Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins.

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell-mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (∼0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.

Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle.

Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.

Differential expression of genes for transcription factors in theca and granulosa cells following selection of a dominant follicle in cattle.

Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells).

Association of the prion protein and its expression with ovarian follicle development in cattle.

The cellular form of the prion protein (PrP(C)) has been detected in many tissues including reproductive tissues. While its function is unclear, it has been suggested to act as a receptor for an unidentified ligand and/or as an antioxidant agent. We tested the hypothesis that PrP(C) is differentially expressed in dominant, growing, compared to subordinate bovine ovarian follicles. Using both microarray analysis and quantitative real-time PCR, the level of prion protein mRNA (Prnp) in both theca and granulosa cells was measured. We found that levels of Prnp were significantly higher in the theca cells of dominant compared to subordinate follicles but similar among granulosa cells from different follicles. This difference was apparent immediately after selection of the dominant follicle and continued to the dominance stage of the follicle wave. Levels of the protein for PrP(C) were also higher (P < 0.05) in theca cells of dominant compared to subordinate follicles. In conclusion, elevated PrP(C) was associated with ovarian follicle growth and development and we suggest that it may play a role in the success of follicle development.

Use of gene expression microarrays for evaluating environmental stress tolerance at the cellular level in cattle.

Selecting domestic animals for tolerance to thermal stress (TS) has been counterproductive, because acclimation involves reducing or diverting metabolizable energy from production to balance heat gain and loss. Ideally, simultaneous selection for increased production and improved thermotolerance is desirable, but to accomplish this at the genomic level the genes associated with acclimation, adaptation, and thermo-tolerance need to be identified. We evaluated the effects of TS on mammary development and gene expression in vitro using a bovine mammary epithelial cell collagen gel culture system. Acute TS was characterized by inhibition and regression of the ductal branches. Gene expression profiling revealed an overall upregulation of genes associated with the stress response and protein repair. In contrast, genes associated with cellular and mammary epithelial cell-specific biosynthesis, metabolism, and morphogenesis were generally downregulated by TS. Future studies will examine the impact of acclimation and adaptation on gene expression to identify those genes associated with acquisition of thermal tolerance.

Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos.

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle.

The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

Investigation of changes in global gene expression in the frontal cortex of early-weaned and socially isolated piglets using microarray and quantitative real-time RT-PCR.

We hypothesize that early-weaned piglets experience aberrant expression of stress-responsive genes in the frontal cortex, a key brain area involved in cognitive function and behavior organization. To test this hypothesis, female early-weaned piglets (EW; n = 6) were weaned 10 days after birth, while non-weaned piglets (NW; n = 6) were left with their dams. Half of EW (n = 3) and NW (n = 3) animals were socially isolated (SI) for 15 min at 12 days of age, when all animals (n = 12) were euthanized and tissue collected. The effects of EW and SI were examined by gene expression profiling using cDNA microarray hybridizations, generated from a porcine brain cDNA library. A total of 103 genes were differentially expressed (P < 0.05, fold change >1.25) among four direct comparisons. Forty-two genes had known functions, from which 24 showed relevant brain-related functions. Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used to confirm regulation of expression of a subset of 6 genes with important brain functions, selected from the microarray outcomes. In non-weaned animals, a significant suppression of mRNA abundance for carboxypeptidase E, 14-3-3 protein and phosphoprotein enriched in astrocytes 15 kDa was observed in response to SI. Also, in early-weaned animals, diazepam binding inhibitor and actin-related protein 2/3 complex mRNA levels were suppressed in response to SI. Results suggest that social isolation of non- and early-weaned piglets may impact expression of genes involved in regulation of neuronal function, development, and protection in the frontal cortex of young pigs.

Gene expression profile of mechanically impacted bovine articular cartilage explants.

Traumatic injury to a joint can initiate cartilage degradation. Blunt trauma increases matrix damage and decreases proteoglycan synthesis in in vitro models. Few studies have investigated gene expression of articular cartilage (AC) following mechanical loading. Recent advances in microarray technology allow analysis of a number of genes, and may elucidate pathways of AC degradation. In the present study, we used a bovine cDNA microarray to determine how acute trauma of cartilage explants in the absence of underlying bone alters gene expression. Results indicate that at least 19 genes were differentially expressed at 3 h after trauma. Fourteen genes were up-regulated and five genes were down-regulated relative to control explants. The up-regulated genes included cytokine and chemokine receptors, enzymes, and molecules involved in signal transduction. Genes of adhesion molecules and apoptosis were down-regulated. The results of this study highlight the potential benefits of using a bovine cDNA microarray to study cartilage metabolism.

Phagocytosis of M. paratuberculosis fails to activate expression of NADH dehydrogenase and nucleolin-related protein in bovine macrophages.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium and causal agent of Johne's disease in cattle. Following phagocytosis, M. paratuberculosis resides and replicates in macrophage phagosomes that fail to mature. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a high throughput initial screen to begin to test the hypothesis that macrophage gene expression patterns would be differentially affected by M. paratuberculosis when compared to readily degraded bacteria or non-degradable latex beads. Gene expression profiles from immortalized bovine macrophage cells (BOMAC) exposed to M. paratuberculosis were compared to gene expression profiles for BOMAC cells exposed to Escherichia coli, latex beads or PBS. Amplicons representing genes specifically activated or repressed during M. paratuberculosis phagocytosis were cloned for further investigation. Northern blot hybridizations preformed using DDRT-PCR-derived amplicons 3-1-4, 5-2-10, 5-4-2 and 4-1-6 confirmed stimuli dependent differential gene expression. Expression pattern observed for amplicon 3-1-4 represents genes that are up-regulated following phagocytosis of E. coli or latex beads, but not M. paratuberculosis. Amplicon 5-2-10 exhibited a pattern of expression representative of genes that are up-regulated strongly following phagocytosis of E. coli or latex beads but only moderately following M. paratuberculosis phagocytosis. Expression pattern of the gene for amplicon 5-4-2 was representative of genes that are specifically suppressed following M. paratuberculosis phagocytosis, while the amplicon 4-1-6 gene expression pattern represented genes that are generally suppressed following phagocytosis of any of the three stimuli. DNA sequencing and Genbank database analysis of these amplicons revealed that amplicon 3-1-4, whose expression failed to activate following M. paratuberculosis phagocytosis, had high levels of similarity to a Rattus norvegicus nucleolin-related protein (NRP). Amplicon 5-2-10, which increased expression moderately following M. paratuberculosis phagocytosis, was a near perfect match to bovine nicotinamide adenine dinucleotide dehydrogenase (FNADH dehydrogenase) subunit 1 (ND1). Failure to activate these two genes at levels observed following phagocytosis of either E. coli or latex beads may uncover new mechanisms for the survival of M. paratuberculosis within bovine macrophage cells.

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle.

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

Survival tactics of M. paratuberculosis in bovine macrophage cells.

Mycobacterium paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium with the ability to survive and proliferate inside the vesicles of macrophage cells. How M. paratuberculosis and other mycobacteria survive in this hostile environment is not well understood. Present research findings can be divided into three possibilities: (1) mycobacteria may interfere with host protein expression and trafficking to stop vesicle maturation, (2) mycobacteria may express proteins that interfere with macrophage activation in a more direct manner, or (3) mycobacteria may enter macrophages in such a way as to avoid the normal process of activation via Toll-like receptors and other, as yet unknown mechanisms. Research thus far has predominately centered on possible macrophage/mycobacteria protein interactions. To more completely define how mycobacteria interfere with the process of phagosome maturation our group has recently taken a functional genomics approach, allowing the macrophage to "tell" us what host genes may be affected by phagocytosis of mycobacteria. We used DDRT-PCR to examine differences in macrophage cell gene expression during phagocytosis of E. coli and M. paratuberculosis. Macrophage cells not exposed to any phagocytosis target and in the process of phagocytosing latex beads were used as negative and positive controls, respectively. To date, we have identified 380 DDRT-PCR amplicons corresponding to transcripts whose expression profiles appear to be altered during the general process of phagocytosis. Dot-blot and Northern blot hybridizations with a subset of these amplicons were performed to confirm results observed with DDRT-PCR. Our preliminary results indicate that macrophage gene expression profiles change dramatically following phagocytosis and that gene expression profiles following phagocytosis of M. paratuberculosis are different than those following phagocytosis of E. coli or latex beads.

Mycobacterium paratuberculosis and the bovine immune system.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a deadly intestinal ailment of ruminants. Johne's disease is of tremendous economic importance to the worldwide dairy industry, causing major losses due to reduced production and early culling of animals. A highly controversial but developing link between exposure to M. paratuberculosis and human Crohn's disease in some individuals has led to the suggestion that M. paratuberculosis is also a potential food safety concern. As with many other mycobacteria, M. paratuberculosis is exquisitely adapted to survival in the host, despite aggressive immune reactions to these organisms. One hallmark of mycobacteria, including M. paratuberculosis, is their propensity to infect macrophages. Inside the macrophage, M. paratuberculosis interferes with the maturation of the phagosome by an unknown mechanism, thereby evading the host's normal first line of defense against bacterial pathogens. The host immune system begins a series of attacks against M. paratuberculosis-infected macrophages, including the rapid deployment of activated gammadelta T cells, CD4+ T cells and cytolytic CD8+ T cells. These cells interact with the persistently infected macrophage and with each other through a complex network of cytokines and receptors. Despite these aggressive efforts to clear the infection, M. paratuberculosis persists and the constant struggle of the immune system leads to pronounced damage to the intestinal epithelial cells. Enhancing our ability to control this important and tenacious pathogen will require a deeper understanding of how M. paratuberculosis interferes with macrophage action, the cell types involved in the immune response, the cytokines these cells use to communicate, and the host genetic factors that control the response to infection.

Generation of EST and cDNA microarray resources for the study of bovine immunobiology.

Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG) at Michigan State University has developed a normalized bovine total leukocyte (BOTL) cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL) and the sequence information plus BLAST results made available through a web-accessible database (http://gowhite.ans.msu.edu). Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls) per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs). In total, hybridization signals on over 90 amplicons showed upregulation (> 3x) in response to Con A stimulation, relative to unstimulated cells. A second experiment with PBMCs from a different group of animals was performed to test reproducibility of microarray results. There was a high correlation between the 2 experiments (r = 0.72, P < 0.001). Resources described in this publication offer a highly efficient and integrated system to study gene expression changes in bovine leukocytes.

An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cows.

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with > 700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with > or = 1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

Evidence that Marek's disease virus exists in a latent state in a sustainable fibroblast cell line.

Previously, we reported the development of two fibroblastic cell lines (MDV OU2.1 and OU2.2) infected with Marek's disease virus (MDV). The two cell lines, in nonconfluent continuous cultures, displayed characteristics consistent with MDV existing in a latent state. However, presence of distinct plaques in confluent cell monolayers and the ability to transfer cytolytic infection to susceptible birds and primary chick embryo fibroblasts, suggest that, if latent, the virus is easily reactivated from MDV OU2 cell lines. In this report, we present evidence which supports the hypothesis that MDV genomes in MDV OU2 cells are latent. PCR analyses and in vivo experiments demonstrate that CHCC-OU2 cells stabilize MDV so that serial in vitro passage does not attenuate the virus. Following two years of active culture, MDV genomes in MDV OU2 cells are still oncogenic, similar to that seen in MDV-lymphoblastoid cell lines. Expansion of the 132-bp repeat within MDV BamHI fragments H and D, typical of highly passaged serotype 1 MDV, has not been observed beyond two copies in MDV OU2 cells. Indirect immunofluorescence assays clearly demonstrate that MDV OU2 cells do not express glycoproteins B and I when subconfluent. However, upon reaching confluence these proteins are expressed in readily detectable amounts. Using RT-PCR we demonstrate that glycoproteins E and D are highly expressed in confluent MDV OU2 cells but absent from subconfluent cells, and MDV latency-associated transcripts (LATs), which are antisense to ICP4 transcripts and have been associated with latent MDV infection, are expressed in subconfluent MDV OU2 cells. Coincident with an increase in ICP4 expression, MDV LAT expression is down regulated when MDV OU2 cells become confluent.

Cloning and sequence analysis of a Marek's disease virus origin binding protein (OBP) reveals strict conservation of structural motifs among OBPs of divergent alphaherpesviruses.

Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In its natural host, MDV induces Marek's disease (MD), a lethal condition characterized by malignant lymphoma of T cells. Although symptoms of MD may be prevented by vaccination, no practical pharmacological method of control has been widely accepted. Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successful. However, this requires detailed knowledge of viral replication proteins. Studies in HSV-1 DNA replication implicate the UL9 protein as a key initiator of replication. For example, binding of UL9 to HSV-1 origins is a prerequisite for assembly of additional replication proteins. In this study, a protein, whose apparent molecular size is similar to that of HSV-1 UL9, was identified in extracts of MDV infected cells by western blot analysis with anti-HSV-1 UL9 antibody. A putative MDV UL9 gene was subsequently identified through sequencing of MDV genome fragments (BamHI G and C). Extended DNA sequence analysis revealed an open reading frame (ORF) which could encode a protein homologous to HSV-1 UL9. The MDV UL9 ORF encodes 841 amino acids, producing a sequence 49% identical to HSV-1 UL9 and 46% identical to VZV gene 51 product (VZV UL9). MDV UL9 shares numerous structural motifs with HSV-1 and VZV UL9 proteins, including six conserved N-terminal helicase motifs, an N-terminal leucine zipper motif, a C-terminal pseudo-leucine zipper sequence, and a putative helix-turn-helix structure.

Development of a sustainable chick cell line infected with Marek's disease virus.

Marek's disease virus (MDV) is a highly infectious and cell-associated avian herpesvirus. Fully productive infections with MDV are restricted to feather follicle epithelium of afflicted birds. In culture, MDV infection of primary chick (CEF) and duck embryo fibroblast cells is semiproductive. Passage of MDV and production of MDV vaccines is limited to these primary cell-associated systems. The finite life span of primary avian cell cultures has hampered efforts to use positive selection in generation of recombinant MDV and complicates studies of temporal gene regulation. In this report, we describe continuous chick fibroblast cell lines (MDV OU2.2 and MDV OU2.1) which support MDV replication. Southern blot and PCR analyses demonstrate that these cell lines harbor MDV DNA. Western blot analyses indicate that MDV OU2.2 cells express at least a limited set of viral proteins, pp38 and pp14, similar to that seen in MDV-lymphoblastoid cells. Presence of distinct plaques in confluent MDV OU2.2 cell monolayers is consistent with cytolytic semiproductive infection, similar to that observed in primary CEF. MDV OU2.2 cells are capable of transferring MDV infection to primary CEF cultures and inducing clinical signs of Marek's disease in susceptible birds. MDV OU2.2 cells have maintained a MDV-positive phenotype for over 16 months of active culture. Southern blot hybridization of MDV OU2.2 cell DNA reveals a distinct expansion of the MDV BamHI H fragment in a subset of viral genomes following long-term cultivation.

A 14-kDa immediate-early phosphoprotein is specifically expressed in cells infected with oncogenic Marek's disease virus strains and their attenuated derivatives.

Previously, we reported two cDNAs derived from the Marek's disease virus (MDV) long internal repeat region. A 14-kDa polypeptide (p14) encoded by two small open reading frames (ORFs) from at least two distinct cDNAs is expressed in cells lytically infected with both oncogenic and attenuated MDV as well as in cells latently infected and transformed by MDV. In this study, we demonstrate that p14 is serotype 1 specific and highly phosphorylated. Given the degree of phosphorylation and lack of homology to known proteins, we propose the name pp14 for the polypeptide encoded by ORF1a and ORF1b. Further analysis reveals that pp14 is predominantly found in cytoplasmic fractions of MDV-infected cells and can be detected in the cytoplasm of MDV-infected cells by immunofluorescence with polyclonal antisera prepared against pp14-glutathione S-transferase fusion protein.