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Identification and characterization of duck plague virus glycoprotein C gene and gene product.

ABSTRACT: BACKGROUND: Viral envelope proteins have been proposed to play significant roles in the process of viral infection. RESULTS: In this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has several features common to other identified herpesvirus gC, and was genetically close to the gallid herpervirus.Antibodies raised in rabbits against the pET32a-gC recombinant protein expressed in Escherichia coli BL21 (DE3) recognized a 45-KDa DPV-specific protein from infected duck embryo fibroblast (DEF) cells. Transcriptional and expression analysis, using real-time fluorescent quantitative PCR (FQ-PCR) and Western blot detection, revealed that the transcripts encoding DPV gC and the protein itself appeared late during infection of DEF cells. Immunofluorescence localization further demonstrated that the gC protein exhibited substantial cytoplasm fluorescence in DPV-infected DEF cells. CONCLUSIONS: In this work, the DPV gC protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gC product for the first time. These properties of the gC protein provided a prerequisite for further functional analysis of this gene.

SUBMITTER: Lian B

PROVIDER: S-EPMC3004831 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Identification and characterization of duck plague virus glycoprotein C gene and gene product.

Lian Bei B Xu Chao C Cheng Anchun A Wang Mingshu M Zhu Dekang D Luo Qihui Q Jia Renyong R Bi Fengjun F Chen Zhengli Z Zhou Yi Y Yang Zexia Z Chen Xiaoyue X

Virology journal 20101127

<h4>Background</h4>Viral envelope proteins have been proposed to play significant roles in the process of viral infection.<h4>Results</h4>In this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has ...[more]

PMID: 21110887

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Project description:BackgroundDuck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh.AimThis study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV.MethodsA total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank.ResultsOf the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity.ConclusionBased on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

Project description:Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1-60 at the N-terminal, and amino acids 1-20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40-50 and 768-777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

Project description:BACKGROUND: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. RESULTS: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-?-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. CONCLUSIONS: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

Dataset Information

Identification and characterization of duck plague virus glycoprotein C gene and gene product.

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Identification and characterization of duck plague virus glycoprotein C gene and gene product.

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