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Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus of chickens that causes lymphomas in various organs. Most MDV genes are conserved among herpesviruses, while others are unique to MDV and may contribute to pathogenesis and/or tumor formation. High transcript levels of the MDV-specific genes MDV082, RLORF11, and SORF6 were recently detected in lytically infected cells; however, it remained elusive if the respective proteins are expressed and if they play a role in MDV pathogenesis. In this study, we first addressed if these proteins are expressed by inserting FLAG tags at their N or C termini. We could demonstrate that among the three genes tested, MDV082 is the only gene that encodes a protein and is expressed very late in MDV plaques

International audience; Viral tropism and transmission of herpesviruses are best studied in their natural host for maximal biological relevance. In the case of alphaherpesviruses, few reports have focused on those aspects, primarily because of the few animal models available as natural hosts that are compatible with such studies. Here, using Marek's disease virus (MDV), a highly contagious and deadly alphaherpesvirus of chickens, we analyze the role of tegument proteins pUL47 and pUL48 in the whole life cycle of the virus. We report that a virus lacking the UL48 gene (vΔUL48) is impaired in growth in cell culture and has diminished virulence in vivo In contrast, a virus lacking UL47 (vΔUL47) is unaffected in its growth in vitro and is as virulent in vivo as the wild-type (WT) virus. Surprisingly, we observed that vΔUL47 was unable to be horizontally transmitted to naive chickens, in contrast to the WT virus. In addition, we show that pUL47 is important for the splicing of UL44 transcripts encoding glycoprotein gC, a protein known as being essential for horizontal transmission of MDV. Importantly, we observed that the levels of gC are lower in the absence of pUL47. Notably, this phenotype is similar to that of another transmission-incompetent mutant ΔUL54, which also affects the splicing of UL44 transcripts. This is the first study describing the role of pUL47 in both viral transmission and the splicing and expression of gC.IMPORTANCE Host-to-host transmission of viruses is ideally studied in vivo in the natural host. Veterinary viruses such as Marek's disease virus (MDV) are, therefore, models of choice to explore these aspects. The natural host of MDV, the chicken, is small, inexpensive, and economically important. MDV is a deadly and contagious herpesvirus that can kill infected animals in less than 4 weeks. The virus naturally infects epithelial cells of the feather follicle epithelium from where it is shed into the environment. In this study, we demonstrate that the viral protein pUL47 is an essential factor for bird-to-bird transmission of the virus. We provide some molecular basis to this function by showing that pUL47 enhances the splicing and the expression of another viral gene, UL44, which is essential for viral transmission. pUL47 may have a similar function in human herpesviruses such as varicella-zoster virus or herpes simplex viruses.

The Herpesviridae encode many conserved genes, including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture to various degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek’s disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken to chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and Western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread and autophosphorylation of CHPK and that mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens, and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread. IMPORTANCE Marek’s disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek’s disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation but do not block interindividual spread from chicken to chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek’s disease while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined that MDV CHPK is not required for cell-to-cell spread, for disease induction, and for oncogenicity. However, it is required for interindividual spread, and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.

Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation., In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek’s disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes. IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Marek’s disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current “gold standard” vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens., Vaccines play a crucial role in the protection of animals and humans from deadly pathogens. The first vaccine that also protected against cancer was developed against the highly oncogenic herpesvirus Marek’s disease virus (MDV). MDV infects chickens and causes severe immunosuppression, neurological signs, and fatal lymphomas, a process that requires the viral oncogene, meq. The most frequently used Marek’s disease vaccine is the live-attenuated CVI988/Rispens (CVI) strain, which efficiently protects chickens and prevents tumorigenesis. Intriguingly, CVI expresses at least two isoforms of meq; however, it remains unknown to what extent these isoforms contribute to virus attenuation. In this study, we individually examined the contribution of the two CVI-meq isoforms to the attenuation of the vaccine. We inserted the respective isoforms into a very virulent MDV (strain RB-1B), thereby replacing its original meq gene. Surprisingly, we could demonstrate that the longer isoform of meq strongly enhanced virus-induced pathogenesis and tumorigenesis, indicating that other mutations in the CVI genome contribute to virus attenuation. On the contrary, the shorter isoform completely abrogated pathogenesis, demonstrating that changes in the meq gene can indeed play a key role in virus attenuation. Taken together, our study provides important evidence on attenuation of one of the most frequently used veterinary vaccines worldwide. IMPORTANCE Marek’s disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current “gold standard” vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens.

Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek’s disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication. IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy., The highly oncogenic alphaherpesvirus Marek’s disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors. IMPORTANCE Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.

Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked using leptomycin B, suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution. IMPORTANCE Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle, which eventually could help us to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important.

Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that causes generalized polyneuritis and T-cell lymphomas in chickens. MDV is able to integrate its genome into host telomeres, but the mechanism of integration is poorly understood. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome: multiple telomeric repeats (mTMR), with a variable number of up to 100 repeats, and short telomeric repeats (sTMR), with a fixed number of 6 repeats. The mTMR have recently been shown to play an important role in MDV integration and tumor formation; however, the functions of the sTMR have remained unknown. In this study, we demonstrate that deletion of the sTMR in the MDV genome abrogates virus replication, while extensive mutation of the sTMR does not, indicating that the presence of the sTMR but not the sTMR sequence itself is important. Furthermore, we generated a panel of truncation mutants to determine the minimal length of the sTMR and observed a direct correlation between sTMR length and MDV replication. To address the role of sTMR in MDV replication, integration, and tumorigenesis, sTMR sequences were replaced by a scrambled repeated sequence (vsTMR_mut). vsTMR_mut replicated comparably to parental and revertant viruses in vitro. In vivo , however, a significant reduction in disease and tumor incidence was observed in chickens infected with vsTMR_mut that also correlated with a reduced number of viral integration sites in tumor cells. Taken together, our data demonstrate that the sTMR play a central role in MDV genome replication, pathogenesis, and MDV-induced tumor formation. IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes high economic losses in the poultry industry. MDV integrates its genetic material into host telomeres, a process that is crucial for efficient tumor formation. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome that are identical to host telomeres and that are termed mTMR and sTMR. mTMR have been recently shown to be involved in MDV integration, while the functions of sTMR remain unknown. Here, we demonstrate that the presence and length of sTMR sequence, but not the exact nucleotide sequence, are crucial for MDV replication. Furthermore, the sTMR contribute to the high integration frequency of MDV and are important for MDV pathogenesis and tumor formation. As a number of herpesviruses harbor arrays of telomeric repeats (TMR), MDV serves as a model to determine the role of the herpesvirus TMR in replication, integration, and pathogenesis.

Marek's disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo .

Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression.

Marek's disease (MD) is a devastating oncogenic viral disease of chickens caused by Gallid herpesvirus 2 , or MD virus (MDV). MDV glycoprotein C (gC) is encoded by the alphaherpesvirus UL44 homolog and is essential for the horizontal transmission of MDV (K. W. Jarosinski and N. Osterrieder, J. Virol. 84:7911-7916, 2010). Alphaherpesvirus gC proteins are type 1 membrane proteins and are generally anchored in cellular membranes and the virion envelope by a short transmembrane domain. However, the majority of MDV gC is secreted in vitro , although secondary-structure analyses predict a carboxy-terminal transmembrane domain. In this report, two alternative mRNA splice variants were identified by reverse transcription (RT)-PCR analyses, and the encoded proteins were predicted to specify premature stop codons that would lead to gC proteins that lack the transmembrane domain. Based on the size of the intron removed for each UL44 (gC) transcript, they were termed gC104 and gC145. Recombinant MDV viruses were generated in which only full-length viral gC (vgCfull), gC104 (vgC104), or gC145 (vgC145) was expressed. Predictably, gCfull was expressed predominantly as a membrane-associated protein, while both gC104 and gC145 were secreted, suggesting that the dominant gC variants expressed in vitro are the spliced variants. In experimentally infected chickens, the expression of each of the gC variants individually did not alter replication or disease induction. However, horizontal transmission was reduced compared to that of wild-type or revertant viruses when the expression of only a single gC was allowed, indicating that all three forms of gC are required for the efficient transmission of MDV in chickens.

Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo , with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo , vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo , while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo , as well as production of free virus in FFE cells.

Viruses cause about 15% of the cancers that are still the leading causes of human mortality. The discovery of viral oncogenes has enhanced our understanding of viral oncogenesis. However, the underlying molecular mechanisms of virus-induced cancers are complex and require further investigation. The present study has attempted to investigate the effects of the microRNAs (miRNAs) encoded by Marek's disease virus 1 (MDV1), a chicken herpesvirus causing acute T-cell lymphomas and solid visceral tumors in chickens, on anti-cancer drug-induced apoptosis and identify the targets of the miRNAs. The results showed that of the total 14 miRNAs encoded by MDV1, MDV1-miR-M3 significantly promoted cell survival under treatment with cisplatin, a widely used chemotherapy drug. MDV1-miR-M3 suppressed cisplatin-induced apoptosis by directly downregulating expression at the protein but not the mRNA level of Smad2, a critical component in the transforming growth factor β signal pathway. Our data suggest that latent/oncogenic viruses may encode miRNAs to directly target cellular factors involved in antiviral processes including apoptosis, thus proactively creating a cellular environment beneficial to viral latency and oncogenesis. Furthermore, the knowledge of the apoptosis resistance conferred by viral miRNAs has great practical implications for improving the efficacy of chemotherapies for treating cancers, especially those induced by oncogenic viruses.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B Meq BZIP/BZIP ) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B Meq Hom/Hom ). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 ( meqGCN ). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the U S 2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of U S 2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the U L 13 , U L 44 , and U S 6 genes encoding the U L 13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that U S 2 , U L 13 , and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.

Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1BΔRLORF5a (rRB-1BΔRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BΔRLORF4 (rRB-1BΔRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BΔRLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1BΔRLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1BΔRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BΔRLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U S 2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

Marek's disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Marek's disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-α. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/ΔvIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/ΔvIL-8. However, the rMd5/ΔvIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/ΔvIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.

The production of cytokine mRNAs, in addition to viral DNA, was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) (cytokines) or PCR (virus) in splenocytes during the course of Marek's disease virus (MDV) infection in four inbred chicken lines: two resistant (lines 61and N) and two susceptible (lines 72and P). Virus loads were only different after 10 days postinfection (dpi), increasing in susceptible lines and decreasing in resistant lines. Gamma interferon (IFN-γ) mRNA was expressed by splenocytes from all infected birds between 3 and 10 dpi, associated with increasing MDV loads. For other cytokines, differences between lines were only seen for interleukin-6 (IL-6) and IL-18, with splenocytes from susceptible birds expressing high levels of both transcripts during the cytolytic phase of infection, whereas splenocytes from resistant birds expressed neither transcript. These results indicate that these two cytokines could play a crucial role in driving immune responses, which in resistant lines maintain MDV latency but in susceptible lines result in lymphomas.

Experiments were conducted to investigate the roles of Marek's disease virus serotype 1 (MDV-1) major tegument proteins VP11/12, VP13/14, VP16, and VP22 in viral growth in cultured cells. Based on a bacterial artificial chromosome clone of MDV-1 (BAC20), mutant viruses were constructed in which the MDV-1 homologs of UL46, UL47, UL48, or UL49 were deleted alone and in various combinations. It could be demonstrated that the UL46, UL47, and UL48 genes are dispensable for MDV-1 growth in chicken embryonic skin and quail muscle QM7 cells, although the generated virus mutants exhibited reduced plaque sizes in all cell types investigated. In contrast, a UL49-negative MDV-1 (20Δ49) and a UL48-UL49 (20Δ48-49) doubly negative mutant were not able to produce MDV-1-specific plaques on either cell type. It was confirmed that this growth restriction is dependent on the absence of VP22 expression, because growth of these mutant viruses could be partially restored on cells that were cotransfected with a UL49 expression plasmid. In addition, we were able to demonstrate that cell-to-cell spread of MDV-1 conferred by VP22 is dependent on the expression of amino acids 37 to 187 of MDV-1 VP22, because expression plasmids containing MDV-1 UL49 mutant genes with deletions of amino acids 1 to 37 or 188 to 250 were still able to restore partial growth of the 20Δ49 and 20Δ48-49 viruses. These results demonstrate for the first time that an alphaherpesvirus UL49-homologous gene is essential for virus growth in cell culture.

The various alphaherpesviruses, including Marek's disease virus (MDV), have both common and unique features of gene content and expression. The entire MDV U s region has been sequenced in our laboratory (P. Brunovskis and L. F. Velicar, Virology 206:324–338, 1995). Genes encoding the MDV glycoprotein D (gD), glycoprotein I (gI), and glycoprotein E (gE) homologs have been found in this region, although no gG homolog was found. In this work, transcription of the tandem MDV gD, gI, and gE genes was studied and found to have both unique characteristics and also features in common with other alphaherpesviruses. MDV gD could not be immunoprecipitated from MDV GA-infected duck embryo fibroblast cells by antisera reactive to its TrpE fusion proteins, while gI and gE could be. When the gD gene was subjected to in vitro-coupled transcription-translation, the precursor polypeptide was produced and could be immunoprecipitated by anti-gD. Northern blot, reverse transcriptase PCR, and RNase protection analyses have shown that (i) no mRNA initiating directly from the gD gene could be detected; (ii) a large but low-abundance 7.5-kb transcript spanning five genes, including the one encoding gD, was seen on longer exposure; and (iii) transcription of the gI and gE genes formed an abundant bicistronic 3.5-kb mRNA, as well as an abundant 2.0-kb gE-specific mRNA. Therefore, the MDV gD gene expression is down-regulated at the transcription level in MDV-infected cell culture, which may be related to the cell-associated nature of MDV in fibroblast cells. Compared to the highly gD-dependent herpes simplex virus and the other extreme of the varicella-zoster virus which lacks the gD gene, MDV is an intermediate type of alphaherpesvirus.

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.

An earlier report (M. Sakaguchi et al., Vaccine 16:472–479, 1998) showed that recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion (F) protein of Newcastle disease virus (NDV-F) under the control of the simian virus 40 late promoter [rMDV1-US10L(F)] protected specific pathogen-free chickens from NDV challenge, but not commercial chickens with maternal antibodies against NDV and MDV1. In the present study, we constructed an improved polyvalent vaccine based on MDV1 against MDV and NDV in commercial chickens with maternal antibodies. The study can be summarized as follows. (i) We constructed rMDV1 expressing NDV-F under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10P(F)]. (ii) Much less NDV-F protein was expressed in cells infected with rMDV1-US10P(F) than in those infected with rMDV1-US10L(F). (iii) The antibody response against NDV-F and MDV1 antigens of commercial chickens vaccinated with rMDV1-US10P(F) was much stronger and faster than with rMDV1-US10L(F), and a high level of antibody against NDV-F persisted for over 80 weeks postvaccination. (iv) rMDV1-US10P(F) was readily reisolated from the vaccinated chickens, and the recovered viruses were found to express NDV-F. (v) Vaccination of commercial chickens having maternal antibodies to rMDV1-US10P(F) completely protected them from NDV challenge. (vi) rMDV1-US10P(F) offered the same degree of protection against very virulent MDV1 as the parental MDV1 and commercial vaccines. These results indicate that rMDV1-US10P(F) is an effective and stable polyvalent vaccine against both Marek's and Newcastle diseases even in the presence of maternal antibodies.

RB1BUS6 lacgpt , a Marek’s disease virus (MDV) mutant having a disrupted glycoprotein D (gD) homolog gene, established infection and induced tumors in chickens exposed to it by inoculation or by contact. Lymphoblastoid cell lines derived from RB1BUS6 lacgpt -induced tumors harbored only the mutant virus. These results provide strong evidence that an intact gD homolog gene is not essential for oncogenicity or horizontal transmission of MDV.

Marek’s disease virus (MDV) induces the rapid development of overwhelming T-cell lymphomas in chickens. One of its candidate oncogenes, meq (MDV Eco Q) which encodes a bZIP protein, has been biochemically characterized as a transcription factor. Interestingly, MEQ proteins are expressed not only in the nucleoplasm but also in the coiled bodies and the nucleolus. Its novel subcellular localization suggests that MEQ may be involved in other functions beyond its transcriptional potential. In this report we show that MEQ proteins are expressed ubiquitously and abundantly in MDV tumor cell lines. Overexpression of MEQ results in transformation of a rodent fibroblast cell line, Rat-2. The criteria of transformation are based on morphological transfiguration, anchorage-independent growth, and serum-independent growth. Furthermore, MEQ is able to distend the transforming capacity of MEQ-transformed Rat-2 cells through inhibition of apoptosis. Specifically, MEQ can efficiently protect Rat-2 cells from cell death induced by multiple modes including tumor necrosis factor alpha, C2-ceramide, UV irradiation, and serum deprivation. Its antiapoptotic function requires new protein synthesis, as treatment with a protein synthesis inhibitor, cycloheximide, partially reversed MEQ’s antiapoptotic effect. Coincidentally, transcriptional induction of bcl-2 and suppression of bax are also observed in MEQ-transformed Rat-2 cells. Taken together, our results suggest that MEQ antagonizes apoptosis through regulation of its downstream target genes involved in apoptotic and/or antiapoptotic pathways.

A loop-mediated isothermal amplification (LAMP) method for the rapid detection of serotype 1 Marek's disease virus (MDV) was developed. The method used a set of three pairs of primers to amplify the MEQ gene for detecting serotype 1 MDV. The MDV LAMP method did not cross-react with serotype 2 and serotype 3, nor did the LAMP primers have binding sites for the common avian DNA viruses (reticuloendotheliosis virus, chicken anemia virus, subgroup J of the avian leukosis virus). Additionally, the assay could detect up to 10 copies of the MEQ gene in the MD viral genome, and it had 10 times higher sensitivity than the traditional PCR methods. The LAMP master mix was stable for 90 days at −20°C. Furthermore, the efficiency of LAMP for detection of serotype 1 MDV in clinical samples was comparable to those of PCR and viral isolation. The LAMP procedure is simple and does not rely on any special equipment. The detection of serotype 1 MDV by LAMP will be useful for detecting and controlling oncogenic Marek's disease.

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR −/− cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.

Marek’s disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.

The Chinese virulent (CHv) strain of duck enteritis virus (DEV) has a genome of approximately 162,175 nucleotides with a GC content of 44.89%. Here we report the complete genomic sequence and annotation of DEV CHv, which offer an effective platform for providing authentic research experiences to novice scientists. In addition, knowledge of this virus will extend our general knowledge of DEV and will be useful for further studies of the mechanisms of virus replication and pathogenesis.

Three distinct subpopulations of cells with suppressor activity were separated by Ficoll density gradient centrifugation methods from the spleens of 3- to 4- week-old chickens infected with cultured lymphoblastoid cells (JM-VLC), derived from JM-V leukemia: (i) a subpopulation of nonadherent cells, which separated in the T-cell-rich gradient fraction of leukemic chicken spleen, inhibited proliferative responses to concanavalin A in mixed cultures with normal chicken spleen cells; (ii) phagocytic cells (macrophages), which were the most effective suppressor cells of all subpopulations in the mixed culture assay, were recovered among the cells of greatest density in the spleens of both normal and leukemic chickens; (iii) JM-VLC cells in the buoyant gradient fractions of leukemic chicken spleens also effected suppression. In the later stages of lymphoproliferative disease, the number of spleen cells of buoyant density was increased, apparently as a result of infiltration of the spleens with JM-VLC cells.

Four monoclonal antibodies, A35, B94, EB29, and G152, against Marek's disease tumor-associated surface antigen have been developed and their specificities studied against a panel of Marek's disease and lymphoid leukosis primary tumors; Marek's disease, and lymphoid leukosis, and reticuloendotheliosis lymphoblastoid cell lines; and normal chicken cells. A35 and G152 are of the immunoglobulin M class, and B94 and EB29 are of the immunoglobulin G1 subclass.

We incubated 31 newly established Marek's disease tumor cell lines at 41 degrees C for 48 h after subculturing and then examined them to determine the spontaneous rates of expression of viral internal antigen(s), viral membrane antigen(s), and virus isolation. All but two of the lines were isolated from tumors induced by clone-purified Marek's disease virus strain JM-10, GA-5, RB-1B, and BC-1A in nine different genetic strains of chickens with defined histocompatibility antigens. The line-to-line variations in the rates of spontaneous expression for the antigens or virus rescue were great, but the levels of expression were very low in most cases. The median rates of expression for viral internal antigen, viral membrane antigen, and virus isolation were 32, 8, and 2 positive cells per 10(5) cells, respectively (ranges, 0 to 20,280, 0 to 22,990, and 0 to 220 positive cells per 10(5) cells, respectively). The ratio of viral internal antigen expression to virus isolation was extremely variable and often high, whereas the ratio of viral internal antigen to viral membrane antigen expression was more consistent and generally low. The virus strain which induced the cell line influenced the level of virus genome expression, but the cell genotype did not. Cell lines transformed by JM-10 virus, which exhibited low oncogenicity, had significantly (p less than 0.01) higher rates of expression than cell lines transformed by CA-5 and RB-1B viruses, which exhibited high oncogenicity. Treatment with iododeoxyuridine or incubation at 37 degrees C induced increased rates of expression in most lines but not in all lines. The degree of enhanced expression was inversely proportional to the rate of spontaneous expression.

The effect of infection with low-virulence, tissue culture-propagated strains of reticuloendotheliosis virus (REV) on protective vaccinal immunity against Marek's disease (MD) lymphomas was investigated. Vaccinated chickens inoculated at hatching with greater than 10(4) focus-forming units of REV and challenged with MD virus were poorly protected against MD lesion development as indicated by protective indices of 53 to 79% for strain CS (P less than 0.05) and 42 to 49% for strain T (P less than 0.01) compared to 78 to 100% for REV-free controls. Furthermore, the response of blood lymphocytes to mitogen stimulation and the antibody response to sheep erythrocytes and Brucella abortus were less in REV-inoculated chickens than in controls. The REV-induced depression of immune responses was more severe in chickens infected with mildly pathogenic strain T than in chickens infected with the apathogenic strain CS and was generally transient with both virus strains. Little or no depression of immune responses was observed in chickens inoculated with less than 10(3) focus-forming units of REV. These studies extend knowledge on the immunodepressive ability of low-virulence REV strains and establish that infection with these viruses depresses certain parameters of MD vaccinal immunity, an important model for cellular immunity against virus-induced neoplasia in the chicken.

Use of Marek's disease virus (MDV) in a neutralization test presents several problems, which are described, making this potentially useful test difficult. To obviate these difficulties, a plaque reduction test has been designed based on cross-neutralization of turkey herpesvirus (HVT) by serum-neutralizing MDV. The technique for such a neutralization test is outlined. Kinetics of development of neutralizing antibodies in chickens inoculated with HVT and MDV are described. The neutralization test can be used to evaluate viability of HVT vaccines and the possible role of neutralizing antibodies in the protection afforded by vaccination against MDV-induced tumors.

The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 7(2) chickens and in HVT-inoculated line 7(2) chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response.

Spleen cells from chickens inoculated 7 to 8 days previously with Marek's disease virus were cytotoxic for 51Cr-labeled cells of a Marek's disease lymphoblastoid cell line (MSB-1 line) in a 4-h in vitro cytotoxic assay. The cytotoxic activity of spleen cells was inhibited by pretreatment with antithymocyte serum and complement, but not with complement alone or in combination with anti-bursa cell serum or normal preimmune serum. The conclusion was that the effector cell in the above cytotoxic assay was a thymus-derived lymphocyte. Also, pretreatment of target cells with Vibrio cholerae neuraminidase enhanced in vitro cytotoxic activity of effector cells. Similar enzymatic treatment of effector cells had a negligible effect on cytotoxicity.

Adherent cells isolated from spleen of normal specific pathogen-free chickens inhibited mitogen-induced blastogenesis of autochthonous, syngeneic, or allogeneic lymphocytes. The adherent cells were also inhibitory to in vitro proliferation of cells of a rapidly dividing tumor line, MDCC-MSB-1, derived from a lymphoma induced by Marek's disease virus. The effector cell of suppression of both lymphoprolifrative functions appeared to be a macrophage because the suppressive activity of adherent cells could be abrogated by pretreatment with carrageenan but not with antisera specific to chicken T or B cells. The proportion of macrophages needed for effective suppression was substantially higher than the proportion of macrophages ordinarily present in spleen of normal, unstimulated chickens. This heretofore unrecognized suppressive capability of normal, presumably resting macrophages have been detected in certain infections.

The infection of young chickens with Marek's disease herpesvirus (MDHV) leads to the early appearance of viral antigen in the bursa Fabricii, in the kidney tubular epithelium, and particularly in the epithelial cells of growing feather follicles (Calnek and Hitchner, 1969; Purchase, 1970). Viral antigen may persist in feather follicles over several months, whereas in neural lesions or in lymphoid tumors induced by MDHV viral antigen is either lacking or present only in a few cells (Calnek and Hitchner, 1969; Spencer and Calnek, 1970; Purchase, 1970). Haider et al. (1976) have pointed out the high diagnostic value of the double-diffusion agar-gel participitation test (Chubb and Churchill, 1968) using growing feathers of MDHV-infected chickens as antigen.

Transcription of the Marek's disease virus (MDV) genome in tumor tissues from MDV-infected chickens has been studied by analyzing the hybridization kinetics of 3 H-labeled MDV DNA with unlabeled RNA extracted from these tissues. Lymphoid tumors of ovary, spleen, liver, and kidney contained MDV genomes, but the virus-specific RNA sequences were transcribed from less than 15% of the viral DNA. A virus nonproductive lymphoblastoid cell line, designated MKT-1, has been established from a kidney lymphoma and contains 15 MDV genomes per cell. In these cells, 12 to 14% of the viral DNA was transcribed. Thus transcription of the MDV genome was restricted both in tumor tissues and MKT-1 cells. A hybridization experiment where RNA extracted from MKT-1 cells and RNA extracted from a spleen tumor were mixed and hybridized to 3 H-labeled MDV DNA indicated that the virus-specific RNAs from the two sources were encoded by the same DNA sequences. The polyribosomal fractions of MKT-1 cells and this spleen tumor contained only a portion of the virus-specific RNA sequences found in whole-cell extracts, indicating the existence of a posttranscriptional control mechanism which prevents the transfer of certain viral RNA transcripts to the polyribosomes. The data suggest that the repressed expression of the viral genome in lymphoid tumor tissues and MKT-1 cells may be the result of precise controls within the cell at the transcriptional and posttranscriptional levels.

Marek's disease virus (MDV) and pseudorabies virus (PRV) cross-reacted by fluorescent antibody test but not by gel diffusion, virus neutralization, or cross-protection tests. Herpesvirus of turkeys cross-reacted with MDV but not with PRV. Other herpesviruses tested did not have cross-reactions.

Chickens of various age levels, free from prior infection, were simultaneously exposed to Marek's disease virus, and the response of each age group was recorded. Four- and 20-week-old chickens of lines 15×7 and CM (commercial source) had substantial resistance to mortality and gross lesions. In contrast, in line 7, which was tested at 1-day, 2-, 4-, 8-, 12- and 16-week age levels, 4-week-old chickens were fully susceptible to clinical Marek's disease (MD), although resistance was demonstrated at 8-week and older age levels. Genetically resistant chickens of line 6 maintained their resistance at all age levels tested. Pathogenesis of MD was compared in 12-week-old and 1-day-old chickens of line 15×7. Within the 1-day-old group, 23% of the chickens died because of MD, whereas there were no deaths in the 12-week-old group. Both groups developed viremia although duration, incidence, and levels of virus in the 1-day-old group were higher than in the 12-week-old group. Although initially the 12-week-old group responded by producing higher levels of antibody, the long term incidence of agar gel precipitin, immunofluorescent, and virus neutralization antibody in the two groups was similar. Gross and microscopic lesions of MD developed in both groups, but lesions regressed in the 12-week-old group and persisted in the 1-day-old group. It was concluded that age resistance to MD was expressed through lesion regression.