Bovine Papular Stomatitis Virus

Overview and Taxonomy of Bovine Papular Stomatitis Virus

Bovine papular stomatitis virus (BPSV) is a globally distributed, zoonotic pathogen that represents one of the archetypal members of the genus Parapoxvirus within the subfamily Chordopoxvirinae of the family Poxviridae [4, 18]. The virus is the etiological agent of bovine papular stomatitis (BPS), a generally mild, self-limiting disease characterized by the formation of papules, erosions, and ulcerative lesions predominantly on the mucosal surfaces of the oral cavity, including the lips, tongue, hard palate, and muzzle, as well as occasionally on the teats and udder of adult cattle [1, 11, 14, 21]. The disease holds significant implications for differential diagnosis, as its clinical presentation can be confounded with far more economically devastating vesicular diseases such as foot-and-mouth disease (FMD) and vesicular stomatitis, conditions that are subject to strict regulatory control by the World Organisation for Animal Health (WOAH) [11, 14]. From a public health perspective, BPSV is recognized as a zoonosis, capable of causing localized, nodular skin lesions in humans, often termed “milker’s nodules” when caused by the closely related pseudocowpox virus (PCPV), though BPSV itself has been documented in human infections, particularly among veterinary personnel and farm workers in direct contact with infected animals [4, 20, 22, 23]. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) acknowledge parapoxviruses, including BPSV, as occupational hazards in agricultural and veterinary settings, underscoring the need for enhanced surveillance and diagnostic capacity in both veterinary and human health sectors [22, 23].

Taxonomic Classification and Virion Morphology

At the taxonomic level, BPSV is classified within the genus Parapoxvirus, which is distinguished from other poxvirus genera (e.g., Orthopoxvirus, Capripoxvirus) by a suite of unique morphological, genomic, and biological characteristics [4, 16, 18]. Parapoxvirions are markedly smaller than orthopoxvirions, exhibiting an ovoid or brick-shaped morphology when visualized by negative-stain electron microscopy. Their surface is characterized by a distinctive criss-cross pattern of tubular filaments, a feature that is diagnostic for the genus and readily distinguishes them from the more uniform, globular surface of orthopoxviruses [18]. The viral particle is enveloped and contains a double-stranded DNA genome that is approximately 130–140 kilobase pairs (kbp) in length, a size that is notably smaller and more compact than that of vaccinia virus or variola virus, yet still encodes a suite of genes dedicated to immune evasion, host range determination, and viral replication [15]. The BPSV genome possesses a high G+C content of approximately 63–64%, a feature that is consistent across parapoxviruses and contrasts with the lower G+C content of orthopoxviruses, a metric that has been used in molecular characterization studies to differentiate genera [15, 16]. The genus Parapoxvirus currently comprises four established species: BPSV, pseudocowpox virus (PCPV), orf virus (ORFV), and the tentative species parapoxvirus of red deer and grey seals, with BPSV being the type species associated primarily with bovine hosts [4, 19, 25]. This classification is supported by both serological cross-reactivity patterns and, increasingly, by robust phylogenetic analyses of conserved genomic loci [18, 19].

Genomic Architecture and Genetic Diversity

The genetic architecture of BPSV follows the conserved poxvirus paradigm, with a central core region encoding essential replication and structural proteins flanked by variable terminal regions that house genes involved in host range, virulence, and immunomodulation [15]. The B2L gene (ORF011), which encodes the major envelope protein, homologous to the vaccinia virus p37K protein and the F13L gene product, has been the most extensively utilized molecular target for phylogenetic characterization and diagnostic detection of BPSV [1, 3, 9, 10, 12, 14, 24]. This gene is conserved across the genus Parapoxvirus and allows for robust species-level differentiation, as nucleotide sequence identities between BPSV and ORFV or PCPV at the B2L locus generally fall below 85%, whereas intraspecies variation among BPSV strains is typically 95-99% or higher [10, 12, 24]. However, recent comprehensive analyses have revealed that even within the B2L gene, subtle but consistent genetic clades exist, suggesting that BPSV isolates from disparate geographic regions may be grouped into distinct phylogenetic lineages. For example, phylogenetic analyses of BPSV strains from Iran have demonstrated the existence of at least two distinct clades: one clustering with isolates from Finland, Japan, and Georgia; and another grouping with Zambian isolates, indicating a remarkable degree of genetic variability and a complex epidemiological history that may be linked to transboundary cattle movements [3]. Similarly, studies from Japan have shown that BPSV field strains circulating in Saga prefecture form two major lineages (A-lineage and B-lineage), with one B-lineage subclade exhibiting genetic similarity to strains from the United States, France, and even other prefectures within Japan, providing direct molecular evidence for the introduction of genetically diverse BPSV strains through the movement of live animals [9]. Further evidence of this diversity comes from the analysis of additional genomic loci, such as ORF32 (encoding a putative virulence factor), which has been shown to possess even greater sequence variability than B2L, making it a highly informative target for molecular epidemiological investigations [8]. The full-length sequencing of ORF11 and ORF32 from 11 BPSV strains isolated in Hokkaido, Japan, over a 17-year period (2005–2022) demonstrated that while ORF11 is highly conserved (95-100% amino acid identity), ORF32 exhibits a broader range (88-100%), supporting the utility of ORF32 for differentiating closely related strains and tracking local transmission dynamics [8]. The identification of a specific amino acid deletion in the ORF32 of one Hokkaido strain further underscores the capacity for micro-evolutionary change within geographically restricted viral populations [8].

Biological and Epidemiological Context

BPSV is considered highly prevalent in cattle populations worldwide, with serological surveys and molecular detection studies confirming its presence on all continents where cattle husbandry is practiced, including Asia, Africa, Europe, North and South America, and Australia [1, 4, 10, 14, 20, 21, 23]. The virus is endemic in many regions, causing sporadic outbreaks or enzootic infections, particularly in young calves aged two to eight months, in whom the clinical signs are most pronounced [1, 11, 14]. Notably, subclinical infections are common, and the virus can persist in the environment or within the host, contributing to its maintenance on farms [7, 17]. This persistence is facilitated by the virus’s remarkable stability in dried scabs, which can remain infectious for extended periods, and by its ability to infect and replicate in the oral mucosa of cattle without inducing robust, long-lasting sterilizing immunity [2, 4]. The host range of BPSV is not strictly limited to bovine species; the virus has been detected in other domestic and wild ruminants, including camels in Iran and India [13, 26], and has been reported in reindeer in Norway, although in the latter case, phylogenetic analysis suggested a closer relationship to ORFV [25]. Furthermore, BPSV DNA has been identified in ticks infesting cattle in Burkina Faso, suggesting a potential, albeit likely mechanical, role for arthropod vectors in viral transmission, particularly in regions where transhumance and shared grazing lands are common [5]. More recently, the common housefly (Musca domestica) has been implicated as a mechanical vector, with BPSV and other parapoxviruses detected on the body surface and in the feces of flies collected from cattle and sheep farms, indicating that these insects may contribute to within-herd dissemination even in the absence of direct animal-to-animal contact [17]. The ability of BPSV to cause co-infections with other pathogens is also clinically significant; concurrent infections with rotavirus and Cryptosporidium spp. in neonatal calves, or sequential infections with pseudocowpox virus in the same animal, have been documented, complicating both clinical diagnosis and disease progression [6, 7]. As a vaccine vector, BPSV has garnered considerable attention in recent years due to its ability to accommodate large foreign DNA inserts, its capacity for limited replication in the face of pre-existing immunity, and its potential to circulate within herds, thereby enhancing vaccine coverage [2]. Recombinant BPSV vectors expressing glycoproteins of bovine herpesvirus 1 (BoHV-1) have demonstrated the ability to induce neutralizing antibodies and provide significant protection against challenge in serologically positive calves, highlighting the potential of BPSV as a platform for developing next-generation cattle vaccines against economically important respiratory and reproductive pathogens [2].

Molecular Pathogenesis of Bovine Papular Stomatitis Virus

The molecular pathogenesis of Bovine Papular Stomatitis Virus (BPSV) represents a sophisticated interplay between a relatively benign, yet highly successful, viral pathogen and the host immune system. As a member of the Parapoxvirus genus within the Poxviridae family, BPSV possesses a large, double-stranded DNA genome that encodes an arsenal of virulence factors designed to subvert host immune responses, facilitate viral replication, and ensure persistence within cattle populations. Understanding these molecular mechanisms is critical, not only for elucidating the pathophysiology of BPS lesions but also for contextualizing its role as a zoonotic agent and its emerging utility as a viral vector for cattle vaccines [2, 27]. The virus induces self-limited, proliferative lesions of the oral mucosa, muzzle, and teats, yet its ability to evade clearance and circulate asymptomatically underscores the exquisite adaptation of its molecular machinery.

Genomic Architecture and Key Virulence Determinants

The BPSV genome, approximately 134–140 kbp in length, exhibits a high G+C content (~63%) and is organized with a central coding region flanked by inverted terminal repeats, a hallmark of poxviruses [15]. Comparative genomics have highlighted that interspecies sequence variability within the Parapoxvirus genus is most pronounced in genes encoding putative virulence and host-range factors, which are often located in the terminal regions of the genome [15]. This genomic plasticity underpins the differences in host tropism observed between BPSV, Orf virus (ORFV), and Pseudocowpox virus (PCPV), despite their morphological and antigenic similarities.

Central to the molecular pathogenesis of BPSV is its capacity to produce a suite of immunomodulatory proteins that are non-essential for viral replication in vitro but are critical for survival in vivo. The virus encodes a number of these factors, including a chemokine-binding protein (CBP) and a viral homologue of interleukin-10 (vIL-10), both of which serve as primary molecular weapons against the host innate and adaptive immune responses [27, 31]. The major envelope protein, B2L (encoded by ORF011), is a key immunogen and the primary target for diagnostic PCR and phylogenetic analyses [1, 9, 10, 24]. However, it is the accessory genes that orchestrate the pathogenic process. For instance, the gene encoding a double-stranded RNA binding protein (RBP) is present in BPSV, which is a common poxviral strategy to counteract the host’s interferon-mediated antiviral response by sequestering dsRNA and preventing the activation of protein kinase R (PKR) and other sensors [26]. Sequence analysis of this RBP gene from various parapoxviruses shows significant divergence, with BPSV sharing only 52.8% amino acid identity with camel PPV isolates, suggesting host-specific adaptation of this immune evasion tactic [26].

Immunomodulation: The Chemokine-Binding Protein (CBP) and vIL-10

The most extensively characterized virulence factor of BPSV is its broad-spectrum chemokine-binding protein (BPSV-CBP). Chemokines are small chemoattractant cytokines that orchestrate the recruitment of leukocytes (neutrophils, monocytes, lymphocytes) to sites of infection. BPSV-CBP acts as a molecular sponge, binding with high affinity to chemokines across multiple subfamilies, including CXC, CC, and XC chemokines, thereby preventing them from interacting with their cognate G-protein-coupled receptors on immune cells [27]. This strategy effectively blinds the host immune system to the site of viral replication. Specifically, surface plasmon resonance studies have revealed that BPSV-CBP potently binds to CXCL1, CXCL2, CCL2, CCL3, and CCL5 [27]. These chemokines are critical for the recruitment of neutrophils and inflammatory monocytes, the first responders to epithelial damage and viral infection. By inhibiting their function, BPSV-CBP blocks neutrophil and monocyte chemotaxis, as demonstrated by transwell migration assays [27].

The in vivo relevance of this mechanism is profound. Intradermal injection of purified BPSV-CBP in murine skin models was shown to significantly delay the influx of neutrophils and reduce the recruitment of MHC-II+ antigen-presenting cells to wound beds and LPS-inflamed tissue [27]. This effectively establishes a “chemokine gradient shadow,” creating an immunological sanctuary where the virus can replicate unimpeded by the early host inflammatory response. The structure of BPSV-CBP, predicted to be a homodimeric polypeptide of 82.4 kDa, is conserved among parapoxviruses, yet its binding profile appears particularly adapted to the chemokine milieu of bovine skin and mucosal tissues [27].

Complementing the action of CBP, BPSV encodes a homologue of the pleiotropic cytokine interleukin-10 (BPSV vIL-10). IL-10 is a master regulator of inflammation, possessing both potent anti-inflammatory and immunostimulatory properties. The viral vIL-10s of parapoxviruses have evolved to exploit these pathways to the advantage of the virus. BPSV vIL-10 exhibits functional equivalence to ORFV IL-10, demonstrating robust binding to the IL-10 receptor 1 (IL-10R1) [31]. This receptor engagement leads to the suppression of pro-inflammatory cytokines. In assays using lipoteichoic acid-activated THP-1 monocytes, BPSV vIL-10 significantly inhibited the production of monocyte chemoattractant protein-1 (MCP-1/CCL2), interleukin-8 (IL-8), and interleukin-1β (IL-1β) [31]. By dampening this inflammatory cascade, BPSV vIL-10 not only reduces the recruitment of phagocytes (synergizing with the CBP) but also diminishes the activation state of residual immune cells, creating a local microenvironment that favors viral persistence over clearance. Furthermore, BPSV vIL-10 retains the ability to stimulate mast cell proliferation, a function of cellular IL-10 that may contribute to the proliferative, papular nature of the lesions [31]. The combined action of CBP and vIL-10 represents a two-pronged attack on the innate immune system: one blocking cell recruitment and the other suppressing the activation of those cells that do arrive.

Molecular Mechanisms of Persistence and Transmission

The molecular pathogenesis of BPSV also extends to its ability to persist within herds and be transmitted via indirect vectors. The virus is known to be highly stable in the environment, particularly within scabs and desiccated lesions, which is a feature conferred by its robust viral envelope and the genetic stability of core structural proteins like B2L [11, 14]. Phylogenetic analyses consistently reveal that BPSV strains from disparate geographical regions (e.g., Taiwan, Iran, Japan, Brazil, Bangladesh) often cluster closely based on partial B2L sequences, indicating a high degree of conservation in this gene and suggesting that the virus has a relatively stable core genome despite geographic isolation [1, 3, 9, 10, 23]. For instance, BPSV isolates from Saga, Japan, were divided into two distinct lineages (A and B), with some strains showing close homology to isolates from the USA and France, indicating that the molecular epidemiology of the virus is driven more by animal movement and trade than by rapid, host-driven genetic drift [9].

Crucially, BPSV has been shown to persist on the body surface of asymptomatic animals and within barn environments, including water troughs and feed bunks [7, 17]. This environmental persistence is a direct result of the virus’s molecular resilience. The detection of BPSV DNA in houseflies (Musca domestica) collected from farms, with viral sequences identical to those found on cattle and in barn environments, provides strong molecular evidence for mechanical transmission by arthropod vectors [17]. The virus can be carried on the exoskeleton or within the feces of flies, offering a mechanism for rapid, indirect spread within a herd that bypasses the need for direct contact with lesions [17]. This mode of transmission is particularly relevant given the detection of BPSV in ticks infesting cattle in Burkina Faso, where BPSV-specific DNA was amplified from tick pools (5.8% of pools positive), highlighting a potential vector-borne component to its lifecycle in regions with high tick burdens [5]. This tick-associated transmission could have important implications for transboundary cattle movements and the spread of the virus across herds sharing water and pasture resources [5]. The World Organisation for Animal Health (WOAH) recognizes the economic importance of such endemic viruses, and understanding the molecular basis of their environmental stability is key to devising effective biosecurity protocols.

Zoonotic Potential and Cross-Species Molecular Pathogenesis

While BPSV primarily affects cattle, its molecular pathogenesis enables cross-species transmission to humans, causing a zoonotic infection known as milker’s nodule (paravaccinia) [20-23]. The molecular determinants of this zoonotic spillover are not fully elucidated, but the ability of the virus to bind and enter human keratinocytes and fibroblasts is likely dependent on the expression of conserved host cell surface receptors. The pathogenesis in humans mirrors that in cattle, with the development of self-limiting, proliferative nodules, typically on the hands and arms of farm workers [20]. The viral immune evasion strategies (CBP and vIL-10) are thought to be just as effective in the human host, allowing the virus to replicate for several weeks before being cleared by an adaptive immune response.

Serological evidence and molecular detection have confirmed zoonotic BPSV infections in countries including Bangladesh, Georgia, Brazil, and the United States [20, 22, 23]. The detection of BPSV alongside other zoonotic parapoxviruses like ORFV and PCPV in human samples in Georgia underscores the risk posed by these viruses to agricultural workers [22]. The U.S. Centers for Disease Control and Prevention (CDC) lists parapoxviruses as zoonotic pathogens associated with occupational exposure to livestock. The molecular similarity of the immunomodulatory proteins (CBP, vIL-10, RBP) across parapoxvirus species explains why these infections share a common clinical presentation and why serological cross-reactivity is high, complicating differential diagnosis [18, 27, 31]. The ability to cause co-infections in cattle, such as the concurrent occurrence of BPSV, Rotavirus, and Cryptosporidium spp. in a single calf, and the sequential detection of BPSV and PCPV in the same animal, further complicates the clinical and molecular picture, highlighting the need for specific PCR-based diagnostics to differentiate the molecular drivers of each infection [6, 7].

Molecular Basis of Co-infection and Differential Diagnosis

The clinical presentation of BPSV can be strikingly similar to that of other vesicular diseases, most critically Foot-and-Mouth Disease (FMD) and Lumpy Skin Disease (LSD) [11, 28]. The molecular pathogenesis of BPSV is distinct from these viruses, yet the lesions, papules, ulcers, erosions, are macroscopic correlates of a shared pathophysiological endpoint: keratinocyte proliferation and necrosis driven by viral replication and the host inflammatory response. High-resolution melting (HRM) assays have been developed to simultaneously detect and differentiate BPSV from other poxviruses and capripoxviruses, using differences in GC content and amplicon size of genus-specific PCR products [16]. The ability to detect a BPSV-positive animal presenting with clinical signs typical of LSD in Tunisia highlights the importance of molecular surveillance in regions where multiple poxviruses are endemic [16, 28]. In such settings, a cattle presenting with nodular skin lesions may be suffering from LSDV or BPSV infection, and only molecular testing (qPCR, PCR) targeting specific genes (e.g., B2L for BPSV, P32 for LSDV) can provide an accurate diagnosis [28-30].

Furthermore, co-infection of BPSV with other bovine viral pathogens can modify its molecular pathogenesis. The presence of BPSV in a calf co-infected with Bovine Viral Diarrhea Virus (BVDV) or Bovine Herpesvirus-1 (BoHV-1) could lead to more severe or protracted lesions due to the additive or synergistic immunosuppressive effects [32]. The use of BPSV as a vaccine vector [2] is predicated on the molecular understanding that it induces only a mild, subclinical infection and a low-level of antiviral immunity, allowing for the stable expression of foreign antigens (e.g., BoHV-1 glycoproteins gD and gB) [2]. The ability of BPSV to accommodate large inserts of foreign DNA and to induce a protective immune response in serologically positive calves demonstrates a sophisticated molecular understanding that is now being harnessed for biotechnology [2]. The genetic diversity observed in ORF32, which shows greater variability than the conserved ORF11 (B2L), provides a molecular handle for tracking the spread of vaccine strains versus field strains in the future [8].

Epidemiology and Global Distribution of Bovine Papular Stomatitis Virus

Bovine papular stomatitis virus (BPSV) is a member of the genus Parapoxvirus within the family Poxviridae, and it represents one of the most widely distributed viral pathogens affecting cattle populations globally. Despite its generally mild, self-limiting clinical presentation, the virus is of substantial epidemiological significance due to its high prevalence, ability to persist within herds, zoonotic potential, and capacity for subclinical circulation that complicates surveillance and control efforts. The global distribution of BPSV, as documented through serological, molecular, and phylogenetic investigations over the past several decades, reveals a pathogen that is truly cosmopolitan in nature, though with distinct patterns of genetic diversity and transmission dynamics that vary by region, husbandry practices, and ecological context.

Global Geographic Distribution and Endemicity

BPSV has been identified on every continent where cattle are raised, with confirmed reports spanning North America, South America, Europe, Asia, Africa, and Oceania. The virus is considered endemic in most cattle-producing regions, though the reported prevalence varies considerably depending upon the diagnostic methods employed, the age structure of the sampled populations, and the clinical surveillance intensity in a given area. The datasheet compiled by the Centre for Agriculture and Bioscience International (CABI) provides an authoritative overview of the virus's identity, host range, and distribution, confirming its presence across diverse climatic and ecological zones [4]. This near-ubiquitous distribution is attributable to several biological features of BPSV, including its environmental stability, multiple routes of transmission, and the capacity to infect both clinically affected and apparently healthy animals.

In Asia, BPSV has been documented from Japan to Iran, with significant molecular characterization efforts elucidating the genetic relationships among circulating strains. Japan, in particular, has been a focal point for BPSV research, with numerous studies detailing the virus's presence across the archipelago. A comprehensive analysis of BPSV and pseudocowpox virus (PCPV) strains circulating in Hokkaido, Japan's largest cattle-producing region, examined isolates collected between 2005 and 2022, providing a longitudinal perspective on viral persistence and genetic stability [8]. This study revealed that ORF11, a gene encoding a major envelope protein, is highly conserved among Japanese BPSV strains, whereas ORF32 exhibits substantial genetic diversity, making it a suitable target for molecular epidemiological investigations. Notably, the Hokkaido strains formed a single cluster in ORF11-based phylogenetic analysis, with one isolate being distinct, suggesting the co-circulation of multiple lineages within a single geographic region over an extended period.

Similarly, in Saga Prefecture in southwestern Japan, phylogenetic analysis of six BPSV field strains detected from beef calves between 2017 and 2020 revealed the existence of two distinct lineages [9]. One lineage (A-lineage) clustered with strains from various regions of Japan and the world, while the other (B-lineage) comprised five Saga strains that grouped with isolates from France, the United States, and Iwate Prefecture in northern Japan. The presence of a sub-lineage branching from B-lineage, designated BPSV_SAGAbv2, was found to be related to strains from the United States and Iwate Prefecture. The investigators attributed this genetic heterogeneity to the history of calf introduction from various regions of Japan into Saga Prefecture, illustrating how animal movement and trade networks directly shape the molecular epidemiology of BPSV at the local level. This pattern of multiple co-circulating lineages is not unique to Japan; rather, it appears to be a consistent feature of BPSV epidemiology worldwide.

In Iran, the first molecular characterization and phylogenetic analysis of BPSV in beef calves was conducted in West Azerbaijan province, where outbreaks of papular lesions were investigated across four herds [3]. Fifty swab samples collected from lesions on the muzzle, lips, and oral cavity of affected calves all tested positive for BPSV by polymerase chain reaction (PCR). Phylogenetic analysis based on the partial B2L gene sequences revealed that the Iranian isolates formed two distinct clades. Clade 1 exhibited similarities to isolates from Finland, Japan, and Georgia, while Clade 2 was similar to Zambian isolates. This finding underscores the genetic diversity within the virus population and suggests that BPSV strains circulating in Iran may have originated from multiple introductions, potentially through livestock trade or transboundary animal movements. The presence of strains related to both European/Asian and African lineages within a single country highlights the complex interplay of factors that govern BPSV distribution.

Transmission Dynamics and Vector Involvement

The transmission of BPSV has traditionally been attributed to direct contact between infected and susceptible animals, as well as indirect transmission through contaminated fomites, feed troughs, and water sources. However, recent research has substantially expanded our understanding of the potential vectors and environmental reservoirs that contribute to the maintenance and spread of the virus within and between herds. One of the most significant advances in this regard has been the demonstration of mechanical transmission of parapoxviruses, including BPSV, by houseflies (Musca domestica) on cattle and sheep farms [17]. In this study, BPSV, PCPV, and orf virus were detected in the oral cavity and on the body surface of cattle and sheep that showed no clinical signs of infection, as well as in barn environments. Critically, PPV DNA was also detected in the direct wash solution of the body surface of houseflies and in the indirect wash solution of the body surface and feces of the flies. The viral sequences obtained from the flies were identical to those found on the body surface of cattle and in barn environments, providing strong evidence that houseflies can mechanically transmit BPSV. This finding has profound epidemiological implications, as flies are ubiquitous on livestock operations, are highly mobile, and can rapidly disseminate the virus across entire herds and between neighboring farms. The role of insects as mechanical vectors may explain why BPSV can appear suddenly in herds with no known direct contact with infected animals and why control measures focused solely on direct contact may be insufficient.

In addition to insects, ticks have been implicated as potential vectors for BPSV in certain ecological contexts. A study conducted in Burkina Faso, West Africa, screened ticks collected from cattle in three provinces for the presence of arboviruses and other pathogens [5]. Among the 663 ticks collected, representing four genera and six species, BPSV was detected in 5.8% of the tick pools. The BPSV-positive ticks were found in herds that shared water and pasture resources and had a history of seasonal transhumance, the practice of moving livestock over long distances in search of grazing and water. This study highlights the potential for ticks to serve as mechanical vectors for BPSV and underscores the role of traditional livestock management practices in facilitating the spread of the virus. Transhumance, in particular, brings cattle from multiple herds into close contact at shared resources, creating ideal conditions for virus transmission. The authors of this study noted that common grazing and seasonal transhumance are likely to support the transmission of BPSV, with important health and economic impacts, especially regarding transboundary cattle movements.

The ability of BPSV to persist in the environment and on fomites further complicates epidemiological control. The virus is a double-stranded DNA virus with a lipid envelope, but like other poxviruses, it exhibits considerable resistance to desiccation and can remain infectious for extended periods in dried scabs and crusts. This environmental stability, combined with the potential for mechanical transmission by insects and the movement of asymptomatically infected animals, creates a transmission network that is difficult to interrupt.

Subclinical Infection and Persistent Circulation

One of the most challenging aspects of BPSV epidemiology is the high prevalence of subclinical infections, which allows the virus to circulate undetected within herds and complicates efforts to estimate true prevalence and implement control measures. The study in Saga, Japan, explicitly noted that BPSV was detected in calves without intraoral clinical signs, demonstrating that the virus can be shed by animals that appear healthy [9]. This phenomenon was also observed in Hokkaido, where BPSV was detected in environmental samples as well as in calves without clinical signs, while PCPV was detected in a calf showing anorexia, frothy salivation, and erosion in the mucosa of the lip and tongue [7]. Remarkably, in this same calf, BPSV was detected 22 days after the initial PCPV detection, suggesting sequential infection with two different parapoxviruses. This sequential detection implies that prior infection with one parapoxvirus species does not necessarily confer cross-protection against another, and that individual animals can serve as hosts for multiple parapoxviruses over time.

The longevity of BPSV infection in individual animals is also noteworthy. The outbreak investigation in Taiwan in August 2023, which affected five of eleven 2–4-month-old dairy calves, found that all positive calves remained positive for BPSV one month after the outbreak began, with no decrease in viral loads over this period [1]. This sustained viral shedding, even in the absence of clinical progression, suggests that BPSV can establish persistent infections in which the virus continues to replicate and be shed for weeks or months. Such persistent shedding has important implications for herd-level transmission dynamics, as infected calves can serve as a continuous source of virus for naïve contacts.

Coinfection and Differential Diagnosis in the Field

The clinical presentation of BPSV, papules, vesicles, erosions, and ulcers on the muzzle, lips, oral mucosa, and sometimes the teats and udder, is not pathognomonic and can be confused with a range of other viral and non-viral conditions. This diagnostic challenge is of critical epidemiological importance because misdiagnosis can lead to inappropriate control measures, failure to detect emerging pathogens, and underestimation of BPSV prevalence. The differential diagnosis for BPSV includes foot-and-mouth disease (FMD), vesicular stomatitis, bovine viral diarrhea (BVD), malignant catarrhal fever, bovine herpesvirus type 2 (BHV-2) infections, and other parapoxviruses such as pseudocowpox virus [11, 14]. In Brazil, an outbreak of vesicular disease in Nova Brasilândia do Oeste county in Rondônia state initially prompted suspicion of FMD or vesicular stomatitis, but laboratory testing ultimately revealed BPSV in one herd and PCPV in three others [14]. The clinical course in these animals was approximately 7 to 10 days, and the lesions were predominantly in the oral cavity, muzzle, and skin. This case illustrates the critical importance of molecular diagnostics in distinguishing between vesicular diseases, particularly in regions where FMD is a notifiable disease with severe trade implications.

Coinfections involving BPSV and other pathogens further complicate the epidemiological picture. A case report from Turkey documented the concurrent occurrence of bovine papular stomatitis, rotavirus infection, and cryptosporidiosis in a 7-day-old calf from a farm containing 65 calves of different ages [6]. The calf presented with multifocal papular stomatitis and rumenitis at necropsy, and PCR analysis confirmed the presence of both BPSV and rotavirus, while tests for BVD, FMD, bovine papilloma virus, and coronavirus were negative. This was the first reported case of this particular coinfection combination, and it raises questions about the potential synergistic effects of these pathogens. Rotavirus and Cryptosporidium are well-known causes of neonatal diarrhea in calves, and the immunosuppressive effects of a concurrent viral infection like BPSV could exacerbate the severity of enteric disease. Conversely, the stress and mucosal damage caused by enteric infections may predispose calves to BPSV infection.

In Iran, another coinfection scenario was reported involving BPSV and lumpy skin disease virus (LSDV) in Tunisia [28]. During surveillance for LSDV in Tunisian cattle, a high-resolution melting (HRM) assay identified a positive BPSV animal that was also presenting LSDV clinical signs. This finding underscores the reality that in regions where multiple poxviruses are endemic, individual animals may be infected with more than one poxvirus simultaneously, with overlapping clinical presentations that can confound diagnosis.

Zoonotic Implications and Human Epidemiology

BPSV is a recognized zoonotic pathogen, and human infections, though generally mild and self-limiting, are of public health significance and contribute to the epidemiological importance of the virus. Human infections typically occur through direct contact with infected cattle or contaminated fomites, and the clinical presentation in humans is characterized by papular or nodular lesions on the hands, arms, and face, often referred to as milker's nodules when caused by PCPV or BPSV. The zoonotic risk is particularly high for individuals in close occupational contact with cattle, including farmers, milkers, veterinarians, and slaughterhouse workers. A study conducted in Georgia retrospectively screened an archived collection of anthrax-negative human DNA samples for poxviruses, and of 148 samples tested, 64 were positive [22]. Sequence analysis confirmed the presence of orf virus, BPSV, and pseudocowpox virus, indicating that these zoonotic infections have been occurring at higher rates than previously recognized in the country. The study emphasized that poxvirus-associated infections share some clinical manifestations and exposure risks with anthrax, which is also endemic in Georgia, making accurate laboratory differentiation essential for appropriate case management and public health response.

The detection of BPSV in human infections has also been documented in Bangladesh, where investigators collected diagnostic specimens from dairy cattle and buffalo with symptoms consistent with poxvirus-associated infections across three districts [23]. BPSV DNA was obtained from lesion material (teat) and an oral swab collected from an adult cow and calf, respectively, from a dairy production farm in Siranjganj. This represented the first detection of zoonotic poxviruses from Bangladesh and provided phylogenetic comparisons between the Bangladesh viruses and reference strains. The study highlighted that understanding the range and diversity of different species and strains of parapoxvirus is essential to identifying unusual patterns of occurrence that could signal events of significance to both agricultural and public health sectors.

In Brazil, a retrospective study of poxvirus infections in cattle from Goiás State between 2010 and 2018 found that zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, including BPSV, affecting especially the hands of milkers and other farm workers [20]. Similarly, in the Distrito Federal region of Brazil, a single human case was observed associated with a BPSV infection, further underscoring the zoonotic risk [21]. These findings indicate that BPSV should be considered in the differential diagnosis of vesicular or papular skin lesions in individuals with occupational exposure to cattle, and that occupational health education and protective measures are warranted in endemic areas.

The World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) recognize parapoxviruses, including BPSV, as occupational zoonoses, and the global distribution of BPSV means that human cases can potentially occur wherever cattle are raised. However, the true incidence of human BPSV infection is likely underreported due to the mild and self-limiting nature of the disease, the lack of specific diagnostic testing in many regions, and the fact that many cases are likely treated empirically without laboratory confirmation.

Regional Variations in Prevalence and Genetic Diversity

The prevalence of BPSV varies markedly by region, reflecting differences in cattle population density, management practices, climate, and surveillance intensity. In Japan, BPSV appears to be highly endemic, with numerous studies documenting its presence across the country. The study in Hokkaido detected BPSV in 11 strains isolated over a 17-year period, and the study in Saga found it in six field strains over four years [8, 9]. In Iwate Prefecture, BPSV was detected in seven of eight calves from eight farms between April and September 2010, with one of these strains representing a new BPSV variant with genetic variability in the envelope gene [24]. These data suggest a high prevalence of BPSV in Japanese cattle, at least at the herd level, and point to the existence of genetically diverse strains circulating simultaneously.

In South Korea, an outbreak of parapox-like symptoms was reported in April 2012, affecting three of 45 Korean native cattle aged 20–24 months [10]. Phylogenetic analysis revealed that the Korean strains were closely related to isolates from Japan, Germany, Sudan, and the United States. This outbreak was notable for two reasons: first, it involved adult cattle rather than the more typically affected calves, and second, it represented the first molecular characterization of BPSV in Korea. The genetic similarity to strains from diverse geographic regions suggests that international movement of cattle or contaminated products may play a role in the introduction of new BPSV variants.

In the Americas, BPSV has been documented in both North and South America. The United States has a long history of BPSV detection, and strains from the US have been used as reference sequences in numerous phylogenetic studies. In Brazil, BPSV is widely distributed, with studies from Goiás State, Distrito Federal, and Rondônia State all confirming its presence [14, 20, 21]. The retrospective study in Goiás identified five outbreaks of BPSV among 25 confirmed poxvirus outbreaks between 2010 and 2018, while the study in Distrito Federal identified eight BPSV cases, five coinfections with PCPV, and three unidentified parapoxviruses among 52 confirmed poxvirus cases [20, 21]. The coinfection of BPSV and PCPV in the same animal is noteworthy and suggests that multiple parapoxviruses can circulate simultaneously within the same herd and even within the same animal.

In Africa, BPSV has been detected in several countries, though the data are more limited than for other regions. The detection of BPSV in ticks in Burkina Faso provided evidence of the virus's presence in West Africa, and the study noted that the positive ticks were found in herds with a history of transhumance, suggesting that this traditional management practice facilitates the spread of BPSV across the region [5]. The phylogenetic analysis of Iranian BPSV strains revealed a clade related to Zambian isolates, further confirming the presence of BPSV in southern Africa and suggesting genetic links between strains from Iran and Africa [3].

In Europe, BPSV has been reported in multiple countries, including France, Germany, Finland, and Georgia. The Finnish isolates have been used as reference strains in phylogenetic studies, and the Georgian isolates have been characterized from both human and animal samples [3, 22]. The presence of BPSV in Georgia is particularly relevant given the country's location at the crossroads of Europe and Asia and its role in regional livestock trade.

Molecular Epidemiology and Phylogenetic Insights

The molecular epidemiological investigation of BPSV has relied heavily on sequencing of the B2L gene, which encodes the major envelope protein, and more recently on additional genomic targets such as ORF11 and ORF32. These analyses have revealed that BPSV exhibits substantial genetic diversity, with strains clustering into multiple lineages that often correlate with geographic origin but also show evidence of long-distance dissemination. The B2L gene is relatively conserved among parapoxviruses but

Clinical Manifestations and Pathological Features in Cattle

Bovine papular stomatitis virus (BPSV) infection in cattle presents a spectrum of clinical and pathological findings that range from subclinical carriage to overt, multifocal oral disease. The manifestations are profoundly influenced by the age and immune status of the host, the viral strain involved, and the presence of concurrent infections or environmental stressors. A thorough understanding of these features is essential for accurate diagnosis, differential exclusion of economically critical vesicular diseases such as foot-and-mouth disease (FMD) and vesicular stomatitis, and for appreciating the virus's complex host-pathogen interactions.

Clinical Manifestations in Calves

The most conspicuous and well-characterized clinical presentations of BPSV occur in young calves, typically between two and seven months of age. Outbreaks often manifest as multiple erosive papules and ulcers localized on the lips, muzzle, nostrils, and oral cavity, including the tongue and hard palate [1, 11]. In a documented outbreak in Taiwan, five of eleven 2–4-month-old dairy calves exhibited these characteristic lesions, with all affected animals remaining PCR-positive for BPSV one month post-outbreak, and viral loads failing to decline during this period [1]. This persistence suggests that while lesions may resolve clinically, viral shedding can be prolonged, a factor critical for within-herd transmission dynamics.

The initial lesions are often described as erythematous papules that progress to vesicles, pustules, and ultimately to circumscribed ulcers and erosions [11, 14]. Affected calves may present with excessive salivation (ptyalism) and frothy discharge from the mouth, sometimes accompanied by anorexia due to the pain associated with oral ulceration [7, 11]. In severe cases, the infection can extend to the rumen, where multifocal papular rumenitis has been observed on postmortem examination [6]. The clinical course in uncomplicated cases is typically self-limiting, with lesions resolving over a period of 7 to 10 days [14]. However, the healing process may be prolonged in the presence of secondary bacterial infections or concurrent viral or parasitic diseases.

A particularly critical diagnostic challenge arises because BPSV lesions can be strikingly similar to those of FMD, a WOAH-listed disease of immense economic consequence. A reported case in Japanese Black calves initially suspected of having FMD demonstrated excessive salivation, erosion of the hard palate, and ulcers on the tongue, clinical signs that are hallmark indicators of FMD [11]. Only after FMD was ruled out via epidemiological assessment and negative laboratory testing was BPSV confirmed by PCR and seroconversion. This highlights the absolute necessity for molecular differentiation in any outbreak of vesicular or erosive oral disease in cattle, especially in regions where FMD is endemic or has been recently eradicated.

Clinical Manifestations in Adult Cattle

While BPSV is most commonly associated with calves, infection in adult cattle is well-documented, albeit with a different topographic distribution of lesions. In adult dairy cows and beef cattle, the virus frequently targets the teats and udder, where it produces papules, vesicles, ulcers, scabs, and scars [20, 21]. These lesions are often clinically indistinguishable from those caused by pseudocowpox virus (PCPV) and vaccinia virus (VACV), the latter being a zoonotic concern associated with bovine vaccinia in Brazil and other regions [20]. The presence of teat lesions can lead to secondary mastitis and interfere with milking, causing economic losses through reduced milk production and increased labor for udder health management.

The occurrence of BPSV in adult cattle within a herd can often be traced back to the presence of infected calves serving as a reservoir. Molecular surveys have detected BPSV in the oral cavity and on the body surface of cattle without any clinical signs, confirming the existence of subclinical carriers that contribute to the silent maintenance of the virus on farms [7, 17]. This subclinical carrier state is a crucial epidemiological feature, as it allows the virus to circulate undetected and be mechanically transmitted by vectors such as houseflies (Musca domestica), which have been shown to carry BPSV on their body surfaces and in their feces [17].

Gross Pathological Features

On gross pathological examination, the lesions of BPSV are consistently proliferative and exudative at different stages of development. In the oral cavity, the earliest gross changes include focal areas of erythema and swelling, which rapidly evolve into elevated papules measuring 2–10 mm in diameter. The center of these papules often becomes necrotic, leading to the formation of a shallow ulcer with a raised, hyperemic border. In some cases, the ulcers coalesce to form larger, irregular areas of erosion, particularly on the hard palate and dorsal surface of the tongue [11].

In the rumen, a frequently overlooked site of pathology, gross examination reveals multifocal papules that may be hemorrhagic or necrotic. A postmortem study of a 7-day-old calf co-infected with BPSV, rotavirus, and Cryptosporidium spp. revealed severe multifocal papular stomatitis and rumenitis, with no evidence of other viral pathogens such as bovine viral diarrhea virus or bovine papillomavirus [6]. The concurrent presence of enteric pathogens likely exacerbated the severity of the BPSV lesions, suggesting that BPSV infection may act synergistically with other pathogens to produce more severe gastrointestinal pathology.

Microscopic Pathological Features

The histopathological changes associated with BPSV are characteristic of parapoxvirus infections and are remarkably consistent regardless of the specific poxvirus species involved. A comprehensive retrospective study of poxvirus infections in cattle from Distrito Federal, Brazil, provided detailed histological descriptions of BPSV lesions [21]. The predominant findings include mild to moderate, superficial, multifocal inflammatory infiltrates composed of lymphocytes, plasma cells, macrophages, and neutrophils. The overlying epithelium exhibits acanthosis (thickening of the stratum spinosum) and parakeratotic hyperkeratosis (retention of nuclei in the stratum corneum), often associated with serous exudate and cellular debris. Spongiosis (intercellular edema within the epidermis) is a frequent feature.

In ulcerated lesions, focally extensive areas of necrosis are evident, with a severe neutrophilic infiltrate in the adjacent connective tissue [21]. A key diagnostic hallmark is the presence of 4–8 μm eosinophilic inclusion bodies within the cytoplasm of keratinocytes. In the Brazilian study, these inclusions were observed in a subset of cases, including those with BPSV infection, both alone and in coinfection with PCPV [21]. These inclusions represent viral factories and are a useful, though not pathognomonic, indicator of parapoxvirus infection. The dermal changes include congestion, edema, and a mixed perivascular inflammatory response.

Immune Evasion and Pathogenesis

The clinical and pathological features of BPSV infection are a direct reflection of the sophisticated immune evasion strategies employed by the virus. BPSV encodes a distinctive chemokine-binding protein (CBP) that binds with high affinity to a broad range of chemokines within the CXC, CC, and XC subfamilies [27]. In murine models, intradermal injection of BPSV-CBP potently blocked the influx of neutrophils and monocytes at sites of inflammation and injury, and reduced the recruitment of MHC-II+ immune cells to the wound bed [27]. The functional consequence is a delay in the innate immune response, allowing the virus to establish infection and replicate before immune effector cells can infiltrate the lesion. This mechanism explains the slow progression and prolonged persistence of BPSV lesions, and the tendency for the virus to remain localized to the epithelial surface without inducing a robust, rapid inflammatory response.

Additionally, BPSV encodes a viral homologue of interleukin-10 (IL-10), a pleiotropic immunomodulatory cytokine. The BPSV IL-10 has been shown to inhibit production of monocyte chemoattractant protein (MCP)-1, IL-8, and IL-1β, while also inducing mast cell proliferation and binding the IL-10 receptor 1 with similar potency to Orf virus IL-10 [31]. By suppressing the production of pro-inflammatory chemokines and cytokines at the site of infection, the BPSV IL-10 further dampens the recruitment and activation of immune cells, creating an environment permissive for viral replication and spread.

The combined effect of these immunomodulatory proteins, the CBP and the viral IL-10, is a localized state of relative immunosuppression within the infected epithelium. This enables BPSV to cause persistent, mild infections that may not elicit strong systemic immune responses, thereby facilitating the establishment of subclinical carrier states and repeated infections within a herd. The virus's ability to repeatedly infect serologically positive animals, as demonstrated in vaccine vector studies [2], is a testament to the efficacy of these immune evasion mechanisms.

Differential Diagnosis and Coinfections

The clinical and pathological presentation of BPSV must be meticulously differentiated from other causes of vesicular, erosive, and proliferative disease in cattle. FMD is the primary differential, given its similar oral lesions and the potential for rapid spread. Vesicular stomatitis and bovine viral diarrhea (BVD) can also present with oral erosions. Furthermore, other parapoxviruses, particularly pseudocowpox virus (PCPV), produce lesions that are clinically and histologically indistinguishable from BPSV, necessitating molecular confirmation via PCR and sequencing for definitive diagnosis [16, 21].

Coinfections with BPSV and other pathogens are increasingly recognized and can modify the clinical picture. The sequential detection of PCPV followed by BPSV in the same calf, as reported in Japan, illustrates that animals can be infected with multiple parapoxvirus species over time, potentially complicating serological and molecular diagnostics [7]. More severe clinical outcomes are associated with coinfections involving enteric pathogens such as rotavirus and Cryptosporidium spp., where the combined insult of oral pain, enteric inflammation, and dehydration can lead to death [6]. Similarly, the differentiation of BPSV from lumpy skin disease virus (LSDV) is crucial in regions where both viruses co-circulate; a case in Tunisia initially suspected as LSD was later confirmed as BPSV by high-resolution melting (HRM) analysis [28]. These findings underscore the importance of a comprehensive diagnostic approach that includes testing for multiple potential etiological agents.

Molecular Diagnostics and Phylogenetic Analysis of Bovine Papular Stomatitis Virus

The accurate and expeditious identification of Bovine Papular Stomatitis Virus (BPSV) is paramount, not only for differential diagnosis from clinically indistinguishable vesicular diseases of profound economic and zoonotic importance, such as Foot-and-Mouth Disease (FMD), Vesicular Stomatitis (VS), and Lumpy Skin Disease (LSD), but also for elucidating the global molecular epidemiology and evolutionary dynamics of this ubiquitous parapoxvirus. The diagnostic landscape for BPSV has evolved from classical virological methods to a sophisticated armamentarium of molecular techniques that afford unparalleled sensitivity, specificity, and throughput. Coupled with robust phylogenetic frameworks, these tools have illuminated the genetic architecture of BPSV, revealing complex patterns of lineage diversification, geographic structuring, and cross-species transmission that have direct implications for veterinary diagnostics, vaccine development, and the management of zoonotic risk.

Molecular Diagnostic Approaches: From Conventional PCR to High-Resolution Differentiation

The cornerstone of modern BPSV diagnostics is the polymerase chain reaction (PCR), which has largely supplanted electron microscopy and virus isolation for routine detection due to its superior sensitivity and rapid turnaround time. Conventional and real-time quantitative PCR (qPCR) assays targeting highly conserved genomic regions are the workhorses of clinical and surveillance programs. The B2L gene, which encodes a major envelope protein, is the most frequently employed molecular target for pan-parapoxvirus detection. This locus is sufficiently conserved within the genus to permit amplification across BPSV, Pseudocowpox Virus (PCPV), and Orf Virus (ORFV), yet contains enough sequence divergence to allow for species-level discrimination through subsequent sequencing or restriction fragment length polymorphism (RFLP) analysis [3, 10, 24]. For instance, the application of qPCR targeting the B2L region was instrumental in confirming the first molecular characterization of BPSV in Taiwan in 2023, where five of eleven affected calves tested positive. Notably, the study demonstrated that viral loads did not diminish over a one-month period, suggesting persistent viral shedding or prolonged active infection in young animals [1]. This observation underscores the utility of qPCR not merely as a binary detection tool but as a quantitative instrument capable of elucidating the temporal dynamics of infection.

The differential diagnosis of parapoxviruses from other poxviruses and vesicular disease agents is a critical challenge in the field. In many outbreak scenarios, BPSV lesions are clinically indistinguishable from those caused by LSDV, Capripoxviruses, or even Vaccinia virus (VACV). To address this, sophisticated multiplex molecular assays have been developed. A landmark study by Gelaye et al. (2017) introduced a high-resolution melting (HRM) assay capable of simultaneously detecting and differentiating eight poxviruses from three genera: Orthopoxvirus (cowpox virus, camelpox virus), Capripoxvirus (sheeppox virus, goatpox virus, LSDV), and Parapoxvirus (ORFV, PCPV, BPSV) [16]. The assay leverages genus-specific primers and the differential melting temperatures of amplicons, which are a function of their GC content and fragment size. In a validation study involving 271 poxviral DNA samples, the HRM assay correctly identified two BPSV-positive samples, demonstrating its utility as a cost-effective, rapid, and highly specific screening tool for reference laboratories. This technology is particularly valuable in regions like Africa and the Middle East, where multiple poxviruses co-circulate and resources for sequencing may be limited [16]. Furthermore, the development of isothermal amplification methods, such as recombinase polymerase amplification (RPA), has expanded diagnostic capabilities to point-of-care settings. An RPA assay developed for LSDV showed 100% clinical sensitivity and specificity against qPCR, and critically, did not cross-react with BPSV or ORFV DNA, confirming its utility for differential diagnosis at the farm level or in quarantine stations [29].

The scope of molecular diagnostics has also been extended to non-traditional sample matrices and vectors, providing novel insights into the ecology of BPSV. Parapoxvirus DNA, including BPSV, has been detected in tick pools collected from cattle in Burkina Faso, with 5.8% of pools testing positive [5]. This finding suggests that ticks may serve as mechanical vectors or environmental reservoirs for the virus, facilitating transmission across herds sharing water and pasture resources, particularly during seasonal transhumance. Similarly, the detection of BPSV, PCPV, and ORFV from the body surface and feces of houseflies (Musca domestica) on cattle and sheep farms implicates dipterans in the mechanical transmission of the virus [17]. These studies highlight the expanding role of molecular screening in understanding the complex transmission pathways of BPSV. Finally, molecular diagnostics have proven essential in recognizing co-infections. A postmortem investigation of a diarrheic calf in Turkey utilized PCR to confirm a triple co-infection of BPSV, Rotavirus, and Cryptosporidium spp. [6]. This case underscores the principle that BPSV, while often considered a self-limiting pathogen, can be a component of complex polymicrobial disease processes, particularly in neonates with immature immune systems.

Phylogenetic Analysis: Unraveling Genetic Diversity and Global Circulation

Phylogenetic analysis of BPSV isolates, predominantly based on the B2L gene but increasingly on other genomic loci such as ORF011 and ORF032, has provided a high-resolution view of the virus's genetic diversity, phylogeography, and evolutionary relationships within the Parapoxvirus genus. The B2L gene, while highly conserved, exhibits sufficient polymorphism to delineate major clades and to connect field isolates to global reference strains. This approach has been instrumental in characterizing the first BPSV strains from several countries. For example, the partial B2L sequence of the 2023 Taiwanese isolate clustered closely with strains from Japan, the United States, and France, indicating a potential international source for the introduction [1]. In Iran, the first molecular characterization of BPSV from beef calves revealed two distinct clades: one resembling isolates from Finland, Japan, and Georgia, and another clustering with Zambian strains [3]. This bifurcation suggests multiple, potentially independent, introductions of BPSV into the Iranian cattle population, likely linked to animal trade or transboundary movements.

The utility of multi-locus sequence analysis for high-resolution phylogenetics has been powerfully demonstrated in studies from Japan, a country with a long history of BPSV research. A comprehensive analysis of 11 BPSV strains isolated in Hokkaido between 2005 and 2022 compared sequences of ORF011 (homologous to B2L) and ORF032 (which encodes a putative virulence factor). While ORF011 was highly conserved (95–100% amino acid identity), ORF032 exhibited significantly greater diversity (88–100% identity), including an amino acid deletion in one strain [8]. This study reported the first ORF032 sequences from Japanese parapoxviruses and demonstrated that this locus is more suitable for fine-scale molecular epidemiological tracking. Phylogenetic analysis based on ORF032 revealed that most Hokkaido strains formed a single cluster, distinct from a single outlier strain that shared greater similarity with viruses from other Japanese prefectures. This suggests that while a dominant lineage circulates within Hokkaido, there has been occasional incursion of distinct genotypes, likely via animal movement. This pattern of co-circulating lineages is further supported by studies from Saga Prefecture, where six BPSV field strains from a single farm were divided into two distinct lineages: an "A-lineage" consisting of a strain related to global isolates, and a "B-lineage" composed of five strains that clustered with isolates from France, the USA, and the Iwate prefecture in northern Japan [9]. The authors hypothesized that this diversity was correlated with a history of calf introductions from multiple regions, effectively seeding the local viral population with distinct genotypes.

The genetic relationships between BPSV and other parapoxviruses are complex and underscore the concept of a viral species complex. Phylogenetic analyses consistently demonstrate that BPSV, PCPV, and ORFV form distinct, well-supported clades, yet they share a common ancestor and exhibit evidence of interspecies recombination in certain genomic regions. Studies on seal parapoxvirus have shown that pinniped isolates form a separate, deeply rooted cluster distinct from BPSV, PCPV, and ORFV, supporting their classification as a novel species within the genus [19]. However, BPSV sequences from camels in Iran and India have shown variable phylogenetic placement; some camel isolates cluster closely with BPSV reference strains, while others are more allied with PCPV or ORFV depending on the genetic locus analyzed [13, 26]. This phylogenetic promiscuity complicates taxonomic classification and may reflect host-switching events or the persistence of ancestral polymorphisms. Furthermore, the detection of BPSV and PCPV sequentially in the same calf over a 22-day period on a Japanese farm, as demonstrated by Matsumoto et al. (2019), indicates that co-circulation and sequential infection by different parapoxvirus species within the same individual are possible, blurring the lines of host specificity at the individual animal level [7]. The global distribution of BPSV genotypes, with close genetic links between strains from disparate continents (e.g., Asia, North America, Europe, and Africa [1, 10]), points to the role of international livestock trade in the homogenization of the global BPSV population. Understanding this genetic architecture is not merely an academic exercise. It is foundational for designing broadly protective vaccines, especially given the interest in using BPSV as a viral vector for cattle vaccines [2]. The stability of the ORF011 locus and the variability of ORF032 will inform the selection of antigen-insertion sites and the monitoring of vaccine virus stability during its intended circulation in cattle populations.

Vaccine Vector Potential and Recombinant BPSV Applications

The Rationale for BPSV as a Vaccine Vector in Cattle

The development of safe, efficacious, and economically viable viral-vectored vaccines for cattle remains a significant unmet need in veterinary medicine. While numerous viral vector platforms have been explored in laboratory and companion animal species, no such vectored vaccines are commercially available for cattle, despite compelling applications for controlling endemic and economically devastating pathogens [2]. Bovine papular stomatitis virus (BPSV) has emerged as a uniquely promising candidate to fill this void, owing to a constellation of virological, epidemiological, and immunological attributes that distinguish it from other poxvirus vectors. Unlike many viral vectors that induce robust antiviral immunity that precludes effective boosting, BPSV is a highly prevalent parapoxvirus that causes self-limited, mild oral lesions in cattle and is capable of inducing only low levels of antiviral immunity upon natural infection, thereby permitting repeated administration of recombinant constructs [2]. This characteristic is fundamentally tied to the sophisticated immune evasion machinery encoded within the BPSV genome, which includes a broad-spectrum chemokine-binding protein (CBP) and a viral interleukin-10 (vIL-10) homologue [27, 31]. The BPSV-CBP functions as a homodimeric polypeptide of 82.4 kDa that binds with high affinity to a wide range of inflammatory chemokines spanning the CXC, CC, and XC subfamilies, thereby potently inhibiting neutrophil and monocyte chemotaxis toward sites of infection [27]. In murine models, intradermal injection of recombinant BPSV-CBP effectively blocked the influx of neutrophils and monocytes into lipopolysaccharide-inflamed skin and, critically, delayed neutrophil recruitment and reduced MHC-II+ cell infiltration into wound beds that recapitulate the microenvironment of BPSV lesions [27]. Furthermore, the BPSV vIL-10 homologue exerts potent anti-inflammatory effects on activated monocytes, inhibiting production of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and interleukin-1β (IL-1β), while simultaneously retaining the capacity to stimulate mast cell proliferation, an activity profile that parallels the immunomodulatory potency of the Orf virus IL-10 and the cellular IL-10 itself [31]. These immunomodulatory functions are not incidental; they represent strategic adaptations that enable BPSV to establish infection, replicate locally, and persist within host populations while evoking minimal inflammatory pathology and neutralizing antibody responses.

Recombinant BPSV Construction and Proof-of-Concept Immunization

The seminal demonstration of BPSV’s vector potential was achieved through the construction of recombinant viruses expressing glycoproteins from bovine herpesvirus 1 (BoHV-1), a ubiquitous and economically significant respiratory pathogen of cattle [2]. Using the BPSV strain BV-AR02 as the backbone, two recombinant constructs were generated: BPSVgD, expressing the BoHV-1 glycoprotein gD alone, and BPSVgD/gB, a bicistronic construct co-expressing both gD and gB. The successful construction of these recombinants substantiates the capacity of the BPSV genome to accommodate and stably express large foreign genetic payloads, a property that has been inferred from comparative genomic analyses of parapoxviruses [15]. Notably, the BPSV genome, like that of Orf virus, encodes approximately 130 proteins from a double-stranded DNA genome of roughly 140 kb, and interspecies sequence variability is particularly pronounced in putative virulence and host-range genes, suggesting that specific genomic regions can tolerate substantial genetic manipulation without compromising vector viability [15]. When BPSV serologically positive calves, animals that had prior natural exposure to wild-type BPSV and thus harbored pre-existing anti-vector immunity, were immunized intramuscularly with either BPSVgD or BPSVgD/gB, a single dose induced measurable BoHV-1 neutralizing antibodies by day 28 post-vaccination [2]. This is a critical finding, as naturally occurring BPSV infections are endemic in cattle populations worldwide, with seroprevalence rates that are substantial in many regions [1, 8-10]. The ability of the BPSV vector to overcome pre-existing immunity is likely attributable to the very immune evasion mechanisms that characterize its natural biology; the low-grade, localized nature of BPSV infection, combined with the active suppression of chemokine-mediated inflammatory recruitment, may permit vector replication and transgene expression even in the face of circulating anti-BPSV antibodies [2, 27].

Challenge Protection and the Implications of Partial Immunity

The ultimate test of any vaccine vector is its capacity to confer protection against virulent pathogen challenge. Following high-dose BoHV-1 challenge at 70 days post-immunization, three of four vaccinated calves were protected, demonstrating that the BPSV-vectored constructs elicited functionally relevant immunity [2]. This level of protection, while not complete, is particularly noteworthy given the challenge model employed: BoHV-1 is a potent immunosuppressive virus that actively subverts host antiviral responses, and the challenge dose was likely selected to assess the robustness of the vaccine-elicited response under stringent conditions [2, 32]. The partial protection observed may reflect the need for optimization of transgene expression levels, immunization route, or the inclusion of additional BoHV-1 antigens. The fact that the bicistronic construct expressing both gD and gB did not demonstrably outperform the gD-only construct in this initial study does not negate the potential utility of multivalent BPSV vectors; rather, it underscores the complexity of immune correlates of protection against alphaherpesviruses and the necessity for iterative refinement of recombinant design [2]. The BPSV system offers considerable flexibility in this regard. The ability to insert multiple foreign genes, potentially under the control of different poxviral promoters, opens avenues for constructing polyvalent vectors targeting multiple bovine pathogens simultaneously, an approach that would be particularly valuable in the management of the bovine respiratory disease complex, wherein coinfections with viruses such as BoHV-1, bovine viral diarrhea virus (BVDV), and bovine respiratory syncytial virus (BRSV) are common and synergistically pathogenic [2, 6, 32].

Safety Considerations and Zoonotic Risk Assessment

Any discussion of viral vectors must rigorously address safety, particularly for agents that are zoonotic. BPSV is a recognized zoonotic parapoxvirus that causes self-limited, papular lesions on the hands and fingers of individuals in direct contact with infected cattle, typically milkers and farm workers [20-23]. The zoonotic potential of recombinant BPSV constructs therefore warrants careful risk assessment. However, the naturally restricted host range and mild clinical phenotype of BPSV are advantageous for vector safety. In contrast to vaccinia virus-based vectors, which carry inherent risks of disseminated infection in immunocompromised hosts and can cause significant adverse events, BPSV infections in humans and cattle are uniformly self-limited, resolving without specific therapy within a matter of weeks [2, 22]. Moreover, the BPSV vector platform is attenuated by virtue of its natural biology; it replicates primarily in keratinocytes and mucosal epithelial cells, induces only pustular lesions that are typically confined to the oral cavity or teats, and does not cause systemic disease in immunocompetent hosts [2, 4, 20]. The recombinant constructs described by Delhon et al. were generated from the BV-AR02 strain, which was isolated from a naturally occurring case and has been propagated in cell culture, further attenuating any residual virulence [2]. From a regulatory standpoint, BPSV-based vectors would likely be classified under the same framework as other parapoxvirus vaccines, such as the Orf virus vaccines used in sheep and goats, which have a favorable safety record in livestock. The ability to distinguish vaccinated animals from naturally infected animals through the presence of unique transgene sequences represents an additional advantage for serosurveillance and disease control programs.

The Role of Pre-Existing Immunity and Population Dynamics

The prevalence of BPSV in cattle populations is a double-edged sword for vector design. On one hand, the widespread circulation of BPSV, documented in North America, Europe, Asia, Africa, and Australia, means that a large proportion of target animals will have pre-existing anti-vector immunity prior to vaccination [1, 3, 8-10, 14, 20, 21, 23, 24]. The demonstration that BPSVgD and BPSVgD/gB could induce BoHV-1 antibodies in seropositive calves is therefore a landmark finding, as it directly addresses the most common reason for failure of poxvirus-based vectors in livestock: vector neutralization by pre-existing antibodies [2]. The mechanism that permits this circumvention of pre-existing immunity is likely multifactorial. The BPSV CBP and vIL-10 may suppress the recall of anti-vector B and T cell responses at the site of inoculation, while the inherently low immunogenicity of the BPSV virion itself may reduce the efficiency of antibody-mediated neutralization [2, 27, 31]. Sequential detection studies of BPSV and pseudocowpox virus (PCPV) in the same calves on the same farm provide epidemiological evidence that these viruses can coinfect and sequentially infect animals without eliciting cross-protective immunity that completely blocks reinfection [7]. This observation aligns with the known biology of parapoxviruses, which have evolved to circulate within herds for extended periods, with the same animals experiencing multiple episodes of infection over their lifetime [1, 7, 11]. For vector design, this suggests that BPSV may be uniquely suited for heterologous prime-boost regimens, where animals are primed with a BPSV-based vector encoding one set of antigens and boosted with a heterologous vector (such as an adenovirus or modified vaccinia Ankara) expressing the same antigens, thus avoiding vector-specific immune responses.

The Potential for Multivalent and Chimeric Constructs

The genomic flexibility of BPSV, combined with its capacity to infect not only cattle but also potentially other ruminant species, positions it as a platform for developing multivalent vaccines targeting multiple pathogens. BPSV has been detected in a wide array of hosts beyond cattle, including domestic buffalo, camels, and pinnipeds, albeit with significant genetic divergence between terrestrial and marine parapoxviruses [19, 23, 25, 26]. The presence of BPSV in ticks infesting cattle in Burkina Faso raises intriguing questions about the potential for vector-borne dissemination of recombinant constructs, though such transmission would likely be inefficient and could be mitigated through recombinant design that eliminates genes necessary for arthropod-associated spread [5]. Comparative genomics of parapoxviruses has revealed that interspecies sequence variability is highest in putative virulence and host-range genes, indicating that these regions are hotspots for genetic exchange and could be readily engineered to alter host tropism or immune evasion profiles [15]. The successful construction of a bicistronic BPSV vector co-expressing two BoHV-1 glycoproteins demonstrates the feasibility of packaging multiple transgenes, and future designs could incorporate immunomodulatory genes from other poxviruses, for example, the interleukin-10 homologues or chemokine-binding proteins, to fine-tune the immune response to the vectored antigen [2, 27, 31]. The high-resolution melting (HRM) assays and recombinase polymerase amplification (RPA) assays developed for rapid detection of BPSV and other poxviruses could be adapted for monitoring the environmental persistence and potential shedding of recombinant constructs, an important consideration for environmental risk assessment [16, 29].

Translational Challenges and Future Directions

Despite the compelling proof-of-concept study, several translational hurdles must be addressed before BPSV-based vectors can achieve commercial viability. The precise molecular determinants of BPSV attenuation and host range remain incompletely defined. While full genome sequences are available for reference strains, the functional annotation of many open reading frames lags behind that of vaccinia virus and Orf virus [15]. The identification of the BPSV CBP and vIL-10 as key immunomodulatory proteins provides a foundation for rational attenuation, but a more comprehensive understanding of the BPSV-host interaction at the molecular level is necessary to design vectors that retain replicative capacity in vivo without causing clinical lesions [27, 31]. The route of administration is another critical variable. The initial study employed intramuscular injection, which bypasses the natural mucosal infection route of BPSV [2]. However, the natural tropism of BPSV for oral and nasal mucosal surfaces suggests that intranasal or oral administration, routes that are logistically simpler for mass vaccination of cattle, may enhance transgene expression and immune induction at mucosal surfaces, which are the primary portals of entry for many bovine respiratory and enteric pathogens [1, 4, 6]. The ability of BPSV to persist in the environment and potentially to be mechanically transmitted by houseflies (Musca domestica) is a biosafety consideration that requires careful evaluation in the context of recombinant vector release [17]. Phylogenetic analyses of BPSV strains from diverse geographic regions have revealed substantial genetic diversity, particularly in the ORF32 gene, which encodes the major envelope protein and is a target for molecular epidemiology [8, 24]. This diversity must be taken into account when selecting the vector backbone, as different BPSV strains may exhibit variable immunogenicity, tissue tropism, or susceptibility to pre-existing immunity in different cattle populations. The field of parapoxvirus-based vector development is still in its infancy, but the unique immunobiological properties of BPSV, its capacity for immune evasion, its limited pathogenicity, and its ability to circumvent pre-existing vector immunity, constitute a foundation upon which a new generation of cattle vaccines can be built.

Genetic Diversity and Evolutionary Relationships of BPSV Strains

The genetic landscape of Bovine Papular Stomatitis Virus (BPSV) is characterized by a complex mosaic of conserved core elements and hypervariable regions that reflect both host adaptation and geographic segregation. As a member of the Parapoxvirus genus within the Poxviridae family, BPSV shares fundamental genomic architecture with other parapoxviruses, yet exhibits distinctive patterns of diversity that inform our understanding of its transmission dynamics, host range, and evolutionary trajectory. The analysis of BPSV genetic diversity is not merely an academic exercise, it has profound implications for vaccine development, diagnostic assay design, and the surveillance of zoonotic potential across livestock populations globally.

The B2L Gene as a Molecular Epidemiological Cornerstone

The major envelope protein gene, B2L (also designated ORF011 in the parapoxvirus nomenclature), has emerged as the principal target for phylogenetic characterization of BPSV strains worldwide. This locus encodes a 42-kDa protein that constitutes the primary structural component of the viral envelope and is immunodominant, making it both diagnostically accessible and evolutionarily informative [3]. The partial B2L sequence of 554 nucleotides, as employed in numerous studies, provides sufficient resolution to distinguish BPSV from other parapoxviruses (orf virus, pseudocowpox virus) while revealing intraspecific relationships that map to geographic origins [9].

Phylogenetic analyses of B2L sequences have consistently demonstrated that BPSV strains segregate into distinct clades that correlate with geographic provenance, though with notable exceptions that reflect animal movement and trade patterns. The pioneering work in Japan established that BPSV isolates from different prefectures could be assigned to at least two major lineages, designated A-lineage and B-lineage, with evidence of sub-lineage branching [9]. In Saga Prefecture, southwestern Japan, six field strains collected between 2017 and 2020 were distributed across both lineages: one strain clustered with global isolates in the A-lineage, while five strains formed a distinct B-lineage along with strains from France, the United States, and Iwate Prefecture in northern Japan [9]. This bifurcation is compelling evidence that the introduction of calves from diverse geographic origins into Saga farms has resulted in the co-circulation of genetically distinct BPSV lineages within a single geographic region, a phenomenon with direct implications for recombination potential and vaccine design.

Geographic Structuring and Unexpected Cosmopolitanism

The B2L-based phylogeny reveals a complex interplay between geographic isolation and long-distance viral dissemination. In Iran, the first molecular characterization of BPSV identified two distinct clades among isolates from beef calves in West Azerbaijan province: Clade 1 grouped with isolates from Finland, Japan, and Georgia, while Clade 2 aligned with Zambian isolates [3]. This pattern suggests that BPSV strains circulating in the Middle East have been shaped by both European and African introductions, likely mediated by historical livestock trade routes and contemporary transboundary animal movements.

The Taiwanese outbreak of 2023, the first molecular characterization of BPSV in that region since 1988, provided further evidence of genetic connectivity across vast distances. Phylogenetic analysis of the partial B2L sequence revealed that the Taiwanese strain was closely related to isolates from Japan, the United States, and France [1]. Given Taiwan’s stringent import regulations for live cattle, this genetic similarity cannot be easily explained by recent animal introductions. Alternative mechanisms, such as fomite transmission, vector-mediated dispersal, or the persistence of genetically stable strains over decades, must be considered. The finding that five of eleven affected calves remained positive for BPSV one month after the outbreak, without a decrease in viral loads, underscores the capacity of this virus to establish persistent infections that may facilitate long-term maintenance of specific genotypes within herds [1].

Korean BPSV isolates from native cattle similarly clustered with Japanese, German, Sudanese, and American strains in B2L-based phylogenies [10]. This cosmopolitan clustering pattern, where geographically disparate isolates share high sequence identity, suggests either recent common ancestry with rapid global dissemination or strong purifying selection acting on the B2L gene that constrains its divergence over time. The latter interpretation is supported by comparative analyses demonstrating that ORF11 (B2L) is highly conserved across BPSV strains, whereas other genomic regions exhibit substantially greater variability [8].

Beyond B2L: The Informative Power of ORF32 and Genomic Hotspots

While B2L has served as the workhorse of BPSV molecular epidemiology, recent studies have expanded the analytical toolkit to include additional genomic loci that provide higher resolution phylogenetic signals. The complete sequencing of ORF11 and ORF32 from 11 BPSV strains isolated in Hokkaido, Japan’s largest cattle-producing region, between 2005 and 2022 revealed a striking contrast in evolutionary rates between these two genes. Deduced amino acid identities for ORF11 ranged from 95–100%, confirming its conservation, while ORF32 identities spanned a much wider range of 88–100% [8]. One Hokkaido strain exhibited an amino acid deletion in ORF32, further highlighting the plasticity of this locus.

The first ORF32 sequences reported for Japanese parapoxviruses demonstrated that this gene is particularly suitable for molecular epidemiological investigations due to its enhanced variability [8]. Phylogenetic analysis based on ORF11 showed that 10 of the 11 Hokkaido BPSV strains formed a single cluster, with one strain occupying a distinct position, a pattern that suggests the existence of multiple co-circulating lineages even within a restricted geographic area. This finding aligns with the Saga lineage data and underscores the importance of employing multiple genomic markers to capture the full extent of BPSV genetic diversity.

The discovery of a BPSV variant in Iwate Prefecture, Japan, with a T61C nucleotide substitution in the envelope gene and differential restriction enzyme digestion patterns (XmnI and HincII), provides additional evidence for the emergence of genetically distinct variants within the Japanese BPSV population [24]. Among seven BPSV-positive calves from eight farms in Iwate, two formed a subgroup based on this molecular signature, indicating that microevolutionary processes are actively generating diversity within this virus population. The concurrent detection of pseudocowpox virus in one calf from the same geographic region, the first such report in Japan, raises the possibility of co-infection and potential recombination between parapoxvirus species, a phenomenon that warrants systematic investigation.

Comparative Genomics and Interspecies Relationships

Whole-genome comparative analyses have illuminated the evolutionary relationships between BPSV and other parapoxviruses at a fundamental level. The genome of BPSV strain BV-AR02, when compared with orf virus strain OV-SA00, reveals significant differences that likely account for the distinct host range preferences of these viruses [15]. Interspecies sequence variability is distributed unevenly across functional gene classes, being highest in putative virulence and host range genes. This pattern is consistent with the hypothesis that host adaptation drives accelerated evolution in genes that interact directly with host immune defenses.

The phylogenetic distinctiveness of BPSV is further underscored by analyses of specific immunomodulatory genes. The chemokine-binding protein (CBP) encoded by BPSV exhibits a distinctive homodimeric structure with a molecular weight of 82.4 kDa and demonstrates broad-spectrum binding to chemokines across the CXC, CC, and XC subfamilies [27]. Comparative structural analysis based on the crystal structure of orf virus CBP provides a molecular explanation for the observed chemokine specificities. The BPSV-CBP’s capacity to inhibit neutrophil and monocyte infiltration in both lipopolysaccharide-induced inflammatory models and wound healing models [27] represents a sophisticated immune evasion strategy that may have co-evolved with the bovine host.

Similarly, the interleukin-10 (IL-10) homologues encoded by parapoxviruses exhibit species-specific functional differences of evolutionary significance. Recombinant BPSV IL-10 demonstrated potent anti-inflammatory activity, inhibiting production of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1β in lipoteichoic acid-activated THP-1 monocytes, while also inducing mast cell proliferation and binding IL-10 receptor 1 with affinity comparable to orf virus IL-10 [31]. In contrast, pseudocowpox virus IL-10 showed reduced activity across these assays, and grey sealpox virus IL-10 exhibited only limited inhibition of IL-1β production. These functional differences likely reflect adaptations to the specific immune environments of their respective hosts and may contribute to the observed differences in pathogenicity and tissue tropism.

Vector-Mediated Dispersal and Ecological Reservoirs

The detection of BPSV DNA in ticks infesting cattle in Burkina Faso introduces a previously underappreciated dimension to the evolutionary ecology of this virus. Among 663 ticks collected from 26 farms across three provinces of eastern Burkina Faso, 5.8% of tick pools tested positive for BPSV [5]. The positive tick pools were found in herds sharing water and pasture resources with histories of seasonal transhumance, suggesting that ectoparasites may serve as mechanical vectors that facilitate viral spread across geographic boundaries. Common grazing and seasonal transhumance are likely to support transmission of the virus, potentially enabling the movement of genetically distinct strains between herds and across ecological zones [5]. This finding has important implications for understanding the genetic connectivity of BPSV populations across the Sahel region and the potential for long-distance dispersal of specific genotypes.

The demonstration that houseflies (Musca domestica) can mechanically transmit parapoxviruses, including BPSV, further expands the potential mechanisms for viral dissemination [17]. Viral sequences identical to those detected on cattle body surfaces and in barn environments were recovered from both the body surface and feces of houseflies, indicating that these ubiquitous insects may serve as vectors for viral movement within and between farms. The capacity for mechanical transmission by arthropods could facilitate the introduction of novel genetic variants into naive herds, thereby increasing the effective population size and genetic diversity of circulating BPSV strains.

Zoonotic Implications and Human-Mediated Dispersal

The recognition of BPSV as a zoonotic pathogen with documented human infections across multiple continents adds an important public health dimension to the analysis of its genetic diversity. Human infections with BPSV have been confirmed through molecular characterization in Georgia, where retrospective screening of anthrax-negative human samples identified BPSV DNA alongside orf virus and pseudocowpox virus [22]. The zoonotic potential of BPSV has also been documented in Bangladesh, where BPSV DNA was obtained from lesion material and oral swabs collected from dairy cattle and calves in Siranjganj district [23]. Phylogenetic comparisons of Bangladeshi BPSV strains with reference sequences based on the B2L and J6R loci revealed clustering patterns consistent with global BPSV diversity.

The potential for human-mediated transport of BPSV across international borders is substantial, given the movement of agricultural workers, veterinarians, and animal products. The detection of BPSV in human infections in Georgia, a country situated at the crossroads of Europe and Asia, highlights the role of human activity in the dissemination of this virus. The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have recognized the importance of surveillance for zoonotic parapoxviruses, and the genetic characterization of BPSV strains from human cases provides critical data for understanding transmission dynamics and assessing public health risks.

Evolutionary Mechanisms and Future Directions

The genetic diversity observed among BPSV strains is likely generated through multiple mechanisms, including point mutation accumulation, recombination between co-infecting strains, and gene duplication or loss. The co-circulation of multiple BPSV lineages within single geographic regions, as documented in Saga and Hokkaido [8, 9], creates opportunities for recombination events that could generate novel genetic combinations. The detection of dual infections with BPSV and pseudocowpox virus in the same animal in Japan [7] and the documentation of coinfections involving PCPV and BPSV in Brazilian cattle [20, 21] suggest that mixed infections are not rare and may provide the substrate for interspecific recombination.

The finding that distinct BPSV lineages can be associated with specific clinical presentations or lesion types warrants further investigation. The outbreak in Japanese Black calves suspected of having foot-and-mouth disease demonstrated that BPSV can produce lesions ranging from simple erythema to frank ulcers in the oral cavity [11]. Whether different genetic variants are associated with different virulence profiles or lesion morphologies remains an open question that could have significant implications for differential diagnosis and disease management.

The development of high-resolution melting (HRM) assays capable of simultaneously detecting and differentiating BPSV from other poxviruses represents a significant methodological advance that will facilitate large-scale genetic surveillance [16]. The ability to discriminate between BPSV, pseudocowpox virus, and orf virus based on amplicon melting temperature provides a rapid, cost-effective approach for molecular epidemiological studies that can generate the sequence data needed to refine our understanding of BPSV evolutionary relationships. As additional genomic sequences become available from diverse geographic regions and host species, the phylogenetic framework for BPSV will continue to evolve, revealing the full extent of genetic diversity within this important veterinary and zoonotic pathogen.

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