The Threat of Marek s Disease Virus Is Expanding

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1 The Threat of Marek s Disease Virus Is Expanding The virus responsible for this lymphoproliferative disease is growing more virulent and evading control by available vaccines Kyle S. MacLea and Hans H. Cheng Kyle S. MacLea is a postdoctoral fellow and Hans H. Cheng is a supervisory research scientist in the Avian Disease and Oncology Laboratory, Agricultural Research Service, United States Department of Agriculture, East Lansing, Mich. espite the attention being paid to D avian influenza and its potential to cause a pandemic among humans, the threat of avian diseases extends well beyond the influenza virus. Of particular interest to poultry producers are foodborne infectious bacteria such as Salmonella and Campylobacter spp., as well as several viruses, including those responsible for causing Exotic Newcastle Disease, avian leukosis, and Marek s disease (MD). Indeed, Marek s disease virus (MDV), an oncogenic avian herpesvirus that causes the lymphoproliferative disease characteristic of MD, is becoming more virulent and evading control by available vaccines thus posing an increasing challenge for poultry producers. Poultry plays an important global role nutritionally and economically, making the control of these and other bird diseases an increasingly important challenge. Poultry raised for meat mainly chickens, turkeys, and ducks, but also other specialty bird species and for eggs is an important source of protein for humans worldwide. Because of its increasing market share and low production costs, economists predict that poultry will overtake pork by the year 2020 as the leading meat consumed across the world. In the United States, poultry accounts for 41% and thus is already the top meat consumed, and it is the thirdlargest agricultural commodity overall, topping every plant species, including corn. American consumer demand leads to the processing of 1 million birds and the consumption of 10 million eggs on average per hour! Meeting this amazing demand for meat and eggs challenges producers. To improve the economic efficiency of rearing birds and producing eggs, the poultry industry has bred special chickens and also developed methods for greatly increasing the numbers of birds being produced (Fig. 1). Vaccines Help To Control Marek s Disease in Chickens With high-intensity breeding, large-scale closed rearing of birds, and increased global shipping of poultry, epidemic disease becomes a major concern. A pathogen infecting one or a few chickens in a large-scale production facility can very quickly infect hundreds of other birds there and lead to the condemnation of thousands more in short order. In such settings, MD looms as a serious concern to chicken producers, especially for egglaying and breeding operations. Clinical signs of the disease include blindness and transient paralysis. Once infected, birds are unfit for eating, while egg-laying chickens show poor productiv- Summary Marek s disease looms as a serious concern to chicken producers, particularly in the context of high-intensity production facilities. Researchers are beginning to breed poultry for genetic resistance to Marek s disease as a way of complementing available vaccines and biosecurity programs. Because the Marek s disease virus appears to be growing more virulent, researchers are taking several approaches to identify disease-resistance genes in host poultry species. 238 Y Microbe / Volume 2, Number 5, 2007

ity. The serotype 1, oncogenic versions of MDV are antigenically related to other nononcogenic avian herpesviruses, including serotype 2 viruses that infect chickens and serotype 3 viruses that

2 ity. The serotype 1, oncogenic versions of MDV are antigenically related to other nononcogenic avian herpesviruses, including serotype 2 viruses that infect chickens and serotype 3 viruses that infect turkeys. MDV is found throughout the environment, meaning nearly all chickens are exposed to the virus within days of hatching. The virus is transmitted when birds inhale cell-free viral particles that accumulate in dust and dander from birds reared in the same space. Lung macrophages phagocytize the virus particles, carrying them to immune cells in lymphoid organs, including the spleen, thymus, and bursa of Fabricius, which is the site of hematopoiesis and B-cell development. After MDV infects B cells, the virus targets activated T-cells and the infection becomes latent, a hallmark of herpesviruses. Infected lymphocytes spread the virus to other tissues throughout the body, resulting in chronic inflammation and frank lymphomas. Meanwhile, once formed in the epithelium of feather follicles, infectious MDV can be shed as dander and feathers, becoming a part of the poultry house dust and allowing further spread. As with many other poultry diseases, vaccines are used to control MD in chickens. The first such vaccine, which was developed in our Agricultural Research Service lab and was introduced to the U.S. poultry industry in 1970, is based on HVT, a nonpathogenic herpesvirus of turkeys. This vaccine prevents cancer in chickens that become infected with MDV, making it the first vaccine used to prevent cancer in any animal. When they were introduced, these vaccines dramatically reduced bird losses. Rising Virulence of Marek s Disease Virus However, because the HVT vaccine and other newer serotype 3 vaccines do not provide sterilizing immunity, strains of serotype 1 MDV continue to infect chickens, sometimes developing enough virulence to overcome the protective effects of the vaccines. In response, researchers in this field developed alternative vaccines, such as a bivalent vaccine incorporating HVT as well as a serotype 2 virus, SB-1. FIGURE 1 A typical chicken house. (USDA photo.) This bivalent product proved effective until very virulent plus strains arose in the early 1990s. To counteract them, researchers incorporated an attenuated serotype 1 strain, known as CVI988 or Rispens, to control MDV in the field. Subsequent to Rispens usage, reports of even more virulent strains of the virus began to appear, and even greater virulence is anticipated (Fig. 2). However, newer vaccines are proving no more effective at stopping disease than the Rispens product. Even worse, some of these emerging strains are giving rise to very severe symptoms. For example, although older strains of MDV sometimes caused transient paralysis, some of the extremely virulent strains that now are circulating give rise to a particularly acute paralysis with very high mortality, along with greater lymphoid organ and skin pathology. Identifying Disease Resistance Genes Using the Chicken Genome To combat these problems, we are seeking ways to enhance Marek s disease resistance and vaccine responses among chickens. To identify disease resistance genes in chickens, we are taking a threefold integrated genomics approach (Fig. 3). First, at the DNA level, we conduct quantitative trait locus (QTL) scans, using genetic markers to Volume 2, Number 5, 2007 / Microbe Y 239

3 FIGURE 2 where progeny, which span the entire range of MD susceptibility, were evaluated for MD phenotype. After viral challenge, we compared the genomes of diseased and resistant chickens, enabling us to identify 14 MD-associated QTL. Unfortunately, the precision of this analysis is low, making it impossible to associate QTLs with specific genes and usually necessitating finer mapping. Typical QTL scans identify genetic intervals that are as large as centimorgans (cm), or even as high as 50 cm in some cases. Increasing virulence of the chicken oncogenic herpesvirus, Marek s disease virus (MDV), over time. (Based on the original virulence graph of R. L. Witter.) identify regions within the genome where resistance traits might be found. Second, at the RNA level, we use DNA microarrays to identify genes that are differentially expressed between disease-resistant and susceptible birds. Third, at the protein level, we try to identify points where key viral and chicken proteins interact. Without candidate resistance genes in hand, we prepared to conduct a genome-wide QTL scan by developing a population of birds containing pedigreed individuals that can be exposed to MDV and analyzed for their responses to this disease. After these birds are genotyped, statistics are used to scan the chicken genome to determine whether certain sections (intervals between genetic markers) are associated with disease resistance. We relied on two lines of chickens that we produced specifically for studying MD. Individual chickens from our inbred line 6, which are resistant to MD, and line 7, which are susceptible, were mated to create a resource F 2 population Harnessing Microarray Technology To Search for Resistance Traits Apart from genome-wide QTL scans, there are other more precise methods for identifying candidate disease-resistance genes. For instance, we are using DNA microarrays to compare patterns of gene expression under various conditions and thus to develop a picture of the many genes associated with those phenotypes, including some associated with resistance to MD. Our first chicken DNA microarray contained genes from a spleen T-cell cdna library. It entailed preparing reversetranscribed RNA from peripheral blood lymphocytes from infected or uninfected line 6 (resistant) and line 7 (susceptible) birds, and then hybridizing those transcripts to genes in the library. Despite the limited power of this version of a chicken chip, we identified 105 genes from a set of 1,200 that had twofold or greater expression differences, of which 25 genes behaved this way reproducibly. In a second experiment, we looked for differences in the expression of chicken genes over the course of MDV infections, and identified 50 genes of interest. Of these genes, 11 showed twofold or greater differences in expression. Subsequently, we used more comprehensive microarrays to extend the number of promising genes. However, these results include not only genes that are directly involved in resistance and susceptibility, but also those that are secondarily regulated by those genes, complicating our task of identifying the important genes for MD resistance. 240 Y Microbe / Volume 2, Number 5, 2007

To sort through these genes, we mapped and then compared them with our QTL scan results and what we knew of immunologically relevant genes in other species.

4 To sort through these genes, we mapped and then compared them with our QTL scan results and what we knew of immunologically relevant genes in other species. Of the 15 genes examined, 12 mapped with human homologs, and another 7 were located using the chicken-human comparative gene map. Through these analyses, we determined that one gene, GH1 (chicken growth hormone), is associated with MD resistance, while another gene, SCYC1, maps near a QTL site on chromosome 1. This latter gene, SCYC1, encodes chicken lymphotactin, a cytokine involved in recruiting CD4-positive and CD8-positive T-cells. Thus, by combining findings from our QTL scan and microarray experiments, we helped to implicate lymphotactin as playing a role in MDV infections. Improving Our View of Virus-Host Protein Interactions Another method for identifying candidate resistance genes for MD takes advantage of viral interactions with host-cell components. For example, we use two-hybrid screening to find host proteins that interact with viral proteins of interest. In using both yeast and bacterial twohybrid screens, we take an open reading frame (ORF) protein from a virulent MDV to bind host proteins from a chicken spleen T-cell cdna library. Each of the host gene products that interacted with viral ORFs was also subjected to coimmunoprecipitation and/or in vitro binding assays. Of the confirmed protein-protein interactions, several are particularly interesting because they were identified independently as putative resistance genes. One of them, chicken growth hormone, or GH, that is encoded by GH1, was also identified in our microarray experiments; it binds to the viral protein SORF2. Although we did not survey the region of the chicken genome that encodes GH1 in our QTL scan, a separate mapping project involving commercial chickens linked GH1 with MD-associated traits. Through these two-hybrid assays, we also FIGURE 3 An integrated genomics approach to identifying genes involved in MDV resistance. learned that the chicken lymphocyte antigen 6 complex, locus E (LY6E, also called stem cell antigen 2), specifically interacts with viral protein US10. We also identified the LY6E gene through our microarray screens and in separate mapping experiments using commercial chickens. It, too, is associated with MD resistance. Yet another chicken protein, the major histocompatibility complex (MHC) class II beta chain, is one of seven host proteins that interact specifically with virus proteins from virulent strains, in this case interacting with a MDV gene called LORF4. The MHC class II beta chain gene, BLB, is a positional candidate for MD resistance because it is located within the 19- gene cluster of the chicken MHC. Although we cannot determine which of the genes within the MHC is responsible, this immunologically important gene region is strongly associated with MD resistance in chickens. Convergence of Several Lines of Analysis and Viral Genomics With the help of three independent analytic methods, we implicated three genes GH1, LY6E/SCA2, and BLB as possible MD-resistance genes. We also identified several additional candidates from this integrated genomics approach. All these candidates are being further tested for their role in the MDV lifecycle. Even though preliminary, these findings have promising implications for MD-resistant chicken Volume 2, Number 5, 2007 / Microbe Y 241

5 FIGURE 4 Upregulation of MHC Class II in response to MDV infection. Uninfected (A and C) and MDV-infected (B and D) bursa are shown; light microscopy (A and B) shows MDV (brown) and MHC Class II (pink), and fluorescence microscopy (C and D) shows MHC class II (red). breeding and for our understanding of how viruses spread in these poultry populations. First, because GH modulates immune responses, breeding ever-larger and faster-growing chickens inadvertently may also be selecting for birds that are more susceptible to viral infections, including with MD. Like all herpesviruses, MDV follows a biochemical program when infecting and spreading among cells and between individual animals, while evading the host immune system. Our results with the MHC class II beta chain and invariant chain, which was also identified in our screens, dovetail with another of our studies of the role of MHC class II in MDV infection. Like other herpesviruses, MDV downregulates MHC class I to avoid destroying infected cells. However, unlike other herpesviruses, MDV upregulates cell-surface expression of MHC class II in infected chicken cells, both in vitro and in vivo (Fig. 4). We suspect that upregulating MHC class II augments viral spread within the host by increasing the chance of contact between productively infected cells and uninfected, but susceptible, activated T-cells. These other associated host proteins, namely MHC class II beta chain and invariant chain, may also play a role in augmenting viral spread. Meanwhile, we are also using reverse genetics, specifically by developing molecular clones of the virus, to manipulate the viral genome and assess the effects of such changes on disease pathogenesis. Our findings are also helping us to appreciate the genetic complexities of MDV. Although the virus was known to carry mutated copies of the chicken telomerase RNA subunit and IL-8 chemokine genes, by applying reverse genetics, we learned that MDV can actively acquire additional host genes. Thus, in cloning the Md11 strain of MDV, we determined that the virus also pirated a region of its host genome in this particular case, from a duck rather than a chicken. ACKNOWLEDGMENTS Our work was supported by funding from the United States Department of Agriculture (USDA) Agricultural Research Service and National Research Initiative Competitive Grants Program. SUGGESTED READING Davison, F., and V. Nair Use of Marek s disease vaccines: could they be driving the virus to increasing virulence? Expert Rev. Vaccines 4: Liu, H. C., H. J. Kung, J. E. Fulton, R. W. Morgan, and H. H. Cheng Growth hormone interacts with the Marek s disease virus SORF2 protein and is associated with disease resistance in chicken. Proc. Natl. Acad. Sci. USA 98: Liu, H. C., M. Niikura, J. E. Fulton, and H. H. Cheng Identification of chicken lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek s disease resistance gene via a virus-host protein interaction screen. Cytogenet. Genome Res. 102: Niikura, M., H. C. Liu, J. B. Dodgson, and H. H. Cheng A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek s disease virus. Poultry Sci. 83: Y Microbe / Volume 2, Number 5, 2007

Niikura, M., J. Dodgson, and H. Cheng. 2006. Direct evidence of host genome acquisition by the alphaherpesvirus Marek s disease virus. Arch. Virol. 151:537 549. Niikura, M., T. Kim, H. D. Hunt, J.

Virology 359:212 219. Osterrieder, N., J. P. Kamil, D. Schumacher, B. K. Tischer, and S. Trapp. 2006. Marek s disease virus: from miasma to model. Nature Rev. Microbiol. 4:283 294. Vallejo, R. L., L.

Genetic mapping of quantitative trait loci affecting susceptibility to Marek s disease virus induced tumors in F2 intercross chickens. Genetics 148:349 60. Witter, R. L., B. W. Calnek, C.

6 Niikura, M., J. Dodgson, and H. Cheng Direct evidence of host genome acquisition by the alphaherpesvirus Marek s disease virus. Arch. Virol. 151: Niikura, M., T. Kim, H. D. Hunt, J. Burnside, R. W. Morgan, J. B. Dodgson, and H. H. Cheng Marek s disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells. Virology 359: Osterrieder, N., J. P. Kamil, D. Schumacher, B. K. Tischer, and S. Trapp Marek s disease virus: from miasma to model. Nature Rev. Microbiol. 4: Vallejo, R. L., L. D. Bacon, H. C. Liu, R. L. Witter, M. A. Groenen, J. Hillel, and H. H. Cheng Genetic mapping of quantitative trait loci affecting susceptibility to Marek s disease virus induced tumors in F2 intercross chickens. Genetics 148: Witter, R. L., B. W. Calnek, C. Buscaglia, I. M. Gimeno, and K. A. Schat Classification of Marek s disease viruses according to pathotype: philosophy and methodology. Avian Pathol. 34: ASM accepts VISA, Mastercard, American Express, purchase orders, and checks drawn on U.S. banks in U.S. currency. Canadian orders must include appropriate GST. WEB: CALL: or WRITE TO: ASM Press, P. O. Box 605, Herndon, VA 20172, USA FAX: Volume 2, Number 5, 2007 / Microbe Y 243

Host Genetic Resistance Sustains HVT Protective Efficacy Comparable to CVI988/Rispens in Lines of Chickens Relatively Resistant to Marek s Disease

Host Genetic Resistance Sustains HVT Protective Efficacy Comparable to CVI988/Rispens in Lines of Chickens Relatively Resistant to Marek s Disease Huanmin Zhang 1, Shuang Chang 1, 2, John R. Dunn 1 Mohammad