Avian Leukosis Virus

Overview

Avian leukosis virus (ALV) is an enveloped retrovirus with a single-stranded RNA genome that has been responsible for significant economic losses in the global poultry industry. The virus causes a spectrum of diseases in chickens, ranging from neoplastic conditions such as lymphoid leukosis and hemangiomas to immunosuppression that predisposes birds to secondary infections. ALV continues to be a subject of intensive research due to its impact on poultry health and production, as well as its potential to serve as a model for studying retroviral biology [2, 7]. The virus has been extensively monitored, and its persistence in various chicken populations, from commercial flocks to indigenous and backyard birds, still raises concerns among animal health organizations, including authorities like the Centers for Disease Control and Prevention (CDC) and the World Organisation for Animal Health (WOAH), which emphasize rigorous surveillance to prevent outbreaks that jeopardize food security and animal welfare.

Vertical and horizontal transmission of ALV further complicates eradication efforts; hens can pass the virus to their offspring through eggs, while direct contact facilitates spread among birds [12]. Notably, several studies have pinpointed the role of live vaccines and contaminated materials as additional, albeit less common, sources of ALV infection in poultry flocks [10, 24]. Moreover, the virus’s ability to evade host immune responses, partly by modulating key molecular signaling pathways, underscores the challenges facing conventional control measures and the urgent need for innovative strategies, including genetic manipulation and vaccine development [1, 18]. International bodies such as the Food and Agriculture Organization (FAO) also monitor such enzootic diseases to secure the global poultry supply chain and mitigate the economic burden on producers.

Taxonomy

In the taxonomic hierarchy, ALV is classified within the family Retroviridae and belongs to the genus Alpharetrovirus [2]. This classification is grounded in the virus’s molecular architecture, particularly the organization of its genome and its envelope glycoproteins. The viral genome typically contains genes that encode the structural proteins (gag), the enzymes necessary for viral replication (pol), and the envelope proteins (env), among which the gp85 surface protein and gp37 transmembrane protein are key determinants for subgroup classification [7].

The diversity of ALV is underscored by the existence of multiple subgroups. Historically, the virus has been categorized into subgroups A, B, C, D, and E on the basis of serological reactions and differences in cellular receptor usage. More recently, subgroup J has emerged as the most pathogenic and economically deleterious form, particularly in China where it has caused re-emergence events in commercial and indigenous flocks [2, 7, 15]. Additionally, the identification of a novel subgroup, designated ALV-K, has expanded the classification further, reflecting rapid viral evolution through mutation and recombination [4, 11, 22]. These subgroups are not only defined by their antigenic properties but also by the specific cellular receptors they exploit. For example, subgroup J ALV, which predominantly affects commercial poultry, utilizes the chicken Na^+/H^+ exchanger type 1 (chNHE1) as its entry receptor, a molecular interaction that has been exploited in recent gene‐editing studies aimed at conferring resistance to infection [3, 5]. Similarly, other subgroups, such as ALV-A and the newly recognized ALV-K, have been associated with receptors like Tva, further demonstrating the complex interplay between viral evolution and host susceptibility [6, 11].

The phylogenetic relationships among the various ALV strains have been investigated using envelope gene sequencing and whole-genome comparisons. Phylogenetic analyses consistently reveal clustering of strains by subgroup, with ALV-J strains forming distinct monophyletic clades that correlate with geographic origin and host species diversity [4, 15]. Discrepancies in nucleotide and amino acid homologies, particularly in key regions such as the gp85 gene, not only facilitate molecular classification but also offer insight into the virus’s evolutionary trajectory and mechanisms of immune escape [9, 19]. The ongoing evolution of ALV, driven by high mutation rates inherent to retroviral replication, recombination between exogenous and endogenous viral sequences, and selection pressures from host immune responses, renders its taxonomy dynamic and necessitates continuous molecular monitoring [16, 22].

Molecular Biology

The molecular biology of ALV is defined by its retroviral life cycle, which involves reverse transcription, integration into the host genome, and the subsequent expression of viral proteins that commandeer host cellular machinery. The env gene, encoding the surface glycoprotein gp85 and the transmembrane protein gp37, plays a critical role in determining host range and tissue tropism [19]. Gp85 mediates the initial binding of the virus to its specific cellular receptor, such as chNHE1 for subgroup J [3, 5], thereby triggering a cascade of conformational changes that facilitate membrane fusion and viral entry. The fidelity of these interactions is crucial, and even minor mutations in gp85 or in the untranslated regions (UTRs) of the viral genome can lead to significant changes in pathogenicity and replication kinetics [9, 13].

Central to the viral replication process is the reverse transcription step, wherein the viral RNA genome is converted into DNA by the viral reverse transcriptase enzyme. This newly formed viral DNA integrates into the host genome as a provirus, establishing a persistent infection that can be transmitted vertically through germ-line integration [2, 7, 12]. The integrated provirus exploits the host cellular transcriptional machinery, resulting in the synthesis of viral mRNA and proteins required for assembling new virions. Studies have demonstrated that host cell signaling pathways, including those mediated by Wnt/β-catenin and the MAPK cascade, can modulate the replication efficiency of ALV, serving as potential targets for antiviral intervention [8, 17, 21]. For instance, inhibition of the Wnt/β-catenin pathway has been shown to reduce ALV-J gene expression and virus production, highlighting a possible therapeutic avenue for controlling viral replication [8].

Another layer of regulation in ALV molecular biology is provided by host microRNAs (miRNAs), which can either restrict or promote viral replication by targeting key viral or host factors. Differentially expressed miRNAs, such as miR-23b and miR-375, have been implicated in modulating host immune responses and viral oncogenesis through interactions with factors like IRF1 and YAP1; these interactions consequently affect the balance between viral proliferation and apoptosis in infected cells [20, 23]. Furthermore, the rapid evolution of ALV, driven by recombination events between exogenous and endogenous sequences, contributes to the emergence of novel strains with altered virulence profiles, as observed in the evolution of ALV-J and the emergence of ALV-K [9, 11, 14].

At the genomic level, the long terminal repeat (LTR) regions of ALV are crucial regulatory elements that dictate viral gene expression and integration site selection. Variations in the LTR sequences, as well as deletions in specific regions such as the 3′ UTR, have been associated with differences in viral pathogenicity and tumorigenic potential [9, 13]. These molecular insights, derived from sequencing studies and reverse genetic analyses, underscore the complex interplay between viral genetic determinants and host cellular responses, ultimately shaping the epidemiology, virulence, and transmission dynamics of ALV.

The intricate molecular biology of ALV continues to be a focus of research that not only informs our understanding of retroviral replication and evolution but also guides the development of innovative strategies to mitigate its impact on the poultry industry. In light of the economic importance of ALV and the rigorous surveillance programs endorsed by international organizations such as the CDC, WHO, and WOAH, ongoing molecular studies are critical for designing effective control measures and informing global animal health policies [2, 7].

Molecular Pathogenesis and Oncogenic Mechanisms of Avian Leukosis Virus

Avian Leukosis Virus (ALV) is an enveloped retrovirus that has evolved complex molecular mechanisms to induce tumorigenesis and persist in host cells through integration into the chicken genome. As an oncogenic retrovirus, ALV employs a series of molecular strategies to dysregulate cellular signaling pathways, induce mutations, and subvert immune responses, ultimately leading to cell transformation and tumor formation [7, 18]. The molecular pathogenesis involves critical interactions between viral proteins and host cellular machinery that facilitate viral entry, replication, integration, and eventual oncogenic transformation.

Viral Entry and Receptor Usage

The initial events in ALV infection are marked by receptor binding and membrane fusion. ALV subgroups, notably subgroup J, utilize distinct cellular receptors to gain entry into host cells. The primary receptor for ALV-J is chicken Na⁺/H⁺ exchanger type 1 (chNHE1), which plays an indispensable role in virus entry; mutations in chNHE1 have been correlated with resistance to viral infection, emphasizing its importance in pathogenesis [3, 5, 28]. The high conservation of cellular receptors such as chNHE1 facilitates a broad host range and contributes to the persistent infections observed in diverse chicken populations [28]. In contrast, other subgroups use receptors such as Tva (for subgroups A and K) and distinct cysteine-rich domains in tvb (for subgroup B) [6, 29]. These receptor interactions not only dictate the host cell tropism but also influence subsequent viral replication kinetics and pathogenic outcomes.

Integration and Insertional Mutagenesis

Once internalized, ALV reverse transcribes its RNA genome into DNA and integrates into the host genome. This integration event is random yet exhibits a preference for transcriptionally active regions, thereby increasing the potential for insertional mutagenesis [32]. Viral integration near proto-oncogenes or tumor suppressor genes can perturb their normal regulatory functions and lead to uncontrolled cell proliferation [32]. The process of integration is central to the virus’s oncogenic potential, as it may result in the deregulated expression of genes involved in cell cycle control and apoptosis. Studies using high-throughput sequencing have revealed common viral integration sites in ALV-induced B-cell lymphomas, thereby establishing a direct link between integration events and the development of lymphoid tumors [16, 32]. This stealthy method of oncogenesis allows ALV to remain latent yet potent in its ability to trigger tumor formation over time.

Signal Transduction Pathway Modulation

ALV infection has been shown to manipulate several intrinsic cellular signaling pathways to promote viral replication and transformation. One key pathway implicated in ALV pathogenesis is the Wnt/β-catenin signaling cascade [8]. Activation of this pathway by pharmacological inhibition of GSK-3 can result in elevated ALV-J mRNA and protein expression, underscoring the importance of dysregulated cell proliferation signals in the viral life cycle [8]. Additionally, ALV has been shown to stimulate the extracellular signal-regulated kinase (ERK)/AP1 pathway. This activation leads to enhanced expression of viral proteins such as gp85 and gag, concurrently promoting cell survival and transformation [21]. The simultaneous activation of multiple signaling cascades, including those that regulate cell proliferation, differentiation, and apoptosis, creates an intracellular environment conducive to oncogenic transformation.

Another critical host factor in ALV pathogenesis is the tripartite motif-containing protein (TRIM62), which normally restricts ALV replication. The antiviral function of TRIM62 relies heavily on its SPRY domain, and its down-regulation or functional impairment can facilitate enhanced viral replication and contribute to tumor development [26]. This interplay between viral evasion strategies and host restriction factors further complicates the host’s ability to clear the infection, thereby predis­posing infected cells to undergo neoplastic transformation.

Epigenetic Regulation and MicroRNA Dysregulation

Beyond direct genetic alterations, ALV manipulates epigenetic mechanisms and microRNA (miRNA) expression profiles to modulate host gene expression. Changes in miRNA expression patterns have been repeatedly observed in ALV-infected cells and are linked to key pathways governing cell cycle progression, apoptosis, and immune responses [30, 31]. For example, down-regulation of tumor-suppressor miRNAs such as gga-miR-375 has been associated with increased expression of oncogenic factors like YAP1, which plays a role in cell proliferation and survival [23]. Conversely, the up-regulation of miRNAs such as miR-23b can suppress critical antiviral factors like IRF1, thus promoting ALV replication and contributing to tumorigenesis [20]. In addition, miR-125b suppression has been shown to inhibit apoptosis and elevate levels of pro-oncogenic proteins such as Semaphorin 4D (Sema4D) [27]. This fine-tuning of cellular gene expression by miRNAs not only facilitates viral persistence by dampening antiviral responses but also contributes directly to the oncogenic transformation of infected cells.

Moreover, the deregulation of the piRNA pathway in domestic chickens has been implicated in the response to ALV integration, thereby serving as a genomic defense mechanism that is perturbed during infection [16]. The disruption of normal epigenetic regulation, involving both miRNAs and piRNAs, can lead to altered transcriptional programs that favor cell survival and proliferation, further enhancing the probability of tumor formation.

Recombination and Mutation-Driven Evolution

The inherent error-prone nature of reverse transcriptase activity in retroviruses, combined with recombination events between exogenous and endogenous retroviral sequences, leads to a high degree of genetic variability in ALV. This genetic diversity is particularly evident in the envelope glycoprotein (gp85), which is subject to continuous mutation and recombination [15, 25]. Such genetic variations can alter viral receptor usage, antigenicity, and pathogenicity, ultimately leading to the emergence of new oncogenic variants [15, 25]. These recombination events have significant epidemiological implications, as evidenced by the emergence of novel subgroups with enhanced oncogenic properties that challenge current eradication and control strategies [2, 33]. Governmental agencies such as the CDC, WHO, and WOAH recognize the economic implications of retroviral infections in livestock, urging continued surveillance and research into curtailing the spread of such pathogens.

The intertwining of receptor mutations, signal pathway modulation, and insertional mutagenesis encapsulates a multifaceted molecular strategy that ALV exploits for its oncogenic progression. These detailed molecular interactions underscore the importance of understanding ALV pathogenesis not only for developing robust diagnostic and therapeutic strategies but also for informing biosecurity measures in poultry production systems globally.

Epidemiology, Economic Impact, and Global Distribution of Avian Leukosis Virus

Avian leukosis virus (ALV) represents a group of retroviruses that have caused considerable concern in the poultry industry worldwide. The epidemiology of ALV reflects a complex interaction between virus evolution, host genetics, and diverse transmission pathways. Both vertical and horizontal transmission routes are well documented, and recent evidence indicates that non-commercial flocks, including fancy, backyard, and indigenous chickens, continue to serve as persistent reservoirs for ALV spread [2, 7, 33]. The global distribution of ALV is directly related to the interconnection of local chicken populations and the inadvertent dissemination through contaminated vaccines and live animal trade, as evidenced by studies from China and other regions [10, 24, 33].

The epidemiological landscape of ALV is characterized by frequent recombination events and the emergence of novel subgroups. For instance, the subgroup J of ALV (ALV-J) has been associated with increased pathogenicity, tumor formation, and immunosuppression that consequently leads to severe economic losses in several countries [2, 36]. Recent molecular epidemiological studies conducted in China highlight that local indigenous breeds are not only susceptible to ALV-J but also appear to harbor a broad range of viral quasispecies, indicating long-term co-evolution between the host and virus [4, 35]. These studies have demonstrated high genetic diversity among isolates, where even within the same flock, distinct ALV strains show significant nucleotide variation in key viral proteins such as gp85 [4, 35]. Such genetic drift results in continuous evolution of the virus, posing challenges to effective surveillance and control programs.

Economic impacts of ALV infections are significant. Outbreaks cause reduced productivity due to immunosuppression in chickens, increased mortality rates, and decreased egg production, ultimately resulting in financial losses for commercial producers [2, 33]. The detrimental effects on production performance are particularly pronounced in flocks where ALV infections are prevalent. In some countries, stringent eradication programs have effectively reduced the incidence of ALV in commercial operations; however, in less-regulated sectors such as in backyard poultry or indigenous breeds, infection persists and maintains the risk of spillover into high-value commercial populations [2, 33, 34]. In addition, the contamination of live virus vaccines with ALV, as reported in retrospective investigations, underlines an additional transmission route that can compromise biosecurity measures and further compound economic repercussions [10, 38]. Recognized international organizations such as the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) emphasize the critical need for continuous monitoring and robust control measures in order to protect both national economies and global poultry markets.

Global distribution patterns of ALV are complex and dynamic. In regions such as China, where diverse chicken breeds are reared, often with less intensive biosecurity compared to industrial operations, epidemiological surveys have revealed the circulation of several ALV subgroups, including ALV-J, ALV-K, and classical subgroups A and B [2, 9, 22]. Phylogenetic analyses have demonstrated that certain strains even cluster into distinct evolutionary branches that are unique to local chicken populations [4, 9]. Similar studies in Europe have indicated that ALV infections are particularly prevalent among fancy-chicken flocks in regions such as Saxony, Germany, where detection rates can reach as high as 56% at the flock level, albeit with variable clinical outcomes [34]. Such findings illustrate that ALV is not confined to a single geographical region, and its spread is influenced by factors such as trade, migration of wild birds, and even laboratory practices in vaccine production.

Furthermore, emerging research has identified additional host factors that contribute to ALV susceptibility and resistance. Genetic modifications using CRISPR/Cas9 editing approaches have successfully conferred resistance in chickens by targeting key viral entry receptors, providing a promising tool to mitigate the economic burden of ALV infections [3, 5, 6]. Such advances are particularly relevant in the context of global poultry health management, where economic losses due to ALV continue despite advanced biosecurity measures. The interplay between host genetic predisposition and ALV’s capacity for rapid mutation underscores the importance of integrating both traditional eradication programs and modern genetic interventions to control virus spread [3, 6, 28].

ALV’s capacity for recombination and cross subgroup interference complicates epidemiological monitoring. For instance, co-infection studies have demonstrated that ALV, especially when present in combination with other immunosuppressive viruses such as Marek’s disease virus or reticuloendotheliosis virus, leads to synergistic increases in viral replication and tumor formation, thereby exacerbating economic impacts in commercial settings [36, 37]. This synergy not only enhances pathogenicity but also creates conditions under which new recombinant viruses may emerge, further challenging existing vaccine and eradication strategies [10, 14]. Hence, surveillance programs must adopt multiplex molecular diagnostic assays that can simultaneously detect multiple subgroups of ALV, providing critical insights into virus circulation and assisting in timely intervention measures [39, 40].

Monitoring efforts by authoritative bodies, including the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), although primarily focused on zoonotic infectious diseases, underscore the importance of continued vigilance over pathogens with significant economic impacts. While ALV is not a zoonotic agent, its economic significance and potential impact on food security align with global health initiatives provided by organizations such as the WOAH and FAO [33].

In summary, the contemporary epidemiology of ALV is marked by a widespread global distribution coupled with significant economic consequences for the poultry industry. The virus’s ability to recombine, adapt, and co-infect alongside other pathogens, along with its persistence in non-commercial flocks, necessitates a multifaceted control approach that integrates advanced molecular detection techniques, gene-editing strategies for enhanced resistance, and rigorous surveillance across all sectors of poultry production [2, 9, 22, 33-35].

Innovative Immunoinformatics and Vaccine Development Strategies Against Avian Leukosis Virus

Innovative approaches integrating immunoinformatics and modern vaccine development methodologies have gained significant traction in combating avian leukosis virus (ALV), a pathogen that continues to severely impact poultry production worldwide. Given that ALV infections result in neoplastic diseases and immunosuppression, which contribute to considerable economic losses as noted by organizations such as the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO), the development of effective vaccines remains an urgent priority. Recent studies have demonstrated that the utilization of advanced computational tools to design multi-epitope vaccines represents a promising frontier in animal health management against ALV [1].

Immunoinformatics Approaches in ALV Vaccine Design

Immunoinformatics leverages bioinformatics tools to predict and analyze epitopes – the specific parts of an antigen recognized by immune cells. In the context of ALV, immunoinformatics platforms such as ABCpred and the Immune Epitope Database (IEDB) servers have been employed to identify potent B and T lymphocyte epitopes from viral proteins [1]. This high-throughput computational screening facilitates the selection of epitopes with high antigenicity and minimal allergenicity and toxicity, thereby streamlining the vaccine design process. By computationally assessing the antigen landscape of ALV, researchers can produce well-characterized multi-epitope constructs that promise to elicit robust immune responses.

The immunoinformatics workflow begins with the retrieval of viral protein sequences from genetically characterized ALV strains. The selected proteins are then analyzed for immunogenic segments using sequence alignment algorithms and machine learning–based prediction models. These approaches not only shorten the time required to identify vaccine candidates but also enhance the precision of targeting immunodominant regions responsible for the activation of both the humoral and cellular arms of the immune system [1]. This methodology is critical in light of the genetic variability and recombination events among ALV subgroups, which have been extensively documented in poultry populations [1, 2, 34, 41].

Multi-Epitope Peptide Vaccine Constructs

One of the cutting-edge outcomes of immunoinformatics in ALV research is the construction of multi-epitope peptide vaccines. These vaccines integrate multiple epitope sequences into a single chimeric protein, intended to stimulate broad-spectrum and long-lasting immunity. The ideal multi-epitope vaccine candidate is designed by linking functionally validated epitopes with appropriate molecular linkers and incorporating adjuvants, molecular compounds known to boost the immune response. This modular design enables the vaccine to target diverse immune mechanisms while reducing the risk of immune escape due to viral mutations or genetic recombination.

Studies have illustrated that the integration of B-cell and T-cell epitopes within one vaccine construct can significantly enhance immunogenic synergy. Detailed in silico analyses, ranging from secondary structure predictions to three-dimensional molecular modeling, enable researchers to forecast the stability, solubility, and hydrophilicity of the multi-epitope vaccine [1]. Following model refinement, rigorous validation steps are implemented using Ramachandran plot analysis and ProsA server assessments to ensure that the tertiary structure of the vaccine does not present steric clashes or unfavorable conformations that could impede immune recognition.

Molecular Docking, Immune Simulation, and In Silico Cloning

The application of molecular docking strategies is pivotal in evaluating the interaction between the proposed vaccine candidates and immune system receptors. For instance, docking studies against Toll-like receptor 7 (TLR7) in chickens have revealed competent binding energies, suggesting that the proposed multi-epitope constructs can effectively trigger innate immune pathways critical for downstream adaptive responses [1]. Beyond docking, immune simulation software enables predictive modeling of the immunological outcomes upon vaccine administration. Such simulations provide valuable insights into the dynamics of immunoglobulin production, cytokine profiles, and lymphocyte proliferation, offering a preliminary yet comprehensive understanding of the vaccine’s potential efficacy.

In silico cloning further extends this approach by predicting the optimal expression of the vaccine candidate in common production platforms, such as Escherichia coli. This computational cloning process ensures that once the candidate is transitioned to experimental validations, the synthetic gene sequences are well-optimized for high-yield production without inducing unexpected immunogenicity or destabilizing alterations.

Recombinant Vector-Based Vaccines and Synergistic Applications

While peptide vaccines represent a promising avenue, recent advancements also emphasize the development of recombinant vaccines utilizing viral vectors. One innovative strategy involves the engineering of recombinant Marek’s disease virus (MDV) to express ALV antigens, thereby exploiting the strong immunogenic properties of MDV as a vaccine vector [42]. This approach not only offers protection against ALV but also holds potential to confer dual protection against MDV co-infections, a scenario particularly critical in poultry where multiple oncogenic viruses may co-circulate.

The integration of immunoinformatics with recombinant vector design signifies a convergence of computational precision and traditional virological techniques. By incorporating epitope predictions and structural validations into vector design, researchers can tailor vaccines that are both highly effective and safe. This multi-pronged approach is in line with global initiatives from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) to enhance the arsenal of veterinary vaccines, reflecting a translational leap in the fight against economically critical pathogens such as ALV.

Enhancing Vaccine Efficacy Through Structural Engineering and Host Immune Modulation

Beyond epitope prediction and vaccine vector design, immunoinformatics provides critical insights into the molecular determinants of host–virus interactions. For example, structural modeling of ALV antigens combined with disulfide engineering can further optimize vaccine stability and immunogenicity. By introducing specific mutations or modifying disulfide bridges within the epitope regions, researchers have demonstrated enhanced structural rigidity, which is vital for the preservation of conformational epitopes that are recognized during natural infections [1].

Moreover, simulated immune responses indicate significant upregulation of key cytokines and immunoglobulin isotypes upon vaccine administration, suggesting that these rationally designed vaccines can prime not only a robust antibody response but also potent cellular immunity. This dual activation is essential in controlling ALV, as the virus is known to induce immunosuppression and tumorigenesis through complex interactions with host immune regulators [1, 42].

The integration of these advanced immunoinformatics techniques into ALV vaccine development effectively bridges the gap between computational design and practical immunization strategies. This innovative paradigm is paving the way for future prophylactic interventions that could dramatically reduce ALV incidence and its associated economic burden in poultry industries globally, aligning with the mandates of international animal health agencies such as the CDC, WHO, and FAO.

Diagnostics and Molecular Detection Techniques for Avian Leukosis Virus

The diagnosis and molecular detection of Avian Leukosis Virus (ALV) are critical in both preventing economic losses and maintaining the biosecurity of poultry populations worldwide. Given ALV’s classification as an oncogenic retrovirus that can induce immunosuppression and neoplastic transformation in chickens, it is imperative that diagnostic methods offer speed, sensitivity, and specificity. The integration of classical virological assays, immunological methods, and modern molecular techniques has significantly enhanced our ability to detect ALV infections at both the individual and flock levels [2, 10].

Traditional Serological and Antigen-Capture Techniques

Historically, diagnostic methodologies for ALV have relied on antigen detection using enzyme-linked immunosorbent assays (ELISAs) to identify viral proteins such as the p27 capsid protein. This antigen-capture ELISA approach has been prominently deployed in routine surveillance programs due to its ease of application and reasonable sensitivity in detecting ALV at the individual-animal and flock levels [34, 44]. For instance, in one study employing this method, cloacal swabs from fancy-chicken flocks were analyzed, and a significant detection rate at both individual and flock levels was recorded, highlighting the antigen detection technique’s robustness in field diagnostics [34]. Although effective, these techniques sometimes require confirmatory tests to discriminate between different ALV subgroups and to detect low-level infections.

Polymerase Chain Reaction (PCR)-Based Assays

Molecular amplification techniques have transformed the diagnostic landscape for ALV by offering enhanced sensitivity and specificity. Conventional reverse transcription-PCR (RT-PCR) and its variations have been particularly useful in amplifying viral gene targets, such as the env gene’s gp85 region or the gag gene, which are critical for subgroup differentiation and epidemiological investigations [9, 47]. These PCR-based approaches facilitate the detection of low copy numbers of ALV nucleic acids in clinical samples and possess the capability of differentiating between exogenous and endogenous viral sequences, an important consideration given the recombination events that can occur in ALV [10, 12].

Real-Time PCR and SYBR Green-Based Assays

The advent of quantitative real-time PCR (qPCR) has further refined ALV diagnostics by enabling not only the identification but also the quantification of viral load in various tissue samples. SYBR Green-based real-time PCR assays have been developed for the separate detection of ALV-J and multiplex detection of ALV subgroups A and B, offering limits of detection orders of magnitude lower than those achieved by conventional PCR [39, 45]. In one notable example, a SYBR Green I-based assay was able to detect viral loads in organ tissues, revealing that the highest copy numbers of ALV-J were consistently found in the heart and kidney at later stages post-infection. These sensitive detections allow for a more refined understanding of virus dissemination, a factor that is vital for formulating effective surveillance programs in regions where ALV remains endemic [39, 45]. In addition, multiplex quantitative PCR assays have recently been introduced to distinguish among ALV-A, ALV-B, ALV-J, and the emerging ALV-K simultaneously, thereby streamlining the detection process in field samples with potential coinfections. The multiplex qPCR’s high sensitivity and discriminative capability are essential for both monitoring clinical outbreaks and enforcing eradication strategies, as outlined by global entities like the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) [40].

Immunochromatographic Test Strips and Electrochemical Sensors

Rapid detection methods based on immunochromatographic test strips have also been devised to address the need for on-site testing. Fluorescent microsphere immunochromatographic test strip assays employ monoclonal antibodies against ALV’s p27 protein, coupled with innovative labeling techniques such as β-cyclodextrin-nanogold-ferrocene complexes to amplify the detection signal [43]. This method not only provides the advantage of rapid, visible detection under UV light but also offers quantitative analysis when paired with a fluorescence analyzer. The integration of these host–guest complex labels and conductive graphene nanocomposites results in an ultrasensitive platform that is ideally suited for point-of-care screening in both commercial and backyard poultry settings [43]. Given the economic impact of ALV on the poultry industry, such portable and user-friendly systems have the potential to complement laboratory-based assays, ensuring that early interventions can be implemented.

Metagenomic and Next-Generation Sequencing Approaches

Beyond traditional PCR and immunoassays, next-generation sequencing (NGS) and metagenomic approaches have emerged as powerful tools for the comprehensive detection and molecular characterization of ALV. After virus isolation in cell cultures or embryonated eggs, metagenomic analysis can be employed to generate complete genome sequences that not only confirm the presence of ALV but also provide detailed insights into its genetic diversity and recombinant nature [46]. By scrutinizing the complete viral genome, researchers can identify mutations, deletion patterns, and novel recombinant strains that may elude standard diagnostic tests [46, 48]. NGS-based diagnostics allow for the simultaneous detection of coinfecting agents, such as other oncogenic viruses, and provide critical data to understand the epidemiology and evolutionary trajectory of ALV across different chicken breeds and geographical regions [46, 48]. This level of genomic surveillance is particularly emphasized by regulatory agencies like the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) for economically significant animal pathogens, as rapid detection and characterization are key to preventing widespread outbreaks.

Host Gene Expression Analysis and Reference Gene Selection

An additional layer of diagnostic scrutiny involves the measurement of host gene expression responses following ALV infection. Quantitative real-time PCR analyses, which require the selection of stable reference genes, have been developed specifically for chicken embryo fibroblasts infected with ALV-J [49]. The validation of reference genes such as RPL30 and SDHA ensures that studies analyzing host mRNA profiles are reproducible and accurate, thereby enabling researchers to assess viral impact on cellular pathways and immune responses. This is critical not only for pathogen detection but also for understanding host-virus interactions that may reveal novel targets for therapeutic intervention or inform the design of genetically resistant poultry lines.

Integration with Biosecurity Surveillance Programs

Current diagnostic methodologies are increasingly being integrated with comprehensive biosecurity measures and surveillance programs recommended by international bodies such as the CDC, WHO, and FAO. Such integration ensures that both commercial and non-commercial poultry populations are routinely screened using a combination of rapid immunoassays, highly sensitive molecular techniques, and genomic characterization methods. These multifaceted strategies not only facilitate the timely detection of ALV infections but also enable the monitoring of novel variants and recombinant strains, thereby informing control strategies on both local and global scales [2, 10, 40].

By harnessing a combination of classic virological, serological, and advanced molecular diagnostic techniques, the field of ALV detection has evolved into an integrated system capable of addressing both immediate outbreak responses and long-term epidemiological studies.

Host-Virus Interactions and Immunosuppression in ALV-Infected Poultry

Avian leukosis virus (ALV) exerts profound effects on infected poultry by establishing intricate host-virus interactions that not only promote viral replication but also lead to severe immunosuppression. These interactions occur at multiple levels, ranging from viral entry via specific cellular receptors to downstream modulation of intracellular signaling cascades. The virus employs a multifaceted strategy that both evades immune surveillance and disrupts normal immune function, thereby heightening the risk of secondary infections and exacerbating tumorigenesis in the host, an issue of significant concern for global animal health authorities such as the CDC, WHO, and FAO when monitoring economically critical pathogens.

Mechanisms of Viral Entry and Receptor-Mediated Infection

At the outset of infection, ALV utilizes a range of cellular receptors to gain entry into host cells. For example, the subgroup J virus specifically targets receptors such as the chicken Na⁺/H⁺ exchanger type 1 (chNHE1), a process that is critical for efficient viral entry and subsequent replication [5]. The viral envelope proteins, particularly gp85, are pivotal in mediating the binding to these receptors. A strong conservation of receptor structure across different breeds of poultry has been observed, which is likely a major factor contributing to the broad host range of ALV-J, especially among local chicken populations [28]. The high conservation of these receptors minimizes species-specific immune barriers and allows ALV to efficiently cross infect diverse genetic backgrounds, further compounding its epidemiological impact within both commercial and non-commercial settings.

Modulation of Host Cellular Signaling Pathways

Following cellular entry, ALV manipulates several host intracellular signaling pathways that facilitate viral replication and subvert immune defense mechanisms. Studies have revealed that early during infection, ALV triggers activation of the MAPK/ERK and AP-1 signaling cascades, thereby increasing viral protein production and promoting neoplastic transformation [21]. The activation of ERK, in particular, not only leads to enhanced replication but also establishes a link with oncogenesis by modulating gene expression patterns that fortify the malignant transformation of lymphoid and myeloid lineages. This activation is believed to result in part from the direct contributions of viral structural proteins, such as gp85 and gag, to the host signal transduction mechanisms. Such interventions in cellular signaling contribute to the dysregulation of normal cell cycle progression and ultimately result in the immunosuppressive phenotype associated with ALV infection.

Impact on Dendritic Cells and Apoptotic Pathways

A critical component of the host’s innate immune response is the dendritic cell (DC) population, which orchestrates antigen processing and presentation. However, ALV infection of chicken dendritic cells has been shown to induce apoptosis through aberrant microRNA expression profiles. Research indicates that infection with ALV-J in DCs triggers the widespread upregulation of various microRNAs, over one hundred differentially expressed miRNAs have been noted, with a concurrent suppression of key genes important for cell viability and antigen processing [30]. The apoptosis of DCs not only disrupts the early interferon response but also compromises the bridge between innate and adaptive immunity. As a result, the host becomes less capable of mounting an effective immune response against the invading virus, paving the way for persistent infection and subsequent tumor development.

Role of microRNAs in Immune Modulation and Cell Proliferation

In addition to their role in inducing apoptosis in dendritic cells, microRNAs represent a vital regulatory mechanism through which ALV modulates host immunity. For instance, the upregulation of miR-34b-5p has been associated with the suppression of melanoma differentiation-associated gene 5 (MDA5), a crucial sensor in the innate antiviral response [17]. Downregulation of MDA5 disrupts the host’s ability to detect viral RNA, thus limiting the production of antiviral cytokines such as interferon and facilitating robust viral replication. Similarly, other microRNAs, including miR-125b and miR-23b, have been implicated in the alteration of apoptotic pathways and cell cycle regulation. These miRNAs target genes involved in immune signaling and apoptosis, effectively creating an environment favorable for unchecked cellular proliferation and neoplastic transformation [20, 27]. Such fine-tuned regulation by viral-induced microRNAs underscores a sophisticated mechanism of immunosuppression that not only allows for enhanced viral replication but also initiates oncogenic processes.

Disruption of Interferon Responses and Innate Immunity

Interferon responses constitute the first line of defense in viral infections. ALV has evolved strategies to dampen these responses, thereby weakening the overall immune capacity of the host. Alterations in the expression of interferon regulatory factors (IRFs), such as IRF1, have been documented in ALV-infected tissues, a change mediated by virus-induced microRNAs that target these signaling molecules directly [20]. With the attenuation of interferon signaling, antiviral effector functions are diminished, leading to a state of immunosuppression that renders the host vulnerable not only to ALV replication but also to coinfections with other immunosuppressive viruses such as chicken infectious anemia virus (CIAV) and Marek’s disease virus (MDV) [36, 37]. The synergistic effects of coinfection further intensify the immunosuppressive state, contributing to severe clinical outcomes including tumor formation, organ atrophy, and increased mortality rates.

Alteration of Host Gene Expression and Epigenetic Modifications

Beyond the direct interference with cell signaling and immune detection, ALV infection is associated with broad alterations in host gene expression and epigenetic landscapes. Changes in the expression profiles of genes involved in immune regulation, cell cycle control, and cytokine production have been observed in infected birds [30]. The virus–host interaction consequently results in sustained immunosuppression, which not only facilitates viral persistence but also exacerbates tumorigenic processes. Epigenetic modifications, including those mediated by circular RNAs, further disrupt normal cellular responses and may lead to resistance against apoptosis, a hallmark of transformed cells [50]. This reprogramming of the host cellular environment underlines the complexity of ALV-induced immunosuppression and highlights the interconnected nature of viral replication, immune evasion, and tumorigenicity.

Synergistic Infections and the Economic Impact on Poultry Production

The immunosuppressive effects induced by ALV have wide-reaching implications for poultry health and production. Infected birds exhibit diminished responses to both natural infections and vaccination, leading to higher incidences of secondary infections and elevated mortality rates. Epidemiological studies underscore that even with eradication programs in place for commercial flocks, immunosuppressed populations in backyard, hobby, and indigenous breeds continue to serve as reservoirs for ALV [2]. This persistence of ALV in less scrutinized environments heightens the risk of spillover events into commercial flocks, thus amplifying economic losses and complicating control efforts as emphasized by regulatory bodies such as WHO and FAO.

The cooperative pathogenicity observed in coinfections, where ALV-J and other immunosuppressive viruses such as CIAV or MDV interact, further illustrates the detrimental impact on the immune system and overall poultry health [36, 37]. Such synergistic effects not only promote higher viral loads and prolonged viremia but also lead to more severe tissue damage and immunodeficiency, thereby compromising vaccine efficacy. The resultant decrease in poultry productivity and the increased demand for intensive monitoring and control measures necessitate the use of advanced diagnostic and molecular epidemiology tools, such as multiplex quantitative PCR assays, to detect and manage ALV infections efficiently [40].

Altogether, these complex host-virus dynamics and the profound immunosuppressive effects orchestrated by ALV underpin the significant challenges faced in controlling this pathogen in diverse poultry populations. Such insights into the molecular and cellular interplay between ALV and the host immune system are critical for informing future strategies aimed at both genetic manipulation for resistance and the development of innovative diagnostic tools to mitigate the impact of this virus on global poultry production.

Emerging Control Measures and Eradication Strategies for Avian Leukosis Virus

The evolving landscape of avian leukosis virus (ALV) control requires multi-pronged approaches that incorporate state-of-the-art immunoinformatics, gene editing, advanced diagnostic methodologies, and vigilant biosecurity measures. Emerging strategies in the eradication of ALV are taking advantage of both traditional epidemiological control measures and novel molecular techniques, each aiming to reduce viral prevalence among susceptible poultry populations and ultimately safeguard economic productivity in the global poultry industry. Authorities such as the CDC and WOAH have underscored the importance of integrating innovative vaccination, genetic modification, and improved surveillance into comprehensive control programs for economically critical pathogens.

Advanced Vaccine Development and Immunoinformatics Approaches

One promising avenue in ALV control is the development of multi-epitope peptide vaccines using advanced immunoinformatics techniques. Researchers have exploited computational tools to design vaccines that incorporate multiple B- and T-cell epitopes from ALV proteins, ensuring robust antigenicity while minimizing allergenic and toxic responses [1]. These vaccines are modeled to elicit potent humoral and cellular immune responses, a critical factor considering that ALV infection typically leads to immunosuppression and tumor formation in chickens. Structural refinement of vaccine candidates has also enhanced their stability and solubility, while in silico docking studies confirm their efficient binding to specific receptors such as TLR7 in chickens, which are essential for initiating innate immune responses. This approach not only holds the promise of rapid vaccine production but also offers a cost-effective solution that can be scaled in response to outbreaks across various geographical regions, aligning with surveillance directives issued by international bodies like the FAO.

Gene Editing Strategies to Confer Viral Resistance

Concurrently, gene editing techniques have emerged as a powerful tool in the creation of virus-resistant poultry lines. A notable breakthrough is the precise CRISPR/Cas9-mediated deletion of specific amino acids in the cellular receptors that ALV exploits for entry. For instance, deletion of the tryptophan residue (W38) in the Na+/H+ exchanger type 1 (NHE1) gene has resulted in chickens that are resistant to ALV subgroup J, thereby preventing the initial step in viral replication [3]. Similarly, targeted knockout of the tva gene, essential for the entry of ALV subgroups A and K, has successfully generated poultry lines with complete resistance in both in vitro and in vivo settings [6]. These gene editing strategies leverage our growing understanding of receptor-virus interactions and represent a paradigm shift from conventional vaccination toward the development of inherently resistant genetic stocks. Moreover, the application of CRISPR/Cas9 technology in primordial germ cells ensures that these protective mutations are stably inherited, providing a long-term solution to interrupting ALV spread in both commercial and indigenous chicken populations [5, 29].

Enhancements in Diagnostic Technologies and Surveillance Tools

Rapid and precise detection of ALV is crucial for effective disease management and eradication. Recent developments in electrochemical immunosensors and multiplex quantitative polymerase chain reaction (qPCR) assays have significantly reduced the detection threshold of ALV antigens and nucleic acids. The use of a novel β-cyclodextrin-nanogold-ferrocene host-guest label in immunosensor platforms has allowed for ultrasensitive detection of ALV-J, with detection limits reaching femtomolar levels [43]. In addition, multiplex quantitative PCR assays enable simultaneous detection and differential diagnosis of various ALV subgroups, including A, B, J, and the emerging K subgroup, with high sensitivity and specificity [40]. With such enhanced diagnostic capacities, endemic infection foci can be identified rapidly, allowing for timely culling or targeted vaccination programs under strict biosecurity protocols. These diagnostic improvements are critical in regions where ALV remains prevalent, such as among hobby, backyard, and indigenous chickens, which may serve as reservoirs for the virus and pose a threat to commercial production systems.

Mitigation of Vaccine Contamination and Secure Vaccine Development

Another emerging control measure is the stringent monitoring of live vaccines for contamination with ALV. Historical evidence has shown that contaminated live vaccines can serve as inadvertent vectors for virus transmission [10, 24, 38]. As a result, current eradication strategies emphasize the need for rigorous quality control and enhanced screening protocols during vaccine production. Implementation of sensitive diagnostic assays, as described above, ensures that only ALV-free vaccines are distributed, significantly reducing the risk of vaccine-mediated outbreaks. Ensuring the sterility and purity of live vaccines aligns with guidelines from organizations such as the WOAH and WHO, further reinforcing international efforts to eliminate ALV from poultry populations.

Modulation of Host Factors and Antiviral Agents

In addition to genetic and immunologic interventions, other promising measures include the modulation of host signaling pathways to inhibit ALV replication. Investigations into compounds such as Sargassum fusiforme polysaccharide (SFP) have disclosed its ability to exert virustatic effects by interfering with viral attachment and reducing gene expression levels of ALV at both transcriptional and translational stages [51]. Furthermore, modulation of cellular pathways like Wnt/β-catenin signaling has shown potential in reducing viral replication, thereby representing an adjunct strategy to traditional prophylactic measures [8]. Such therapeutic approaches, while in the earlier stages of research, may complement genetic modifications and immunization campaigns by providing an additional layer of defense against viral spread in infected flocks.

Integrated Biosecurity and Surveillance Programs

Finally, integrating these emerging modalities into a cohesive biosecurity strategy is pivotal. Modern eradication programs combine early detection through advanced diagnostics, immediate culling of infected specimens, rigorous vaccination protocols, and the strategic breeding of genetically resistant birds. Continuous monitoring of viral evolution through molecular epidemiological surveys ensures that new variants, especially those arising from recombination events, can be detected and managed promptly [2, 33]. In regions where ALV is endemic, particularly within diverse indigenous populations, multi-layered surveillance programs complemented by strict movement controls and improved sanitation practices are essential. These comprehensive measures, developed in response to both technological advances and evolving viral ecology, are designed to significantly diminish the incidence of ALV across all levels of poultry production, while enabling rapid outbreak response consistent with global standards recommended by agencies such as the CDC and FAO.

By harnessing cutting-edge vaccine design, gene editing, and precision diagnostics within a robust biosecurity framework, the field is steadily progressing toward the comprehensive control and potential eradication of avian leukosis virus in poultry populations.

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