Identification of a novel linear B-cell epitope in the UL26 and UL26.5 proteins of Duck Enteritis Virus

Abstract

BackgroundThe Unique Long 26 (UL26) and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV), which is an unassigned member of the family Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c), which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb). The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV.ResultsA mAb (designated 1C8) was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, 520IYYPGE525, which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The 520IYYPGE525 motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif 521YYPGE525 in the epitope sequence was conserved among the alphaherpesviruses.ConclusionA mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was 520IYYPGE525. The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV.

Adaptation and Attenuation of Duck Tembusu Virus Strain Du/CH/LSD/110128 following Serial Passage in Chicken Embryos

“…In the fully attenuated P90 virus, one mutation occurred in the 5= UTR and two mutations and a 72-nt deletion occurred in the 3= UTR. Previous studies have shown that the 5= and 3= UTRs form secondary structures that influence the transcription and translation of the flavivirus genome (19,27,28). We were unable to determine whether these changes in the UTRs of the Du/CH/LSD/ 110128 genome contributed to the attenuation or adaptation of the DTMUV in the chicken embryos.…”

“…The protein band was excised from the gel. The gel slices were crushed and suspended in an appropriate volume of sterilized PBS for Western blotting, as described previously (19).…”

Evaluation of a Recombinant Multiepitope Peptide for Serodiagnosis of Toxoplasma gondii Infection

“…The identification of human immunodominant B-cell epitopes within the T. gondii antigens can help to find those antigens involved in the specific B-cell response, and they will be useful for immunoassays to detect anti-Toxoplasma antibodies (7,18,21). Using the B-cell epitopes of those antigens for the serodiagnosis of toxoplasmosis present several advantages, such as the composition of diagnostic antigen being precisely known, being able to use more than one identified B-cell epitope, and easy standardization of the method.…”

Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4

“…Attempts to develop a peptide-based vaccine for T. gondii have been encouraging because they have demonstrated significant protection in murine models [ 22 - 25 ]. Using B cell epitopes for the serodiagnosis of toxoplasmosis presents several advantages, such as precise knowledge of the composition of the diagnostic antigen, the ability to use more than one identified B cell epitope, and easy standardization of the method [ 26 ]. The newly synthesized multiepitope antigen is one of the most promising antigens for the development of diagnostic kits for routine toxoplasmosis screening [ 27 ].…”