Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks

Abstract

Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to ²²¹LD/NLPW²²⁵ and ⁸⁷YAEYI⁹¹ by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas ²²¹LD/NLPW²²⁵ was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.

Keywords: E protein 3D structure; E protein epitopes; diagnosis; duck Tembusu virus; type specific and cross-reactive epitopes.

Figure 1

Detection of selected phages for monoclonal antibody (mAb) binding in the phage enzyme-linked immunosorbent assay (ELISA). The selected phage clones were detected by 1F3 and 1A5 or the anti-porcine interferon (IFN)-c mAb (negative control) after three rounds of biopanning. OD, optical density.

Figure 2

Identification of the E protein epitopes based on mAbs IF3 (A) and 1A5 (B) reactivity with the synthesized peptides in the dot blotting assay. YIRTPACWD and the E protein were used as the negative and positive control, respectively.

Figure 3

The reactivity of the synthesized mutations of the LD/NLPW (A) and YAEYI (B) peptides to duck anti-duck Tembusu virus (DTMUV) serum by Western blot. YIRTPACWD and the E protein were used as the negative (N) and positive (P) controls, respectively. M: Protein marker; N: GST negative control; (A) lane 1, GST-DLNLPWT; lane 2, GST-DLDLPWT; lane 3, GST-DLNLPW; lane 4, GST-LNLPW; lane 5, GST-LNLP; lane 6, NLPW; (B) lane 1, GST-YAEYI; lane 2, GST-AEYI; lane 3, GST-YAEY.

Figure 4

Sequence alignment of the epitope-coding regions LNLPW (A) and YAEYI (B) in the DTMUV strain E proteins. The amino acid positions for each sequence are numbered on both sides. The DTMUV TA strain sequence is shown at the top; the dashes indicate identical amino acids. The identified epitope region is boxed in grey.

Figure 5

Sequence alignment of the epitope-coding regions LNLPW (A) and YAEYI (B) in the flavivirus strain E proteins. The amino acid positions for each sequence are numbered on both sides. The DTMUV TA strain sequence is shown at the top; the dashes indicate identical amino acids. The identified epitope region is boxed in grey.

Figure 6

The cross-reactivity of the epitopes to the Japanese encephalitis virus (JEV)-, West Nile virus (WNV)-, and dengue virus (DENV)-positive sera in the dot blotting assay. YIRTPACWD and the E protein were used as the negative and positive controls, respectively.

Figure 7

The locations of epitopes on the DTMUV E protein dimer. The DTMUV E protein structure is modeled based on the JEV E crystal structure protein using MODELLER [34]. Domains I, II and III in are colored in magenta, yellow and blue, respectively, in one monomer. The other monomer is colored grey. The locations of the two epitopes are depicted as spheres and labeled. The locations of two epitopes are depicted as spheres and labeled. Two predicted N-glycosylation sites by GlycoEP [32] and NGlycPred [33] are colored as cyan.

Figure 8

Competitive inhibition of synthetic peptide YAEYI binding to mAb 1F3. A competitive ELISA was performed using the antigen peptide YAEYI as the competitor for the E protein. Values represent three independent experiments with triplicate determinations included in each experiment (p < 0.05, Student’s t-test).

Figure 9

ELISA reactivity of the DTMUV synthetic peptide against serum samples from 25 DTMUV-infected duck (A) versus sera from 25 healthy ducks (B). The cutoff value (dashed lines) was calculated as 0.375. Solid line, mean OD405.