Molecular analysis of US10, S3, and US2 in duck enteritis virus

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Aims: Duck enteritis virus (DEV) is a member of the family Herpesviridae, the genome of which is not completely analyzed. In order to provide further insights into the genomic organization, we decided to amplify the unknown sequence in the US region since three key glycoproteins are coded in this region. Glycoprotein D encoded by US6 gene is essential for virus entry and plays an important role in protecting animals from lethal challenge. Glycoprotein I and E encoded by US7 and US8 gene are required for cell-to-cell spread. Methods: Single oligonucleotide nested polymerase chain reaction was adopted to amplify the unknown sequence. The open reading frames (ORF) were predicted by Editseq program. Then the sequence was submitted to an online program to search promoter, polyadenylation signal and potential N-glycosylation sites. The phylogenetic analysis was performed by MEGA4.1, while multiple alignments were performed by ClustalX. Results: Three complete ORFs including US6, US7 and US8 were determined. The three genes shared the same polyadenylation signal. Phylogenetic analysis showed that DEV was closed to mardivirus of alphaherpesvirus. Conclusion: These results extend our understanding of the DEV genome and its classification, and also provide further basic knowledge for functional investigations on the proteins in the US region.

“…Color version available online 143 PCR was conducted as described previously [18,19] . The 25-l reaction volume containing 2.5 l 10 !…”

Section: Polymerase Chain Reaction Cloning and Sequencingmentioning

confidence: 99%

Characterization of Duck Enteritis Virus US6, US7 and US8 Gene

Zhao

Wang²

2010

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“…DEV has a genome of approximately 180 kb [9] . The complete gene sequences of the unique long (UL) region have been sequenced and determined in our laboratory [10][11][12][13][14] , and several other genes in the unique short (US) region have also been reported [15][16][17] . The results demonstrate that DEV has a similar, but not the same, genomic organization as other herpesviruses.…”

Section: Introductionmentioning

confidence: 99%

Unique Sequence Characteristics of Genes in the Leftmost Region of Unique Long Region in Duck Enteritis Virus

Liu

Liu²,

Li³

et al. 2009

Objective: Our objective was to further understand the evolutionary position of duck enteritis virus (DEV) in the family Herpesviridae and to determine the genomic structure in the leftmost region of the unique long region (UL) of DEV. Methods: ‘Targeted gene walking PCR’ was used to amplify the unknown gene of DEV clone-03 strain adjacent to known sequences in the UL region. Open reading frames (ORFs) were determined by Gene Runner and an online transcriptional element search engine. The phylogenetic tree was constructed by DNAStar based on the deduced amino acid sequences. Results: A fragment of 13,947 bp, containing 5 ORFs (designated ICP4, ORF1, ORF2, LORF2 and LORF3, respectively), was amplified from the DEV clone-03 strain genome. In addition, a tandem repeat region of α-type-like sequences was detected between ICP4 and LORF2. Phylogenetic analysis of ICP4 indicated that DEV ICP4 should be clustered into Varicellovirus. DEV ORF1 and ORF2 were unique among herpesviruses. Additionally, the existence and characteristics of LORF2 and LORF3 indicated a close relationship with Mardivirus members. Conclusion: The characteristics and gene content in the leftmost end of the UL region of the DEV genome showed that the DEV genome is different from those of other herpesviruses.

“…DEFs were either mock infected or DPV infected as described above, and they were harvested by lowspeed centrifugation at indicated times (2,4,8,12,24,36,48 and 72 h after infection). The cells were lysed on ice for 30 min with an equal volume of radioimmunoprecipitation assay buffer (50 m M ofTris-HCl, pH 7.4, 150 m M of NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 m M of phenylmethylsulfonyl fluoride) [42] .…”

Section: Western Blot Analysis For the Expression Kineticsmentioning

confidence: 99%

“…In brief, DEFs, grown in a coverslip in 6-well plates, were either mock infected or DPV infected as described above. The cells were harvested at different times after infection (2,4,8,12,24,36,48 and 72 h) and fixed with 4% paraformaldehyde for 15 min at room temperature. Next, the cells were washed once with PBS and with 0.2% (v/v) TrionX-100 in PBS for an additional 10 min at 25 ° to allow permeabilization.…”

Section: Subcellular Localizationmentioning

confidence: 99%

“…With many similarities and a few differences, accumulating evidence indicates that the UL35 protein and its homologues of Alphaherpesvirus , Betaherpesvirus and Gammaherpesvirus may play similar roles in viral assembly and maturation. To date, the majority of reported DPV sequences are limited to single open reading frames, which include UL5-UL6 [16,18,19] , UL22-UL24 [20][21][22] , UL25-UL30 [23] , UL31-UL35 [24][25][26][27] , UL44 [28,29] , UL50-UL51 [30][31][32] , US3-US5 [33,34] , US8 [35] , US2 and US10 [36] . However, no reports have been published concerning the characterization of transcription, expression, intracellular localization and the dependence of DPV VP26 on viral DNA synthesis.…”

mentioning

confidence: 99%

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Characterization of the Duck Plague Virus UL35 Gene

Cai

Cheng²,

Wang³

et al. 2010

Objective: Previous study has demonstrated that the duck plague virus (DPV) UL35 gene can be expressed as a recombinant fusion protein, and the prepared antiserum has a high reactivity and specificity against the purified recombinant protein. In the present study, to elucidate the properties and functions of its encoding protein, the UL35 gene product (VP26) was identified by using the prepared rabbit polyclonal antiserum. Methods: Real-time PCR, Western blot and immunofluorescence analysis were used to determine the transcription and expression kinetics and subcellular localization of DPV VP26 in DPV-infected cells. Results: A protein of approximately 13 kDa that reacted with the antiserum was detected in immunoblot of DPV-infected cellular lysates. Real-time PCR and Western blot analysis of DPV-infected cells showed that VP26 was produced predominantly at the late stage of infection, its production was highly dependent on viral DNA synthesis, and the UL35 gene was regulated as a late viral gene, suggesting that the gene should be categorized as γ2 class. Additionally, analysis of the association of DPV VP26 with purified virions revealed that VP26 was a component of extracellular mature DPV virions. Subcellular localization demonstrated that VP26 firstly localized in cytoplasm, then it transferred to the nucleus and aggregated in the punctate region of the nucleus in DPV-infected cells. Conclusion: Taken together, these results will provide a foundation for further functional analysis of the DPV UL35 gene.