Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota

Abstract

Matrix (M) protein of Newcastle disease virus (NDV) is an abundant protein that can induce a robust humoral immune response. However, its antigenic epitopes remain unknown. In this study, we used a pepscan approach to map linear B cell immunodominant epitopes (IDEs) of M protein with NDV-specific chicken antisera. The six epitopes with the highest reactivity by peptide scanning were obtained as IDE candidates. Among them, aa71–85 and aa349–363 were identified by immunological assays with NDV-specific or IDE-specific antisera. The minimal antigenic epitopes of the two IDEs were further characterized as ⁷⁷MIDDKP⁸² and ³⁵⁴HTLAKYNPFK³⁶³. Moreover, an amino acid sequence alignment and immunoblot analysis revealed the conservation of the two IDEs in the M protein of strains of different genotypes. These two IDEs of M protein could be genetically eliminated as negative markers in recombinant NDV for serologically differential diagnosis in the development of marker vaccines.

Introduction

Newcastle disease is a highly contagious disease caused by the virulent strain of Newcastle disease virus (NDV) in avian species; it causes economic losses in the poultry industry (24). NDV, also named avian paramyxovirus serotype 1 (APMV-1), belongs to the genus Avulavirus, family Paramyxoviridae, and order Mononegavirales (37). It has a nonsegmented, single-stranded, negative-sense RNA genome that encodes six major structural proteins (from 3′ to 5′): nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L) (7,16).

M protein has 364 amino acids (aa) with a molecular weight of ∼40 kDa (3,10). It is an abundant protein that interacts with the host cell membrane, viral RNP, and glycoprotein in the inner cytomembrane to promote virus particle assembly and budding (27,38). It is also virulence related and inhibits host cell RNA transcription and protein translation (1,26). In addition, M protein completes import and export from the nucleus through one nuclear localization signal (NLS) and three nuclear explore signals (NES) to assist with the reproduction of virus particles (11,13,14).

M protein elicits considerable serum antibody titers after the immunization of chickens with NDV (17), and anti-NDV chicken serum can recognize M protein in virus-like particles (29). These reports indicate that viral M protein is able to induce strong humoral immunity during NDV infection in animals, even though the M protein-specific antibody has no neutralization activity against NDV (30). However, the antigenic determinants of M protein remain elusive.

Epitope characterization is crucial for the understanding of antibody functions in recognition and binding (5,25). Detailed analyses of epitopes are essential for understanding immunological events and for the development of diagnostic tools for various diseases and epitope-oriented vaccines (21,33). Furthermore, the M epitopes would be useful as potential markers that can be modified in recombinant virus for the differential immunodiagnosis in differentiating infected from vaccinated animals (DIVA) vaccine.

The pepscan technique, also known as overlapping peptide scan or array-based oligopeptide scanning (12), has facilitated the identification of a linear epitope in the fusion (F) protein of bovine respiratory syncytial virus (BRSV) characterized by neutralization activity, which made it feasible to develop an epitope-based vaccine (19). A pepscan analysis has also been used to identify linear B cell epitopes on the NP protein of NDV to develop a marker vaccine candidate (23). This epitope mapping technology is fast and low cost for practical applications (28,34). Briefly, it involves the use of an array of unique, overlapping synthetic peptides based on the primary amino acid sequence of an antigen bound to individual pins to detect serologically accurate epitopes.

In this study, two accurate linear B cell epitopes, ⁷⁷MIDDKP⁸² and ³⁵⁴HTLAKYNPFK³⁶³, of NDV M protein were identified by a pepscan analysis and site-directed mutagenesis. Our results clarify antigenic properties of the M protein and could genetically eliminate these immunodominant epitopes (IDEs) in recombinant virus that serve as negative markers for the development of DIVA or marker vaccines for NDV.

Materials and Methods

Cells and virus

HEp-2, DF-1, and BHK21 lines were obtained from the ATCC (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/high glucose (Hyclone, Thermo Scientific, Logan, UT) in a humidified 5% CO₂ atmosphere at 37°C. All culture media were supplemented with 10% inactivated fetal bovine serum (GIBCO, Invitrogen, Gaithersburg, MD) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). The F48E9 strain of NDV was obtained from the China Institute of Veterinary Drug Control. The inactivated and attenuated LaSota vaccines were purchased from Yebio (Qingdao, China). The strains SX10, JS17, and Pigeon/QH were isolated from China in our laboratory.

Preparation of anti-LaSota chicken hyperimmune serum and anti-IDE peptide mouse antisera

Four 4-week-old specific pathogen-free (SPF) white leghorn chickens (SAIS Poultry) were raised in animal isolators. They were inoculated through the intraocular–nasal route with 0.2 mL per chicken of LaSota live vaccine on day 1. Then, the chickens were immunized with 0.2 mL per chicken of inactivated vaccine through subcutaneous injection on days 14 and 28. Blood samples were collected at day 35, and the antisera were prepared.

Three 6-week-old SPF BALB/c mice were immunized by subcutaneous injection with 100 μg per mouse of each synthetic IDE peptide emulsified by complete Freund's adjuvant (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS) at day 1. Then, the mice were boosted with each peptide emulsified by incomplete Freund's adjuvant (Sigma) at days 14 and 28. Finally, the blood samples were collected at day 38. The antisera of selected IDEs (IDE1, IDE2, IDE5, and IDE6) were prepared. All experimental procedures were approved by the Animal Care and Use Committee of the Northwest A&F University, China (Approval No. 2015ZX0512008-012 to chicken, No. 2016XN0928010-068 to mouse).

Pepscan analysis

The pepscan analysis was performed by the Novasnow Science & Technology China with anti-LaSota hyperimmune chicken serum. The matrix protein sequences were linked and elongated with neutral GSGSGSG linkers at the C- and N-termini to avoid truncated peptides. The elongated antigen sequences were translated into 15 amino acid peptides with a peptide–peptide overlap of 14 amino acids. The resulting PEPperCHIP^® Peptide Microarrays contained a series of different peptides printed in duplicate and framed by additional HA (YPYDVPDYAG, 100 spots) and c-myc (EQKLISEEDL, 100 spots) control peptides.

After 15 min of preswelling in washing buffer and 30 min of incubation in blocking buffer, peptide microarrays were initially incubated with the secondary antibody goat anti-chicken IgG (H+L) DyLight680 (1:5,000) and with control antibody mouse monoclonal anti-HA (12CA5) DyLight800 (1:2,000) for 45 min at 37°C to analyze background interactions with the antigen-derived peptides. Then, the peptide microarray analysis was performed with anti-LaSota chicken hyperimmune serum diluted at 1:500 in incubation buffer for 16 h at 4°C and shaking at 140 rpm. The LI-COR Odyssey Imaging System was used to scan the preprocessed peptide microarrays at scanning intensities of 7/7 (red = 700 nm/green = 800 nm).

Construction of the truncated M protein, IDE-RFP, and IDE mutants

To confirm whether NDV-specific antisera can recognize viral M protein, we constructed the M protein and its truncations. M protein was divided into two parts, aa1–177 (M1) and aa178–365 (M2), based on B cell epitope prediction using the Immune Epitope Database and DNAstar-protean (Fig. 1A).Viral RNA was extracted from the purified NDV using TRIzol according to the manufacturer's protocol (TaKaRa, Kusatsu, Japan). The extracted RNA was reverse transcribed with random primers to generate complementary DNA (cDNA) of LaSota. The fragments of the M, M1, and M2 genes were amplified by polymerase chain reaction (PCR) (GenStar, Beijing, China) with the primers 1 and 2, primers 1 and 3, and primers 4 and 2, respectively (Supplementary Table S1). The reverse transcription-polymerase chain reaction (RT-PCR) products were cloned into the pMD19-T vector (TaKaRa) and sequenced (GENEWIZ, South Plainfield, NJ). The gene fragments in the pMD-19T vectors were digested with the restriction enzymes EcoRI and BglII (Thermo, Waltham, MA). Then, they were cloned into the eukaryotic expression vector pCAGGS with T4 ligase (TaKaRa) to obtain the constructs pCAGGS-M, pCAGGS-M1, and pCAGGS-M2.

FIG. 1.

Pepscan analysis of M protein in the LaSota strain. (A) Schematic diagram of M protein. Functional late-domain (L-domain) and NLS and three NES are shown. The M protein was divided into two parts, M1 and M2, for construction and expression. The aa number is indicated. (B) Recognition of M protein by NDV-specific chicken antisera. The 293T cells were transfected with pCAGGS-M, pCAGGS-M1, and pCAGGS-M2 constructs. After 36 h, the cell lysates were evaluated by western blotting with NDV-specific chicken antisera produced by LaSota vaccination. (C) IFA of M protein. DF-1 cells were transfected with M, M1, and M2 expression constructs, or infected with LaSota at a multiplicity of infection of 1 for 1 h. After incubation for 48 h, the cells were fixed and stained with NDV-specific chicken antisera. (D) Intensity plot of M protein by a pepscan analysis with NDV-specific chicken antisera. The x-axis indicates the partial peptide sequences of M protein from the N-terminus (left) to C-terminus (right). The six selected IDEs with highly reactive peaks are marked. aa, Amino acid; IDEs, immunodominant epitopes; IFA, immunofluorescence assay; M, matrix protein; NDV, Newcastle disease virus; NES, nuclear explore signals.

Six IDE fusions with red fluorescent protein (RFP) were constructed by overlapping PCR with primers for IDE-RFP (Supplementary Table S2). Upstream primers were combined with IDE sequences and the 3′ terminus of the RFP gene. The downstream primer was located at the 5′ terminus of the RFP gene. Each PCR product was cloned into pMD-19T for sequencing. Then, the IDE-RFP fragments were digested by restriction enzymes, XhoI and BglII, and ligated into pCAGGS vectors.

M_IDE2 and M_IDE6 mutants were generated by overlapping PCR (Supplementary Tables S3 and S4). Amino acids at the mutated sites were replaced with alanine. The fragments of M_IDE2 and M_IDE6 mutants fused with RFP were cloned into pCAGGS with the restriction enzymes XholI and BglII.

Immunofluorescence assay

Cells in 12-well plates were fixed with 4% paraformaldehyde for 10 min at 4°C. Then, they were washed three times with PBS and incubated for 1 h at 37°C with primary antibodies. After washing three times with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-chick antibody (Abcam, Cambridge, United Kingdom) for 1 h at 37°C and then further washed with PBS. The cells were observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Western blotting

The 293T cells were transfected by TurboFect (Thermo Scientific) with expression plasmids. After 48 h posttransfection, the cell lysates were prepared with 2 × sodium dodecyl sulfate (SDS)-loading buffer and subjected to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) after denaturation at 100°C for about 10 min. After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA), and blocked in 5% skim milk in PBS at 4°C overnight. After washing with PBST (0.5% Tween-20 in PBS), the membrane was incubated with primary antibodies for 2 h at 37°C. After washing, the protein bands were detected with an horseradish peroxidase (HRP)-conjugated IgG antibody (Abcam).

Dot blot

The IDE peptides (15 amino acids) were synthesized by Ontores Biotechnology (Shanghai, China). Each peptide (2 μg) was dropped onto the nitrocellulose membrane and incubated at 37°C for 15 min. The membrane was blocked with 5% skim milk at 37°C for 2 h. After washing three times with TBS, the membrane was incubated with the LaSota antiserum or anti-IDE antibody at 37°C for 1 h. After washing with TBST, the membrane was incubated with the corresponding HRP-conjugated secondary antibody. The ECL peroxidase substrate (Millipore) was used for detection.

Enzyme-linked immunosorbent assay

A 96-well plate was coated with synthesized epitopes in 0.1 M carbonate buffer (pH 9.6) and incubated at 4°C overnight. After washing three times with PBST, plates were blocked with 5% skim milk for 2 h at 37°C. After washing three times with PBST, a rabbit anti-mouse HRP-conjugated polyclonal serum diluted 1:5,000 with 5% skim milk (Abcam) was added and incubated for 1 h at 37°C. After washing, TMB (Solarbio, Beijing, China) was added to the wells as a substrate for HRP and incubated in the dark for 15 min. Then, 2 M H₂SO₄ was added to stop the reaction and absorbance was read at 450 nm using a Microplate Absorbance Reader (Bio-Rad, Hercules, CA). Data are presented as the average of triplicate assays.

Conservation analysis of IDEs

To determine whether IDE amino acid sequences are conserved in the M protein of NDV strains, the sequences of NDV strains belonging to different genotypes were downloaded from GenBank, and sequence alignments among them were generated using DNAstar MegAlign.

Results

Epitope mapping by peptide scanning

NDV infection or vaccination can induce a humoral immune response in chickens. First, to test whether the M protein of NDV can be recognized by NDV-specific chicken antiserum, the 293T cell lysates transfected with the M constructs were analyzed by western blotting using chicken hyperimmune NDV-specific antiserum prepared by LaSota vaccination. The NDV-specific antisera was able to recognize the expressed M, M1, and M2 proteins (Fig. 1B). This result was confirmed by an immunofluorescence assay (IFA) using transfected BHK21 cells (Fig. 1C). Subsequently, the NDV-specific antiserum was used to scan the linear B cell epitopes of M protein by the pepscan approach. Six epitopes with high reactivity in the intensity plot were selected as IDE candidates, marked M_IDE1 to M_IDE6 from the N-terminus to C-terminus of the M protein (Fig. 1D). Each IDE contained 15 amino acids (Table 1).

Table 1.

Six Potential Epitopes Identified by Pepscan

Epitope	Sequence (15 aa)
M_IDE1	40-QYRIQRLDLWTDSKE-54
M_IDE2	71-EEATVGMIDDKPKRE-85
M_IDE3	171-YKVNFVSLTVVPKKD-185
M_IDE4	181-VPKKDVYKIPAAVLK-195
M_IDE5	213-EVDPRSPLVKSLSKS-227
M_IDE6	349-KLEKGHTLAKYNPFK-363

aa, Amino acid.

Identification of IDEs

To express the IDEs in mammalian cells, the six candidates were each fused to RFP. M_IDE2-RPF and M_IDE6-RFP were clearly detected by IFA with NDV-specific antisera in the transfected BHK21 cells, but the others were not (Fig. 2A). Similarly, M_IDE2-RFP and M_IDE6-RFP were positively detected by western blotting (Fig. 2B). These results indicated that the epitopes M_IDE2 and M_IDE6 could be recognized by NDV-specific antisera.

FIG. 2.

Identification of IDEs. (A) Detection of IDEs by IFA. HEp2 cells were transfected with IDE-RFP constructs. After 48 h, the cells were fixed and incubated with NDV-specific chicken antisera and subsequently incubated with an FITC-conjugated rabbit anti-chicken antibody. The cells were observed by fluorescence microscopy. Scale bar = 50 μm. (B) Analysis of IDEs by western blotting. The 293T cells were transfected with IDEs-RFP plasmids, except for M_IDE4. After 48 h, the cell lysates were subjected to 15% SDS-PAGE. The RFP plasmid was used as a negative control. The assay was performed with NDV-specific chicken antisera as a primary antibody. (C) Analysis of IDE peptides by a dot blot assay. The synthetic IDE peptides were probed with NDV-specific chick antisera on a nitrocellulose membrane. The expressed M protein from transfected 293T cells and the LaSota virus served as positive controls. KLH served as a negative control. (D) Sensitivity of the M_IDE2 and M_IDE6 peptide reactions to the antisera by a dot blot assay. Serial twofold dilutions were obtained. (1) 2 μg; (2) 1 μg; (3) 0.5 μg; (4) 0.25 μg; (5) 0.125 μg; (6) 0.0625 μg; (7) 0.0313 μg; (8) 0.0156 μg. (E) Reactivity of IDE peptides with the antisera, as detected by ELISA. The 96-well plates were coated with 200 ng of each synthetic peptide and incubated with NDV-specific chicken antisera. KLH was used as a negative control. *p < 0.05; **0.05 < p < 0.01; ***p < 0.001. ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; RFP, red fluorescent protein; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

To confirm the IDEs, the peptides of four epitope candidates conjugated to KLH were synthesized. Using a dot-blot analysis, M_IDE2 and M_IDE6 were obviously detected with NDV-specific antisera and M_IDE5 was slightly detected (Fig. 2C). The serial dilution of synthetic M_IDE2 and M_IDE6 showed a reduction in reactivity (Fig. 2D), indicating that both IDEs were highly sensitive to NDV-specific antisera. In addition, enzyme-linked immunosorbent assay further revealed that M_IDE2 and M_IDE6 exhibit high reactivity, even though M_IDE1 and M_IDE5 were also positively detected (Fig. 2E). Taken together, these results suggested that the M_IDE2 at residues 71–85 (M71–85) and M_IDE6 at residues 349–363 (M349–363) are IDEs of the M protein.

Immunogenicity of the IDE peptides

To examine the immunogenicity of the IDEs, anti-IDE mouse antisera were prepared. Using a dot-blot assay, antisera against M_IDE1, M_IDE2, M_IDE5, and M_IDE6 were able to recognize the LaSota virus and M protein. The antisera of M_IDE2 and M_IDE6 strongly reacted with their own peptides M_IDE2 and M_IDE6, respectively. However, the antisera against M_IDE1 and M_IDE5 were unable to recognize their own peptides (Fig. 3A). Antisera to the IDE peptides did not exhibit crossreactivity with each other. Moreover, these data were confirmed by western blotting (Fig. 3B). These results indicated that M_IDE2 and M_IDE6 could specifically elicit a humoral response.

FIG. 3.

Immunogenicity of the IDE peptides. (A) Dot blot analysis. Anti-IDE mouse antisera were prepared by immunization with synthetic IDE peptides. The synthetic peptides (2 μg) were dropped on a nitrocellulose membrane and incubated with anti-IDE mouse antisera. The M protein-expressing cell lysates and LaSota virus were used as positive controls. KLH was used as a negative control. (B) Western blot analysis. The LaSota virus and M protein cell lysates were subjected to 15% SDS-PAGE and reacted with anti-IDE mouse antisera.

Minimization of M_IDE2 and M_IDE6

To examine whether all 15 amino acids of M_IDE2 ⁷¹EEATVGMIDDKPKRE⁸⁵ and M_IDE6 ³⁴⁹KLEKGHTLAKYNPFK³⁶³ are required (i.e., to identify minimal epitopes), a series of amino acid mutations of M_IDE2 and M_IDE6 fused to RFP were constructed and expressed in transfected 293T cells. Based on detailed data from a pepscan analysis (Supplement 2), the residues 71–74 and 83–85 of M_IDE2 as well as 349–351 of M_IDE6 were not important for the two epitopes. Therefore, M_IDE2 ⁷⁵VGMIDDKP⁸² and M_IDE6 ³⁵²KGHTLAKYNPFK³⁶³ were analyzed by mutagenesis. The M_IDE2 N-terminal mutants AAAIDDKP (V75A, G76A, M77A) and AAAADDKP (V75A, G76A, M77A, I78A) exhibited significantly decreased or a lack of recognition by anti-M_IDE2 mouse antiserum (Fig. 4A, left). The M_IDE2 C-terminal mutants VGMIDDKA (K81A), VGMIDDAA (K81A, P82A), and VGMIDAAA (K81A, P82A, K81A) failed to be recognized by the anti-M_IDE2 mouse serum (Fig. 4A, right). These data indicated that ⁷⁷MIDDK⁸² was a minimal epitope of M_IDE2 responsible for recognition by the antiserum. Similarly, the M_IDE6 N-terminal mutants AAAALAKYNPFK and AAAAAAKYNPFK (Fig. 4B, left) as well as the C-terminal mutants KGHTLAKYNPFA, AAHTLAKYNPFA, and KGHTLAKYNPAA (Fig. 4B, right) exhibited dramatically reduced or failed reactions with anti-M_IDE6 serum, suggesting that ³⁵⁴HTLAKYNPFK³⁶³ was a minimal epitope of M_IDE6.

FIG. 4.

Minimization of M_IDE2 and M_IDE6. (A) Identification of the minimal M_IDE2. Mutations at the N- and C-termini of M_IDE2 ⁷⁵VGMIDDKP⁸² were fused to RFP. The 293T cells were transfected with M_IDE2-RFP and its mutant-RFP constructs. The expressed fusion proteins were analyzed by western blotting with anti-M_IDE2 mouse antisera. (B) Identification of the minimal M_IDE6. The construction and analysis methods for M_IDE6 ³⁵⁴KGHTLAKYNPFK³⁶³ mutants fused to RFP were the same as those in (A). RFP expression was a negative control. β-Tubulin was an internal control for protein expression.

Conservation of IDEs among strains

Among various strains with different genotypes, the linear epitope of M_IDE2 ⁷¹EEATVGMIDDKPKRE⁸⁵ was mostly conserved. In the minimal epitope ⁷⁷MIDDKP⁸², the residues 78I, 80D, and 82P were particularly highly conserved. M_IDE6 was also conserved, and the region ³⁵⁸KYNPF³⁶² had the highest conservation among selected 14 strains (Supplementary Tables S5 and S6). Furthermore, the M protein of the strains LaSota (genotype II), SX10 (genotype VI), JS17 (genotype VII), and F48E9 (genotype IX) of Class II as well as Pigeon/QH (Class I) were positively detected by western blotting with anti-M_IDE2 (Fig. 5A) and anti-M_IDE6 (Fig. 5B) mouse antisera in the infected DF-1 cell lysates, indicating that the M_IDE2 and M_IDE6 peptide antisera were able to recognize the M protein of multiple NDV strains. These results suggested that M_IDE2 and M_IDE6 of NDV were not only conserved with respect to amino acid sequences but also with respect to serological characteristics.

FIG. 5.

Serological recognition of the M protein in multiple NDV strains by anti-M_IDE2 and anti-M_IDE6 mouse antisera. DF-1 cells were infected with NDV strains. The cells were collected at 36 h for a western blot analysis with anti-M_IDE2 (A) and anti-M_IDE6 (B) mouse antisera.

Spatial location of the minimal M_IDE2 and M_IDE6 in M protein

We established the crystal model of LaSota M protein using SWISS-MODEL and performed a three-dimensional structure analysis using PyMOL (2), as shown in Figure 6. M_IDE2 (aa77–82) and M_IDE6 (aa354–363) were located at the random coil of the surface and were not in close proximity (Fig. 6). These characteristics suggest that they could feasibly form linear antigenic epitopes and have little interaction effects (18). These data further suggested that the refined motifs ⁷⁷MIDDKP⁸² and ³⁵⁴HTLAKYNPFK³⁶³ are potential epitopes.

FIG. 6.

Three-dimensional localization of the minimal M_IDE2 and M_IDE6 in the M protein structure. The spatial structure of M protein was visualized using PyMol (Schrodinger, Inc.). ⁷⁷MIDDKP⁸² is M_IDE2 and ³⁵⁴HTLAKYNPFK³⁶³ is M_IDE6.

Discussion

The identification of epitopes, as antigenic determinants, is critical for understanding antibody recognition and binding (20,31). As an abundant viral protein of NDV, M protein elicits high antibody titers after vaccination or infection in poultry (17,29). Although the antibody of M protein has no neutralization activity against NDV (30), it would be useful for the differential immunodiagnosis of various strains. To identify the epitopes of M protein, we used pepscan technology to screen immunodominant linear B cell epitopes. This approach has been widely applied to many viral proteins (4,35,39). It has various advantages over other methods, for example, it has high-throughput systematic scanning ability, is rapid, and is inexpensive (22). Its disadvantages are that it fails to identify previously confirmed epitopes and short linear epitopes are often not recognized with high affinity by antibodies, along with low accuracy for scanned data (15). In our study, M_IDE1, M_IDE3, M_IDE4, and M_IDE5 failed to react highly with NDV-specific chicken serum, despite significant signal peaks in the pepscan analysis (Supplement 2). Only M_IDE2 and M_IDE6 were positively confirmed among six candidates by multiple methods with NDV-specific chicken serum.

The M protein plays important roles in the infection, assembly, and budding of NDV (2,6,26). The NLS and NES signals of M protein are required for nuclear shuttling in cells (13,14). The L-domain is critical for NDV budding (8). The arginine residue at position 42 (42R) of M protein contributes to NDV replication and infection (9). M_IDE2 and M_IDE6 identified in our study were distant from those critical regions or sites of M protein (Fig. 1A). Hence, these two epitopes may have no effect on the main functions of M protein.

The M protein is highly conserved in NDV strains (32,36). M_IDE2 and M_IDE6 were highly conserved in the sequence alignment of 14 NDV strains, especially 78–82 aa and 358–362 aa in M protein. Furthermore, mouse anti-M_IDE2 and anti-M_IDE6 sera have distinct reactivity with different strains. These data further confirm the conservation of M protein 71–85 aa and 349–363 aa with respect to serological characteristics. Interestingly, 79D, 81K, and 356L in the minimal epitopes were not conserved among the NDV strains. These residues likely do not affect M protein antigenicity. Moreover, 77M and 82P of M_IDE2 and 354H, 355T, 362F, and 363K of M_IDE6 were important for IDE2 antigenicity.

Conclusion

In this study, we identified two immunodominant linear B cell epitopes, M_IDE2 ⁷⁷MIDDKP⁸² and M_IDE6 ³⁵⁴HTLAKYNPFK³⁶³, of NDV M protein using a pepscan approach with vaccinated chicken antisera. All peptides of six IDE candidates (Table 1) exhibited effective immunogenicity in animals. These data provide fundamental information about the antigenic features of M protein and these IDEs could be genetically eliminated as negative markers in recombinant virus for serologically differential diagnosis in the development of DIVA or marker vaccines for NDV.

Footnotes

Acknowledgments

This work was supported by the Faculty Support Fund of the Northwest A&F University (grant No. Z111021401 to S.X.) and National Key Research and Development Program (grant No. 2017YFD0500702 to H.C.).

Authors' Contributions

Y.B., and S.X. designed the experiments. Y.B., Z.J., Y.W., and S.L. performed experiments. Y.B., S.M., W.W., Q.W., N.H., X.W., and Z.Y. analyzed the data. Y.B., S.X., and H.C. wrote and edited the article. All authors read and approved the final article.

Author Disclosure Statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the article entitled.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

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Supplementary Material

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Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota

Abstract

Introduction

Materials and Methods

Cells and virus

Preparation of anti-LaSota chicken hyperimmune serum and anti-IDE peptide mouse antisera

Pepscan analysis

Construction of the truncated M protein, IDE-RFP, and IDE mutants

Immunofluorescence assay

Western blotting

Dot blot

Enzyme-linked immunosorbent assay

Conservation analysis of IDEs

Results

Epitope mapping by peptide scanning

Identification of IDEs

Immunogenicity of the IDE peptides

Minimization of MIDE2 and MIDE6

Conservation of IDEs among strains

Spatial location of the minimal MIDE2 and MIDE6 in M protein

Discussion

Conclusion

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

Supplementary Material

References

Supplementary Material

Minimization of M_IDE2 and M_IDE6

Spatial location of the minimal M_IDE2 and M_IDE6 in M protein