Deletion of Double Copies of the US1 Gene Reduces the Infectivity of Recombinant Duck Plague Virus In Vitro and In Vivo.

Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.

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.

Two nuclear localization signals regulate intracellular localization of the duck enteritis virus UL13 protein.

Duck enteritis virus (DEV) multifunctional tegument protein UL13 is predicted to be a conserved herpesvirus protein kinase; however, little is known about its subcellular localization signal. In this study, through transfection of 2 predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein, 2 bipartite nuclear localization signals (NLS) were identified. We found that ivermectin blocked the NLS-mediated nuclear import of DEV UL13, showing that the nuclear localization signal of DEV UL13 is a classical importin α- and ß-dependent process. We constructed a DEV UL13 mutant strain in which the NLS of DEV UL13 was deleted to explore whether deletion of the NLS affects viral replication. Amino acids 4 to 7 and 90 to 96 were predicted to be NLSs, further proving that nuclear import occurs via a classical importin α- and ß-dependent process. We also found that the NLS of pUL13 had no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results provide significant information regarding the biological function of pUL13 during DEV infection.

The bZIP Proteins of Oncogenic Viruses.

Basic leucine zipper (bZIP) transcription factors (TFs) govern diverse cellular processes and cell fate decisions. The hallmark of the leucine zipper domain is the heptad repeat, with leucine residues at every seventh position in the domain. These leucine residues enable homo- and heterodimerization between ZIP domain α-helices, generating coiled-coil structures that stabilize interactions between adjacent DNA-binding domains and target DNA substrates. Several cancer-causing viruses encode viral bZIP TFs, including human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) and the herpesviruses Marek's disease virus (MDV), Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we provide a comprehensive review of these viral bZIP TFs and their impact on viral replication, host cell responses and cell fate.

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

Duck plague virus gE serves essential functions during the virion final envelopment through influence capsids budding into the cytoplasmic vesicles.

Duck plague virus (DPV), a member of the alphaherpesviruses subfamily, causes massive ducks death and results in a devastating hit to duck industries in China. It is of great significance for us to analyze the functions of DPV genes for controlling the outbreak of duck plague. Thus, glycoproteins E (gE) of DPV, which requires viral cell-to-cell spreading and the final envelopment in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), was chosen herein. The gE mutant virus BAC-CHv-ΔgE was constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Viral plaques on duck embryo fibroblast (DEF) cells of BAC-CHv-ΔgE were on average approximately 60% smaller than those produced by BAC-CHv virus. Viral replication kinetics showed that BAC-CHv-ΔgE grew to lower titers than BAC-CHv virus did in DEF cells. Electron microscopy confirmed that deleting of DPV gE resulted in a large number of capsids accumulating around vesicles and very few of them could bud into vesicles. The drastic inhibition of virion formation in the absence of the DPV gE indicated that it played an important role in virion morphogenesis before the final envelopment of intracytoplasmic nucleocapsids.

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16.

BACKGROUND: pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. RESULTS: We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. CONCLUSIONS: The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek's Disease Virus.

Viruses may hijack glycolysis, glutaminolysis, or fatty acid ß-oxidation of host cells to provide the energy and macromolecules required for efficient viral replication. Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism, such as glutamic acid, alanine, glycine, pyrimidine, and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (malonyl-CoA) inhibitors, did not reduce MDV replication, indicating that MDV replication does not require fatty acid ß-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial ß-oxidation.IMPORTANCE Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek's disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid ß-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumor cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.

US10 Protein Is Crucial but not Indispensable for Duck Enteritis Virus Infection in Vitro.

To investigate the function of the duck enteritis virus (DEV) tegument protein US10, we generated US10 deletion and revertant mutants (ΔUS10 and US10FRT) via two-step RED recombination based on an infectious BAC clone of DEV CHv-BAC-G (BAC-G). In multistep growth kinetic analyses, ΔUS10 showed an approximately 100-fold reduction in viral titer, while the genome copies decreased only 4-fold compared to those of BAC-G. In one-step growth kinetic analyses, there were no significant differences in genome copies among BAC-G, ΔUS10 and US10FRT, but ΔUS10 still showed a 5- to 20-fold reduction in viral titer, and the replication defect of ΔUS10 was partially reversed by infection of US10-expressing cells. The transcription levels of Mx, OASL, IL-4, IL-6 and IL-10 in ΔUS10-infected duck embryo fibroblasts (DEFs) were significantly upregulated, while TLR3 was downregulated compared with those in BAC-G-infected DEFs. Taken together, these data indicated that US10 is vital for DEV replication and is associated with transcription of some immunity genes.

Oncogenic Marek's disease herpesvirus encodes an isoform of the conserved regulatory immediate early protein ICP27 generated by alternative promoter usage.

Herpesvirus gene expression is temporally regulated, with immediate early (IE), early (E) and late (L) genes. ICP27, which is involved in post-transcriptional regulation, is the only IE gene product conserved in all herpesviruses. We show here that the ICP27 transcript of the oncogenic Marek's disease virus shares the same polyadenylation signal as the bicistronic glycoprotein K-ICP27 transcript and is regulated by alternative promoter usage, with transcription from its own promoter (pICP27) or that of gK (pgK). The pgK can generate a spliced ICP27 transcript yielding an N-terminal-deleted ICP27 isoform (ICP27ΔN) that, like ICP27, co-localizes with the SR protein in infected cells, but with a diffuse nuclear distribution. The pICP27 includes functional responsive elements (REs) for SP1, AP1 and CREB, is essentially active during the lytic phase and leads to exclusive expression of the native form of ICP27. The alternative promoter, pgK, including active REs for GATA, P53 and CREB, preferentially generates the gK transcript during the lytic phase and the spliced ICP27 transcript (ICP27ΔN) during the latent phase. An analysis of the DNA methylation marks of each promoter showed that pgK was systematically demethylated, whereas pICP27 was methylated during latency and demethylated during the lytic stage. Thus, MDV ICP27 gene expression is dependent on alternative promoters, the usage of which is regulated by DNA methylation, which differs between viral stages.

An MHC class I immune evasion gene of Marek׳s disease virus.

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years.

Marek's disease virus-induced transient cecal tonsil atrophy.

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens that is caused by a highly cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. MD is characterized by bursal and thymic atrophy and rapid onset of T cell lymphomas that infiltrate lymphoid tissues, visceral organs, and peripheral nerves with severe clinical symptoms that include transient paralysis, anemia, weight loss, and neurologic disorders. The cecal tonsils (CT) are considered the largest lymphoid aggregates of avian gut-associated lymphoid tissue (GALT). Along with Peyer's patches, CT elicits protective immune responses against bacterial and viral pathogens in the intestinal tract of avian species. In this study we investigated the effect of MDV infection on CT structural changes and cytokine gene expression in two MD-susceptible and resistant chicken lines. The histopathologic analysis revealed that MDV causes the loss of germinal follicular centers within the CT of the resistant line while inducing a severe, near-total lymphoid depletion in the susceptible line during cytolytic infection. The lymphoid depletion, however, recovered approximately 2 wk postinfection but the loss of germinal centers persisted during the latent phase of infection in both lines. The atrophy of this important GALT was transient and there were no visible differences between the CT of the infected and control birds of either line by 21 days postinfection. Of the genes tested, IFN-beta and IFN-gamma were up regulated in the CT of both infected lines during lytic infection. The expression levels of both genes were much higher in the susceptible line than in the resistant line. A similar pattern of expression was observed for IL-6, IL-10, IL-13, and iNOS. IL-12 was up regulated in the CT of birds of the susceptible line during all three phases of infection. An over expression of IL-18 was also observed in CT of the susceptible line during lytic and latent phases of infection. IL-8 was the only cytokine expressed at higher levels in the CT of the resistant line during the lytic and reactivation phases of infection. The histopathologic observations and gene expression profiling are further discussed.

Cell cycle modulation by Marek's disease virus: the tegument protein VP22 triggers S-phase arrest and DNA damage in proliferating cells.

Marek's disease is one of the most common viral diseases of poultry affecting chicken flocks worldwide. The disease is caused by an alphaherpesvirus, the Marek's disease virus (MDV), and is characterized by the rapid onset of multifocal aggressive T-cell lymphoma in the chicken host. Although several viral oncogenes have been identified, the detailed mechanisms underlying MDV-induced lymphomagenesis are still poorly understood. Many viruses modulate cell cycle progression to enhance their replication and persistence in the host cell, in the case of some oncogenic viruses ultimately leading to cellular transformation and oncogenesis. In the present study, we found that MDV, like other viruses, is able to subvert the cell cycle progression by triggering the proliferation of low proliferating chicken cells and a subsequent delay of the cell cycle progression into S-phase. We further identified the tegument protein VP22 (pUL49) as a major MDV-encoded cell cycle regulator, as its vector-driven overexpression in cells lead to a dramatic cell cycle arrest in S-phase. This striking functional feature of VP22 appears to depend on its ability to associate with histones in the nucleus. Finally, we established that VP22 expression triggers the induction of massive and severe DNA damages in cells, which might cause the observed intra S-phase arrest. Taken together, our results provide the first evidence for a hitherto unknown function of the VP22 tegument protein in herpesviral reprogramming of the cell cycle of the host cell and its potential implication in the generation of DNA damages.

Current state of Marek's disease virus microRNA research.

MicroRNA (miRNA) is a major family of small RNAs that posttranscriptionally regulate gene expression. Small RNA profiling studies have revealed that some viruses, particularly large DNA viruses, such as Marek's disease virus (MDV), encode their own set of miRNAs. There are currently 406 viral miRNAs in miRBase, of which 392 are encoded by herpesviruses. To date, 26 MDV-1 miRNAs, 36 MDV-2 miRNAs, and 28 herpesvirus of turkeys miRNAs have been identified. Interestingly, herpesvirus miRNAs appear to have spatial conservation, located in clusters within repeat regions, but lack sequence conservation. Two clusters of MDV-1 miRNA have been identified, one located near the MEQ gene and one within the latency-associated transcript (LAT). miRNA profiling studies have shown that MDV miRNA are differentially expressed between strains and stages of infection. For example, mdv1-miR-M4 and mdv1-miR-M2-3p are three- and sixfold higher, expressed, respectively, in vv+ strains compared to vv strains. A recent study found that deletion or seed region mutation of mdv1-miR-M4 reduces viral oncogenicity, suggesting a link between mdv1-mir-M4 and lymphoma development in MDV-infected birds. Taken together, current research suggests that viral miRNAs are a key component of MDV pathogenesis.

Proteomics applied to avian herpesviruses.

Proteomics is the application of rapidly evolving high-throughput technologies that enable analysis of proteins on a large scale. Recent advances in instrumentation have allowed detection, identification, and quantification of proteins with unparalleled precision and reproducibility, and this, in combination with novel bioinformatics tools, has helped to move proteomics from the simple cataloging of expressed proteins toward discovery of operating mechanisms in the biological systems. Proteomics holds great promise for advancing the understanding of viral pathogenesis, immunity, and the dynamics of virus-host protein interactions. Nevertheless, only a small number of proteomic studies have been done on animal viruses and avian herpesviruses in particular. This review summarizes the basic concepts and technologies used in proteomics and highlights the most successful applications of different proteomic approaches that resulted in identification of new virus-host protein interactions, mechanisms of genetic resistance and susceptibility to Marek's disease in chickens, and profiling and analysis of proteomes of Gallid herpesvirus 2 (GaHV-2), GaHV-3, and Meleagrid herpesvirus 1 infected or transformed cells. This review also discusses current limitations and potential future applications of proteomic methods in avian herpesvirus research.

DNA methylation down-regulates EGFR expression in chickens.

The epidermal growth factor receptor (EGFR), a growth-factor-receptor tyrosine kinase, is up-regulated in numerous tumors, which provides a good target for cancer therapy. Although it has been documented that oncoviruses are responsible for the activation of EGFR in tumors, the impact of Marek's disease virus (MDV) infection on EGFR has not yet been studied. We performed quantitative reverse transcriptase (RT)-PCR to check EGFR expression and found that it was significantly down-regulated after MDV infection. To explore the mechanism of EGFR repression, we examined the level of methylation of the EGFR promoter. The methylation level was significantly increased at 21 days postinfection, indicating a potential role of promoter methylation in EGFR repression.

Importance of differential expression of Marek's disease virus gene pp38 for the pathogenesis of Marek's disease.

The role of pp38 in the pathogenesis of Marek's disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B-based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Sp1 A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated.

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Characterization of Meq proteins from field isolates of Marek's disease virus in Japan.

Serotype 1 strains of Marek's disease virus (MDV-1) cause malignant lymphomas in chickens (Marek's disease; MD). Although MD has been controlled by vaccination, field isolates of MDV-1 have tended to increase in virulence and cause MD even in vaccinated chickens. Meq, a putative MDV-1 oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. Sequencing analysis of some of the viral genes of various MDV-1 strains revealed a distinct diversity of and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and, consequently, to increases in MDV-1 oncogenicity. However, few reports have characterized MDV-1 strains isolated in Japan. In this study, we established the amino acid sequences of MDV-1 field isolates from Japan in order to determine whether they display a distinct diversity of and point mutations in Meq. In addition, we analyzed the transactivation activities of the Meq proteins in order to evaluate whether the observed mutations affect their functions. Japanese MDV-1 isolates displayed the distinct mutations in basic region 2 (BR2) and proline-rich repeats (PRRs) of the Meq proteins as well as some unique mutations. Reporter assays revealed that the amino acid substitutions in BR2 and the PRRs affected the Meq transactivation activity. These results suggest that the distinct mutations are also present in the Meq proteins of MDV-1 isolates from Japan and affect their transactivation activities.

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.