, none of the lymph node samples tested positive for viral DNA (Figure 3(F)).

Figure 4.

MPXV isolation from samples of the MPXV Clade Ib and Clade IIb.A.2.1-infected rabbits. Vero cells showing cytopathic effect after infection with the A) nasal swab samples collected on day 8, C) testes homogenate collected on day 14, E) skin scabs collected on 9 days from MPXV Clade IIb.A.2.1 infected rabbits. Vero cells showing cytopathic effect after infection with the B) nasal swab samples collected on day 8, D) testes homogenate collected on day 14, F) skin scabs collected on 9 days from MPXV Clade Ib infected rabbits. G&H) Vero cells kept as a cell control for the assay, showing normal cellular morphology.

The skin lesions observed in infections caused by both Clade Ib and IIb.A.2.1 viruses were similar in appearance. Initially, they manifested as rashes, which progressed to erythematous papules that then evolved into dark red papules. In larger lesions, crust formation occurred, along with scab development (Figure 5(A-C)). Skin scabs collected from rabbits that developed lesions also showed the presence of MPXV DNA (Range: 4.6 × 10⁵ to 3.1 × 10¹⁰ DNA copies/ml). Virus isolation could also be obtained (Figure 4(E,F), Table 2). Histopathological examination of the skin samples revealed dense infiltration of inflammatory cells within the dermis and crust formation in the epidermis. The crusts were composed of necrotic keratinocytes, neutrophils, and red blood cells. Depending on the healing stage, some crusts were found attached to the dermis without any underlying epidermal layers, while fully healed lesions had crusts adhering to the stratum corneum (Figure 5(D-F)). At the site of intradermal inoculation, a significant infiltration of inflammatory cells was noted in the dermis. Positive immunostaining was observed in the skin crusts and the epidermal cells of the infected rabbits (Figure 5(G-I)). The loss of spermatogenic epithelium and spermatocytes of varying degrees was observed in 4 out of 5 rabbits from the Clade Ib infected groups and 3 out of 5 rabbits from the IIb.A.2.1 infected group (Figure 5(J-L)). The testicular tissue showed normal morphology in 2 of the Clade IIb.A.2.1-infected rabbits. Immunostaining was observed in the spermatogonial cells and spermatocytes lining the seminiferous tubules of the MPXV PCR-positive rabbits (Figure 5(M-O)). The lungs and liver sections from all the rabbits did not show any histological changes.

Figure 5.

Gross lesions on the skin, histopathological and immunohistochemical evaluation of skin and testis samples of rabbits infected with MPXV Clade Ib and IIb.A.2.1. erythematous papules of varying size on the dorsum of rabbits after 1-week post-virus infection with A, B) Clade Ib and C) Clade IIb.A.2.1 lineage. D) Skin section showing crust attached to the dermis in a Clade IIb.A.2.1 infected rabbit, H&E, 200 μm. E) Skin section from a Clade IIb.A.2.1 infected rabbit showing inflammatory cell infiltration in the dermis, with engorged blood vessels and thickened epidermal layer with keratinocytes and inflammatory cells forming crusts, H&E, 200 μm. F) Skin section from a Clade Ib infected rabbit showing crusts composed of keratinocytes and inflammatory cells separated from the outer layer of epidermis, H&E, 200 μm. G) Skin section from a Clade Ib infected rabbit showing the immunostaining in the outer layer of skin crust, DAB, 100 μm. H) Skin section from a Clade Ib infected rabbit showing the immunostaining in the epithelial cells of the hair follicles, DAB, 200 μm. I) Skin section from a Clade Ib infected rabbit showing the immunostaining in the cells of the epidermis and the epithelial cells of the hair follicles, DAB, 200 μm. Testis section from the J) mock control rabbit showing the normal arrangement of cells in the seminiferous tubules, H&E, 200 μm, K) Clade Ib infected and L) Clade IIb.A.2.1 infected rabbit showing loss of cells from the seminiferous tubules, H&E, 200 μm. Immunohistochemical staining of testis sections from MPXV-infected rabbits showing M) positive immunostaining of spermatocytes, DAB, 100 μm.N) positive immunostaining of spermatocytes, DAB, 200 μm and O) negative immunostaining of the control section, DAB, 200 μm.

Discussion

We developed a laboratory rabbit model, employing a combined intradermal intravenous inoculation method and conducted a comparative study on the pathogenicity of Clade Ib and Clade IIb.A.2.1 variants of the MPXV. The model showed the development of erythematous skin papules that progressed to dark red papules, followed by crusting and scabbing within two weeks. We successfully isolated the virus from the upper respiratory tract, skin lesions, and testicular tissue. Additionally, the model exhibited similar Mpox disease associated with both the Clade Ib and Clade IIb.A.2.1 variants of the MPXV.

In humans, mpox disease is characterized by fever, rash, and lymphadenopathy. Rare complications, such as pneumonitis, encephalitis, keratitis, and secondary bacterial infections, have also been reported [15,33]. Additionally, involvement of the digestive tract and lungs has been reported in fatal mpox cases among immunocompromised humans [34]. The lesions primarily affect the skin, especially in the perigenital area, in cases of human Clade Ib and IIb.A.2.1 mpox disease [3,7,33,35]. Viremia and virus shedding have been detected in human mpox cases, both through skin lesions and upper respiratory tract swabs [33]. We observed cutaneous rashes, papules, crust formation, and scabbing of lesions in rabbits, consistent with findings from earlier studies in humans, although we did not observe a pustular stage. Notably, we only found perigenital lesions in a single male rabbit. However, we isolated MPXV from the testicular tissue of Clade Ib and IIb.A.2.1 infected rabbits and also antigen localization to the spermatocytes could be demonstrated. MPXV antigen has been detected in the interstitial cells and seminiferous tubules of MPXV-infected macaques [36]. Previous studies in rabbits have shown MPXV spreads through the bloodstream, affecting multiple organs during the acute phase of infection [24]. Other than skin and testes, we could detect MPXV DNA in a few lung, spleen and liver samples in the study. Here, we have terminated the study at the end of the 2nd week of infection, by the time antibody response could already be detected in these rabbits, which could have limited the MPXV detection in different organs. The spleen and liver samples from only Clade Ib (3 out of 5) infected rabbits showed DNA detection, which also had a lower IgG titer when compared to the Clade IIb infected group. The virus isolation could be obtained from samples with even high Ct values up to 37, indicating that PCR threshold values may not correlate with infectiousness. Similar observations were reported from Mpox patients [37].

The susceptibility of rabbit models to the MPXV varies based on the age of the animals, the dose administered, and the route of infection [5,25]. Previous studies have demonstrated this variability. Young rabbits, specifically those 10 days old, were found to be more susceptible, often leading to fatal disease. In contrast, adult rabbits show comparatively lower susceptibility. Hence, we have used sub-adult rabbits of 3–4 months of age for the studies. Among the different routes studied in adult rabbits, only intravenous administration resulted in skin rashes [5,25]. In our study, we observed generalized skin lesions in 3 out of 8 rabbits infected intradermally, contrary to earlier studies in adult rabbits demonstrating dense infiltration and necrosis at the site of inoculation. When the MPXV Copenhagen strain and Clade IIb virus were administered intravenously, the adult rabbits developed generalized disease characterized by skin rashes, conjunctivitis, and weight loss. In the first study, adult Chinchilla rabbits weighing between 2.5 and 3 kg were used. The specific breed of rabbits used in the second study was not mentioned, but the rabbits were 8 months old.

Previous studies have indicated that a high viral dose is required to induce disease in adult rabbits. As we observed that only a few rabbits showed extensive skin lesions by the intradermal route alone, we have opted to use a combination of the intradermal and intravenous routes for infection. The intravenous only group was not included in our study for comparison, as the rabbits used were sub-adults (<4 months of age), in which the administration of larger volumes (>0.2 ml) was found difficult. The intradermal route provides an anatomically favorable site for immune stimulation. Clinical trials with the modified vaccinia Ankara (ACAM3000) have demonstrated that intradermal immunization offers a dose-sparing effect compared to subcutaneous and intramuscular administration [38]. In our observations, the group that received only intradermal injections showed a higher IgG response and fewer rabbits with cutaneous lesions or virus shedding than those in the combined intradermal and intravenous group. This suggests that the intradermal route may lead to a more rapid immune response. In contrast, direct intravenous administration may allow the virus to bypass the host’s immune defenses, potentially facilitating the development of disease.

A previous study conducted in 1976 with the Copenhagen strain on adult rabbits found the virus in the blood, lymph nodes, kidneys, and testicles of some infected animals, even in those showing no other signs of disease [5]. In our study, we also observed one rabbit each from the Clade Ib and IIb.A.2.1 infected groups shedding viral DNA without displaying any observable clinical signs. More recent research using the MPXV Clade IIb C.1.1 virus strain in a rabbit model demonstrated viral replication in the skin, lungs, and testes, leading to severe pathological damage by six days post-infection [25]. In our study, we noted viral shedding through skin lesions, the nasal cavity, and the testes. However, MPXV DNA was only detected in the lungs of two animals. Notably, the lung pathology seen in earlier studies during the first week of infection was absent in our model on day 14. This discrepancy may be attributed to the later termination point of our study or differences in the virus strains used. The detection window for viral DNA in throat swabs was found to be between 2 to 8 days’ post-infection in the same study. Nevertheless, we observed a longer duration of viral DNA presence in nasal swabs and testes within our rabbit model. These findings were consistent across both Clade Ib and Clade IIb.A.2.1 MPXV strains. The variations in virus detection windows in samples across studies could also be attributed to sampling method differences, strain differences and age of the animals used. Future studies should include more contemporary isolates to account for ongoing viral evolution.

A higher virulence of the Clade I virus was demonstrated compared to Clade II using the lethal Cast/EiJ mouse, non-human primates and prairie dog model [27,39–41]. A recent study has reported increased virulence of Clade Ib lineage compared to Clade IIb using the Cast/EiJ mouse model [42]. In the rabbit model, we found no differences in disease manifestation or virulence between the Clade Ib and Clade IIb.A.2.1 variants. More detailed studies will be required to understand the utility of this model for pathogenicity differentiation between different virus strains. Pathogenicity comparison studies with different MPXV strains in rabbit models are not available. The studies in rabbit models itself are very few. Two studies which have used adult rabbit models have used Clade I (Copenhagen strain) and Clade IIb C.1.1. at high virus doses for intravenous administration to develop generalized skin rashes.

In conclusion, we successfully established a rabbit model for Mpox disease, which demonstrated susceptibility to both the MPXV Clade Ib and Clade IIb A.2.1 variant. We found no significant differences in Mpox disease signs, virus shedding, or organ tropism when comparing the two variants in the rabbit model. These observations should be verified with a large sample size study. Additional studies with different doses and multiple time point evaluation of the viral load and pathology in organs will be required to have a deeper understanding of the rabbit model utility in assessing pathogenicity differences of MPXV clades. These findings suggest that this model could be effectively used to evaluate medical countermeasures.

Limitations of the study

The current study has some limitations, such as a small sample size, assessing organ pathology and virus tropism based on a single time point. The study of virus and antibody kinetics will provide a more comprehensive understanding of the tissue predilection, associated pathology and the recovery. To account for genetic evolution, it would be beneficial to include multiple virus isolates in future evaluations of the model.

Funding Statement

This study was supported by the Indian Council of Medical Research as an intramural grant to ICMR-National Institute of Virology, Pune.

Disclosure statement

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

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and the supplementary materials. The sequences of the virus isolates used in the study, as provided in the Methods, are available in the Pathoplexus database (Accession no. PP_004G599.1, PP_004G68A.1). The raw data files are uploaded to the Mendeley Data repository (DOI: https://doi.org/10.17632/8dbypy3p92.1).

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2026.2728484