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. 2026 May 27;16:24120. doi: 10.1038/s41598-026-55484-y

Investigating the emergence of the Brazilian porcupinepox poxvirus in wild Brazilian porcupines (Coendou longicaudatus)

Liz de Albuquerque Cerqueira 1,2, Isabel Luana de Macêdo 1,2, Davi Emanuel Ribeiro de Sousa 1,2, Yasmin Nunes Godoy da Fonseca 1,2, Thiago Marques de Lima 3, Paula Luiza Cezário da Nobrega 3, Sônia Náir Bao 4, Bergmann Morais Ribeiro 5, Valéria Dutra 6, Luciano Nakazato 6, Liria Queiroz Luz Hirano 1,7, Márcio Botelho de Castro 1,2,✉
PMCID: PMC13439539  PMID: 42192143

Abstract

Brazilian porcupinepox virus (BPoPV) has been associated with fatal infections in free-ranging Brazilian porcupines (Coendou longicaudatus), raising concerns regarding its epidemiology and conservation impact. This study comprehensively investigates BPoPV infection, pathology, and mortality in a free-living population from the Federal District, Brazil. Between 2021 and 2024, 30 porcupines submitted for postmortem examination were evaluated using gross and histopathology, PCR, epidermal morphometric analysis, and transmission electron microscopy. BPoPV infection was detected in 70% of animals (21/30), including all ten fatal cases and eleven individuals that died from unrelated traumatic injuries. Six PCR-positive animals without gross lesions showed significant epidermal thickening on microscopy, revealing a previously unrecognized subclinical infection. Fatal cases consistently exhibited severe proliferative and degenerative cutaneous lesions, intracytoplasmic inclusions, marked dermal inflammation, and visceral involvement, particularly interstitial pneumonia, esophageal pustules, and lymphoid depletion, suggesting systemic dissemination. Ultrastructural analysis confirmed abundant poxvirus-like particles within keratinocytes. Traumatic injuries, mainly dog attacks and electrocution, represented the most frequent noninfectious cause of death. These findings demonstrate a high frequency of clinical and subclinical BPoPV infection, identify the virus as a major infectious threat, and provide essential baseline data to support wildlife disease surveillance and conservation strategies for C. longicaudatus.

Keywords: Infectious disease, BPoPV, Spillover, Pathology, Conservation, Environment

Subject terms: Diseases, Medical research, Microbiology, Pathogenesis

Introduction

Infectious disease emergence is frequently driven by multifactorial processes influenced by ecological, genetic, and environmental determinants that allow microorganisms to cross species barriers1. Although small rodents and marsupials are widely recognized reservoirs of zoonotic agents, other arboreal wild mammals, such as porcupines (Coendou spp.), remain underrepresented in surveillance and research2,3.

Poxviruses warrant particular attention in the context of spillover events4. Variola virus, the etiological agent of human smallpox, exemplifies the profound impact that Orthopoxviruses can exert on host populations, having caused substantial global mortality for centuries prior to its eradication in 1980. The biological attributes of Orthopoxviruses, including high genomic plasticity, environmental resilience, and broad host tropism, underscore the importance of wildlife monitoring for the early detection of emerging viral threats5.

In 2021, the first fatal infection associated with the Brazilian porcupinepox virus (BPoPV), a still-unclassified member of the Poxviridae and phylogenetically close to other rodent-associated poxviruses, was reported in a free-ranging Coendou longicaudatus in Minas Gerais, southeastern Brazil6. Since then, additional fatal cases have been described, all exhibiting severe cutaneous proliferative, inflammatory lesions, and cytopathic changes typical of poxviral infections7–9. These lesions share some pathological similarities with those observed in human smallpox caused by the variola virus10,11. Such findings raise concerns regarding the potential role of porcupines as reservoirs or highly susceptible hosts of emerging poxviruses and highlight the possibility of substantial ecological impacts on wild populations.

The Brazilian porcupine (BP) C. longicaudatus, previously named as C. prehensilis12, is an arboreal rodent of the family Erethizontidae, distributed from northwestern South America to Paraguay and Argentina2,13–16. Since 2021, the species has been classified as Near Threatened, primarily due to deforestation, habitat fragmentation, and escalating anthropogenic pressures17. C. longicaudatus commonly inhabits peri-urban and urban areas2, where chronic environmental stressors may impair immunocompetence and increase susceptibility to infectious agents18,19.

Despite these concerns, information on mortality patterns, disease progression, and the epidemiology of BPoPV emergence in BP wild populations remains extremely limited. The present study aims to address these knowledge gaps: characterizing the principal lesions associated with fatal BPoPV infection; determining the frequency of viral infection in the examined population through molecular testing; assessing the frequency of BPoPV infection in animals lacking gross or histologic evidence of poxviral disease (individuals that died from unrelated causes); and evaluating whether asymptomatic infection induces detectable microscopic alterations in the skin.

Additionally, the study includes an investigation of the leading causes of mortality in porcupines submitted to the Veterinary Pathology and Forensic Laboratory (LVPF) of the University of Brasília between January 2021 and December 2024. Collectively, these findings provide essential baseline data to advance understanding of BPoPV epidemiology, pathology, and potential conservation impacts on wild populations of C. longicaudatus.

Results

Epidemiology and spatial distribution

Between January 2021 and December 2024, a total of 30 BP (C. longicaudatus) were submitted to the VPFL-UnB for postmortem examination. All individuals were free-ranging and had been found dead or rescued in urban and peri-urban areas of the Federal District (FD), Midwestern Brazil (Fig. 1). Of these, 10 animals evidenced BPoPV fatal infection (33.4%, n = 10/30), whereas the remaining 20 (66.6%, n = 20/30) died from traumatic conditions (TC). Of the 30 porcupines in this study, 14 were females (46.6%, n = 14/30) and 16 were males (53.3%, n = 16/30). Of the 10 cases of fatal BPoPV infection, 60% were females (n = 6/10). Epidemiological data regarding disease category and sex are presented in Table 1.

Fig. 1.

Fig. 1

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Spatial distribution of collected BP with fatal BPoPV and asymptomatic infection and non-infected animals. CETAS n = 11 animals without geographic location.

Table 1.

Causes of death, sex, and PCR assay results for Brazilian porcupinepox virus (BPoPV) in Coendou longicaudatus from Midwestern Brazil.

Cause of death Nº (%) Sex PCR positivity
Female Male
Infectious disease 10 (33.3%) 6 4 10 (33.3%)
BPoPV 10 (100%) 6 4 10 (100%)
Traumatic condition 20 (66.6%) 8 12 11 (36.6%)
Dog attack 12 (60%) 6 6 5 (25%)
Electrocution 5 (25%) 0 5 5 (25%)
Polytrauma 2 (10%) 1 1 1 (5%)
Traumatic brain injury 1 (5%) 1 0 0
Total 30 14 16 21 (70%)
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Molecular detection of BPoPV infection

PCR analysis using a pan-Chordopoxvirinae assay revealed viral DNA amplification in 70.0% (n = 21/30) of the BP examined, encompassing all 10 cases of fatal BPoPV infection as well as 11 asymptomatic individuals that died from traumatic causes. The assay targeted a 231-bp region of the poxvirus genome, confirming the presence of a poxvirus consistent with BPoPV. The remaining 30.0% (n = 9/30) showed no viral DNA amplification and had no lesions consistent with poxviral infection (Table 1). Annual PCR positivity for BPoPV was 100% in 2021 (n = 1/1, one fatal infection), 100% in 2022 (n = 6/6, four fatal infections), 30% in 2023 (n = 3/10, one fatal infection), and 84.6% in 2024 (n = 11/13, four fatal infections). A comparison of four PCR-positive amplicons shows 100% identity with Porcinepox virus (GenBank ON322740). Phylogenetic analysis of this partial sequence placed the detected virus within the rodent-associated poxvirus clade, which includes BPoPV (Fig. 2), supporting its identification.

Fig. 2.

Fig. 2

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Partial poxvirus protease sequences (180 bp) were aligned using MAFFT v7, and phylogenetic analysis was conducted in MrBayes v3.2 with the GTR+equal rates substitution model. The analysis was run for 100,000 generations with sampling every 500 generations, resulting in 200 posterior samples after discarding the first 25% as burn-in. Convergence was confirmed by inspection of trace plots and PSRF values. The resulting phylogenetic tree was visualized in iTOL, and the corresponding GenBank accession numbers are provided. Sequences generated in this study are indicated in red.

Clinical and pathological findings of BPoPV infection and skin thickness morphometry

The main clinical signs observed in the 10 fatally infected animals with BPoPV were apathy (6/10), followed by dyspnea (5/10), anorexia (3/10), hypothermia (2/10), and dehydration. The clinical findings, as well as the clinical course, are individually detailed in Table 2. Gross examination of porcupines with BPoPV fatal infection revealed marked skin thickening with fissures, erythema, erythematous papules, edema, and erosive areas covered by dry crusts. These lesions predominantly affected the muzzle, periocular region (Fig. 3A), external ears, lips, perianal region (Fig. 3B), and eyelids, occasionally resulting in palpebral fissure occlusion and copious purulent ocular discharge. Similar skin lesions were observed on the lateral and inner surface of the limbs (Fig. 3C) in 30% (n = 3/10) of cases, on the ventral thoracic region (Fig. 3D) in 10% (n = 1/10), and on the tail in 10% (n = 1/10).

Table 2.

Clinical findings in Coendou longicaudatus with fatal BPoPV infection, Midwestern Brazil.

Animal Sex Clinical signs Clinical course
A1 Female Apathy and anorexia. Hyperemic and thickened skin with erosions and a moist appearance on the thoracic and pelvic limbs, muzzle, eyelids, and perianal regions. Papules and purulent ocular discharge 3 days
A2 Female Anorexia. Thickened skin with dry crusts and pustules. Skin of the periocular, frontal, and perineal regions with an irregular surface, along papules and purulent ocular discharge 23 days
A3 Female Dyspnea and right lateral recumbency. Thickened skin with an irregular surface and dry crusts on the muzzle, periocular, and perianal regions, and papules 1 day
A4 Female Thickened and edematous skin with an irregular surface covered by dry crusts in the periocular, periauricular, muzzle, and perineal regions, inner surfaces of thoracic and pelvic limbs, and ventral thoracic region. Papules and purulent ocular discharge 2 days
A5 Female Recumbency and dyspnea. Thickened skin with an irregular surface and moist crusts on the muzzle, periocular, and perianal regions, and papules 2 days
A6 Male Apathy. Thickened skin with an irregular surface on the muzzle, periocular, and perianal regions, and papules Not reported
A7 Male Apathy, hypothermia, and dyspnea. Thickened skin with an irregular surface on the muzzle, periocular, periauricular, and perianal regions 1 day
A8 Female Apathy and dyspnea. Thickened skin with an irregular surface on the muzzle, periocular, tail, and perianal region 10 days
A9 Male Apathy, hypothermia, and dyspnea. Thickened skin with an irregular surface on the muzzle, periocular, periauricular, and perianal regions, and inner surfaces of thoracic and pelvic limbs Not reported
A10 Male Apathy, anorexia, and dehydration. Hyperemic and thickened skin with erosions at the muzzle, eyelids, and perianal region. Papules and purulent ocular discharge 2 days
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Fig. 3.

Fig. 3

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Gross findings in Coendou longicaudatus with BPoPV fatal infections. Marked skin thickening, papules, erythema, and edema. (A) Muzzle. (B) Perianal region. (C) Inner surface of a hind limb. (D) Ventral surface of the thorax.

Internal examination revealed multifocal pustules with mucosal ulceration in the esophagus of one animal (n = 1/10). Pulmonary involvement was observed in 50% of the BPoPV-infected individuals (n = 5/10). Affected BPs presented interstitial pneumonia characterized by diffusely congested, non-collapsed lungs with a rubbery consistency and multifocal alveolar emphysema. No other significant gross pathological abnormalities were identified in the remaining organs and tissues.

Microscopically, the cutaneous lesions were characterized by marked diffuse thickening of the stratum corneum (Fig. 4A), accompanied by the accumulation of nucleated keratin lamellae, consistent with parakeratotic hyperkeratosis. Severe acanthosis with elongation of the rete ridges was evident, reflecting pronounced irregular proliferation of the stratum spinosum and the formation of digitiform projections extending into the dermis, accompanied by pronounced dermal edema (Fig. 4B). Hallmark features of poxviral infection included intense ballooning degeneration of keratinocytes within the spinous layer (Fig. 4C), individual keratinocyte necrosis, and eosinophilic intracytoplasmic viral inclusions of variable size and distribution (Fig. 4D). At the dermo-epidermal junction, hemorrhage was observed, along with a lymphohistiocytic inflammatory infiltrate extending into both the superficial and deep dermal layers, permeating vessels and adnexal structures. Occasional areas of skin ulceration demonstrated bacterial myriads and severe polymorphonuclear inflammation. A summary of the most relevant skin histological findings in BPoPV-infected animals is presented in Table 3.

Fig. 4.

Fig. 4

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Microscopic findings at the skin in Coendou longicaudatus with BPoPV fatal infections. (A) Severe thickening of the stratum corneum (asterisks), muzzle (H&E, 4x objective). (B) Marked epidermal hyperplasia (asterisks) and edema in the dermis (dots), perianal region (H&E, 4x objective). (C) Ballooning degeneration of keratinocytes (arrowheads) with marked thickness of epidermis, muzzle (H&E, 20x objective). (D) Numerous eosinophilic intracytoplasmic poxviral inclusions of variable size (arrowheads), muzzle (H&E, 40x objective).

Table 3.

Most relevant dermatopathological findings in Coendou longicaudatus with BPoPV fatal infection.

Animal Epidermis Dermis
Crust SE PH AC LE BD Espongiosis IC IKN VP BM Edema LHI NI HEM
A1 ++ − +++ +++ ++ +++ +++ + +++ + +++ +++ ++ + +++
A2 +++ + + + ++ ++ + + +++ ++ +++ +++ +++ + −
A3 − − +++ +++ +++ ++ ++ + ++ + ++ ++ ++ + ++
A4 − − +++ +++ +++ + + + + + +++ ++ ++ + ++
A5 + + ++ +++ +++ ++ ++ + ++ ++ +++ +++ ++ + ++
A6 − − + + − + + + + +++ − + + + −
A7 + - + ++ ++ + + + + − +++ ++ + ++ −
A8 ++ - +++ + + ++ + ++ + − + + ++ + +
A9 +++ + +++ + ++ + + + + − +++ +++ + +++ −
A10 − − + +++ + +++ + ++ ++ ++ + + + + −
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Lesions: SE: Skin erosion; PH: parakeratotic hyperkeratosis; AC: acanthosis; LE: leukocyte exocytosis; BD: ballooning degeneration of keratinocytes; IC: intracytoplasmic viral inclusions; IKN: individual keratinocyte necrosis; VP: vesicles and/or pustules; PS: bacterial myriads; LHI: lympho-histiocytic inflammatory infiltrate; NI: neutrophilic inflammatory infiltrate; HEM: hemorrhage.

Intensity of lesions: (−) absent; (+) mild; (++) moderate; (+++) severe. Intracytoplasmic viral inclusions: (-) absent or (+) detected.

Measurements of epidermal thickness across all groups are presented in Fig. 5. Significant differences (p < 0.05) were identified between BPoPV-infected and non-infected C. longicaudatus within the same anatomical region (muzzle or perianal skin), encompassing both the interpapillary and rete ridge epidermal regions (Fig. 6). In asymptomatic C. longicaudatus infected with BPoPV, the observed increase in epidermal thickness was likely associated with subtle acanthosis. No significant differences (p > 0.05) were observed in the epidermal thickness between most of the muzzle and perianal regions in the BPoPV-infected and non-infected BP. However, in animals with fatal BPoPV infection, the perianal rete ridge epidermal region exhibited significantly greater thickness compared with the corresponding region of the muzzle (p < 0.05).

Fig. 5.

Fig. 5

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Thickness (mean ± SD) of interpapillary (green) and rete ridge (blue) epidermis from the muzzle and perianal skin in non-infected (PCR −) and infected (PCR +) Coendou longicaudatus by BPoPV, without skin lesions (WL) and with poxvirus-induced skin lesions (PL). Different lowercases in the same anatomical region (muzzle or perianal) and epidermal area (interpapillary or rete ridges), p < 0.05. Different capital letters, p < 0.05.

Fig. 6.

Fig. 6

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Coendou longicaudatus. Skin. Morphometric analysis of epidermal thickness (bars) (H&E, 20x objective). (A) non-infected (PCR −) animal, exhibiting normal epidermal thickness. (B) BPoPV subclinical infection (PCR +), without gross skin lesions, showing a mild increase in epidermal thickness. (C) BPoPV fatal infection (PCR +), with poxvirus-induced skin lesions, demonstrating a marked increase in epidermal thickness.

One BP with BPoPV-associated lesions exhibited multifocal esophageal pustules and ulceration characterized microscopically by intraepithelial pustulation, epithelial ballooning degeneration, necrosis, and eosinophilic intracytoplasmic viral inclusions, surrounded by neutrophils and cellular debris (Fig. 7A). In addition, severe pulmonary involvement was one of the most impressive systemic manifestations affecting 50% of the fatal cases of BPoPV infection. These animals evidenced moderate to marked interstitial pneumonia, characterized by severe histiocytic and lymphoplasmacytic infiltration and cellular vacuolation within alveolar septa, alveolar collapse, moderate type II pneumocyte hyperplasia, congestion, multifocal alveolar emphysema, and occasional scattered intracytoplasmic eosinophilic poxvirus-like inclusions (Fig. 7B).

Fig. 7.

Fig. 7

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Coendou longicaudatus. BPoPV fatal infection. (A) Esophageal pustule (asterisks) and ulceration (dots) (H&E, 4x objective). Inset: Intracytoplasmic eosinophilic viral inclusions (arrowhead). (B) Marked interstitial pneumonia characterized by severe mononuclear inflammation and cellular vacuolation within alveolar septa (asterisks), and alveolar emphysema (dots) (H&E, 20x objective). Inset: Intracytoplasmic poxvirus-like viral inclusions (arrowhead). (C) Depletion of lymph node lymphoid follicles, characterized by a decreased cellularity and loss of germinal centers (arrowheads) (H&E, 10x objective). (D) Numerous elliptical viral particles characterized by a biconcave core and distinct lateral bodies (arrowheads), ultrastructural features consistent with poxviruses (TEM).

Other visceral alterations observed were splenic congestion in 60% of animals with fatal BPoPV infection (n = 6/10), while 80% (n = 8/10) exhibited mild to moderate depletion of splenic lymphoid follicles. Sinusal histiocytosis was identified in the lymph nodes of 60% of affected animals (n = 6/10), four of which also displayed lymphoid follicle depletion, characterized by decreased cellularity and loss of germinal centers (Fig. 7C). Furthermore, hepatic sinusoidal congestion was observed in 5 of 10 infected individuals.

Ultrastructural BPoPV detection and characterization

Transmission electron microscopy of the skin from BPoPV fatal infections in BP revealed numerous elliptical viral particles measuring approximately 190–300 nm in length, characterized by a biconcave core and distinct lateral bodies, ultrastructural features consistent with poxviruses (Fig. 7D).

Other causes of death of C. longicaudatus

Traumatic injuries were the most frequent cause of death among the porcupines examined in this study (66.6%, n = 20/30). These cases included dog attacks (DA, Fig. 8A) in 60% (n = 12/20), electrocution (Fig. 8B) in 25% (n = 5/20), polytrauma (PL, Fig. 8C) in 10% (n = 2/20), and traumatic brain injury (TBI, Fig. 8D) in 5% (n = 1/20). Macroscopic findings in animals subjected to DA consisted of multifocal puncture-contusion wounds (bite marks), hemorrhage, and lacerations of the skin, musculature, and adjacent soft tissues, primarily affecting the tail, abdomen, and thorax.

Fig. 8.

Fig. 8

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Gross aspect of traumatic injuries in Coendou longicaudatus. (A) Skin hematoma (asterisk) and blunt-perforation lesions (arrowheads), resulting from a dog attack. (B) Severe electrothermal injury with charred tissues at the tail (arrowheads). (C) Rupture of the skin (arrowheads) due to severe traumatic impact in a polytraumatized animal. (D) Traumatic brain injury with multifocal bleeding and a blood clot covering the cerebellum and spinal cord (asterisk).

Electrocution resulted in electrothermal lesions characterized by ulcerated and charred skin, exposure of underlying skeletal muscle and bone, multifocal hemorrhage, necrosis, and, in some cases, amputations involving the tail and both pelvic and thoracic limbs. Gross lesions associated with PL included abdominal abrasions with subcutaneous ecchymoses, multifocal hemorrhage of internal organs, and complete or comminuted fractures. The single TBI case showed a focally extensive hemorrhage within the right ventral brain, extending from the piriform lobe into the cervical spinal cord.

Discussion

The emergence of a new pathogen such as BPoPV demands critical assessment of its potential impact on wild BP populations, drawing on past experiences with high-impact poxvirus outbreaks, such as smallpox, and their devastating effects on human populations. This study reveals a highly concerning, unprecedented, and alarming scenario regarding the BPoPV infection in free-ranging BP populations in the Federal District of Brazil, emphasizing the elevated frequency of asymptomatic or subclinical infections, as well as its role as the leading infectious cause of mortality.

Up to one-third of the animals in this study died from fatal BPoPV infection. Even more concerning, an additional 36.6% of asymptomatic BPs that died from unrelated causes were also infected with BPoPV, resulting in an overall infection rate of 70.0%. Although BPoPV disease has been reported in different regions of Brazil6–9, the frequency of infection in wild BP populations has not been established. In this study, the novel insights generated regarding BPoPV infection and the causes of death in free-living Brazilian porcupines inhabiting anthropogenic environments are subject to inherent limitations. These include a relatively small sample size, which may not be fully representative of populations in undisturbed habitats, the absence of clinical histories or directly observed deaths, and the lack of comprehensive laboratory evaluations for the affected animals.

Despite its limitations, the present study reveals a highly concerning and alarming epidemiological scenario. Although precise frequency and mortality rates cannot be definitively established from the current dataset, the findings strongly suggest that infection levels in the studied population are very high and may reflect the broader situation in free-ranging C. longicaudatus within the region.

Clinical signs observed in affected Brazilian porcupines (BPs) in this study included the characteristic cutaneous lesions associated with BPoPV infection6–9. In addition, nonspecific manifestations such as anorexia, apathy, and dyspnea were documented, and to our knowledge, these have not been previously reported in fatal cases of BPoPV infection. These findings likely reflect generalized debilitation associated with disease progression and pulmonary involvement, which contribute to the clinical presentation in some animals.

Of the 21 PCR-positive animals, 10 BP with BPoPV fatal infections presented gross poxvirus-associated lesions, characterized by marked skin thickening and crust formation at the face, muzzle, mucocutaneous, and perineal regions, as described in other reports6–8. Only one animal in our study had lesions on the tail, similarly observed in a hairy dwarf porcupine (C. spinosus)7. These findings reinforce the head/face and perianal regions as the most grossly affected areas by the viral infection. Gross manifestation in the smallpox virus infection in humans, despite different clinical presentations, likely evidences a centrifugal distribution of skin vesiculopapular and pustular lesions and petechyation, hallmarks of the disease10,11, contrastingly with BPoPV skin lesions in BPs, but similarly, evidence erythematous papules affecting mucocutaneous junctions6–8, as observed in this study.

Esophageal lesions detected in one animal in this survey and not reported in other cases of BPoPV infection7–9 suggest a likely systemic involvement, similar to the gross appearance of smallpox cases in humans, with involvement of the esophagus10,11. Despite being generically described previously in cases of BPoPV infection in BP6,9, this study evidenced severe pulmonary lesions in 50% of fatally infected BP. This finding in fatal cases of BPoPV infection possibly contributed to the clinical deterioration and mortality of animals.

Microscopic examination of the 10 BPs with fatal BPoPV infection confirmed the most severe and characteristic pathological hallmarks of poxviral disease. These included marked epidermal hyperplasia with parakeratotic hyperkeratosis, acanthosis, hydropic degeneration of keratinocytes within the stratum spinosum, and numerous intracytoplasmic eosinophilic inclusion bodies consistent with poxviral replication, as previously described in the species6–9.

Notably, the morphometric analysis of epidermal thickness revealed an unreported increase in epidermal thickness in six randomly selected free-ranging C longicaudatus that tested PCR-positive for BPoPV but were initially considered grossly asymptomatic and died from unrelated causes. Remarkably, these subtle microscopic alterations occurred in the same anatomical regions (muzzle and perianal skin) where the most severe cutaneous lesions were observed in fatal cases. These findings indicate that the six BPs represent subclinical cases of BPoPV infection rather than truly asymptomatic individuals, providing a novel and important contribution to understanding the clinical spectrum and manifestations of this disease.

Poxviral pathogenesis disrupts epidermal turnover and modulates local inflammatory responses, leading to hyperplasia and epidermal thickening even in the absence of overt clinical signs20–22. The epidermal thickening observed in this study likely reflects direct cytopathic effects, including increased keratinocyte proliferation (acanthosis) and architectural disorganization. Given that the progression of BPoPV infection remains poorly characterized, these changes may represent an early stage of disease, preceding the development of the gross lesions typically described in fatal cases. Conversely, considering the high frequency of BPoPV infection detected in this study, yet-undefined environmental and/or immunosuppressive factors in anthropogenic settings may play a critical role in driving progression to clinically apparent disease and influencing case outcomes.

Insights from other poxviruses provide a framework for interpretation. Poxviral skin lesions typically result from intense viral replication, immune evasion, and host inflammatory responses20–23. Growth factor homologs such as EGF-like proteins activate MAPK/ERK signaling and drive epidermal hyperplasia, while anti-apoptotic proteins extend keratinocyte survival and viral replication21,23. Poxviruses also stimulate extracellular matrix deposition and induce inflammation, necrosis, and pustule or ulcer formation20,22. The marked epidermal hyperplasia, cytopathic changes, inclusion bodies, and dermal inflammation in C. longicaudatus6–9 may share substantial mechanistic overlap with other virulent poxviruses, including variola virus, particularly with regard to lesion severity, pathogenic patterns, and associated lethality10,11.

A possible visceral involvement in fatally infected porcupines, including esophageal pustules, severe interstitial pneumonia, splenic congestion with lymphoid depletion, lymph node follicular depletion, and hepatic congestion, demonstrates that BPoPV likely produces systemic disease rather than exclusively cutaneous infection. These lesions possibly contribute significantly to morbidity and mortality. Although interstitial pneumonia has been reported previously in porcupines from Minas Gerais and Mato Grosso, additional visceral lesions had not been documented6,8. As with other poxviruses, primary viremia may disseminate BPoPV to major organs, where extensive replication precedes secondary viremia and the development of both cutaneous and visceral lesions21.

Transmission electron microscopy (TEM) was essential for confirming the infection and for characterizing the ultrastructural features of BPoPV. Examination of infected keratinocytes from affected skin lesions revealed viral particles consistent in size and morphology with members of the Poxviridae family7,9. In addition, phylogenetic analysis further placed the BPoPV detected in this study within a newly proposed genus of the family Poxviridae, subfamily Chordopoxvirinae, the only poxviruses known to infect vertebrates and a group that includes four genera capable of causing disease in humans, three of which have recognized zoonotic potential6,24. BPoPV is closely related to other rodent-associated poxviruses, such as Cotia virus6,9, raising additional concern in light of the increasing interface among humans, domestic animals, and wildlife7.

While the partial protease gene sequence used here confirms BPoPV’s placement within this rodent-associated clade, it provides limited resolution for detailed evolutionary or epidemiological inference. Whole-genome sequences of BPoPV isolates would be necessary for higher-resolution phylogenetic analysis and a better understanding of its genomic plasticity and host-adaptation mechanisms. Poxviruses possess genomic features that facilitate host switching and spillover25, underscoring the importance of continued surveillance and genomic characterization of emerging poxviruses at the wildlife-human interface. Furthermore, the association of visceral lesions with BPoPV infection, while suggestive of systemic dissemination, requires definitive confirmation through complementary techniques such as immunohistochemistry, in situ hybridization, or tissue-specific PCR on affected organs. Future studies incorporating these methods are essential to establish viral tropism and pathogenesis beyond the integumentary system conclusively.

Poxviruses possess genomic features that facilitate host switching and cross-species transmission25,26. The transmission history of other poxviruses, such as Monkeypox virus, illustrates how pathogens initially confined to wildlife populations can adapt to new hosts and, in some cases, precipitate widespread outbreaks27,28. Although the zoonotic potential of BPoPV remains undetermined, its recent characterization underscores the need for further investigation to assess the risk of spillover to other species, including humans. This is particularly relevant given that previous Orthopoxvirus outbreaks have had substantial impacts on both public health and wildlife conservation25.

In addition to fatal BPoPV infections, traumatic causes (TC) constituted the leading mortality factor, accounting for 70% (n = 20/30) of all cases. Among TC, dog attacks (DA) were the predominant cause (60%), followed by electrocutions (25%), with fewer cases attributed to polytrauma (PL; n = 2/20) and traumatic brain injury (TBI; n = 1/20). Given that most free-ranging BPs were found in anthropized environments, particularly urban and peri-urban areas, this high proportion of TC likely reflects the impact of environmental pressures on the species.

Dog attacks and electrocution emerged as major causes of mortality in this study, underscoring the impact of habitat modification and other anthropogenic pressures on arboreal species2,3,29–32. Comparable traumatic injuries have been documented in wild birds in the same region33 and are frequently reported in medium-sized mammals, including BPs in Brazil34, often associated with environmental disturbance and modifications.

Furthermore, 11 of the traumatically injured BPs (55%, n = 11/20) were concurrently infected with BPoPV in subclinical or asymptomatic forms. These concurrent infections may have further contributed to population-level impacts, which likely evidenced the combined influence of infectious disease and anthropogenic threats on the survival and conservation of this species.

Conclusion

Overall, this study reveals an alarming epidemiological scenario for wild Coendou longicaudatus, demonstrating that BPoPV infection is not only widespread but also a major cause of mortality in the Federal District of Brazil. The exceptionally high infection rate, including a substantial proportion of individuals previously classified as asymptomatic, suggests that BPoPV may represent one important emerging pathogen currently affecting Brazilian wildlife. The identification of subclinical infections with subtle yet measurable microscopic lesions indicates that the true disease burden is likely underestimated when relying solely on gross examination, underscoring the urgent need for enhanced diagnostic and surveillance efforts.

Moreover, the convergence of infectious disease with prominent anthropogenic mortality factors, particularly dog attacks and electrocutions, may contribute to local population declines in increasingly urbanized and fragmented landscapes. Insights from historically significant poxvirus outbreaks, such as smallpox, underscore the potential for severe ecological disruption. Mitigating these risks for BP wild populations will require integrated strategies, including systematic wildlife monitoring, expanded genomic surveillance, targeted studies on transmission, public education to reduce dog-wildlife interactions, and urban planning measures to minimize traumatic injuries. Strengthened One Health-oriented policies are also crucial to anticipate impacts on wild populations. Together, these actions are essential to limit the spread of BPoPV, sustain porcupine populations, and prevent broader ecological consequences from this emerging poxvirus.

Methods

Animals, data collection, epidemiology, and spatial distribution

We conducted an archival study using postmortem examination records of Brazilian porcupines (C. longicaudatus) submitted to the Veterinary Pathology and Forensics Laboratory (VPFL) at the College of Agronomy and Veterinary Medicine, University of Brasília, FD, Brazil, between January 2021 and December 2024. In the FD, wild animals found dead, injured, or ill by residents are reported to the Environmental Military Police or the Local Environmental Service, who collect the animals and direct them either for medical treatment or necropsy.

In this study, deceased C. longicaudatus were submitted for postmortem evaluation at VPFL-UnB by the Wildlife Screening and Rehabilitation Center of the Brazilian Institute of Environment and Renewable Natural Resources (CETAS/IBAMA-DF), the Wildlife Hospital of the Brasília Environmental Institute (HFAUS/IBRAM-DF), and the Wildlife Medicine Service (WMS) of the Veterinary Teaching Hospital, University of Brasília. This study used exclusively the archives of C. longicaudatus necropsy records from the VPFL-UnB, including data and samples. Therefore, ethical approval was not required for the study according to the National Council for the Control of Animal Experimentation (CONCEA)36 and ARRIVE guidelines 2.036.

Retrieved data included sex, available clinical history, geographic location, cause of death (fatal BPoPV infection or other causes), and pathological findings. Cases lacking information on the exact location of death or collection but referred by CETAS/IBAMA-DF were mapped to this referral site for spatial analysis. All georeferenced data were processed using QGIS (3.40.7-Bratislava) software.

Clinical and pathological findings of BPoPV infection and skin thickness morphometry

During postmortem examination, tissue samples from all major organs, with special attention to the skin, were collected, fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for histopathological evaluation. Fresh tissue samples from skin and internal organs were also collected and stored at − 80 °C for molecular analyses.

In all BPoPV-infected porcupines, histopathological lesions at the skin were recorded and semi-quantitatively graded as absent (–), mild (+), moderate (++), or severe (+++). Skin samples from the muzzle and perineal regions, sites most frequently affected in previously reported cases6–9, were randomly selected for morphometric analysis from six animals with fatal BPoPV infection and gross and microscopic lesions; six asymptomatic, PCR-positive animals without macroscopic alterations; and six PCR-negative animals that died from unrelated causes and had no cutaneous lesions. Epidermal thickness was measured using a calibrated light microscope (20x objective) equipped with an eyepiece micrometer. Ten non-overlapping microscopic fields were evaluated per sample to measure the thickness (µm) of the interpapillary (IP) and rete ridge (RR) regions in 4 μm H&E-stained skin histological sections. Measurements included the granular, spinous, and basal layers, and the stratum corneum was excluded to avoid artifacts from tissue processing (Fig. 9).

Fig. 9.

Fig. 9

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Diagram illustrating the interpapillary (IP) and rete ridge (RR) regions of the epidermis used for thickness measurements (µm). Abbreviations: epidermis (ep), stratum corneum (sc), dermis (dr), and panniculus (pan).

Molecular detection of BPoPV infection

Molecular detection of BPoPV was conducted using a well-established pan-Chordopoxvirinae PCR assay37 targeting a conserved 231-bp region of the poxvirus genome, which is routinely used for the screening and preliminary identification of poxviruses in clinical and surveillance contexts. DNA was extracted from an individual pool of frozen muzzle and perianal skin samples, using phenol-chloroform protocols38. This approach was selected to maximize sensitivity for detecting cutaneous infection, which is the hallmark of BPoPV disease. Amplicons were purified (Agencourt AMPure XP; Beckman Coulter), sequenced using an ABI Prism 3500 genetic analyzer (Life Technologies, USA). Sequence identity was confirmed by BLAST analysis, and the generated sequence was deposited in GenBank (Accession Number: ON322740).

Ultrastructural BPoPV detection and characterization

For Transmission Electron Microscopy (TEM), skin samples were fixed in Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2) for 2 h, post-fixed in 1% osmium tetroxide for 1 h, stained with 0.5% aqueous uranyl acetate, dehydrated in a graded acetone series (30 to 100%), embedded in Spurr’s resin, sectioned into ultra-thin slices (Leica EM ultramicrotome, Wetzlar, Germany), contrasted with uranyl acetate and lead citrate, and observed in a JEOL 1011 TEM at 80 kV.

Data analysis

Descriptive data were summarized as frequencies of observations and pathological findings. Mean epidermal thickness and standard deviations (SD) were calculated for each animal group, anatomical and epidermal regions. Comparisons among groups were performed using one-way analysis of variance (ANOVA) in GraphPad Prism version 8.0.

Acknowledgements

We thank CAPES, Brazil, for partial financial support (Finance Code 001) in the doctoral scholarship of Liz de Albuquerque Cerqueira, Isabel Luana de Macêdo, Davi Emanuel Ribeiro de Sousa, and Yasmin Nunes Godoy da Fonseca, and to the CNPq for the Research Productivity Grant (PQ).

Author contributions

Conceived the study: LAC, ILM, DERS, YNGF, MBC. Supervised the study: ILM, DERS, MBC. Data acquisition and analysis: LAC, ILM, DERS, YNGF, TML, PLCN, SNB, BMR, VD, LN, LQLH, MBC. Interpretation of data: SNB, BMR, VD, LN, LQLH, MBC. Formal analysis and investigation: LAC, ILM, DERS, YNGF, MBC. Writing original draft: LAC, ILM, DERS, MBC. Writing review and editing: LAC, ILM, DERS, YNGF, TML, PLCN, SNB, BMR, VD, LN, LQLH, MBC. All authors have read and approved the final version of the manuscript.

Funding

The authors declare that no specific funding was received for this study.

Data availability

The data that support the findings of this study are available in GenBank under accession number ON322740. These data were derived from the following resources available in the public domain: GenBank accession ON322740, https://www.ncbi.nlm.nih.gov/nuccore/ON322740.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available in GenBank under accession number ON322740. These data were derived from the following resources available in the public domain: GenBank accession ON322740, https://www.ncbi.nlm.nih.gov/nuccore/ON322740.

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