Cells and viruses
The chicken embryonic fibroblast cell line DF-1, BHK-21 and HEK293T were purchased from the ATCC (USA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin–streptomycin (100x, Macgene, China) in a humidified 5% CO2 atmosphere at 37 °C.
The F48E9 and LaSota strains of NDV were obtained from the China Institute of Veterinary Drug Control. The class II strains SX10 and JS17 and the class I strain QH-1 were isolated from China by our laboratory. All viruses were propagated in 9- to 11-day-old specific-pathogen-free chicken embryonated eggs. Fresh allantoic fluid was harvested from embryonated eggs dead between 24 and 120 h after inoculation and kept at − 80 °C.
The pepscan analysis was performed by the Novasnow Science & Technology China with anti-LaSota hyperimmune chicken serum as described previously . The HN 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 HA (YPYDVPDYAG,100 spots) and c-myc (EQKLISEEDL, 100 spots) were used as 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 antichicken IgG (H + L) DyLight680 (1:5000) and with control antibody mouse monoclonal anti-HA (12CA5) DyLight800 (1:2000) 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).
Preparation of anti-LaSota chicken hyperimmune sera, anti-LaSota mouse hyperimmune sera and anti-IDE peptide mouse sera
Four-week-old specific pathogen-free (SPF) white leghorn chickens (SAIS Poultry) were inoculated through the intraocular–nasal route with 0.2 ml per chicken of LaSota live vaccine on day one. 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 on day 35 .
Six-week-old SPF female BALB/c mice (purchased from DaShuo, China) were subcutaneously immunized with 0.2 ml per mouse with inactivated LaSota vaccine (Yebio Qingdao, China) at days 1, 14 and 28. The sera were collected at day 35 and kept at − 80 °C.
For preparation of an anti-HN guinea pig polyclonal antibody (pAb), one-month-old guinea pigs were immunized with HN, which was obtained by prokaryotic expression, at 50 μg per animal. The guinea pigs were subcutaneously injected with a mixture of the protein and complete Freund’s adjuvant (Sigma). The animals were subcutaneously injected with a mixture of the protein and incomplete Freund’s adjuvant (Sigma) at 14 and 28 days. After the guinea pigs were anesthetized with ketamine and xylazine, their antisera were collected and stored at − 80 °C .
Due to 15 amino acid peptides are usually too short to provoke a sufficient immune response, the IDE peptides and an HN341-355 peptide of the previously reported HN neutralizing epitope 346–353 , were synthesized and conjugated with KLH by Ontores Biotechnology (Shanghai, China). Each peptide was dissolved to 1 mg/ml with phosphate-buffered saline (PBS). Four six-week-old SPF female BALB/c mice with peptide emulsified by complete Freund’s adjuvant (Sigma) were subcutaneously immunized with 50 μg per mouse. The mice were boosted twice with the peptide emulsified by incomplete Freund’s adjuvant (Sigma) at day 14 and 28. Finally, blood samples were collected at day 38. The antisera of five IDEs and HN341-355 were prepared.
Construction of the IDE-RFP plasmids
Five IDEs and HN341-355 fusion with red fluorescent protein (RFP) were constructed by overlapping PCR. 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. The primers are listed in Additional file 1: Table S1. After overlapping PCR, the IDE-RFP and HN341-355-RFP fragments were digested by the restriction enzymes XhoI and BglII and ligated into the pCAGGS vector. The products were confirmed by DNA sequencing.
Western blot analysis
HEK293T cells were cultured at approximately 70–80% confluence in 12-well plates and transfected with 2 μg each of expression plasmids (IDEs-RFP and HN341-355-RFP) by using Tuborfect Transfection Reagent (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s guidelines. The cells were harvested at 48 h post transfection. The cell samples were washed with PBS and lysed with lysis buffer (2% SDS, 10% glycerin, 5% β-mercaptoethanol and 0.1% bromophenol blue). The lysates were collected in 1.5 ml tubes on ice for 30 min and centrifuged at 12,000 rpm at 4 °C for 15 min for clarification. After incubation at 100 °C for 10 min, an equal amount of these prepared samples was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a nitrocellulose blotting membrane (Millipore, USA) and blocked in 10% skim milk in PBS at 4 °C overnight. After the membrane was washed with PBST (PBS containing 0.5% Tween-20), it was incubated with primary antibodies for 2 h at 37 °C. After washing, the protein bands were detected with an HRP-conjugated IgG antibody (Abcam). Finally, after three more washes, the antibody-antigen complex was exposed with a chemiluminescence (ECL) reagent solution kit (Share-bio Biotechnology, China) by using a Tanon 5200 multichemiluminescence image analysis system (China).
Each synthesized peptide (1 µg) was dropped onto the nitrocellulose membrane and incubated at 37 °C for 30 min. The membrane was blocked with 5% skim milk at 37 °C for 2 h. After the membrane was washed three times with TBS, it was incubated with the 1:2000 anti-IDE mouse antibody diluted with TBS at 37 °C for 1 h. After the membrane was washed with TBST, it was incubated with the corresponding HRP-conjugated goat anti-mouse secondary antibody. The ECL peroxidase substrate (Millipore) was used for detection.
Indirect immunofluorescence assay (IFA)
BHK-21 cells were cultured at approximately 60–70% confluence in 48-well plates and transfected with 1 μg each of the expression plasmids by using Tuborfect Transfection Reagent (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s guidelines. DF-1 cells were cultured at 60% confluence in 48-cell plates and infected with LaSota at an MOI of 1. The cells were washed with PBS and fixed with 4% methanol for 10 min at room temperature. After the cells were washed three times with PBS, they were blocked with 1% BSA in PBS at 37 °C for 2 h, and then, the cells were inoculated with a primary antibody at 4 °C overnight. After the cells were washed thrice with PBST, they were incubated with secondary antibody conjugated FITC (Abcam) in a moist container in the dark at 37 °C. After the cells were washed thrice with PBST, they were stained with DAPI (0.1 μg/ml) for 8 min at room temperature. Finally, fluorescent images were visualized and captured on a confocal fluorescence microscope (Olympus, Japan).
Enzyme-linked immunosorbent assay
One hundred nanograms of synthetic peptides was coated on ELISA plates in 100 μl of 0.05 M carbonated bicarbonate (pH 9.6) by overnight incubation at 4 °C. After the samples were washed four times with PBST, the plate was blocked with 10% skim milk at 37 °C for 2 h and washed with PBST. A total of 100 μl of antisera of the IDEs diluted 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400 with PBS was added to the indicated wells and incubated at 37 °C for 2 h. The anti-LaSota mouse serum was diluted 1:500 with PBS. The plate was washed again. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam) was added to each well and incubated at 37 °C for 45 min. After another wash, 100 μl of TMB substrate was added to each well. The reaction was stopped with 50 μl of 0.5 M H2SO4 after incubation at room temperature for 30 min. The OD450 value of each well was immediately read with a Microplate Absorbance Reader (Bio-Rad, USA).
The different diluted antisera (anti-IDE, anti-HN341-355 mouse sera, HN guinea pig polyclonal antiserum) and normal BALB/C mouse serum as controls were used in BHK-21 cells to perform the neutralization test. Twelve microliters of the antisera treated at 56 °C for 30 min was serially diluted twofold from 1/4 to 1/512. Then, the diluted antisera were mixed with three viruses, LaSota (50 μl, 200 TCID50), JS17 (50 μl, 50 TCID50) and F48E9 (50 μl, 10 TCID50), and incubated at 37 °C for 2 h in a 96-well plate. Subsequently, 100 μl of the mixtures was transferred to BHK-21 cells in a 96-well plate and incubated at 37 °C for 1 h. After three washes with PBS, DMEM containing 2% FBS was added. After incubation for 12 h, the LaSota- and JS17-infected BHK-21 plates were fixed for 20 min with methanol. After the plates were washed again, the HN pAb from guinea pigs and the anti-NDV HN monoclonal antibody were used to identify the number of infected cells by IFA. The number of infected cells was calculated and analyzed by ImageJ. Data are presented as the average of triplicates. The F48E9-infected BHK-21 cells were calculated by the average number of syncytia.
Statistical analyses were performed using GraphPad Prism 5. Data from three independent experiments are presented as the mean ± standard deviation (SD) from triplicate samples (n = 3); P < 0.05 with Student’s t-test was considered to be statistically significant. Statistical significance was set at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).