Abstract
A 4554-bp fragment was amplified from the DEV C-KCE vaccine strain by single oligonucleotide nested polymerase chain reaction with partially known sequences for the DEV US1 and US10 genes. Three open reading frames containing the genes encoding US10, S3, and US2 were predicted using the Editseq program (DNAStar). The S3 and US2 genes have the same transcription orientation but are oriented head-to-head with respect to US10. The promoters and polyadenylation signals were predicted. Two poly A sequences were predicted in S3, but none were predicted in US2. These results provide partial sequence of US region for the physical map of the DEV genome. Phylogenetic analysis suggests that the DEV C-KCE strain is more closely related to Mardivirus in the alphaherpesvirus subfamily of the Herpesviridae.
Avoid common mistakes on your manuscript.
Introduction
Duck viral enteritis (DVE) is an acute contagious disease that is highly lethal in all ages of birds from the order Anseriformes (ducks, geese, and swans) [1]. Duck enteritis virus (DEV), a member of the family Herpesviridae, is the causative agent for DVE. DVE was first known as an acute hemorrhagic disease found in domestic ducks in Holland as early as 1923 [2]. DVE was first reported in China in 1957 [3]. Several studies indicate that DVE is difficult to monitor and control, because DEV establishes an asymptomatic carrier state in both farmed and wild waterfowl and it is only detectable during the intermittent shedding period of the virus [4].
The genome of DVE is a linear, double-stranded DNA molecule of approximately 180 kb, consisting of unique long (UL) and unique short (US) regions with a structure similar to that of other alphaherpesviruses. The G + C content of the genome is 64.3%, which is higher than any other reported avian herpesvirus in the subfamily Alphaherpesvirinae [5]. DEV was classified as an unassigned virus in the family Herpesviridae according to the Eighth International Committee on Taxonomy of Viruses (ICTV) [6], although it was previously grouped in the subfamily Alphaherpesvirinae [7, 8]. The majority of DEV sequences are limited in the UL region; however, only partial sequences of US1 and US10 have been published and other sequences in the US region were not clear. In order to clarify the genomic organization, we have amplified the unknown sequences in the US region of DEV.
Here, we report the cloning of the full-length US10, S3, and US2 genes by combining single oligonucleotide nested polymerase chain reaction (SON-PCR) [9] with common PCR. We have characterized the acquired sequences and performed phylogenetic analyses of the evolutionary relationship of DEV with reference strains of Alphaherpesvirinae. These results further supported the phylogenetic classification of DEV. Our findings help in illustrating the structure and function of these genes in DEV.
Materials and methods
Virus propagation and preparation of viral genomic DNA
DEV C-KCE was obtained from the station of veterinary drugs censorship (Beijing, China). The virus was propagated in chicken embryo fibroblasts (CEF) in Dulbecco’s Minimal Essential Medium. Viral particles were harvested when the cytopathic effect reached 80%. After three frozen-thawed cycles cell lysate was then centrifuged to remove cell debris and stored at −70°C until use.
Viral DNA preparation was described previously [10]. The presence of DEV was confirmed by using PCR, The primers, D1 (+) 5′-GTAGACGAAGGCGGGTATG-3′ and D2 (−) 5′-CGTATTGGTTTCTGAGTTGG-3′, were designed according to the partial sequence available for UL30 (AF064639).
Primer design and PCR amplification of US10, S3, and US2
The procedure for amplifying the DEV genomic fragment is shown in Fig. 1. Several specific primers were designed according to the DEV US1 and US10 partial sequence, and synthesized as reported previously [11]. All the primers are listed in Table 1.
PCR was carried out in a reaction mixture containing 2.5 μl 10 × reaction buffer, 2.0 μl dNTPs (2.5 mM for each of the four dNTPs), 0.5 μl of each primer (10 pmol each), 2.0 μl DNA template, 0.3 μl ExTaq DNA polymerase (5 U/μl), and water up to 25 μl (all the reagents were purchased from TaKaRa). PCR conditions used are shown in Table 2.
Cloning and sequencing of PCR products
PCR products were analyzed on 1% agarose gels and purified using a DNA Gel Extraction Kit (Bioteke, Peking). They were then cloned into the pMDl8-T vector (TaKaRa) according to the manufacturer’s instructions. Each fragment was sequenced by Sangon.
Analysis of DNA sequence
The full-length assembled sequence was analyzed using the Editseq program (DNAStar) to search for open reading frames (ORFs), and then submitted to GenBank for BLAST search analysis. The predicted amino acid sequences of these ORFs were compared with those from other alphaherpesviruses using the DNAStar program (version 7.1, DNAStar, Inc.). The promoters of these genes were analyzed by the program of Berkeley Drosophila Genome project’s Neural Network Promoter Prediction, which is a eukaryotic (human) core promoter search engine (http://www.fruitfly.org/seq_tools/promoter.html) [12]. The core promoters were examined for the presence of TATA box consensus sites using the TRANSFACFind search engine (http://motif.genome.jp/). POLYADQ, analyzed by a eukaryotic (human) polyadenylation (poly A) signal search engine (Cold Spring Harbor Laboratory, [http://rulai.cshl.org/tools/polyadq/polyadq_form.html]). The potential N-glycosylation sites transmembrane region, and signal peptide were analyzed using the program of NetNGlyc 1.0 Server, TMHMM, and SignalP 3.0 Server from the search engine (http://www.cbs.dtu.dk/services/). The DNA sequence identity to the Kozak consensus sequence (GCCGCCRCCATGG, R = A/G) [13] around the initiator ATG of each protein was also measured. Phylogenetic analysis (maximum likelihood method) was performed by using the MEGA4.1, while multiple alignment was performed by Clustal X.
Results
ORF determination and molecular characteristics of three genes
A 4554-bp sequence was amplified from DEV C-KCE genome by SON-PCR. Three complete ORFs were predicted, containing the genes US10, S3, and US2 homolog of MDV-1. The ORF sizes were 507, 888, and 720-bp, respectively. The sequence obtained in this study is available from GenBank under the accession number (EF619046). The gene arrangement of the three ORFs in DEV was similar to MDV-1. The relative positions of the three genes in the DEV genome are shown in Fig. 1.
These predicted functional regions are shown in Table 3. The canonical polyadenylation signal AATAAA was found have an overlap of 5 nucleotides with US10, while two potential polyadenylation signals were predicted downstream of S3, but none was found for US2. The three genes were found to have a Kozak ribosome-binding sequence of 5, 6, and 7, respectively, at their translation start sequence.
The US10 protein is encoded by 168 amino acids (aa) and possesses a putative zinc finger motif, 93-C-X3-C-X3-H-X3-C-105, where C = cysteine, H = histidine, X = any amino acid. Comparing the putative US10 protein with other alphaherpesvirus strains revealed a highly conserved stretch of 13 amino acid residues, which lies in the putative zinc finger domain.
The S3 ORF was predicted to encode 295 aa. Only avian herpesviruses have been shown to contain this gene. This gene is conserved, with sequence homology of as high as 41.84%, but its functions are unknown.
The US2 protein is composed of 239 aa. It possesses two potential N-linked glycosylation sites at amino acid residues 85 and 127. Only VZV was absent from the multiple alignment; the result predicted two conserved regions mainly centered on the N-terminal region. The first region, a 14 hydrophobic residue domain was conserved in all Alphaherpesvirinae (Fig. 2).
Phylogenetic analysis
The phylogenetic tree was performed by comparing the three putative protein of DEV with that of 11 reference strains of closely related herpesviruses (Fig. 3), DEV is closely related to members of Alphaherpesvirinae, consistent with that reported previously [14, 15]. Furthermore, the phylogenetic distance showed that DEV is closer to Mardivirus of the Alphaherpesvirinae. The three genes and deduced amino acid sequence of DEV shared high homology with Mardivirus of Alphaherpesvirinae (Table 4). These results will provide evidence for the taxonomic classification of DEV.
Discussion
Phylogenetic trees based on the three putative proteins show that DEV C-KCE is evolutionarily most closely related to the Avian herpesviruses. Our findings are quite similar to those who proposed that this virus should be classified as an alphaherpesvirus [14, 15]. All the three phylogenetic analyses we have performed indicate that DEV forms a single cluster, but is more closely related to Avian herpesviruses in the alphaherpesvirus subfamily of the Herpesviridae.
US10 homologs of EHV-1 and HSV-1 are known to possess a sequence of 13 amino acids (C-X3-C-X3-H-X3-C), which is a perfect match to the consensus zinc finger motif [16], which was also present in DEV 93-C-X3-C-X3-H-X3-C-105. The US10 gene encodes a protein in HVT which is non-essential for virus replication in vitro and in vivo and has been used as a site for the insertion and expression of foreign sequences [17]. There is very little absolute sequence conservation in the N-terminal 70% of the polypeptide. In contrast, the C-terminal region of US10 is highly conserved among all of the primate viruses.
Among the reference strains of Alphaherpesvirinae, only Avian herpesviruses have the S3 gene, which has no orthologue among HSVs or any other mammalian herpesvirus. The function of HVT SORF3 or its MDV homolog, SORF3 [18], is not currently known.
The conservation of the US2 ORF among diverse herpesviral genomes is consistent with the proposal that this gene appeared early in the herpesvirus lineage. The gene encoding the US2 protein had no obvious N-terminal signal sequence or transmembrane domain, and only two N-linked glycosylation sites. It is conserved among alphaherpesviruses family members with the notable exception of the varicella-zoster virus; despite its wide conservation of sequence, no alphaherpesvirus requires the US2 protein for replication in cultured cells [19]. Even in animals, US2-null viruses show only modest attenuation. US2 contributes to virus penetration and efficient cell-to-cell spreading. Furthermore, it plays a role in sustained virus replication in vivo [20]. All US2 homologs share a highly conserved stretch of 14 hydrophobic amino acids at the extreme amino terminus. It is suggested that the US2 homologs may encode either a secreted or an N-terminally anchored transmembrane (glyco) protein [21, 22] and perhaps a common structure or function will be found in US2.
References
S. Davison, K.A. Converse, A.N. Hamir, R.J. Eckroade, Avian Dis. 37, 1142 (1993). doi:https://doi.org/10.2307/1591927
A.E.R.F. Baudet, Tijdschr. Diergeneeskd. 50, 455 (1923)
Y.X. Huang, S. China Agric. Univ. J. 1, 1 (1959)
E.C. Burgess, J. Ossa, M. Yuill, Avian Dis. 23, 940 (1979). doi:https://doi.org/10.2307/1589610
R. Gardner, J. Wilkerson, J.C. Johnson, Intervirology 36, 99 (1993)
C.M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger, L.A. Ball, in Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, Elsevier Academic Press, California, 2005
E.F. Kaleta, Avian Pathol. 19, 193 (1990). doi:https://doi.org/10.1080/03079459008418673
S. Shawky, K. Schat, Avian Dis. 46, 308 (2002). doi:https://doi.org/10.1637/0005-2086(2002)046[0308:LSAROD]2.0.CO;2
Z. Antal, Curr. Genet. 46, 240 (2004). doi:https://doi.org/10.1007/s00294-004-0524-6
F.Y. Liu, B. Ma, Y. Zhao, Y. Zhang, Y.H. Wu, X.M. Liu, J.W. Wang, Virus Genes 37, 328–332 (2008). doi:https://doi.org/10.1007/s11262-008-0266-5
Y. Li, W.J. OuYang, H.C. Yang, Chin. J. Vet. Med. 43, 5–7 (2007)
M.G. Reese, N.L. Harris, F.H. Eeckman, in Biocomputing: proceedings of the 1996 Pacific Symposium, World Scientific Publishing, Singapore, 1996, ed. by L. Hunter, T.E. Klein, pp. 2–7
M. Kozak, Cell 44, 283 (1986). doi:https://doi.org/10.1016/0092-8674(86)90762-2
P.J. Plummer, T. Alefantis, S. Kaplan, P. O’Connell, S. Shawky, S.A. Schat, Avian Dis. 42, 554 (1998). doi:https://doi.org/10.2307/1592682
H. Li, S. Liu, X. Kong, Virus Genes 33, 221 (2006). doi:https://doi.org/10.1007/s11262-005-0060-6
R.V. Holden, R.R. Yalamanchili, R.N. Harty, D.J. O’Callaghan, Virology 188, 704 (1992). doi:https://doi.org/10.1016/0042-6822(92)90525-T
R.W. Morgan, J. Gelb, C.S. Schreurs, J.K. Roseerg, P.J.A. Ondermeiger, Avian Dis. 36, 858 (1992). doi:https://doi.org/10.2307/1591544
P. Brunovskis, L.F. Velicer, in Proceedings of 19th World’s Poultry Congress, Ponsen & Looijen, The Netherlands, 1992, vol. 1, pp. 74–78
A.C. Clase, M.G. Lyman, T. del Rio, J.A. Randall, C.M. Calton, L.W. Enquist, B.W. Banfield, Virology 77, 12285 (2003). doi:https://doi.org/10.1128/JVI.77.22.12285-12298.2003
A. Meindl, N. Osterrieder, Virology 73, 3430 (1999)
C.A. Breeden, R.R. Yalamanchili, C.F. Colle, D.J. O’Callaghan, Virology 191, 649 (1992). doi:https://doi.org/10.1016/0042-6822(92)90240-P
D.J. McGeoch, Virus Res. 3, 271 (1985). doi:https://doi.org/10.1016/0168-1702(85)90051-6
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhao, Y., Wang, J.W., Liu, F. et al. Molecular analysis of US10, S3, and US2 in duck enteritis virus. Virus Genes 38, 243–248 (2009). https://doi.org/10.1007/s11262-008-0315-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11262-008-0315-0