Fine-Scale Genetic Structure in the United Arab Emirates Reflects Endogamous and Consanguineous Culture, Population History, and Geography.

The indigenous population of the United Arab Emirates (UAE) has a unique demographic and cultural history. Its tradition of endogamy and consanguinity is expected to produce genetic homogeneity and partitioning of gene pools while population movements and intercontinental trade are likely to have contributed to genetic diversity. Emiratis and neighboring populations of the Middle East have been underrepresented in the population genetics literature with few studies covering the broader genetic history of the Arabian Peninsula. Here, we genotyped 1,198 individuals from the seven Emirates using 1.7 million markers and by employing haplotype-based algorithms and admixture analyses, we reveal the fine-scale genetic structure of the Emirati population. Shared ancestry and gene flow with neighboring populations display their unique geographic position while increased intra- versus inter-Emirati kinship and sharing of uniparental haplogroups, reflect the endogamous and consanguineous cultural traditions of the Emirates and their tribes.

Identification of a neurovirulence factor from Marek's disease virus.

In addition to tumors, Marek's disease (MD) virus (MDV) can induce a variety of syndromes linked to the central nervous system. In fact, early descriptions of MD suggested that it was a condition affecting mainly the nervous system. Cytokines and other immune-related genes have been suggested to play a crucial role in MDV-mediated neuropathology, but the mechanisms behind the viral-induced neurologic dysfunction are still poorly understood. In the present study we have used reverse genetic strategies to show that pp14 is not involved in the oncogenic phenotype of MDV1 and is not required for viral replication; however, we provide evidence indicating that the absence of pp14 expression is correlated with increased survival of MDV1-infected chickens, and that its expression is associated with enhanced viral neurovirulence. Our data identify for the first time pp14 as a neurovirulence factor from MDV1 and open the possibility to investigate the molecular mechanisms by which pp14 mediates the damage to the avian nervous system.

Pathogenicity of a very virulent strain of Marek's disease herpesvirus cloned as infectious bacterial artificial chromosomes.

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.

Identification of an intercistronic internal ribosome entry site in a Marek's disease virus immediate-early gene.

In this study, we have identified an internal ribosome entry site (IRES) from the highly infectious herpesvirus Marek's disease virus (MDV). The IRES was mapped to the intercistronic region (ICR) of a bicistronic mRNA that we cloned from the MDV-transformed CD4(+) T-cell line MSB-1. The transcript is a member of a family of mRNAs expressed as immediate-early genes with two open reading frames (ORF). The first ORF encodes a 14-kDa polypeptide with two N-terminal splice variants, whereas the second ORF is contained entirely within a single exon and encodes a 12-kDa protein also known as RLORF9. We have shown that the ICR that separates the two ORFs functions as an IRES that controls the translation of RLORF9 when cap-dependent translation is inhibited. Deletion analysis revealed that there are two potential IRES elements within the ICR. Reverse genetic experiments with the oncogenic strain of MDV type 1 indicated that deletion of IRES-controlled RLORF9 does not significantly affect viral replication or MDV-induced mortality.

Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek's disease.

Herpesvirus of turkey (HVT) is an alphaherpesvirus that is widely used as a live vaccine against Marek's disease because of its antigenic relationship with Marek's disease virus (MDV). In spite of a similar genome structure, HVT has several unique genes, the functions of which are not completely understood. As a first step in carrying out detailed analysis of the functions of the HVT genes, a full-length infectious bacterial artificial chromosome (BAC) clone of HVT was constructed. DNA from two independent BAC clones, upon transfection into chicken embryo fibroblasts, produced plaques similar to those produced by the wild-type virus. Viruses derived from the BAC clones were stable during in vitro passage, but showed differences in in vitro growth kinetics compared with the wild-type virus. Using a one-step mutagenesis protocol to delete the essential glycoprotein B gene from the HVT genome, followed by construction of the revertant virus, BAC clones of HVT were shown to be amenable to standard mutagenesis techniques. In spite of the difference in in vitro growth, viruses from both clones induced 100 % protection against infection by the virulent MDV strain RB-1B, indicating that the BAC-derived viruses could be used as vaccines with efficacies similar to that of the parental virus. The construction of HVT BAC is a major step in understanding the functions of HVT genes by exploiting the power of BAC technology. Furthermore, the availability of the BAC clones enables use of HVT as a vector for expressing foreign genes.

Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek's disease virus.

Marek's disease virus (MDV) is an oncogenic herpesvirus that induces fatal T cell lymphomas in chickens. With more than 20 billion doses of vaccine used annually, vaccination constitutes the cornerstone of Marek's disease control. Despite the success of vaccination, evolution of virulence among MDV strains continues to threaten the effectiveness of the current Marek's disease vaccines. MDV-encoded protein MEQ (MDV EcoRI Q) probably acts as a transcription factor and is considered to be the major MDV oncoprotein. MEQ sequence shows a Pro-Leu-Asp-Leu-Ser (PLDLS) motif known to bind C-terminal-binding protein (CtBP), a highly conserved cellular transcriptional corepressor with roles in the regulation of development, proliferation, and apoptosis. Here we show that MEQ can physically and functionally interact with CtBP through this motif and that this interaction is critical for oncogenesis because mutations in the CtBP-interaction domain completely abolished oncogenicity. This direct role for MEQ-CtBP interaction in MDV oncogenicity highlights the convergent evolution of molecular mechanisms of neoplastic transformation by herpesviruses because Epstein-Barr virus oncoproteins EBNA 3A and 3C also interact with CtBP. We also demonstrate that the nononcogenic MDV generated by mutagenesis of the CtBP-interaction domain of MEQ has the potential to be an improved vaccine against virulent MDV infection. Engineering MDV with precisely defined attenuating mutations, therefore, represents an effective strategy for generating new vaccines against this major poultry disease.

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR.

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.