Avian Hepatitis E Virus in Chickens

Overview and Taxonomy of Avian Hepatitis E Virus in Chickens

Avian hepatitis E virus (aHEV) is a significant viral pathogen within poultry populations worldwide. It is responsible for a range of hepatic and splenic diseases in chickens, such as big liver and spleen disease (BLS), hepatitis-splenomegaly syndrome (HSS), and hepatic rupture hemorrhage syndrome (HRHS) [1, 3, 5]. Taxonomically, aHEV is classified within the family Hepeviridae and belongs to the genus Avihepevirus, as outlined in the latest ICTV report [22]. Within this family, aHEV is distinct from its mammalian counterparts by several biological, genetic, and epidemiological characteristics. The virus possesses a positive-sense, single-stranded RNA genome of approximately 6.6 kilobases that is organized into three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encodes nonstructural proteins responsible for viral replication, while ORF2 encodes the capsid protein that plays a critical role in host cell attachment and immune recognition; ORF3 is believed to modulate viral egress and host interactions [12, 13].

Genetic Diversity and Genotypic Classification

Since its first characterization, aHEV has been recognized to exhibit a broad genetic heterogeneity that has led to the identification of multiple genotypes. Early studies documented the existence of several distinct genotypes based on partial gene sequences of ORF1 and ORF2, with nucleotide sequence identity often ranging between 79% and 86% when compared to classical strains [1, 4]. For instance, phylogenetic analyses have grouped circulating strains into genotypes such as genotype 2, prevalent in areas like Poland [3, 7], and genotype 3, which has been identified in several regions across China [9-11]. More recently, novel genotypes have been proposed, including a divergent cluster designated as genotype 7, which was first identified in outbreaks involving both chickens and common pheasants [4]. In addition, isolates from Nigeria have provided evidence for the existence of additional, genetically distinct genotypes, with some strains exhibiting only 79.0–83.5% nucleotide identity to other known aHEV strains [6]. These findings underscore the notion that aHEV is not a monolithic entity but rather a diverse population of viruses with ongoing evolutionary divergence.

The variability observed among different aHEV strains is highlighted by the differing amino acid substitutions in functional regions such as the helicase, capsid, and parts of the ORF1 polyprotein [5, 14]. This genetic variability likely contributes to differences in pathogenicity and host-virus interactions, as observed in studies where distinct genotypes were associated with variations in clinical manifestations, infection kinetics, and mortality rates [5, 15, 16]. It is this diversity that not only complicates diagnostic and control measures but also poses challenges for developing effective vaccines and antivirals [2]. The continuous evolution and expansion of genotypes are therefore of paramount importance, as each novel clade may bear unique pathogenic traits that affect the economic stability of poultry farms globally.

Molecular Characteristics and Functional Domains

At the molecular level, aHEV’s genetic organization is a subject of intense study. The genome’s ORF2 encodes a capsid protein that has been central to investigations into viral entry and immune evasion. Detailed epitope mapping has demonstrated that crucial amino acid residues, particularly those found in the C-terminal region (amino acids 471–507), are essential for binding to both avian and human cells [17]. This suggests that while avian HEV is not known to be zoonotic, the molecular interactions it employs for cell attachment might be partially conserved, offering insights into evolutionary links with mammalian HEV types [12, 20]. Additionally, domains within ORF1, such as the helicase and RNA-dependent RNA polymerase motifs, show unique evolutionary pressures, with reports of potential recombination and positive selection events that may facilitate adaptation to different host cellular environments [11, 14].

A comprehensive taxonomic assessment requires not only sequence-based phylogeny but also an understanding of the virus’s structural and functional properties. The genomic similarities and differences among diverse aHEV genotypes contribute to their classification within the Orthohepevirinae subfamily. In contrast with mammalian HEV, which is responsible for both waterborne epidemics and sporadic zoonotic infections in humans as noted by public health agencies like the CDC, FAO, and WHO, aHEV maintains a relatively restricted host range predominantly in avian species [19, 20]. This distinction informs international surveillance programs and biosecurity measures that are implemented by organizations such as the World Organisation for Animal Health (WOAH) to mitigate economic losses caused by viral hepatitis in chickens.

Epidemiology, Host Range, and Economic Impact

From an epidemiological standpoint, avian HEV exhibits a global distribution with variable prevalence across different regions and production systems [1, 8, 18]. Serological and molecular studies across Asia, Europe, and Africa have identified high seroprevalence rates in chicken flocks, with many subclinical infections complicating routine surveillance [1, 7, 18]. For instance, a recent study from Taiwan reported the endemic circulation of an aHEV strain in chickens, with evidence suggesting possible asymptomatic transmission within flocks [1]. Similarly, investigations in Nigeria and Poland have confirmed the wide occurrence of aHEV, illustrating that aHEV circulates undetected in many commercial layers and broilers [3, 6, 21].

The economic ramifications of aHEV infections are significant. Affected flocks can experience decreased egg production, increased mortality, and liver diseases which necessitate costly interventions and management adjustments [2, 5]. Furthermore, the evolving genetic diversity of aHEV continues to challenge diagnostic assays and vaccine design efforts, underscoring the need for the integration of genomics into routine monitoring practices. This multifaceted approach is essential to inform regulations and disease control strategies endorsed by international bodies such as the CDC and FAO.

Moreover, while aHEV itself has not demonstrated zoonotic potential, its close genetic and antigenic relations to mammalian HEV render it an important model for understanding HEV evolution and host adaptation mechanisms [12, 20]. Comprehensive studies into aHEV have thus expanded our insight into the broader genus, providing data that may eventually benefit both veterinary and human public health sectors.

Taxonomic Implications and Future Directions

In summary, the taxonomy of avian hepatitis E virus, as defined by its phylogenetic position and genomic properties, continues to evolve with ongoing surveillance and molecular characterization studies. The discovery of novel genotypes and the elucidation of critical functional domains in the viral genome have provided new insights into aHEV’s biology and epidemiology [4, 6, 11]. These insights are fundamental to refining diagnostic methodologies and improving our understanding of the virus’s mechanisms of replication, dissemination, and pathogenicity.

With advances in next-generation sequencing and bioinformatics, researchers are now better equipped to delineate the subtle genetic variations and evolutionary trajectories that define the complex taxonomy of aHEV. Continued collaborative research efforts, in line with global standards set by organizations like WHO and WOAH, will be critical to developing effective strategies to monitor, control, and eventually mitigate the impacts of avian hepatitis E virus in the poultry industry.

Molecular Pathogenesis of Avian Hepatitis E Virus in Chickens

Avian hepatitis E virus (aHEV) is a small, positive-sense RNA virus belonging to the Hepeviridae family that exhibits a complex interplay with its host at the molecular level. Its genome, approximately 6.6–6.7 kb in size, encodes three major open reading frames (ORFs). ORF1 encompasses nonstructural proteins required for viral replication, including methyltransferase, helicase, and RNA-dependent RNA polymerase, while ORF2 encodes the viral capsid protein and ORF3 encodes a multifunctional phosphoprotein. The divergences observed in nucleotide and amino acid sequences across the several aHEV genotypes have crucial implications for viral replication capacity, tissue tropism, and clinical outcomes in infected chickens [4, 13, 14].

Genomic Organization, Replication, and Viral Protein Function

The genomic organization of aHEV underscores its ability to commandeer host cellular machinery. The central role of ORF1 in viral replication is evident from its inclusion of domains necessary for RNA synthesis and processing. Studies have demonstrated that viral polymerases and helicases from different aHEV genotypes exhibit variations in activity that correlate with pathogenic manifestations such as hepatic rupture hemorrhage syndrome (HRHS) and hepatitis-splenomegaly syndrome (HSS) [5, 14]. Meanwhile, the ORF2 protein is critical not only for virus assembly but also for mediating initial interactions with susceptible host cells. Mapping studies have revealed that the C-terminal segment between amino acids 471 and 507 within ORF2 is indispensable for binding to both avian and human cells, thereby elucidating a fundamental step in the initiation of infection [17].

Once the virus enters the host cell, replication complexes are established, facilitating a rapid increase in viral RNA levels. Experimental infection models using RNA transcript inoculation in chicken liver cells have confirmed the efficient replication of aHEV in vitro [16]. The interplay between the viral nonstructural proteins and host cell factors is central to the control of viral synthesis, with alterations in these interactions possibly underlying strain-specific pathogenic variations [15, 24].

Host-Cell Entry and Receptor Interactions

The onset of aHEV infection hinges on the interaction between the viral capsid protein and specific cellular receptors. A critical breakthrough in understanding aHEV cellular entry was the identification of the organic anion-transporting polypeptide 1A2 (OATP1A2) as a novel receptor that binds directly to a truncated region of the ORF2 protein (amino acids 313–549) [26]. Overexpression of OATP1A2 in chicken liver cells markedly enhances viral attachment and infection, while the inhibition of this receptor using competitive substrates or antibodies significantly reduces viral entry. This finding not only reinforces the essential role of ORF2 in mediating host-cell recognition but also highlights a potential target for antiviral intervention in poultry farming, a matter of significant economic and food safety relevance as outlined by authoritative organizations such as the CDC and FAO.

Parallel to receptor-mediated binding, aHEV entry is further facilitated by the engagement of additional host proteins. For instance, the capsid protein has been shown to interact with RAS-related protein 1b (Rap1b), a key regulator of cell adhesion and cytoskeletal dynamics [23]. This interaction triggers a cascade of downstream events, including the membrane recruitment of Rap1-interacting adapter molecule (RIAM) and Talin-1, culminating in the activation of integrin α5/β1 complexes. The resultant integrin and focal adhesion kinase (FAK) signaling leads to the activation of small Rho GTPases such as RAC1 and CDC42, which are instrumental in orchestrating actin remodeling at the plasma membrane [17, 23]. Such cytoskeletal rearrangements are pivotal for promoting efficient viral internalization and subsequent intracellular trafficking.

Intracellular Signaling, Cytoskeletal Remodeling, and Viral Dissemination

Following receptor binding, the virus leverages host intracellular machinery to establish a productive infection. The intimate interplay between viral proteins and the host cytoskeleton is a critical determinant of viral dissemination within infected tissues. The activation of Rap1b, as induced by direct interaction with the viral ORF2 protein, not only enhances viral entry but also recruits downstream effectors that subsequently activate non-muscle myosin and modulate F-actin dynamics [23]. Simultaneously, evidence suggests that the capsid protein’s interaction with host CDC42 further amplifies intracellular signaling, promoting the activation of p21-activated kinase 1 (PAK1) and cofilin via the CDC42-PAK1-LIMK1 axis [29]. Such pathways culminate in dramatic cytoskeletal reorganization, facilitating not only efficient viral internalization but also promoting cell-to-cell spread, a hallmark of robust viral pathogenesis.

In infected hepatocytes, the disruption of normal cellular processes is profound. Ultrastructural studies have revealed that mitochondria become swollen and exhibit lost or disrupted cristae, while the endoplasmic reticulum appears distended, indicating that aHEV infection induces significant cellular stress and may trigger apoptotic cascades [24]. Moreover, these cellular perturbations likely contribute to the liver pathology observed in clinical cases of hepatitis-splenomegaly syndrome, where gross liver hemorrhages and hepatomegaly are common [5]. The capacity of aHEV to replicate within liver cells and to promote cytoskeletal and organelle dysfunction underscores its potent pathogenic potential in chickens.

Modulation of Host Cellular Processes and Immune Responses

Beyond simple replication and cellular entry, aHEV has evolved strategies to modulate host immune responses. The engagement of host receptors and subsequent activation of integrin and GTPase pathways not only enhance viral internalization but may also influence the cellular inflammatory response, thereby altering the local immune milieu [23, 26]. This modulation of host cell signaling is further reflected in differential immune responses to various aHEV strains, with certain genotypes inducing more robust liver lesions and a higher incidence of viremia [5, 25]. Although aHEV primarily infects poultry and is not considered zoonotic, these molecular interactions are crucial in understanding the virus’s economic impact on the poultry industry, as well as guiding biosecurity measures recommended by international bodies such as the WHO and the WOAH.

Experimental infection studies have highlighted that cellular immune components, especially CD8+ lymphocytes, are critical for clearing the virus and mitigating liver damage. In contrast, humoral responses, while detectable, appear less effective at preventing viral spread and may even contribute to the development of liver lesions in some scenarios [28]. Continued investigations into the balance between viral evasion strategies and host immune defenses are essential in explaining the variable pathogenic outcomes observed in different chicken populations.

In summary, the molecular pathogenesis of aHEV in chickens is driven by a complex network of viral and host factors. From the intricacies of viral genome organization and replication to the sophisticated mechanisms of receptor binding, intracellular signaling, and immune modulation, each step of the viral life cycle contributes to the overall disease process. The detailed understanding of these molecular interactions not only elucidates how aHEV causes liver and extrahepatic lesions, such as those observed in ovarian tissues with consequent reduced egg production [27], but also provides a valuable roadmap for the development of targeted antiviral strategies and effective vaccines in the poultry industry.

Epidemiology and Transmission Dynamics of Avian Hepatitis E Virus in Chickens

Avian hepatitis E virus (aHEV) exhibits a complex epidemiological pattern in chickens, with a distribution that spans multiple continents and diverse production systems. Initial studies in Taiwan revealed the endemic presence of aHEV in chicken populations, with phylogenetic studies indicating significant divergence from other HEV strains [1]. Subsequent investigations across Asia, Europe, and Africa have consistently demonstrated that aHEV, responsible for conditions such as big liver and spleen disease and hepatitis-splenomegaly syndrome, is widely circulating within both commercial and backyard flocks [2, 3, 7, 30].

Geographical Distribution and Genetic Diversity

Research conducted in Poland confirmed that chicken flocks harbor aHEV, predominantly of genotype 2, although further studies noted the emergence of novel genotypes and subtypes in various outbreaks [3, 7]. In China, multiple studies have documented the co-circulation of diverse genotypes, including novel ones associated with severe disease manifestations such as hepatic rupture hemorrhage syndrome [5, 9, 10, 30]. In Nigeria, evidence of both known and unique aHEV genotypes has been identified in apparently healthy chickens, highlighting a silent reservoir that could contribute to sporadic disease outbreaks if conditions favor viral transmission [6]. This polymorphism, when linked with observations from Europe and Africa [21, 34], reflects the virus’s evolutionary dynamics and its adaptation to varying ecological niches in chicken production systems.

Transmission Routes and Mechanisms in Chicken Flocks

The primary mode of transmission documented for aHEV in chickens is the fecal-oral route. Studies comparing housing arrangements have shown that chickens reared in a cage-free environment tend to exhibit a significantly higher infection rate than those housed in cages. In a controlled study, serological and molecular assessments demonstrated that contact with fecal material is a critical determinant of viral spread, where cage-free conditions facilitate the contamination of shared environments and subsequent viral detection in cloacal swabs and serum samples [31]. This finding underscores the importance of biosecurity measures in intensive poultry production settings and supports recommendations by international bodies such as the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) for meticulous management practices to mitigate fecal–oral transmission.

Vertical transmission has also been highlighted as a potential route, albeit less frequently documented than horizontal spread. Evidence of aHEV in hatching eggs, live and dead embryos, and newly hatched chicks suggests that the virus can be transmitted from breeders to their progeny under certain conditions [30]. This vertical transmission not only perpetuates the virus within affected flocks but also complicates eradication strategies, as infected chicks may become asymptomatic carriers.

Asymptomatic Infections and Carrier Status

A substantial aspect of the epidemiology of aHEV is the occurrence of asymptomatic infections. Several studies have reported that chickens with no overt clinical symptoms can nonetheless harbor and shed the virus, thereby acting as reservoirs in the poultry population. For example, the detection of aHEV RNA and the demonstration of seropositivity in clinically healthy flocks indicate that subclinical carriers contribute significantly to viral maintenance and spread [1, 6, 21]. These cases have been associated with gradual declines in egg production and increased mortality during periods of stress or concurrent infections, even though the infected birds may show no immediate signs of disease. This silent circulation places aHEV alongside other economically significant poultry pathogens that require broad surveillance and regular monitoring by national regulatory agencies such as the Centers for Disease Control and Prevention (CDC) and WHO, particularly given the economic implications for the global poultry industry.

Co-Infection and Environmental Influences

The epidemiology of aHEV is further complicated by the frequent occurrence of co-infections with other avian pathogens. Investigations have shown that aHEV often co-circulates with immunosuppressive viruses such as avian leucosis virus, reticuloendotheliosis virus, and chicken infectious anemia virus in flocks experiencing hepatic rupture hemorrhage or hepatitis-splenomegaly syndromes [5, 25, 32]. These co-infections may exacerbate clinical outcomes by compromising the immune response of the host, thereby facilitating higher viral loads and enhanced dissemination within the flock. Such interactions underscore the need for integrated disease management strategies, as recommended by global animal health organizations, to prevent compounded losses in poultry production.

Environmental factors play a critical role in the transmission dynamics of aHEV. The stability of the virus outside the host and its ability to contaminate feed, water, and litter materials underscore the importance of maintaining rigorous hygiene standards in poultry houses. Studies have shown that modifications in housing conditions, such as reducing exposure to contaminated feces through cage-based systems, significantly lower the incidence of infection [31]. Additionally, the persistence of viral RNA in environmental samples necessitates regular decontamination protocols to break the cycle of transmission. These findings align with guidelines from the FAO and WHO on biosecurity measures in agricultural settings, emphasizing that environmental control is as crucial as direct interventions in live birds.

Host and Molecular Determinants of Virus Persistence

At the molecular level, the interaction between viral structural components and host cell receptors contributes to the virus’s ability to persist and propagate within chicken flocks. The binding of the capsid protein to specific cellular receptors such as OATP1A2, as documented in recent studies, facilitates efficient entry of aHEV into host cells, thereby enhancing viral replication and spread [26]. This receptor-mediated internalization is complemented by the virus’s capacity to induce cytoskeletal changes, further optimizing conditions for viral entry and replication [23]. Such insights into the molecular dynamics of aHEV infection not only fill gaps in our understanding of its pathogenesis but also offer potential targets for the development of novel antiviral interventions and vaccines, which are critical given the virus’s impact on poultry health and production economics.

Implications for Surveillance in Commercial and Backyard Settings

Owing to the diverse transmission dynamics and the wide geographical distribution of aHEV, constant surveillance is paramount. The high seroprevalence rates observed in both emerging and established poultry production regions underline the importance of implementing molecular diagnostic tools, such as real-time RT-PCR assays, for early detection and quantification of viral load in flocks [33]. Integration of these diagnostic techniques with traditional serological monitoring provides a robust framework for tracking aHEV in various production systems, thereby aiding national veterinary services and international bodies in formulating appropriate control measures.

Given the potential for subtle clinical signs and subclinical infections to compromise productivity, surveillance programs must prioritize both large commercial poultry operations as well as smaller backyard farms. This is particularly critical in developing regions where biosecurity practices may not be as rigorously enforced, thereby increasing the risk of unnoticed viral spread. The combined emphasis on molecular epidemiology, biosecurity, and integrated disease management remains central to mitigating the significant economic ramifications associated with aHEV outbreaks in the global poultry industry.

Diagnostic Methodologies and Molecular Detection Techniques for AHEV in Chickens

The detection of avian hepatitis E virus (aHEV) in chicken populations has emerged as a crucial component in managing the health of poultry flocks, especially given the virus’s role in hepatitis–splenomegaly syndrome, big liver and spleen disease, and hepatic rupture hemorrhage syndrome. The molecular detection strategies and diagnostic methodologies developed over the past decade incorporate advanced reverse transcription polymerase chain reaction (RT-PCR) techniques, real-time quantitative methods, and targeted sequencing approaches. These techniques allow researchers and veterinary diagnosticians to identify the virus with high specificity and sensitivity, guide epidemiological investigations, and implement biosecurity measures recommended by international organizations such as the CDC, WHO, and WOAH.

Nucleic Acid-Based Detection and RT-PCR Strategies

A critical element in the diagnosis of aHEV is the detection of viral RNA using RT-PCR based methodologies. Traditional RT-PCR methods have been extensively utilized to amplify conserved genomic regions such as ORF1, which encodes non-structural proteins like the helicase, and ORF2, which encodes the capsid protein. For example, initial studies in Taiwan identified an aHEV strain via conventional reverse transcriptase–PCR assays targeting these regions, providing essential insights into viral similarity and transmission dynamics ([1]). Meanwhile, nested RT-PCR protocols have also been implemented to increase the sensitivity of viral RNA detection in clinical samples, as evidenced by screening studies in various countries that identified aHEV in serum, fecal samples, and tissue biopsies ([3], [8]). The nested approach helps overcome limitations associated with low viral load in subclinical infections and enhances diagnostic accuracy, making it an invaluable tool in routine surveillance programs.

Quantitative Real-Time RT-PCR Using SYBR Green Chemistry

To address the need for rapid, sensitive, and quantitative detection of aHEV, researchers have developed a SYBR Green-based real-time RT-PCR assay. In this assay, primers are designed according to the most conserved segments of the viral genome, often within the ORF3 region, to ensure broad applicability across different aHEV genotypes ([33]). This method establishes a dynamic linear range of detection, with reported sensitivities reaching as low as 10 copies per microliter of viral RNA. The quantitative nature of real-time RT-PCR not only enables the detection of viral RNA but also offers the possibility of viral load monitoring, which assists in assessing infection severity, monitoring viremia and viral shedding in both symptomatic and asymptomatic flocks, and subsequently evaluating the effectiveness of biosecurity measures in reducing virus transmission. Such methodologies have become crucial for poultry health agencies and international bodies like the FAO to monitor pathogen load and guide mitigation efforts in large-scale poultry operations.

Targeted Gene Amplification and Sequencing for Genotyping

Molecular diagnostics of aHEV have further been refined through the amplification and sequencing of specific genomic fragments. The ORF1 gene, particularly segments encoding enzyme domains such as the helicase, serves as a reliable target for phylogenetic analysis and genotype determination ([13], [18]). Sequencing of these amplicons has allowed researchers to classify circulating strains effectively and has led to the identification of novel genotypes that differ markedly from previously known variants ([4], [6]). Such genotypic classification is central not only to tracking virus evolution and epidemiologic patterns but also to understanding the potential for cross-species transmission, a subject of global concern for other zoonotic hepatitis viruses. These molecular characterization methods, often performed in conjunction with phylogenetic analyses, provide a comprehensive approach to unraveling the genetic diversity of aHEV in chickens.

Serological and Molecular Combined Approaches

While molecular detection is paramount for early and accurate diagnosis, serological assays complement nucleic acid techniques to capture the full spectrum of aHEV infection in chicken populations. Enzyme-linked immunosorbent assays (ELISAs) have been used to measure seroconversion and detect anti-aHEV antibody responses in infected flocks ([8], [32]). These seroepidemiological studies, combined with molecular techniques like RT-PCR, provide a dual-layer of diagnostic confirmation. For instance, the detection of high seroprevalence in conjunction with PCR-confirmed viral RNA in serum and fecal samples establishes a more nuanced epidemiological picture and supports the implementation of targeted control measures.

Advanced Techniques: Meta-Transcriptomics and Next Generation Sequencing

In addition to traditional and real-time PCR-based approaches, meta‐transcriptomic analyses and next generation sequencing (NGS) have been increasingly employed to broaden the diagnostic landscape. Meta‐transcriptomics allows for the simultaneous detection of multiple viral pathogens within a given sample, thereby uncovering potential coinfections and the complex interplay between aHEV and other viruses endemic to flocks ([9]). Although these high-throughput sequencing approaches require further standardization for routine diagnostics, their application has unveiled new subtypes and expanded the known host range of aHEV, reinforcing the need for continuous surveillance in poultry farming.

Sample Types and Practical Considerations

The success of molecular detection of aHEV significantly depends on the type and quality of the clinical specimens collected. Fecal samples, serum, liver, and spleen tissues have all been utilized with varying degrees of success. Studies have demonstrated that cloacal swabs and yolk samples can yield positive results, particularly when investigating vertical transmission in breeder flocks ([30]). The strategic selection of specimen types, combined with the robustness of the RT-PCR protocols, ensures that even low-grade or asymptomatic infections are not overlooked. Such comprehensive diagnostic methodologies are critical in regions where aHEV infection can lead to significant economic losses through decreased egg production and increased mortality rates.

Integration in Surveillance and Outbreak Management

International agencies such as the CDC and WOAH have underscored the importance of integrating molecular diagnostic techniques into national surveillance programs for economically important diseases such as aHEV. The rapid turnaround times, high sensitivity, and specificity of these molecular assays allow for early detection and the prompt implementation of control measures, thereby mitigating the spread of infection within and between flocks ([33]). Moreover, the genetic information provided by sequencing adds an additional layer of surveillance, enabling researchers to monitor virus evolution and detect emerging variants that may impact vaccine efficacy or require revised biosecurity protocols.

Collectively, the combination of conventional RT-PCR, nested PCR, SYBR Green real-time RT-PCR, targeted gene sequencing, and meta-transcriptomic approaches represents a highly sophisticated diagnostic framework for aHEV. These methodologies not only facilitate effective outbreak management and epidemiological tracking but also support the development of strategic interventions aimed at safeguarding poultry health on a global scale.

Genotypic Diversity and Molecular Characterization of Avian Hepatitis E Virus

Avian hepatitis E virus (aHEV) is characterized by an impressive genetic heterogeneity that not only defines its various genotypes but also appears to influence its pathogenic potential and host range. Molecular characterization studies have demonstrated that aHEV exhibits nucleotide sequence identities in the range of approximately 81.5% to 86.5% when compared with other aHEV strains, thereby establishing a fundamental basis for the recognition of novel genotypes within the virus species [1]. Detailed molecular investigations, including amplification and sequencing of conserved regions such as the ORF1 helicase and ORF2 capsid proteins, have paved the way for refined phylogenetic clustering of aHEVs into multiple distinct genotypes.

Phylogenetic Analysis and Genotype Delineation

Comprehensive phylogenetic analyses based on partial and complete genome sequences have led to the classification of aHEVs into at least four to seven genotypic groups, with several studies providing evidence for novel genotypes that deviate significantly from previously established clusters. For example, a study conducted during outbreaks in Poland detected sequences belonging primarily to genotype 2 and genotype 4, while also revealing a strain clustering as genotype 3 [3]. Similarly, outbreaks of hepatitis-splenomegaly syndrome in Poland have not only demonstrated typical clinical manifestations, but also provided the molecular evidence for a distinct genotype, proposed as genotype 7, with nucleotide sequence identities of only 79.6–83.2% relative to other aHEVs [4]. Moreover, reports from Nigeria have expanded the known genotypic diversity by identifying co-circulation of established genotype 2 sequences and strains that exhibited only 79–83.5% identity, suggesting the emergence of a novel clade unique to the region [6]. Such findings underscore the importance of regional surveillance and molecular epidemiology in understanding the global diversity of aHEV.

Genomic Organization and Key Molecular Markers

The aHEV genome, approximately 6.6 kb in size, is organized into three major open reading frames (ORF1, ORF2, and ORF3) that encode non-structural proteins responsible for viral replication, the capsid protein, and a small multifunctional phosphoprotein, respectively [12, 22]. Comparative analyses of the ORF1 region, particularly the helicase domain, have proven instrumental in delineating genotypic variation; small differences in amino acid sequences are often associated with functional divergence in the virus’s replication machinery. Concurrently, molecular characterization of the ORF2 gene has revealed distinct antigenic determinants that may correlate with host cell recognition and viral internalization. For instance, sequence variations in the C-terminal region of the ORF2 protein, particularly within amino acids 471–507, have been identified as critical for mediating the binding to both avian and mammalian cells, an observation that not only informs vaccine design strategies but also enhances our understanding of interspecies transmission mechanisms [17, 23]. This dual focus on ORF1 and ORF2 provides a multifaceted view of viral evolution, as both regions appear to concurrently adapt to immune pressures and host-specific factors.

Evolutionary Dynamics and Molecular Evolution

The evolutionary rate of aHEV has been estimated to be in the order of 10⁻³ substitutions per site per year, suggesting a rapid diversification that may be driven by immune selection pressures and interspecies transmission events [11]. Bayesian evolutionary analyses have revealed that the genetic diversity of genotype 3 strains, for example, may trace back to origins in the early 20th century, with ongoing evolution marked by both point mutations and potential recombination events. Such molecular dynamics are critical to understanding how new genotypes emerge and how viral populations shift over time, especially in the context of outbreaks that result in severe clinical syndromes such as hepatic rupture hemorrhage syndrome (HRHS) [5, 14]. It is evident that the combination of antigenic shifts in the ORF2 region and functional alterations in non-structural proteins encoded by ORF1 may collectively influence the virus’s pathogenicity, transmission efficiency, and host tropism.

Molecular Tools and Diagnostic Implications

Accurate detection and genotyping of aHEV have relied on the development of sensitive molecular diagnostic assays, including nested RT-PCR and SYBR Green real-time RT-PCR assays, which target conserved regions such as ORF3 and portions of ORF1 [33]. These molecular diagnostic tools have facilitated high-resolution mapping of aHEV distribution in diverse regions and host species, enhancing the ability to detect co-infections and minor sequence variants within clinical samples [8, 18]. The integration of these techniques in both diagnostic and epidemiological studies is critical for monitoring viral evolution, especially considering the potential for interspecies transmission and the economic implications for poultry production, as noted by internationally recognized organizations such as the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO).

Structural and Functional Insights from Molecular Characterization

Structural analyses of the aHEV capsid protein have provided further insights into viral entry mechanisms and host interactions. It has been demonstrated that specific amino acid sequences within the capsid, notably within the C-terminal region of ORF2, are essential for binding to host cell receptors and may determine cell tropism [17, 23]. The detailed mapping of antigenic regions, combined with findings from recombinant expression studies, has allowed researchers to correlate structural motifs with immunogenicity and viral infectivity. These investigations reveal that modifications within the viral capsid, whether due to point mutations or deletions, can significantly alter the virus’s ability to interact with cellular receptors, thereby affecting both viral dissemination and pathogenic outcomes.

Furthermore, molecular characterizations of intergenic regions, such as the conserved cis-reactive element (CRE) and stem-loop structures observed in the intergenic regions between ORF1 and ORF3, add another layer of complexity to our understanding of aHEV replication and transcription regulation [35]. Such elements are thought to play a role in the regulation of subgenomic RNA synthesis, influencing the balance between viral replication and protein translation. This balance is essential for maintaining efficient virus propagation in host cells, as demonstrated by experimental models which also explore the roles of various host proteins interacting with viral components [26, 29].

Implications for Viral Pathogenesis and Cross-Species Transmission

The expanding genotypic diversity of aHEV is not merely an academic observation; it carries significant implications for viral pathogenesis and the potential emergence of cross-species transmission events. Variations in genotypes, particularly within functionally critical domains such as those involved in viral entry and replication, may determine whether aHEV infection remains subclinical or progresses to overt syndromes such as hepatitis-splenomegaly syndrome, big liver and spleen disease, and HRHS [5, 24]. Moreover, the ability of certain genotypes to infect different avian hosts and the identification of novel genotypes in species beyond chickens, such as common pheasants and ducks, underscore the need for vigilance in monitoring interspecies transmission. This is particularly relevant for governmental and international bodies like the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), which, while primarily focused on zoonotic potential, must consider the economic ramifications of these outbreaks in the global poultry industry [12, 22].

Collectively, the detailed molecular characterization and genotypic diversity documented across numerous studies provide a robust framework for understanding the evolution, epidemiology, and pathogenesis of avian HEV. These insights are crucial for the development of effective control strategies, including diagnostics, vaccines, and breeding practices designed to mitigate the significant economic losses associated with aHEV outbreaks in poultry farms worldwide.

Vaccine Development and Antiviral Strategy Advances against AHEV in Chickens

Avian hepatitis E virus (aHEV) poses significant economic challenges for the poultry sector through reduced egg production, increased mortality, and complex disease syndromes such as big liver and spleen disease and hepatitis-splenomegaly syndrome [1, 12]. In the absence of a commercial vaccine in many endemic regions and given the continuously emerging genetic diversity among aHEV isolates [1, 4, 6], novel vaccine candidates and antiviral strategies are critical to control this pathogen. Advances in molecular virology, host–virus interaction studies, and immunogenic epitope mapping have fostered promising innovations in both prophylactic and therapeutic interventions against aHEV in chickens.

Synthetic Peptide Vaccines and Epitope-Based Approaches

One of the foremost breakthroughs in vaccine research against aHEV has been the evaluation of synthetic peptides derived from the ORF2 region, the major capsid protein known for harboring critical neutralizing epitopes [2]. In a study evaluating novel synthetic peptides, the peptide sequence RLLDRLSRTFPS demonstrated effective protection against aHEV infection in chickens. This result underscores the potential of targeting the ORF2 protein to elicit a robust immune response. The rationale for this approach lies in the immunodominant nature of ORF2, which interacts with both humoral and cell-mediated immune responses. Utilizing synthetic peptides not only allows for the precise identification of immunogenic determinants but also facilitates the design of epitope-based vaccines that can specifically target conserved regions among the genetically diverse aHEV strains [2]. Given that multiple genotypes have emerged over recent years [1, 4, 6], vaccine designs that incorporate conserved epitopes are particularly promising for cross-protective immunity, a need also recognized by international organizations such as the FAO when addressing economically significant pathogens.

Infectious cDNA Clones and Recombinant Platforms

The generation of an infectious cDNA clone of aHEV has provided a robust platform for both vaccine development and antiviral research [16]. By reconstructing an infectious viral genome in vitro, researchers are able to study viral replication dynamics, assess the immunogenicity of different viral proteins, and evaluate possible attenuation strategies. Such an infectious clone can be employed in reverse genetics systems to create live attenuated vaccines. Although cell culture propagation of aHEV remains challenging, the availability of an infectious clone offers a promising avenue to engineer viruses with specific mutations in immunodominant regions, for example, modifications in the ORF2 capsid protein that could destabilize viral entry without compromising antigenicity. This strategy not only paves the way for safer live vaccines but also aids in elucidating the intricate molecular mechanisms of aHEV pathogenesis.

Viral-Like Particles and Subunit Vaccines

In parallel with peptide-based approaches, the construction of virus-like particles (VLPs) presenting the aHEV capsid protein has been explored. VLPs mimic the native conformation of the viral capsid and can induce potent immune responses without the risks associated with replication-competent viruses. Although the direct application of such VLPs for aHEV has been less documented than in mammalian HEV research, the principles underlying VLP technology are highly applicable. By expressing recombinant ORF2 in suitable heterologous systems, it is possible to generate VLPs that display conformational epitopes critical for virus neutralization. Such platforms are particularly advantageous given the antigenic diversity of aHEV, as they can be engineered to display conserved epitopes identified through comprehensive genomic and proteomic analyses [2, 10].

Targeting Host–Virus Interactions for Antiviral Therapy

Beyond traditional vaccine development, recent research has significantly advanced our understanding of the molecular mechanisms governing aHEV entry into host cells. Studies have identified key host proteins involved in viral attachment and internalization. For instance, the interaction between the aHEV ORF2 protein and RAS-related protein Rap1b has been shown to induce cytoskeletal rearrangement, facilitating viral internalization [23]. This interaction offers a compelling target for antiviral interventions. Small molecule inhibitors, designed to disrupt the binding of ORF2 to Rap1b, could consequently block the internalization process, thereby reducing viral load and subsequent disease severity. Additionally, the pivotal role of the organic anion-transporting polypeptide 1A2 (OATP1A2) in mediating viral adsorption to host cells has been elucidated [26]. In vitro studies have demonstrated that blocking OATP1A2 function, via competitive substrates or specific inhibitors, effectively reduces viral attachment. These host-targeted strategies are promising, as they bypass the challenges posed by rapid viral mutation and genotype diversity, offering a broad-spectrum approach to antiviral therapy.

Pathway-Specific Antiviral Approaches

Additional advances have emerged from dissecting virus-induced signaling cascades within the host. For example, the downstream activation of integrin-linked focal adhesion kinase (FAK) and subsequent cytoskeletal rearrangements are critical for aHEV internalization [23]. Inhibitors targeting kinases or signaling mediators such as CDC42, which also interacts directly with the HEV capsid [29], represent innovative antiviral strategies. By interrupting these pathways, researchers can hinder efficient viral replication. Such host-directed interventions are increasingly relevant in the context of zoonotic and economically impactful viruses, as underscored by global health agencies like the CDC and WHO.

Integration of Immunomodulatory Strategies

It is also important to consider the role of cellular immunity in controlling aHEV infections. Recent studies on the role of CD8+ lymphocytes underscore their importance in clearing viral infections in chickens [28]. Vaccine formulations that not only induce strong neutralizing antibody responses but also stimulate robust cytotoxic T cell responses could be particularly effective. Combining traditional antigen-based vaccination with novel immunomodulatory adjuvants may enhance both the breadth and durability of the immune response, effectively curtailing the spread of aHEV in commercial flocks.

Collectively, these vaccine and antiviral strategies are poised to significantly influence the management of aHEV in chickens. By leveraging insights gained from synthetic peptide evaluation, recombinant technologies, and detailed studies of host–virus interactions, future interventions may offer both prophylactic and therapeutic benefits that mitigate the substantial economic impact of aHEV. Such advancements are crucial given the pathogen's dynamic genetic landscape and the ever-present risk of strain diversity within poultry populations, a concern that is already highlighted by international organizations such as the WOAH and FAO.

Economic Impact and Biosecurity Considerations in Poultry Management

The economic impact of avian hepatitis E virus (aHEV) on the poultry industry is multifaceted, affecting production metrics, bird health, and overall farm sustainability. The clinical manifestations of aHEV, including decreased egg production, increased mortality, and organ damage, translate into significant financial losses for producers. At the same time, the broadening host range and multiple viral genotypes complicate both epidemiological surveillance and farm management practices, necessitating robust biosecurity measures to mitigate economic risks.

Economic Impact on Production and Profitability

From a production standpoint, aHEV infections contribute to substantial financial burdens by directly reducing output. Several studies have highlighted that outbreaks of aHEV can cause egg production declines ranging from 10% to 40% in affected layer flocks [8, 27]. Reduced egg output not only diminishes the immediate revenue from sales but also affects the long-term productivity of flocks. In addition, infections leading to conditions such as hepatic rupture hemorrhage syndrome (HRHS) in broilers and layer chickens can result in mortality rates as high as 40% in acute cases [5]. The loss of birds, along with the costs associated with increased veterinary interventions, quarantine measures, and potential culling, severely impacts the bottom line for commercial operations.

In regions such as Taiwan, Poland, and Nigeria, where aHEV has been documented to circulate widely [1, 3, 6], the economic consequences are compounded by subclinical infections and the repeated introductions of novel genotypes. The hidden nature of these infections can extend the economic downturn over time due to chronic underperformance and inefficient flock management. Furthermore, the economic ramifications extend beyond the immediate production losses, as they include costly diagnostic procedures and the implementation of preventative measures such as vaccination campaigns, which are being actively researched [2]. The cumulative burden of these factors reinforces the critical importance of integrating economic risk assessments with proactive health monitoring strategies on poultry farms.

Biosecurity Measures and Farm Management Practices

Given the profound economic implications associated with aHEV outbreaks, stringent biosecurity and flock management practices remain central to mitigating financial losses. In one study comparing caged versus cage-free housing arrangements, it was evident that the housing system itself can influence transmission dynamics, with cage-free systems exhibiting significantly higher rates of infection due to increased contact with fecal material [31]. This underscores the necessity for a re-evaluation of housing designs in outbreak-prone regions. On farms where aHEV is endemic, strategies such as reduced density, enhanced cleaning protocols, and controlled access to poultry areas are essential to reduce the risk of horizontal transmission.

Biosecurity protocols must include rigorous sanitation procedures, effective disinfection of housing units, and strict control of farm traffic. Vaccination research, as illustrated in recent studies, offers promising avenues for reducing infection rates [2]. However, until widely available and cost-effective vaccines are developed, conventional measures such as regular serological and molecular testing remain paramount. The rapid, sensitive SYBR Green real-time RT-PCR assay developed for aHEV [33] provides an important tool for early detection, allowing farmers to implement containment measures before widespread transmission occurs. International bodies like the CDC, WHO, and FAO emphasize the critical role of biosecurity in managing zoonotic and economically significant diseases, and their guidelines serve as a benchmark for poultry producers worldwide.

In addition to housing modifications, the segregation of poultry based on age and production type can limit pathogen spread. For instance, segregating broiler breeders from layers may reduce cross-infection rates and improve the overall efficacy of intervention strategies. Mixed infections, common in farms where aHEV co-occurs with other immunosuppressive viruses such as avian leukosis virus or chicken infectious anemia virus, further emphasize the need for integrated biosecurity plans that address concurrent pathogens [25, 32]. The economic losses are not solely attributable to the virus itself but also stem from the compounded effects of multiple pathogens, which stress the importance of multi-pathogen surveillance programs integrated within biosecurity protocols.

Farm-Level Interventions and Preventative Strategies

At the farm level, preventive strategies extend beyond routine cleaning and disinfection. Biosecurity also involves the proper handling and disposal of potentially contaminated materials, rigorous control of personnel movement, and the implementation of visitor restrictions. For example, the adaptation of biosecurity measures analogous to those recommended by organizations such as WOAH (World Organisation for Animal Health) can help curtail the virus’s spread and reduce associated economic losses. Effective monitoring systems, which employ both serological and molecular diagnostic tools, must be implemented regularly to ensure early detection and rapid isolation of infected flocks.

Furthermore, housing designs that minimize contact with contaminated fecal matter have been shown to significantly reduce transmission rates [31]. Biosecurity protocols should also address the environmental aspects of poultry management, including water quality, ventilation, and waste management. Given that aHEV has been documented in subclinically infected flocks, improving overall environmental hygiene not only reduces the risk of viral spread but also curtails the transmission of other economically significant pathogens.

Economic losses can further be alleviated by investing in research and development aimed at understanding the molecular mechanisms underlying aHEV infection. Studies that explore the interaction between the viral ORF2 protein and host cellular components [17, 23, 26] enhance our understanding of the pathology of aHEV and facilitate the design of targeted therapies and preventative strategies. Insights into immune response mechanisms are equally vital. For instance, the interaction between aHEV and host CD8+ lymphocytes has implications for both viral clearance and the extent of liver lesions [28]. Such information is crucial when devising biosecurity measures that not only prevent the spread of the virus but also support the development of more resistant poultry strains through selective breeding programs.

The Role of Policy and International Guidelines

Economic sustainability in the poultry industry requires cohesive policy actions that incorporate rigorous biosecurity standards. National and international guidelines, such as those endorsed by the FAO, CDC, and WHO, should be integrated into local farming practices to ensure that prevention, early detection, and rapid response protocols are in place. By adhering to these standards, producers can mitigate the risk of significant outbreaks, safeguard their flocks, and minimize economic disruption. These guidelines also encourage the collaborative sharing of diagnostic technologies and best practices across international borders, ultimately contributing to a more resilient global poultry industry.

In summary, the integration of robust biosecurity measures into poultry management practices not only has the potential to significantly reduce the economic burden posed by aHEV but also serves as a critical line of defense against the extensive losses associated with multifactorial viral infections. The interplay between housing arrangements, environmental controls, rapid diagnostic technologies, and targeted vaccination strategies underscores the need for comprehensive farm management practices that align with international biosecurity standards.

References

[1] Hsu I, Tsai H. Avian Hepatitis E Virus in Chickens, Taiwan, 2013. Emerging Infectious Diseases. 2014. DOI: https://doi.org/10.3201/eid2001.131224

[2] Chen Y, Tang Y, Zhang S, Tian Y, Xu S, Zhang C, et al.. Evaluation of novel synthetic peptides of avian hepatitis E virus ORF2 as vaccine candidate in chickens. Virus Research. 2024. DOI: https://doi.org/10.1016/j.virusres.2024.199459

[3] Siedlecka M, Kublicka A, Wieliczko A, Matczuk A. Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland. PLoS ONE. 2022. DOI: https://doi.org/10.1371/journal.pone.0269854

[4] Matos M, Bilic I, Tvarogová J, Palmieri N, Furmanek D, Gotowiecka M, et al.. A novel genotype of avian hepatitis E virus identified in chickens and common pheasants (Phasianus colchicus), extending its host range. Scientific Reports. 2022. DOI: https://doi.org/10.1038/s41598-022-26103-3

[5] Su Q, Li Y, Meng F, Cui Z, Chang S, Zhao P. Hepatic rupture hemorrhage syndrome in chickens caused by a novel genotype avian hepatitis E virus.. Veterinary Microbiology. 2018. DOI: https://doi.org/10.1016/j.vetmic.2018.06.019

[6] Osamudiamen FT, Akanbi O, Zander S, Oluwayelu D, Bock C, Klink P. Identification of a Putative Novel Genotype of Avian Hepatitis E Virus from Apparently Healthy Chickens in Southwestern Nigeria. Viruses. 2021. DOI: https://doi.org/10.3390/v13060954

[7] Matczuk A, Ćwiek K, Wieliczko A. Avian hepatitis E virus is widespread among chickens in Poland and belongs to genotype 2. Archives of Virology. 2018. DOI: https://doi.org/10.1007/s00705-018-4089-y

[8] Razmyar J, Abbasi M, Mirsalimi SM, Baghkheirati AA, Ahmadian G, Yazdani A. Serologic and Molecular Evidence of Widespread Infection of Avian Hepatitis E Virus in Poultry Farms of Iran. Avian diseases. 2021. DOI: https://doi.org/10.1637/aviandiseases-D-21-00077

[9] Zhang X, Li W, Yuan S, Guo J, Li Z, Chi S, et al.. Meta-transcriptomic analysis reveals a new subtype of genotype 3 avian hepatitis E virus in chicken flocks with high mortality in Guangdong, China. BMC Veterinary Research. 2019. DOI: https://doi.org/10.1186/s12917-019-1884-y

[10] Zhang Y, Zhao H, Chi Z, Cui Z, Chang S, Wang Y, et al.. Isolation, identification and genome analysis of an avian hepatitis E virus from white-feathered broilers in China. Poultry Science. 2021. DOI: https://doi.org/10.1016/j.psj.2021.101633

[11] Fu F, Deng Q, Li Q, Zhu W, Guo J, Wei P. Emergence and Molecular Characterization of an Avian Hepatitis E Virus From Donglan Black Chicken in Southern China. Frontiers in Veterinary Science. 2022. DOI: https://doi.org/10.3389/fvets.2022.901292

[12] Sun P, Lin S, He S, Zhou E, Zhao Q. Avian Hepatitis E Virus: With the Trend of Genotypes and Host Expansion. Frontiers in Microbiology. 2019. DOI: https://doi.org/10.3389/fmicb.2019.01696

[13] Zhao Q, Zhou E, Dong S, Qiu H, Zhang L, Hu S, et al.. Analysis of Avian Hepatitis E Virus from Chickens, China. Emerging Infectious Diseases. 2010. DOI: https://doi.org/10.3201/eid1609.100626

[14] Su Q, Zhang Z, Zhang Y, Cui Z, Chang S, Zhao P. Complete genome analysis of avian hepatitis E virus from chicken with hepatic rupture hemorrhage syndrome.. Veterinary Microbiology. 2020. DOI: https://doi.org/10.1016/j.vetmic.2020.108577

[15] Billam P, LeRoith T, Pudupakam RS, Pierson F, Duncan R, Meng X. Comparative pathogenesis in specific-pathogen-free chickens of two strains of avian hepatitis E virus recovered from a chicken with Hepatitis-Splenomegaly syndrome and from a clinically healthy chicken, respectively. Veterinary Microbiology. 2009. DOI: https://doi.org/10.1016/j.vetmic.2009.06.008

[16] Kwon H, LeRoith T, Pudupakam RS, Pierson F, Huang Y, Dryman B, et al.. Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens. Veterinary Microbiology. 2010. DOI: https://doi.org/10.1016/j.vetmic.2010.07.016

[17] Zhang X, Bilic I, Marek A, Glösmann M, Hess M. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells. PLoS ONE. 2016. DOI: https://doi.org/10.1371/journal.pone.0153723

[18] Liu K, Zhao Y, Zhao J, Geng N, Meng F, Wang S, et al.. The diagnosis and molecular epidemiology investigation of avian hepatitis E in Shandong province, China. BMC Veterinary Research. 2021. DOI: https://doi.org/10.1186/s12917-021-03079-2

[19] Turlewicz-Podbielska H, Augustyniak A, Wojciechowski J, Pomorska-Mól M. Hepatitis E Virus in Livestock, Update on Its Epidemiology and Risk of Infection to Humans. Animals. 2023. DOI: https://doi.org/10.3390/ani13203239

[20] Meng X. Hepatitis E virus: Animal Reservoirs and Zoonotic Risk. Veterinary Microbiology. 2009. DOI: https://doi.org/10.1016/j.vetmic.2009.03.017

[21] Osamudiamen FT, Akanbi O, Oluwayelu D, Bock C, Klink P. Serological evidence of avian HEV antibodies in apparently healthy chickens in southwest Nigeria. PLoS ONE. 2021. DOI: https://doi.org/10.1371/journal.pone.0247889

[22] Purdy M, Drexler J, Meng X, Norder H, Okamoto H, Poel WVDvd, et al.. ICTV Virus Taxonomy Profile: Hepeviridae 2022. Journal of General Virology. 2022. DOI: https://doi.org/10.1099/jgv.0.001778

[23] Zhang B, Fan M, Fan J, Luo Y, Wang J, Wang Y, et al.. Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization. Microbiology spectrum. 2022. DOI: https://doi.org/10.1128/spectrum.02265-21

[24] Liu B, Chen Y, Zhao L, Zhang M, Ren X, Zhang Y, et al.. Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus).. Veterinary Microbiology. 2020. DOI: https://doi.org/10.1016/j.vetmic.2020.108688

[25] Su Q, Zhang Y, Li Y, Cui Z, Chang S, Zhao P. Epidemiological investigation of the novel genotype avian hepatitis E virus and co‐infected immunosuppressive viruses in farms with hepatic rupture haemorrhage syndrome, recently emerged in China. Transboundary and Emerging Diseases. 2018. DOI: https://doi.org/10.1111/tbed.13082

[26] Li H, Fan M, Liu B, Ji P, Chen Y, Zhang B, et al.. Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection. Journal of Virology. 2019. DOI: https://doi.org/10.1128/JVI.02205-18

[27] Zhang Y, Gao X, Cao M, Xu H, Liu H, Zhao Q, et al.. Hepatitis E virus causes apoptosis of ovarian cells in hens and resulting in a decrease in egg production. Poultry Science. 2024. DOI: https://doi.org/10.1016/j.psj.2024.103501

[28] Rogers E, Todd S, Pierson F, Kenney SP, Heffron CL, Yugo DM, et al.. CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens. Journal of Medical Virology. 2019. DOI: https://doi.org/10.1002/jmv.25548

[29] Fan M, Luo Y, Zhang B, Wang J, Chen T, Liu B, et al.. Cell Division Control Protein 42 Interacts With Hepatitis E Virus Capsid Protein and Participates in Hepatitis E Virus Infection. Frontiers in Microbiology. 2021. DOI: https://doi.org/10.3389/fmicb.2021.775083

[30] Liu K, Meng F, Zhao J, Zhao Y, Geng N, Wang S, et al.. Research Note: The prevalence and vertical transmission of avian hepatitis E virus novel genotypes in Tai'an city, China. Poultry Science. 2022. DOI: https://doi.org/10.1016/j.psj.2022.102103

[31] Liu B, Sun Y, Chen Y, Du T, Nan Y, Wang X, et al.. Effect of housing arrangement on fecal-oral transmission of avian hepatitis E virus in chicken flocks. BMC Veterinary Research. 2017. DOI: https://doi.org/10.1186/s12917-017-1203-4

[32] Sun Y, Du T, Liu B, Syed SF, Chen Y, Li H, et al.. Seroprevalence of avian hepatitis E virus and avian leucosis virus subgroup J in chicken flocks with hepatitis syndrome, China. BMC Veterinary Research. 2016. DOI: https://doi.org/10.1186/s12917-016-0892-4

[33] Zhao Q, Xie S, Sun Y, Chen Y, Gao J, Li H, et al.. Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus. BMC Veterinary Research. 2015. DOI: https://doi.org/10.1186/s12917-015-0507-5

[34] Ouoba J, Traore KA, M’Bengue A, Ngazoa S, Rouamba H, Doumbia M, et al.. Distribution and molecular characterization of avian hepatitis E virus (aHEV) in domestic and wild birds in Burkina Faso. Journal of Veterinary Medicine and Animal Health. 2019. DOI: https://doi.org/10.5897/JVMAH2018.0741

[35] Iqbal T, Rashid U, Idrees M, Afroz A, Kamili S, Purdy M. A novel avian isolate of hepatitis E virus from Pakistan. Virology Journal. 2019. DOI: https://doi.org/10.1186/s12985-019-1247-0