Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against the Capsid Protein of Goose Astrovirus Genotype 2

Abstract

Since 2015, outbreaks of a disease causing severe visceral gout in goslings had resulted in substantial economic losses to the goose farming industry in China. Subsequently, the disease, characterized by extensive visceral urate deposition and renal swelling, was determined to be caused by a novel astrovirus, designated as goose astrovirus (GAstV). The capsid protein (Cap) of GAstV, encoded by ORF2, is the sole structural protein of the virus and holds potential for developing therapeutic antibodies and diagnostic tools. Based on genetic divergence in the ORF2 gene, GAstV is classified into two serotypes: GAstV-1 and GAstV-2. Despite the critical role of the GAstV Cap in viral pathogenesis, research on generating and characterizing monoclonal antibodies (mAb) against this antigen remains scarce. In this study, six mAbs (1B3, 1B4, 1B6, 1D4, 1E2, 1F1) specifically recognizing GAstV Cap were screened using Western blotting (WB), indirect immunofluorescence assay (IFA), and indirect enzyme-linked immunosorbent assay (ELISA). For epitope mapping, sequential truncations of the GAstV Cap protein fused to glutathione S-transferase (GST) were generated using bacterial expression systems. Ultimately, antigenicity analysis of the prokaryotically expressed, GST-tagged Cap truncations via indirect ELISA and WB delineated two minimal linear epitopes: epitope ⁶³⁰TDPEED⁶³⁵, recognized by mAbs 1B3, 1B4, 1B6, 1D4, and 1E2, and epitope ³DRAVAPREK¹¹, recognized by mAb 1F1. Amino acid sequence alignment revealed that the sequences of epitopes ⁶³⁰TDPEED⁶³⁵ and ³DRAVAPREK¹¹ are highly conserved in GAstV-2 but exhibit significant divergence in the GAstV-1 serotype. This study provides essential tools for both fundamental research and diagnostics of GAstV-2.

INTRODUCTION

China is the largest poultry farming country globally and the leading producer of geese. Goose astrovirus (GAstV) has emerged as a critical viral pathogen threatening the goose industry in recent years. GAstV infection induces pathological changes such as extensive urate deposition in the viscera, joint cavities, and ureteral surfaces of goslings, with mortality rates reaching up to 50%. GAstV has been demonstrated to transmit both horizontally and vertically(Wei, Yang, Wang, et al., 2020a), primarily affecting goslings under three weeks of age(An et al., 2020). The highly lethal gout disease caused by GAstV has severely impacted commercial gosling flocks in certain regions, leading to substantial economic losses and hampering industry development(Lu et al., 2025; Ren et al., 2025). Additionally, studies indicate that GAstV exhibits cross-species transmission to chickens and ducks(He, Jiang, et al., 2023; Wei, Yang, Wang, et al., 2020b). Currently, the pathogenic mechanism of GAstV and its associated immune responses remain poorly characterized, and there is a notable absence of highly effective serological detection methods specifically targeting GAstV.

Goose astrovirus belongs to the family Astroviridae and genus Avastrovirus. Its genome comprises two non-coding regions (5′UTR and 3′UTR), three open reading frames (ORF1a, ORF1b, and ORF2), and a poly(A) tail, with a total length of approximately 7,200 nucleotides(L. Xu et al., 2023). ORF1a and ORF1b encode non-structural proteins (nsp1a and nsp1b), which regulate viral replication and transcription. GAstV ORF2 encodes the capsid protein (Cap), the antigenic determinant responsible for eliciting host immune responses and inducing protective antibodies. Cap is critically involved in viral receptor recognition, host innate immunity, and cellular proliferation(Ren et al., 2020). GAstV virions exhibit a characteristic star-shaped morphology formed by surface spike structures. The viral capsid is composed of a single type of structural protein, forming a robust shell that protects the viral genome, referred to as the Cap. GAstV particles typically measure 28–30 nm in diameter(L. Xu et al., 2023). According to the International Committee on Taxonomy of Viruses (ICTV), GAstV is classified into genotypes 1 and 2 (GAstV-1 and GAstV-2) based on ORF2 (Cap) gene divergence(Ji et al., 2024; L. Xu et al., 2024). These genotypes exhibit distinct pathogenicity and host adaptability due to genomic variations(A. Wang et al., 2025; Zhai et al., 2023).

GAstV can be transmitted not only via the digestive tract but also vertically within goose populations, posing significant challenges for its prevention and control(Wei, Yang, He, et al., 2020a). Therefore, controlling transmission routes and host infections remains the primary strategy for mitigating GAstV outbreaks. Studies have shown that disinfection with 1 mg/mL free chlorine for 2 hours demonstrates substantial efficacy against AstV, though variations may exist among different strains. Proper disposal of infected goose byproducts and strict segregation of diseased and healthy goslings are critical to preventing GAstV transmission(L. Wang et al., 2025). Currently, no vaccines or therapeutic agents for GAstV are available for widespread application(Ren et al., 2025). Monoclonal antibodies (mAb) are homogeneous antibodies derived from a single B cell clone fused with myeloma cells. They specifically target defined antigenic epitopes and play pivotal roles in therapeutic development and molecular diagnostics.

In this study, splenocytes isolated from GAstV Cap-immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cell lines. Following subcloning and cellular screening via Western blotting (WB), indirect immunofluorescence assay (IFA), and indirect enzyme-linked immunosorbent assay (ELISA), six hybridomas producing GAstV Cap-specific mAbs were identified and designated as 1B3, 1B4, 1B6, 1D4, 1E2, and 1F1. To map the epitopes recognized by these mAbs, truncated forms of the full-length GAstV Cap protein were constructed as glutathione S-transferase (GST) fusion proteins in a prokaryotic expression system. The minimal epitope-containing fragments were determined through WB. Sequence alignment analysis was performed to assess the conservation of these epitopes. Furthermore, the spatial localization of the identified epitopes on the GAstV Cap dimer was visualized and mapped. Collectively, six mAbs specifically recognizing GAstV Cap were successfully generated, and their linear epitopes were systematically identified. The preparation of GAstV-specific mAbs with well-characterized epitopes provides essential tools for epitope-based vaccine design and antigen detection.

MATERIALS AND METHODS

Cells and Virus Templates

The Baby hamster kidney 21 (BHK-21) cells were propagated in high glucose Dulbecco's Modified Eagle's Medium (DMEM; Yuanpei, Shanghai, China) enriched with 10% heat-inactivated fetal bovine serum (FBS; Gibco, America). Hybridoma cell lines (commercially procured from Wuhan Jisa Biological Engineering Co., Ltd.) were cultured in DMEM medium supplemented with 20% fetal bovine serum and 0.1% penicillin-streptomycin antibiotic cocktail. All cell lines were incubated under standard conditions (37°C, 5% CO₂, humidified atmosphere). Hybridoma culture supernatants were collected at a density of 2 × 10⁶cells/mL for downstream applications. The genotype 2 goose astrovirus strain SCG3 (GenBank accession number: OQ909424.1) was preserved by the Research Center of Avian Disease at Sichuan Agricultural University, from a deceased gosling at a poultry farm in Guanghan District, Deyang City, Sichuan Province. Both virus-negative and virus-positive tissues of Type 2 goose astrovirus (GAstV-2) were provided by the Research Center of Avian Disease at Sichuan Agricultural University, China.

Plasmids Construction

The pET-28a(+) plasmid was linearized using restriction enzymes Xho I and BamH I. The full-length gene fragment encoding the Cap protein was amplified using primers SEQ No.69 and SEQ No.70(Table 1), with cDNA derived from viral RNA (template: GAstV strain isolated by the Avian Disease Center of Sichuan Agricultural University, GenBank: OQ909424.1) as the template. The amplified fragment was ligated into the linearized pET-28a(+) vector via homologous recombination. The His tag sequence encoded by the pET-28a(+) vector is located at the 5′ end of the amplified fragment. The ligation product was subsequently transformed into DH5α competent cells. The recombinant plasmid verified by Sanger sequencing was designated as pET-28a-Cap-His.

Table 1.

Primers used in this study for plasmid construction and truncated protein generation.

Primer ID	Primer Name	Primer Sequence (5′→3′)	Amplified Fragment
SEQ No.1	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap(1-704aa)
SEQ No.2	Cap-R	TCAGTCACGATGCGGCCGTCACTCATGTCCACCCTTCTCAAA
SEQ No.3	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap(1-352aa)
SEQ No.4	Cap-A-R	TCAGTCACGATGCGGCCGTCATGAAGCAGCACCAAACAATTTTCT
SEQ No.5	Cap-B-F	GTTCCGCGTGGATCCCCGAATTCTGGTTCCACTTATCTGATCTACTCC	Cap(353-704)
SEQ No.6	Cap-R	TCAGTCACGATGCGGCCGTCACTCATGTCCACCCTTCTCAAA
SEQ No.7	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap(1-176aa)
SEQ No.8	Cap-A1-R	TCAGTCACGATGCGGCCGTCATTTTGAGCCAATCGGGAGTTCAAGATG
SEQ No.9	Cap-A2-F	GTTCCGCGTGGATCCCCGCATCTTTGGAGGGTTCAACCCAGG	Cap(177-352aa)
SEQ No.10	Cap-A-R	TCAGTCACGATGCGGCCGTCATGAAGCAGCACCAAACAATTTTCT
SEQ No.11	Cap-B-F	GTTCCGCGTGGATCCCCGAATTCTGGTTCCACTTATCTGATCTACTCC	Cap (353-528aa)
SEQ No.12	Cap-B1-R	TCAGTCACGATGCGGCCGTCACAGGACTGCTGTTTCAAGCTCAGG
SEQ No.13	Cap-B2-F	GTTCCGCGTGGATCCCCGCGTGTGAATACCAGTACAACATCTACT	Cap (529-704aa)
SEQ No.14	Cap-R	TCAGTCACGATGCGGCCGTCACTCATGTCCACCCTTCTCAAA
SEQ No.15	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap (1-88aa)
SEQ No.16	Cap-A1a-R	TCAGTCACGATGCGGCCGTCACCGGTCTAGTGTGTCTGTTGAATT
SEQ No.17	Cap-A1b-F	GTTCCGCGTGGATCCCCGAAGCATAAATACTTCACAAATCCACTCATG	Cap (89-176aa)
SEQ No.18	Cap-A1-R	TCAGTCACGATGCGGCCGTCATTTTGAGCCAATCGGGAGTTCAAGATG
SEQ No.19	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap (1-44aa)
SEQ No.20	Cap-A1a1-R	TCAGTCACGATGCGGCCGTCACTTCATGGGTAATTTTTGGGGCTT
SEQ No.21	Cap-A1a2-F	GTTCCGCGTGGATCCCCGGCCGAGAGGAAGCTTGAGAAAGAA	Cap (45-88aa)
SEQ No.22	Cap-A1a-R	TCAGTCACGATGCGGCCGTCACCGGTCTAGTGTGTCTGTTGAATT
SEQ No.23	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap (1-22aa)
SEQ No.24	A1a1-1-R	TCAGTCACGATGCGGCCGTCAAACGGTGACCACTTTTGTAACCTT
SEQ No.25	A1a1-2-F	GTTCCGCGTGGATCCCCGACCAAGAAGGTTACAAAAGTGGTCACC	Cap (13-32aa)
SEQ No.26	A1a1-2-R	TCAGTCACGATGCGGCCGTCACTTTGGTTTCTTTTTTGGGTGTTTTTTCTT
SEQ No.27	A1a1-3-F	GTTCCGCGTGGATCCCCGAAGAAAAAACACCCAAAAAAGAAACCAAAGCAG	Cap (23-44aa)
SEQ No.28	Cap-A1a1-R	TCAGTCACGATGCGGCCGTCACTTCATGGGTAATTTTTGGGGCTT
SEQ No.29	Cap-F	GTTCCGCGTGGATCCCCGATGGCAGACAGGGCGGTG	Cap (1-12aa)
SEQ No.30	A1a1-P1-R	TCAGTCACGATGCGGCCGTCACACCTTCTCGCGCGGG
SEQ No.31	A1a1-P2-F	GTTCCGCGTGGATCCCCGACCAAGAAGGTTACAAAAGTGGTCACC	Cap (13-22aa)
SEQ No.32	A1a1-1-R	TCAGTCACGATGCGGCCGTCAAACGGTGACCACTTTTGTAACCTT
SEQ No.33	A1a1-P3-F	GTTCCGCGTGGATCCCCGGCAGACAGGGCGGTGGC	Cap (2-11aa)
SEQ No.34	A1a1-P3-R	TCAGTCACGATGCGGCCGTCACTTCTCGCGCGGGGCCA
SEQ No.35	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap (3-10aa)
SEQ No.36	A1a1-P4GST-R	CAGTCACGATGCGGCCGTCACTCGCGCGGGGCCACCGCCCTGTCCGGGGATCCACGCGG
SEQ No.37	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap (4-9aa)
SEQ No.38	A1a1-P5GST-R	TCAGTCACGATGCGGCCGTCAGCGCGGGGCCACCGCCCTCGGGGATCCACGCGGAAC
SEQ No.39	A1a1-P3L-F	GTTCCGCGTGGATCCCCGGACAGGGCGGTGGCCC	Cap (3-11aa)
SEQ No.40	A1a1-P3-R	TCAGTCACGATGCGGCCGTCACTTCTCGCGCGGGGCCA
SEQ No.41	A1a1-P3-F	GTTCCGCGTGGATCCCCGGCAGACAGGGCGGTGGC	Cap (2-10aa)
SEQ No.42	A1a1-P3R-R	TCAGTCACGATGCGGCCGTCACTCGCGCGGGGCCAC
SEQ No.43	Cap-B2-F	GTTCCGCGTGGATCCCCGCGTGTGAATACCAGTACAACATCTACT	Cap (529-616aa)
SEQ No.44	Cap-B2a-R	TCAGTCACGATGCGGCCGTCAGACTCTATTATTATATTGGTCCTGGGAATC
SEQ No.45	Cap-B2b-F	GTTCCGCGTGGATCCCCGAGGATGGTACAGTATGCTAATGCACAA	Cap (617-704aa)
SEQ No.46	Cap-R	TCAGTCACGATGCGGCCGTCACTCATGTCCACCCTTCTCAAA
SEQ No.47	Cap-B2b-F	GTTCCGCGTGGATCCCCGAGGATGGTACAGTATGCTAATGCACAA	Cap (617-660)
SEQ No.48	Cap-B2b1-R	TCAGTCACGATGCGGCCGTCAGACTGCTAGGTGGAAGTCAGTCTC
SEQ No.49	Cap-B2b2-F	GTTCCGCGTGGATCCCCGTCGCTCAAGACCTCTGACTAT	Cap (661-704)
SEQ No.50	Cap-R	TCAGTCACGATGCGGCCGTCACTCATGTCCACCCTTCTCAAA
SEQ No.51	B2b1-1-F	GTTCCGCGTGGATCCCCGAGGATGGTACAGTATGCTAATGCACAA	Cap (617-638aa)
SEQ No.52	B2b1-1-R	TCAGTCACGATGCGGCCGTCAGGGATCATCATCTTCCTCAGG
SEQ No.53	B2b1-2-F	GTTCCGCGTGGATCCCCGACTTTGACAGACCCTGAGGAA	Cap (628-649aa)
SEQ No.54	B2b1-2-R	TCAGTCACGATGCGGCCGTCATGTTGGATCAAAAAGCGAAGTGACATC
SEQ No.55	B2b1-3-F	GTTCCGCGTGGATCCCCGCTTTCTGATGTCACTTCGCTTTTTGAT	Cap (639-660aa)
SEQ No.56	B2b1-3-R	TCAGTCACGATGCGGCCGTCAGACTGCTAGGTGGAAGTCAGTCTC
SEQ No.57	B2b1-P1-F	GTTCCGCGTGGATCCCCGACTTTGACAGACCCTGAGGAAGATGATGATCCCTGA	Cap (628-638aa)
SEQ No.58	B2b1-P1-R	TCAGTCACGATGCGGCCGTCAGGGATCATCATCTTCCTCAGGGTCTGTCAAAGT
SEQ No.59	B2b1-P2-F	GTTCCGCGTGGATCCCCGTTGACAGACCCTGAGGAAGATGATGATTGA	Cap (629-637aa)
SEQ No.60	B2b1-P2-R	TCAGTCACGATGCGGCCGTCAATCATCATCTTCCTCAGGGTCTGTCAA
SEQ No.61	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap(630-636aa)
SEQ No.62	B2b1-P3GST-R	CCGCTCGAGTCGACCCGGTCAATCATCTTCCTCAGGGTCTGTCGGGGATCCACGCGG
SEQ No.63	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap (631-635aa)
SEQ No.64	B2b1-P4GST-R	CCGCTCGAGTCGACCCGGTCAATCTTCCTCAGGGTCCGGGGATCCACGCGG
SEQ No.65	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap (631-636aa)
SEQ No.66	B2b1-P3LGST-R	CCGCTCGAGTCGACCCGGTCAATCATCTTCCTCAGGGTCCGGGGATCCACGCGG
SEQ No.67	Cap-GST-F	CAGGAAACAGTATTCATGTCCCCTATACTAGGT	Cap (630-635aa)
SEQ No.68	B2b1-P3RGST-R	CCGCTCGAGTCGACCCGGTCAATCTTCCTCAGGGTCTGTCGGGGATCCACGCGG
SEQ No.69	Cap-His-F	CAAATGGGTCGCGGATCCATGGCAGACAGGGCGGTG	Cap
SEQ No.70	Cap-His-R	GTGGTGGTGGTGCTCGAGTCACTCATGTCCACCCTTCTCAAA
SEQ No.71	PC-Cap-F	CATCATTTTGGCAAAGAATTCGCCACCATGGAGCAGAAACTCATCTCTGAAGAGGATCTGATGGCAGACAGGGCGGTG	Cap
SEQ No.72	PC-Cap-R	TTGGCAGAGGGAAAAAGATCTCTATCACTCATGTCCACCCTTCTCAAA

The pCAGGS plasmid was digested with EcoR I and Bgl Ⅱ restriction enzymes to generate a linearized vector. The full-length Cap gene fragment was amplified from the constructed pET-28a-Cap-His plasmid template using primers SEQ No.71 and SEQ No.72 (Table 1), with a Myelocytomatosis (Myc) tag incorporated at the N-terminus of the recombinant fragment. The amplified fragment was then ligated into the linearized pCAGGS vector via homologous recombination enzymes. The ligation product was transformed into DH5α competent cells, and the recombinant plasmid confirmed by Sanger sequencing was designated as pCAGGS-Cap.

Construction of Fusion Truncated Proteins

To preliminarily map the epitope recognized by the mAbs, the full-length GAstV Cap protein was divided into six truncated fragments using a dichotomous truncation strategy (sequential bisection: dividing into halves and then quarters). These fragments were cloned into the prokaryotic expression vector pGEX-4T-1 to generate GST-tagged recombinant fusion proteins. The expression of GST-tagged truncated fusion proteins was verified using anti-GST antibodies. Successfully constructed fusion proteins were induced for expression and analyzed by WB to identify the mAbs recognition region.

To determine the minimal linear epitope recognized by the mAbs, the initial positive fragment was subjected to iterative dichotomous truncation. Truncated fragments were cloned into the GST-tagged fusion protein system for repeated screening. When fragments reached approximately 10 amino acids in length, sequential deletions were performed from both termini to generate progressively shorter truncations. These truncations were screened via WB until the minimal B-cell epitope was defined.

All recombinant plasmids were introduced into Escherichia coli BL21 (DE3) competent cells via heat shock transformation. Protein expression was initiated by supplementing the bacterial culture with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Following an 8-hour induction period at 37°C with vigorous agitation (180 rpm), bacterial cells were pelleted through centrifugation at 7,000 xg for 10 min at 4°C. The pelleted prokaryotically expressed fusion proteins were resuspended in phosphate-buffered saline (PBS).

Western Blot

Liver tissue specimens (100 mg) obtained from GAstV-infected goose in Suining, Sichuan Province (see Figure S1 for detection results, see Table S1 for detection primers), were homogenized in 1 mL RIPA lysis buffer (Beyotime Biotechnology, Cat# P0038,China) and incubated on ice for 5 min to achieve complete cellular lysis. The lysate was centrifuged at 10,000 xg for 5 min, and the supernatant was collected for subsequent analysis. For recombinant pCAGGS-Cap-transfected BHK-21 cells, the cell culture supernatant and cell pellet mixture were harvested at 48 hours post-transfection (hpt). The sample underwent centrifugation at 10,000 xg (8 min, 4°C) for pellet collection. Subsequent lysis was performed by resuspending the pellet in RIPA buffer followed by 30-minute incubation on ice. Following lysis, the cellular debris was removed by centrifugation (10,000 xg, 8 min) and the clarified supernatant was collected as aliquots. Protein separation was achieved through 12.5% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), followed by semi-dry electrophoretic transfer (Bio-Rad) onto PVDF membranes. Membranes were probed with hybridoma-derived primary antibodies and subsequently incubated with HRP-conjugated goat anti-mouse IgG (Abcam, America) for immunodetection. Protein signals were detected using an e-BLOT electronic pressure imaging system.

Indirect ELISA

The recombinant GAstV Cap fusion protein served as the coating antigen, with GST protein derived from the pGEX-4T-1 vector utilized as the negative control. Both antigens were immobilized on 96-well microtiter plates via overnight adsorption at 4°C in 0.1 M carbonate-bicarbonate buffer (pH-9.6). Post-coating, plates were blocked with 5% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20 (PBST) at 37°C for 1 h to mitigate nonspecific binding. Hybridoma supernatants (100 μL/well) were then applied as primary antibodies and incubated at 37°C for 2 h. Following three PBST washes (5 min each), HRP-conjugated goat anti-mouse IgG (Abcam; 1:5,000 dilution in PBST) was administered as the secondary antibody for 1 h at 37°C. Post-incubation, plates underwent three additional PBST washes. For chromogenic detection, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Tiangen Biotech) was added per well under light-protected conditions. The enzymatic reaction was terminated after 15 min by adding 50 μL of 2 M H₂SO₄, and absorbance at 450 nm was quantified using a SpectraMax microplate reader (Bio-Rad, America).

Indirect Immunofluorescence Assays

BHK-21 cells were cultured on sterile glass coverslips pre-positioned in 24-well plates. At ∼80% confluency, cells were transfected with pCAGGS-Cap plasmid using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. At 48 hpt, cells were washed thrice with PBST and fixed with freshly prepared 4% paraformaldehyde in PBS at 4°C for 12 h. Following fixation, cells were permeabilized with 0.3% Triton X-100 in PBS at 4°C for 20 min to enable antibody penetration. After three PBST washes, nonspecific bindings were blocked with 5% bovine serum albumin (BSA) in PBS at 37°C for 1 h. Hybridoma supernatant (undiluted) was applied as the primary antibody and incubated at 37°C for 2 h. Cells were then washed three times with PBST and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG at 37°C for 1 hour under light-protected conditions. After three PBST washes, nuclei were counterstained with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Coolaber, SL7101) in PBS for 15 min at room temperature. Following three final PBST washes, coverslips were air-dried in the dark, mounted onto glass slides with 6 to 7 μL glycerol, and visualized using fluorescence microscopy (Nikon, Tokyo, Japan) for image acquisition.

Structural Analysis of Epitopes

As no experimentally resolved 3D structure of GAstV Cap is available, the tertiary structure was predicted using AlphaFold3 (https://www.alphafoldserver.com). The linear B-cell epitopes identified in this study were mapped onto the predicted structure using PyMOL software. For sequence alignment, Cap sequences of GAstV genotypes were retrieved from GenBank and aligned using Geneious software.

Statistical Analysis

All statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software). Experiments were performed in triplicate across three independent replicates. Indirect ELISA data are presented as mean ± standard error of the mean (SEM).

RESULTS

Hybridoma Cells Screening

SDS-PAGE analysis confirmed predominant expression of GAstV Cap in the insoluble fraction (inclusion bodies). Subsequent purification of the pelleted inclusion bodies yielded GAstV Cap protein with approximately 85% purity (Fig. 1A). To induce antigen-specific immune responses, female BALB/c mice (6 weeks old) were primed via subcutaneous injection with purified Cap recombinant protein formulated in Freund’s complete adjuvant. Following a standardized immunization protocol, splenocytes were aseptically isolated from the immunized mice and subjected to polyethylene glycol (PEG)-mediated fusion with SP2/0 murine myeloma cells, enabling the establishment of monoclonal hybridoma cell lines. Six hybridoma clones (1B3, 1B4, 1B6, 1D4, 1E2, 1F1) that reacted to GAstV Cap were selected from 36 candidate hybridomas via indirect ELISA. The supernatants from these six hybridomas exhibited specific recognition of the Cap coating antigen (Fig. 1B). Specificity and efficiency of the six mAbs were further validated by WB and IFA. IFA confirmed that all six mAbs specifically recognized Cap protein (Fig. 1C). Subtyping using an antibody isotyping kit revealed the immunoglobulin classes of the six mAbs (Fig. 1D). ‌To validate the efficacy of mAbs in virus recognition. WB demonstrated their ability to detect GAstV and lysates of transfected cells compared to controls (Fig. 2A). Protein expression was analyzed using a β-tubulin loading control antibody and a Myc-tag-specific antibody (Fig. 2B), confirming proper expression levels. These results confirmed the successful isolation of six hybridoma cell lines stably producing GAstV Cap-specific antibodies.

Fig. 1.

Expression and purification of goose astrovirus capsid protein (GAstV Cap) and hybridoma screening.‌ ‌(A) SDS-PAGE analysis of GAstV Cap protein expression (left panel) and purification efficiency (right panel), with lanes labeled as follows: Pellet, Supernatant, Marker, purified GAstV Cap protein and Marker. (B) Preliminary screening of high-sensitivity mAbs via indirect ELISA. Full-length GAstV Cap fused with GST was used as the coating antigen, with empty pGEX-4T-1 GST protein as the negative control. Supernatants from six hybridoma clones served as primary antibodies. (C) IFA of BHK-21 cells at 48 hpt. GAstV Cap-specific fluorescence (green) was detected using FITC-conjugated goat anti-mouse IgG under fluorescence microscopy. (D)‌ Subtype identification of mAbs.

Fig. 2.

Validation of monoclonal antibodies (mAb) recognition efficacy against the virus.‌(A)‌ Western blotting analysis of mAbs. Lanes pCAGGS and pCAGGS-Cap represent lysates of BHK-21 cells transfected with the respective plasmids, probed with six mAbs as primary antibodies. Protein expression was analyzed using an anti-Myc tag antibody targeting the Myc-tagged pCAGGS-Cap construct. (B)‌ Western blotting of goose negative control (NC) tissue lysates and goose astrovirus-positive tissue lysates, probed with six mAbs and β-tubulin as primary antibodies. The Myc-tagged pCAGGS-Cap fusion protein was detected at approximately 81 kDa, while the infectious viral particle protein of goose astrovirus migrated at approximately 34 kDa or 29 kDa. β-Tubulin, serving as a loading control, was recognized by the corresponding antibody at ∼50 kDa.

mAbs Epitope Mapping

Full-length Cap was initially truncated into six fragments to localize the recognition regions of the six mAbs via WB. mAbs 1B3, 1B4, 1B6, 1D4, and 1E2 reacted with Cap(529-704aa) (amino acids 529–704; Fig. 3), while mAb 1F1 recognized Cap(1-176aa) (amino acids 1–176; Fig. 4). Further truncations were performed to identify the minimal linear epitopes. Briefly, the reactive peptide segments were bisected iteratively. If truncation to 44 amino acids or bisection eliminated reactivity, overlapping peptides (e.g., 1–22aa, 12–33aa, 22–44aa) were synthesized for validation. Progressive truncation from both ends of the reactive fragments ultimately identified the minimal epitopes: ⁶³⁰TDPEED⁶³⁵ for mAbs 1B3, 1B4, 1B6, 1D4, and 1E2 (Fig. 3), and ³DRAVAPREK¹¹ for mAb 1F1 (Fig. 4). To further validate the identified epitopes, eukaryotic expression plasmids harboring epitopes ³DRAVAPREK¹¹ and ⁶³⁰TDPEED⁶³⁵ were constructed using the pCDNA3.1 vector as the backbone, with the mCherry fluorescent protein sequence fused to the N-terminal ends of the epitope sequences for expression detection. GAstV Cap-specific epitopes were subsequently detected in BHK-21 cells at 30 hpt via IFA (Figure S2).

Fig. 3.

Epitope identification using monoclonal antibodies (mAb) 1B3, 1B4, 1B6, 1D4, and 1E2. The empty vector pGEX-4T-1 (containing GST-tagged protein) and GST-tagged full-length goose astrovirus capsid protein (GAstV Cap) (Cap (1-704 aa)) served as negative and positive controls, respectively, with antibody-identified proteins subsequently used as additional positive controls. Truncated GAstV Cap fusion proteins were screened via Western blotting. Schematic for constructing truncated fusion proteins targeting the epitopes recognized by mAbs 1B3, 1B4, 1B6, 1D4, and 1E2. Green: Recognition regions; Blue: Non-reactive regions; Gray: Non-expressed regions(A). Expression of truncated fusion proteins was validated using GST-tag-specific antibodies (B). Western blot analysis of mAbs specificity: mAb 1B3 (C), mAb 1B4 (D), mAb 1B6 (E), mAb 1D4 (F), and mAb 1E2 (G). The molecular weight (MW) of the empty GST vector was approximately 26 kDa, while the full-length Cap (1-704 aa) exhibited an MW of ∼104 kDa. Truncated proteins had MWs ranging from 26 to 65 kDa.

Fig. 4.

Epitope identification using monoclonal antibody (mAb) 1F1.Truncated goose astrovirus capsid protein (GAstV Cap) fusion proteins were screened via Western blotting. The empty vector pGEX-4T-1 (GST-tagged) and GST-tagged full-length GAstV Cap (Cap(1-704aa)) served as negative and positive controls, respectively, with antibody-recognized proteins subsequently used as positive controls. Schematic for constructing truncated fusion proteins targeting the epitope recognized by mAb 1F1. Purple: Recognition regions; Orange: Non-reactive regions; Gray: Non-expressed regions(A). Expression of truncated fusion proteins was validated using a GST-tag antibody (B). Western blot analysis of mAb specificity: mAb 1F1 (C). The molecular weight (MW) of the empty GST vector was approximately 26 kDa, while the full-length Cap (1-704 aa) exhibited an MW of ∼104 kDa. Truncated proteins had MWs ranging from 26 to 65 kDa.

Structural Visualization of mAb Epitopes

To predict functional implications, the two epitopes were mapped onto a predicted Cap dimer structure (Figs. 5A and 5B). Structural analysis revealed that the 1F1 epitope resides within the shell domain of the capsid, while the epitopes recognized by 1B3, 1B4, 1B6, 1D4, and 1E2 localize to the spike domain on the capsid surface (Fig. 5A). Surface rendering further illustrated the spatial positioning of these epitopes at the molecular level (Fig. 5B).

Fig. 5.

Epitope mapping of monoclonal antibodies (mAb) against goose astrovirus capsid protein (GAstV Cap). The crystal structure of GAstV Cap protein predicted by AlphaFold3 was utilized as the display template, and its three-dimensional model was modified using PyMOL software. (A) Two antigenic epitopes screened from GAstV Cap are displayed. The selected epitopes (³DRAVAPREK¹¹ and ⁶³⁰TDPEED⁶³⁵) are highlighted in red and green, respectively. The central panel demonstrates the spatial correspondence between different antibody-recognized epitopes and their respective color codes. (B) Surface and cartoon representations illustrate the spatial distribution of these two epitopes at the molecular level, presented from multiple viewing angles. (C) Conservation analysis of the identified epitopes across evolutionary lineages.

Conservation Analysis of Epitopes

To assess the conservation of the two epitopes across GAstV-2 and other avian astroviruses (AAstVs), ORF2 sequences of representative GAstV-2 and AAstV strains were retrieved from NCBI and aligned using Geneious software. The epitopes ⁶³⁰TDPEED⁶³⁵ and ³DRAVAPREK¹¹ were highly conserved in GAstV-2 strains (Fig. 5C). However, significant divergence was observed in GAstV-1 and AAstV strains, despite their shared association with gosling gout.

DISCUSSION

China is the world’s largest producer of poultry, particularly geese. Goose astrovirus (GAstV) has emerged as a critical pathogen threatening the goose industry, causing visceral gout, urate deposition in joints and ureters(Ren et al., 2025), and mortality rates up to 50% in goslings(Zhang, Lv, et al., 2022). The high lethality of GAstV-associated gout has inflicted severe economic losses on commercial goose farms(Chen et al., 2024; Li et al., 2024; A. Wang et al., 2023). Despite its impact, the pathogenic mechanisms and immune evasion strategies of GAstV remain poorly understood, and robust serological diagnostics are lacking. Developing early detection methods for GAstV infection is imperative. Besides, Epitope-based vaccines, targeting protective epitopes that elicit immune responses without adverse effects, represent a promising strategy. Therefore, we aim to generate mAbs to provide essential tools for establishing relevant detection assays and advancing epitope-based vaccine development.

To acquire infectivity, GAstV requires proteolytic processing by host extracellular proteases. During this process, the Cap protein is cleaved by trypsin-like proteases to generate highly infectious particles with molecular weights of approximately 34 kDa or 27 kDa(Méndez et al., 2002; Xiang et al., 2024), consistent with our detection results (Fig. 2B). The Cap protein elicits robust immune responses by mediating receptor recognition, viral attachment and entry, and host immune interactions, and is critical for inducing protective antibodies(He, Sun, et al., 2023; Zhang et al., 2023). Notably, as the surface-exposed structural component of the virion, the Cap protein governs GAstV serotype classification and represents a dominant target for neutralizing antibody induction (Espinosa et al., 2019; Z. Wang et al., 2022). Studies have demonstrated that cellular vimentin interacts with the Cap protein and promotes viral replication(Xiang et al., 2024). While the pathogenicity, epidemiology, and transmission dynamics of GAstV-2 have been well characterized(Wei, Yang, He, et al., 2020b; Wu et al., 2020; P. Xu et al., 2024; Zhang et al., 2022), epitope mapping of the mAbs against GAstV-2 Cap protein remains poorly explored. Several studies have investigated antigenic epitopes of GAstV, predominantly focusing on the generation of a single monoclonal antibody (mAb) followed by characterization of its recognized epitope. In contrast, this study successfully generated multiple mAbs and systematically identified their corresponding antigenic epitopes(Dai et al., 2022; Ren et al., 2025). Notably, the epitopes identified in this work represent novel findings that have not been previously reported in previous studies. In this study, we developed mAbs demonstrating high specificity and sensitivity for GAstV Cap protein in infected cells, positioning them as reliable diagnostic tools for GAstV infection surveillance.

Using a prokaryotic expression system, we expressed GAstV-2 Cap as inclusion bodies, which were subsequently refolded via dialysis to restore antigenicity. The refolded protein served as an immunogen to produce six mAbs (1B3, 1B4, 1B6, 1D4, 1E2, 1F1). Subtyping revealed IgG2b (1B3), IgG2a (1B4, 1D4), and IgG1 (1B6, 1E2, 1F1) heavy chains, with all light chains being kappa-type. IFA and WB confirmed their specific binding to GAstV-2. During truncation analysis, Cap(1-352aa) and Cap(177-352aa) failed to express, likely due to rare codon usage. Fortunately, mAb 1F1 recognized Cap(1-176aa), enabling epitope mapping. Notably, truncation to 9aa (for 1B3 group) or 12aa (for 1F1) reduced reactivity, likely due to loss of critical residues. Nonetheless, further truncation successfully identified minimal epitopes (Fig. 3, Fig. 4). Specificity was further validated using lysates from BHK-21 cells transfected with eukaryotic GAstV-2 Cap expression plasmids.The six mAbs recognize two distinct epitopes, enabling future development of sandwich ELISA assays.

CONCLUSIONS

In this study, we successfully generated six mAbs—1B3, 1B4, 1B6, 1D4, 1E2, and 1F1—and identified two antigenic epitopes recognized by them: ³DRAVAPREK¹¹ and ⁶³⁰TDPEED⁶³⁵. Collectively, the development of these GAstV-2 Cap-specific mAbs and the characterization of their linear epitopes lay a foundation for rapid detection of GAstV-2 infection and further exploration of its molecular mechanisms and pathogenesis.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The experiments were approved by the Institutional Animal Care and Use Committee of Sichuan Agriculture University in Sichuan, China (Protocol Permit Number: SYXK(川) 2024-0187).

AUTHORS' CONTRIBUTIONS

Writing—original draft: WL. Data analysis: LX, HT, TW, ZW, YH. Methodology: MW, RJ, DZ, ML, XZ, QY, YW, SZ, JH, XO, DS, BT. Writing—review and editing:AC, SC. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declared that there are no competing financial interests regarding the publication of this paper.