Identification of a novel monoclonal antibody derived from the lumpy skin disease virus ORF123 that confers cross-binding and cross-neutralizing activity against Capripoxvirus members

TL;DR

A new monoclonal antibody from lumpy skin disease virus shows cross-protection against related poxviruses, offering potential for universal vaccines.

Contribution

A novel monoclonal antibody with cross-binding and neutralizing activity against multiple capripoxviruses is identified.

Findings

A monoclonal antibody derived from LSDV ORF123 cross-reacts with GTPV and SPPV.

The epitope 85PYFLKN90 is conserved across LSDV and GTPV, with a mutation affecting SPPV recognition.

The mAb neutralizes LSDV, GTPV, and extracellular SPPV replication.

Abstract

Capripoxviruses cause diseases (e.g., lumpy skin disease, sheep pox, and goat pox) that significantly hinder the growth of livestock production in endemic areas. Here, we systemically describe a B-cell monoclonal antibody (mAb) derived from the lumpy skin disease virus (LSDV) ORF123, which exhibits cross-reactivity with goat poxvirus (GTPV) and sheep poxvirus (SPPV). A novel continuous linear and conformational epitope, 85PYFLKN90, of LSDV and GTPV was first identified using bioinformatics, western blotting, and indirect immunofluorescence methods. Furthermore, the linear epitope recognition of SPPV by this LSDV ORF123 mAb was determined by the natural point mutation from P to Q at amino acid 85. Moreover, through alanine-scanning mutagenesis analysis, we demonstrated that the critical amino acid of this conserved linear and conformational epitope of LSDV ORF123 slightly differs from that of the GTPV homologue. Importantly, this mAb retained cross-neutralizing activity against LSDV, GTPV, and extracellular SPPV replication. The cross-binding and neutralizing activity of this LSDV ORF123 mAb strengthens our understanding of the pathogenic mechanisms and antigenic similarities among different members of the genus Capripoxviruses. These findings provide theoretical and experimental evidence for the development of universal pan-vaccines and therapeutic antibodies.

The online version contains supplementary material available at 10.1186/s13567-025-01501-3.

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Full text

Introduction

Ruminants, including cattle, sheep, and goats, are natural hosts for capripoxviruses (CaPVs). According to the host species of CaPVs, the subfamily Chordopoxvirinae and the family Poxviridae are subdivided into SPPV, GTPV, and LSDV [1]. Lumpy skin disease (LSD) is caused by LSDV, a rapidly emerging pox virus that infects cattle and wild animals [2]. LSD is an infectious cattle disease characterized by multifocal nodules on the skin, generalized lymphadenitis, and fever [1]. LSD causes significant economic losses and can devastate livelihoods and food security in endemic areas [2]. LSD is becoming increasingly severe and epidemic worldwide owing to the lack of timely vaccines and effective drug treatments. The first LSD outbreak was officially recorded in 1929 in Zambia, sub-Saharan Africa [2, 3]. In the past century, the geographical spread of LSD has extended from Africa to the Middle East, Eastern Europe, and Asia [2, 4, 5]. LSDV is a rapidly emerging pathogen in Asia, including in China. Initial outbreaks of LSD were officially reported in Qapqal Xibe Autonomous County, Ili Kazakh Autonomous Prefecture, Xinjiang, and Northwest China [6]. Since then, several LSD epidemics have been formally reported in 14 provinces or municipalities [6–8].

Although the first LSD outbreak was formally recorded in 1929 [2, 3], the whole-genome characterization of LSDV was not performed until 2001 [1]. The LSDV genome is a linear dsDNA molecule of approximately 150,000 base pairs (bp) in length, with a central coding region and 2.4 identical inverted terminal repeats encoding 156 putative open reading frames (ORFs) [1]. Unlike most DNA viruses, poxviruses replicate in the cytoplasm of infected cells to complete their lifecycle [9–12]. The viral morphology of LSDV has a barrel and brick shape consisting of an internal nucleoprotein core flanked by protein structures and an outer membrane envelope, which is similar to the morphology of vaccinia virus (VACV) [8, 13, 14]. Most of the understanding of the viral genome of CaPVs has been gained from research on VACVs [9, 12, 15–18].

The discovery of hybridoma technology has promoted the development of antibody-based drugs, immunotherapeutics, and epitope-based vaccines. Many monoclonal antibodies (mAbs) (e.g., RSV 199 [19], 7D6/6D6 [20], 2G12 [21], AP33 [22], and C12H5 [23]) have been discovered and characterized. These mAbs display broad neutralization activity by targeting viral surface envelope proteins such as respiratory syncytial virus (RSV) [19], severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [20], human immunodeficiency virus type 1 (HIV-1) [21, 24], hepatitis C virus (HCV) [22], and influenza A virus [23, 25]. Several cross-neutralizing mAbs that target different envelope proteins of VACV, such as D8 [26], H3 [27], L1 [28, 29], A27, D8, D13, A14, B5, and A33 [30–32], have also been identified. However, compared with RNA virus infections, poxvirus infections, such as VACV infection, elicit a more complex immune response that is reactive to diverse antigens [33]. Efficient protective immunity against poxvirus infections is achieved using a mixture of diverse mAbs rather than a single mAb that targets multiple viral proteins [33].

Compared with VACVs, a few specific mAbs targeting LSDV-encoded proteins have been developed and identified. VACV A33, a type II integral membrane viral glycoprotein, is located on the surface of the enveloped virion and host cell membrane [34–36] and can be used as a component of subunit vaccination [30–32]. ORF123 of LSDV is a putative extracellular enveloped viral membrane glycoprotein [8], similar to the homologues of VACV A33 [34, 37]. The role of LSDV ORF123 in the viral replication cycle and the activation of host immunity must be elucidated. Using traditional hybridoma technology, we aimed to obtain an experimental mAb against the LSDV ORF123 protein to investigate the biological function of the LSDV ORF123 gene and its involvement in pathogenesis. This is the first study describing the mAbs derived from LSDV, which have cross-binding and neutralizing abilities against LSDV, GTPV, and SPPV infections.

Materials and methods

Cells, reagents, viruses, and virus infections

The MDBK, Vero, and 293 T cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 37 °C incubator with 5% CO_2_. The hybridoma cell line that secretes monoclonal antibody (mAb) was cultured in DMEM supplemented with 20% FBS (Nulen, CF602) (Shanghai, China). Mycoplasma detection was performed using the GMyc-PCR Mycoplasma Test Kit (YEASEN, Shanghai, China), which was negative in all the cell lines.

Primary antibodies, including mouse anti-ORF123 and anti-ORF29 antibodies against LSDV, were prepared in our laboratory. Anti-LSDV polyclonal serum samples were prepared and stored in our laboratory [8]. The anti-β-actin antibody was purchased from Nulen Biotechnology (Shanghai, China). The secondary antibodies, including horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG, were purchased from CWBio (Jiangsu, China). Goat IgG (H + L) cross-adsorbed secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The goat anti-mouse and goat anti-rabbit Alexa Fluor secondary antibodies used for indirect immunofluorescence, including Alexa Fluor 488 (Molecular Probes), were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 2 × Phanta Flash Master Mix and Phanta Max Super-Fidelity DNA Polymerase used for polymerase chain reaction (PCR) were purchased from Vazyme Biotechnology (Nanjing, China). The Clone Express Ultra One Step Cloning Kit V2 and Mut Express Multis Fast Mutagenesis Kit V2 used to construct the plasmids were purchased from Vazyme Biotechnology (Nanjing, China).

The LSDV/FJ/CHA/2021 strain (GenBank: OP752701) was isolated and maintained in our laboratory at the LVRI of CAAS [8]. LSDV-EGFP and LSDV-RFP strains were constructed using the homologous recombination technique [38]. GTPV (AV41 strain) was isolated from a commercial vaccine. GTPV-EGFP and GTPV-RFP strains were constructed using homologous recombination on the basis of the viral genome sequence (GenBank: MH381810) [38]. SPPV (SPPV/NX/CHA/2022 strain, unpublished) was isolated and stored in our laboratory. The Orf virus (OrfV) (SC1 strain, GenBank: ON932452) and vaccinia virus (VACV) (Ankara strain, GenBank: U94848) were obtained from Haotai Chen Associate Researcher. Purified virus titres were determined using the 50% tissue culture infectious dose (TCID_50_) assay in MDBK and Vero cells. Virus titres were calculated using the Reed‒Muench method by determining the dilution that produced 50% of the cells showing cytopathic effects.

For the viral infection experiment, MDBK and Vero cells were infected with viruses (LSDV, GTPV, SPPV, OrfV, and VACV) at a multiplicity of infection (MOI) of 1 in a 37 °C cell culture incubator. After 4 h of absorption, the unbound virus was removed, and the cells were washed three times with phosphate-buffered saline (PBS) and cultured in complete medium. The cell samples were used for western blotting, flow cytometry, and immunofluorescence analyses after viral infection.

Plasmid construction and protein purification

To construct the prokaryotic pET28a-ORF123 plasmid, a primer pair with BamH I and EcoR I enzyme sites, ORF123-For: 5′-CAGCAAATGGGTCGCGGATCCGCCACCATGTTAGTTGATATTCCAAAGAGTGGAACTGAAAC-3′; ORF123-Rev: 5′-TTGTCGACGGAGCTCGAATTCGTGGTGGTGGTGGTGGTGGCTTCCTCCTCCAAAAAAAGATCTTACACAGTAATAGCTTCTC-3′, was designed and synthesized by AZENTA (Suzhou, China). The constructed plasmid was verified by sequencing (Additional file 1A).

For purification of the LSDV ORF123 protein, the recombinant N-terminal ORF123 protein plasmid with a 6-His tag was transformed into Rosetta (DE3) competent cells. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added for induction of expression at 28 °C for 24 h. The sediment was dialyzed with carbamide and finally purified by nickel‒nitrilotriacetic acid affinity chromatography (GE Healthcare) by elution with 0.25 M imidazole, 20 mM Tris, pH 8.0, and 0.3 M NaCl.

Production of monoclonal antibodies (mAbs) against LSDV ORF123

Briefly, the recombinant prokaryotic pET28a-ORF123 protein that had been removed was used as an immunogen to produce the mAb (Additional file 1B). Next, 25 µg of purified recombinant pET28a-ORF123 protein emulsified with the same amount of complete Freund’s adjuvant (Sigma‒Aldrich, USA) was used for the first subcutaneous immunization of three 6-week-old BALB/c mice. The mice were booster immunized intraperitoneally with purified recombinant pET28a-ORF123 protein emulsified with an equal volume of incomplete Freund’s adjuvant 10–14 days after the first immunization injection. After the last immunization with 25 µg of pET28a-ORF123 protein, cells from the spleens of the best responders were removed and mixed with cells from SP2/0 myeloma using polyethylene glycol 2000 (Sigma‒Aldrich).

Fused cells were selected in hypoxanthine-aminopterin-thymidine (HAT) medium. After incubation, the medium was diluted in multi-well plates such that each well contained only one cell. Finally, the hybridoma culture supernatant of the positive clone (^#^1G1-1G12) was screened using ELISA and western blotting with the recombinant ORF123 protein as an antigen. The mAb was produced by injecting hybridoma cells into the peritoneal cavity of 6-week-old BALB/c mice. Ascitic fluid was collected and purified using affinity chromatography on protein A agarose (GE Healthcare) after 10–15 days.

Surface plasmon resonance (SPR) and biolayer interferometry (BLI) analysis

For SPR analysis, the binding affinity of the LSDV ORF123 mAb was measured using a Biacore T200 instrument (GE Healthcare). Briefly, for affinity measurements, 1 mg/mL LSDV ORF123 mAb was dissolved in 10 mM acetate buffer (pH 5.5) and immobilized on a CM5 sensor chip (GE Healthcare). The synthesized epitope polypeptide (PYFLKN) was diluted in HBS-EP + buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) (pH 7.4) to the indicated concentrations (125, 250, 500, 1000, 2000, and 4000 nM) and measured at a flow rate of 30 μL/min for a contact time of 60 s and a dissociation time of 60 s. Regeneration was performed using 100 mM glycine (pH 3.0) for 30 s. The difference in the RUs between the ligand and reference flow cells resulted in a binding signal that was monitored as a function of time, represented as sensorgrams. Biacore T200 evaluation software was employed to evaluate the kinetics of the binding response using reference-subtracted sensorgrams. Apparent K_D_ values were calculated in a steady-state affinity model using BIAcore evaluation software.

BLI was performed using a GatorBio Plus instrument (GatarBio, Shanghai, China). For the quantitative analysis of the purified LSDV ORF123 mAb, a standard antibody sample was prepared in advance to construct a standard curve. The purified hybridoma of the LSDV ORF123 mAb was subsequently immobilized on anti-mouse IgG Fc biosensors to determine its concentration. Moreover, the LSDV ORF123 mAb was immobilized on anti-mouse IgG Fc biosensors and used to capture viruses (LSDV, SPPV, GTPV, and VACV) for specific binding between the virus and the LSDV ORF123mAb. Finally, the raw BLI response traces were processed using Gator Software v2.7.

Western blot (WB) analysis

The cell samples were washed with PBS and lysed with lysis buffer (2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) for denaturing polyacrylamide gel electrophoresis (PAGE) separation. The lysates were collected in 1.5 mL tubes, kept on ice for 30 min, and centrifuged at 12 000 rpm at 4 °C for 15 min for clarification. The lysate was diluted in 5 × SDS buffer to a final concentration of 1 × SDS. Denaturation was performed at 100 °C for 10 min. Equal amounts of these prepared samples were subjected to SDS‒polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (GE Healthcare Life Sciences, Amersham, Protran 0.2, or 0.45 NC, Germany).

For native PAGE protein separation, viral samples (LSDV, GTPV, SPPV, Orfv, and VACV) were collected without a reducing agent. The Omni-PAGE gel was loaded with samples and run for separation of proteins in neutral pH Tris-HEPES-native buffer. The proteins were subsequently transferred to a nitrocellulose membrane following the manufacturer’s instructions (LK205, Shanghai, China).

The membranes were blocked for 30 min with 5% skim milk in 0.05% Tris-buffered saline containing 0.05% Tween 20 (TBST). The membranes were washed three times with 0.05% TBST (5 min each) and incubated with primary antibodies for at least 6 h at 4 °C. The membranes were washed three times with 0.05% TBST (5 min/wash) and incubated with HRP-conjugated secondary antibodies for two hours at room temperature. Finally, after three washes, the antibody-antigen complex was exposed to a chemiluminescence reagent solution kit (US EVERBRIGHT, Suzhou, China) using a multichem luminescence image analysis system (GE, Amersham Imager 600, New York, USA). All the above steps were performed according to Ren et al. [39].

Indirect immunofluorescence assay (IFA) analysis

MDBK and Vero cells were grown and transfected on coverslips in a six-well plate for IFA analysis. The coverslips were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature after plasmid transfection or viral infection. The cells were permeabilized with 0.5% Triton X-100 for 10 min at room temperature and washed three times with PBS. The cells were blocked with 3% bovine serum albumin (BSA) for 30 min at 37 °C, incubated with a primary antibody for two hours, and washed three times with PBS. The antibodies were detected by incubation with an Alexa Fluor 488-conjugated secondary antibody for one hour in a moist container at 37 °C in the dark. The cells were stained with DAPI (0.1 μg/mL) for eight minutes at 37 °C. Finally, the coverslips were mounted on microscope slides and allowed to air dry. Fluorescence images were recorded with a confocal fluorescence microscope (ZEISS LSM880, Germany). The above steps were performed as previously described [39].

Neutralization activity of the LSDV ORF123 mAb against LSDV

To determine the neutralizing activity of the prepared LSDV ORF123 mAb, monolayers of Vero or MDBK cells were seeded at 90–100% confluence in a 96- or 48-well plate one day in advance. LSDV (10^7.015^TCID_50_), LSDV-EGFP (10^5.5^TCID_50_), LSDV-RFP (10^6.25^TCID_50_), GTPV (10^5.2^TCID_50_), GTPV-EGFP (10^5.2^TCID_50_), GTPV-RFP (10^4.38^TCID_50_), and SPPV (10^4.38^TCID_50_) and diluted mAbs with DMEM were co-incubated for four hours at 37 °C in an incubator. After incubation, the same medium volume was added to monolayers of Vero or MDBK cells in four wells. After 4 h of adsorption at 37 °C, the monolayer was washed with PBS, supplemented with DMEM supplemented with 10% FBS, and incubated for 48, 72, and 96 h. Western blot samples were collected, and representative images were captured using the EVOS M5000 imaging system.

Bioinformatic analysis

For sequence alignment and phylogenetic analyses, nucleotide sequence homology was determined using the CLUSTAL W multiple alignment algorithm of DNASTAR, Snap Gene, Jalview, and CLUSTAL W. The phylogenetic tree was constructed using MEGA-X and modified using iTOL online software. For 3D structural modelling, amnio acid was submitted to the SWISS-MODEL and AlphaFold protein structure databases and saved in PDB format. Structural simulation and visualization were subsequently performed using the open-source PyMOL 2.5 software.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). All values represent the means of at least three independent experiments. The standard deviation and standard error of the mean were calculated and determined using at least three technical replicates (unless otherwise stated). Three or more means were analysed using one-way analysis of variance (ANOVA). Statistical significance was evaluated by determining the* p* values using a two-tailed Student’s t test (two-tailed distribution). ns, p value > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001.

Results

Identification of the cross-reactivity of this linear and conformational LSDV ORF123 mAb with LSDV, GTPV, and SPPV

A hybridoma cell line (^#^1G1-1G12) that secretes a mAb against the LSDV ORF123 protein was obtained for further research using the traditional hybridoma technology. Quantitative analysis of the purified LSDV ORF123 mAb was performed using the biolayer interferometry (BLI) technique on the basis of the standard antibody sample (Additional file 1C). Western blot analysis of the data in Additional file 1D suggested that this ORF123 mAb retained good reactivity and specificity against LSDV. Given that LSDV, SPPV, and GTPV belong to the same genera, capripoxvirus, subfamily Chordopoxvirinae, and family Poxviridae [1], the potential cross-reactivity of the LSDV ORF123 mAb against GTPV and SSPV was further evaluated. As shown in Additional file 2, the phylogenetic analysis also suggested that ORF123 of LSDV/FJ/CHA/2021 was closely related to the ORF123 analogue of GTPV 122/A33R. Figure 1A and Additional file 3A show that this mAb against LSDV ORF123 could recognize LSDV and GTPV rather than SPPV, Orfv, or VACV. Furthermore, this LSDV ORF123 mAb enables the maintenance of reactivity with the mature virion (MV) and immature virion (IMV) of LSDV and GTPV rather than with SPPV (Additional files 3B-3C). However, as shown in Figure 1B and Additional file 3D, this mAb of LSDV ORF123 could recognize LSDV, GTPV, and SPPV rather than VACV and OrfV in virus-infected MDBK cells. We performed WB analysis without a reducing agent to further determine the cross-reactivity between LSDV, GTPV, and SPPV and this LSDV ORF123 mAb. As shown in Additional file 3E, compared with the ORF29 group (horizontal line 2), native PAGE analyses suggested that this LSDV ORF123 mAb recognized LSDV, GTPV, and SPPV under nondenaturing conditions. Moreover, biolayer interferometry analysis further revealed the cross-reactivity between LSDV, GTPV, and SPPV and this LSDV ORF123 mAb. As shown in Figure 1C, compared with those of the VACV group, specific binding shift curves were observed among the LSDV, GTPV, and SPPV groups. The specific binding shift curve of the SPPV group presented a dose-dependent pattern in response to the LSDV ORF123 mAb (Figure 1D). On the basis of the above WB, IFA, and BLI results in Figures 1A–D and Additional file 3, we verified that this LSDV ORF123 mAb enables recognition of LSDV, SPPV, and GTPV and does not cross-react with OrfV and VACV.Figure 1Cross-reactivity identification of this linear and conformational LSDV ORF123 mAb to LSDV, GTPV****, and SPPV. A Western blot analysis of cross-reactivity with the anti-ORF123 mAb against LSDV, GTPV, SPPV, and VACV. B Immunofluorescence analysis of cross-reactivity with anti-ORF123 and ORF29 mAbs against LSDV, GTPV, SPPV, and VACV. MDBK cells were infected with LSDV, GTPV, SPPV, or VACV for 48 h. LSDV ORF123 (green); LSDV ORF29 mAb (green); nuclei (blue). C BLI binding assays of the LSDV ORF123 mAb with LSDV, SPPV, GTPV, and VACV. The cell culture supernatant of virus-infected Vero cells was collected and centrifuged at 2500 rpm for approximately 15 min to remove cell debris. The collected viruses were subsequently subjected to BLI following the procedure described in the Materials and methods. Western blot analysis of anti-ORF123 and ORF29 effects on LSDV, GTPV, SPPV, and VACV.** D** BLI binding assays of LSDV ORF123 mAb with serially diluted SPPV. SPPV was diluted with PBS (2 ×) and used in the BLI binding assay.** E** Western blot analysis of the cross-reactivity of anti-ORF123 against ORF123 and its analogue proteins. 293 T cells were transfected with pCAGGS,* pCAGGS-ORF123-Flag, pCAGGS-A33R/122/ORF123(GTPV)-Flag, or pCAGGS-117/ORF123(SPPV)-Flag. After 24 h, western blotting samples were collected and analysed.** F** Immunofluorescence analysis of cross-reactivity with the anti-ORF123 mAb. Vero cells were transfected with pCAGGS, p*CAGGS-ORF123-Flag, pCAGGS-A33R/122/ORF123(GTPV)-Flag, or pCAGGS-117/ORF123(SPPV)-Flag. LSDV ORF123 mAb (green), nuclei (blue).

To further confirm the cross-binding activity of the LSDV ORF123 mAb, GTPV A33R/ORF123 and SPPV 117/ORF123 were constructed using a specific primer pair (Additional file 4). As shown in Figure 1E, the mAb against LSDV ORF123 recognized the exogenous ORF123 analogue of LSDV and GTPV rather than SPPV (Figure 1E, vertical line 3). As expected, compared with the positive control group (Flag group) (horizontal lines 1 and 2), the IFA analysis suggested that the LSDV ORF123 mAbs enabled the recognition of exogenous ORF123 and the homologues of LSDV, GTPV, and SPPV (Figure 1F, horizontal lines 3 and 4). Moreover, as shown in Figure 1F, the subcellular location of LSDV ORF123 was slightly different from those of GTPV and SPPV. Compared with those of GTPV/122/A33R and SPPV 117, the cellular location of LSDV ORF123 is located on the cell membrane and around the central nucleus, similar to that of VACV A33, which is located on the surface of the enveloped virion and host cell membrane [34–36].

Peptide mapping of the linear and conformational epitopes of this mAb against LSDV ORF123

B-cell epitopes can be classified into two categories: linear epitopes, which consist of a continuous sequence of amino acid residues, and conformational epitopes, which consist of non-contiguous residues, meaning that they may be far apart in sequence but are brought into proximity by protein folding [40, 41]. Given the WB and IFA results shown in Figure 2 and Additional file 5, we further mapped the linear and conformational antigenic epitopes against the LSDV ORF123 mAb using specific primers (Additional file 6). Compared with traditional methods of epitope identification, predicting linear and conformational B-cell epitopes using reliable bioinformatics platforms as a complementary method is necessary and time-saving [40, 42, 43]. We first predicted these linear and conformational antigenic epitopes against LSDV ORF123 using the IEDB online analysis resource website (Additional file 5A). According to the results of the bioinformatics analysis, the LSDV ORF123 protein was divided into two fragments (aa 1–98 and aa 99–196) (Figure 2A and Additional file 5A). As shown in Figure 2B and Additional file 5B, the results of WB and IFA suggested that the linear and conformational epitopes of LSDV ORF123 were located in the 1–98 aa region rather than in other fragments. On the basis of IEDB analysis (Additional file 5C), the 1–98 aa fragments of the LSDV ORF123 protein were subsequently further subdivided into 1–50 and 51–98 aa (Figure 4C). On the basis of the WB and IFA results (Figure 2D and Additional file 5D), we concluded that this linear and conformational epitope overlaps from amino acids 51 to 98. To further narrow down the antigenic epitopes, the F5 peptide fragment was deleted into four fragments, namely, F6, F7, F8, and F9 (Figure 2E and Additional file 5E). Figure 2F and Additional file 5F show that we could further identify this linear and conformational epitope between amino acids 76 and 96. Single amino acid truncation analysis of the C- and N-termini was performed on the basis of the schematic diagram (Figure 2G and Additional file 5G) to map the epitope sequence accurately. As shown in Figure 2H, we concluded that the C-terminus of this linear epitope is located at 85 aa, whereas the N-terminus of the linear epitope is located at 90 aa. Moreover, the IFA results (Additional file 5H) indicated that the C- and N-termini of this conformational epitope were also located at amino acids 85 and 90, respectively. Finally, a continuous rather than discontinuous linear and conformational epitope was progressively identified as ^85^PYFLKN^90^ via WB and IFA analysis on the basis of multiple rounds of truncated and overlapping expression (Figure 2I). Notably, this continuous linear epitope is the same as the conformational epitope against LSDV ORF123, indicating that this conformational epitope is continuous rather than a traditional discontinuous pattern [40, 41].Figure 2Peptide mapping of the linear and conformational epitopes to the LSDV ORF123 mAb. A, C, and E Schematic diagrams of the first, second, and third rounds of truncation for peptide mapping against LSDV ORF123. B, D, F Western blot analysis of the first, second, and third rounds of truncation for peptide mapping of LSDV ORF123. 293 T cells were transfected with* pCAGGS-F1(1-196aa)-Flag, pCAGGS-F2(1-98aa)-Flag, pCAGGS-F3(99-1196aa)-Flag, pCAGGS-F4(1-50aa)-Flag, pCAGGS-F5(51-98aa)-Flag, pCAGGS-F5(Δ51-98aa)-Flag, pCAGGS-F6(Δ51-54aa)-Flag, pCAGGS-F7(Δ66-75aa)-Flag, pCAGGS-F8(Δ76-87aa)-Flag, pCAGGS-F9(Δ88-96aa)-FLAG, and pCAGGS. After 24 h, western blotting samples were collected and analysed. G Schematic diagram of the fourth round of truncation for peptide mapping of the N-terminal and C-terminal regions of the LSDV ORF123 epitope. H Western blot analysis of the fourth round of truncation for C-terminal peptide mapping against the LSDV ORF123. 293 T cells were transfected with pCAGGS-F8(76-87aa)-Flag, pCAGGS-F11(76-90aa)-Flag, pCAGGS-F10(76-93aa)-Flag, pCAGGS-F8 + 9(76-96aa)-FLAG, pCAGGS-F9(88-96aa)-Flag, pCAGGS-F14(85-96aa)-Flag, pCAGGS-F13(82-96aa)-Flag, pCAGGS-F12(79-96aa)-Flag, pCAGGS-F5(51-98aa)-Flag, pCAGGS-F15(86-96aa)-Flag, p*CAGGS-F16(76-89aa)-Flag, and pCAGGS.** I** Amino acid labelling analysis of LSDV ORF123 for peptide mapping. The nucleotides of LSDV ORF123 were converted into an amino acid sequence using the DNASTAR software. After the fourth round of truncation, the critical amino acids are marked with different colors.

The natural point mutation of the SPPV 117/ORF123 analogue from P to Q at 85 aa determines the linear epitope recognition of this LSDV ORF123 mAb

Given the cross-reactivity of this mAb against GTPV and SPPV (Figure 1 and Additional file 3), the conservativeness of this contiguous linear and conformational epitope against LSDV ORF123 among LSDV, GTPV, and SPPV was investigated. ORF123 and analogue proteins of twenty different CaPVs were downloaded from the GenBank database for amino acid sequence alignment. As shown in Figures 3A and B, the results of multiple sequence alignment indicated that the PYFLKN motif is highly conserved between LSDV ORF123 and GTPV 122/A33R. In contrast, the epitope motif of SPPV 117/ORF123 is QYFLKN, which maintains a natural point mutation from Q to P at amino acid 85. To confirm the conserved characteristics of this antigenic peptide among GTPV, LSDV, and SPPV, epitope deletion plasmids, including LSDV ORF123 (Δ85-90aa), GTPV A33R/122 (Δ86-91aa), and SPPV 117 (Δ85-90aa), were constructed using specific primers (Additional file 7). Compared with those of the positive control groups (LSDV ORF123 and GTPV A33R/122 groups) and the negative control groups (pCAGGS and SPPV 117/ORF123), the reactivities of the LSDV ORF123 (Δ85–90aa), GTPV A33R/122 (Δ86–91aa) groups, and SPPV 117/ORF123 (Δ85–90aa) groups were negative (Figures 3C and 3D). Moreover, compared with those of the flag-tag control group (Figure 3F, vertical line 1) and positive control group (Figure 3F, horizontal line 3), the results of the IFA assay of LSDV ORF123 (Δ85–90aa), GTPV A33R/122 (Δ86–91aa), and SPPV 117 (Δ85–90aa) were negative (Figure 3F, vertical line 2) (solid red border). WB (Figures 3C–E) and IFA (Figure 3F) indicated that this continuous linear and conformational peptide is the same and conserved between LSDV, GTPV, and SPPV.Figure 3The natural point mutation of the SPPV 117/ORF123 analogue from P to Q at 85 aa determines the linear epitope recognition ability of this LSDV ORF123 mAb. A Sequence alignment of ORF123 and homologous amino acids of LSDV, GTPV, and SPPV. The sequences were downloaded from the GenBank database and analysed via DNASTAR and Jalview software. B Statistical analysis of the reactivity of the LSDV ORF123 mAb against LSDV, GTPV, and SPPV. C–E Western blot analysis of the epitope deletion of LSDV ORF123 and analogues against this LSDV ORF123 mAb. 293 T cells were transfected with pCAGGS-LSDV ORF123-Flag,* pCAGGS-LSDV ORF123(Δ85-90aa)-Flag, pCAGGS-GTPV A33R/122-Flag, pCAGGS-GTPV A33R/122(Δ86-91aa)-Flag, pCAGGS-SPPV 117-Flag, pCAGGS-SPPV 117/(Δ85-90aa)-Flag, and pCAGGS. Twenty-four hours post-transfection (hpt), Western blot samples were collected and analysed. F Indirect immunofluorescence analysis of the epitope deletion of LSDV ORF123 and its analogues against the LSDV ORF123 mAb. Vero cells were transfected with pCAGGS-LSDV ORF123(Δ85-90aa)-Flag, pCAGGS-GTPV A33R/122(Δ86-91aa)-Flag, pCAGGS-SPPV 117-Flag, and pCAGGS-SPPV 117/(Δ85-90aa)-Flag. LSDV ORF123 mAb, FLAG (green), nuclei (blue). G Western blot analysis of the mutated plasmids of LSDV ORF123 and SPPV 117 against the LSDV ORF123 mAb. 293 T cells were transfected with pCAGGS-LSDV ORF123(85P-Q)-Flag, pCAGGS-SPPV 117/ORF123(85Q-P)-Flag, or pCAGGS. H Expression of reverse-mutated LSDV ORF123 and SPPV 117 plasmids against the LSDV ORF123 mAb. Vero cells were transfected with p*CAGGS-LSDV ORF123(85P-Q)-FLAG, pCAGGS-SPPV 117/ORF123(85Q-P)-FLAG, or pCAGGS. LSDV ORF123 mAb (green), FLAG (green), nuclei (blue).

We further confirmed the role of 85P and 85Q in the cross-reactivity of the antigen‒antibody complexes among GTPV, LSDV, and SPPV. We mutated LSDV ORF123 from P to Q at 85 aa according to the sequence alignment results between LSDV, GTPV, and SPPV (Figure 3A). As shown in Figure 3G (vertical line 1), the WB results suggested that the point mutation of LSDV ORF123 from P to Q at amino acid 85 was crucial for linear epitope recognition of the antigen‒antibody complex. Moreover, the point mutation of LSDV ORF123 from P to Q at 85 did not significantly affect the conformational epitope recognition of LSDV ORF123. We subsequently mutated Q to P at 85 aa of SPPV 117 to reverse verify the influence of 85 aa on antigen‒antibody complex recognition by this LSDV ORF123 mAb. As shown in Figure 3G and H (vertical line 2), WB and IFA analyses of the SPPV 117/ORF123 (85Q to P) mutated group were positive for this LSDV ORF123 mAb. These results suggest that the natural point mutation of SPPV 117 from P to Q at 85 aa determines the linear rather than conformational epitope recognition of the SPPV analogue against this LSVD ORF123 mAb.

The critical amino acid of this conserved linear and conformational epitope of LSDV ORF123 slightly differs from that of the GTPV homologue

To gain insight into the critical amino acids of this linear and conformational epitope against LSDV ORF123, alanine scanning substitutions were performed with specific primers (Additional file 8), as shown in the schematic diagram in Figures 4A and C. As shown in Figures 4B and D, WB analysis suggested that 86Y and 87F play decisive roles in recognizing the antigen‒antibody complex. In contrast, 85P, 88 L, 89 K, and 90 N also played a vital role in the reactivity against the LSDV ORF123 mAb, especially 88 L and 90 N. Notably, as shown in Figure 4D (vertical line 1), WB analysis indicated that the point mutation from P to Q at 85 aa determines the recognition of the antigen‒antibody complex, which is in accordance with the results in Figures 3G and H. On the basis of the alanine-substituted residue analysis shown in Figures 4B and D, all amino acids affected the binding ability of the linear epitopes, whose degrees differed. The IFA results also revealed that 87F (blue square border) is essential for recognizing the conformational epitope of the antigen‒antibody complex (Figure 4E). As shown in Figure 4E, the 86Y amino acid is not a key amino acid for this conformational epitope, which differs from the role of linear epitope recognition (Figure 4B). Notably, 85P in the epitope was not a critical amino acid for the conformational recognition of LSDV ORF123, which is in accordance with the results shown in Figure 3.Figure 4Identification of the critical amino acids of the continuous linear and conformational epitopes of LSDV ORF123 and GTPV A33R/122. (A, C, and F) Alanine scanning mutagenesis analysis of the linear and conformational epitopes. The amino acid residues of the epitope against LSDV ORF123 and GTPV A33R/122 were mutated to alanine (A). The 85 proline (P) amino acid residues of the LSDV ORF 123 epitope were mutated to glutamine (Q). The 86 proline (P) amino acid residues of the GTPV A33R/122 epitope were mutated to glutamine (Q). B, D and G Western blot analysis of the mutated plasmids of LSDV ORF123 and GTPV A33R/122 against the LSDV ORF123 mAb. 293 T cells were transfected with pCAGGS-LSDV ORF123(85P-A)-Flag, pCAGGS-LSDV ORF123(85P-Q)-Flag, pCAGGS-LSDV ORF123(86Y-A)-Flag*, pCAGGS-LSDV ORF123(87F-A)-Flag, pCAGGS-LSDV ORF123(88L-A)-Flag, pCAGGS-LSDV ORF123(89 K-A)-Flag, pCAGGS-LSDV ORF123(90N-A)-Flag, pCAGGS-GTPV A33R/122(86P-Q)-Flag, pCAGGS-GTPV A33R/122(87Y-A)-Flag, p*CAGGS-GTPV A33R/122(88F-A)-Flag, pCAGGS-GTPV A33R/122(89L-A)-Flag, pCAGGS-GTPV A33R/122(90 K-A)-Flag, or pCAGGS-GTPV ORF123(91N-A)-Flag. After 24 h, the samples were collected for western blot analysis. E and H Immunofluorescence analysis of conformational epitopes of the LSDV ORF123 and GTPV A33R/122 anti-ORF123 mAbs. Vero cells were transfected with the mutant plasmids LSDV ORF123 and GTPV A33R/122. Fluorescence images were visualized and captured via a confocal fluorescence microscope (ZEISS LSM880, Germany). LSDV ORF123 mAb (green); Flag (green); nuclei (blue).

Next, we performed alanine mutation analysis to identify the critical amino acids of this linear and conformational epitope against GTPV 122/A33R (Figure 4F). As shown in Figure 4G, WB analysis revealed that four amino acids, 86P, 87Y, 88F, and 89 L, play critical roles in linear epitope recognition of the antigen‒antibody complex. This amino acid differed slightly from the critical amino acid of the linear epitope of LSDV ORF123 (Figures 4A–D). Moreover, as shown in Figure 4H and Additional file 9C, three amino acids (87Y, 88F, and 89 L) (blue square border) play crucial roles in the recognition of the conformational epitope of the antigen‒antibody complex. These three amino acids are also critical for the linear epitope of GTPV 122/A33R (Figure 4G). Notably, the 86P amino acid is not a key amino acid for this conformational epitope of GTPV 122/A33R.

To better understand the structural characteristics of these critical amino acids, we predicted and simulated the structure of LSDV ORF123 using the PyMOL software. As shown in Additional file 9A, on the basis of the WB analysis of Figures 4A–D, we concluded that the random coil located at 85P, 86Y, and 87F of LSDV ORF123 located at the random coil is crucial for the recognition of the linear epitope. On the basis of the IFA results (Figure 4E), 87F (blue square border) was required for conformational epitope recognition (Additional file 9C). We also predicted and simulated the structure of A33R/122 of the GTPV using PyMOL software. As shown in Additional file 9B and C, the structural characteristics of GTPV A33R/ORF123 were located on a random coil, similar to those of LSDV ORF123.

The LSDV ORF123 mAb retains cross-neutralizing antibody activity against CaPVs

Next, we examined the affinity and avidity of this LSDV ORF123 mAb by surface plasmon resonance. As shown in Figure 5A, SPR analysis indicated that this synthetic epitope polypeptide bound to the LSDV ORF123 mAb with good affinity and avidity. Moreover, the IC_50_ value of the LSDV ORF123 mAb for LSDV was 3.421 μg/mL (Figure 5B).Figure 5The LSDV ORF123 mAb retains neutralizing activity against LSDV. A SPR kinetics and affinities of this LSDV ORF123 mAb to a synthetic epitope polypeptide against the LSDV ORF123 mAb. The binding affinity and kinetic evaluation of the binding response were measured with a Biacore T200 instrument following the procedures described in the Materials and methods. The synthetic polypeptide was serially diluted (2 ×) (125, 250, 500, 1000, 2000, and 4000 nm), and each curve represents a separate biosensor bound to an equivalent of the LSDV ORF123 mAb. B IC_50_ determination of the LSDV ORF123 mAb against LSDV. The purified LSDV ORF123 mAb (100 µg/mL) was diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128. The x-axis shows the amount of monoclonal antibody tested, and the y-axis indicates the percent inhibition in the assay. A nonlinear curve fit was performed for all the antibody dilution series. C Neutralizing activity of the LSDV ORF123 mAb against LSDV infection. A representative image was taken using the EVOS M5000 imaging system.** D** Western blot analysis of the neutralizing activity of the LSDV ORF123 mAb against LSDV infection.** E** Determination of extracellular LSDV titres with diluted ORF123 monoclonal antibodies. Extracellular LSDV titres were determined in MDBK cells as TCID_50_ values via the Reed–Muench method. Statistical significance was evaluated by determining the* p* values using a two-tailed Student’s t test. ns, p > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001.

To assess whether the LSDV ORF123 mAb retained neutralizing activity against LSDV, we used EGFP- and RFP-fluorescently labelled LSDVs (EGFP-LSDV and RFP-LSDV, respectively) to test the ability of anti-ORF123 mAbs to neutralize LSDV replication. As demonstrated in Figure 5C and Additional file 10A, compared with the positive control groups (EGFP- and RFP-LSDV, vertical line 1), the observed EGFP and RFP fluorescence intensities suggested that the LSDV ORF123 mAb retained neutralizing activity against EGFP-LSDV and RFP-LSDV in a dose-dependent manner at 48, 72, and 96 h post-infection (hpi). We then performed WB and TCID_50_ assays to determine the intracellular and extracellular virus titres during the neutralization activity assay. As shown in Figures 5D, E, and Additional file 10B, C, this mAb retained neutralizing activity against intracellular and extracellular viral replication in a dose-dependent manner compared with the positive control group (EGFP-LSDV and RFP-LSDV, vertical line 1). These results indicated a strong correlation between antibody valency and neutralization efficiency.

To further determine whether the LSDV ORF123 mAb retained neutralizing activity against GPTV and SPPV infections, we determined the intracellular and extracellular titres of GTPV and SPPV using WB and TCID_50_ assays. As shown in Additional files 11A-11D, this LSDV ORF123 mAb retained neutralizing activity against GTPV in a dose- and time-dependent manner, suggesting a strong correlation between antibody valency and neutralization efficiency. Compared with the WB analysis in Additional files 11E-11G, this LSDV ORF123 mAb only maintained neutralizing activity against extracellular rather than intracellular SPPV infection. On the basis of the above results, we conclude that this LSDV ORF123 mAb retains broad-spectrum and cross-neutralizing antibody activity against LSDV, GTPV, and SPPV.

Discussion

In the present study, an LSDV ORF123 mAb was first obtained using hybridoma technology to elucidate the pathogenesis of LSDV infection. Interestingly, we found that the screened LSDV ORF123 mAb could recognize not only the linear and conformational epitopes of LSDV and GTPV but also the conformational epitope of SPPV. A novel continuous linear and conformational epitope of LSDV ORF123 and GTPV 122/A33R was subsequently identified. The critical amino acids of these conserved linear and conformational epitopes of LSDV ORF123 and GTPV 122/A33R, which differ slightly, were further identified by alanine-scanning mutagenesis. Moreover, a natural point mutation from P to Q at 85 aa determines the recognition of the linear epitope of the SPPV analogue (117/ORF123) against this LSDV ORF123 mAb. This mAb possesses cross-neutralizing activities against LSDV, GTPV, and SPPV. The present study broadens our understanding of how immunity is connected among different viruses of the genus CaPVs and provides valuable insights for developing universal vaccines, antibody treatments, and antiviral drugs.

Identifying and mapping B-cell epitopes is essential for understanding host immune responses to pathogens and developing epitope-based vaccines [40, 41]. Generally, linear B-cell epitopes comprise sequential/continuous residues, whereas conformational B-cell epitopes contain scattered/discontinuous residues along their sequence [40]. Intriguingly, contrary to the conventional opinion [40, 41], the linear and conformational antigen epitope of the LSDV ORF 123 mAb is characterized as continuous rather than discontinuous, which is conserved between LSDV ORF123, GTPV A33R/122, and SPPV 117. Notably, the recognition of the linear epitope of the SPPV analogue by this LSDV ORF123 mAb is determined by the natural point mutation from P to Q at amino acid 85 of SPPV 117. Viruses evade the host immune system and avoid elimination via rapid antigenic shifts and drift in the viral antigenic protein [44, 45]. Under endogenous and exogenous selective pressures, RNA and DNA viruses undergo unique evolutionary patterns to survive and produce progeny [46]. Here, we inferred that the naturally occurring mutation of this epitope in SPPV 117 from P-to-Q at 85 aa is a strategy for the adaptive evolution of the virus. The linear and conformational epitopes of LSDV ORF123 are continuously conserved among LSDV ORF123, GTPV A33R/122, and SPPV117. Furthermore, alanine-scanning mutagenesis revealed that this conserved linear epitope’s critical amino acid slightly differs from the essential amino acid of the conformational epitopes on the ORF123 analogues of LSDV and GTPV. This is a sign of slow evolution among LSDV, GTPV, and SPPV.

B-cell-mediated humoral immunity is a vital part of the adaptive immune system, as it can provide long-term protection and immunological memory against viral infections [41]. B-cell neutralizing mAbs are essential for many biomedical and immunological applications, including immunotherapy, diagnostic testing, and vaccine or drug development [25, 47]. For example, the broadly neutralizing antibody 2G12 effectively protects against SHIV challenge at the mucosal level even when serum neutralizing titres are low [21]. The roles of VACA L1 [28, 29] and A33 [30–32] in the application of multicomponent subunit vaccines have been identified. These examples highlight the enormous potential for the application and design of neutralizing mAbs and pan-vaccines. On the basis of the antigenic homology and cross-protection among SPPV, GTPV, and LSDV [4], two live attenuated strains of CaPVs (LSDV Neethling and SPPV RM-65) have been specifically recommended and used as vaccines for the control and prevention of LSD [48, 49]. However, the cross-protective efficacy of homologous live attenuated vaccines of SPPV and GTPV against LSDV infection still needs to be re-evaluated [49, 50]. Given that VACV A33, an analogue of LSDV ORF123, can be used as a component of subunit vaccination [30–32], the identification of a conserved neutralizing linear and conformational epitope offers valuable insights into the future development of universal epitope-targeted pan-vaccines against LSDV, GTPV, and SPPV. Here, we provide experimental evidence and a theoretical basis for the cross-protection of heterogeneous pan-vaccines. Admittedly, the application of mAbs as therapeutics or vaccines is not straightforward. Several critical attributes must be optimized [47].

Virus infection-induced neutralizing antibodies offer protection against viral infection by targeting viral surface proteins and Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity, mast cell activation, opsonization, and complement activation [25, 51]. For example, 7D6/6D6 mAbs of SARS-CoV-2 resist circulating variants and maintain cross-neutralizing activity by binding to the cryptic site of the receptor-binding domain [20]. RSV-199, a potent cross-neutralizing antibody, potently recognizes, neutralizes, and protects against RSV and hMPV by targeting antigenic site III of prefusion F [19]. The mAb AP33 broadly neutralizes HCV infectivity by targeting the viral E2 envelope glycoprotein [22]. Recently, C12H5, a chimeric monoclonal antibody, provided neutralization against seasonal and pandemic H1N1 viruses and cross-protection against some H5N1 viruses by binding to viral hemagglutinin and controlling viral entry and exit [23]. In this case, the detection of neutralizing activity revealed that this LSDV ORF123 mAb allowed partial inhibition of intracellular and extracellular LSDV and GTPV replication rather than of intracellular SPPV replication. We speculated that the natural point mutation from P to Q at amino acid 85 of the SPPV 117 analogue not only determines linear epitope recognition but also influences the neutralizing activity of this LSDV ORF123 mAb against SPPV infection. Notably, the neutralizing potency of this mAb is lower than that of mAbs (e.g., RSV 199 [19], 7D6/6D6 [20], AP33 [22], and C12H5 [23]) against RNA viruses. Compared with those of RNA viruses (e.g., RSV [19], SARS-CoV-2 [20], HIV-1 [21, 24], HCV [22], and IAV [23]), the entry/fusion complex of VACV consists of at least eight viral proteins that together participate in the viral entry step [29]. Protective immunity against LSDV, GTPV, and SPPV requires the cooperation of antibodies that target different viral proteins in the entry/fusion complex of CaPVs. Owing to the lack of a suitable animal model and the high cost of cows (Bos taurus), we did not conduct an animal protection experiment with the LSDV ORF123 mAb, which is necessary and should be further assessed in host animals (cattle, sheep, and goats) in the future.

Supplementary Information

** Additional file 1. Generation of monoclonal antibodies (mAbs) against LSDV ORF 123. A** PCR amplification of the LSDV ORF123 gene. B Inducible expression and purification of the LSDV ORF123 protein. C BLI quantitative analysis of the LSDV ORF123 mAb. This purified LSDV ORF123 mAb was determined using the BLI technique following the procedures described in the Materials and Methods. Standard antibody samples were serially diluted (3 ×) to obtain a standard curve. Two independent experiments were conducted to determine the concentration of the LSDV ORF123 mAb. D Determination of the reactivity of the purified hybridoma ascites with the LSDV ORF123 mAb (1G1–1G12). The LSDV sample was collected and analysed following the procedures described in the Materials and methods section.** Additional file 2. The phylogenetic analysis is based on the complete sequence of the LSDV ORF123 gene and analogue genes of other reference strains.** A phylogenetic tree was drawn to scale with the highest log-likelihood, with branch lengths measured as the number of substitutions per site. No discrete gamma distribution was used to model the evolutionary rate differences among the sites (uniform rates). The first, second, third, and noncoding positions are included. Branch lengths are shown below the branches. The solid black squares indicate the LSDV strains analysed in this study.** Additional file 3. Determination of the cross-reactivity of the LSDV ORF 123 mAb and LSDV polyclonal serum against SPPV, GTPV, LSDV, and VACV. A** Western blot analysis of the cross-reactivity of the anti-ORF123 mAb against LSDV, OrfV, GTPV, and SPPV.** B** A flow diagram was used to prepare the crude cell lysate and supernatant samples.** C** Western blot analysis of the cell lysate and collected infection supernatants of LSDV, GTPV, and SPPV. D Immunofluorescence analysis of cross-reactivity with the anti-ORF123 mAb against LSDV, OrfV, GTPV, and SPPV. MDBK cells were infected with LSDV, OrfV, GTPV, or SPPV for 48 h. The cells were fixed, permeabilized, and stained with the indicated anti-ORF123 mAb. Fluorescence images were visualized and captured using a confocal fluorescence microscope (ZEISS LSM880, Germany). LSDV ORF123 mAb (green); nuclei (blue). E Native PAGE analysis of the cross-reactivity between GTPV, LSDV, SPPV, VACV, and Orfv and the LSDV ORF123 and ORF29 mAbs.Additional file 4. Primers were used to construct ORF123 and analogues of LSDV, SPPV, and GTPV.Additional file 5. Peptide mapping of the conformational epitope against the LSDV ORF123 mAb. A, C and E Bioinformatics analysis of the first, second, and third rounds of truncation for peptide mapping against the LSDV ORF123 mAb. The full-length LSDV ORF123 was truncated into two segments. Amino acids 1–98 of LSDV ORF123 were truncated into two segments. The B-cell epitope was predicted via the online IEDB analysis resource website. G Schematic diagram of the fourth round of truncation for peptide mapping of the N-terminal and C-terminal regions of the LSDV ORF123 epitope. B, D, F and H Expression of truncated LSDV ORF123 plasmids. The Vero cells were transfected with pCAGGS-F1(1-196aa)-Flag, pCAGGS-F2(1-98aa)-Flag, pCAGGS-F3(99-196aa)-Flag, pCAGGS-F4(1-50aa)-Flag, pCAGGS-F5(51-98aa)-Flag, pCAGGS-F6(51-54aa)-Flag, pCAGGS-F7(66-75aa)-Flag,* pCAGGS-F8(76-87aa)-Flag, pCAGGS-F9(88-96aa)-Flag, pCAGGS-F8 + 9(76-96aa)-Flag, pCAGGS-F10(76-93aa)-Flag, pCAGGS-F11(76-90aa)-Flag, pCAGGS-F8(76-87aa)-Flag, pCAGGS-12(79-96aa)-Flag, pCAGGS-F13(82-96aa)-Flag, pCAGGS-14(85-96aa)-Flag, pCAGGS-F9(88-96aa)-Flag, pCAGGS-F15(86-96aa)-Flag, pCAGGS-F16(76-89aa)-Flag, pCAGGS-ORF123(Δ85-90aa)-Flag, and p*CAGGS-ORF123(85-90aa)-Flag. A positive result against this LSDV ORF123 mAb is marked with a solid red line. LSDV ORF123 (green), FLAG (green), nuclei (blue).**Additional file 6. Primers were used to construct these truncated plasmids of LSDV ORF123.****Additional file 7.Primers were used to construct these truncated and mutant plasmids of ORF123 and analogues of LSDV and GTPV.**Additional file 8. The primers used to construct these mutant plasmids were LSDV ORF123, GTPV A33R/122, and SPPV 117/ORF123.Additional file 9. Spatial structure simulation and prediction of the critical amino acids of the LSDV ORF123 and GTPV A33R/122 proteins. A and B Amino acid was submitted to SWISS-MODEL online software and saved in PDB format. The structural simulation and visualization were subsequently performed with open-source PyMOL 2.5 software. The epitope residues of LSDV ORF123 and GTPV A33R/122 are shown in green. The key epitope residues 85P and 86Y of LSDV ORF123 are shown in red, whereas amino acid 87F is shown in blue. The key epitope residues 86P of GTPV A33R/122/ORF123 are shown in red, whereas amino acids 87Y, 88F, and 88 L are shown in blue. C Reactivity analysis of the LSDV ORF123 and GTPV A33R/122/ORF123LSDV ORF123 mutation plasmids with the LSDV ORF123 mAb.Additional file 10. This LSDV ORF123 mAb retains neutralizing activity against LSDV infection. A Determination of the neutralizing activity of the LSDV ORF123 mAb against EGFP-LSDV infection. A representative image was taken using an EVOS M5000 imaging system. B Western blot analysis of the neutralizing activity of the LSDV ORF123 mAb against LSDV infection. C Determination of extracellular LSDV titres against diluted ORF123 mAbs. Statistical significance was evaluated by determining p values using the two-tailed Student’s t tests. ns, p > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001.Additional file 11. This LSDV ORF29 mAb retains cross-neutralizing activity to GTPV and SPPV. A Western blot analysis of the neutralizing activity of the LSDV ORF123 mAb against GTPV infection. Western blot samples were collected and analysed following the procedures described in the Materials and methods section. B Determination of extracellular GTPV titres against the diluted LSDV ORF123 mAb. The cell culture supernatants were collected, and the extracellular GTPV titres were determined using Vero cells as the TCID_50_ on the basis of the Reed–Muench method. Statistical significance was evaluated by determining the p values using a two-tailed Student’s t test. ns, p > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001. C and D Determination of the neutralizing activity of the LSDV ORF123 mAb against GTPV infection. A representative image was taken using an EVOS M5000 imaging system. E Western blot analysis of the neutralizing activity of the LSDV ORF123 mAb against SPPV infection. Western blot samples were collected and analysed following the procedures described in the Materials and methods section. F The band intensity ratio of ORF29 to β-actin. The data are presented as the means from three independent statistical experiments. The intensities of the protein bands were quantified via ImageJ. Significance was analysed via two-tailed Student’s t test. ns, p > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001. G Determination of extracellular SPPV titres against the diluted LSDV ORF123 mAb. Extracellular SPPV titres were determined via the Reed-Muench method with Vero cells as the TCID_50_. Statistical significance was evaluated by determining p values via two-tailed Student’s t tests. ns, p > 0.05; *p value < 0.05; **p value < 0.01; ***p value < 0.001.