Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence.

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

Methods for the Manipulation of Herpesvirus Genome and the Application to Marek's Disease Virus Research.

Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek's disease virus research.

Latest Insights into Unique Open Reading Frames Encoded by Unique Long (UL) and Short (US) Regions of Marek's Disease Virus.

Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus whose genome consists of unique long (UL) and short (US) regions that are flanked by inverted repeat regions. More than 100 open reading frames (ORFs) have been annotated in the MDV genome, and are involved in various aspects of MDV biology and pathogenesis. Within UL and US regions of MDV, there are several unique ORFs, some of which have recently been shown to be important for MDV replication and pathogenesis. In this review, we will summarize the current knowledge on these ORFs and compare their location in different MDV strains.

Manipulation of Promyelocytic Leukemia Protein Nuclear Bodies by Marek's Disease Virus Encoded US3 Protein Kinase.

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are dynamic nuclear structures, shown to be important for herpesvirus replication; however, their role in regulating Marek's disease virus (MDV) infection has not been studied. MDV is an oncogenic alphaherpesvirus that causes lymphoproliferative disease in chickens. MDV encodes a US3 serine/threonine protein kinase that is important for MDV replication and gene expression. In this study, we studied the role of MDV US3 in regulating PML-NBs. Using an immunofluorescence assay, we found that MDV US3 disrupts PML and SP100 in a kinase dependent manner. In addition, treatment with MG-132 (a proteasome inhibitor) could partially restore the levels of PML and SP100, suggesting that a cellular proteasome dependent degradation pathway is involved in MDV US3 induced disruption of PML and SP100. These findings provide the first evidence for the interplay between MDV proteins and PML-NBs.

US3 Serine/Threonine Protein Kinase from MDV-1, MDV-2, and HVT Differentially Regulate Viral Gene Expression and Replication.

Gallid alphaherpesvirus 2 (GaHV-2), commonly known as Marek's disease virus type 1 (MDV-1), is an oncogenic avian alphaherpesvirus, and along with its close relatives-Gallid alphaherpesvirus 3 (GaHV-3) or MDV-2 and Meleagrid alphaherpesvirus 1 (MeHV-1) or turkey herpesvirus (HVT)-belongs to the Mardivirus genus. We and others previously showed that MDV-1 US3 protein kinase plays an important role in viral replication and pathogenesis, which could be partially compensated by MDV-2 and HVT US3. In this study, we further studied the differential roles of MDV-1, MDV-2 and HVT US3 in regulating viral gene expression and replication. Our results showed that MDV-2 and HVT US3 could differentially compensate MDV-1 US3 regulation of viral gene expression in vitro. MDV-2 and HVT US3 could also partially rescue the replication deficiency of MDV-1 US3 null virus in the spleen and thymus, as determined by immunohistochemistry analysis of MDV-1 pp38 protein. Importantly, using immunohistochemistry and dual immunofluorescence assays, we found that MDV-2 US3, but not HVT US3, fully compensated MDV-1 US3 regulation of MDV-1 replication in bursal B lymphocytes. In conclusion, our study provides the first comparative analysis of US3 from MDV-1, MDV-2 and HVT in regulating viral gene expression in cell culture and MDV-1 replication in lymphocytes.

A Novel Effective and Safe Vaccine for Prevention of Marek's Disease Caused by Infection with a Very Virulent Plus (vv+) Marek's Disease Virus.

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset lymphoma in chickens. Marek's disease (MD) is effectively controlled using vaccination; however, MDV continues to break through vaccinal immunity, due to the emergence of highly virulent field strains. Earlier studies revealed that deletion of the meq gene from MDV resulted in an attenuated virus that protects against MD in chickens challenged with highly virulent field strains. However, the meq deleted virus retains the ability to induce significant lymphoid organ atrophy. In a different study, we found that the deletion of the vIL8 gene resulted in the loss of lymphoid organ atrophy in inoculated chickens. Here, we describe the generation of a recombinant MDV from which both meq and vIL8 genes were deleted. In vitro studies revealed that the meq and vIL8 double deletion virus replicated at levels similar to the parental very virulent plus (vv+) virus. In addition, in vivo studies showed that the double deletion mutant virus (686BAC-ΔMeqΔvIL8) conferred protection comparable to CVI988, a commercial vaccine strain, when challenged with a vv+ MDV virus, and significantly reduced lymphoid organ atrophy, when compared to meq null virus, in chickens. In conclusion, our study describes the development of a safe and effective vaccine candidate for prevention of MD in chickens.

Marek's disease virus US3 protein kinase phosphorylates chicken HDAC 1 and 2 and regulates viral replication and pathogenesis.

Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus that elicits a rapid onset of malignant T-cell lymphomas in chickens. Three MDV types, including GaHV-2 (MDV-1), GaHV-3 (MDV-2) and MeHV-1 (HVT), have been identified and all encode a US3 protein kinase. MDV-1 US3 is important for efficient virus growth in vitro. To study the role of US3 in MDV replication and pathogenicity, we generated an MDV-1 US3-null virus and chimeric viruses by replacing MDV-1 US3 with MDV-2 or HVT US3. Using MD as a natural virus-host model, we showed that both MDV-2 and HVT US3 partially rescued the growth deficiency of MDV-1 US3-null virus. In addition, deletion of MDV-1 US3 attenuated the virus resulting in higher survival rate and lower MDV specific tumor incidence, which could be partially compensated by MDV-2 and HVT US3. We also identified chicken histone deacetylase 1 (chHDAC1) as a common US3 substrate for all three MDV types while only US3 of MDV-1 and MDV-2 phosphorylate chHDAC2. We further determined that US3 of MDV-1 and HVT phosphorylate chHDAC1 at serine 406 (S406), while MDV-2 US3 phosphorylates S406, S410, and S415. In addition, MDV-1 US3 phosphorylates chHDAC2 at S407, while MDV-2 US3 targets S407 and S411. Furthermore, biochemical studies show that MDV US3 mediated phosphorylation of chHDAC1 and 2 affect their stability, transcriptional regulation activity, and interaction network. Using a class I HDAC specific inhibitor, we showed that MDV US3 mediated phosphorylation of chHDAC1 and 2 is involved in regulation of virus replication. Overall, we identified novel substrates for MDV US3 and characterized the role of MDV US3 in MDV pathogenesis.

Marek's disease virus Meq oncoprotein interacts with chicken HDAC 1 and 2 and mediates their degradation via proteasome dependent pathway.

Marek's disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

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.

Marek's Disease Virus Cluster 3 miRNAs Restrict Virus' Early Cytolytic Replication and Pathogenesis.

Herpesvirus-encoded microRNAs (miRNAs) have been discovered in infected cells; however, lack of a suitable animal model has hampered functional analyses of viral miRNAs in vivo. Marek's disease virus (MDV) (Gallid alphaherpesvirus 2, GaHV-2) genome contains 14 miRNA precursors, which encode 26 mature miRNAs, grouped into three clusters. In this study, the role of MDV-encoded cluster 3 miRNAs, also known as mdv1-miR-M8-M10, in pathogenesis was evaluated in chickens, the natural host of MDV. Our results show that deletion of cluster 3 miRNAs did not affect virus replication and plaque size in cell culture, but increased early cytolytic replication of MDV in chickens. We also observed that deletion of cluster 3 miRNAs resulted in significantly higher virus reactivation from peripheral blood lymphocytes. In addition, pathogenesis studies showed that deletion of cluster 3 miRNAs resulted in more severe atrophy of lymphoid organs and reduced mean death time, but did not affect the incidence of MDV-associated visceral tumors. We confirmed these results by generating a cluster 3 miRNA revertant virus in which the parental MDV phenotype was restored. To the best of our knowledge, our study provides the first evidence that MDV cluster 3 miRNAs play an important role in modulating MDV pathogenesis.

Role of Marek's Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation.

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.

Effects of combined viral-bacterial challenge with or without supplementation of Saccharomyces cerevisiae boulardii strain CNCM I-1079 on immune upregulation and DMI in beef heifers.

Objectives were to determine whether live yeast (LY) supplementation would affect daily dry matter feed intake, body weight (BW), immune, and febrile responses to a viral-bacterial (VB) respiratory challenge. Crossbred heifers (N = 38, BW = 230 ± 16.4 kg) were allocated into a 2 × 2 factorial treatment arrangement: Factor 1 = roughage-based diet with or without LY (Saccharomyces cerevisiae boulardii CNCM I-1079, 62.5 g/hd/d), Factor 2 = VB, intranasal administration of bovine herpesvirus-1 (BHV-1, 2 ×108, PFU) on day 0 and endobronchial inoculation with Mannheimia haemolytica (5.4 × 1010, CFU) on day 3, or intranasal saline administration followed by inoculation with phosphate buffer solution (PBS). Heifers were fed their respective diets for 27 d prior to VB challenge on day 0. Heifers were housed by treatment and group-fed using electronic feedbunks. Thermo-boluses (Medria; Châteaubourg, FR) measured rumen temperature (RUT) at 5-min intervals and rectal temperature and whole blood samples were collected on days 0, 3 to 8, 10, 13, and 15. Data were analyzed using repeated measures in the mixed procedure of SAS with fixed effects of day, diet, inoculation, and their interactions. Animals fed LY exhibited a 16% increase (P = 0.02) in neutrophils relative to CON. Diet × inoculation × day interactions were detected for monocytes and haptoglobin. The VB-LY had the greatest (P < 0.05) concentration of monocytes on day 4, followed by VB-CON which was greater (P < 0.05) than PBS treatments. Haptoglobin concentration was greatest (P < 0.02) for VB-CON on day 5, followed by VB-LY which was greater (P < 0.05) than PBS. Heifers supplemented with LY had less (P < 0.05) haptoglobin production than CON. The VB challenge produced nasal lesions that increased (P < 0.01) with day, reaching a zenith on day 6 with 70% of the nares covered with plaques, and increased (P < 0.05) neutrophils on days 3 to 5. The VB challenge increased RUT (P < 0.05) days 2 to 7 and rectal temperature (P < 0.05) on days 0 and 3 to 6. The increased rectal temperature on day 0 was likely due to increased ambient temperature at time of challenge, as VB heifers were processed after the PBS heifers to avoid contamination. The VB challenge was effective at stimulating immune responses, and RUT was effective for measuring febrile responses. These results indicate that prior LY supplementation altered the leukogram in response to VB challenge, suggestive of increased innate immune response.

Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

Evaluation and Identification of Marek's Disease Virus BAC Clones as Standardized Reagents for Research.

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease (MD), a lymphoproliferative disease in chickens. Understanding of MDV gene function advanced significantly following the cloning of the MDV genome as either a series of overlapping cosmids or as a bacterial artificial chromosome (BAC), both of which could produce viable MDV. The objectives of this study were to compare multiple virulent MDV BAC clones using the Avian Disease and Oncology Laboratory's pathotyping assay, and to demonstrate the use of these clones as standardized reagents for a modified pathotyping assay by other laboratories. To date, MDV BAC clones have been produced for at least 10 MDV strains from all three serotypes including several virulent serotype 1 strains. We determined that MDV BAC clones exist for each virulent pathotype, despite the fact that these clones are not always equal in virulence to their corresponding parental strains. One clone from each pathotype was further evaluated in commercial specific-pathogen-free (SPF) chickens and found suitable for use in assays such as best-fit pathotyping, although results were variable based on the source of the SPF birds. The benefits of using BAC clones, which include easy shipping, ability to more easily manipulate, and long-term ability to use at a low passage level, are likely to result in the use of BAC clones as standard reagents for MD research. The use of the defined set of clones should allow side-by-side comparison, allowing researchers to better interpret results produced in different laboratories using different MDV field strains. Furthermore, a modified best-fit pathotyping assay has been proposed using these clones and reduced bird numbers.

Marek's disease vaccines: Current status, and strategies for improvement and development of vector vaccines.

Marek's disease (MD) is a lymphoproliferative viral disease of chickens, which has been controlled through vaccination since 1969. MD vaccines protect against tumors but do not provide sterilizing immunity, and thus it is generally believed that their use has contributed to increase virulence of field strains with the ability to cause MD in vaccinated chickens. Traditional methods of developing vaccines, like cell culture attenuation, have proved unsuccessful for the development of improved vaccines to protect against highly virulent MD virus (MDV) field strains. With the advent of recombinant DNA technology, it is now possible to study MDV gene function and develop rational vaccines that protect against highly pathogenic strains. In addition, the long term protection conferred by MD vaccines, their excellent safety profile, their efficacy when administered early (at hatch or in ovo), and their ability to overcome maternal antibodies, has made MDV an excellent candidate vector to protect not only against MD but also against other important viral poultry diseases. In this review we will discuss the current status of MD vaccines and their use as vector vaccines to control important viral poultry diseases.

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.

Deletion of Marek's disease virus large subunit of ribonucleotide reductase impairs virus growth in vitro and in vivo.

Marek's disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens called Marek's disease (MD). In the unique long (UL) region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of the ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present in chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5deltaRR1) in which RR1 was deleted. PCR amplification of the RR gene in Md5deltaRR1-infected duck embryo fibroblasts (DEF) confirmed the deletion of the 2.4 kb RR1 gene with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed a UL39 deletion and the absence of gross rearrangement. The biologic characteristics of Md5deltaRR1 virus were studied in vitro and in vivo. The Md5deltaRR1 replicated in DEF, but significantly slower than parental Md5-BAC, suggesting that RR is important but not essential for replication in fibroblasts. In vivo studies, however, showed that the RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of specific-pathogen-free (SPF) chickens with Md5deltaRR1 showed the mutant virus is nonpathogenic and does not induce MD in birds. A revertant virus, Md5deltaRR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that the Md5deltaRR1 virus is not a candidate vaccine against MD.

Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

Properties of a meq-deleted rmd5 Marek's disease vaccine: protection against virulent MDV challenge and induction of lymphoid organ atrophy are simultaneously attenuated by serial passage in vitro.

We have previously shown that deletion of the meq gene from the genome of Cosmid-cloned rMd5 strain of Marek's disease virus (MDV-1) resulted in loss of transformation and oncogenic capacity of the virus. The rMd5deltaMeq (Meq null) virus has been shown to be an excellent vaccine in maternal antibody positive (MAb+) chickens challenged with a very virulent plus (vv+) strain of MDV, 648A. The only drawback was that it retained its ability to induce bursa and thymus atrophy (BTA) like that of the parental rMd5 in maternal antibody negative (MAb-) chickens. We recently reported that the attenuated Meq null virus did not induce BTA at the 40th cell culture passage onward. Its protective ability against challenge with vv+ MDV, strain 686 was similar to the original virus at the 19th passage in MAb- chickens. In this study, we compared the same series of attenuated meq null viruses in commercial chickens. In commercial chickens with MAb, the attenuated viruses quickly lost protection with increasing cell culture attenuation. These data suggest that although attenuation of these meq null viruses eliminated BTA, it had no influence on their protective efficacy in MAb- chickens. However, in commercial chickens (MAb+), the best protection was provided by the original 19th passage; the attenuated 40th passage was as good as one of the currently commercial CVI988/Rispens vaccine, and it did not induce BTA. Therefore, protection against virulent MDV challenge and induction of lymphoid organ atrophy are simultaneously attenuated by serial passage in vitro.