The intracellular domain of duck plague virus glycoprotein E affects UL11 protein incorporation into viral particles.

Studies have shown that proteins in the tegument (located between the viral capsid and envelope layer) play critical roles in the assembly and budding of herpesviruses. The UL11 protein of herpesviruses is important in the process of virus particle cell entry, release, assembly and secondary envelopment. Herpesvirus glycoprotein E (gE) is involved in syncytia formation, transmission between cells and nerve invasion. In herpes simplex virus, UL11 has been shown to interact with gE. However, little is known about the relationship of duck plague virus (DPV) pUL11 and gE. In this study, we constructed DPV cytoplasmic domain (CT)-gE, and extracellular domain (ET)-gE deletion mutants, pCMV-gE, CT-gE, and ET-gE and UL11 recombinant plasmids. We found that pUL11 can interact and colocalize with gE, CT-gE and ET-gE. Together, these results highlight an important role for UL11 in the function of gE, and may also have important implications for the role of pUL11 and gE.

Role of cholesterol in anatid herpesvirus 1 infections in vitro.

Cholesterol is an essential constituent of the cell membrane that modulates several physiological events, including virus entry into the host. Duck virus enteritis (DVE) is a contagious and lethal infection that attacks several species of waterfowl. Anatid herpesvirus 1 (AnHV-1) is the causative agent of duck viral enteritis and classified under subfamily Alphaherpesvirinae. In this study, the effect of cholesterol depletion in both host cell membrane and viral envelope on the infectivity of AnHV-1 was explored. Cholesterol depletion of chicken embryo fibroblast cells (DF-1) by methyl-ß-cyclodextrin (MßCD) inhibited the infectivity of AnHV-1. This inhibitory effect was moderately reversed by the exogenous replenishment of cholesterol in the cells. Furthermore, the inhibition of endogenous cholesterol synthesis by a statin drug also inhibited the infectivity of AnHV-1. Presumably, the removal of cholesterol from AnHV-1 envelope might be disrupting the viral envelope resulting in its diminished infectivity. The presence of a relatively hydrophobic cavity in MßCD can be used to extract cholesterol from the cell membrane. Loss of infectivity of the virus might be due to the effects of MßCD mediated cholesterol depletion from the cell membrane. The results implicate that the cell membrane cholesterol is vital for the infectivity of AnHV-1 in DF-1 cells, and its depletion from virion curtails the infectivity by destabilizing the envelope.

Complete genome sequence and evolution analysis of a columbid herpesvirus type 1 from feral pigeon in China.

Here, we report the genome sequence of a feral pigeon alphaherpesvirus (columbid herpesvirus type 1, CoHV-1), strain HLJ, and compare it with other avian alphaherpesviruses. The CoHV-1 strain HLJ genome is 204,237 bp in length and encodes approximately 130 putative protein-coding genes. Phylogenetically, CoHV-1 complete genome resides in a monophyletic group with the falconid herpesvirus type 1 (FaHV-1) genome, distant from other alphaherpesviruses. Interestingly, the evolutionary analysis of partial genes of CoHV-1 isolated from different organisms and areas (currently accessible on GenBank) indicates that the CoHV-1 HLJ strain isolated from pigeon (Columba livia) is closely related to the strains isolated from peregrine falcon (Falco peregrinus) in Poland and owl (Bubo virginianus) in USA. These results may suggest possible transmission of the virus between different organisms and different geographic areas.

Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

Prokaryotic expression and characteristics of duck enteritis virus UL29 gene.

Duck enteritis virus (DEV) causes a contagious, acute and highly lethal disease in all ages of birds from the order Anseriformes. DEV leads to heavy economic losses to the commercial duck industry due to its high mortality rate and decrease in egg production. With development of molecular biology, more information about DEV genes is reported, nonetheless little information is known about DEV UL29 gene and its product major DNA-binding protein or infected-cell protein 8 (ICP8). The sequence characteristics of DEV UL29 gene was initially showed in our article. Phylogenetic tree analysis provided useful proof that DEV belongs to the subfamily Alphaherpesvirinae. The predicted characteristics of ICP8 amino acid sequence showed that ICP8 possesses good immunogenicity and is a hydrophobic protein. These findings correlate with the experimental results that ICP8 protein forms inclusion bodies in the prokaryotic expression system. By immunofluorescence we have identified ICP8 as nuclear protein. All the fundamental data in this article contribute to understanding the functions of DEV UL29 gene and its product ICP8.

Replication kinetics of duck enteritis virus UL16 gene in vitro.

BACKGROUND: The function and kinetics of some herpsvirus UL16 gene have been reported. But there was no any report of duck enteritis virus (DEV) UL16 gene. FINDINGS: The kinetics of DEV UL16 gene was examined in DEV CHv infected duck embryo fibroblasts (DEFs) by establishment of real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) and western-blotting. In this study, UL16 mRNA was transcript at a low level from 0-18 h post-infection (p.i), and peaked at 36 h p.i. It can't be detected in the presence of acyclovir (ACV). Besides, western-blotting analysis showed that UL16 gene expressed as an apparent 40-KDa in DEV infected cell lysate from 12 h p.i, and rose to peak level at 48 h p.i consistent with the qRT-PCR result. CONCLUSIONS: These results provided the first evidence of the kinetics of DEV UL16 gene. DEV UL16 gene was a late gene and dependent on viral DNA synthesis.

Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis.

A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.

Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

BACKGROUND: In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. RESULTS: DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. CONCLUSIONS: DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and most similar to the genus Mardivirus. The UL15 and/or UL15.5 accumulate(s) in the cytoplasm during early times post-infection and then are translocated to the nucleus at late times.

Homodimerization of the Meq viral oncoprotein is necessary for induction of T-cell lymphoma by Marek's disease virus.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B Meq(BZIP/BZIP)) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B Meq(Hom/Hom)). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

The UL31 to UL35 gene sequences of Duck enteritis virus correspond to their homologs in herpes simplex virus 1.

Five ORFs in the genome of Duck enteritis virus (DEV) corresponding to UL31, UL32, UL33, UL34, and UL35 genes of Herpes simplex virus 1 (HSV-1) were amplified by a modified "targeted gene walking" PCR, cloned, and sequenced. UL33, UL34, and UL35 genes were oriented from the left to the right of genome, while UL31 and UL32 had an opposite orientation. A comparison of deduced amino acid sequences of the DEV ORFs with their alphaherpesvirus homologs showed well-conserved regions except for the UL34 and UL35 genes. Phylogenetic analysis revealed that DEV was closer to the genus Mardivirus than to any other genus of the subfamily Alphaherpesvirinae. Based on this evidence, we proposed to assign DEV to the subfamily Alphaherpesvirinae.

A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek's disease virus.

Genetic resistance to Marek's disease (MD) has been proposed as a method to augment current vaccinal control of MD. Although it is possible to identify QTL and candidate genes that are associated with MD resistance, it is necessary to integrate functional screens with linkage analysis to confirm the identity of true MD resistance genes. To help achieve this objective, a comprehensive 2-hybrid screen was conducted using genes unique to virulent Marek's disease virus (MDV) strains. Potential MDV-host protein interactions were tested by an in vitro binding assay to confirm the initial two-hybrid results. As a result, 7 new MDV-chicken protein interactions were identified and included the chicken proteins MHC class II beta (BLB) and invariant (Ii) chain (CD74), growth-related translationally controlled tumor protein (TPT1), complement component Clq-binding protein (C1QBP), retinoblastoma-binding protein 4 (RBBP4), and alpha-enolase (ENO1). Mapping of the encoding chicken genes suggests that BLB, the gene for MHC class II beta chain, is a positional candidate gene. In addition, the known functions of the chicken proteins suggest mechanisms that MDV might use to evade the chicken immune system and alter host gene regulation. Taken together, our results indicate that integrated genomic methods provide a powerful strategy to gain insights on complex biological processes and yield a manageable number of genes and pathways for further characterization.