Virus-encoded microRNA-M7 restricts early cytolytic replication and pathogenesis of Marek's disease virus.

MicroRNAs (miRNAs) are a class of ∼22 nucleotides non-coding RNAs that are encoded by a wide range of hosts. Viruses, especially herpesviruses, encode a variety of miRNAs that involved in disease progression. Recently, a cluster of virus-encoded miRNAs, miR-M8-M10, have been shown to restrict early cytolytic replication and pathogenesis of Marek's disease virus (MDV), an oncogenic avian alphaherpesvirus that causes lymphoproliferative disease in chickens. In this study, we specifically dissected the role of miR-M7, a member of cluster miR-M8-M10, in regulating MDV replication and pathogenesis. We found that deletion of miR-M7-5p did not affect the virus plaque size and growth in cell culture. However, compared to parental virus, infection of miR-M7-5p deletion virus significantly increased MDV genome copy number at 5 days post infection, suggesting that miR-M7 plays a role to restrict MDV replication during early cytolytic phase. In addition, our results showed that infection of miR-M7-5p deletion virus significantly enhanced the mortality of chickens, even it induced lymphoid organ atrophy similar to parental and revertant viruses. Taken together, our study revealed that the miR-M7 acts as a repressive factor of MDV replication and pathogenesis.

Deletion of LORF9 but not LORF10 attenuates Marek's disease virus pathogenesis.

Marek's disease virus (MDV) genome contains a number of uncharacterized long open reading frames (LORF) and their role in viral pathogenesis has not been fully investigated. Among them, LORF9 (MDV069) and LORF10 (MDV071) are locate at the right terminus of the MDV genome unique long region (UL). To investigate their role in MDV pathogenesis, we generated LORF9 or LORF10 deletion and revertant viruses. In vitro growth kinetics results show that both LORF9 and LORF10 are not essential for virus growth in cell culture. However, LORF9, but not LORF10, is involved in MDV early cytolytic replication in vivo, as evidenced by limited viral antigen expression in lymphoid organs of LORF9 deletion virus inoculated chickens. MDV genome copy number data further confirmed that LORF9 is important for MDV replication in spleen during early cytolytic phase. Deletion of LORF9 also partially impairs the replication of MDV in feather follicle epithelium (FFE); however, it can still establish latency and transformation. In addition, pathogenesis studies show that deletion of LORF9, but not LORF10, result in significant reduction of MDV induced mortality and slightly reduce MDV associated tumors of inoculated chickens. Importantly, we confirmed these results with the generation of LORF9 and LORF10 revertant viruses that fully restore the phenotypes of parental MDV. In conclusion, our results show that deletion of LORF9, but not LORF10, significantly impair viral replication in lymphoid organs during early cytolytic phase and attenuate Marek's disease virus pathogenesis.

Identification of Marek's Disease Virus VP22 Tegument Protein Domains Essential for Virus Cell-to-Cell Spread, Nuclear Localization, Histone Association and Cell-Cycle Arrest.

VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.

Suspension culture of Marek's disease virus and evaluation of its immunological effects.

Marek's disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76 ± 2.64 × 106 PFU/flask in a working volume of 100 ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5 g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.

Reduction of Marek's Disease Virus Infection by Toll-Like Receptor Ligands in Chicken Embryo Fibroblast Cells.

Evolutionarily conserved pattern recognition receptors, including Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) that are present in microbes. PAMPs induce several pathways downstream of TLRs that lead to induction of antiviral responses. The objective of this study was to investigate the stimulatory effect of various PAMPs (in the form of TLR ligands) in reducing Marek's disease virus (MDV) infection in chicken embryo fibroblast cells (CEFs). To this end, CEFs were pretreated with Pam3CSK4, Poly(IC), lipopolysaccharide (LPS), and CpG ODN as TLR2, TLR3, TLR4, and TLR21 ligands, respectively for 24 h followed by infection with MDV. The results indicated that pretreatment with Poly(IC) resulted in a robust reduction (by about 81%) of MDV infection in CEFs at 96 h postinfection while a moderate reduction was observed with treatment of Pam3CSK4 (35%), LPS (26%), and CpG ODN (23%) PAMPs. Transcriptional analysis of gene expression in CEFs demonstrated that all TLR ligand treatments and MDV infection significantly increased the expression of type I interferons, interleukin (IL)-1ß, interferon regulatory factor 7 (IRF7), interferon induced protein with tetratricopeptide repeats 5 (IFIT5), and myxoma-resistance protein (Mx). Further studies are needed to explore the mechanism by which PAMPs, particularly the TLR3 ligands could reduce MDV infection in CEFs, which may play an important role in controlling the replication of MDV in chicken.

Attenuation of Marek's disease virus by codon pair deoptimization of a core gene.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of Gallus gallus, the domesticated chicken. Control strategies rely upon vaccination with live attenuated viruses of antigenically similar avian herpesviruses or attenuated strains of MDV. Recent studies in other viruses have shown that recoding certain viral genes to employ synonymous but rarely-used codon pairs resulted in viral attenuation. We deoptimized two MDV proteins, UL54/ICP27 and UL49/VP22, and demonstrate that the more severely deoptimized variant of UL54 accumulates significantly less gene product in vitro. Using these UL54 deoptimized mutants, we further demonstrate that animals infected with the UL54-recoded recombinant virus exhibited decreased viral genome copy number in lymphocytes, reduced lymphoid atrophy and reduced tumor incidence. This study demonstrates that codon pair deoptimization of a single viral gene can produce attenuated strains of MDV. This approach may be useful as a rational way of making novel live attenuated virus vaccines for MDV.

Marek's disease virus and skin interactions.

Marek's disease virus (MDV) is a highly contagious herpesvirus which induces T-cell lymphoma in the chicken. This virus is still spreading in flocks despite forty years of vaccination, with important economical losses worldwide. The feather follicles, which anchor feathers into the skin and allow their morphogenesis, are considered as the unique source of MDV excretion, causing environmental contamination and disease transmission. Epithelial cells from the feather follicles are the only known cells in which high levels of infectious mature virions have been observed by transmission electron microscopy and from which cell-free infectious virions have been purified. Finally, feathers harvested on animals and dust are today considered excellent materials to monitor vaccination, spread of pathogenic viruses, and environmental contamination. This article reviews the current knowledge on MDV-skin interactions and discusses new approaches that could solve important issues in the future.

Marek's disease virus morphogenesis.

Marek's disease virus (MDV) is a highly contagious virus that induces T-lymphoma in chicken. This viral infection still circulates in poultry flocks despite the use of vaccines. With the emergence of new virulent strains in the field over time, MDV remains a serious threat to the poultry industry. More than 40 yr after MDV identification as a herpesvirus, the visualization and purification of fully enveloped infectious particles remain a challenge for biologists. The various strategies used to detect such hidden particles by electron microscopy are reviewed herein. It is now generally accepted that the production of cell-free virions only occurs in the feather follicle epithelium and is associated with viral, cellular, or both molecular determinants expressed in this tissue. This tissue is considered the only source of efficient virus shedding into the environment and therefore the origin of successful transmission in birds. In other avian tissues or permissive cell cultures, MDV replication only leads to a very low number of intracellular enveloped virions. In the absence of detectable extracellular enveloped virions in cell culture, the nature of the transmitted infectious material and its mechanisms of spread from cell to cell remain to be deciphered. An attempt is made to bring together the current knowledge on MDV morphogenesis and spread, and new approaches that could help understand MDV morphogenesis are discussed.

Latency and tumorigenesis in Marek's disease.

Despite the remarkable progress in our understanding of Marek's disease (MD) and the causative Marek's disease virus (MDV) biology, a number of major features of this complex viral disease remain unknown. Significant information on critical aspects of virus latency in lymphoid cells, and the virus-host interaction in MDV-induced lymphoma, remains to be identified. Moreover, the nature of the unique milieu of the feather follicle epithelial cell that allows cytolytic infection to continue, despite maintaining the latent infection in the lymphoid cells, is not fully understood. Although there has been significant progress in our understanding of the functions of a number of viral genes in the pathogenesis of the disease, the characteristics of the latent infection, how it differs from tumor phase, and whether latency is a prerequisite for the tumor phase are all important questions still to be answered. Reticuloendotheliosis virus-transformed cell lines have been shown to support MDV latency in a manner almost identical to that seen in MDV-transformed cell lines. There are increasing data on the role of epigenetic regulation, including DNA methylation and histone modifications, in maintaining viral latency. Onset of MD tumor is relatively rapid, and recent studies based on chromosomal integration and T-cell repertoire analysis demonstrated the clonal nature of MD lymphomas. Among the viral determinants of oncogenicity, the basic leucine zipper protein Meq is considered to be the most important and the most extensively studied. Deleting the Meq proteins or abolishing some of the important interactions does affect the oncogenicity of the virus. In addition, the noncoding sequences in the viral genome, such as the viral telomerase RNA and the virus-encoded microRNAs, also have significant influence on MDV-encoded oncogenesis.

Insertion of reticuloendotheliosis virus long terminal repeat into the genome of CVI988 strain of Marek's disease virus results in enhanced growth and protection.

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by serotype 1 MD virus (MDV). Vaccination of commercial poultry has drastically reduced losses from MD, and the poultry industry cannot be sustained without the use of vaccines. Retrovirus insertion into herpesvirus genomes is an efficient process that alters the biological properties of herpesviruses. RM1, a virus derived from the virulent JM strain of MDV, by insertion of the reticuloendotheliosis (REV) long terminal repeat (LTR), was attenuated for oncogenicity but retains properties of the parental virus, such as lymphoid organ atrophy. Here we show that insertion of the REV LTR into the genome of vaccine strain CVI988 resulted in a virus (CVRM) that replicated to higher levels than parental CVI988 in cell culture and that remained apathogenic for chickens. In addition, CVRM showed protection indices similar or superior to those afforded by CVI988 virus in laboratory and field protection trials, indicating that it could be developed as a safe and efficacious vaccine to protect against very virulent plus MDV.

Microsatellite instability in chicken lymphoma induced by gallid herpesvirus 2.

Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek's disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken.

Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas.

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

Efficacy and safety of cell associated vaccines against Marek's disease virus grown in a continuous cell line from chickens.

The Marek's disease virus (MDV) vaccine strains CVI 988 and herpes virus of turkeys (HVT) strain FC126, usually are grown in primary chicken embryo fibroblasts (CEF). We found that the strains could be grown also in the so-called JBJ-1 cell line to titres in the same range as when chicken embryo fibroblasts were used. The JBJ-1 cell line is a fibroblast-like continuous chicken cell line, which can be grown in flat bottom tissue culture flasks, roller bottles and on micro carriers. We investigated the efficacy of experimental CVI 988 vaccines grown in JBJ-1 cells and the efficacy of combinations of CVI 988 grown in JBJ-1 cells with HVT FC 126 also grown in JBJ-1 cells. The study was performed in accordance with European Pharmacopoeia monograph 0589 for live MDV disease vaccines. Groups of 1-day-old SPF chicks were vaccinated subcutaneously or intramuscularly, with 10(2.5) TCID50 per dose of CVI 988 alone or in combination with 500PFU per dose of HVT. As a control a group vaccinated with CVI 988 grown in CEF was included. One group was not vaccinated. Five days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of Marek's disease (MD). The protection induced by CVI 988 grown in JBJ-1 cells and the combination of CVI 988 and HVT-FC126 both grown in JBJ-1 cells, amply complied with the requirements of the European Pharmacopoeia which prescribes that the protection index should be at least 80%. The safety of the vaccines grown in JBJ-1 cells was tested in a field study in commercial layer chickens. No signs of MD were noticed during the study and no other signs attributable to the vaccine. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Deletion of the Marek's disease virus UL41 gene (vhs) has no measurable effect on latency or pathogenesis.

Marek's disease is an economically important disease of poultry and is caused by an alphaherpesvirus, Marek's disease virus (MDV). The predicted protein product of the MDV UL41 open reading frame has significant protein sequence identity with the virion host shutoff (vhs) protein gene in other alphaherpesviruses. To determine whether the MDV UL41 gene functions as a vhs protein, we utilized a transient co-transfection assay and demonstrated that the MDV UL41 protein was as active in degrading RNA as the vhs protein of herpes simplex virus type 1. To evaluate whether the MDV UL41 gene was involved in pathogenesis, we deleted the MDV UL41 gene. The UL41 deletion mutant replicated in cell culture as well as the parental MDV. The deletion mutant was inoculated into susceptible day-old chicks. The pattern and degree of tumor lesions and neurovirulence produced by the deletion mutant was same as the pattern of lesions induced by the parental virus. The only observable difference between the inoculation of MDV and the MDV deletion mutant was that the early in vivo cytolytic infection with the deletion mutant was of a longer duration than in the non-mutant MDV.

MicroRNA profile of Marek's disease virus-transformed T-cell line MSB-1: predominance of virus-encoded microRNAs.

Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the R(L)/R(S) region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 "miRNAome." Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.

Enzymatically inactive U(S)3 protein kinase of Marek's disease virus (MDV) is capable of depolymerizing F-actin but results in accumulation of virions in perinuclear invaginations and reduced virus growth.

Marek's disease (MD) is a highly contagious, lymphoproliferative disease of chickens caused by the cell-associated MD virus (MDV), a member of the alphaherpesvirus subfamily. In a previous study we showed that the absence of the serine/threonine protein kinase (pU(S)3) encoded in the MDV unique-short region resulted in accumulation of primarily enveloped virions in the perinuclear space and significant impairment of virus growth in vitro. It was also shown that pU(S)3 is involved in actin stress fiber breakdown [Schumacher, D., Tischer, B. K., Trapp, S., and Osterrieder, N. (2005). Here, we constructed a recombinant virus to test the importance of pU(S)3 kinase activity for MDV replication and its functions in actin rearrangement. Disruption of the kinase active site was achieved by substituting a lysine at position 220 with an alanine (K220A). Titers of a kinase-negative MDV mutant, 20U(S)3()K220A, were reduced when compared to parental virus similar to those of the U(S)3 deletion mutant. We were also able to demonstrate complete absence of phosphorylation of MDV-specific phosphoprotein pp38 in cells infected with the kinase-deficient virus, indicating that pp38 phosphorylation depends entirely on the kinase activity of pU(S)3. Enzymatically inactive pU(S)3()K220A was, however, still capable of mediating breakdown of the actin cytoskeleton in transfection studies, and this activity was indistinguishable from that of wild-type pU(S)3(). Furthermore, we demonstrated that pU(S)3 possesses anti-apoptotic activity, which is dependent on its kinase activity. Taken together, our results demonstrate that pU(S)3 and MDV-specific phosphoprotein pp38 represent a kinase-substrate pair and that growth impairment in the absence of pU(S)3 is caused by the absence of kinase activity. The unaltered disruption of F-actin by the K220A pU(S)3 mutant suggests that F-actin disassembly is unrelated to MDV growth restrictions in the absence of the unique-short protein kinase.

Optimization of methods for the isolation of Marek's disease viruses in primary chicken cell cultures.

A real-time PCR was used to measure increases in viral DNA in Marek's disease virus (MDV)-infected primary chicken cell cultures in order to optimize methods for viral isolation. Serotype-1 and -3 vaccine and serotype-1 challenge strains exhibited similar growth characteristics, with increases in viral DNA being proportional to inoculum size. Studies of viral growth revealed a linear relationship between increase in MDV copy number and infectious titre, although the rate of increase for copy number was greater. Using real-time PCR, viral DNA yields of the virulent Woodlands strain in infected chicken kidney cultures were shown to be slightly, but not significantly, higher than in chicken embryo kidney cultures and significantly higher than in chicken embryo fibroblast cultures. Viral DNA levels in freshly trypsinised cells suspended in growth medium and infected with the Woodlands strain were higher than levels obtained following the inoculation of monolayer cultures. For cells infected in suspension, no significant enhancement of yield was observed following a medium change after 2-3 days. Peak yields were obtained at days 6-8 after inoculation of all cultures. Findings obtained from the optimization of viral DNA levels were applied to a program for the isolation of Australian strains of serotype-1 viruses from problem flocks over 3 years. Significant improvements were obtained in the isolation rate of strains capable of growing to high titre (>10(4) plaque-forming units/mL) for use in challenge studies.

Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay.

Marek's disease, a T cell lymphoma, is an economically important disease of poultry caused by the Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. A greater understanding of viral gene function and the contribution of sequence variation to virulence should facilitate efforts to control Marek's disease in chickens. To characterize a naturally occurring single nucleotide polymorphism (SNP; AY510475:g.108,206C>T) in the MDV UL41 gene that results in a missense mutation (AAS01683:p.Arg377Cys), bacterial artificial chromosome (BAC)-derived MDVs that differed only in the UL41 SNP were evaluated using a head-to-head competition assay in vitro. Monitoring the frequency of each SNP by pyrosequencing during virus passage determined the ratio of each viral genome in a single monolayer, which is a very sensitive method to monitor viral fitness. MDV with the UL41*Cys allele showed enhanced fitness in vitro. To evaluate the mechanism of altered viral fitness caused by this SNP, the virion-associated host shutoff (vhs) activity of both UL41 alleles was determined. The UL41*Cys allele had no vhs activity, which suggests that enhanced fitness in vitro for MDV with inactive vhs was due to reduced degradation of viral transcripts. The in vitro competition assay should be applicable to other MDV genes and mutations.

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES.

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.

Stability of Marek's disease virus 132-bp repeats during serial in vitro passages.

The Marek's disease virus (MDV) genome contains 2 sets of 132-bp tandem repeat sequences. An increase in 132-bp repeat units has been associated with attenuation of oncogenicity during in vitro passage. By cloning entire genomes, we demonstrated that the copy number of 132-bp repeats can differ within an individual MDV genome. The stability of the 132-bp repeats during cell passage depended on the initial copy number. When both sets of repeats contained 2 copies, the copy number remained stable, while if even 1 set of repeats contained 6 copies, repeat expansion occurred relatively quickly. This expansion did not affect the in vitro growth curve. However, when MDV clones with low and high copy numbers were passed together, genomes with expanded repeats rapidly predominated, mimicking the behavior of naturally-occurring MDV. These results suggest that the preponderance of high-copy repeats after passage reflects intracellular copy number within individual infected cells rather than an influence on the spread of the virus.