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Marek's disease virus (MDV) or Gallid herpesvirus 2 (GaHV-2) is a lymphotropic alphaherpesvirus and causes Marek's disease. Former studies have demonstrated that MDV is spread from chicken to chicken about 2 wk postexposure as infectious dander shed from infected chickens. More recent reports, using highly sensitive quantitative PCR analyses of dander from infected chickens, suggested that MDV replicates and is shed from the chicken much earlier (5-7 days). However, detection of viral DNA in chicken dander does not indicate whether fully infectious virus is present. To determine if viral replication is present in the skin of infected chickens at these early times, expression of a late viral protein indicative of fully productive virus replication was evaluated using fluorescent microscopy. To do this, highly virulent and attenuated recombinant (r)MDV was generated that abundantly expresses the monomeric red fluorescent protein fused to the late UL47 (VP13/14) protein in feather follicle epithelial cells. Detection of viral DNA could be detected in the skin of infected chickens as early as 6 days postinfection (p.i.), consistent with previous reports detecting viral DNA in dander shed from infected chickens. Replication of virulent rMDV was evident in the feather follicles as early as 8 days p.i., while attenuated rMDV replication in the feather follicles was delayed 1-2 days. Former studies, using less sensitive techniques, suggested viral protein expression to occur about 10-12 days p.i. Undoubtedly differences in time of detection can partly be explained by multiple factors including the pathotype of virus, the route of infection, and the age and genetic line of the infected chickens used in different studies. In summary, though viral DNA can be detected as early as 6 days p.i., late viral protein expression, indicative of infectious virus production, occurs 2-3 days after DNA detection, but earlier than previously thought.

Attenuation of Gallid herpesvirus-2 (GaHV-2), the causative agent of Marek's disease, can occur through serial passage of a virulent field isolate in avian embryo fibroblasts. In order to gain a better understanding of the genes involved in attenuation and associate observed changes in phenotype with specific genetic variations, the genomic DNA sequence of a single GaHV-2 virulent strain (648A) was determined at defined passage intervals. Biological characterization of these "interval-isolates" in chickens previously indicated that the ability to induce transient paralysis was lost by passages 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesion formation. Deep sequencing of the interval-isolates allowed for a detailed cataloguing of the mutations that exist within a single passage population and the frequency with which a given mutation occurs across passages. Gross genetic alterations were identified in both novel and well-characterized genes and cis-acting regions involved in replication and cleavage/packaging. Deletions in genes encoding the virulence factors vLipase, vIL8, and RLORF4, as well as a deletion in the promoter of ICP4, appeared between passages 61 and 101. Three mutations in the virus-encoded telomerase which predominated in late passages were also identified. Overall, the frequency of mutations fluctuated greatly during serial passage and few genetic changes were absolute. This indicates that serial passage of GaHV-2 results in the generation of a collection of genomes with limited sequence heterogeneity.

Marek's disease (MD) is a neoplastic and neurodegenerative disease of chickens, which is caused by the Gallid herpesvirus type 2 (GaHV-2). Although vaccination has been used widely in China, MD still occurs frequently. Some molecular epidemiologic studies have shown that Chinese GaHV-2 isolates seem to constitute a separate clade from strains isolated from other regions. However, more of a genomic background of the Chinese strains is necessary. In 2007, a virulent GaHV-2 field strain, named LMS, was isolated from diseased chicken flocks in the southwest of China. The whole genome sequence of LMS was determined to evaluate its genetic property. The genome of LMS is 177,526 bp long, and 197 open reading frames (ORFs) were predicted. Most of the ORFs have high sequence identity with homologous ORFs of reference strains. Two regions in the LMS genome are grossly different from other strains: the α-like region and the latency-associated transcripts (LATs) promoters. Evolutionary analysis demonstrated that LMS has a larger phylogenetic distance from most American isolated strains but a closer relationship with 648Ap80 and the European pC12/130 strain. The characterised genome of LMS provides further insight into the genetics of the Chinese GaHV-2 field strains, which is useful for the control of MD in China.

Marek's disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5' end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3' end and allowed identification of the 5' end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3' UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

Marek's disease virus serotype 1 (MDV-1) strains cause malignant lymphoma in chickens. MDV-1 has been previously reported to be widespread in white-fronted geese (Anser albifrons); however, the prevalence of MDV-1 in other wild birds has not been determined. In this study, we investigated the prevalence of MDV-1 in various wild birds in Hokkaido, Japan. The MDV-1 genome was widespread in geese and ducks, but was not detected in other birds. MDV-1 was detected in both sedentary and migratory species. These results suggest that, in Japan, MDV-1 is widespread in wild goose and duck populations, and that resident ducks may be significant carriers and reservoirs of MDV-1., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv(+)) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4(+) cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.

The complete DNA sequence of Marek's disease virus (MDV) serotype 1 vaccine strain 814 was determined. It consisted of 172,541 bp, with an overall gene organization identical to that of the MDV-1 type strains. Comparative genomic analysis of vaccine strains (814 and CVI988) and other strains (CU-2, Md5, and Md11) showed that 814 was most similar to CVI988. Several unique insertions, deletions, and substitutions were identified in strain 814. Of note, a 177-bp insertion in the overlapping genes encoding the Meq, RLORF6, and 23-kDa proteins of strain 814 was identified, and a 69-bp deletion was also located in the origin of replication site (Ori) in the gene encoding RLORF12. Compared to the CVI988 vaccine strain, a deletion of 510 bp was identified in the UL36 gene. These analyses identified key mutations in the 814 strain and the vaccine strain that could be exploited for future MDV vaccine design.

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.

The main aim of the present study were to investigate sequence diversity in the Meq gene of Marek's disease viruses (MDV) isolated in China and to determine the most prevalent MDV strains. The 19 MDV strains were isolated from dead or diseased chickens from different chicken farms in China during 2006-2008, and the Meq gene was sequenced from each of these strains. Sequence analysis showed that all of the isolates contained an open reading frame of 1020 nucleotides, which encoded a 339 amino acid peptide. Compared with reference MDV strains, 12 of the 19 MDV isolates possessed two amino acid substitutions, (T → A) at position 139 and (P → R) at position 176, one isolate shared sequence similarity with the attenuated strain CVI988, and five of the other six isolates exhibited one amino acid change (P → T) at position 177 or 176. The 19 MDV isolates shared between 99.0 and 100% nucleotide sequence homology, and between 97.7 and 100% amino acid sequence homology. The nucleotide and amino acid sequence identity between the 19 MDV isolates and the 25 reference MDV strains varied from 97.6 to 100% and 94.4 to 100%, respectively. Based on the phylogenetic relationships between Meq gene sequences, Chinese MDV isolates constituted a separate clade to MDV reference strains, demonstrating that a different genotype of MDV was prevalent in China between 2006 and 2008.

MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.

Marek's disease (MD) is a disease of chickens that occurs worldwide and has serious economic consequences. MD can present as one of several forms, with the most commonly occurring forms being the lymphoproliferative diseases. Under experimental conditions, an early mortality syndrome has been recognized following infection by some but not all strains of MD virus (MDV). This is the first report of a confirmed case of mortality due to naturally occurring MDV infection in 1-week-old, nonvaccinated, chickens. Necrotizing lesions were observed in the bursa of Fabricius, lung, duodenum, jejunum, and proventriculus, and large intranuclear inclusion bodies were a striking feature in tissues with lesions in all birds. Immunohistochemical staining for the pp38 protein of MDV revealed abundant pp38 antigen in the affected tissues, confirming the presence of MDV within the lesions. PCR yielded an amplicon with 97% homology to the meq gene of MDV. No evidence of co-infection by either of the immunosuppressive agents chicken anemia virus and infectious bursal disease virus was detected.

Marek's disease (MD) remains a serious problem in the production of poultry. The disease is caused by Marek's disease virus (MDV), and despite the ubiquitous use of vaccination to control losses, MD still affects poultry farming worldwide. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple and inexpensive detection of MDV in feather tips of chickens. Two pairs of specific primers complementary to the meq oncogene of MDV were designed, targeting the sequence of the very virulent MDV strain, RB1B. Bst polymerase was used for the isothermal amplification of viral DNA at 65 C for 90 min in a water bath. The fluorescence signal was identified in MDV-positive samples after the addition of SYBR Green and ultraviolet (UV) illumination. The sensitivity of LAMP was 2 log 10 plaque-forming units (PFU)/ml of HPRS16 and 10(3) copies/il of plasmid containing the target gene (meq) and was equal in sensitivity to PCR amplification. Due to the use of three sets of primers, LAMP was highly specific for MDV-1 DNA. The developed LAMP technique is a rapid and simple tool for the specific detection of MDV in samples of feathers taken from live chickens. Since the use of thermocyclers is not necessary for LAMP assay, it can be conducted by small laboratories and even field veterinarians.

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands., (Copyright © 2010 John Wiley & Sons, Ltd.)

All herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Marek's disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Marek's disease.

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.

Researchers reported that co-cultivating the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in an REV long terminal repeat (LTR) being inserted into the internal repeat short (IRS) region of JM/102W. When the resulting recombinant virus was serially passed in cell culture, the initial LTR was duplicated and a second LTR spontaneously appeared in the terminal repeat short (TRS) region of the MDV genome. The virus, designated RM1, was significantly attenuated but still induced severe bursal and thymic atrophy (Isfort et al. PNAS 89:991-995). To determine whether the altered phenotype was due solely to the LTR, we cloned the LTR from the RM1 IRS region and inserted it into the IRS region of a very virulent bacterial artificial clone (BAC) of the Md5 strain of MDV, which we designated rMd5-RM1-LTR. During blind passage in duck embryo fibroblast cultures, the initial LTR in the rMd5-RM1-LTR was also duplicated, with LTRs appearing in both IRS and TRS regions of the MDV genome. The inserted LTR sequences and transcripts associated with the MDV open reading frames MDV085, MDV086, SORF2, US1, and US10 were molecularly characterized. The parental Md5 BAC contains a family of transcripts of 3, 2, and 1 kb that all terminate at the end of the US10 gene. The rMd5-RM1-LTR and RM1 viruses both express an additional 4 kb transcript that originates in the LTR and also terminates after US10. Collectively, the data suggest that our engineered rMd5-RM1-LTR virus very closely resembles the RM1 virus in its structure and transcription patterns.

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Marek's disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5μg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT+empty vector group and 80% in HVT group versus 33.3% in the HVT+chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Marek's disease virus (vvMDV) in chickens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces a rapid onset T-cell lymphoma and demyelinating disease in chickens. Since the 1970s the disease has been controlled through mass vaccination with herpesvirus of turkeys [meleagrid herpesvirus type 1 (MeHV-1)]. Over time this vaccine's efficacy decreased, and in the 1980s a bivalent vaccine consisting of MeHV-1 and a non-oncogenic gallid herpesvirus type 3 (GaHV-3) strain known as SB-1 was introduced. The complete DNA sequence (165,994 bp) of this GaHV-3 strain was determined using 454 pyrosequencing. A total of 524 open reading frames (ORFs) were examined for homology to protein sequences present in GenBank using BLAST (E-values <0.9). Of the 128 ORF hits, 75 ORFs showed homology to well-characterized alphaherpesviral proteins. Phylogenetically, this strain partitions in its own branch along with the GaHV-3 strain HPRS24 and shows more relatedness to MeHV-1 than gallid herpesvirus type 2 (GaHV-2, Marek's disease virus). When comparing the GaHV-3 ORFs to their homologues in MeHV-1 and GaHV-2, a greater percentage of amino acid similarity was found with homologous ORFs in the genome of SB-1 than with those in the HPRS24 genome. Overall, twice as many of the 75 ORFs within the SB-1 genome showed greater sequence identities and similarities to homologous ORFs in the Marek's disease genome than those within the HPRS24 genome. This paper describes the sequence difference between the two GaHV-3 genomes. Overall 19 ORFs differ in the number of predicted amino acids; of these, eight (U(L)3.5, U(L)5, U(L)9, U(L)28, U(L)30, U(L)36, U(L)37, and U(L)50) encode well-characterized alphaherpesviral proteins A sequence within the unique short region of the SB-1 genome exhibited significant sequence homology to long terminal repeat (LTR) sequences of avian retroviruses. This sequence was only found in the SB-1 genome and not the HPRS24 genome.

Background: Marek's disease (MD) is an economically important viral disease of chickens caused by Marek's disease virus (MDV), an oncogenic herpesvirus. This disease was well controlled since the widespread use of commercial vaccines, but field MDVs have shown continuous increasing in virulence and acquired the ability to overcome the immune response induced by vaccines. Nowadays, MD continues to be a serious threat to poultry industry, isolation and characterization of MDVs are essential for monitoring changes of viruses and evaluating the effectiveness of existing vaccines., Results: Between 2008 and 2010, 18 field MDV strains were isolated from vaccinated chicken flocks in Sichuan province, China. Three oncogenic genes including Meq, pp38 and vIL-8 genes of the 18 isolates were amplified and sequenced. Homology analysis showed that the deduced amino acid sequences of these three genes exhibit 95.0-98.8%, 99.3-100% and 97.0-98.5% homology respectively with these of other reference strains published in GenBank. Alignment analysis of the nucleotide and deduced amino acid sequences showed that four amino acid mutations in Meq gene and two amino acid mutations in vIL-8 gene displayed perfect regularity in MDVs circulating in China, which could be considered as features of field MDVs prevalent in recent years in China. In addition, one amino acid mutation in pp38 gene can be considered as a feature of virulent MDVs from USA, and three amino acid mutations in Meq gene were identified and unique in very virulent plus (vv+) MDVs. Phylogenetic analysis based on Meq and vIL-8 protein sequences revealed that field MDVs in China evolved independently. Virulence studies showed that CVI988 could provide efficient protection against the field MDVs epidemic recently in China., Conclusions: This study and other published data in the GenBank have demonstrated the features of Meq, pp38 and vIL-8 genes of MDVs circulating in recent years in Sichuan, China. Mutations, deletions or insertions were observed in these three genes, and some mutations could be considered as the unique marks of the MDVs circulating presently in China. The paper supplies some valuable information concerning the evolution of MDV which is useful for the vaccine development and control of MD in China.

Some herpesviruses, particularly lymphotropic viruses such as Marek's disease virus (MDV) and human herpesvirus 6 (HHV-6), integrate their DNA into host chromosomes. MDV and HHV-6, among other herpesviruses, harbor telomeric repeats (TMRs) identical to host telomeres at either end of their linear genomes. Using MDV as a natural virus-host model, we show that herpesvirus TMRs facilitate viral genome integration into host telomeres and that integration is important for establishment of latency and lymphoma formation. Integration into host telomeres also aids in reactivation from the quiescent state of infection. Our results and the presence of TMRs in many herpesviruses suggest that integration mediated by viral TMRs is a conserved mechanism, which ensures faithful virus genome maintenance in host cells during cell division and allows efficient mobilization of dormant viral genomes. This finding is of particular importance as reactivation is critical for virus spread between susceptible individuals and is necessary for continued herpesvirus evolution and survival.

Background: Studies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV)., Methods: The VP1 and VP2 genes of CAV isolate SMSC-1 were amplified and inserted into eukaryotic co-expression vector, pBudCE4.1 to construct pBudVP2-VP1. We also constructed pBudVP2-VP1/VP22 encoding CAV VP2 and the VP22 of MDV-1 linked to the CAV VP1. In vitro expression of the genes was confirmed by using RT-PCR, Western blot and indirect immunofluorescence. The vaccines were then tested in 2-week-old SPF chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. After in vivo expression studies, immune responses of the immunized chickens were evaluated pre- and post-immunization., Results: Chickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV and also proliferation induction of splenocytes in comparison to the chickens vaccinated with pBudVP2-VP1. Furthermore, the pBudVP2-VP1/VP22-vaccinated group showed higher level of the Th1 cytokines IL-2 and IFN-γ., Conclusions: This study showed that MDV-1 VP22 gene is capable of enhancing the potency of DNA vaccine against CAV when fused with the CAV VP1 gene., (© 2011 Moeini et al; licensee BioMed Central Ltd.)

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

Background: Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in immunosuppression, which is considered to be an integral aspect of the pathogenesis of Marek's disease (MD). A recent study showed that deletion of the Meq gene resulted in loss of transformation of T-cells in chickens and a Meq-null virus, rMd5ΔMeq, could provide protection superior to CVI988/Rispens., Results: In the present study, to investigate whether the Meq-null virus could be a safe vaccine candidate, we constructed a Meq deletion strain, GX0101ΔMeq, by deleting both copies of the Meq gene from a pathogenic MDV, GX0101 strain, which was isolated in China. Pathogenesis experiments showed that the GX0101ΔMeq virus was fully attenuated in specific pathogen-free chickens because none of the infected chickens developed Marek's disease-associated lymphomas. The study also evaluated the effects of GX0101ΔMeq on the immune system in chickens after infection with GX0101ΔMeq virus. Immune system variables, including relative lymphoid organ weight, blood lymphocytes and antibody production following vaccination against AIV and NDV were used to assess the immune status of chickens. Experimental infection with GX0101ΔMeq showed that deletion of the Meq gene significantly decreased immunosuppression in chickens caused by pathogenic MDV., Conclusion: These findings suggested that the Meq gene played an important role not only in tumor formation but also in inducing immunosuppressive effects in MDV-infected chickens.

Bacterial artificial chromosome (BAC) vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs) are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130) of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).

Viruses cause about 15% of the cancers that are still the leading causes of human mortality. The discovery of viral oncogenes has enhanced our understanding of viral oncogenesis. However, the underlying molecular mechanisms of virus-induced cancers are complex and require further investigation. The present study has attempted to investigate the effects of the microRNAs (miRNAs) encoded by Marek's disease virus 1 (MDV1), a chicken herpesvirus causing acute T-cell lymphomas and solid visceral tumors in chickens, on anti-cancer drug-induced apoptosis and identify the targets of the miRNAs. The results showed that of the total 14 miRNAs encoded by MDV1, MDV1-miR-M3 significantly promoted cell survival under treatment with cisplatin, a widely used chemotherapy drug. MDV1-miR-M3 suppressed cisplatin-induced apoptosis by directly downregulating expression at the protein but not the mRNA level of Smad2, a critical component in the transforming growth factor β signal pathway. Our data suggest that latent/oncogenic viruses may encode miRNAs to directly target cellular factors involved in antiviral processes including apoptosis, thus proactively creating a cellular environment beneficial to viral latency and oncogenesis. Furthermore, the knowledge of the apoptosis resistance conferred by viral miRNAs has great practical implications for improving the efficacy of chemotherapies for treating cancers, especially those induced by oncogenic viruses.

Marek's disease is a highly contagious disease of poultry characterized by rapid-on set of T-cell lymphomas, which is caused by Marek's disease virus (MDV), but its pathogenic mechanism is still not very clear. Recently, some new progress were achieved in molecular character of MDV. Along with the genomic sequencing of MDV serotype 1, some novel open reading frames (ORFs) were predicted, and ORF72.2 was one of them which have no homologues in other MDV serotypes or in other alphaherpesvirus. In the study, ORF72.2 was firstly identified as a protein-coding gene by the method of reverse transcription polymerase chain reaction (RT-PCR), western blotting and indirect immunofluorescence assay. This study paved the way to conduct further studies to determine whether ORF72.2 plays a role in MDV replication and pathogenicity.

Herpesviruses are members of a diverse family of viruses that colonize all vertebrates from fish to mammals. Although more than one hundred herpesviruses exist, all are nearly identical architecturally, with a genome consisting of a linear double-stranded DNA molecule (100 to 225 kbp) protected by an icosahedral capsid made up of 162 hollow-centered capsomeres, a tegument surrounding the nucleocapsid, and a viral envelope derived from host membranes. Upon infection, the linear viral DNA is delivered to the nucleus, where it circularizes to form the viral episome. Depending on several factors, the viral cycle can proceed either to a productive infection or to a state of latency. In either case, the viral genetic information is maintained as extrachromosomal circular DNA. Interestingly, however, certain oncogenic herpesviruses such as Marek's disease virus and Epstein-Barr virus can be found integrated at low frequencies in the host's chromosomes. These findings have mostly been viewed as anecdotal and considered exceptions rather than properties of herpesviruses. In recent years, the consistent and rather frequent detection (in approximately 1% of the human population) of human herpesvirus 6 (HHV-6) viral DNA integrated into human chromosomes has spurred renewed interest in our understanding of how these viruses infect, replicate, and propagate themselves. In this review, we provide a historical perspective on chromosomal integration by herpesviruses and present the current state of knowledge on integration by HHV-6 with the possible clinical implications associated with viral integration.

The tumor suppressor protein p53 plays a role in cellular responses to cancer-initiating events by regulating progress through the cell cycle. Several recent studies have shown that p53 transactivates expression of the members of the proapoptotic microRNA-34 family, which are underexpressed in several cancers. We demonstrate here that the latency-associated cluster of microRNAs (miRNA) encoded by an oncogenic herpesvirus, gallid herpesvirus 2 (GaHV-2), is a direct target of p53. Robust transcriptional activity was induced in three avian cell lines by a sequence mapping 600 base pairs (bp) upstream of the cluster of miRNAs. We found transcription start sites for the pri-miRNA transcript at the 3' end of this transcription-inducing sequence. The promoter has no consensus core promoter element, but is organized into a variable number of tandem repeats of 60-bp harboring p53-responsive elements (RE). The minimal functional construct consists of two tandem repeats. Mutagenesis to change the sequence of the p53 RE abolished transcriptional activity, whereas p53 induction enhanced mature miRNA expression. The identification of a viral miRNA promoter regulated by p53 is biologically significant, because all avirulent GaHV-2 strains described to date lack the corresponding regulatory sequence, whereas all virulent, very virulent, and hypervirulent strains possess at least two tandem repeats harboring the p53 RE.

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek's disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek's disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

Marek's disease is a devastating neoplastic disease of chickens caused by Marek's disease virus (MDV). MDV down-regulates surface expression of MHC class I molecules, although the mechanism has remained elusive. MDV harbors a UL49.5 homolog that has been shown to down-regulate MHC class I expression in other Varicelloviruses. Using in vitro assays, we showed that MDV pUL49.5 down-regulates MHC class I directly and identified its cytoplasmic tail as essential for this function. In vivo, viruses lacking the cytoplasmic tail of pUL49.5 showed no differences in MD pathogenesis compared to revertant viruses in highly susceptible chickens of the B(19)B(19) MHC class I haplotype, while there was a mild reduction in pathogenic potential of the deletion viruses in chickens more resistant to MD pathogenesis (MHC:B(21)B(21)). We concluded that the pathogenic effect of MHC class I down-regulation mediated by pUL49.5 is small because virus immune evasion possibly requires more than one viral protein., (Copyright 2010 Elsevier Inc. All rights reserved.)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)(n). Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity.

Objective: To evaluate the immuno-protective effect of GX0101 deltameq-BAC containing an infectious meq-null Marek's disease virus genome., Method: One-day-old SPF birds were reared separately in isolators with positive filtered air. On day 1 of age, chickens immunized with 101 microg of GX0101A deltameq-BAC suspended in PBS, challenge infection with 500PFU very virulent rMD5 was performed at day 5 and 12 post-immunization separately. During 90 days after challenge, all bird were recorded and checked for necropsy. The samples of heart and liver were collected for histo-sections., Results: The protective index of the two vaccines used was 87 and 33 for CVI988/Rispens and GX0101 deltameq-BAC, respectively, after challenged with the very virulent virus rMd5 at day 5 post-immunization. When challenged with rMd5 at day 12 post-immunization, the protection index of GX0101 deltameq-BAC increased to 53%., Conclusion: Except that GX0101 deltameq-BAC can confer protection against very virulent Marek's disease virus, a delay in the development of Marek's disease could be observed in some chickens vaccinated with GX0101 deltameq-BAC. On the other hand, compared with CVI988/Rispens, the reconstruction of GX0101 deltameq-BAC in the body is a prerequisite for access to protection. Therefore, there is a blank period after immunization, which provides a chance for infection with the wild Marek's disease virus.

The use of avian herpesviruses (Marek's disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10(3)PFU/chicken, whereas 10(4)PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Marek' disease virus serotype-1, also know as Gallid herpesvirus 2 (GaHV-2), elicits T-cell lymphomas in chickens. The GaHV-2 genome encodes an oncoprotein, Meq, with similarity to the Jun/Fos family of proteins. We have previously shown that Meq homodimers are not sufficient to induce lymphomas in chickens. In this study, we investigated the role of Meq heterodimers in the pathogenicity of GaHV-2 by generating a chimeric meq gene, which contains the leucine zipper region of Fos (meqFos). A recombinant virus containing the meqFos gene in place of parental meq, rMd5-MeqFos, was not capable of transforming chicken lymphocytes, indicating that heterodimerization of Meq alone is not sufficient for transformation. In addition, the recovery of the oncogenic phenotype by a recombinant virus encoding one copy each of MeqGCN (homodimer) and MeqFos (heterodimer) conclusively demonstrates that both homo and heterodimerization of Meq are required for oncogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Oxidative stress contributes to the accumulation of genomic abnormalities, prevents cellular apoptosis, and also mediates immunosuppression resulting in tumor formation. Marek's Disease provides excellent opportunities for the study of herpesvirus-induced tumors both in experimental- and natural conditions. The aim of this study was to examine the effects of Marek's Disease (MD) on basal levels of DNA strand breaks and on the oxidative-antioxidative status of chickens with MD. White-Lohmann hens-fifteen infected with Marek's Disease Virus (MDV) and fifteen healthy-of same age and sex were included in this study. MD infection was diagnosed via clinical signs, gross- and micro-pathological findings and also by detection of viral antigens in feather follicle epithelium by the indirect immunoperoxidase method. Compared with healthy controls, DNA damage was greater and levels of malondialdehyde (MDA) and plasma protein carbonyl (PCO), and plasma concentration of nitric oxide metabolites (NOx) higher in the MD group. Furthermore, total antioxidant activities (AOAs) were found lowered and glutathione (GSH) levels reduced in the MD group compared to the control group. Significantly positive correlation was found between DNA damage, MDA, PCO, and NOx in the MD group. DNA strand breaks were found negatively associated with AOA and GSH concentrations in the MD group. Our results demonstrated that oxidative stress markers and DNA damage substantially increased in chickens with MD, which indicated that increased DNA damage levels might be related to the increased oxidative stress and reduced antioxidant activity.

The main goal of the present study was to analyse the nucleotide and amino acid sequence of Meq--the main oncoprotein of Marek's disease virus. The conducted analysis of the nucleotide sequences of MDV has shown a significant similarity between Polish and reference strains of MDV assigned to the vvMDV and vv+MDV pathotypes. Some of the detected nucleotide point mutations were specific only for Polish strains. The sequence analysis has shown a number of single substitutions and two poly-amino acid insertions among strains defined as mild-pathogenic (mMDV) and one attenuated vaccine strain CVI988--Rispens. These results indicate that there is a close relation between changes in nucleotide or amino acid sequence of meq and the virulence of Polish MDV strains.

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces the rapid onset of T-cell lymphomas in poultry. The MDV-encoded oncoprotein Meq plays an important role in oncogenicity, as its deletion abolishes the ability of the virus to induce tumours. It has been shown previously that Meq oncogenicity is linked to its interaction with C-terminal binding protein 1 (CtBP), a property also shared by other virus-encoded oncoproteins such as adenovirus E1A and Epstein-Barr virus EBNA3A and -3C. Therefore, this study examined whether Meq also shares the properties of these viral oncoproteins in interacting with other binding partners such as heat-shock protein 70 (Hsp70), a molecular chaperone protein linked to multiple cellular functions including neoplastic transformation. Confocal microscopic analysis demonstrated that MDV infection induced nuclear accumulation of Hsp70 and its co-localization with Meq. Biochemical evidence of Meq-Hsp70 interaction was obtained by two-way immunoprecipitation with Meq- and Hsp70-specific antibodies. To demonstrate further the Meq-Hsp70 interaction in virus-induced lymphomas, recombinant MDV was generated expressing an N-terminal tandem affinity purification (TAP) tag-fused Meq by mutagenesis of the infectious BAC clone of the oncogenic MDV strain RB-1B. Demonstration of Hsp70 in the TAP-tag affinity purified Meq from tumours induced by the recombinant virus, using quadrupole time-of-flight tandem mass spectrometry analysis, further confirmed the Meq-Hsp70 interaction in the transformed lymphocytes. Given the well-documented evidence of the tumorigenic properties of Hsp70 and its interaction with a number of other known viral oncoproteins, demonstration of the interaction of Meq and Hsp70 is significant in MDV oncogenesis.

Aim: To study the transduction and localization mechanism of Marek's disease virus serotype 1 (MDV-1) CVI988 VP22 in different cells., Methods: VP22 was expressed with recombinant adenovirus and identified by immunofluorescence assay (IFA) and Western blot. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells to identify the transduction property of VP22. AD-293 cells infected with recombinant virus were fixed to investigate localization of VP22 at different time post infection. Transient expression of VP22 in AD-293 cells was carried out for control., Results: The results showed that the VP22 expressed by recombinant adenovirus entered almost all the monolayer cells, which indicate the VP22 remains its transduction property. The VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm of 293 cells infected with recombinant adenovirus, compared with the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells., Conclusion: The VP22 presented a different localization pattern in cells infected with different recombinant virus.

Marek's disease virus type 1 (MDV-1) shows a strict dependency on the direct cell-to-cell spread for its propagation in cell culture. As MDV-1 shows an impaired nuclear egress in cell culture, we wished to address the characterization of capsid/tegument genes which may intervene in the maturation of intranuclear capsids. Orthologs of UL17 are present in all herpesviruses and, in all reported case, were shown to be essential for viral growth, playing a role in capsid maturation and DNA packaging. As only HSV-1 and PrV UL17 proteins have been characterized so far, we wished to examine the role of MDV-1 pUL17 in virus replication. To analyze MDV-1 UL17 gene function, we created deletion mutants or point mutated the open reading frame (ORF) to interrupt its coding phase. We established that a functional ORF UL17 is indispensable for MDV-1 growth. We chose to characterize the virally encoded protein by tagging the 729 amino-acid long protein with a repeat of the HA peptide that was fused to its C-terminus. Protein pUL17 was identified in infected cell extracts as an 82 kDa protein which localized to the nucleus, colocalizing with VP5, the major capsid protein, and VP13/14, a major tegument protein. By using green fluorescent protein fusion and HA tagged proteins expressed under the cytomegalovirus IE gene enhancer/promoter (P(CMV IE)), we showed that MDV-1 pUL17 nuclear distribution in infected cells is not an intrinsic property. Although our results strongly suggest that another viral protein retains (or relocate) pUL17 to the nucleus, we report that none of the tegument protein tested so far were able to mediate pUL17 relocation to the nucleus.

Gallid herpesvirus 2 (GaHV-2), commonly known as Marek's disease virus serotype-1 (MDV-1), causes T cell lymphomas in chickens. Vaccines prepared from the attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988/Rispens is non-oncogenic, it codes for at least two forms of the MDV oncoprotein Meq, and these proteins (CVI-Meq and CVI-LMeq) have not been fully characterized. Here, we report that both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), were capable of transforming Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-LMeq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, Meq proteins of both Md5 and CVI988 bound the meq promoter in a ChIP assay regardless of whether CK-Jun was co-expressed. To understand the role of Meq DNA binding and transactivation/repression domains in transcription, we constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins, unlike CVI-Meq proteins, transactivated the meq promoter, the activation was significantly less than Md5-Meq. To determine the role of individual amino acids, point mutations were introduced corresponding to the amino acid changes of CVI-Meq into Md5-Meq. Amino acid residues at positions 71 and 320 of the Md5-Meq protein were found to be important for transactivation of the meq promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. Although no significant differences were observed, decreased transactivation activity was observed with CVI-Meq proteins when compared to Md5-Meq. Collectively, the data presented here indicate that CVI-Meq proteins are generally weak transactivators, which might contribute to the non-oncogenic phenotype of CVI988 virus in chickens.

Methods for the isolation of DNA from cell-associated herpesviruses have often yielded samples contaminated with host cellular DNA. Because 2nd and 3rd generation nucleotide sequencers do not rely on molecular cloning of viral DNA, there is a need to develop methods for isolating highly pure DNA from these viruses. The cell-associated alphaherpesvirus Marek's disease virus (MDV-1) was chosen as a test virus for the development of such methodologies. The genomes of six MDV-1 strains have previously been sequenced using both Sanger dideoxy sequencing and 454 Life Sciences pyrosequencing. These genomes largely represent cell culture adapted strains due to the difficulty in obtaining large quantities of DNA from true low passage isolates. There are clear advantages in analyzing MDV-1 virus taken directly from infected tissues or low passage isolates since serial passage attenuates the virus. Procedures using an ATP-dependent exonuclease and Phi29 DNA polymerase to degrade host DNA selectively and amplify MDV-1 DNA enzymatically from total DNA preps were attempted without much success. Ultimately, however, a protocol was developed for purification of low passage MDV-1 DNA from infected avian fibroblasts. The method builds upon and extends available protocols based on hypotonic lysis to release virus particles followed by micrococcal nuclease treatment to degrade cellular DNA. Intact high-molecular weight viral DNA is purified away from an excess of degraded cellular DNA using polyethylene glycol precipitation. 454-based pyrosequencing of viral DNA purified in this manner has generated data containing as little as 2.3% host sequence. On average, DNA preparations were 70% (+/-20%) pure yielding a genome coverage range of 25-74-fold.

Objective: To find catalytic center of MDV-1 UL13 and express it in vitro to investigate the function of UL13 kinase., Methods: UL13 gene was amplified by polymerase chain reaction (PCR) from MDV-1 CVI988/Rispens strain. The codon bias and antigenicity of UL13 in Escherichia coli was analyzed by online service GENEART (www.gcua.de)and DNAstar software respectively. Then the UL13 truncated fragments were expressed in Escherichia coli, and mice were immunized with the expressed Glutathione S Transferase fusion protein. The conserved domain was analyzed with protein blast and Cn3D 4.1 online software of National Center for Biotechnology Information., Results: UL13 gene was successfully amplified. The sequence analysis suggests that 259-400 and 431-513 amino acid residues are low abundance for rare codon and strong antigenicity in UL13. Result of conserved domain analysis demonstrated that 152-297 residue iskinase catalytic center of UL13. However, conserved glycin in kinase subdomain VII for most protein kinase was replaced by serine in UL13 and proline in kinase subdomain VIII replaced by cysteine. The serum from mice immunized with truncated fragment, 259-400 amino acids, could react with recombinant UL13 protein expressed in insect cells in immunofluorenscence assay., Conclusion: The 152-297 residue is kinase catalytic center of MDV-1 UL13; UL13 protein expressed in vitro induced specific antibodies against UL13.

Live attenuated herpesvirus vaccines such as herpesvirus of turkey (HVT) have been used since 1970 for the control of Marek's disease (MD), a highly infectious lymphoproliferative disease of poultry. Despite the success of these vaccines in reducing losses from the disease, Marek's disease virus (MDV) strains have shown a continuing increase in virulence, presumably due to the inability of the current vaccines in preventing MDV replication. The highly specific and effective nature of RNA interference (RNAi) makes this technology particularly attractive for new antiviral strategies. In order to exploit the power of RNAi-mediated suppression of MDV replication in vivo delivered through existing vaccines, we engineered recombinant HVT expressing short hairpin RNA (shRNA) against MDV genes gB and UL29. The levels of protection induced by the RNAi-expressing HVT against virulent virus challenge were similar to the parent pHVT3 virus. However, chickens vaccinated with recombinant HVT expressing shRNA showed moderate reduction of challenge virus replication in blood and feather samples. Delivery of RNAi-based gene silencing through live attenuated vaccines for reducing replication of pathogenic viruses is a novel approach for the control of infectious diseases.