The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Molecular characterization and phylogenetic analysis of Marek's disease virus from clinical cases of Marek's disease in Egypt.

In spite of the intensive vaccination policy against the Marek's disease virus (MDV) in Egypt, the Egyptian poultry flocks are still suffering from several tumor and paralysis cases. To investigate if MDV is the possible cause, feather follicle and tumor samples were collected during 2011 from 30 vaccinated chicken flocks experiencing nervous signs, emaciation, and tumor lesions. The samples were screened by PCR amplification of the meq full-length gene. Only five of 30 flocks were positive for MDV. Additionally, we sequenced meq from five samples and gL and gC from three samples. A phylogenetic tree was constructed using the deduced amino acid sequences of the meq gene. The sequence analysis revealed that most of the studied sequences showed > or = 98% identity to the very virulent European ATE and C12/130 isolates and the very virulent Chinese LMS, YA, WS03, and GX070060 MDV isolates. The two glycoproteins, gL and gC, displayed high similarity to the classical MDV strains published in the database regardless of their virulence.

Prevalence of Marek's disease virus in different chicken populations in Iraq and indicative virulence based on sequence variation in the ecoRI-q (meq) gene.

A cross-sectional survey was conducted in six provinces in southern Iraq to determine the point prevalence of Marek's disease virus (MDV) in different chicken populations followed by sequencing the meq gene for phylogenetic analysis and virulence-associated polymorphisms. A total of 109 samples from unvaccinated flocks were analyzed comprising 52 dust and 30 spleen samples from commercial broiler farms and 27 spleens from local layer chickens purchased in the town markets. The overall prevalence of MDV was 49.5% with no significant differences between provinces (P = 0.08) or sample types (P = 0.89). Prevalence ranged from 36.8% in Karbala and Nasiriyah to 65% in Amarah. The percentages of positive samples were 59.1%, 46.7%, and 48.1% in broiler dust, broiler spleen, and layer spleen, respectively. The overall mean (+/- SEM) Log10 MDV viral copy number per milligram of dust or spleen as determined by quantitative PCR was 1.78 +/- 0.19, with no significant differences between provinces (P = 0.10) or sample types (P = 0.38). In positive samples only, the overall mean was 3.43 +/- 0.18. Sequencing of the meq gene from samples that showed high levels of MDV target in qPCR testing was attempted. Nine samples were sequenced. These sequences were compared with meq sequences of MDVs of different pathotype. All the Iraqi MDVs had a short meq gene of 897 base pairs because of the deletion of 123 bp relative to the reference strain Md5. The Iraqi meq sequences also contained single-nucleotide polymorphisms, resulting in differences in the amino acid sequence. All of the nine Iraqi meq genes encoded two repeats of four-proline sequences. The published negative association between four-proline repeat number and MDV virulence suggests that the Iraqi MDVs are likely to be highly virulent, but this needs to be confirmed by in vivo testing. Taken together, these results indicate that MDV is common in unvaccinated commercial and village chickens in southern Iraq, that there is limited meq gene sequence variation, that all sequenced samples had a short meq with two four-proline repeats, and that this is consistent with a high level of virulence.

Sequence analysis of the Meq gene in the predominant Marek's disease virus strains isolated in China during 2006-2008.

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.

Comparative genomic sequence analysis of the Marek's disease vaccine strain SB-1.

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.

Interaction of Marek's disease virus oncoprotein Meq with heat-shock protein 70 in lymphoid tumour cells.

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.

[Kinase domain analysis of MDV-1 CVI988/Rispens UL13 and preferred codon fragments expression in Escherichia coli].

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.

Sequence determination of a mildly virulent strain (CU-2) of Gallid herpesvirus type 2 using 454 pyrosequencing.

The complete DNA sequence of the mildly virulent Gallid herpesvirus type 2 strain CU-2 was determined and consists of 176,922 bp with an overall gene organization typical of class E herpesviruses. Phylogenetically, this strain partitions in its own branch between the virulent strains RB-1B, Md11, and Md5, and the vaccine strain CVI988. Overall, the genome of CU-2 is more similar to that of CVI988, with identically sized unique short regions of 11,651 bp. As in CVI988, an insertion of 177 bp was identified in the overlapping genes encoding the Meq, RLORF6, and 23 kDa proteins within the repeat long region of the genome. A total of 15 single nucleotide polymorphisms (SNPs) common to both CU-2 and CVI988, and not occurring in virulent strains, were identified in the genes encoding UL29, UL45, UL50, UL52, LORF10, RLORF14a, RLORF12, Meq(RLORF7), 23kDa, ICP4, US3, and two hypothetical proteins MDV071.4 and MDV076.4. Each gene encoding UL29 and Meq contained two SNPs. Only one major open reading frame (ORF) encoding UL41, the virus host shutoff (VHS) ribonuclease, was disrupted in the CU-2 genome. An additional cytosine after the 25 codon is predicted to produce a truncated protein of 97 aa. Since GaHV-2 mutants lacking UL41 have been reported to retain their virulence, other factors are likely responsible for the low virulence of CU-2. It is largely suspected that SNPs in common with CVI988 along with the insertions in the Meq loci are responsible for its phenotype. Conversely, we identified 43 nonsynonymous mutations (within 23 genes) that may contribute to the virulence of CU-2. These SNPs are shared exclusively with all sequenced virulent strains (Md5, Md11, and RB-1B) and not present within the CVI988 genome. Although most occur in proteins of unknown function, a significant percentage is in proteins involved in virion assembly.

[The N1-18 terminus of Marek's disease virus VP22 is essential for protein transduction].

We previously showed some differences in Marek's disease virus (MDV) VP22 gene between virulent and avirulent strains, in the deletion from 201aa to 206aa, namely 201TKSERT206. In this study, VP22 genes were amplified from strains: CVI988/Rispens and GA. And then the fragments were subcloned into pcDNA3.1/zeo(+), respectively, which were co-expressed with an enhancer green fluorescent protein (EGFP) after transfection into COS-1 cells. As with both human herpesvirus 1 and bovine herpesvirus 1 VP22-EGFP fusion proteins, the subcellular localization of the three MDV EGFP-VP22 products revealed few differences, which bind to microtubules and nucleus membrane, and then to heterochromatin. In addition, VP22s also bind to centrosomes and inter-membrane. During mitosis, EGFP-VP22s bind to sister chromatids, but dissociates from the centrosomes and the microtubules of the mitotic spindle. In truncated fragments' transfection experiments, stained with the specific monoclonal antibody against VP22, it concluded that the full length of VP22 was required for protein transduction, and N1-18aa was essential to VP22 translocating from cytoplasm to nucleus as a potential nucleus localization site in the absence of other viral factors in MDV-1.

Prevalence and molecular characterization of meq in feather follicular epithelial cells of Korean broiler chickens.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens. Meq is the relevant oncogene and four isoforms, long (L)-meq, meq, short (S)-meq and very short (VS)-meq, have been identified. Although MD is important in the poultry industry, the prevalence and molecular properties of Korean MD virus (MDV) among broiler chickens remain unclear. Therefore, we characterized meq in pooled feather tips sampled at 3- and 5-week-old chickens from 21 unvaccinated and 22 vaccinated broiler farms via nested-PCR and nucleotide sequence analysis. Multiple bands consisting of L-meq, meq, and S-meq amplicons were observed in a commercial vaccine (CVI988 + HVT), 1 (4.8%) and 5 samples (22.7%) from unvaccinated and vaccinated farms, respectively. A strong meq amplicon was observed in a MD-related tumor tissue, 6 (28.6%) and 1 (4.5%) samples from unvaccinated and vaccinated farms, respectively. Six and one amplicons from unvaccinated (28.6%) and vaccinated farms (4.5%), respectively, were differentiated from CVI988 by nucleotide sequence analysis. Therefore, the relatively high rate of meq in the unvaccinated broiler farms constitutes support for vaccination. However, the existence of CVI988-related meq in unvaccinated chickens necessitates further study regarding the origins and pathoimmunological effects of the viruses on chickens.

Characterization of a very virulent Marek's disease virus mutant expressing the pp38 protein from the serotype 1 vaccine strain CVI988/Rispens.

Marek's disease virus (MDV), a highly cell-associated oncogenic chicken herpesvirus, causes Marek's disease in domestic chickens. A unique phosphoprotein of MDV, pp38, has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. However, recently, the biological properties of a deletion mutant virus (rMd5Deltapp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. Thus, pp38 is important for early cytolytic infection and not for transformation. The pp38 protein of the MDV serotype 1 vaccine strain CVI988/Rispens differs by one amino acid when compared to the pathogenic strains of MDV. Monoclonal antibody, H19, recognizes all serotype 1 MDV strains except CVI988/Rispens. Previous studies have also shown that the unique pp38 epitope in CVI988/Rispens induced high antibody response. In order to study the role of this epitope in the protective properties of CVI988/Rispens, we generated a mutant rMd5 virus in which the wild type pp38 gene has been substituted with that of CVI988/Rispens (rMd5/pp38CVI). The replication properties of rMd5/pp38CVI, both in vitro and in vivo, and tumor induction were examined. We found that the biological properties of rMd5/pp38CVI were similar to the wild type rMd5 virus with regards to in vivo replication, antibody response and tumor induction. This shows that the pp38 derived from CVI988/Rispens is not involved in protective properties as was previously suggested.

Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication.

Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5DeltaMeq virus by deleting both copies of the meq gene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable for in vitro virus replication. In vivo replication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5DeltaMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5DeltaMeq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of the meq gene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.

Identification of the Marek's disease virus serotype 2 genes homologous to the glycoprotein B (UL27), ICP18.5 (UL28) and major DNA-binding protein (UL29) genes of herpes simplex virus type 1.

We determined the nucleotide sequence of non-pathogenic Marek's disease virus serotype 2 (MDV2) strain HPRS24 glycoprotein B (gB) (UL27), ICP18.5 (UL28) and major DNA-binding protein (MDBP) (UL29) genes homologous to herpes simplex virus type 1 (HSV-1). The sequence data revealed that important motives in the proteins are conserved in MDV2 ICP18.5 and MDBP, however the sequence of viral DNA replication origin which exists in the regions between the UL29 and UL30 genes of other alphaherpesviruses was not found in the regions of the MDV2 genome. By northern blot analyses, we also demonstrated that 8.9, 5.0 and 2.6 kb transcripts were actually transcribed from the sequenced region in MDV2-infected cells. The MDV2 UL28 and UL29 genes have not been reported in other serotypes of MDV.

Sequence and initial characterization of the U(L)10 (glycoprotein M) and U(L)11 homologous genes of serotype 1 Marek's Disease Virus.

The nucleotide sequence of the U(L)10 (glycoprotein M) and the U(L)11 homologs of Marek's Disease Virus 1 strain GA was determined. The U(L)10 open reading frame encodes a type III membrane protein of 424 amino acids that contains eight hydrophobic domains and two consensus N-linked glycosylation sites. The U(L)11 homologous gene encodes an 84 amino acid polypeptide, and contains a highly conserved myristylation site at its aminoterminus. By analysis of infected-cell RNA with strand-specific RNA probes, transcription of both U(L)10 and U(L)11 in infected cells was demonstrated. Coupled in vitro transcription-translation confirmed that the U(L)10 product is a 47 kD N-glycosylated viral protein that aggregated upon boiling, whereas the U(L)11 protein exhibited a size of 12 kD after in vitro translation.

Identification and characterization of glycoprotein H of MDV-1 GA strain.

A 2439 bp open reading frame (ORF) was identified from the DNA sequence of BamHI-F and -K2 fragments of Marek's disease virus of serotype 1 (MDV-1) GA strain, which predicts an 813 amino acid polypeptide. This peptide is homologous to HSV-1 gH, and has typical glycoprotein features. There are nine potential N-linked glycosylation sites within the extracellular domain. A fragment of the gH ORF was cloned into pGEX vector in frame with glutathione S-transferase (GST) to produce a GST-gH fusion protein in Escherichia coli. The GST-gH fusion protein was used to develop gH monoclonal and polyclonal antibodies. Expression of gH was detected in duck embryo fibroblasts (DEFs) infected with MDV-1 GA strain by immunofluorescence assay (IFA) with these antibodies. Virus neutralization and plaque-forming inhibition analyses were conducted with the gH antiserum. There were no neutralization and plaque-forming inhibition activities of gH antiserum. Comparison of the DNA sequence of gH gene between GA and RB1B strains of MDV-1 revealed major difference in the upstream control elements of gH ORF.

Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain.

A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.