Identification of a Type-Specific Epitope in the ORF2 Protein of Duck Astrovirus Type 1

Simple Summary Duck astrovirus type 1 (DAstV-1) infection constitutes a cause of viral hepatitis in ducklings and little is known about the B-cell epitope of DAstV-1. In this study, using a monoclonal antibody (mAb) 3D2 against ORF2 protein of DAstV-1, a highly conserved linear B-cell epitope of 454 STTESA459 in DAstV-1 ORF2 was identified. The mAb 3D2 showed no neutralizing activity to DAstV-1 and had no cross-reactivity with other DAstV serotypes. Abstract Duck astrovirus type 1 (DAstV-1) infection constitutes a cause of viral hepatitis in ducklings and little is known about the B-cell epitope of DAstV-1. In this study, a monoclonal antibody (mAb) 3D2 against open reading frame 2 (ORF2) protein of DAstV-1 was used to identify the possible epitope in the four serotypes of DAstV. The mAb 3D2 showed no neutralization activity to DAstV-1, and reacted with the conserved linear B-cell epitopes of 454STTESA459 in DAstV-1 ORF2 protein. Sequence analysis, dot blot assay, and cross-reactivity test indicated that the epitope peptide was highly conserved in DAstV-1 sequence and mAb 3D2 had no cross-reactivity with other DAstV serotypes. To the best of our knowledge, this is the first report about identification of the specific conserved linear B-cell epitope of DAstV-1, which will facilitate the serologic diagnosis of DAstV-1 infection.

It is recognized that monoclonal antibodies (mAbs) consisting of one specific antibody molecule are superior to their polyclonal antisera in many facets of immunology [25][26][27]. Their characteristics of sensitive and specificity make hybridoma-derived antibodies the effective immunological reagents in immunoassays, immunotherapy, immunoaffinity chromatography and immune diagnosis. Until now, the application of mAb in DAstV diagnosis has not been reported. In this study, taking the prokaryotic-expressed ORF2 protein as the immunogen, a DAstV-1 ORF2-specific mAb 3D2 was generated using cell hybridization technique, and a highly conserved B-cell epitope in DAstV-1 ORF2 protein was identified with the mAb. These findings will be valuable for developing epitope-based diagnostic kit for DAstV-1 infections.

Viruses, Cells, and Antibodies
DAstV-1 virulent strain D51 (GenBank accession no. MH712856) was isolated from the liver of sick cherry valley ducks in the Shandong province of China in 2012 [28]. The ORF2 gene of DAstV-1 D51 strain was cloned into the prokaryotic expression vectors pET-32a (+) (Novagen, Darmstadt, Germany) and pGEX-6p-1 (GE Healthcare, Amersham, UK) to generate recombinant histidines tagged ORF2 (His-ORF2) and glutathione S-transferase tagged ORF2 (GST-ORF2). The purified His-ORF2 protein was used to immunize BALB/c mice. The hybridoma cell line producing mAb 3D2 was produced by fusion of B-lymphocytes from immunized mice with mouse myeloma cells. Subtype identification revealed that mAb 3D2 was of the IgG2b/kappa type.
Horseradish peroxidase (HRP) labeled goat anti-mouse antibody and fluorescein isothiocyanate (FITC) labeled goat anti-mouse antibody were purchased from KPL (Gaithersburg, MD, USA). The positive anti-DAstV-1 serum was obtained from five mice immunized with purified His-ORF2 protein and stored in the veterinary molecular etiology laboratory of Shandong Agricultural University.

Primers Sequence (5 →3 ) AA Position in ORF2 Protein
Note: EcoR I and Xho I restriction enzyme sites were introduced at the 5 end of the primers with underlined letters.
Indirect immunofluorescence assay (IFA) was used to identify the reactivity of mAb 3D2 with DAstV-1 virus. Briefly, 70-90% confluent BHK-21 cells were transfected with the DNA-launched infectious clone of DAstV-1 or recombinant mutant plasmids. At 48 h post-transfection (hpt), the cells were fixed with an icy mixture of acetone-formaldehyde (1:1 v/v) for 20 min and followed by transparent with 0.2% Triton X-100 in PBS for 3 min. After cells being blocked with PBS buffer containing 1% bovine serum albumin for 1 h, mAb 3D2 (dilution with 1:1000), mouse polyclonal antibody (dilution with 1:500) or SP2/0 supernatant (dilution with 1:50) were added as the primary antibodies. After washing three times with PBST buffer, the secondary antibody, FITC-conjugated goat anti-mouse IgG (dilution with 1:50) was added and incubated at 37 • C for 1 h in the dark. Finally, the stained cells were observed using a fluorescence microscope (Leica AF6000).

Neutralization Assay for mAb 3D2 Against DAstV-1
Virus neutralization assay was used to detect the neutralizing activity of mAb 3D2 against DAstV-1 by IFA. Briefly, 100 µL of serial diluted mAb 3D2 samples (the initial dilution of 1:10; and 2-fold dilutions to 1:160) were incubated with at 1 MOI (multiplicity of infection) of DAstV-1 for 2 h at 37 • C, respectively. Then, 200 µL virus-mAb mixture was added into DEF monolayer cells. At 48 h post infection (hpi), the cells were observed and analyzed by IFA.

Identification of the B-Cell Linear Epitope to 3D2
To preliminarily identify the epitope that mAb 3D2 can recognize, three overlapping fragments of the DAstV-1 ORF2 gene were amplified with specific primers (Table 2) and cloned into plasmid pGEX-6p-1 for construction of the recombinant plasmids ORF2-A, ORF2-B, and ORF2-C. All truncated GST-fusion proteins were expressed and purified, and subsequently were analyzed by dot blot analysis using mAb 3D2 or anti-GST mAb, respectively.  Based on the dot blot detecting results, five fragments (ORF2-I, ORF2-II, ORF2-III, ORF2-IV, and ORF2-V) were designed to further determine the position of the epitope ( Table 2). The recombinant proteins were detected by dot blot assay. Finally, six fragments (ORF2-1, ORF2-2, ORF2-3, ORF2-4, ORF2-5, and ORF2-6) were designed to determine the accurate position of the epitope by cutting down the amino acids from both sides one by one ( Table 2). The recombinant proteins were also detected by dot blot analysis.
Subsequently, we constructed different mutations in DNA-Launched clone of DAstV-1, and they were the clone deleted epitope 'STTESA' followed by the substitution of amino acids in the epitope motif location with 'GDWQSN', 'DSWKSH', 'TNWNGN', 'STAETG', and 'STTETG', respectively ( Figure 1, Table 3). In detail, we firstly amplified the upstream (primers of U-F and U-R) and downstream (primers of D-F and D-R) of the products, respectively. Then, based on the U-F primer, a series of reverse primers (UF-1R, UF-2R, UF-3R, and UF-4R) was used for PCR amplification. At the same time, based on the D-R primer, a series of forward primers (DR-1F, DR-2F, DR-3F, and DR-4F) were used for PCR amplification. These PCR products were finally fused to obtain the mutations mentioned above. When reaching 70-90% confluence, BHK-21 cells were respectively transfected with those mutant plasmids and analyzed by IFA at 48 hpt.

Reactivity Analysis of mAb 3D2
Dot blotting results showed that mAb 3D2 specifically reacted with GST-D51 ORF2, while did not react with GST-SL1 ORF2, GST-CPH ORF2, and GST-YP2 ORF2 (Figure 2A). The IFA result revealed that mAb 3D2 could recognize the native-form of the ORF2 protein, whereas could not recognize GST-SL1 ORF2, GST-CPH ORF2, and GST-YP2 ORF2 ( Figure 2B). Those results suggested that mAb 3D2 had good specificity with ORF2 protein of DAstV-1 and did not react with the ORF2 proteins of other serotypes of DAstV.

Neutralization Analysis
The virus-mAb mixture, a series of diluted mAb 3D2 incubated with DAstV-1 for 1 h at 37 • C, was adsorbed in the DEF cell for 2 h and fresh DMEM with 2% FBS was added after washed with PBS. The IFA result showed that there was still strong green fluorescence whether in the maximum concentration (dilution with 1:10) or the minimum concentration (dilution with 1:160) of the mAb 3D2 treatment group (Figure 3). It indicated that the mAb 3D2 had no neutralizing activity to DAstV-1.

The Accurate Position of the Linear Epitope in DAstV-1 ORF2 to mAb 3D2
A total of three overlapping GST-fused fragments (ORF2-A, ORF2-B, and ORF2-C) of DAstV-1 ORF2 protein were successfully expressed. Dot blotting results showed that mAb 3D2 reacted with ORF2-B, while did not react with ORF2-A and ORF2-C ( Figure 4A). Subsequently, five overlapping GST-fused fragments (ORF2-I, ORF2-II, ORF2-III, ORF2-IV, and ORF2-V) were successfully expressed and dot blotting results showed that mAb 3D2 reacted with ORF2-III, ORF2-IV, and ORF2-V, while not with ORF2-I and ORF2-II ( Figure 4B). Therefore, we initially inferred that the epitope against mAb 3D2 was within the amino acids of 452 RISTTESAAL 461 in DAstV-1 ORF2 protein.

Epitope Mapping of the mAb 3D2
To identify whether the 454 STTESA 459 epitope was conserved in the ORF2 protein among different serotypes of DAstV, we aligned all the ORF2 sequences of DAstV-1, DAstV-2, DAstV-3, and DAstV-4 in GenBank. The result showed that the amino acid sequences in the epitope motif were conserved among all the DAstV-1 strains, but were quite different from the amino acids in the corresponding regions of other DAstV serotypes (Figure 6), indicating that the motif represented a conserved epitope on the ORF2 protein of DAstV-1.

Discussion
DAstV-1 infection causes fatal hepatitis to ducklings [13,18] and is ubiquitous in China [30,31]. As the most prevalent serotype, the overall mortality in 1-to 2-week-old ducklings infected by DAstV-1 was 50%, and livers in the affected ducklings displayed typical multiple and widespread hemorrhagic lesions [13,32]. As an effective antiviral agent and differential diagnostic agent, mAbs of HAstV and CAstV had been used to diagnosis [33,34]. However, there is still a lack of new classes of antibodies for diagnosis of DAstV-1 infection. From a genetic point of view, ORF1b of astrovirus was the least divergent whereas ORF2 appeared to be the most divergent among the different ORFs [35]. Moreover, as viral capsid protein, ORF2 protein serves as the major candidate antigen for vaccine preparation or virus serological diagnosis [36]. In this study, mAb 3D2 against DAstV-1 ORF2 protein was firstly prepared, and IFA showed that mAb 3D2 specifically recognized DAstV-1-transfected BHK-21 cells (Figure 2).
In terms of diagnostic and disease monitoring, epitopes can be used to monitor the response to the particular virus strain during infection. B-cell epitopes could bind to corresponding receptors in humoral and cellular immune responses and contribute to the specific antibody production [37]. Well-defined epitopes against mAbs will contribute to benefit from the antigen structure study and vaccine preparation [38][39][40][41]. Therefore, it is imperative to screen B-cell epitopes in epitope-based diagnosis kits. Among the common antigen-antibody specificity methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot, dot blot is a low-complexity and time-saving technique that is widely used for peptide screening [29,42]. Using dot blotting, the epitope 454 STTESA 459 against the mAb 3D2 was identified in the present study. To the best of our knowledge, this was the first accurate epitope identified in DAstV-1.

Conclusions
In conclusion, a conserved linear B-cell epitope in DAstV-1 ORF2 protein was identified for the first time. The 3D2 was a highly specific mAb without neutralizing activity to DAstV-1, which might play a significant role in the development of a discriminating diagnostic kit for DAstV-1 infection.