Sequences were obtained from the National Center for Biotechnology Information database (http://ncbi.nlm.nih.gov) and the Global Initiative on Sharing Avian Influenza Data (GISAID) database (http://platform.gisaid.org/epi3/frontend). Since AIV-H7 has frequent variation, 24 strains of Euro-Asiatic lineage H7 subtype AIV that had triggered serious poultry pandemics or had infected humans in the past 30 years were selected (13). Of the 24 strains, two represented the latest H7N9 virus, and were selected from 94 of the latest complete records of H7N9 in the GISAID database.

ClustalW (www.ebi.ac.uk/Tools/msa/clustalw2/) was used to compare the HA amino acid sequence of 24 strains and to identify their conserved region. The accession numbers of the HA proteins of the 24 strains are listed in Fig. 1. The HA gene with 560-amino acids from H7N9 (A/tree sparrow/Shanghai/01/2013 (H7N9), gi|546235348| or EPI439486) was selected as the reference sequence as it was the latest HA gene.

Various properties of the 560-amino acid HA protein from H7N9, including the theoretical isoelectric point (pI), amino acid composition and molecular weight, were tested using the ProtParam tool from the ExPASy Bioinformatics Resource Portal (http://web.expasy.org/protparam/). Subsequently, a structural search was performed using the Protein Data Bank database (http://rcsb.org). PDB:4N5J was identified as the three dimensional (3D) structure of AIV-H7.

Linear B-cell epitopes were predicted using the algorithms of ABCped, BepiPred and LBtope. The sequence of HA protein were downloaded and analyzed by each software. ABCpred has developed a systematic method based on a neural network (12). The amino acid length was set at 10, 12, 14, 16, 18 and 20 mer and the scoring threshold at 0.8. BepiPred, which was developed by Larsen et al (14), employs the hidden Markov model and apropensity scale method developed by Parker et al (15). A threshold of 0.350 was selected as it is the point at which sensitivity (0.49)/specificity (0.75) is maximized (14). LBtope is a new method for predicting linear epitopes and was created by Singh et al (16) in 2013. The Lbtope_Confirm dataset was selected since it has previously shown the best performance. The amino acid length was set to 15 mer and the scoring threshold to 60%. The results from the three softwares were assembled and the overlapping regions were considered predicted epitopes.

Using a combination of software prediction and sequence comparisons, the conserved epitopes were predicted and a domain enhanced lookup time accelerated-Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to determine the specificity of the epitopes. Furthermore, the location and structures of the epitopes were denoted in 3D images.

The 235th amino acid of HA from 24 AIV-H7 strains, as well as 100 strains of human H7N9 viruses from the GISAID database, were compared. The 235th amino acid of HA from 24 AIV-H7 strains, as well as 100 strains of human H7N9 viruses from the GISAID database, were distinguished based on whether mutations were present.

Primary sequence analyses revealed that the theoretical pI of HA was pH 6.25 and the molecular weight was 62.062 kDa. The location and variation of HA from 24 AIV-H7 strains are presented in Fig. 1. Following the comparison, the highly conserved sequences included amino acids 18–41, 72–98, 149–164, 253–269, 294–306, 339–356, 358–402, 415–426, 428–454, 463–499 and 507–535. The majority of mutations were located in the N-terminal 339 amino acids, which comprised the HA1 subunit of HA. The HA1 subunit forms the head of HA, which has a vital role in interacting with the environment and host receptor. Thus, the HA1 subunit is readily mutated in order to avoid the attack of antibodies (17).

BepiPred software was used to produce a Linear Epitope Prediction map (Fig. 2) and BepiPred Prediction Details (Table I). BepiPred analyzes each amino acid independently and assigns it a score between −3 and 3. A higher score indicates a higher probability for the existing epitope (14). The threshold was set at 0.35 and scores >0.35 were regarded as positive. From the Fig. 2, beyond the axis, the higher of the peak, the higher scores of prediction. Therefore, the 19 peaks of the map formed by consecutive amino acids were regarded as 19 predicted linear epitopes. They were ranked by mean residue score (mean residue score was averaged by every residue in the predicted peptide), as presented in Table I. The peaks 1–4 were the most likely epitopes to be predicted by BepiPred.

The output of the LBtope analysis is presented in Fig. 3. LBtope assigns scores between 0 and 100% to each epitope it predicts (16). A higher score indicates a higher probability of the epitope existing (16). According to the mean residue score (Table II), the software selected 14 consecutive amino acids that have a possibility of 61 to 100% of being an epitope. As the threshold was set at 60%, these 14 consecutive amino acids were regarded as 14 predicted epitopes. They were ranked by mean residue score, as shown in Table II. The sequences 1–2 were the most likely linear epitopes as their scores were >80%.

ABCpred is able to predict antigens that vary in length from 10 to 20 residues and assigns a score between 0 and 1 to each epitope it predicts. A score that is closer to 1 indicates a higher probability of the epitope existing and a score closer to 0 suggests that the amino acid sequence is unlikely to be an epitope (12). In order to avoid omissions, the amino acid length was set to 10, 12, 14, 16, 18 and 20 mer and the scoring threshold to 0.8. In total, 67 sequences met the requirements.

The overlapping epitopes predicted by the three softwares were considered as potential epitopes. Following a comparison and analysis, 11 epitopes met the requirements. Each of the potential epitopes were above the thresholds set for the 3 software packages, had a suitable length and were highly antigenic. Therefore, they adequately represented the linear epitopes of HA. The 11 epitopes are ranked by ABCpred scores in Table III.

The locations of the 11 potential epitopes were determined. The entire HA protein can be divided into three parts: Amino acids 1–18 form the signal peptide, amino acids 19–339 form the HA1 subunit and amino acids 340–560 form the HA2 subunit (17). Eight of the 11 epitopes were in the HA1 subunit, two were in the HA2 subunit and one was at the junction of the two subunits. These results suggest that the HA1 subunit is the immunodominant antigen and the HA2 subunit is more conserved.

Five epitopes showing minimal variation were selected from the 11 epitopes by observation and comparison (Fig. 1). Two of the five epitopes were from the HA2 subunit and three were from the HA1 subunit. These five epitopes showed high antigenicity and were highly conserved; thus they were called the conserved predicted epitopes. Three of the five epitopes were 20 amino acids in length, one was 16 amino acids and the other was 12 amino acids. The exact position and composition of amino acids are shown in Table IV.

A Venn diagram was used to show the analysis process and overall results (Fig. 4). The 11 epitopes that were detected by all three algorithms were in the overlapping part of the three circles. The middle overlapping part was divided into two parts by a short line. The five dots laying above the line represented the five conserved predicted epitopes among the 11 potential epitopes.

The secondary structures of the five conserved predicted epitopes were analyzed and are presented in Fig. 5. Epitope 6 contains two β-strands and one turn, epitope 8 contains three β-strands and one β-bridge, epitope 9 consists of one turn and two parts of an α-helix, epitope 10 contains one β-strand and one β-bridge and epitope 11 constitutes one β-strand, one complete β-bridge, one partial β-bridge and one turn. All of the five epitopes are on the surface of HA protein, which exposes them to the environment and makes them more likely to be antigenic (18). Previous studies have demonstrated that β-bridges and turns are more likely to form epitopes (18,19) and, in the present study, at least one β-bridge or turn was identified in every conserved predicted epitope. The exact locations of the epitopes are displayed in Fig. 5.

Epitopes 8, 9 and 11 had no significant similarity with other organisms, as demonstrated by a BLAST analysis. Epitope 6 showed 70% similarity with H5N1 and H2N2. Epitope 9 showed 74% similarity with H3N1 and H3N2, 68% with H5N1, H5N2 and H1N1, and 63% with H12N4. Since changing one amino acid in an epitope can markedly decrease the antigen-antibody interaction involved in antibody recognition, it was concluded that all five epitopes were specific to AIV-H7 (9).

The present study demonstrated that the 235th amino acid of HA (Gln) was a highly conserved residue for the majority of H7-AIV, including those associated with previous human outbreaks (Fig. 1). However, in the 2013 H7N9 AIV, the 235th residue had mutated to Leu; in the 100 strains of human H7N9 viruses collected from across China, only 16 carried Gln²³⁵, with the remaining (84%) all carrying Leu²³⁵. These results suggest that the Gln²³⁵-to-Leu²³⁵ mutation exists in the novel H7N9 viruses.