Fowl Pox Virus

Overview and Taxonomy of Fowl Pox Virus

Taxonomic Hierarchy and Phylogenetic Position

Fowl pox virus (FWPV) occupies a definitive position within the virological landscape as the archetypal and most economically consequential member of the genus Avipoxvirus, a taxon nested within the subfamily Chordopoxvirinae of the family Poxviridae [1, 2]. This family comprises large, enveloped, double-stranded DNA viruses characterized by their cytoplasmic replication strategy, a feature that distinguishes them fundamentally from most other DNA viruses that replicate within the host cell nucleus. The genus Avipoxvirus encompasses a diverse array of viruses capable of infecting over 200 species of birds, spanning both domesticated poultry and a wide variety of wild avian species [2]. The taxonomic organization within this genus has historically been challenging, but contemporary phylogenetic analyses, particularly those targeting the highly conserved P4b core protein gene (a homologue of the vaccinia virus A3L gene), have provided a robust framework for classification [1, 5, 9].

Phylogenetic studies consistently delineate at least three major clades: Clade A, Clade B, and Clade C [1]. Clade A, which contains the majority of viruses isolated from gallinaceous birds, including chickens, turkeys, and quail, is further subdivided into subclades A1 through A7 [1, 9]. The FWPV strains responsible for the vast majority of commercial poultry outbreaks globally cluster within subclade A1, a grouping that includes both virulent field isolates and the traditional vaccine strains currently in widespread use [1, 5]. This clustering is not merely a matter of academic classification; it has profound implications for vaccine efficacy and disease control. The close phylogenetic relationship between field strains and vaccine strains within subclade A1 suggests that vaccine-induced immunity theoretically should be protective; however, the emergence of novel variants bearing specific amino acid substitutions, such as the E120K and N121D mutations observed in recent Egyptian isolates, raises critical questions about potential antigenic drift within this subclade [1]. Similarly, isolates from the Giza governorate of Egypt, characterized by analysis of the thymidine kinase (TK) gene, have demonstrated genomically distinct profiles, showing as little as 60% similarity to other published FPV sequences, underscoring the existence of locally circulating, potentially emergent strains that may evade existing immunological protections [3].

Virion Morphology and Genomic Architecture

The FWPV virion exhibits the classic brick-shaped or ovoid morphology characteristic of poxviruses, measuring approximately 200-300 nm in diameter, a size sufficient to be visualized by light microscopy in specially stained preparations [2]. The external envelope, which is not acquired from the host cell membrane but is instead synthesized de novo by the virus, surrounds a complex internal core containing the linear double-stranded DNA genome. Electron microscopic studies of infected chorioallantoic membranes have revealed the typical features of poxvirus replication within the cytoplasm of infected epithelial cells: the formation of viral factories, the progressive vacuolation and granulation of the cytoplasm, and the eventual accumulation of mature virion clumps both at the cell surface and deep within degenerating tissues [8].

The FWPV genome is among the largest known among viruses, spanning approximately 270-290 kilobase pairs and encoding for over 250 open reading frames [2]. This expansive genetic repertoire equips the virus with the machinery necessary for autonomous replication within the cytoplasm, including its own DNA-dependent RNA polymerase, transcription factors, and enzymes for nucleotide metabolism. The terminal regions of the genome are characterized by inverted terminal repeats and hairpin loops, a structural feature common to all poxviruses that facilitates genome replication and encapsidation. Key genes that serve as molecular targets for diagnostic and phylogenetic analyses include the P4b core protein gene (frequently amplified as a 578-base pair fragment for routine PCR detection), the thymidine kinase (TK) gene (a 305-base pair fragment useful for strain differentiation), and the 4b core protein gene utilized in phylogenetic typing [1, 3-5]. The TK gene is particularly noteworthy because its disruption forms the basis for generating recombinant FWPV-based vaccine vectors, a technology that has been exploited extensively in modern veterinary vaccinology [2, 3].

Host Range Specificity and Cross-Species Infectivity

Perhaps the most clinically and epidemiologically significant aspect of FWPV taxonomy is the pattern of host range restriction within the Avipoxvirus genus. Historically, avipoxviruses were classified on the basis of the avian host from which they were isolated, leading to designations such as fowl pox virus (from chickens), turkey pox virus, pigeon pox virus, canary pox virus, and others [2, 5, 9]. However, modern molecular characterization has revealed that this host-based nomenclature is an oversimplification, as viruses from different avian orders may segregate into distinct phylogenetic clades that do not strictly correspond to traditional host names. Three broad host range groupings have been proposed: (a) "fowlpox-like viruses" predominantly isolated from Galliformes (chickens, turkeys, quail), (b) "canarypox-like viruses" from Passeriformes (canaries, finches, sparrows), and (c) viruses associated with Psittaciformes (parrots, macaws) [2]. This classification is biologically meaningful because it reflects genuine barriers to cross-species transmission, although these barriers are not absolute.

The host specificity of FWPV is broader than traditionally appreciated. Experimental infections have demonstrated that FWPV can productively infect pigeons, with one Egyptian isolate (ch-08TK) inducing characteristic "takes" in 75% of inoculated pigeons following feather follicle inoculation, compared to 100% in chickens [3]. Furthermore, pigeon pox virus (PPV) has been historically employed as a heterologous vaccine against fowl pox, a practice dating back to the 1930s, predicated on the observation that PPV induces protective immunity in chickens while causing only mild, localized reactions [7]. Contemporary molecular data confirm that pigeon pox viruses cluster phylogenetically within the same clade A as fowl pox viruses, often showing 100% sequence identity in the P4b gene to certain fowl pox isolates from geographically distant regions, suggesting a shared ancestral lineage and potential for cross-protection [5, 6]. This is further exemplified by a recent outbreak in Libya, where FPV and PPV isolates from the same geographical region exhibited high genetic similarity to strains from Iraq, Iran, Brazil, Egypt, and India, indicating that these viruses are not strictly host-restricted but rather circulate within a broader avian reservoir, potentially facilitated by mechanical vectors such as mosquitoes [5].

Biological Implications of Taxonomic Diversity

The taxonomic diversity within the Avipoxvirus genus carries profound implications for disease pathogenesis, vaccine strategy, and global surveillance. The biological basis for host range restriction lies in the virus's ability to evade or subvert the host innate immune response, a capacity encoded by specific virulence genes that may vary significantly between strains infecting different avian orders. For example, canarypox viruses tend to cause a more severe, systemic, and often fatal disease in their natural hosts compared to the typically self-limiting cutaneous form seen with FWPV in chickens, reflecting fundamental differences in their interaction with the avian immune system [2]. This variation is reflected at the genomic level: complete genome sequencing projects have revealed that avipoxviruses possess a highly plastic genome with extensive regions of recombination, gene duplication, and gene loss, particularly in the terminal variable regions that encode host range factors and immunomodulatory proteins [2].

The emergence of fowl pox outbreaks in vaccinated commercial flocks worldwide, a phenomenon documented across multiple continents, highlights the pressing need for a more nuanced understanding of FWPV taxonomy and evolution [1-3]. The phylogenetic placement of field isolates relative to vaccine strains is not merely an academic exercise; it is a critical determinant of vaccine efficacy. When circulating field strains diverge significantly from vaccine strains at key antigenic epitopes, as suggested by the limited TK gene homology observed in certain Egyptian isolates, or by specific amino acid changes in the P4b protein, the potential for vaccine failure increases [1, 3]. The World Organisation for Animal Health (WOAH) recognizes fowl pox as a disease of significant economic impact, particularly in regions where the poultry industry is expanding rapidly and where vector populations are abundant. The taxonomic insights derived from ongoing molecular surveillance efforts, such as those supported by FAO and national veterinary services, are essential for updating vaccine seed strains and for designing effective disease control programs that account for the genetic flux within this ancient and adaptable genus of viruses.

Molecular Pathogenesis of Fowl Pox Virus

Introduction: A Poxviral Paradigm of Host Cell Manipulation

The molecular pathogenesis of Fowl Pox Virus (FWPV) represents a sophisticated paradigm of viral subversion, cellular remodeling, and immune evasion, resulting in a disease of profound economic significance to the global poultry industry. As a member of the genus Avipoxvirus within the family Poxviridae, FWPV is distinguished by its large, double-stranded DNA genome, its cytoplasmic replication strategy, and its remarkable capacity to induce both proliferative and necrotic lesions depending on the host tissue and viral strain [2]. The World Organisation for Animal Health (WOAH) classifies fowlpox as a notifiable disease due to its capacity to cause severe drops in egg production, growth retardation in young birds, and substantial mortality, particularly in unvaccinated or immunocompromised flocks [2]. Understanding the molecular underpinnings of FWPV pathogenesis is not merely an academic exercise; it is essential for the rational design of next-generation vaccines, the interpretation of emerging field variants, and the elucidation of host-range determinants that allow this virus to cross species barriers with alarming frequency [2].

Viral Entry, Replication, and Cytoplasmic Factories

The molecular pathogenesis of FWPV begins at the portal of entry, typically through abrasions in the skin or mucous membranes, often facilitated by mechanical vectors such as mosquitoes [2]. The virus exhibits a marked tropism for epithelial cells, a property that dictates the clinical course of the disease. Upon attachment to the host cell membrane, FWPV enters via macropinocytosis or direct fusion, a process mediated by a complex of viral surface proteins that remain poorly characterized in avipoxviruses compared to orthopoxviruses. Once inside the cytoplasm, the virus disassembles its core, releasing the genomic DNA into the cytosol. This is a critical juncture: FWPV, like all poxviruses, replicates entirely within the cytoplasm, an extraordinary feat that requires the virus to encode its own machinery for DNA replication, transcription, and RNA processing, thereby bypassing the host cell’s nuclear restriction [8].

The replication cycle proceeds within discrete cytoplasmic foci, termed viral factories or B-type inclusion bodies. Electron microscopy studies of FWPV-infected chorioallantoic membranes (CAM) have provided exquisite detail of these structures, demonstrating extensive vacuolation and granulation within proliferating epithelial cells, and revealing clumps of mature virions both at the cell surface and deep within the diseased epithelium [8]. The assembly of progeny virions proceeds through a series of morphogenetic steps, from crescent membranes to immature virions, then to mature intracellular virions. The release of virus is often cytopathic, but FWPV can also induce cell-to-cell spread via actin-based motility, a mechanism that facilitates local dissemination without exposure to the extracellular environment [10, 11]. This cell-associated nature of the virus is clinically relevant: early after infection, the virus is highly cell-associated for the first 84 hours post-inoculation, before a significant proportion becomes cell-free, reaching maximum titers in the supernatant by 120 hours [11]. This temporal shift from cell-associated to cell-free virus correlates with the progression from localized infection to systemic spread.

Cellular Pathology: From Hyperplasia to Necrosis

The hallmark of FWPV pathogenesis is the induction of two distinct clinical forms: the cutaneous (dry) form, characterized by proliferative, wart-like lesions on featherless skin (comb, wattle, eyelids, legs), and the diphtheritic (wet) form, characterized by necrotic, fibrino-necrotic lesions on the mucous membranes of the upper respiratory tract and digestive system [2, 5]. These divergent pathologies are rooted in the virus's molecular interaction with the host cell.

In the cutaneous form, FWPV drives a pronounced hyperplasia and hypertrophy of epidermal keratinocytes [2]. This proliferative response is orchestrated by viral proteins that mimic host growth factors and manipulate cell cycle checkpoints. Poxviruses are masters of encoding homologues of host cytokines, growth factors, and their receptors. For FWPV, a virally encoded epidermal growth factor (EGF)-like protein is hypothesized to bind to the host EGF receptor, triggering a signaling cascade that leads to unchecked cellular proliferation. This results in the characteristic pock lesions observed on the CAM of embryonated eggs, where field isolates produce large, 3-5 mm pocks that represent foci of intense epithelial proliferation [10]. Histologically, these pocks are composed of acanthotic, hyperkeratotic epidermis with massive ballooning degeneration of keratinocytes, ultimately leading to the formation of intracytoplasmic inclusion bodies (Bollinger bodies), which are the pathognomonic feature of avipoxvirus infection [5, 10].

Conversely, in the diphtheritic form, the virus induces necrosis of mucosal epithelial cells. The formation of pseudomembranes composed of fibrin, necrotic debris, and inflammatory cells is a direct consequence of viral-induced cell death and secondary bacterial invasion. The balance between proliferation and necrosis is exquisitely strain-dependent. Seminal work by Buddingh demonstrated that serial intracerebral passage of FWPV in chicks dramatically alters its molecular pathogenesis: the virus acquires a greatly increased virulence for epithelial cells in vivo, shifting from a proliferative to a rapidly necrotizing phenotype, and concurrently acquires an affinity for mesodermal cells, including vascular endothelium [14, 15]. This suggests that the genetic determinants of tissue tropism and cell fate (proliferation vs. necrosis) are malleable and can be selected for within a few passages, a finding with profound implications for understanding the emergence of virulent field strains.

Genetic Determinants of Pathogenesis: The P4b Core Protein and TK Gene

The molecular dissection of FWPV pathogenesis has largely focused on two key genetic loci: the P4b core protein gene and the thymidine kinase (TK) gene.

The P4b gene, encoding a major core protein of the virion, is the primary target for molecular epidemiology and phylogenetic studies of avipoxviruses [1, 5, 9]. This gene is highly conserved among avipoxviruses but contains variable regions that allow for clade and subclade differentiation. Phylogenetic analysis of the P4b gene consistently places FWPV isolates within Clade A, subclade A1, which encompasses fowlpox viruses from Galliformes [1, 5, 9]. Crucially, specific amino acid substitutions in the P4b protein have been associated with emerging field strains. For example, recent Egyptian isolates from Al-Sharkia Governorate (2021) harbored distinct mutations: Avipoxvirus-Egy-f1086-2-P4b carried mutations E120K and N121D, while Avipoxvirus-Egy-f1086-3-P4b carried H83P and S93N [1]. The functional significance of these mutations is not fully elucidated, but they are located within a region of the protein that may be involved in viral assembly or immune recognition. The presence of such mutations in vaccinated flocks raises the critical question of whether these changes contribute to antigenic drift, allowing the virus to partially escape vaccine-induced immunity [1, 2]. The P4b gene is also a robust diagnostic target, as PCR amplification of a 578 bp fragment of this gene reliably detects FWPV in clinical samples [1].

The thymidine kinase (TK) gene is a second critical determinant of FWPV pathogenesis. TK is a non-essential enzyme for viral replication in actively dividing cells, but it is crucial for replication in quiescent or terminally differentiated cells, such as neurons. The TK gene is also a classic locus for the insertion of foreign genes in the development of recombinant FWPV vaccines [2]. Intriguingly, phylogenetic analysis of the TK gene from an Egyptian field isolate (ch-08TK, Accession No. KF314718) revealed profound genetic divergence, showing no more than 60% similarity to other published FWPV TK sequences from Egypt and other canarypox and pigeonpox viruses [3]. This level of diversity suggests that the TK gene may be under different selective pressures than the P4b gene, possibly reflecting host adaptation or recombination events. The functional consequence of such a divergent TK gene for viral pathogenesis remains unknown, but it could influence the virus's ability to replicate in specific cell types or tissues, thereby altering its tissue tropism and virulence.

Host Range Determinants and Cross-Species Transmission

The molecular basis of host range in avipoxviruses is a complex and evolving paradigm. Historically, FWPV was considered restricted to Galliformes (chickens, turkeys, quail), but mounting evidence reveals a broader host range than previously appreciated, with "fowlpox-like viruses" now isolated from a widening array of avian species [2]. The molecular determinants of this host range are multifactorial and encoded by specific viral genes that counteract host restriction factors.

Poxviruses encode a suite of host range (HR) genes that modulate the interferon (IFN) response, apoptosis, and NF-κB signaling. While the specific HR genes of FWPV are less well-characterized than those of vaccinia virus, it is clear that FWPV is exquisitely sensitive to the host interferon system. Recent investigations into the immune response following FWPV vaccination reveal that the protective immune response is dominated by a Th1-type response, characterized by high levels of IFN-γ production relative to IL-4 [13]. IFN-γ is a potent antiviral cytokine that induces an antiviral state in cells, upregulating MHC class I expression and activating macrophages. FWPV must therefore possess mechanisms to circumvent IFN-γ signaling. The emergence of strains like the one from Giza, Egypt, which showed 75% pathogenicity in pigeons (a non-galliform host) [3], suggests that these viruses may have lost or modified a host range factor that normally restricts replication in other avian species. This molecular plasticity is a major concern for disease control, as it allows for the establishment of viral reservoirs in wild birds that can spill over into domestic poultry [2, 5].

Immunomodulation and Systemic Spread

FWPV has evolved sophisticated strategies to subvert the host immune response. The virus encodes virokines and viroceptors that neutralize cytokines and chemokines, dampening the inflammatory response and delaying the recruitment of effector cells. The observation that FWPV can replicate and cause pathological lesions in the bone marrow of cranial bones, paranasal sinuses, and orbital tissues following intracerebral inoculation [15] indicates that the virus can access and replicate in mesenchymal tissues, a property that is likely mediated by viral genes that suppress antiviral immunity in these compartments.

Systemic spread is further facilitated by the virus's ability to induce cell-associated viremia. Infected cells, particularly monocytes and macrophages, can carry the virus to distant sites, including the liver, kidneys, and central nervous system. Safety evaluations of cell culture-adapted FWPV vaccines have shown that even attenuated strains can induce mild histopathological changes, including renal tubular necrosis, hepatocyte necrosis, hemorrhage, and vascular congestion, as well as polymorphonuclear cell clusters in lung and liver tissues [12]. While these changes are not associated with clinical disease, they demonstrate that even attenuated FWPV retains the capacity to induce molecular pathology in internal organs, underscoring the virus's inherent tissue tropism.

The Emergence of Variants and Molecular Surveillance

The molecular pathogenesis of FWPV is not a static entity. The virus continues to evolve, driven by selective pressures from vaccination, vector ecology, and host genetics. The high degree of genetic similarity observed among recent FWPV isolates from global outbreaks, with Libyan isolates showing 100% identity to strains from Iraq, Iran, and Brazil [5], suggests that certain clonal lineages are circulating widely and are highly successful. Conversely, the identification of novel genetic variants, such as the divergent TK gene isolate from Egypt [3], indicates that the molecular landscape of FWPV is more dynamic than previously thought.

This heterogeneity has direct implications for vaccine efficacy. The detection of P4b mutations in field isolates [1] and the documented occurrence of fowlpox outbreaks in vaccinated flocks [2] signal that antigenic drift or the emergence of vaccine-resistant strains may be occurring. The World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) emphasize the need for continuous molecular surveillance of avipoxviruses to track the emergence of such variants and to inform vaccine strain selection. The development of real-time PCR assays with melting curve analysis capable of distinguishing between different FWPV strains [4] represents a powerful tool for monitoring viral evolution at the molecular level.

Furthermore, the observation from standard curve analyses that some real-time PCR assays exhibit greater than 100% efficiency [4] raises technical considerations about the accuracy of viral quantification, which is critical for both pathogenesis studies and vaccine potency testing. Such anomalies may reflect the presence of inhibitors, the formation of secondary structures in the target sequence, or the amplification of defective interfering particles, all of which can confound molecular analyses of viral load and replication kinetics.

Epidemiology and Global Distribution of Fowl Pox Virus

Fowl pox virus (FWPV), a member of the genus Avipoxvirus within the family Poxviridae, represents one of the most economically burdensome viral pathogens affecting global poultry production. As a notifiable disease of considerable significance to the World Organisation for Animal Health (WOAH) and a pathogen of interest to the Food and Agriculture Organization (FAO) due to its impact on food security in developing nations, FWPV exhibits a truly worldwide distribution. Its epidemiology, however, is far from uniform, characterized by a complex interplay of viral strain diversity, host susceptibility, climatic influences, vector ecology, and anthropogenic factors such as vaccination practices and biosecurity implementation. The disease is frequently described as endemic in many regions, yet its incidence waxes and wanes in response to these dynamic variables, making a comprehensive understanding of its global distribution essential for effective control strategies.

Global Prevalence and Endemicity Patterns

Fowl pox is acknowledged as a disease of global importance, with documented presence on every continent where poultry are raised [2]. However, the status of infection varies dramatically across geographical regions. In industrialized nations with intensive poultry production systems and robust vaccination programs, FWPV often occurs in sporadic, localized outbreaks, typically associated with breakdowns in biosecurity, lapses in vaccination coverage, or the emergence of antigenically divergent strains. Conversely, in many parts of Africa, Asia, and the Middle East, fowl pox remains enzootic, causing continuous, low-level morbidity and periodic epizootics that result in significant economic losses [1, 2]. The disease is frequently observed across all production spectrums, from smallholder backyard flocks to large commercial layer and breeder operations, primarily due to insufficient vaccination coverage, the use of sub-potent vaccines, or the circulation of field strains that are genetically distinct from vaccine strains [2]. This endemicity is particularly pronounced in tropical and subtropical climates, where conditions favor the proliferation of arthropod vectors, primarily mosquitoes and biting flies, which serve as mechanical vectors for FWPV transmission. The virus’s ability to persist in dried scabs for extended periods, months to years, further contributes to its maintenance in environments with poor sanitation and high population densities of susceptible birds.

Regional Epidemiology: A Focus on Africa and the Middle East

The epidemiological landscape of fowl pox in Africa and the Middle East provides a compelling illustration of the disease’s complex dynamics. Recent molecular investigations have revealed a surprising degree of genetic heterogeneity among circulating FWPV strains. In Egypt, for instance, a 2021 study of commercial layer flocks in Al-Sharkia Governorate detected FWPV in five out of six sampled populations using PCR targeting the P4b core protein gene [1]. Phylogenetic analysis of these isolates grouped them within subclade A1 of Clade A, demonstrating 99.99% homology with local reference strains and the vaccinal strain used in Egypt [1]. This high degree of similarity to vaccine strains is paradoxical and raises critical questions about vaccine efficacy. While it may suggest that the circulating field viruses are antigenically similar to the vaccine, clinical outbreaks in vaccinated flocks indicate either insufficient vaccine take, waning immunity, or the presence of other virulence factors not captured by P4b gene analysis. The detection of specific amino acid mutations, such as E120K and N121D in one isolate and H83P and S93N in another, further underscores the potential for antigenic drift, warranting ongoing surveillance to ensure vaccine relevance [1].

A separate investigation in Egypt from 2012, focusing on a molecular identification of a field isolate from Giza governorate, isolated a strain (ch-08TK) from 30-day-old laying chickens manifesting pox lesions [3]. This isolate was successfully propagated on the chorioallantoic membrane (CAM) of specific pathogen-free (SPF) embryonated chicken eggs and demonstrated 100% pathogenic takes in experimentally infected chickens and 75% in pigeons [3]. Critically, phylogenetic analysis of the thymidine kinase (TK) gene of this isolate revealed very limited similarity (not exceeding 60%) with published sequences of other Egyptian FWPV isolates, including those from canarypox and pigeonpox viruses [3]. This finding is epidemiologically significant as it suggests the co-circulation of genetically distinct FWPV lineages within the same geographical region and time period. The presence of such a divergent strain (GenBank Accession No. KF314718) implies that the molecular epidemiology of FWPV is far more complex than previously appreciated, with multiple genetic clades potentially evolving independently and exhibiting different pathogenic or antigenic properties.

Further evidence of the dynamic nature of FWPV in the region comes from the first official record of a fowl pox outbreak in Libya, reported in 2025 [5]. This outbreak, which primarily affected backyard chickens, pigeons, and some commercial layers, was characterized by classical cutaneous lesions (wart-like growths and scabs). Histopathological examination confirmed the presence of eosinophilic intracytoplasmic inclusion bodies (Bollinger bodies), the pathognomonic feature of avipoxvirus infection [5]. Molecular characterization of four FPV isolates from this outbreak, based on the P4b gene, revealed a remarkable 100% genetic identity to selected isolates from Iraq, Iran, and Brazil [5]. This finding has profound epidemiological implications. The high degree of similarity between isolates from Libya and geographically distant countries suggests either a common ancestral origin, long-distance dissemination via migratory birds or the international trade in live poultry and poultry products, or the widespread use of genetically similar vaccine strains that have become established in the field. The Libyan outbreak underscores the need for enhanced surveillance across North Africa to monitor the transboundary spread of FWPV and to assess the impact of these novel introductions on local poultry populations.

Host Range, Transmission, and Vector Ecology

The epidemiology of fowl pox is inextricably linked to its host range and transmission mechanisms. While FWPV is primarily a pathogen of chickens and turkeys, its host specificity appears to be broader than historically assumed. The current taxonomic framework groups avipoxviruses into three major clusters based on host origin: (a) "fowlpox-like viruses" mainly isolated from Galliformes (chickens, turkeys, pheasants), (b) "canarypox-like viruses" from Passeriformes, and (c) viruses from Psittaciformes [2]. However, cross-species transmission events are increasingly documented. The Egyptian isolate ch-08TK, for instance, was able to infect pigeons, albeit with lower pathogenicity (75% takes) compared to chickens (100% takes) [3]. Similarly, pigeon pox virus (PPV) has been shown to be closely related to FPV, and PPV has historically been used as a live vaccine against fowl pox in some regions due to its lower virulence in chickens [7]. This cross-reactivity, while therapeutically useful, complicates the epidemiological picture, as multiple avipoxvirus species can circulate within the same ecological niche, potentially recombining or competing for susceptible hosts.

Transmission of FWPV occurs through two primary routes: mechanical vector-borne transmission and direct or indirect contact with infectious material. Arthropod vectors, particularly mosquitoes (Culex and Aedes spp.) and other blood-feeding dipterans, play a pivotal role in the epizootiology of the disease [2]. The importance of vectors is underscored by the seasonal pattern of outbreaks, which frequently peak during warm, wet months when mosquito populations are highest. The virus is mechanically transmitted when a contaminated mosquito feeds on a susceptible bird, introducing the virus through the skin. This mode of transmission is particularly efficient in tropical and subtropical environments and explains the high incidence of the cutaneous form of the disease in such regions [2]. Direct transmission occurs through contact with virus-laden scabs, dried crusts, or fomites such as contaminated equipment, feeders, and waterers. The virus is remarkably stable in the environment; dried scabs can retain infectivity for months, and contaminated premises can remain a source of infection for extended periods, contributing to the pattern of "recurring" outbreaks on the same farm year after year [7].

Molecular Epidemiology and Phylogenetic Insights

The application of molecular techniques, particularly sequencing and phylogenetic analysis of the P4b core protein gene, has revolutionized the understanding of FWPV epidemiology. The P4b gene is a highly conserved region of the avipoxvirus genome, making it an ideal target for phylogenetic classification. Global studies have consistently grouped FWPV isolates into two major clades: Clade A and Clade B. The vast majority of FWPV isolates from domestic chickens, including those from Egypt, India, Libya, and Brazil, fall within Clade A, and more specifically, within subclade A1 [1, 5, 9]. For example, a 2021 study on turkey pox virus from Maharashtra, India, found that the P4b gene sequences clustered within Clade A, showing 99% homology with Indian fowl pox virus isolates from chickens [9]. This suggests a common evolutionary origin for these Galliforme-adapted avipoxviruses and indicates that host adaptation is not absolute; viruses from turkeys can be genetically nearly indistinguishable from those infecting chickens.

However, the picture is not one of complete homogeneity. The discovery in Egypt of a FPV isolate with only 60% similarity in the TK gene to other local strains highlights the existence of genomically distinct subpopulations [3]. Furthermore, melting curve analysis of pigeon pox vaccine samples has revealed the presence of two distinct viral strains or variants within a single vaccine product, each with a slightly different melting temperature, while FPV samples contained only a single strain [4]. This finding has direct epidemiological and vaccinological implications: the presence of mixed viral populations within vaccines could theoretically lead to the emergence of novel recombinants or to differential immune responses that might not protect against all circulating field strains. The use of a single vaccine strain in the face of multiple circulating genetic clades may be a contributing factor to vaccine failure observed in several parts of the world [2, 11]. Therefore, molecular epidemiological surveillance that goes beyond the P4b gene to include whole-genome sequencing is urgently needed to fully characterize the genetic diversity of FWPV and to design vaccines that provide broad cross-protection. The Centers for Disease Control and Prevention (CDC) and WOAH have emphasized the importance of such genomic surveillance for emerging viral pathogens, and this principle applies with equal force to the control of fowl pox in global poultry production systems.

Drivers of Outbreaks and Vaccine Failure

The persistence of fowl pox as a significant disease despite the availability of effective vaccines is a central epidemiological paradox. Several factors contribute to this dynamic. First, the emergence of antigenic variants, as evidenced by the detection of mutations in the P4b and TK genes of field isolates, may allow the virus to partially evade vaccine-induced immunity [1, 3]. Second, vaccine coverage is often inadequate, particularly in backyard and smallholder production systems common in endemic regions [2]. Third, the use of poorly maintained or improperly stored vaccines can lead to reduced potency and immunogenicity. Fourth, the increasing complexity of host range, with the potential for avipoxviruses adapted to other bird species to infect chickens, provides a reservoir of virus that is not targeted by current FPV-specific vaccines [2]. Finally, the inherent stability of the virus in the environment and the ubiquity of arthropod vectors create a constant pressure of infection that can overwhelm even well-vaccinated flocks, especially if biosecurity measures are lax.

In Nigeria, for example, despite the widespread use of CAM-propagated fowl pox vaccines, outbreaks persist [12, 16]. This has prompted investigations into safer and more immunogenic cell culture-adapted vaccines. Studies have shown that cell culture vaccines produced in chicken embryo fibroblasts (CEF), quail embryo fibroblasts (QEF), duck embryo fibroblasts (DEF), and Vero cells are all sterile and immunogenic, with titers ranging from 10^7.3 TCID50/mL (Vero) to 10^9.3 TCID50/mL (CEF) [16]. All vaccinated birds developed "takes" within 7 days post-vaccination and induced high levels of serum antibodies [16]. Furthermore, safety evaluations of these cell culture-adapted vaccines showed 100% vaccine "takes" with no adverse reactions, disease, or mortality, and histological examination confirmed the absence of Bollinger bodies, indicating no virulence reversal [12]. These findings suggest that the shift towards cell culture-based vaccine production, already a global best practice, could enhance both vaccine quality and immunological protection, thereby helping to mitigate the epidemiological impact of FWPV in endemic regions like Nigeria and beyond.

In conclusion, the epidemiology and global distribution of fowl pox virus are characterized by a persistent and evolving threat. While the virus is present worldwide, its impact is disproportionately felt in regions with warm climates, high vector densities, and limited veterinary infrastructure. The co-circulation of genetically diverse strains, including some that show limited homology to vaccine strains, presents a formidable challenge to control. The increasing recognition of cross-species transmission events and the potential for vaccine-derived strains to become established in the field further complicates the epidemiological landscape. Addressing this problem requires a multi-pronged approach: enhanced molecular surveillance using standardized genetic markers to track viral evolution and spread; the development and deployment of more broadly protective vaccines, potentially incorporating multiple antigenic variants; rigorous quality control of vaccine production; and improved vector control and biosecurity measures at the farm level. Only through such comprehensive, globally coordinated efforts can the burden of this ancient and economically devastating disease be effectively reduced.

Genetic Diversity and Evolution of Fowl Pox Virus

The genetic landscape of Fowl Pox Virus (FWPV) represents a dynamic and continually evolving paradigm that challenges both our understanding of avipoxvirus biology and the efficacy of control measures. As a member of the genus Avipoxvirus within the family Poxviridae, FWPV exhibits a genomic plasticity that has profound implications for host range, pathogenicity, and vaccine breakthrough. The World Organisation for Animal Health (WOAH) classifies fowlpox as a notifiable disease due to its economic impact on global poultry production, yet the molecular epidemiology driving its persistence remains incompletely characterized. Recent molecular investigations have revealed that FWPV is not a static entity but rather a virus undergoing active diversification through point mutations, recombination events, and selective pressures imposed by host immunity and vaccination campaigns [1, 2].

Molecular Phylogeny and Clade Structure

The phylogenetic architecture of FWPV has been elucidated primarily through sequencing of the P4b core protein gene, a highly conserved locus that serves as the gold standard for molecular characterization of avipoxviruses. Comprehensive analyses consistently place FWPV isolates within Clade A, with the majority of field strains from gallinaceous birds clustering within subclade A1 [1, 5, 9]. This phylogenetic framework, however, masks considerable heterogeneity at the nucleotide level. Studies examining a 300-base pair region of the P4b gene from Egyptian isolates collected in 2021 demonstrated that while these strains exhibited 99.99% homology with local reference strains and vaccinal FWPV strains, they displayed significantly lower homology when compared to avipoxviruses originating from other avian species [1]. This pattern suggests that host-driven selection pressures are shaping the genetic architecture of FWPV at a finer scale than previously appreciated.

Notably, the phylogenetic positioning of FWPV isolates from different geographical regions reveals both conservation and divergence. Libyan isolates from a 2025 outbreak demonstrated 100% identity with strains from Iraq, Iran, and Brazil, indicating that certain viral lineages possess remarkable genetic stability across vast geographical distances [5]. Conversely, a thymidine kinase (TK) gene analysis of an Egyptian isolate from 2012 revealed a sequence that shared less than 60% similarity with published FWPV sequences, including strains from the same country [3]. This finding is extraordinary, it suggests that either highly divergent lineages are circulating undetected within poultry populations or that recombination events are generating novel genomic configurations that escape conventional phylogenetic classification. The TK gene, which plays a critical role in nucleotide metabolism and viral DNA synthesis, may be subject to different evolutionary constraints than the P4b gene, potentially explaining this discordance.

Mechanisms of Genetic Diversification

The emergence of genetic variants in FWPV populations is driven by multiple, often synergistic mechanisms. Point mutations constitute the most frequently documented source of genetic variation, with nonsynonymous substitutions accumulating in key genomic regions. Amino acid sequence analysis of recent Egyptian isolates has identified specific mutations in the P4b protein, including E120K and N121D in one isolate and H83P and S93N in another [1]. The functional significance of these mutations warrants careful consideration. The P4b protein, also known as the 4b core protein, is a major structural component of the viral core and is essential for virion morphogenesis. Substitutions at positions 120 and 121 introduce charge alterations (glutamic acid to lysine represents a change from acidic to basic, while asparagine to aspartic acid introduces a negative charge) that could potentially affect protein folding, stability, or interactions with other viral or host proteins. Similarly, the H83P substitution introduces a proline residue, which is known to disrupt alpha-helical structures and introduce conformational rigidity, potentially altering epitope presentation.

The melting curve analysis of pigeon pox virus vaccine samples has revealed the presence of two distinct viral strains or variants within a single vaccine preparation, each exhibiting slightly different melting temperatures [4]. This finding has profound implications for our understanding of FWPV evolution. The coexistence of multiple variants within a single host or vaccine stock provides the raw material for selection and adaptation. In contrast, the FWPV sample examined in the same study contained only a single strain or variant [4], suggesting either that FWPV populations are less heterogeneous than those of pigeon pox virus or that sampling methods have not yet captured the full extent of within-host diversity. The presence of mixed populations in vaccines raises critical questions about the genetic stability of vaccine seeds and the potential for vaccine-derived variants to contribute to field outbreaks.

Host Range Evolution and Species Adaptation

The genetic diversity of FWPV is intimately linked to its host range, which is now recognized as considerably broader than historically assumed. Traditionally, avipoxviruses were classified based on host species: fowlpox-like viruses from Galliformes, canarypox-like viruses from Passeriformes, and viruses from Psittaciformes [2]. However, molecular characterization has challenged this simplistic taxonomy. The P4b gene sequences from turkey pox virus isolates in India cluster within Clade A alongside fowlpox viruses from chickens, showing 99% homology [9]. This genetic proximity raises fundamental questions about host species barriers and the potential for cross-species transmission events.

The evolutionary trajectory of FWPV appears to be influenced by host immunological pressures that drive antigenic variation. The TK gene of a field isolate from Egyptian laying chickens (accession number KF314718) was found to differ substantially from contemporaneous isolates, suggesting that this strain may represent an emerging lineage against which existing vaccines may provide incomplete protection [3]. The amino acid substitutions observed in the P4b gene of recent isolates may affect T-cell epitope recognition, as the P4b protein contains known immunodominant regions. Indeed, the cytokine response following vaccination is dominated by Th1 lymphocytes, with elevated IFN-γ production relative to IL-4 [13]. Mutations that alter peptide presentation on major histocompatibility complex molecules could facilitate immune evasion, particularly in vaccinated populations where cell-mediated immunity is critical for protection.

Recombination and Genomic Plasticity

The poxviral genome is characterized by extensive terminal inverted repeats and a central conserved core flanked by variable regions that encode host range and virulence factors. This genomic architecture is particularly conducive to recombination, both between co-infecting viruses and between vaccine and field strains. The lack of comprehensive whole-genome sequences for most FWPV isolates represents a critical gap in our understanding of viral evolution. Partial gene sequencing, while valuable for phylogenetic placement, cannot capture the full extent of genomic rearrangements, gene duplications, or deletions that may contribute to phenotypic variation [2].

The genetic relatedness between FWPV and pigeon pox virus (PPV) is particularly noteworthy. PPV isolates from Libya demonstrated 100% identity with strains from Egypt and India [5], while Ghanaian PPV isolates showed complete relatedness to the reference PPV isolate FeP2 [6]. The close genetic relationship between these viruses, combined with the historical use of pigeon pox virus as a cutaneous vaccine against fowlpox [7], suggests that cross-protection and potential recombination between these viruses may be occurring in field settings. The 1932 observations that pigeon pox virus could serve as an immunogen against fowlpox [7] indicate that antigenic similarity between these viruses has long been recognized, but the genetic mechanisms underlying this cross-reactivity are only now being elucidated.

Selective Pressures and Vaccine-Driven Evolution

Vaccination exerts a powerful selective pressure on viral populations, and FWPV is no exception. The emergence of outbreaks in vaccinated flocks worldwide [2] indicates that vaccine-driven selection is shaping the genetic diversity of circulating strains. The mutations identified in the P4b gene of Egyptian isolates [1] may represent escape variants that have arisen under immunological pressure from vaccination. The high homology between these isolates and vaccinal strains suggests that recent field strains may be derived from vaccine progenitors that have acquired adaptive mutations over time.

The genetic diversity of FWPV also reflects the ecological and environmental contexts in which the virus circulates. Factors such as vector population dynamics, management practices, and biosecurity measures influence transmission intensity and, consequently, the effective population size of the virus. In regions where vaccination coverage is incomplete or where backyard production systems predominate, viral populations may experience less stringent selective constraints, allowing the accumulation of genetic variation. The isolation of genetically distinct FWPV strains from backyard chickens in Egypt [3], Libya [5], and Ghana [6] underscores the role of these production systems as reservoirs of genetic diversity.

The potential for genetic mutations in cell culture-adapted vaccine strains [12] introduces another dimension to FWPV evolution. Serial passage in heterologous cell systems, such as Baby Grivet Monkey Kidney (BGM) cells [11] or various embryo fibroblast cultures [16], may select for variants with altered receptor tropism or replicative capacity. While the marked increase in virulence following intracerebral passage in chicks [14, 15] demonstrates the phenotypic plasticity of FWPV, the genetic correlates of these adaptations remain largely unexplored. The acquisition of affinity for mesodermal cells and endothelial cells following intracerebral passage [14] suggests that FWPV possesses the genetic capacity to expand its tropism under selective pressure, with obvious implications for pathogenesis and host range.

The absence of comprehensive genomic surveillance programs for FWPV in most regions of the world represents a significant impediment to understanding viral evolution. While partial gene sequencing has provided valuable insights into the phylogenetic relationships among isolates, the resolution afforded by single-gene analyses is insufficient to detect recombination events, gene content variation, or the accumulation of mutations in non-coding regulatory regions. The development of standardized, whole-genome sequencing approaches for FWPV, coupled with robust bioinformatic analyses, is urgently needed to elucidate the molecular epidemiology of this economically significant pathogen and to inform the rational design of next-generation vaccines.

Clinical Manifestations and Pathological Features of Fowl Pox

Fowl pox, a disease of paramount economic significance to the global poultry industry, presents a complex array of clinical manifestations that are fundamentally dictated by the viral strain, host species, age, immune status, and route of infection. As a notifiable disease to the World Organisation for Animal Health (WOAH) in many commercial contexts, understanding its clinical and pathological spectrum is critical for rapid diagnosis, containment, and management. The disease, caused by the Fowlpox virus (FWPV) of the genus Avipoxvirus within the family Poxviridae, is characterized by a slow-spreading but persistent nature, leading to significant production losses, including severe drops in egg production, retarded growth in young birds, and, in severe cases, substantial mortality [2]. Clinical presentations are broadly categorized into three distinct forms: the cutaneous (dry) form, the diphtheritic (wet) form, and a less common systemic or septicemic form, though mixed infections involving both cutaneous and diphtheritic lesions are frequently encountered in field conditions [2, 6].

2.1 Clinical Manifestations of Fowl Pox

The cutaneous form is the most readily identifiable and commonly reported manifestation. It is characterized by the development of proliferative lesions on the featherless or sparsely feathered areas of the body. These lesions typically manifest initially as small, raised, whitish nodules or vesicles that rapidly progress into wart-like growths, scabs, and crusts [2, 5]. The comb, wattles, ear lobes, eyelids, and the skin around the beak and legs are the most frequently affected sites [1, 10]. In layer flocks, lesions on the eyelids can lead to photophobia, conjunctivitis, and, in severe cases, complete closure of the eye due to the accumulation of exudate and scab formation, essentially blinding the bird and preventing it from accessing feed and water. These cutaneous lesions are often itchy and hemorrhagic, and they mature over a period of 1 to 3 weeks, eventually forming thick, dry scabs that slough off, often leaving a scar. The host specificity of Avipoxvirus is broader than once thought; while "fowlpox-like viruses" are primarily isolated from Galliformes, spillover and infection in other orders have been documented, though the clinical presentation remains consistent with cutaneous proliferative lesions [2, 3, 9].

The diphtheritic form is clinically more insidious and pathologically severe, involving the mucous membranes of the upper respiratory and digestive tracts. This form is marked by the formation of caseous, diphtheritic membranes (pseudomembranes) on the mucosa of the mouth, tongue, esophagus, pharynx, and larynx [2, 6, 13]. These lesions appear as yellowish-white, raised plaques or cheesy masses that are firmly adherent to the underlying tissue. Their development can lead to significant mechanical obstruction. Affected birds exhibit pronounced dyspnea, open-mouth breathing, gurgling respiratory sounds, and a characteristic stretching of the neck to facilitate breathing. The diphtheritic form carries a much higher mortality rate than the cutaneous form, primarily due to asphyxiation, starvation, or secondary bacterial infections that exploit the damaged mucosal barrier. A concurrent infection with other respiratory pathogens, such as Avibacterium paragallinarum or Mycoplasma gallisepticum, can dramatically exacerbate the clinical severity and mortality. In some field cases and experimental models, a mixed form is observed where an animal presents with both cutaneous scabs and diphtheritic oral plaques concurrently, representing the most severe presentation of the disease [6].

Atypical clinical manifestations are also recognized, particularly in relation to specific viral passage history or host susceptibility. Intracerebral inoculation of FWPV into young chicks, as documented in classical studies, produces a distinct neurological syndrome. This includes the development of drowsiness and somnolence approximately 4 to 5 days post-inoculation, followed by spastic paralysis and convulsions by days 6 to 7, with the majority of infected chicks succumbing on days 7 or 8 [15]. This neurotropic behavior, while not a standard feature of natural infection, underscores the virus's latent capacity for mesodermal tissue affinity when placed in a permissive environment [14, 15]. In commercial layers, a more gradual but economically devastating clinical sign is the precipitous drop in egg production, which can persist for several weeks and is compounded by reduced feed intake and general malaise [2, 13].

2.2 Pathological Features and Histopathology

The pathological hallmarks of fowl pox are centered on epithelial hyperplasia, cellular hypertrophy, and the formation of characteristic intracytoplasmic inclusion bodies. Grossly, the cutaneous form shows thickened, roughened epidermis with raised papules and scabs. The diphtheritic form reveals firmly attached pseudomembranes overlying ulcerated and inflamed mucosa. Upon removal of these membranes, a raw, bleeding surface is exposed, indicating deep tissue involvement.

Histologically, the most definitive diagnostic feature of fowl pox infection is the presence of large, eosinophilic intracytoplasmic inclusion bodies, known as Bollinger bodies [5, 6, 10, 12]. These inclusions, which represent aggregates of mature virions (elementary bodies or Borrel bodies) embedded in a protein matrix, are found within the cytoplasm of hyperplastic epithelial cells. In the epidermis, the virus induces prominent acanthosis (thickening of the stratum spinosum) and hyperkeratosis. The infected epithelial cells undergo ballooning degeneration; they become enlarged, rounded, and lose their intercellular bridges. The cytoplasm becomes vacuolated and granular, and the nucleus is often pyknotic or karyorrhectic [8]. As the lesions mature, these cells undergo necrosis, leading to the formation of scabs composed of cellular debris, fibrin, and inflammatory cells.

On the chorioallantoic membrane (CAM) of embryonated chicken eggs, a classic experimental model, FWPV produces discrete, focal, raised, white opaque lesions called pocks [10, 17]. The morphology of these pocks can vary between strains. For instance, field isolates have demonstrated the ability to produce larger pocks (3 to 5 mm in diameter) compared to vaccine strains (0.5 to 2.5 mm) [10]. Histopathology of the infected CAM reveals profound epithelial proliferation (ectodermal hyperplasia), cellular hypertrophy, and the presence of characteristic intracytoplasmic inclusion bodies within the ectodermal cells [8, 10]. The mesodermal layer of the CAM often shows a mononuclear inflammatory cell infiltrate and fibroblastic proliferation. Electron microscopy of these infected CAMs has confirmed the presence of clumps of virus particles both at the surface and deep within the diseased, vacuolated epithelium, correlating the light microscopic findings with the ultrastructural presence of virions [8].

The diphtheritic form presents histologically as a severe, fibrinonecrotic inflammation of the mucosa. The pseudomembrane is composed of necrotic epithelium, fibrin, and heterophils and macrophages. The underlying lamina propria is congested, edematous, and heavily infiltrated with inflammatory cells. In these lesions, Bollinger bodies are often more difficult to find than in the cutaneous form, as the necrosis is more extensive, requiring careful examination of the less-damaged epithelial cells at the periphery of the ulcer [12]. In experimental infections using intracerebrally passaged virus, a remarkable tropism for endothelial cells of blood vessels and cells of mesodermal origin is observed, leading to a meningo-encephalitis characterized by perivascular cuffing, meningitis, and choroid plexitis, with all infected cells exhibiting the same spherical transformation and desquamation [14, 15]. Even in modern live-vaccine safety trials, histopathological examinations have revealed mild but observable changes such as renal tubular necrosis, hepatocyte necrosis, hemorrhage, and vascular congestion, alongside the formation of polymorphonuclear cell clusters in lung and liver tissues, though these are considered transient and indicative of the robust host immune response rather than virulent disease [12]. The ability to detect these specific cytopathic effects, including syncytia formation and intracytoplasmic inclusion bodies in cell culture (e.g., Chicken Embryo Fibroblast or BGM cell lines), further confirms the virus's characteristic pathological footprint at the cellular level [10, 11, 16].

Laboratory Diagnosis of Fowl Pox Virus

The definitive diagnosis of Fowl Pox Virus (FPV) necessitates a multifaceted laboratory approach that integrates classical virological techniques with modern molecular and serological methodologies. Given the clinical resemblance of fowlpox to other proliferative and diphtheritic conditions, such as infectious laryngotracheitis, vitamin A deficiency, and Trichomonas or Candida infections, laboratory confirmation is paramount for implementing appropriate control measures and informing vaccination strategies [2]. The diagnostic arsenal ranges from historical methods like histopathological examination and virus isolation in embryonated chicken eggs to contemporary molecular assays and advanced cell culture systems, each offering distinct advantages in sensitivity, specificity, and throughput.

Histopathological Examination

Histopathology remains a cornerstone for the preliminary identification of FPV infection due to its ability to reveal pathognomonic cellular alterations. Tissues collected from suspect lesions, typically scabs from the cutaneous form or diphtheritic membranes from the upper respiratory tract, are fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The hallmark of FPV infection is the presence of eosinophilic intracytoplasmic inclusion bodies, historically termed Bollinger bodies, within hyperplastic and hypertrophic epithelial cells [5, 6, 10]. These inclusions are large, homogenous, and often displace the host cell nucleus to the periphery. Their detection is considered confirmatory for poxvirus infection, as they represent aggregates of mature virions (virus factories) within the cytoplasm [6, 10]. Furthermore, the histopathological section reveals striking epithelial proliferation (acanthosis) and degeneration (ballooning degeneration) of keratinocytes, accompanied by varying degrees of inflammation and necrosis. In cases of the diphtheritic form, microscopic examination of tracheal or oral mucosa may reveal severe necrosis and sloughing of the epithelium. The field isolate studies demonstrate that infected chorioallantoic membranes (CAM) also exhibit these characteristic intracytoplasmic inclusions, confirming the utility of this technique across different sample types [5, 10]. While highly specific, histopathology is limited in its ability to differentiate between various avipoxvirus species (e.g., fowlpox vs. pigeonpox) and cannot quantify viral load or determine genetic lineage.

Virus Isolation in Embryonated Chicken Eggs and Cell Culture

Virus isolation has historically been the gold standard for FPV diagnosis and is essential for obtaining live virus for further characterization and vaccine production.

Inoculation on the Chorioallantoic Membrane (CAM)

The most sensitive and widely employed method for primary isolation involves inoculation of suspect material onto the chorioallantoic membrane (CAM) of 9- to 12-day-old specific-pathogen-free (SPF) embryonated chicken eggs via the dropped CAM technique [1, 10, 17]. Following a 5–7 day incubation period at 37°C, the CAMs are examined for the presence of characteristic pock lesions. Field strains typically produce larger, more necrotic pocks ranging from 3 mm to 5 mm in diameter after the third passage, often accompanied by pronounced edematous thickening, whereas vaccine or attenuated strains produce smaller, discrete pocks (0.5 mm to 2.5 mm) [10]. Histological examination of these pocks confirms the presence of intracytoplasmic inclusion bodies, confirming the identity of the virus [10]. This classic method, pioneered by Burnet and colleagues, allows for titration of the virus by pock counting at serial dilutions, providing a quantitative measure of infectious particles [17]. The CAM is a rich source of virus, as the virus replicates preferentially within the ectodermal and endodermal layers of the membrane [8, 19].

Adaptation and Propagation in Cell Culture

While CAM inoculation is reliable, cell culture systems offer advantages in terms of economy, scalability, and reduced use of live animals [16]. The most traditional and widely accepted cell line for FPV propagation is the Chicken Embryo Fibroblast (CEF) . Adaptation of field isolates and vaccine strains to CEF typically requires serial passaging. At early passages (1–3), a characteristic cytopathic effect (CPE) develops, including cell rounding, aggregation, syncytia formation, and eventual plaque formation [10]. The tissue culture infective dose (TCID50/mL) can then be calculated using the Spearman-Kärber method to quantify viral titers, with CEF-adapted FPV achieving titers as high as 10⁹·³ TCID50/mL, ensuring high immunogenicity in vaccines [11, 16].

Alternative cell lines have been explored to overcome the logistical challenges of producing primary CEF cells (which require continuous supply of embryonated eggs). Notably, Baby Grivet Monkey Kidney (BGM) cells have been identified as a susceptible and efficient system for FPV propagation. Studies adapting the Baudette strain to BGM cells demonstrated that the virus reached titers of 10⁶·² TCID50/mL by the 12th passage, with CPE evolving from rounding and intracytoplasmic inclusion formation (passage 7) to syncytium formation (passages 9–13) [11]. The virus remains highly cell-associated for up to 84 hours post-inoculation, with optimal cell-free virus harvest occurring at 120 hours post-inoculation, providing a critical window for vaccine production [11]. Other cell lines, including Duck Embryo Fibroblast (DEF), Quail Embryo Fibroblast (QEF), and Vero cells, have also been successfully utilized, yielding titers of 10⁷·⁶ TCID50/mL, 10⁸·⁶ TCID50/mL, and 10⁷·³ TCID50/mL, respectively, though Vero cells appear abortive for certain FPV strains [11, 16]. The use of Japanese quail embryo fibroblast systems has been highlighted as a promising, economical, and stable platform for vaccine production, free from extraneous contaminants [20].

Molecular Diagnostics: PCR and Sequencing

Molecular techniques, particularly polymerase chain reaction (PCR), have revolutionized the diagnosis and characterization of FPV due to their superior sensitivity, specificity, and speed compared to virus isolation.

Conventional PCR

The most common molecular target for FPV detection is the P4b core protein gene (also known as the fpv167 locus), a highly conserved region within the avipoxvirus genome. PCR amplification using primers targeting the P4b gene yields a specific amplicon of 578 base pairs (bp) and is widely used for genus-level detection [1, 5]. Studies from recent outbreaks in Egypt and Libya have successfully amplified the P4b gene from field samples, confirming the presence of FPV in clinical cases showing characteristic pock lesions and histopathological inclusions [1, 5]. Another valuable target is the Thymidine Kinase (TK) gene. Amplification of a 305 bp fragment of the TK gene has been employed for molecular identification and phylogenetic analysis, as demonstrated in isolates from Egyptian layer chickens, which showed limited similarity (not exceeding 60%) to other published avipoxvirus sequences, suggesting the emergence of novel strains [3].

Real-Time PCR (qPCR)

Quantitative real-time PCR (qPCR) provides the added benefit of viral quantification. Using a standard curve correlating cycle threshold (Ct) values with known virus titers, researchers can accurately measure the viral load in vaccines or clinical samples [4]. This technique is particularly useful in vaccine quality control, ensuring consistent antigenic mass. Melting curve analysis in qPCR can also differentiate between viral strains; for instance, pigeon pox vaccine samples have shown two distinct melting temperatures, indicating the presence of mixed viral strains, while FPV samples typically yield a single homogeneous peak [4].

Phylogenetic and Sequence Analysis

Following PCR amplification, sequencing of the P4b gene fragment (typically 300–578 bp) allows for robust phylogenetic analysis and classification of the isolate.

• Clade Classification: FPV isolates are typically classified within Clade A of the avipoxvirus phylogeny. More specifically, many recent field isolates from Egypt and Libya cluster within Subclade A1, exhibiting 99–100% homology with local reference and vaccine strains [1, 5, 9].
• Genetic Variation: Although highly conserved, sequence analysis can reveal specific point mutations that may have implications for vaccine efficacy and virulence. For example, isolates from Al-Sharkia, Egypt, have demonstrated amino acid substitutions such as E120K and N121D in one isolate, and H83P and S93N in another, highlighting ongoing evolutionary drift [1]. Such mutations underscore the need for continuous genomic surveillance to monitor the emergence of variant strains that might evade immunity induced by current vaccines [1, 2].
• Host Specificity: Phylogenetic analysis of the P4b gene reliably distinguishes FPV from other avipoxviruses, such as pigeon pox virus (PPV) or canarypox virus. Isolates from pigeons in Ghana and Libya showed 100% identity to reference PPV strains, confirming host-specific clustering even when clinical signs overlap with FPV [5, 6].

Serological Diagnosis

Serological testing is employed to detect antibodies against FPV, primarily for monitoring vaccine take and flock seroprevalence rather than acute diagnosis.

Agar Gel Precipitation Test (AGPT)

The AGPT is a classical, simple, and economical test used to detect circulating antibodies or identify viral antigens from isolates. In AGPT, soluble antigens from FPV-infected CAMs or cell cultures react with specific antisera to form visible precipitation lines [1]. This method has been used for decades to confirm the identity of FPV isolates [1]. While useful for serosurveillance, AGPT is less sensitive than modern immunoassays and may not detect low levels of antibody.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA offers a more sensitive and quantitative alternative for measuring antibody responses. Specifically, ELISA can be used to profile the immune response post-vaccination by quantifying Th1-associated cytokines (e.g., IFN-γ) and Th2-associated cytokines (e.g., IL-4) in peripheral blood mononuclear cells (PBMCs) [13]. Studies have shown that FPV vaccination predominantly stimulates a Th1-type response, with significantly higher IFN-γ levels compared to controls, indicating a robust cell-mediated immune mechanism [13]. This cytokine profiling can help evaluate the immunogenicity of novel vaccines [13, 16].

Advanced and Emerging Techniques

Electron Microscopy

Transmission electron microscopy (EM) of infected CAM sections or cell lysates provides direct visualization of FPV virions. Mature virus particles appear as characteristic brick-shaped structures (approximately 200–300 nm in length) with a complex internal core and lateral bodies [8]. While not a routine diagnostic tool due to cost and technical expertise requirements, electron microscopy is invaluable for confirming atypical isolates and for basic virological research.

Synthetic Silicate Binding Assays

Innovative methods, such as the use of synthetic Aluminum-Magnesium Silicate (AMS), have been explored to enhance virion release for diagnostic titrations. Incubation of FPV vaccines with AMS has been shown to significantly increase detectable virus titers in modified passive hemagglutination tests (from a mean of 2.8 ± 1.10 to 11.2 ± 4.38), potentially improving the sensitivity of serological or antigen detection assays [18].

Diagnostic Algorithm and Best Practices

For comprehensive FPV diagnosis, the World Organisation for Animal Health (WOAH) recommends the following integrated approach:

1. Sample Collection: Scabs, dry crusts, or diphtheritic membranes taken from the head, comb, wattle, or oral cavity. For molecular testing, samples should be stored in sterile PBS or viral transport medium at 4°C.
2. Initial Screening: Histopathological examination of impression smears or tissue sections for Bollinger bodies.
3. Virus Isolation: Inoculation of clarified homogenate onto the CAM of 10–12 day old SPF embryonated eggs, observing for pocks after 5–7 days.
4. Molecular Confirmation: PCR targeting the P4b gene followed by sequencing for genetic characterization and phylogenetic analysis to differentiate FPV from related avipoxviruses and to detect emerging variants.
5. Serological Monitoring: AGPT or ELISA for post-vaccination immune response assessment.

This robust combination of classical and molecular tools ensures accurate diagnosis, facilitates epidemiological tracking, and supports the development of effective vaccines against this economically significant pathogen of global poultry production [2]. The ongoing emergence of genetically distinct FPV strains, as evidenced by TK gene divergence and P4b amino acid substitutions, demands that diagnostic laboratories remain vigilant and update PCR primer sets and reference strains accordingly to avoid false-negative results [1, 3, 5].

Prevention, Control Strategies, and Emerging Challenges

The multifaceted nature of fowl pox virus (FWPV) infection, encompassing its global distribution, its ability to affect diverse avian species across production systems, and the documented emergence of outbreaks in vaccinated flocks, demands a comprehensive, layered approach to prevention and control. The cornerstone of FWPV management remains prophylactic vaccination, but the evolving genetic landscape of the virus and the practical limitations of traditional vaccine platforms necessitate concurrent strategies encompassing biosecurity, vector management, environmental decontamination, and enhanced molecular surveillance. This section provides a deep analysis of these interrelated components, with a particular focus on the mechanistic underpinnings of vaccine-induced immunity, the comparative advantages of modern cell-culture-derived vaccines, and the emerging challenges that threaten the long-term efficacy of current control paradigms.

Vaccination as the Primary Prophylactic Strategy

The use of live-attenuated fowl pox vaccines has been a mainstay of poultry health management for decades, yet the biological basis of their efficacy and the factors governing vaccine “take” are increasingly understood at a cellular and molecular level. Historically, vaccines were propagated on the chorioallantoic membrane (CAM) of embryonated chicken eggs, a method that yields pock lesions of varying size, field isolates typically produce pocks ranging from 3 to 5 mm, whereas lyophilized vaccine strains generate smaller (0.5–2.5 mm) pocks [10]. The CAM-propagated vaccine has been widely used, but its production is labor-intensive, yields variable titers, and carries the risk of extraneous contaminants [12, 16]. In response, the poultry industry has increasingly adopted cell-culture-adapted vaccines, which offer superior standardization, scalability, and immunogenicity. The World Organisation for Animal Health (WOAH) recognizes the importance of quality-controlled vaccine production, and the shift toward cell culture aligns with international best practices for biologicals.

Cell-culture-adapted vaccines: immunogenicity and safety. Recent comparative studies have demonstrated that vaccines propagated on chicken embryo fibroblast (CEF) cells achieve titers as high as 10⁹.³ TCID₅₀/mL, while Vero cell-adapted vaccines yield 10⁷.³ TCID₅₀/mL [16]. These titers are substantially higher than those typically obtained from CAM harvests. Importantly, birds vaccinated via the wing-web route with cell-culture-adapted products develop characteristic “takes” (localized swelling and scab formation) within 7 days, and seroconversion is confirmed by agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) [16]. The immunogenicity of these vaccines is driven predominantly by a Th1-type cellular response, as evidenced by elevated interferon-gamma (IFN-γ) relative to interleukin-4 (IL-4) in peripheral blood mononuclear cells (PBMCs) of vaccinated specific-pathogen-free (SPF) chicks [13]. This Th1 polarization is critical because FWPV, as an intracytoplasmic pathogen, requires robust cell-mediated immunity for clearance.

Safety evaluations using ten-fold field doses of cell-culture-adapted vaccines have confirmed their innocuity: all vaccinated chickens developed takes with no adverse reactions, mortality, or evidence of virulence reversal [12]. Nonetheless, careful histopathological examination revealed mild, non-specific changes, including renal tubular necrosis, hepatocyte necrosis, and vascular congestion, without the detection of Bollinger bodies (eosinophilic intracytoplasmic inclusion bodies indicative of active viral replication) [12]. These findings underscore that while cell-culture-adapted vaccines are safe under laboratory conditions, their persistence in host tissues and potential for genetic mutation, particularly upon serial passage, merits continued monitoring [12]. The adaptation of FWPV to continuous cell lines, such as the Baby Grivet Monkey Kidney (BGM) cell line, offers an alternative for vaccine production; the BGM-adapted virus reaches peak titers of 10⁶.² TCID₅₀/mL at 120 hours post-inoculation, with characteristic cytopathic effects including syncytium formation from passage 9 onward [11]. However, the use of heterologous cell lines raises questions about the retention of immunogenic epitopes and the risk of adventitious agents.

Alternative vaccine approaches: pigeon pox virus and recombinant vectors. Historically, pigeon pox virus (PPV) has been employed as a heterologous vaccine against fowl pox, leveraging the cross-protective immunity induced by closely related avipoxviruses. Early experiments in the 1930s demonstrated that cutaneous vaccination with PPV could confer protection against subsequent fowl pox challenge, though the method was not without drawbacks, including variable take rates and the potential for localized disease [7]. In recent years, modern molecular characterization has confirmed that PPV isolates are genetically distinct but share high homology in key genes such as P4b; for instance, a Libyan PPV isolate showed 100% identity to strains from Egypt and India [5]. The recent discovery of PPV in Ghana, where both cutaneous and diphtheritic forms were observed, highlights the need for targeted surveillance to determine whether PPV-based vaccines remain effective against emergent strains [6]. Concurrently, FWPV has been exploited as a vector for the production of recombinant veterinary vaccines, a strategy that capitalizes on its large genome capacity and ability to elicit durable immune responses [2]. Such recombinant vaccines, expressing protective antigens from other avian pathogens (e.g., Newcastle disease virus, infectious laryngotracheitis virus), offer the potential for multivalent protection but require rigorous evaluation of their impact on FWPV-specific immunity.

Control Strategies: Biosecurity, Hygiene, and Eradication

Beyond vaccination, the prevention of FWPV transmission relies on an integrated biosecurity framework that addresses both direct bird-to-bird contact and vector-borne spread. The virus is mechanically transmitted by arthropod vectors, particularly mosquitoes and biting flies, which amplify its persistence in endemic regions [2]. Therefore, vector control measures, including insecticide application, environmental management to reduce breeding sites, and the use of screened housing, are essential components of a comprehensive control program. The FAO and WOAH emphasize that biosecurity protocols must be tailored to local epidemiological contexts, given that FWPV prevalence varies with climate, vector population density, and husbandry practices.

Environmental decontamination is another critical pillar. FWPV, like other poxviruses, exhibits considerable resistance to desiccation and can remain infectious in dried scabs for months. Formaldehyde fumigation has been historically employed for incubator hygiene; early studies demonstrated that exposure to formaldehyde gas effectively inactivates FWPV on contaminated cotton squares and down of day-old chicks, provided humidity and temperature are controlled [21]. However, modern disinfection protocols increasingly favor peracetic acid, chlorine dioxide, or accelerated hydrogen peroxide, which are less hazardous to personnel and more compatible with automated sanitation systems. It is noteworthy that aluminum-magnesium silicate (AMS) has been shown to both enhance the release of FWPV virions from vaccine preparations and, upon repeated treatment, inhibit viral titers, suggesting a potential dual role in vaccine formulation and disinfection that warrants further investigation [18].

Emerging Challenges: Vaccine Failure, Genetic Drift, and Expanding Host Range

Despite decades of vaccination, fowl pox remains an “evolving disease” with numerous documented outbreaks in immunized commercial flocks worldwide [2]. This phenomenon points to several interrelated challenges that threaten the sustainability of current control strategies.

Genetic diversification and antigenic variation. Phylogenetic analyses of recent FWPV field isolates have revealed significant genetic heterogeneity, particularly in the P4b and thymidine kinase (TK) genes. For example, a 2021 Egyptian isolate (Avipoxvirus-Egy-f1086-2-P4b) harbored amino acid substitutions E120K and N121D in the P4b protein, while another isolate carried H83P and S93N mutations [1]. These changes, though located in a core protein, could alter epitope presentation and reduce the efficacy of vaccines derived from older strains. More strikingly, a 2012 Egyptian isolate (ch-08TK) exhibited only 60% homology in the TK gene compared to published sequences, leading researchers to classify it as a new emerging strain with limited similarity to contemporaneous Egyptian or canarypox viruses [3]. Such divergence raises the specter of immune escape, underscoring the urgent need for continuous genetic surveillance and periodic vaccine strain updates. The WOAH’s Network of Veterinary Laboratories and the FAO’s Emergency Prevention System (EMPRES) advocate for systematic molecular characterization of avipoxviruses to monitor these shifts.

Outbreaks in vaccinated flocks: factors and mechanisms. The occurrence of fowl pox in vaccinated birds can be attributed to several non-exclusive factors: insufficient vaccine coverage, improper administration, waning immunity, and antigenic mismatch. Suboptimal vaccination programs, especially in backyard and small-scale flocks, leave gaps in herd immunity. Even in well-managed commercial operations, the duration of vaccine-induced immunity is not precisely defined; recent studies indicate that antibodies persist for at least several weeks post-vaccination, but the longevity of protective cellular responses, particularly the Th1 arm, requires further study [13, 16]. The ratio of IFN-γ to IL-4 in vaccinated chickens supports a strong Th1 bias, but booster vaccination schedules remain empirically determined in many regions [13]. Moreover, the emergence of variant strains that are not fully neutralized by vaccine-induced antibodies could explain breakthrough infections. In Libya, for example, four FPV isolates from an outbreak were 100% identical to strains from Iraq, Iran, and Brazil, suggesting that international spread of genetically homogeneous but immunologically distinct strains may contribute to vaccine failure [5]. The global trade of live birds and hatching eggs facilitates such introductions, highlighting the need for coordinated international surveillance.

Broadening of host range and cross-species transmission. Traditionally, FWPV was considered largely restricted to Galliformes, but molecular evidence has expanded its host range to include turkey, quail, and even pigeons, while other avipoxvirus clades (e.g., canarypox-like viruses) affect Passeriformes and Psittaciformes [2, 9]. The isolation of turkey poxvirus in India, clustering within clade A alongside chicken isolates (99% homology in P4b), indicates that species barriers are porous [9]. This blurring of host-specificity complicates control: reservoirs in wild birds or non-commercial poultry can sustain transmission, and the use of pigeon pox virus vaccines for fowl pox may not provide adequate protection against strains that have evolved in different avian hosts. The recent detection of PPV in Ghana for the first time, associated with severe diphtheritic lesions, underscores the need for active surveillance across diverse bird populations [6].

Challenges in vaccine production and quality control. The transition from CAM-propagation to cell culture has been uneven globally. In Nigeria, for instance, CAM-propagated vaccines still predominate, and outbreaks persist despite their use [12, 16]. The cost and technical requirements for establishing certified cell-culture facilities remain barriers in low- and middle-income countries. Moreover, even modern cell-culture vaccines require rigorous quality assurance. Real-time PCR (rt-PCR) has been proposed as a rapid method for molecular titration, with the construction of standard curves relating cycle threshold (Ct) values to log₁₀ virus titers [4]. However, anomalous amplification efficiency exceeding 100% observed in some standard curves demands further investigation to ensure accuracy [4]. Additionally, the presence of distinct viral variants within a single vaccine sample, as revealed by melting curve analysis of pigeon pox vaccine [4], highlights the potential for mixed populations that could complicate both safety and efficacy.

Future directions: antivirals, immunomodulators, and next-generation vaccines. While vaccination remains the primary tool, alternative interventions are being explored. The synthetic aluminum-magnesium silicate (AMS) demonstrated a dose-dependent effect on FWPV: at a single incubation, it boosted detectable virus titers, potentially useful for vaccine enrichment, but upon repeated treatment it inhibited the virus [18]. This dual activity invites investigation into AMS as an adjuvant or direct antiviral. The use of cell culture systems derived from Japanese quail embryos offers a promising platform for producing highly immunogenic, contaminant-free vaccines, with stable biological properties that facilitate scale-up [20]. Finally, deeper understanding of the molecular determinants of virulence, such as the enhanced neurotropism observed after intracerebral passage of FWPV [14, 15], may guide rational attenuation of vaccine strains to improve safety profiles without compromising immunogenicity.

In light of these emerging challenges, a proactive, evidence-based control strategy must integrate routine molecular surveillance of field strains, periodic reassessment of vaccine seed viruses, enhanced biosecurity tailored to vector ecology, and international collaboration under the auspices of the WOAH and FAO. Only through such a holistic approach can the poultry industry hope to stay ahead of a virus that has proven remarkably adaptable despite decades of vaccination.

References

[1] Hassan M, Elboraay IM, El-Zanaty AEI, Sharawy S. Genetic features of P4b gene of recent Fowl Pox virus strains from Al-Sharkia Governorate Egypt 2021.. Benha veterinary medical journal. 2024. DOI: https://doi.org/10.21608/bvmj.2024.301667.1845

[2] Minhas SK, Kumar P, Panghal R, Mehtani R, Yadav R, Kalonia S, et al.. Fowl pox virus: a minireview. Worlds Poultry Science Journal. 2023. DOI: https://doi.org/10.1080/00439339.2023.2273353

[3] El-mahdy S, Awaad M, Soliman YA. Molecular identification of local field isolated fowl pox virus strain from Giza governorate of Egypt.. Veterinary World. 2014. DOI: https://doi.org/10.14202/VETWORLD.2014.66-71

[4] Rady‎ DI, Zieny AE, Ali ZM, Ibrahim AI. Tracking quality standard test for tissue culture ‎pigeon pox virus Vaccine. المجلة العربیة للعلوم الزراعیة. 2024. DOI: https://doi.org/10.21608/asajs.2024.386890

[5] Elshwihdi M, Kammon A, Asheg A. Molecular characterization of fowl and pigeon pox viruses: A recent outbreak in Libya. Open Veterinary Journal. 2025. DOI: https://doi.org/10.5455/OVJ.2025.v15.i5.31

[6] Abbiw R, Amoako KK, Enyetornye B, Odoom T, Boateng PA, Abbang SM, et al.. Isolation and Molecular Detection of Pigeonpox Virus in a Pigeon With Both Cutaneous and Diphtheritic Forms of Pigeon Pox Disease in Ghana. Veterinary Medicine International. 2025. DOI: https://doi.org/10.1155/vmi/7523480

[7] Johnson E. Results of Experiments With the Use of Pigeon-pox Virus as Cutaneous Vaccine Against Fowl-pox. Poultry Science. 1932. DOI: https://doi.org/10.3382/PS.0110187

[8] Morgan C, Wyckoff R. The electron microscopy of fowl pox virus within the chorioallantoic membrane.. Journal of Immunology. 1950. DOI: https://doi.org/10.4049/jimmunol.65.2.285

[9] Pawade M, Mhase P, Muglikar D, Lonkar V, Mehere P, Shelke P, et al.. Isolation and Molecular Characterization of Turkey Pox Virus from Maharashtra, India: A Review. Indian Journal of Animal Research. 2021. DOI: https://doi.org/10.18805/ijar.b-4468

[10] Gilhare VR, Hirpurkar S, Kumar A, Naik S, Sahu T. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture. Veterinary World. 2015. DOI: https://doi.org/10.14202/vetworld.2015.245-250

[11] El-Bagoury F, El-Nahas M, Nakhla OE, Hassan N. Baby Grivet Monkey Kidney (BGM) a new cell line for adaptation of fowl pox virus. . 2016. DOI: https://doi.org/10.21608/BVMJ.2016.31239

[12] Courage C, Bwala DG, Adetunji V, Alesa M, Okafor U, Soumah F, et al.. Evaluation of the safety of cell culture-adapted fowl pox vaccines. International Journal of Veterinary Sciences and Animal Husbandry. 2025. DOI: https://doi.org/10.22271/veterinary.2025.v10.i3f.2161

[13] Reza AM, Ismail A. Cytokine Immune Response Following Vaccination against Fowl Pox Disease in Specific Pathogen Free Chickens.. Archives of Razi Institute. 2025. DOI: https://doi.org/10.32592/ari.2025.80.2.525

[14] Buddingh GJ. A STUDY OF THE BEHAVIOR OF FOWL POX VIRUS MODIFIED BY INTRACEREBRAL PASSAGE. Journal of Experimental Medicine. 1938. DOI: https://doi.org/10.1084/jem.67.6.933

[15] Buddingh GJ. A MENINGO-ENCEPHALITIS IN CHICKS PRODUCED BY THE INTRACEREBRAL INJECTION OF FOWL POX VIRUS. Journal of Experimental Medicine. 1938. DOI: https://doi.org/10.1084/jem.67.6.921

[16] Courage C, Bwala DG, Shimilimo A, Adetunji V, Israel S, Killo AO. A comparative evaluation of the immunogenicity of cell culture adapted Fowl Pox vaccines.. Nigerian Veterinary Journal. 2025. DOI: https://doi.org/10.4314/nvj.v46i1.1

[17] Reid D, Crawley J, Rhodes AJ. A study of fowl pox virus titration on the chorioallantosis by the pock counting technique.. Journal of Immunology. 1949. DOI: https://doi.org/10.4049/jimmunol.63.2.165

[18] Ezeibe M, Ekeanyanwu E, Ngene A, Mbuko I. Aluminum-Magnesium Silicate enhances release of virions of cultured Fowl Pox Virus and inhibits the virus. Health. 2013. DOI: https://doi.org/10.4236/HEALTH.2013.59190

[19] Thorning WM, Graham R, Levine ND. Studies on Certain Filtrable Viruses. IV. Immunogenic Properties of Fowl Pox Virus Prepared from the Entire Embryo. Poultry Science. 1943. DOI: https://doi.org/10.3382/PS.0220287

[20] Yusifova K. ASPECTS OF IMMUNIZATION OF BIRDS BY CULTURAL VACCINES AGAINST DISEASES FOWL POX. . 2020. DOI: https://doi.org/10.36359/scivp.2020-21-1.32

[21] Graham R, Barger E. Studies on Incubator Hygiene IV A Note on the Virucidal Effect of Formaldehyde on Fowl Pox Virus. Poultry Science. 1936. DOI: https://doi.org/10.3382/PS.0150048