Sheep Pox Virus

Overview and Taxonomy of Sheep Pox Virus (SPPV)

Sheep pox virus (SPPV) represents one of the most economically consequential pathogens affecting global small ruminant production, classified as a notifiable agent by the World Organisation for Animal Health (WOAH) due to its capacity for rapid transboundary spread and severe impacts on livestock trade and food security [4, 21, 42]. The disease, known as sheep pox (SPP), is an acute, highly contagious viral infection characterized by fever, generalized papular-pustular skin lesions, and internal organ pathology, with morbidity and mortality rates that can approach 100% in naive, susceptible populations [24, 29]. SPPV, together with goat pox virus (GTPV) and lumpy skin disease virus (LSDV), constitutes the genus Capripoxvirus within the subfamily Chordopoxvirinae of the family Poxviridae [12, 30, 42]. This taxonomic placement reflects a shared evolutionary heritage characterized by large, double-stranded DNA genomes, cytoplasmic replication, and a complex virion architecture that includes a core containing the viral genome flanked by lateral bodies, all enveloped by a lipoprotein membrane [19, 42]. The three capripoxvirus species exhibit nucleotide sequence identities of up to 97%, yet they demonstrate distinct, though not absolute, host preferences: SPPV primarily infects sheep, GTPV primarily infects goats, and LSDV primarily infects cattle [12, 37, 45]. This high degree of genetic homology has profound implications for diagnostics, vaccine development, and cross-species protection strategies, as heterologous vaccination using SPPV or GTPV strains is routinely employed to protect cattle against LSDV in endemic regions [5, 11, 15, 27, 34, 39].

Taxonomic Hierarchy and Virion Structure

The taxonomic classification of SPPV is firmly established within the poxvirus family, which encompasses a diverse array of viruses infecting vertebrates and insects. Within the subfamily Chordopoxvirinae, the genus Capripoxvirus is distinguished by its restricted host range to ruminants, a characteristic that differentiates it from other poxvirus genera such as Orthopoxvirus (which includes variola virus, vaccinia virus, and monkeypox virus) and Parapoxvirus (which includes orf virus) [30, 42]. The SPPV virion is a brick-shaped or ovoid particle, approximately 300 nm in length and 270 nm in width, exhibiting the characteristic poxvirus morphology of a complex, multilayered structure [18, 44]. Transmission electron microscopy of negatively stained preparations reveals the presence of an outer envelope surrounding a biconcave core containing the linear double-stranded DNA genome, which ranges from approximately 150 to 155 kilobase pairs in length [19, 36]. The genome encodes for approximately 150 to 160 open reading frames (ORFs), including genes involved in viral replication, host range determination, immune evasion, and structural protein synthesis [1, 19]. Among these, the SPPV14 protein has garnered significant attention as a potent inhibitor of BCL-2-mediated apoptosis, a critical virulence factor that facilitates viral persistence by preventing the premature death of infected cells [1]. The structural proteins of SPPV, including orthologs of vaccinia virus immunodominant proteins such as L1 (SPPV-ORF 060), A4 (SPPV-ORF 095), A27 (SPPV-ORF 117), and A33 (SPPV-ORF 122), are essential for virion assembly, entry, and egress, and they serve as primary targets for neutralizing antibody responses [10, 14, 19, 20].

Genetic Markers and Phylogenetic Relationships

The molecular characterization of SPPV has been advanced significantly through the analysis of several conserved genetic loci that serve as reliable markers for species identification and phylogenetic inference. The P32 gene, encoding a 32-kDa virion envelope protein, is among the most widely utilized targets for PCR-based detection and differentiation of capripoxviruses [2, 9, 12, 17, 25, 38]. This gene exhibits sufficient sequence variability to distinguish SPPV from GTPV and LSDV, with phylogenetic analyses of P32 sequences consistently clustering SPPV isolates into a distinct clade separate from GTPV and LSDV [12, 25]. The RNA polymerase subunit gene RPO30 has also proven invaluable for differential diagnosis, as it contains species-specific deletions and insertions that yield amplicons of different sizes: 172 base pairs for SPPV and 152 base pairs for GTPV [18, 45]. Similarly, the GPCR (G protein-coupled receptor) gene and the PRO30 gene have been employed in multiple phylogenetic studies to elucidate the genetic relationships among capripoxvirus isolates from diverse geographic origins [12, 13]. Sequence analysis of these genes from SPPV strains circulating in Saudi Arabia, India, Iraq, Russia, and Kazakhstan has revealed nucleotide identities ranging from 90% to 100% among worldwide isolates, confirming the overall genetic stability of SPPV while also highlighting the existence of geographically distinct lineages [7, 9, 12, 17, 25, 26]. Whole-genome sequencing of field isolates from recent outbreaks in Central Russia (2018-2019) demonstrated that these viruses cluster with the SPPV-Srinagar strain from India (2000) and the SPPV A strain from Kazakhstan (2000), forming a sister clade to the NISKHI vaccine strain, thereby confirming that the outbreaks were caused by virulent field viruses rather than vaccine-derived strains [7, 26]. This phylogenomic approach has become increasingly important for tracking the transboundary movement of SPPV and for distinguishing between vaccine and field strains in post-vaccination surveillance programs [6, 26, 43].

Host Specificity and Cross-Species Infections

While SPPV is considered to be primarily host-specific for sheep, a growing body of evidence indicates that capripoxviruses are not strictly confined to their namesake hosts, and cross-species infections occur with greater frequency than previously appreciated [12, 30, 45]. Experimental and field observations have documented instances where SPPV causes disease in goats, and conversely, GTPV can infect sheep, although the clinical manifestations may be milder or atypical compared to infections in the natural host [28, 30, 35]. This phenomenon is particularly relevant in regions where sheep and goats are raised in mixed flocks, as it complicates both clinical diagnosis and the implementation of targeted vaccination strategies [16, 21, 28]. The antigenic cross-reactivity among capripoxviruses is so pronounced that serological assays, such as virus neutralization tests and enzyme-linked immunosorbent assays (ELISAs), cannot reliably distinguish between antibodies elicited by SPPV, GTPV, or LSDV [8, 31, 37]. This serological cross-reactivity, however, has been exploited for vaccination purposes, as heterologous vaccines based on SPPV or GTPV strains are widely used to protect cattle against LSDV in Asia, Africa, and the Middle East [5, 11, 15, 27, 32, 34, 39, 41]. The live attenuated SPPV vaccine strains, including the Romanian Fanar, Bakırköy, and NISKHI strains, have demonstrated efficacy in inducing protective immunity against LSDV in cattle, with seroconversion rates exceeding 80% and duration of immunity lasting up to 11 months in adult animals [5, 11, 15, 27, 39]. However, the use of heterologous vaccines necessitates careful molecular characterization of the vaccine seed virus, as misidentification of vaccine strains has occurred historically; for instance, the KSGP O-240 strain, previously believed to be a sheep pox virus, was subsequently identified as a strain of LSDV through molecular analysis [32, 42].

Global Distribution and Epidemiological Significance

Sheep pox is endemic across large swathes of Africa, Asia, and the Middle East, where it poses a persistent threat to small ruminant production systems and rural livelihoods [6, 16, 21, 29, 40]. The disease is responsible for substantial economic losses attributable to mortality, reduced weight gain, decreased milk production, damage to hides and wool, and the costs associated with control measures, including vaccination, movement restrictions, and culling [1, 6, 24, 29]. In recent years, SPPV has demonstrated a concerning capacity for re-emergence in regions where it had been previously eradicated, as exemplified by the 2022-2023 outbreak in Spain, which occurred more than 50 years after the disease was last reported in the country [4]. This outbreak, which infected 30 farms across Andalusia and Castilla-La Mancha over a nine-month period, underscored the vulnerability of naive populations and the critical importance of active clinical surveillance combined with laboratory confirmation for early detection [4]. Similarly, the re-emergence of sheep pox in Greece and Bulgaria during 2024-2025, with unprecedented intensity, has prompted urgent assessments of vaccination strategies and control measures by the European Food Safety Authority (EFSA) [43]. In Central Asia, epizootic monitoring in Kazakhstan from 2021 to 2024 revealed the re-establishment of virus circulation in several regions, including East Kazakhstan, where no cases had been reported for an extended period, highlighting the dynamic and transboundary nature of SPPV transmission [6, 22]. The virus is transmitted primarily through direct contact between infected and susceptible animals via respiratory droplets, contaminated fomites, and skin lesions, although indirect transmission via vectors such as ticks and mechanical transmission by insects may also contribute to spread under certain conditions [5, 29, 33]. The incubation period typically ranges from 4 to 14 days, after which clinical signs including pyrexia, conjunctivitis, anorexia, and the development of characteristic papules and nodules on the skin and mucous membranes become apparent [24, 44]. The pathological hallmark of sheep pox is the formation of eosinophilic intracytoplasmic inclusion bodies in hyperplastic epithelial cells of the skin, rumen, reticulum, and respiratory tract, accompanied by interstitial pneumonia and systemic lymphadenopathy [24, 30]. The severity of disease is influenced by viral strain, host age, breed, nutritional status, and immune competence, with young lambs and animals under stress being particularly susceptible to severe generalized infections and high mortality [3, 16, 23, 40].

Molecular Pathogenesis and Viral Evasion Mechanisms of SPPV

The pathogenesis of Sheep Pox Virus (SPPV), a member of the genus Capripoxvirus within the family Poxviridae, is a complex, multifactorial process that begins at the epithelial barrier and culminates in systemic dissemination, profound immunosuppression, and characteristic proliferative and necrotic lesions. The World Organisation for Animal Health (WOAH) lists sheep pox as a notifiable disease due to its transboundary nature and severe economic impact on small ruminant production [6, 21, 43]. Understanding the molecular mechanisms by which SPPV establishes infection, subverts host defenses, and orchestrates tissue damage is critical for developing rational countermeasures, including antivirals and next-generation vaccines.

Initial Infection and Host Cell Tropism

SPPV exhibits a pronounced tropism for epithelial cells, particularly those of the skin, mucous membranes, and the respiratory and gastrointestinal tracts. The virus is typically transmitted via the respiratory route or through direct contact with infectious scabs and exudates [24, 29]. Following entry, SPPV initiates a lytic infection in permissive cells, a process that is preceded by the expression of early genes. The viral genome, a linear double-stranded DNA molecule of approximately 150 kbp, features inverted terminal repeats (ITRs) that contain essential regulatory elements and genes involved in host range determination and virulence [7, 31]. The ITRs have been exploited for molecular diagnostics, including loop-mediated isothermal amplification (LAMP) assays that distinguish SPPV from the closely related goat pox virus (GTPV) [31]. The virion core protein P4a, encoded by a highly conserved gene, is a structural component essential for viral assembly and is a target for phylogenetic clustering of capripoxviruses [48]. The P32 envelope protein, a major immunodominant antigen, is critical for viral entry and serves as the primary target for PCR-based diagnostics and serological assays [2, 25, 38]. Sequence analysis of the P32 gene from outbreaks in India, Iraq, and Saudi Arabia has revealed high lineage specificity and genetic conservation, underscoring its importance in viral fitness and host adaptation [12, 17, 25].

Viral Replication and Cytopathic Effect

The replication cycle of SPPV is entirely cytoplasmic, a hallmark of poxviruses. Following uncoating, the virus orchestrates a tightly regulated cascade of gene expression: early, intermediate, and late. This process is dependent on a virus-encoded RNA polymerase, whose subunits (e.g., RPO30 and RPO132) are used for species differentiation and vaccine strain identification [18, 26]. The cytopathogenic effect (CPE) in permissive cell lines, such as lamb testis cells (LTC) and Vero cells, is characterized by rounding, detachment, and the formation of syncytia [46, 47]. Adaptation of the NISKHI vaccine strain to Vero cells results in a robust CPE with titers reaching 6.50 lg TCID50/ml, demonstrating the virus's capacity for serial propagation in continuous cell lines [46]. Ultrastructural examination via transmission electron microscopy reveals large, oval-shaped virions with an enveloped structure, consistent with the typical poxvirus morphology [18].

SPPV14: A Master Regulator of Apoptosis Evasion

A cornerstone of SPPV pathogenesis is its ability to subvert intrinsic apoptotic pathways, thereby ensuring prolonged host cell survival for viral replication. The SPPV14 protein has been identified as a potent inhibitor of BCL-2-mediated apoptosis [1]. This viral BCL-2 ortholog functions by sequestering pro-apoptotic BH3-only proteins, preventing them from activating BAX and BAK at the mitochondrial membrane. The structural basis for this interaction involves a canonical binding groove on SPPV14 that mimics the host BH3 motif. A critical residue for the stability of the SPPV14–BH3 complex is Arg84, which forms a salt bridge with the aspartic acid residue of the BH3 ligand. Disruption of this ionic interaction effectively neutralizes the anti-apoptotic function of SPPV14 [1]. In silico docking studies have demonstrated that certain flavonoids, notably isoxanthohumol, can bind with high affinity to the Arg84 residue, displacing the BH3 ligand and potentially restoring apoptotic pathways in infected cells [1]. This highlights the SPPV14–BH3 interface as a high-value druggable target for the development of antiviral therapeutics that function by inducing premature apoptosis of the viral factory. The elimination of this anti-apoptotic blockade would trigger the rapid demise of infected cells, curtailing viral spread and reducing the severity of disease.

Modulation of Host Antiviral Responses

Beyond apoptosis inhibition, SPPV deploys a multifaceted arsenal to neutralize the host immune response. Structural proteins orthologous to those of vaccinia virus (VACV) play key roles in both virion architecture and immune evasion. SPPV-ORF 060 (L1R ortholog), SPPV-ORF 095 (A4L ortholog), SPPV-ORF 117 (A27L ortholog), and SPPV-ORF 122 (A33R ortholog) are immunodominant and have been shown to elicit virus-neutralizing antibodies in experimental animals [19]. The SPPV-117 protein, a structural component of the mature virion, has been successfully expressed in tobacco chloroplasts, retaining its antigenic properties and demonstrating the feasibility of plant-based subunit vaccine production [10, 14]. The SPPV-060 (L1R) protein, a myristoylated membrane protein, is essential for viral entry and is a prime target for neutralizing antibodies [14, 19].

SPPV also interferes with the host's interferon (IFN) and cytokine networks. While specific SPPV-encoded IFN antagonists have not been fully characterized in the provided sources, the virus's ability to replicate despite a robust inflammatory milieu suggests the presence of potent evasion mechanisms. The induction of oxidative stress and altered iron metabolism in infected sheep, as evidenced by elevated proinflammatory cytokines and changes in hematological parameters, indicates that SPPV manipulates the host's redox environment to favor its replication [23]. The presence of single-nucleotide polymorphisms (SNPs) in immune and antioxidant genes (e.g., MBL1, MBL2, TLR4, IL-1β, IL-10, IL-13, SOD1, GPX1) correlates with susceptibility to SPPV infection in Barki ewes, suggesting that host genetic factors directly influence the outcome of infection [23]. This underscores the evolutionary arms race between the virus and the host's immune surveillance, where viral proteins attempt to dampen innate signaling while host polymorphisms dictate the efficiency of the antiviral response.

Systemic Dissemination and Pathology

Following primary replication at the inoculation site, SPPV disseminates via the lymphatics and bloodstream, leading to a secondary viremia and the formation of characteristic pox lesions throughout the skin, mucous membranes, and internal organs. The gross pathology of sheep pox is defined by the development of papules, vesicles, pustules, and scabs on the skin, particularly on hairless areas such as the groin, ventral abdomen, and inner thighs [24]. Histopathological examination reveals hallmark features: hyperplasia and hydropic degeneration of epithelial cells, with large, eosinophilic intracytoplasmic inclusion bodies (Guarnieri-like bodies) representing sites of viral replication [24]. In the respiratory tract, the virus causes interstitial pneumonia, epithelial hyperplasia of the trachea, and the formation of intracytoplasmic inclusions in bronchiolar epithelial cells [24]. Lesions are also observed in the gastrointestinal tract, affecting the rumen and reticulum [24]. The severity of pathology is directly correlated with viral load; real-time PCR assays targeting the P32 gene have demonstrated that high viral titers in skin and blood samples are associated with disseminated disease and a poor prognosis [38].

Antigenic Cross-Reactivity and Implications for Vaccination

A critical aspect of SPPV pathogenesis is its antigenic relatedness to other capripoxviruses, including lumpy skin disease virus (LSDV) and GTPV. The three viruses share up to 97% nucleotide sequence identity, and cross-neutralization has been extensively documented [37]. This antigenic homology has been exploited for vaccination: heterologous sheep pox vaccines are routinely used to protect cattle against LSDV in regions where LSD is endemic [5, 11, 15, 27]. Live attenuated SPPV strains (e.g., Romanian Fanar, Bakırköy, NISKHI) induce robust humoral and cell-mediated immunity in sheep, protecting against homologous challenge and providing cross-protection against LSDV in cattle, albeit with varying efficacy [11, 39, 43, 46]. The duration of immunity in cattle vaccinated with heterologous SPPV is substantial, with population immunity reaching >80% at 30 days post-vaccination, though it wanes more rapidly in young animals, necessitating booster vaccinations [5, 27].

The high degree of sequence conservation, however, presents a major challenge for serological surveillance. Traditional serological tests, such as virus neutralization tests (VNT) and enzyme-linked immunosorbent assays (ELISA), cannot distinguish between animals infected with LSDV and those vaccinated with SPPV or GTPV [8, 37]. This inability to differentiate infected from vaccinated animals (DIVA) hinders outbreak control measures. To address this, researchers have developed DIVA-compatible ELISAs based on synthetic genes unique to LSDV, which show no cross-reactivity with SPPV antibodies [37]. Such tools are essential for effective epidemiological monitoring and eradication campaigns in regions where multiple capripoxviruses co-circulate. The genomic plasticity of capripoxviruses, including the emergence of recombinant strains, further complicates diagnosis and emphasizes the need for continuous molecular surveillance and whole-genome sequencing of field isolates [6, 26, 49].

Epidemiology, Transmission, and Global Distribution of Sheep Pox

Sheep pox virus (SPPV) remains one of the most economically consequential pathogens affecting small ruminant production systems worldwide, classified by the World Organisation for Animal Health (WOAH) as a notifiable disease of significant transboundary importance. The epidemiological landscape of sheep pox is characterized by a complex interplay of host susceptibility, environmental determinants, husbandry practices, and viral genetic diversity that collectively dictate its spatial and temporal distribution across endemic and emerging regions.

Global Distribution and Endemic Patterns

The distribution of sheep pox is not uniform; rather, it reflects historical trade routes, climatic gradients, and the effectiveness of national veterinary services. Currently, the disease is considered endemic across broad swaths of Africa, Asia, and the Middle East, with periodic incursions into Europe that underscore its potential for transboundary spread. Detailed serological surveys conducted in the Kordofan region of Sudan revealed an alarming overall seroprevalence of 73.4% as determined by virus neutralization, with South Kordofan and North Kordofan states exhibiting 85% and 63.6% seropositivity, respectively [40]. Such figures indicate that in the absence of systematic vaccination, virtually the entire population may be exposed, with infection occurring early in life. By contrast, seroprevalence data from the Wolaita Zone of Southern Ethiopia, employing a serum neutralization test, yielded a substantially lower overall rate of 4.95%, though this figure masked a fivefold higher odds of seropositivity in sheep compared to goats (adjusted odds ratio 4.73; 95% CI 1.39–15.99) [16]. This stark disparity between regions highlights the heterogeneity in transmission intensity and the critical influence of management systems and environmental conditions.

In Uganda, a comprehensive cross-sectional study across Sembabule and Nakapiripirit districts detected an overall seroprevalence of 10% (137/1,515) using a double Capripox multispecies antigen ELISA, with seropositivity greater in the pastoral Nakapiripirit district (12%) than in the semi-intensive Sembabule district (6%) [21]. This finding is consistent with the hypothesis that communal grazing, uncontrolled animal movements, and limited veterinary oversight facilitate viral maintenance and spread. The risk factors independently associated with seropositivity at the multivariable level were age (OR 0.43; 95% CI 0.21–0.87) and sex (OR 2.14; 95% CI 1.31–3.5), with female animals demonstrating higher odds of exposure [21]. The former likely reflects waning maternal immunity and subsequent exposure during the vulnerable young adult period, while the latter may be attributable to the prolonged retention of females in the breeding herd and their increased cumulative contact time.

The European epidemiological situation has undergone a dramatic shift. After more than 50 years of freedom, sheep pox re-emerged in Spain in September 2022, with an outbreak lasting nine months that infected 30 farms dispersed across Andalusia and Castilla-La Mancha [4]. Critically, active clinical surveillance combined with laboratory confirmation via real-time PCR on oral swabs was the most sensitive method for farm-level detection, yet even this intensive approach detected only two of six reported outbreaks before clinical signs were observed [4]. This case illustrates the challenges of early detection even in well-resourced veterinary systems. More recently, unprecedented epidemic intensity has been observed in Greece and Bulgaria during 2024–2025, with modeling indicating that nationwide vaccination campaigns combined with standard control measures could achieve eradication one year earlier than non-vaccination strategies, though rapid detection and culling alone could still control epidemics within one to two years [43]. The European experience underscores that sheep pox remains a pathogen capable of exploiting gaps in surveillance even in regions with historically robust animal health infrastructure.

Transmission Pathways and Epidemiological Dynamics

Transmission of SPPV occurs through multiple routes, with direct contact between infected and susceptible animals serving as the primary mechanism. The virus is present in high concentrations in scabs, skin lesions, nasal and oral secretions, and to a lesser extent in milk and semen. Aerosol transmission over short distances has been documented, and the virus can persist in the environment for extended periods, particularly in dried scabs and contaminated bedding. The efficacy of disinfectants such as GAN, Dexid-400, and sodium hydroxide against the capripox virus has been demonstrated in field conditions in Tajikistan, where their application during outbreaks successfully prevented spread and reduced economic losses [33].

Indirect transmission via contaminated fomites, including feed troughs, water sources, vehicles, and personnel, is well established and contributes to the rapid within-flock spread observed in naive populations. The potential role of arthropod vectors in mechanical transmission has been a subject of investigation. In Armenia, testing of six types of Ixodes ticks from LSD-affected regions yielded no evidence of LSDV circulation in these vectors via real-time PCR, suggesting that while mechanical transmission by biting flies cannot be excluded, ticks may not serve as significant biological or mechanical vectors in that ecological context [5]. However, the vector-borne hypothesis remains plausible, particularly in tropical and subtropical environments where high densities of hematophagous insects coincide with peak disease incidence. Indeed, seroprevalence studies in the Kordofan region identified geographic region as a significant risk factor, likely reflecting differences in vector abundance and husbandry practices [40].

Long-distance transmission is predominantly anthropogenic, mediated by the movement of infected animals, often through informal trade or nomadic pastoralism. In India, sheep rearing is frequently associated with landless and economically weaker communities whose owners migrate seasonally in search of grazing, a practice that exposes flocks to novel viral strains and facilitates the geographic expansion of outbreaks [25]. Similarly, in the West Gojjam and Awi zones of Northwest Ethiopia, the absence of free animal movement was associated with a dramatically reduced odds of disease occurrence (OR 0.05; 95% CI 0.02–0.15), reinforcing the primacy of animal movement control as an epidemiological intervention [28].

The incubation period typically ranges from 4 to 14 days, during which susceptible animals may shed virus before clinical signs become apparent. This subclinical shedding represents a critical window for undetected spread, particularly in systems where animals are commingled at markets or during transhumance. Once clinical disease manifests, the characteristic papular-ulcerative lesions on the skin and mucous membranes serve as concentrated sources of infectious virus. The duration of infectivity is prolonged by the persistence of scabs, which can retain viable virus for months under favorable environmental conditions, particularly in cool, dry environments. Outbreak investigations in Central Russia during 2018–2019 identified the SPPV-Srinagar strain and SPPV A strain from Kazakhstan as the closest relatives to circulating field isolates, with phylogenetic clustering separate from the NISKHI vaccine strain used in the country, confirming that recent outbreaks were not attributable to vaccine reversion [7].

Risk Factors and Host Determinants

A thorough understanding of host-level risk factors is essential for targeted surveillance and control. Age represents a consistently identified determinant of infection risk. In the Thatta district of Pakistan, seroprevalence was significantly higher (P < 0.05) in animals aged 4–12 months (young) compared to sucklers (0–4 months) and adults (>12 months) [3]. The lower rate in sucklers is attributable to protection conferred by maternal immunity, the duration of which has been experimentally determined to be approximately 60 days in lambs born to vaccinated ewes [51]. This period of passive protection has profound implications for vaccination schedules: lambs vaccinated before two months of age may fail to seroconvert due to interference from maternally derived antibodies, necessitating a delay in primary immunization until after the waning of passive immunity. The higher risk in young adults likely reflects the convergence of waning maternal immunity with increased environmental exposure and the onset of reproductive and social behaviors that promote contact.

Sex differences in seroprevalence have been documented across multiple geographical contexts. In Pakistan, female animals showed a seroprevalence of 17.85% compared to 7.50% in males, with the difference reaching statistical significance (P < 0.05) [3]. Similarly, in the Sembabule and Nakapiripirit districts of Uganda, female animals exhibited higher odds of seropositivity (OR 2.14; 95% CI 1.31–3.5) at the multivariable level [21]. This disproportionate burden in females may be multifaceted: females are typically retained in the flock for longer periods, increasing cumulative exposure; they experience physiological stress during pregnancy and lactation, which may impair immune function; and management practices often keep females in closer confinement with their offspring, facilitating transmission.

Breed-associated susceptibility has been suggested by several observational studies. In the Kordofan region, breed was identified as a risk factor, though the specific genetic mechanisms remain poorly characterized [40]. Experimental infection studies using Barki ewes in Egypt demonstrated that infected animals exhibited anemia, leukocytosis, elevated proinflammatory cytokines, oxidative stress, and altered iron metabolism, with 23 novel single-nucleotide polymorphisms identified in expressed regions of immune and antioxidant genes [23]. While these findings are preliminary, they raise the intriguing possibility that host genetic variation at these loci may modulate disease susceptibility and severity, offering potential targets for marker-assisted selection in breeding programs.

Body condition and management factors also modulate risk. In the Wolaita Zone of Ethiopia, poorly conditioned animals had a dramatically higher seroprevalence of 31.58% compared to animals in good body condition, while animals with ticks on their skin showed a seroprevalence of 10.38% compared to 4.33% in tick-free animals [16]. Larger flock sizes were associated with higher risk, with flocks of more than 50 animals having 6.73 times the odds of seropositivity compared to small flocks (95% CI 1.58–28.67) [16]. These data underscore the role of management practices, including nutrition, ectoparasite control, and stocking density, in modulating the force of infection.

Seasonal Patterns and Environmental Drivers

The temporal distribution of sheep pox outbreaks exhibits pronounced seasonality, though the precise pattern varies by geographic region and climate. In the Thatta district of Pakistan, the highest prevalence was recorded in January (22.97%, 17/74), while the lowest occurred in March (4.33%, 2/46) [3]. This winter peak may be associated with cooler temperatures that favor viral persistence in the environment, as well as management practices such as winter housing that increase animal density and contact rates. Conversely, in the Kordofan region of Sudan, the disease is reported to show a different seasonal profile, likely influenced by the bimodal rainfall pattern and associated vector activity [40].

Climatic factors also influence the distribution and abundance of potential mechanical vectors. While the role of arthropods in SPPV transmission is less well established than for lumpy skin disease virus, the potential for mechanical transmission by stable flies (Stomoxys calcitrans), mosquitoes, and other biting insects cannot be discounted, particularly in tropical and subtropical environments. The emergence of sheep pox in new geographic areas may therefore be influenced by climate-driven shifts in vector distributions, though this remains an area requiring further investigation.

Surveillance Challenges and Molecular Epidemiology

The accurate detection and characterization of circulating SPPV strains are fundamental to understanding epidemiological dynamics and informing control strategies. Molecular diagnostic tools have revolutionized the capacity for rapid, specific detection. A SYBR Green-based real-time PCR assay targeting the P32 gene demonstrated high sensitivity, enabling quantification over nine orders of magnitude and outperforming conventional gel-based PCR for detection of low viral titers [38]. In Libya, a conventional PCR targeting the P32 gene successfully amplified the expected 390 bp product from all 67 clinically suspected sheep pox samples, with no amplification from contagious ecthyma or healthy controls, confirming the assay's specificity [2]. More sophisticated approaches include a real-time high-resolution melting (HRM) PCR assay that differentiates SPPV from goat pox virus and LSDV based on melting temperature peaks, with SPPV melting at 74.24 ± 0.06°C, allowing species-level discrimination in a single reaction [50]. A multiplex real-time PCR method employing specific probes for each capripoxvirus species has been validated with a limit of detection of 102 genome copies and no cross-reactivity with other pathogens causing similar clinical syndromes [45].

Phylogenetic analyses have provided critical insights into the global spread and genetic relationships among SPPV isolates. Sequencing of the P32, RPO30, and GPCR genes from six capripoxvirus strains identified in Saudi Arabia between 2013 and 2017 classified five as SPPV and one as GTPV, with nucleotide identities ranging from 94% to 99% for P32 among worldwide isolates [12]. In Iraq, the first molecular detection of SPPV was achieved through P32 gene sequencing, with phylogenetic analysis suggesting that the origin of infection may be linked to importation from India, China, and the USA [17]. Whole-genome sequencing of isolates from outbreaks in Central Russia (2018–2019) clustered closely with the SPPV-Srinagar strain from India (2000) and the SPPV A strain from Kazakhstan (2000), forming a sister clade to the NISKHI vaccine strain and confirming that outbreaks were caused by field isolates rather than vaccine reversion [7]. Similarly, analysis of SPPV isolates from Maharashtra, India, based on the ORF 103 gene, revealed 99–100% identity with isolates from Egypt, China, and other Indian states, underscoring the high degree of genetic conservation and the potential for transboundary dissemination [9].

A critical challenge in molecular epidemiology is the difficulty of differentiating between infectious virus and residual non-infectious genomic material following outbreaks. In the Spanish outbreak, a non-infectious genome of the virus could be detected by real-time PCR months after cleaning and disinfection, complicating the interpretation of sentinel animal testing during repopulation [4]. This finding has profound implications for post-outbreak surveillance and the determination of when premises can be safely restocked, and it underscores the need for combination approaches that integrate molecular detection with virus isolation or infectivity assays.

Vaccination as an Epidemiological Modifier

Vaccination profoundly alters the epidemiological landscape of sheep pox, though its impact is contingent upon vaccine efficacy, coverage, and the durability of immunity. Live attenuated vaccines based on SPPV strains such as NISKHI, RM65, Romania, and Bakırköy provide robust protection, typically 80–100%, with substantial reduction in viral replication and shedding [43]. However, the duration of immunity is not uniform across age groups. In cattle vaccinated with a heterologous sheep pox vaccine for protection against lumpy skin disease, population immunity reached 86.09% (95% CI 83.83–87.97%) at 30 days post-vaccination, but a significant drop in antibody levels was observed in young cattle (≤12 months) at six months, suggesting that an additional booster is necessary at 4–6 months after primary vaccination [27]. In sheep, the situation is analogous: the duration of maternal immunity is approximately 60 days, and the optimal immunizing dose must be calibrated to overcome interference from maternal antibodies while avoiding excessive vaccine virus replication [51].

The use of heterologous vaccines, SPPV strains to protect against goat pox and lumpy skin disease, and vice versa, adds complexity to epidemiological interpretation. The vaccine strain G20-LKV of goat pox virus has been shown

Clinical Manifestations and Pathological Features of Sheep Pox

Sheep pox virus (SPPV), a member of the genus Capripoxvirus within the family Poxviridae, induces a systemic, highly contagious disease in ovine populations that is characterized by a spectrum of clinical presentations ranging from mild, localized eruptions to severe, generalized, and frequently fatal infections. The clinical manifestations and pathological findings are the cornerstone of field diagnosis, outbreak investigation, and the assessment of disease severity, and they underpin the official notifiable status of the disease as defined by the World Organisation for Animal Health (WOAH). The economic consequences of an outbreak are devastating, driven by high morbidity and mortality, permanent damage to hides and wool, and strict trade restrictions, making a profound understanding of the disease's clinical and pathological profile essential for effective surveillance, control, and eradication programs.

Cutaneous Manifestations: The Hallmark of Infection

The most conspicuous and diagnostically significant clinical manifestation of sheep pox is the development of a characteristic papular-pustular rash, often referred to as a "pock." The clinical course of the disease typically begins after an incubation period of 4 to 14 days, during which the virus replicates locally at the site of entry, often in the respiratory tract or through abrasions in the skin [29, 44]. The initial prodromal phase is marked by a biphasic pyrexia, with temperatures often exceeding 40°C to 41°C, accompanied by profound depression, anorexia, and serous to mucopurulent ocular and nasal discharges [24, 44]. This acute systemic phase precedes the eruptive stage by one to two days.

The evolution of cutaneous lesions follows a highly predictable sequence. The first visible sign is the appearance of erythematous macules, particularly on sparsely wooled or hairless areas of the body, such as the perineum, ventral abdomen, inner thighs, axillae, face, and around the coronary bands and hooves [4, 24]. These macules rapidly progress into firm, raised, and circumscribed papules. Over the subsequent 48 to 72 hours, the center of these papules undergoes necrosis, leading to vesicle formation (a small fluid-filled blister) and, subsequently, pustules filled with a turbid, purulent exudate [29, 30]. The pustules are deeply embedded in the dermis, a feature that distinguishes them from other vesicular diseases. As the pustules mature, they rupture and desiccate, forming thick, dark brown to black scabs that eventually slough off over a period of 3 to 4 weeks, often leaving a permanent, pitted scar on the hide [30]. The intensity of the skin eruption is a primary indicator of disease severity. In benign forms, lesions may be few and localized, healing without significant complication. However, in severe, malignant cases, the eruption can be confluent, covering a substantial portion of the body surface, leading to extensive tissue necrosis and secondary bacterial infections.

Systemic and Internal Pathological Features

While cutaneous lesions are the hallmark of sheep pox, the disease is fundamentally a systemic infection affecting multiple organ systems. The severity of internal pathology is directly correlated with the extent of the cutaneous rash. In severe, generalized cases, often referred to as the "malignant" form, the virus spreads hematogenously, leading to widespread vascular damage and the development of "pocks" on the mucous membranes and internal organs [9, 29, 44].

The respiratory tract is a primary target for internal pathology. Affected sheep frequently exhibit severe respiratory distress, including labored breathing, coughing, and nasal discharge, which is a significant contributor to mortality. On post-mortem examination, the trachea shows marked mucosal congestion and subacute tracheitis with epithelial hyperplasia [24]. The lungs are heavily affected, presenting as a characteristic "pock pneumonia." Grossly, the lung parenchyma is studded with numerous, discrete, reddish-brown, circular, firm foci, which correspond to areas of interstitial pneumonia [24, 30]. Histologically, these lesions are characterized by severe congestion, edema, and the infiltration of mononuclear cells, predominantly macrophages and lymphocytes, into the interalveolar septa. Alveolar epithelial cells undergo hyperplasia and hydropic degeneration, and eosinophilic intracytoplasmic inclusion bodies, a pathognomonic feature of poxvirus infections, can be identified within these cells [24]. The involvement of the respiratory tract is so characteristic that it serves as a key differential from other, less severe pox diseases.

The gastrointestinal tract and its associated organs are also frequently affected. The mucosal surfaces of the oral cavity, tongue, pharynx, and esophagus may develop papules, pustules, and shallow erosions, causing dysphagia and drooling [24]. Furthermore, pock lesions are commonly observed on the serosal surfaces of the rumen and reticulum, and they can extend into the mucosa, causing epithelial hyperplasia and hydropic degeneration [24]. This alimentary involvement contributes to anorexia, poor nutrient absorption, and rapid weight loss. Lymph nodes, particularly those draining the primary sites of viral replication, undergo marked enlargement, edema, and congestion, reflecting a robust immunological response [30].

Histopathological Hallmarks: The Basis of Lesion Development

The histopathological changes in sheep pox are a direct consequence of the virus's cytopathic effect and its interaction with the host's vascular and immune systems. A defining feature is the proliferation and degeneration of epithelial cells. The earliest microscopic change in the skin and mucous membranes is hyperplasia of the stratum spinosum (acanthosis), followed by pronounced hydropic (ballooning) degeneration of the keratinocytes [24]. This cellular swelling and vacuolization culminate in cell lysis and the formation of intraepidermal vesicles and pustules.

Intracytoplasmic inclusion bodies are the definitive histopathological finding for the diagnosis of sheep pox. These are large, eosinophilic, homogenous structures that fill the cytoplasm of infected epithelial cells [9, 24]. Often referred to as "cellule pox" or "Borrel bodies," these inclusions represent the sites of viral replication and assembly within the cell. Their presence is considered pathognomonic for infection with capripoxviruses.

A prominent vascular component is observed in the deep dermis. Blood vessel walls undergo a smoldering vasculitis, characterized by swelling and proliferation of endothelial cells, leading to thrombosis of local capillaries and venules [30]. This ischemia is the primary cause of the necrosis seen at the center of the pustules, which gives them their characteristic "button-like" appearance. The dermal inflammatory infiltrate is predominantly composed of lymphocytes, macrophages, and plasma cells, reflecting a mixed cellular and humoral immune response. The highly efficient immune evasion strategies of SPPV, notably the expression of the SPPV14 protein, which inhibits BCL-2-mediated apoptosis, allow for prolonged viral survival within infected cells, exacerbating the severity and duration of these pathological changes [1].

Pathogenesis and the Role of Secondary Infections

The severity of clinical disease is profoundly influenced by the host's immune status, age, and breed. Young lambs and animals with poor nutritional status are at a significantly higher risk of developing the severe, generalized form of the disease, which carries a mortality rate that can approach 100% in naive populations [29, 51]. The duration of maternal immunity is a critical factor in the epidemiology of the disease in lambs. Passive immunity transferred via colostrum from vaccinated ewes can protect lambs for up to approximately 60 days of age, but this wanes significantly by 90 days, creating a window of susceptibility that coincides with the recommended age for primary vaccination [51].

The extensive tissue damage caused by the primary viral infection creates a favorable environment for secondary bacterial infections, which significantly complicate the clinical picture and contribute to mortality. The skin barrier is compromised, leading to bacterial dermatoses. More critically, secondary bacterial pneumonia is a major cause of death in advanced cases. Metagenomic profiling of pox lesions has revealed a characteristic shift in the skin and lesion microbiome, with the emergence of opportunistic and pathogenic bacteria. The most common and abundant secondary invaders identified from SPPV lesions include Fusobacterium necrophorum, a known cause of necrotic lesions, and Trueperella pyogenes, a common agent of suppurative infections in ruminants [36]. Other commonly identified genera include Streptococcus, Staphylococcus, and Corynebacterium [36]. This bacterial superinfection is a key factor driving the transition from a primarily viral pathogenesis to a severe, often fatal, suppurative and necrotic process. Clinical signs of this superinfection include purulent nasal discharge, a foul odor emanating from the skin lesions, and a rapid worsening of the animal's systemic condition, characterized by severe dehydration, toxemia, and profound anemia, as indicated by a significant drop in packed cell volume and hemoglobin concentration [23]. The presence of these secondary pathogens is a critical consideration for therapeutic intervention, as the use of appropriate antimicrobials can be a life-saving measure in the management of an outbreak.

Molecular Diagnostics and PCR Optimization for SPPV Detection

The accurate and rapid molecular detection of Sheep pox virus (SPPV) is a cornerstone of effective outbreak control, surveillance, and eradication programs, as recognised by the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) in their guidelines for transboundary animal diseases. SPPV, together with Goat pox virus (GTPV) and Lumpy skin disease virus (LSDV), forms the genus Capripoxvirus within the Poxviridae family, and these three viruses exhibit nucleotide sequence similarities exceeding 97% [37]. This high genetic homology, coupled with the clinical similarity of capripox diseases, necessitates the use of exquisitely specific molecular tools for unambiguous SPPV identification and differentiation from closely related pathogens. Over the past two decades, a suite of polymerase chain reaction (PCR)-based assays has been developed and refined, targeting various genomic loci with distinct advantages in sensitivity, specificity, throughput, and field applicability. The optimisation of these assays, ranging from conventional gel-based PCR to real-time quantitative and high-resolution melting (HRM) platforms, as well as isothermal amplification methods, has been driven by the need to detect SPPV in diverse sample matrices, to differentiate vaccine from field strains, and to provide rapid results under resource-limited settings.

Target Gene Selection and Primer Design

The selection of the appropriate genomic target is the most critical determinant of assay performance. The most widely exploited locus for SPPV detection is the P32 gene (also referred to as the virion envelope protein gene), which encodes a 32-kDa structural protein that is highly conserved across capripoxviruses but possesses species-specific polymorphisms. Alhudiri and Abukreba [2] optimised a conventional PCR targeting the P32 gene for the detection of SPPV in Alzawiyah City, Libya, using oral swab samples. Their assay, employing specific primers designed to amplify a 390-bp fragment, demonstrated 100% sensitivity and specificity when tested against 67 clinically suspected sheep pox cases, with no cross-reactivity with contagious ecthyma or healthy controls. This work underscores the robustness of the P32 target for routine diagnostic applications in endemic settings. Similarly, Agarwal et al. [25] used P32-based PCR for molecular characterisation of an outbreak in Uttar Pradesh, India, followed by full-length sequencing of the amplicon to establish lineage-specificity and phylogenetic relatedness to Indian and neighbouring country isolates.

Another frequently employed target is the RPO30 gene, encoding the 30-kDa RNA polymerase subunit. Mahmoud and Khafagi [18] demonstrated that an RPO30-based PCR could simultaneously detect, identify, and differentiate SPPV from GTPV based on amplicon size differences: SPPV yielded a 172-bp fragment whereas GTPV produced a 152-bp fragment. This size-based differentiation, while not as precise as sequencing, provided a rapid and accessible method for species-level identification during outbreaks in Egypt. The GPCR gene (G protein-coupled receptor homologue) has also been exploited for phylogenetic and discriminatory purposes. El-Sabagh et al. [12] sequenced the full-length GPCR, P32, and PRO30 genes from Saudi Arabian isolates and found that GPCR exhibited the highest variability, with nucleotide identity among capripoxviruses ranging from 90 to 99%, making it a suitable marker for molecular epidemiology and strain differentiation. Additionally, the inverted terminal repeats (ITRs) , which contain species-specific sequences, have been targeted for both conventional and loop-mediated isothermal amplification (LAMP). Sasikala et al. [24] used ITR-targeted PCR to confirm SPPV in tissue samples from an outbreak in Mecheri sheep, generating a 289-bp amplicon, while Zhao et al. [31] exploited ITR sequences to design LAMP primers capable of differentiating SPPV from GTPV with 98.8% detection rate for SPPV. The choice of target must balance conservation (for pan-capripox detection) with variability (for differentiation). For differential diagnostics, the ORF 103 gene [9] and the LSDV010 ORF [50] have also been employed.

Optimisation of Conventional PCR

Optimisation of the polymerase chain reaction involves fine-tuning several parameters including annealing temperature, primer concentration, magnesium ion concentration, cycling conditions, and the choice of DNA polymerase. Alhudiri and Abukreba [2] systematically optimised their P32-based assay by testing a gradient of annealing temperatures (55–65°C) and determined that 58°C provided the most specific amplification with minimal non-specific bands. They also evaluated the effect of different extraction methods on PCR performance, noting that high-quality DNA extraction from oral swabs was essential for consistent results. Sample type significantly influences PCR success: Villalba et al. [4] demonstrated that oral swabs were the sample of choice for early detection in the absence of scabs, as they allowed testing in pools of five without extensive loss in sensitivity. The pooling strategy, validated during the 2022–2023 SPPV outbreak in Spain, is a critical optimisation for cost-effective surveillance of large populations.

Inhibitor removal is another vital aspect of PCR optimisation. Many field samples, especially scabs, skin lesions, and faecal-contaminated swabs, contain compounds such as haemoglobin, polysaccharides, and humic acids that inhibit Taq polymerase. The use of commercial DNA extraction kits that incorporate inhibitor-removal steps (e.g., silica membrane columns or magnetic bead-based methods) is recommended. Tian et al. [38] developed a SYBR Green real-time PCR method using a plasmid construct carrying the P32 gene and reported that the assay tolerated a wide range of DNA template concentrations, achieving quantification over nine orders of magnitude (from 10¹ to 10⁹ copies). The addition of bovine serum albumin (BSA) or betaine to the PCR mix can further alleviate inhibition in problematic samples.

Real-Time PCR and High-Resolution Melting (HRM) Assays

Real-time PCR has largely supplanted conventional PCR for SPPV detection due to its superior sensitivity, quantification capability, and reduced risk of amplicon contamination. Tian et al. [38] established that SYBR Green-based real-time PCR was more sensitive than conventional gel-based PCR, allowing detection of low viral titers in infected sheep. However, SYBR Green assays lack specificity for differentiating between capripoxvirus species unless combined with post-amplification melting curve analysis. This limitation is overcome by high-resolution melting (HRM) PCR, which exploits minute sequence differences within the amplicon to produce distinct melting temperature (Tm) profiles.

Pestova et al. [50] developed an HRM assay targeting a 111-bp region of the LSDV010 ORF that uniquely discriminates SPPV, GTPV, field LSDV, and vaccine LSDV strains. The unique melting temperatures were: 74.24 ± 0.06°C for SPPV, 73.61 ± 0.04°C for GTPV, 74.56 ± 0.04°C for field LSDV, and 74.95 ± 0.08°C for vaccine LSDV. This assay, validated on a Rotor-Gene Q platform, demonstrated a detection limit of 0.1 TCID₅₀/mL using LSDV template and was reproducible across replicates and operators. The ability to differentiate vaccine from field strains is particularly valuable in regions where homologous or heterologous capripox vaccines are used, as it enables DIVA (differentiating infected from vaccinated animals) capabilities.

Sprygin et al. [26] tailored an HRM real-time PCR specifically for the NISKHI SPPV vaccine strain, targeting the RPO132 gene. A unique single nucleotide polymorphism (SNP) in the NISKHI strain altered the melting curve: the Tm for field isolates ranged from 75.47 ± 0.04 to 75.86 ± 0.08°C, while the vaccine strain averaged 76.46 ± 0.12°C. This assay was used to confirm that recent SPP outbreaks in central Russia were caused by virulent field isolates and not by reversion of the vaccine strain, a critical safety check for live attenuated vaccines. Similarly, a real-time PCR targeting the LW032 ORF was developed by Sprygin et al. [49] to specifically detect KSGP-related LSDV isolates and recombinant strains, providing an additional tool for epidemiological surveys in regions where multiple capripox lineages co-circulate.

Multiplex and Differential Diagnostic Platforms

Given the clinical overlap of sheep pox, goat pox, and lumpy skin disease, simultaneous detection and differentiation of the three capripoxviruses is a practical need. Wang et al. [45] developed a multiplex real-time PCR using universal primers for all capripoxviruses combined with species-specific probes for LSDV, SPPV, and GTPV. The probes, labelled with different fluorophores, allowed single-tube discrimination with a limit of detection of 10² copies of target genome DNA. The assay was validated on 557 clinical samples from local and Ethiopian outbreaks, and results were 100% concordant with conventional RFLP-PCR of P32 and RPO30 genes as well as sequencing. This multiplex approach reduces reagent costs, turnaround time, and sample volume requirement, advantages that are particularly important for high-throughput surveillance.

A different multiplexing strategy was employed by Uzar et al. [39], who used a combination of SYBR Green real-time PCR and virus neutralisation tests to evaluate the efficacy of heterologous SPP vaccines against LSDV in cattle. While not a diagnostic per se, the study highlights how multiplex molecular tools can be integrated with serological methods for comprehensive vaccine efficacy assessment. The choice of fluorophore and probe design must account for potential cross-talk and require rigorous optimisation of annealing temperature and primer/probe concentrations.

Isothermal Amplification: LAMP Assays

For field deployment and point-of-care diagnostics, isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) offer distinct advantages over PCR because they do not require thermal cyclers and can be performed using a simple heat block or water bath. Zhao et al. [31] described a LAMP assay based on ITR sequences that could differentiate SPPV from GTPV within 45–60 minutes at 62°C. Using three sets of LAMP primers (universal GSPV, GTPV-specific, and SPPV-specific), the assay achieved 100% detection for GTPV and 98.8% for SPPV when tested against 135 preserved epidemic materials. Importantly, no cross-reactivity was observed with Orf virus, foot-and-mouth disease virus, Anaplasma marginale, Mycoplasma mycoides, or various protozoan parasites. The LAMP assay also showed higher detection rates than conventional RFLP-PCR, likely due to its tolerance of inhibitors in crude sample lysates. The simplicity of LAMP makes it an ideal tool for low-resource laboratories and for rapid on-farm screening during outbreak investigations, though careful primer design is required to avoid false positives from non-specific amplification.

Sample Types, Pooling, and Pre-analytical Considerations

The success of any molecular diagnostic assay depends heavily on the quality and type of sample collected. For SPPV detection, skin scabs, papular lesions, oral swabs, and blood are the most common matrices. Villalba et al. [4] conducted an extensive surveillance during the Spanish outbreak, testing over 35,000 oral swabs from 335 farms by real-time PCR in pools of five. Their data demonstrated that pooling did not result in extensive sensitivity loss, provided that adequate mixing and a sufficiently sensitive PCR platform were used. However, they cautioned that a non-infectious genome of the virus could be detected months after cleaning and disinfection of premises, meaning that real-time PCR results from sentinel animals during repopulation must be interpreted with caution, a critical lesson for outbreak recovery phases.

For skin lesions, scab material contains high viral loads, but the presence of necrotic tissue and contaminating bacteria can inhibit PCR. Metagenomic profiling of pox lesions by Sharko et al. [36] revealed high bacterial diversity, including Fusobacterium necrophorum, Streptococcus dysgalactiae, and Trueperella pyogenes, which may interfere with nucleic acid extraction and amplification. Therefore, thorough homogenisation, proteinase K digestion, and optional pre-filtration steps are recommended for scab samples. Blood samples are less sensitive for early detection because viremia may be transient; serological testing (ELISA) is often preferred for herd-level immunity assessment [4, 27]. Nonetheless, PCR on blood can be useful in acute cases, especially if combined with quantitative analysis to monitor viral load [38].

Quality Control, Validation, and Interpretation Challenges

Rigorous validation of PCR assays for SPPV detection must include assessment of analytical sensitivity (limit of detection), analytical specificity (cross-reactivity panel), repeatability, and reproducibility. The WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals provides guidelines for validation, including the use of well-characterised reference strains and field isolates. For the HRM and real-time assays described by Pestova et al. [50] and Sprygin et al. [26], acceptance criteria included a coefficient of variation for cycle threshold (Ct) values below 5% across intra- and inter-run replicates. The inclusion of internal amplification controls (e.g., exogenous DNA spiked into each sample) is essential to detect inhibition.

One significant challenge is the interpretation of positive results in the context of vaccination. Since many countries use live attenuated SPPV vaccines (e.g., NISKHI, RM65, Bakırköy) [26, 39, 43], PCR assays that cannot differentiate vaccine from field strains may yield false-positive outbreak reports. The HRM assay of Sprygin et al. [26] addressed this by targeting the RPO132 SNP, but such DIVA capabilities are not yet standardised across all diagnostic laboratories. Furthermore, the risk of vaccine virus transmission is considered minimal [43], but molecular detection alone cannot distinguish between residual vaccine nucleic acid and active infection. Therefore, PCR results should be integrated with clinical signs, serology, and epidemiological context.

Another pitfall is the detection of non-infectious viral DNA in the environment. Villalba et al. [4] found that after cleaning and disinfection, real-time PCR could still amplify SPPV DNA from surfaces and sentinel animal swabs for months, long after viral infectivity had been lost. This highlights the need for combined molecular and virological (e.g., virus isolation or cell culture) approaches for confirmation when assessing post-outbreak freedom from infection. From a One Health perspective, while SPPV is not zoonotic, its economic impact on small ruminant production, particularly in Africa, Asia, and the Middle East, warrants the development of robust, fit-for-purpose molecular tools that can be deployed under field conditions to support rapid response and eradication programmes, as emphasised by WOAH and FAO in their global strategies for transboundary animal diseases.

Antiviral Strategies and In Silico Discovery of SPPV14 Inhibitors

Strategic Context for Antiviral Development Against Sheep Pox Virus

Sheep pox virus (SPPV) remains a formidable pathogen of small ruminants, listed as a notifiable disease by the World Organisation for Animal Health (WOAH) and recognized by the Food and Agriculture Organization (FAO) as a major constraint to livestock productivity in endemic regions spanning Africa, Asia, and the Middle East. The disease exacts a profound economic toll, manifesting in mortality rates that can approach 100% in susceptible flocks, permanent hide damage that devastates the tanning industry, and severe restrictions on international animal movement and trade [1, 3, 29]. Traditional control strategies have relied almost exclusively on vaccination with live attenuated strains, such as the NISKHI strain used extensively across Russia and Central Asia, the Romanian Fanar strain, the Bakırköy strain employed in Turkey, and various homologous and heterologous capripoxvirus vaccines deployed against both SPPV and lumpy skin disease virus [5, 7, 11, 26, 27, 39, 46, 52]. While these vaccines have demonstrated efficacy in reducing clinical disease and viral shedding, they are not without limitations. Concerns regarding potential reversion to virulence, interference with serological surveillance, cold-chain requirements in resource-limited settings, and the inability to protect against heterologous strains in certain epidemiological contexts underscore the pressing need for alternative or adjunctive antiviral strategies [1, 14]. The emergence of recombinant field strains, documented in recent outbreaks across Kazakhstan and Central Russia, further complicates the landscape and demands a diversified therapeutic arsenal [6, 7].

The SPPV14 Protein: A Critical Viral Antiapoptotic Target

At the molecular level, SPPV encodes a repertoire of proteins designed to subvert host antiviral defenses, and among these, the SPPV14 protein has emerged as a particularly compelling target for therapeutic intervention. SPPV14 functions as a potent inhibitor of BCL-2-mediated apoptosis, a fundamental host defense mechanism that eliminates virus-infected cells and curtails viral propagation [1]. The BCL-2 family of proteins governs the intrinsic apoptotic pathway through a delicate balance of pro-apoptotic (BAX, BAK, BH3-only proteins) and anti-apoptotic (BCL-2, BCL-XL, MCL-1) members. In response to viral infection, host cells upregulate BH3-only proteins, such as BIM, BID, and PUMA, which engage and neutralize anti-apoptotic BCL-2 homologs, thereby liberating BAX and BAK to permeabilize the mitochondrial outer membrane and initiate the caspase cascade. SPPV14 subverts this checkpoint by mimicking host BCL-2 proteins, binding with high affinity to BH3 motifs and sequestering them away from their pro-apoptotic targets, thus effectively disabling the host's capacity to execute the infected cell [1]. This functional mimicry is evolutionarily conserved among poxviruses, but SPPV14 exhibits unique structural features that distinguish it from host homologs and from the antiapoptotic proteins encoded by other capripoxviruses. Specifically, the Arg84 residue within SPPV14 has been identified as essential for protein stability and for mediating the canonical ionic interaction with BH3 motif ligands [1]. Disruption of this key residue or of its interaction interface therefore represents a rational, structure-based strategy for restoring apoptosis in SPPV-infected cells and limiting viral dissemination.

Computational Screening of Flavonoids as SPPV14 Inhibitors

The recent in silico exploration of natural product libraries against SPPV14 marks a significant advance in the search for direct-acting antiviral agents against sheep pox [1]. Flavonoids, a diverse class of polyphenolic secondary metabolites ubiquitously distributed in fruits, vegetables, and medicinal plants, have long been recognized for their broad-spectrum antiviral properties, including activity against influenza virus, hepatitis C virus, human immunodeficiency virus, and numerous coronaviruses. Their mechanisms of action are multifaceted, encompassing inhibition of viral polymerases, proteases, and entry/fusion processes, as well as modulation of host inflammatory and oxidative stress responses. The application of these compounds to capripoxvirus infections, however, had remained largely unexplored until the rigorous computational investigation conducted by Krishna and colleagues in 2025 [1].

Using the AutoDock Vina molecular docking platform, a curated panel of ten flavonoids was screened for binding affinity to the active site of SPPV14, with particular emphasis on interactions involving the Arg84 residue. The computational methodology employed is predicated on the assumption that high-affinity ligand-receptor interactions, as quantified by predicted binding free energies (ΔG) and dissociation constants (Ki), correlate with functional inhibition. All ten flavonoids evaluated demonstrated significant binding affinities to SPPV14, with values in the range typically associated with potent small-molecule inhibitors. Notably, isoxanthohumol, a prenylated flavonoid abundant in hops (Humulus lupulus) and beer, exhibited the most remarkable interaction profile, forming stable contacts with Arg84 that were predicted to be far more energetically favorable than those observed for the other tested compounds [1]. This is a critical finding, because the stability of the SPPV14:Arg84 interaction is paramount for the integrity of the viral protein's antiapoptotic function; any ligand that competes with or disrupts this site would be expected to abrogate BH3 motif binding and thereby relieve the blockade on host apoptosis.

Mechanistic Implications of Flavonoid-SPPV14 Interactions

Perhaps the most striking observation from the docking simulations was that the selected flavonoids comprehensively eliminated the canonical ionic interaction observed in the native SPPV14:BH3 motif complex [1]. In the absence of inhibitor, the BH3 motif of pro-apoptotic host proteins forms a salt bridge and hydrogen bond network with Arg84 of SPPV14, an interaction that is essential for high-affinity binding and for the subsequent neutralization of the pro-apoptotic signal. The flavonoids, by contrast, appeared to occupy the same binding pocket but through a distinct set of non-covalent forces, predominantly hydrophobic contacts, π-stacking interactions with aromatic residues, and hydrogen bonds with neighboring polar amino acids, that effectively occluded the BH3 binding groove without engaging in the Arg84-mediated ionic clamp [1]. This differential binding mode has profound functional consequences: it suggests that the flavonoids may act as competitive antagonists of BH3 motif recognition, preventing SPPV14 from sequestering endogenous pro-apoptotic proteins and thereby preserving the host cell's ability to undergo apoptosis in response to infection. From a virological standpoint, restoration of the apoptotic program in SPPV-infected cells would be expected to limit viral replication, reduce the duration and severity of clinical disease, and potentially decrease the quantity of virus shed into the environment, thereby interrupting transmission chains at the individual animal and flock level.

Broader Significance and the Path to Validation

The significance of this in silico discovery extends beyond the immediate identification of flavonoid leads. It establishes a robust computational framework for the rational design of SPPV14 inhibitors, a protein target that had received little attention prior to this work despite its centrality to viral pathogenesis [1]. The focus on Arg84 as a critical hotspot for inhibitor binding is particularly astute, as it mirrors successful strategies employed against other viral antiapoptotic proteins, such as the BCL-2 homologs encoded by vaccinia virus (F1L) and myxoma virus (M11L). Moreover, the use of naturally occurring flavonoids as a chemical starting point offers practical advantages: they are generally recognized as safe, readily available from dietary sources or commercial suppliers, and amenable to medicinal chemistry optimization through prenylation, glycosylation, or methylation to improve potency, selectivity, and pharmacokinetic properties. Isoxanthohumol, for instance, can be metabolized in vivo to 8-prenylnaringenin, a compound with even greater estrogenic and antiangiogenic activity, raising the possibility that both parent and metabolite could contribute to antiviral efficacy.

However, it is imperative to acknowledge the limitations inherent in computational predictions. Molecular docking provides a static, simplified representation of the dynamic and solvated environment in which protein-ligand interactions occur; binding affinity predictions are sensitive to scoring function parameterization, protonation states, and conformational flexibility of both receptor and ligand. False positives are not uncommon, and the docked poses must be validated through rigorous molecular dynamics simulations to assess the stability of the predicted complexes over time and in the presence of explicit water molecules. Furthermore, flavonoids are notorious for promiscuous binding, they can interact with multiple cellular targets, including kinases, transcription factors, and membrane receptors, and their antiviral activity in silico may not translate to selective inhibition of SPPV14 in the context of a live virus infection [1]. The concentrations required for antiviral efficacy in cell culture may exceed achievable plasma or tissue levels following oral administration, and off-target effects could lead to toxicity or immunosuppression that negates any therapeutic benefit.

Future Directions for Antiviral Development Against SPPV

The path forward demands a systematic, multi-tiered validation pipeline. First, the top-ranking flavonoid hits, particularly isoxanthohumol, should be tested in vitro using plaque reduction assays in permissive cell lines, such as lamb testis cells, Vero cells, or primary kidney cells, against both vaccine strains (e.g., NISKHI, Romanian Fanar) and virulent field isolates of SPPV [19, 46, 47]. Cytotoxicity must be evaluated in parallel using standard MTT or neutral red uptake assays to establish a therapeutic index. Mechanistic confirmation of apoptosis restoration can be achieved through flow cytometric detection of Annexin V/propidium iodide staining, caspase-3/7 activation assays, and western blot analysis of PARP cleavage in flavonoid-treated, SPPV-infected cells compared to infected controls. Co-immunoprecipitation experiments could further demonstrate whether flavonoid treatment disrupts the physical association between SPPV14 and host BH3-only proteins in lysates from infected cells. Second, promising candidates should advance to pharmacokinetic and toxicity assessment in rodent models to determine oral bioavailability, half-life, tissue distribution, and maximum tolerated dose. Third, efficacy trials in the natural ovine host are ultimately required: experimentally infected lambs, challenged intradermally with a virulent SPPV strain (e.g., the "Afghanistan" strain or locally circulating isolates), would be treated orally or parenterally with the lead flavonoid, and outcomes measured include reduction in lesion number and size, duration of pyrexia, viral load in blood and swabs by quantitative PCR [38, 44, 51], and survival [51]. A concurrent vaccine efficacy group could explore whether flavonoid therapy augments or interferes with the immune response to vaccination [43]. Finally, structure-activity relationship studies around the isoxanthohumol scaffold could identify synthetic analogs with improved potency, metabolic stability, and reduced off-target interactions, potentially yielding a clinical candidate suitable for field use in endemic regions.

The in silico discovery of flavonoids as SPPV14 inhibitors represents a foundational step toward the development of the first direct-acting antiviral agents against sheep pox virus. By targeting the viral antiapoptotic machinery rather than host factors, this approach minimizes the risk of host toxicity while maximizing the barrier to viral resistance, as mutations that restore BH3 binding would likely compromise the structural integrity of SPPV14 itself. If validated through the rigorous experimental framework outlined above, flavonoid-based therapeutics could complement existing vaccination programs, provide an intervention option for outbreak settings where vaccination is contraindicated or logistically impractical, and reduce reliance on culling as a control measure, a particularly important consideration for smallholder farmers and pastoralist communities where sheep represent both livelihood and food security [3, 6, 16, 21, 40]. The convergence of computational chemistry, structural virology, and natural product pharmacology holds substantial promise for transforming the management of this devastating transboundary disease.

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