Bioinformatics Analysis of the Duck Enteritis Virus UL 54 Gene

In this study, we analyze the Duck Enteritis Virus (DEV) UL54 gene, which has been isolated and identified in our lab (GenBank accession NO EU071033), to help deeply research on DEV. DNA sequence analysis showed that the identified ORF which composed of 1377 bp nucleotides encoded 458 amino acids with a predicted Mr. of 51.75 kDa. Multiple sequence alignment suggested that the UL54 gene was highly conserved in Alphaherpesvirinae and was similar to the other herpesviral UL54 gene. Phylogenetic analysis of the DEV UL54 gene revealed that DEV had a close evolutionary relationship with Gallid, Herpesvirus 2 (GaHV-2), Gallid Herpesvirus 3 (GaHV-3), Meleagrid Herpesvirus1 (MeHV-1) and should belong to a single cluster within the Alphaherpesvirinae subfamily.


INTRODUCTION
Duck Viral Enteritis (DVE), also called duck plague, is an acute, contagious and lethal disease of ducks, geese and other water flows.Since it was reported in Holland ducks in 1923 for the first time, more outbreaks were reported all over the world and produced significant economic losses in both domestic and wild waterfowl due to mortality, elimination and decreased egg production.DVE caused some typical pathologic features, vascular damage, tissue hemorrhage, digestive mucosal eruptions, lesions of lymphoid organs and degenerative changes in parenchymatous organs (Sandhu and Leibovitz, 2008).The pathogens of DEV is Duck Enteritis Virus (DEV), which has been classified as belonging to Alphaherpesvirinae subfamily of Herpesviridae based on the report of the 9 th International Committee on Taxonomy of Viruses (ICTV), but it has not been grouped into any genus (King et al., 2011).
The putative sub-cellular localization showed that the protein was located mainly into nuclear (98%) and the rest was located in mitochondrial matrix space and lysosome (lumen) 1.0%, respectively.
Whether existed in endoplasmic reticulum (membrane) or not is still unknown.The analysis result shows no classic NLS and a Leu-rich located at 339-348 aa in the protein.
A phylogenetic tree (Fig. 6) was constructed by Clustal X and Treeview according to the amino acid 0.1 of DPV UL54 by NetNES (http://www.cbs.dtu.dk).A leu-rich NES was located at 339 Phylogenetic analysis of UL54 in 22 herpesviruses.Phylogenetic tree of the protein in the herpesvirusess was generated dependence Protein kinase phosphorylation sites.Forecasting the secondary structure revealed alpha helix 22.05%, extended strand 5.46% and loop 72.49% in the cellular localization showed that d mainly into nuclear (98%) and the rest was located in mitochondrial matrix space and

Whether existed in endoplasmic reticulum
The analysis result rich NES (Fig. 5) A phylogenetic tree (Fig. 6) was constructed by Clustal X and Treeview according to the amino acid sequences of the UL54 gene in DEV and other 21 herpesviruses.The result is in accordance with the subtype classification of DEV proposed previously and the DEV UL54 gene was closed to the Gallid Herpesvirus 2, Gallid Herpesvirus3, Meleagrid Herpesvirus1, although DEV was branched independently to others.

DISCUSSION
Up to now, the specific characteristics and functions of the UL54 protein in DEV are still unknown.In this study, we analyzed DEV UL54 gene by bioinformatics methods.UL 54 gene was selected DEV GaHV-3 MeHV-1 GaHV-2 HHV-3 CeHV-9 PsHV-1 ed at 339-348 aa in the Phylogenetic analysis of UL54 in 22 herpesviruses.Phylogenetic tree of the protein in the herpesvirusess was generated sequences of the UL54 gene in DEV and other 21 The result is in accordance with the pe classification of DEV proposed previously and the DEV UL54 gene was closed to the Gallid erpesvirus3, Meleagrid although DEV was branched

DISCUSSION
Up to now, the specific characteristics and functions of the UL54 protein in DEV are still unknown.In this study, we analyzed DEV UL54 gene by bioinformatics methods.UL 54 gene was selected from the completed DEV gene libraries and identified by comparing and analyzing the nucleotide and amino acids sequences.The gene was analyzed by NCBI ORF Finder programs and sequence results revealed a single ORF of 1377 nucleotides encoded a protein of 458 amino acids with predicted Mr of 51.75 kDa.
Previously studies indicated that UL54 protein was the unique alpha-protein which can find its homology in all Herpesviridaes (Judith et al., 1995).Consequently, the UL54 protein was a conserved protein, but the genus has not been determined yet (Andrew and Davison, 2010).To help know clearly the classification of DEV, phylogenetic tree was constructed on the putative proteins of UL54 (Fig. 6).The result showed that the DEV CHv strain was more similar to alphaherpervirus than beta-herpervirus or gammaherpervirus.The DEV CHv formed an independent branch and was closed to GaHV-2, GaHV-3 and MeHV-1.
The HSV-1 UL54 protein, known as the ICP27, was identified as a multi-function protein.Due to the high similarity among the three alphaherpesviruses, HSV-1, PRV and DEV, we anticipated that DEV UL54 product has the same role as HSV-1 or PRV.The function of a protein was fundamentally rested with its spatial structure, so it was important to explore the structural characteristics of UL54 protein.
The analysis of signal peptide and transmembrane region suggested that the UL54 protein was not a membrane protein, which provided some information for its expression.The contact may play a key role in regulating the DEV gene expression.From the secondary structure and hydrophobicity analysis, we can see that the protein contained alpha helix 22.05%, extended strand 5.46%, loop 72.49% and the domain located 373-384 aa was the highest hydrophobicity regions.
Many function sites were found in the UL54 protein of DEV-CHv, including phosphorylation sites, Nutmeg acylation sites, Amidation site and RGD.These potential sites may provide more chances to interact with other proteins and regulate the functions.The antigenic determinants analysis results can improve knowledge of the antigenic and structural properties of DEV UL54 protein, as well as revealed the immunologic mechanism and yield methods for developing new vaccine.The protein was located mainly into nuclear without a classic NLS indicated that a new type of NLS should be identified.The NES also help the protein shuttle between the nuclear and cytoplasm (Yaju et al., 2005).
In conclusion, we complete the characterization analysis of DEV UL54 gene in the present study.The results of bioinformatics analysis, coupled with the previously published data strongly suggested that the DEV UL54 protein also served as a multi-function protein, but more experiments should be needed to elucidate its specific function and mechanism in the future study.

Fig. 3 :
Fig. 3: The hydrophobicity analysis of DPV UL54 by the online program of EXPASY.The defining hydrophobicity regions located 373-384 amino acids

Table 1
Fig. 1: The signal peptide analysis of DPV UL54 by SignalP V4.0.The protein contained no signal peptide Fig. 2: The transmembrane region of DPV UL54 was analyzed by http://genome.cbs.dtu.dk/services/TMHMM/.And there is no transmembrane region in the protein RESULTS Analysis of DEV UL54 gene nucleotides sequences: The UL54 gene owns a 1377 bp long nucleotides sequence and contained a single ORF, which composed of 415 adenine (30.1%), 307 cytosine (22.3%), 329 guanine (23.9%), 326 thymine (23.7%) and a GC content 46.19%.The NC value of the nucleotides sequence of DEV UL54 gene was 57.04, less than the