ORF3 Antibody
Description
Characteristics and Structure of ORF3 Antibodies
ORF3 antibodies are immunoglobulins specifically designed to recognize epitopes within the ORF3 protein, which is a small multifunctional phosphoprotein expressed by HEV and certain other viruses. These antibodies are available in both polyclonal and monoclonal formats, each offering distinct advantages for research and diagnostic applications.
Polyclonal ORF3 Antibodies
Polyclonal ORF3 antibodies are typically raised in rabbits using recombinant ORF3 protein or synthetic peptides as immunogens. The commercially available polyclonal antibody from Cepham Life Sciences, for example, is produced using the full-length recombinant Hepatitis E virus genotype 1 ORF3 protein (1-114 amino acids) . These antibodies recognize multiple epitopes on the ORF3 protein, providing robust detection capabilities across various applications. Polyclonal antibodies are often purified using techniques such as Protein A affinity chromatography or caprylic acid ammonium sulfate precipitation to ensure high specificity and minimal cross-reactivity .
Monoclonal ORF3 Antibodies
Monoclonal antibodies against ORF3 offer enhanced specificity by targeting distinct epitopes within the protein. For instance, the monoclonal antibody 7D3 binds specifically to the ORF3 peptide region spanning residues 35-66 and effectively detects native ORF3 protein in virus-infected peripheral blood mononuclear cells (PBMCs) . These antibodies are typically generated by immunizing mice with ORF3 protein or peptides, followed by hybridoma technology to isolate and propagate B cell clones producing the desired antibody.
Functions of the ORF3 Protein Targeted by Antibodies
Understanding the functions of the ORF3 protein is essential for appreciating the utility of ORF3 antibodies in viral research. Research using these antibodies has revealed several critical roles of the ORF3 protein in viral pathogenesis.
Ion Channel Activity (Viroporin)
Recent research has demonstrated that HEV ORF3 functions as a viroporin, forming multimeric complexes that act as ion channels. These structures are associated with intracellular endoplasmic reticulum (ER)-derived membranes and share structural features with class I viroporins . Studies utilizing ORF3 antibodies have shown that this ion channel activity is essential for viral release from infected cells. Remarkably, the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein, further confirming its role as an ion channel .
Immune Evasion Mechanisms
ORF3 antibodies have been instrumental in uncovering how the ORF3 protein helps HEV evade host immune responses. Research has revealed that ORF3 downregulates Toll-like receptor 3 (TLR3)-mediated NF-κB signaling via tumor necrosis factor receptor 1-associated death domain protein (TRADD) and receptor-interacting protein kinase 1 (RIP1) . Additionally, ORF3 inhibits TLR7 to suppress the generation of type I interferons, further dampening the host antiviral response . These findings highlight the sophisticated strategies employed by HEV to establish infection and potentially contribute to chronic disease in susceptible individuals.
Virion Morphogenesis and Egress
The ORF3 protein plays critical roles in the final steps of viral release by interacting with host tumor susceptibility gene 101 (TSG101), a member of the vacuolar protein-sorting pathway . This interaction, along with ORF3's engagement of other cellular host proteins involved in vesicle formation, facilitates the egress of viral particles from infected cells. Research using ORF3 antibodies has demonstrated that ORF3 is essential for this process, as deletion of ORF3 abrogates the release of infectious virions .
Cell Signaling Modulation
ORF3 antibodies have helped elucidate how this viral protein modifies host cell signaling to create a favorable environment for viral replication. Studies have shown that ORF3 interacts with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases . This interaction delays the trafficking and degradation of activated growth factor receptors, potentially prolonging endomembrane growth factor signaling and promoting cell survival to support viral replication .
Applications of ORF3 Antibodies in Research and Diagnostics
ORF3 antibodies serve as versatile tools in both research and diagnostic settings, enabling the detection, localization, and characterization of ORF3 proteins in various experimental systems.
Western Blotting
Western blotting represents one of the primary applications for ORF3 antibodies, allowing for the detection and quantification of ORF3 protein in cell lysates. As indicated by the specifications of commercial antibodies like bs-0212R, ORF3 antibodies can effectively detect the protein in Western blot applications . This technique has been used to confirm successful expression of ORF3 in transfected cells and to study protein expression levels during infection .
Immunofluorescence Assays
Immunofluorescence assays using ORF3 antibodies enable the visualization of ORF3 protein localization within cells, providing valuable insights into its distribution and potential interactions with other cellular or viral components. Research has employed this approach to demonstrate that ORF3 colocalizes with both the viral capsid protein and cellular p53 protein in infected PBMCs . These studies have shown that approximately 3-5% of PBMCs may be positive for ORF3 protein expression during infection, compared to 78-82% positivity for the capsid protein .
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA techniques using ORF3 antibodies have been developed for both research and diagnostic applications. Double-antibody sandwich ELISAs have been created using rabbit anti-ORF3 peptide antibodies as capture antibodies and monoclonal anti-ORF3 antibodies as detection reagents . These assays enable the quantification of ORF3 protein in blood samples and have been used to study the correlation between ORF3 and capsid protein levels during infection.
In a comparative study of ORF3 and ORF2 (capsid) immunogenicity, researchers found that while 85% of HEV-positive swine sera reacted against specific regions of ORF2 (particularly amino acids 461-544), only 5-12% reacted against the ORF3 antigen . This finding suggests differential immunogenicity of these viral proteins during natural infection, which has implications for diagnostic test design and vaccine development.
Immunohistochemistry
ORF3 antibodies such as bs-0212R are suitable for immunohistochemistry applications on paraffin-embedded tissues (IHC-P), with recommended dilutions typically in the range of 1:200-400 . This application allows for the detection and localization of ORF3 protein in tissue sections, providing valuable information about viral distribution in infected organs.
Neutralization Assays
Anti-ORF3 antibodies have been used in neutralization assays to evaluate their potential to inhibit viral infection. Research has shown that serum from ORF3-immunized mice exhibits a modest inhibitory effect on the infection of quasi-enveloped HEV in vitro . These findings suggest that anti-ORF3 antibodies might complement traditional anti-capsid (ORF2) antibodies in providing protection against HEV infection, particularly against the quasi-enveloped form of the virus that circulates in the bloodstream.
Recent Research Advances Using ORF3 Antibodies
ORF3 antibodies have facilitated several recent breakthroughs in understanding viral pathogenesis and developing novel therapeutic approaches.
Vector-Based Vaccine Development
Innovative research has explored the use of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express HEV ORF3 in the musculature of mice, resulting in robust and dose-dependent formation of anti-ORF3 antibodies . This approach represents a potential vector-based vaccine strategy that targets ORF3 rather than the conventional capsid protein (ORF2). The study demonstrated that the AAVMYO3 capsid could serve as a versatile platform for vaccine development against HEV and potentially other infectious agents .
Evolutionary Relationships of Viral Ion Channels
Research employing sequence analysis and structural studies, likely facilitated by antibody-based detection methods, has revealed evolutionary relationships between HEV ORF3 and proteins from other viruses. A comprehensive analysis unified the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs and the Matrix (M) proteins from coronaviruses . This research demonstrated that these diverse viral families might utilize specific conserved polar residues to constitute an aqueous pore within membrane-spanning regions, suggesting a conserved role for ion channels in virion assembly and membrane budding across different virus families .
ORF3 in Quasi-Enveloped HEV
Studies employing ORF3 antibodies have enhanced our understanding of the quasi-enveloped form of HEV (eHEV) that circulates in the bloodstream. Unlike the non-enveloped form shed in feces, eHEV bears both ORF2 and ORF3 proteins on its quasi-envelope . This membrane coating protects the virus from neutralizing anti-capsid antibodies in the serum, which may explain why conventional vaccines targeting only the capsid protein might have limited efficacy against the circulating form of the virus. ORF3 antibodies have been shown to capture viral particles from serum samples of HEV patients and partially neutralize eHEV infections in vitro .
Data on ORF3 Protein Functions Detected by Antibodies
Research using ORF3 antibodies has uncovered multiple functions of this viral protein, as summarized in the following table:
Applications and Detection Methods Using ORF3 Antibodies
The versatility of ORF3 antibodies is reflected in the range of applications and detection methods they enable:
Comparison of ORF3 and ORF2 Immunogenicity
Studies comparing the immunogenicity of HEV ORF3 and ORF2 proteins have yielded important insights for diagnostic and vaccine development:
Future Directions in ORF3 Antibody Research
The identification of ORF3 as a multifunctional viral protein with essential roles in viral replication, immune evasion, and virion release suggests several promising directions for future research using ORF3 antibodies.
Therapeutic Development
Given the essential role of ORF3 as an ion channel required for viral release, antibodies or small molecules targeting this function represent attractive candidates for antiviral development . Future research may focus on developing therapeutic antibodies that specifically block the viroporin activity of ORF3 or its interactions with host factors critical for viral egress.
Improved Diagnostics
While ORF3 appears less immunogenic than ORF2 during natural infection, the development of diagnostic assays incorporating both antigens might improve sensitivity and specificity, particularly for detecting different viral variants or genotypes . Future diagnostic approaches may leverage the distinct properties of ORF3 to detect quasi-enveloped forms of the virus or to distinguish between acute and chronic infections.
Combination Vaccine Strategies
The modest neutralizing activity of anti-ORF3 antibodies against quasi-enveloped HEV suggests that combining ORF3 with traditional ORF2-based vaccines might provide more comprehensive protection . Future vaccine strategies might explore such combination approaches, particularly using vector-based platforms like the AAVMYO3 system described in recent research.
As research into HEV pathogenesis continues to advance, ORF3 antibodies will likely remain essential tools for understanding viral biology and developing effective interventions against this globally significant pathogen.
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What is the HEV ORF3 protein and why are antibodies against it significant?
ORF3 is a small phosphoprotein encoded by the hepatitis E virus that has multiple functions critical to viral pathogenesis. The protein forms multimeric complexes associated with endoplasmic reticulum-derived membranes and functions as a viroporin (ion channel), which is essential for viral particle release . Anti-ORF3 antibodies are significant because they can capture and partially neutralize quasi-enveloped HEV particles circulating in the bloodstream, providing a potential mechanism for controlling infection that differs from anti-ORF2 (capsid) antibodies . These antibodies are particularly important since ORF3 protein appears on the surface of released HEV virions but not on fecal virions, making them useful for distinguishing between different forms of the virus in clinical and research settings .
How does ORF3 contribute to HEV pathogenesis?
ORF3 contributes to HEV pathogenesis through multiple mechanisms:
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Viroporin activity: ORF3 forms ion channels that mediate ionic currents across membranes, a function critical for viral release .
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Viral egress: ORF3 is required specifically for apical release of HEV particles from polarized hepatocytes, which is essential for fecal shedding and transmission .
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Persistent infection: Studies using human liver chimeric mouse models demonstrate that ORF3 is necessary for establishing persistent HEV infection; viruses lacking ORF3 expression (ORF3null) fail to sustain infection despite transient replication .
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Immune modulation: ORF3 affects various host pathways including ERK signaling, growth factor responses, and immune system interactions .
The multifunctional nature of ORF3 makes it an attractive target for both diagnostic antibodies and therapeutic interventions.
What techniques are most effective for detecting ORF3 expression in infected cells?
For detecting ORF3 expression in infected cells, immunofluorescence assay (IFA) using specific anti-ORF3 monoclonal antibodies has proven highly effective. Research demonstrates that this approach can reveal the accumulation of ORF3 protein in the cytoplasm of infected cells .
Methodology:
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Grow susceptible cells (e.g., PLC/PRF/5 or HepG2/C3A) on appropriate substrates
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Infect with HEV or transfect with ORF3-expressing plasmids
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Fix cells at appropriate timepoints (typically 48-72 hours post-infection)
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Permeabilize cells and block non-specific binding
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Incubate with anti-ORF3 primary antibodies
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Detect using fluorophore-conjugated secondary antibodies
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Visualize using fluorescence microscopy
Western blotting provides a complementary approach for quantitative assessment of ORF3 expression. When performing Western blotting, cell lysates from HEV-infected cells or cells transfected with ORF3-expressing constructs can be analyzed using RIPA buffer extraction followed by SDS-PAGE separation and immunoblotting with anti-ORF3 antibodies .
How can researchers effectively detect anti-ORF3 antibodies in serum samples?
Detecting anti-ORF3 antibodies in serum samples requires careful methodological considerations. Based on published protocols, the following approach has been validated:
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Generate ORF3-containing cell lysates:
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Transfect HEK-293 cells with an ORF3-expressing plasmid
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Prepare parallel mock-transfected controls
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Harvest cells 48 hours post-transfection
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Lyse in RIPA buffer with protease inhibitors
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Separate proteins by SDS-PAGE
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Western blot detection method:
This method allows for specific detection of anti-ORF3 antibodies while controlling for non-specific binding.
How can researchers evaluate the neutralizing capacity of anti-ORF3 antibodies against quasi-enveloped HEV?
Evaluating the neutralizing capacity of anti-ORF3 antibodies requires specialized techniques due to the unique properties of quasi-enveloped HEV particles. The following methodology has been validated:
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Neutralization assay protocol:
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Prepare quasi-enveloped HEV (eHEV) from cell culture supernatants
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Seed susceptible cells (e.g., HepG2/C3A derivatives) in multi-well plates
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Dilute test sera or purified antibodies in cell culture medium
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Incubate diluted antibodies with eHEV at a defined MOI (e.g., 1×10^-3) for 60 minutes at 37°C
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Add the antibody-virus mixture to cells
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Remove inoculum after appropriate incubation time (e.g., 8 hours)
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Replenish with fresh medium
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Assess infection by immunofluorescence or other detection methods
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Controls to include:
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Known neutralizing anti-ORF3 antibodies (e.g., RB198 and RB200 mixed 1:1 at 1:50 dilution)
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Anti-ORF2 antibodies (as comparative controls)
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Pre-immune sera
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Research indicates that anti-ORF3 monoclonal antibodies can partially neutralize HEV infection, with efficiency varying by antibody clone. The maximum neutralization efficiency reported is approximately 61% for culture medium-derived virus and 49% for serum-derived virus .
What are the key experimental considerations when studying ORF3's role in polarized hepatocyte systems?
Studying ORF3's role in polarized hepatocyte systems requires specific methodological approaches to accurately model the apical-basolateral polarity relevant to HEV infection:
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Experimental setup:
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Culture hepatocytes (e.g., HepG2/C3A derivatives) on transwell inserts
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Allow cells to polarize fully (typically 7-10 days)
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Verify polarization through tight junction markers and/or transepithelial electrical resistance (TEER)
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Infect or transfect cells with wild-type or ORF3null HEV constructs
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Separate collection and analysis:
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Collect supernatants separately from apical and basolateral chambers
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Quantify viral RNA by RT-qPCR
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Assess infectivity through focus-forming assays
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Complementation experiments:
Research demonstrates that ORF3 deletion substantially decreases HEV replication and virion release from the apical surface but not the basolateral surface of polarized hepatocytes, indicating a specific role in directional viral egress .
What approaches can generate robust anti-ORF3 antibody responses in vivo?
Multiple approaches have been validated for generating anti-ORF3 antibody responses in vivo, each with specific advantages:
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Synthetic peptide immunization:
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AAV vector-based expression:
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Using myotropic Adeno-associated virus vectors (AAVMYO3) expressing ORF3
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Systemic administration targeting muscular expression
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Demonstrates dose-dependent antibody production
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Comparative antibody induction results:
The AAV vector approach offers particular advantages for in vivo studies as it leads to robust antibody production through muscle-specific expression and mimics natural infection presentation of antigens .
How do anti-ORF3 antibodies compare to anti-ORF2 antibodies for diagnostic and therapeutic applications?
Anti-ORF3 and anti-ORF2 antibodies have distinct properties relevant to their diagnostic and therapeutic applications:
| Property | Anti-ORF3 Antibodies | Anti-ORF2 Antibodies | Clinical Implications |
|---|---|---|---|
| Target location | Surface of quasi-enveloped particles | Capsid protein (exposed in non-enveloped virions) | Complementary detection capabilities |
| Sample type utility | Serum/blood and cell culture (but not feces) | All sample types including feces | Different diagnostic niches |
| Neutralization mechanism | Partial neutralization of quasi-enveloped HEV | Strong neutralization of non-enveloped HEV | Combined approach may be beneficial |
| Diagnostic value | Indicates active infection (circulating virus) | Indicates exposure or infection | Different interpretation required |
Anti-ORF3 antibodies uniquely capture quasi-enveloped HEV particles in blood and culture medium but not in feces, while anti-ORF2 antibodies recognize both enveloped and non-enveloped forms . This distinction suggests that a combined approach targeting both proteins might provide comprehensive protection against different forms of the virus, particularly relevant for vaccine design strategies .
What experimental approaches can differentiate between the multiple functions of ORF3?
Differentiating between ORF3's various functions requires sophisticated experimental approaches:
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Targeted mutagenesis:
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Identify key residues within ORF3 responsible for specific functions
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Create point mutations rather than complete deletion
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Assess each mutant for:
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Multimeric complex formation
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Ion channel activity
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Virus release capacity
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Interaction with host factors
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Functional complementation:
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Heterologous expression systems:
Such methodological approaches have revealed that ORF3's ion channel activity is directly linked to its role in particle release, providing potential targets for antiviral development .
What are the challenges and solutions for studying ORF3 antibodies in persistent infection models?
Studying ORF3 antibodies in persistent infection models presents several challenges with corresponding methodological solutions:
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Challenges:
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HEV naturally causes persistent infections only in specific populations
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Limited animal models that recapitulate human disease
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Difficulty maintaining long-term cultures for in vitro studies
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Variable antibody responses in different hosts
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Solutions and approaches:
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Human liver chimeric mouse models:
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Long-term polarized cell culture systems:
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Adaptive immune response assessment:
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Analyze antibody responses longitudinally
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Compare antibody functionality between acute and persistent phases
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Evaluate epitope recognition patterns and neutralization effectiveness
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This research demonstrates that targeting ORF3 might be particularly effective for treating persistent HEV infections, providing a rationale for therapeutic development .
