Investigation of a herpesvirus outbreak in mixed breeds of adult domestic ducks using next generation sequencing.

This report characterizes the first lethal outbreak of Marek's disease on a large farm of mixed-breed adult ducks (>18,000) and identifies the pathogen that resulted in high mortality (35%). Clinical signs included inappetence, respiratory distress, depression, muscle weakness, and ataxia. Post mortem revealed enlarged fragile liver mottled with miliary whitish spots and an enlarged spleen. Histopathology revealed hepatocellular necrosis with eosinophilic intra-nuclear inclusion bodies, necrosis of splenic follicles and degeneration/necrosis of renal tubules. The disease was tentatively diagnosed as a herpesvirus infection, confirmed by virus isolation from the liver. DNA was isolated from 15-year-old archival formalin-fixed tissues from infected ducks and subjected to next generation sequencing (NGS). Despite highly degraded DNA, short stretches of G- and C-rich repeats (TTAGGG and TAACCC) were identified as telomeric repeats frequently found in herpesviruses. Megablast and further investigative bioinformatics identified presence of Marek's disease virus (MDV), a Gallid alphaherpesvirus type 2 (GAHV-2), as the cause of the acute fatal infection. The source of infection may be attributed to a dead migratory flamingo found close to the duck enclosures three days prior to the outbreak; hence, GAHV-2 may also be responsible for the fatal infection of the flamingo accentuated by heat stress. Considering the possible spread of this highly contagious and lethal virus from a flamingo to the ducks, and the increasing zoonosis of animal viruses into humans, such as monkey B alphaherpesvirus transmission from macaques to humans with ~80% fatality, this observation has important ramifications for human health and safety of the poultry industry.

MDV-induced differential microRNA expression in the primary lymphoid organ of thymus.

Marek's disease virus (MDV), a highly contagious cell associated virus, is the etiological agent of Marek's disease (MD), a lymphoproliferative and neuropathic disease of domestic chickens. Clinical signs of MD include transient paralysis, bursal/thymic atrophy, and T cell lymphomas. MicroRNAs (miRNAs) are short single-stranded non-coding RNAs that regulate gene expression by transcriptional suppression or mRNA degradation. Herpesviruses, including MDV, encode for miRNAs that are known to play essential roles in viral pathogenicity, oncogenesis, and evasion of immune responses. In this study, we performed miRNA sequencing in thymuses of control and MDV-infected chickens of MD-resistant (63) and susceptible (72) lines at 21 days post infection (dpi). The thymus is a lymphoid organ that undergoes severe atrophy due to MDV-induced apoptotic mediated destruction of T cells. Sequence analysis identified 658 total chicken miRNAs in the thymuses of control and MDV-infected birds of both lines. Of these, 453 were novel and 205 were known microRNAs. All novel miRNAs mapped to chicken genome with no sequence homology to existing miRNAs in the chicken miRbase. Comparative analysis between the thymuses of control and infected birds of resistant and susceptible lines identified 78 differentially expressed microRNAs that might provide insights into mechanisms of thymus atrophy.

Marek's Disease in an Indian Peafowl (Pavo cristatus) with Clinical Ocular Disease and Paraparesis.

Marek's disease (MD) is caused by virulent strains of Gallid alphaherpesvirus type 2 (MD virus serotype 1; MDV 1) and frequently causes a lymphoproliferative disorder in poultry and other galliform birds worldwide. However, within the peafowl (Phasianinae) subfamily, there are only rare confirmed reports of MD. Here we report MD in an Indian peafowl (Pavo cristatus), which clinically presented with hindlimb paraparesis and intraocular swelling of the right eye. Soft, off-white to tan masses within the right eye, sciatic nerves and coelomic cavity were identified at post-mortem examination which effaced the cranial pole of the kidneys and diffusely effaced the testes. Lymphoid neoplasia was identified histologically at all of these sites and there was extensive hepatic lymphoid cell infiltration, which had not been grossly evident. The T-cell origin of the lymphoid cells was confirmed by immunohistochemistry for CD3 antigen. A virulent strain of MDV 1 was detected by real-time polymerase chain reaction in DNA samples extracted from the kidney and testes. As MD is rare in peafowl it should be considered as a differential diagnosis for intraocular and coelomic masses with associated clinical signs.

Simultaneous detection and differentiation of three oncogenic viral diseases of chicken by use of multiplex PCR.

Avian oncogenic or tumor diseases are common in poultry industry causing significant economic loss. Marek's disease (MD), avian leukosis (AL) and Reticuloendotheliosis (RE) are the three major viral oncogenic infections that are difficult to differentiate with gross lesions. Multiplex PCR for simultaneous detection and differentiation of these three viruses was developed and validated. The primers targeting the genes of pp38, pol and LTR for MDV, ALV and REV were designed to yield 206, 429, and 128 bp, respectively. The sensitivity of the PCR primers was checked with serial dilution of positive template DNA for each virus and found to be in the range of 10-5 to 10-7 of 1 µg/µl of initial template DNA. Out of 114 suspected tumor samples screened, 8 samples were positive for MDV, 13 samples were positive for ALV and 31 samples positive for REV. Five samples were positive for both MD and ALV; 3 samples were positive for MD and REV and 25 samples were positive for ALV and REV. Eight samples were positive for all three viruses. Multiplex PCR demonstrated to be a useful technique for simultaneous, rapid detection and differentiation of major tumor causing and immunosuppressive viral diseases of chicken.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

Marek's Disease Virus Telomeric Integration Profiles of Neoplastic Host Tissues Reveal Unbiased Chromosomal Selection and Loss of Cellular Diversity during Tumorigenesis.

The avian α-herpesvirus known as Marek's disease virus (MDV) linearly integrates its genomic DNA into host telomeres during infection. The resulting disease, Marek's disease (MD), is characterized by virally-induced lymphomas with high mortality. The temporal dynamics of MDV-positive (MDV+) transformed cells and expansion of MD lymphomas remain targets for further understanding. It also remains to be determined whether specific host chromosomal sites of MDV telomere integration confer an advantage to MDV-transformed cells during tumorigenesis. We applied MDV-specific fluorescence in situ hybridization (MDV FISH) to investigate virus-host cytogenomic interactions within and among a total of 37 gonad lymphomas and neoplastic splenic samples in birds infected with virulent MDV. We also determined single-cell, chromosome-specific MDV integration profiles within and among transformed tissue samples, including multiple samples from the same bird. Most mitotically-dividing cells within neoplastic samples had the cytogenomic phenotype of 'MDV telomere-integrated only', and tissue-specific, temporal changes in phenotype frequencies were detected. Transformed cell populations composing gonad lymphomas exhibited significantly lower diversity, in terms of heterogeneity of MDV integration profiles, at the latest stages of tumorigenesis (>50 days post-infection (dpi)). We further report high interindividual and lower intraindividual variation in MDV integration profiles of lymphoma cells. There was no evidence of integration hotspots into a specific host chromosome(s). Collectively, our data suggests that very few transformed MDV+ T cell populations present earlier in MDV-induced lymphomas (32-50 dpi), survive, and expand to become the dominant clonal population in more advanced MD lymphomas (51-62 dpi) and establish metastatic lymphomas.

Pathologic Characterization of Coinfection with Histomonas meleagridis, Marek's Disease Virus, and Subtype J Avian Leukosis Virus in Chickens.

Histomonas meleagridis is a trichomonad protozoan parasite that can cause an important poultry disease known as histomoniasis; Marek's disease virus (MDV) and subtype J avian leukosis virus (ALV-J) usually cause avian oncogenic diseases. Although these diseases have been reported in a single pathogen infection, information about their coinfection is scarce. This study reports a naturally occurring case of coinfection with H. meleagridis, MDV, and ALV-J in a local chicken flock at the age of 150 days. Necropsy revealed necrosis and swelling in the liver and spleen. Histologic analysis showed large areas of mild to severe necrosis of hepatocytes, with numerous intralesional trophozoites of H. meleagridis by H&E and periodic acid-Schiff staining; H&E staining showed pleomorphic and neoplastic lymphoid tumor cells in the liver and myeloid cells with eosinophilic cytoplasmic granules in the spleen. Coexpression of MDV and ALV-J antigens was detected in the liver by fluorescence multiplex immunohistochemistry staining. The 18S rRNA gene of H. meleagridis, meq gene of MDV, and gp85 gene of ALV-J were identified in mixed liver and spleen tissues by PCR and sequencing, respectively.

Reporte de caso­Caracterización patológica de la coinfección con Histomonas meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en pollos Histomonas meleagridis es un parásito protozoario tricomonial que puede causar una enfermedad avícola importante conocida como histomoniasis; El virus de la enfermedad de Marek (MDV) y el virus de la leucosis aviar subtipo J (ALV-J) suelen causar enfermedades oncogénicas aviares. Aunque estas enfermedades se han reportado como infecciones patógenas separadas, la información sobre coinfección es escasa. Este estudio reporta un caso natural de coinfección con H. meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en una parvada de pollos local a la edad de 150 días. La necropsia reveló necrosis e inflamación del hígado y el bazo. El análisis histológico mostró grandes áreas de necrosis de hepatocitos de leve a severa, con numerosos trofozoítos intralesionales de H. meleagridis por tinción de hematoxilina y eosina y por tinción de ácido periódico-Schiff. La tinción de hematoxilina y eosina mostró células linfoides neoplásicas y pleomórficas en el hígado y en el bazo presencia de células mieloides con gránulos citoplásmicos eosinofílicos. La coexpresión de antígenos del virus de Marek y de la leucosis aviar subtipo J se detectó en el hígado mediante tinción inmunohistoquímica de fluorescencia múltiple. El gene de ARNr 18S de H. meleagridis, el gene meq del virus de Marek y el gene gp85 del virus de la leucosis aviar subtipo J se identificaron en tejidos mixtos de hígado y bazo mediante PCR y secuenciación, respectivamente.

Cytokine expression in the eye and brain of chickens following infection with a very virulent plus Marek's disease virus strain.

Cytokine transcripts were evaluated chronologically in the brain and in the eye of chickens infected with the very virulent plus Marek's disease virus (vv + MDV) strain 648A. Brain and eye samples were collected from chickens that were either suffering from transient paralysis (TP) (11 days post inoculation, dpi) or had completely recovered from TP but started developing clinical signs of persistent neurological disease (PND) (18-31 dpi). Results obtained from samples collected at 11 dpi are referred as EL (early lesions) and results obtained from samples collected at later times (18-31 dpi) are referred as LL (late lesions). Marked differences were found in the cytokine transcripts in brain and eye. While proinflammatory cytokines (IL-1ß, IL-8, IL-18), iNOS, IFN-α, IFN-γ, and IL-15 were upregulated in the brain during EL and LL, only IL-8 and IFN-γ were upregulated in the eye at both times (EL and LL). The two evaluated viral transcripts (gB and meq) were found in both eye and brain during EL and LL. Levels of the two viral transcripts evaluated were higher at LL than at EL in both brain and eye. No differences were found in any of the viral transcripts between eye and brain during EL. However, during the LL, the levels of meq transcripts were higher in the eye than in the brain. Our results suggest that MDV elicits different immune responses in the brain and in the eye of infected chickens. Because immune responses in the eye of chickens have been poorly studied, further studies on the pathogenesis of MDV in the eye could greatly contribute to our knowledge on the chicken eye immunity.

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.

Role of microRNA and long non-coding RNA in Marek's disease tumorigenesis in chicken.

Marek's disease virus (MDV), the causative agent of Marek's disease (MD), results in highly infectious phymatosis, lymphatic tissue hyperplasia, and neoplasia. MD is associated with high morbidity and mortality rate. Non-coding RNAs (ncRNAs) entails long non-coding RNA (lncRNA) and microRNA (miRNA). Numerous studies have reported that specific miRNAs and lncRNAs participate in multiple cellular processes, such as proliferation, migration, and tumor cell invasion. Specialized miRNAs and lncRNAs militate a similar role in MD tumor oncogenesis. Despite its growing popularity, only a few reviews are available on ncRNA in MDV tumor oncogenes. Herein, we summarized the role of the miRNAs and lncRNAs in MD tumorigenesis. Altogether, we brought forth the research issues, such as MD prevention, screening, regulatory network formation, novel miRNAs, and lncRNAs analysis in MD that needs to be explored further. This review provides a theoretical platform for the further analysis of miRNAs and lncRNAs functions and the prevention and control of MD and malignancies in domestic animals.

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.

Marek's Disease Virus Requires Both Copies of the Inverted Repeat Regions for Efficient In Vivo Replication and Pathogenesis.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of chickens. The MDV genome consists of two unique regions that are both flanked by inverted repeat regions. These repeats harbor several genes involved in virus replication and pathogenesis, but it remains unclear why MDV and other herpesviruses harbor these large sequence duplications. In this study, we set to determine if both copies of these repeat regions are required for MDV replication and pathogenesis. Our results demonstrate that MDV mutants lacking the entire internal repeat region (ΔIRLS) efficiently replicate and spread from cell-to-cell in vitro However, ΔIRLS replication was severely impaired in infected chickens and the virus caused significantly less frequent disease and tumors compared to the controls. In addition, we also generated recombinant viruses that harbor a deletion of most of the internal repeat region, leaving only short terminal sequences behind (ΔIRLS-HR). These remaining homologous sequences facilitated rapid restoration of the deleted repeat region, resulting in a virus that caused disease and tumors comparable to the wild type. Therefore, ΔIRLS-HR represents an excellent platform for rapid genetic manipulation of the virus genome in the repeat regions. Taken together, our study demonstrates that MDV requires both copies of the repeats for efficient replication and pathogenesis in its natural host.IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes losses in the poultry industry of up to $2 billion per year. The virus is also widely used as a model to study alphaherpesvirus pathogenesis and virus-induced tumor development in a natural host. MDV and most other herpesviruses harbor direct or inverted repeats regions in their genome. However, the role of these sequence duplications in MDV remains elusive and has never been investigated in a natural virus-host model for any herpesvirus. Here, we demonstrate that both copies of the repeats are needed for efficient MDV replication and pathogenesis in vivo, while replication was not affected in cell culture. With this, we further dissect herpesvirus genome biology and the role of repeat regions in Marek's disease virus replication and pathogenesis.

An outbreak in three-yellow chickens with clinical tumors of high mortality caused by the coinfection of reticuloendotheliosis virus and Marek's disease virus: a speculated reticuloendotheliosis virus contamination plays an important role in the case.

Both reticuloendotheliosis and Marek's disease are neoplastic diseases of chickens caused by reticuloendotheliosis virus (REV) and Marek's disease virus (MDV), respectively. The infection of REV or MDV may lead to clinical tumors and also result in immunosuppression and easily allow secondary infection by other pathogens. Here, we investigated a breeder flock of three-yellow chickens in southern China that had been vaccinated with CVI988/Rispens at hatching and had experienced depression, weakness, reduction in weight gain, and an increased death rate after 120 d of age. The morbidity and mortality were 20% and 10%, respectively, at 140 d of age when this infection was diagnosed. The necropsy of the birds revealed significant tumor-like lesions in the heart, liver, spleen, and ceca. Peripheral blood lymphocytes and tumor-like tissues were sampled for PCR detection and for histopathological observation, for virus isolation and the subsequent immunofluorescent assay on the cell cultures and for gene sequencing of the isolated viruses. A REV isolate GX18NNR1 and a MDV isolate GX18NNM5 were both recovered from the sampled bird. Further phylogenetic analysis based on the env gene of REV and the meq gene of MDV demonstrated that GX18NNR1 was closely related to the reference REV strain MD-2, which was isolated from a contaminated commercial turkey herpesvirus vaccine. In addition, the GX18NNM5 was found to belong to the Chinese very virulent MDV strains' cluster. The coinfection of REV and MDV may contribute to tumor outbreaks with high morbidity and mortality in three-yellow chicken flocks.

The Emergence of a vv + MDV Can Break through the Protections Provided by the Current Vaccines.

Marek's disease (MD) is an infectious malignant T-cell lymphoma proliferative disease caused by Marek's disease virus (MDV). In recent years, the emergence of very virulent (vv) and/or very virulent plus (vv +) strains of MDV in the field has been suggested as one of the causes of vaccination failure. The pathogenicity of the MDV strain GX18NNM4, isolated from a clinical outbreak in a broiler breeder flock that was vaccinated with CVI988/Rispens, was investigated. In the vaccination-challenge test, GX18NNM4 was able to break through the protections provided by the vaccines CVI988 and 814. It also significantly reduced body weight gain and caused marked gross lesions and a large area of infiltration of neoplastic lymphocyte cells in the heart, liver, pancreas, etc. of the infected birds. In addition, the expressions of programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), in the spleens and cecal tonsils (CTs) of the unvaccinated challenged birds were significantly increased compared to those in the vaccinated challenged birds, indicating that the PD-1/PD-L1 pathway is related to immune evasion mechanisms. The results showed that the GX18NNM4 strain could cause severe immunosuppression and significantly decrease the protections provided by the current commercial vaccines, thus showing GX18NNM4 to be a vv + MDV strain.

Molecular characterization of a Marek's disease virus strain detected in tumour-bearing turkeys.

Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2), which primarily affects chickens. However, the virus is also able to induce tumours in turkeys, albeit less frequently than in chickens. This study reports the molecular characterization of a GaHV-2 strain detected in a flock of Italian meat-type turkeys exhibiting visceral lymphomas. Sequencing and phylogenetic analysis of the meq gene revealed that the turkey GaHV-2 has molecular features of high virulence and genetic similarity with GaHV-2 strains recently detected in Italian commercial and backyard chickens. GaHV-2 is ubiquitous among chickens despite vaccination, and chicken-to-turkey transmission is hypothesized due to the presence of broilers in neighbouring pens.RESEARCH HIGHLIGHTS A GaHV-2 strain from Italian turkeys was molecularly characterized.The turkey strain presented molecular characteristics of high virulence in its meq gene.The turkey strain was closely related to previously detected chicken strains.

IFNα and IFNγ Impede Marek's Disease Progression.

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek's disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus.

Histologic findings and viral antigen distribution in natural coinfection of layer hens with subgroup J avian leukosis virus, Marek's disease virus, and reticuloendotheliosis virus.

We investigated the histologic findings and viral antigen distribution in 3 cases of natural coinfection of layer hens with subgroup J avian leukosis virus (ALV-J), Marek's disease virus (MDV), and reticuloendotheliosis virus (REV) in hens. At autopsy, diseased hens were found to have hepatosplenomegaly and thickened proventriculi, with white tumor nodules in the liver, spleen, lung, kidney, and ovary. Microscopically, most tissues had been infiltrated by neoplastic lymphocytes; the spleen, lung, proventriculus, heart, and liver had been infiltrated by both neoplastic lymphocytes and myeloblastic cells and/or primitive reticular cells. Fluorescence multiplex immunohistochemistry staining revealed ALV-J, MDV, and REV antigens co-expressed in the same tissue, even the same cell.

Lorf9 deletion significantly eliminated lymphoid organ atrophy induced by meq-deleted very virulent Marek's disease virus.

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset of T cell lymphomas in chickens. 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 results in attenuated vaccines that protect against disease when chickens are infected with highly virulent strains. However, meq-deleted viruses still retain the ability to induce lymphoid organ atrophy, which raises safety concerns. In an earlier study, we found that deletion of lorf9 counteracts this lymphoid organ atrophy. Here, we describe the generation of a double deletion mutant virus lacking virus-encoded meq and lorf9. In vitro studies revealed that during replication, the mutant virus had kinetic characteristics similar to the parental virus; however, in vivo the replication capability was significantly reduced. Results of animal studies revealed no obvious MDV-specific symptoms and lesions. Importantly, the double deletion mutant virus lost the capacity to induce lymphoid organ atrophy, which has been the main obstacle during development of a good vaccine candidate.

Replication of Marek's Disease Virus Is Dependent on Synthesis of De Novo Fatty Acid and Prostaglandin E2.

Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates the fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEFs). In addition, MDV-infected cells contained high levels of fatty acids and showed increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titers by approximately 30-fold. Addition of the downstream metabolites, including malonyl-coenzyme A and palmitic acid, completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates the COX-2/prostaglandin E2 (PGE2) pathway, as evident by increased levels of arachidonic acid, COX-2 expression, and PGE2 synthesis. Inhibition of the COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using short hairpin RNA reduced MDV titers, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is a link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.IMPORTANCE Disturbances of the lipid metabolism in chickens infected with MDV contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates FAS and induces LD formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway, which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signaling pathways.

Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3.

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.