William S. Payne

Multi-platform next-generation sequencing of the domestic Turkey (Meleagris gallopavo): Genome assembly and analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference.

RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short-hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In this study, similar RNAi vectors are shown to inhibit replication of the avian herpesvirus, Mareks disease virus (MDV, also known as gallid herpesvirus type 2), and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. We also describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and employ this vector with multiple targets within the MDV gB gene or within both the gB and ICP4 genes. The use of targets within multiple genes potentially can provide a larger antiviral effect and/or make it more difficult for viral escape mutations to evolve.

Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

ABSTRACT We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing theenv-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 × 106 CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey ( Meleagris gallopavo ) and chicken ( Gallus gallus ) genomes

BackgroundA robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species.ResultsThe turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes.ConclusionThe BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Telomerase Activity and Differential Expression of Telomerase Genes and c-myc in Chicken Cells In Vitro

This study examined telomerase activity and gene expression profiles for three genes in Gallus gallus domesticus: telomerase reverse transcriptase (chTERT), telomerase RNA (chTR), and c‐myc. Expression of these genes was studied in chicken embryonic stem (chES) cells, chicken embryo fibroblasts (CEFs), and DT40 cells using quantitative real‐time polymerase chain reaction. Our results establish that, relative to transcription levels in telomerase‐negative CEFs, chTERT and chTR are up‐regulated in telomerase‐positive chES cells. Transcription levels of chTERT, chTR, and c‐myc are dramatically up‐regulated in telomerase‐positive DT40 cells, relative to CEFs and chES cells. These results are consistent with a model in which telomerase activity is up‐regulated in proliferating embryonic stem cells requiring stable telomeres to endure multiple rounds of cell division; down‐regulated in differentiated, lifespan‐limited cells; and dramatically up‐regulated in immortalized, transformed cells for which uncontrolled proliferation is correlated with c‐myc dysregulation and telomerase activity. Developmental Dynamics 231:14–21, 2004.

Retroviral delivery of RNA interference against Marek’s disease virus in vivo

The process of RNA interference (RNAi) has been exploited in cultured chicken cells and in chick embryos to assess the effect of specific gene inhibition on phenotypes related to development and disease. We previously demonstrated that avian leukosis virus-based retroviral vectors are capable of delivering effective RNAi against Mareks disease virus (MDV) in cell culture. In this study, similar RNAi vectors are shown to reduce the replication of MDV in live chickens. Retroviral vectors were introduced into d 0 chick embryos, followed by incubation until hatching. Chicks were challenged with 500 pfu of strain 648A MDV at day of hatch, followed by assays for viremia at 14 d postinfection. Birds were monitored for signs of Mareks disease for 8 wk. A stem-loop PCR assay was developed to measure siRNA expression levels in birds. Delivery of RNAi co-targeting the MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduced MDV viremia in vivo, although to lesser extents than were observed in cell culture. Concomitant reductions in disease incidence also were observed, and the extent of this effect depended on the potency of the MDV challenge virus inoculum. Successful modification of phenotypic traits in live birds with retroviral RNAi vectors opens up the possibility that such approaches could be used to alter the expression of candidate genes hypothesized to influence a variety of quantitative traits including disease susceptibility.

A new inherited ocular anomaly in pigmented White Leghorn chickens

Inherited ocular anomalies in chickens include several types of microphthalmia, retinal dysplasia, retinal degeneration, cataract, buphthalmos and pop-eye, or keratoconus in White Leghorns.2–5,7,8,12,15–18 In this report, a new ocular anomaly that appeared in pigmented White Leghorns homozygous for a mutation at the dominant white (I) locus is described with emphasis on clinical, gross, and histologic findings to aid in the diagnosis of the ocular lesions. An incompletely dominant mutation, called Smoky Joe (SJ), allows the production of feather pigment, which the I allele inhibits. The SJ mutation originally appeared in ADOL Line 0, a noninbred White Leghorn line maintained at the USDA Avian Disease and Oncology Laboratory (ADOL) in East Lansing, Michigan. Females homozygous for SJ have dark grey feathers with barring and homozygous males have much lighter colored plumage. This dimorphism in color is the result of the dilution that occurs with two copies of sexlinked barring in the males. The plumage of heterozygous birds (I, SJ) is intermediate in color compared to either parental type. Phthisis bulbi was noticed in adult females of the second generation of the homozygous SJ population. None of the parents of these females had apparent ocular lesions. Approximately 30% of the 85 surviving females had phthisis bulbi, while none of the 100 surviving males were affected. There was no clinical evidence of bacterial or viral infection, and the chickens were housed in pens isolated from chickens with experimental viral infections. Because the ocular anomaly appeared to be recessive and sex-linked, 4 test matings were designed to further study the inheritance. In matings 1 and 2, sighted SJ males, which were suspected of being carriers of the syndrome, were mated to either affected or sighted SJ females. These same male birds were then mated to either sighted females of Line 0 (II) (mating 3) or sighted females of ADOL Line 15I5 (II) and Line 0 (II) (mating 4). All chicks had ophthalmic examinations at hatch and 2, 4, and 8 weeks of age. In mating 1,197 chicks were examined. Of the females, 62% were affected, and 10% of the males were affected.