Marek's Disease Virus Replication Research Articles

Immunopotentiating effect of lentinan on chicks and its inhibitory effect on Marek's disease virus infection

Marek's disease virus (MDV) is a significant tumorigenic virus that causes severe immunosuppression in chickens. Lentinan (LNT) is an immunomodulator containing β-glucans and is widely used in areas such as antiviral, anticancer, and immune regulation. To investigate the immunomodulatory effects of LNT on specific pathogen-free (SPF) chicks and its potential to inhibit MDV infection, we conducted an MDV challenge experiment and observed the immune-enhancing effect of LNT on SPF chicks. The results showed that LNT promoted the growth and development of SPF chicks and induced the upregulation of cytokines such as Mx protein, interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2). The specific gravity of CD4+ T-lymphocytes and CD8+ T-lymphocytes and their ratios were also significantly upregulated. Prophylactic use of LNT inhibited MDV replication in lymphocytes, liver, and spleen. It also alleviated MDV-induced weight loss and hepatosplenomegaly in SPF chicks. The present study confirms that LNT can enhance the levels of innate and cellular immunity in SPF chicks and contributes to the inhibition of MDV replication in vivo and mitigation of immune organ damage in chicks due to MDV infection. This provides an adjunctive measure for better control of MDV infection.

The nature of expression of avian immunity genes during infection with GALLID ALPHAHERPESVIRUS 2 and MELEAGRID ALPHAHERPESVIRUS 1

Marek's disease is a dangerous immunosuppressive viral disease of poultry. Most egg poultry farm in the Russian Federation vaccinate their chickens on the first day of life against this viral disease. Thanks to this vaccine prevention tactic, poultry farms protect themselves from the outbreak of this disease and economic damage. Traditionally, among the viruses related to this disease, there are the 1st serotype of the Marek's disease virus (Gallid alphaherpesvirus 2) and the 3rd serotype of the Marek's disease virus, which is not such in a strict taxonomic sense (Meleagrid alphaherpesvirus 1, also called turkey herpesvirus). When poultry is infected, this virus causes a significant change in the expression of the main immunity genes (IL6, PTGS2, IRF7). Based on the results obtained, it was concluded that the expression of the IL6 and PTGS2 genes was slightly higher than the level of expression of these genes in the control group. It is possible that local immunity was suppressed by the immunosuppressive properties of the Marek's disease virus. When studying the expression of the IRF7 gene, a strong effect of viruses of both serotypes on this gene was found, and Gallid alphaherpesvirus 2 exceeded the level of this effect compared to the effect of Meleagrid alphaherpesvirus 1 by 4 times. These results may indicate that sharply increasing expression of the IRF7 gene may be a sign of Marek's disease virus replication (since Gallid alphaherpesvirus 2 and Meleagrid alphaherpesvirus 1 interact with the same cellular receptor, the vaccine effect of Meleagrid alphaherpesvirus 1 is based on this). On the other hand, we can conclude that the introduction of these viruses does not cause inflammatory and immune reactions in the ileocecal tonsils of birds.

Metabolomic profiling of Marek's disease virus infection in host cell based on untargeted LC-MS.

Marek's disease (MD) caused by Marek's disease virus (MDV), poses a serious threat to the poultry industry by inducing neurological disease and malignant lymphoma in infected chickens. However, the underlying mechanisms how MDV disrupts host cells and causes damage still remain elusive. Recently, the application of metabolomics has shown great potential for uncovering the complex mechanisms during virus-host interactions. In this study, chicken embryo fibroblasts (CEFs) infected with MDV were subjected to ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) and multivariate statistical analysis. The results showed that 261 metabolites were significantly altered upon MDV infection, with most changes occurring in amino acid metabolism, energy metabolism, nucleotide metabolism, and lipid metabolism. Notably, MDV infection induces an up-regulation of amino acids in host cells during the early stages of infection to provide the energy and intermediary metabolites necessary for efficient multiplication of its own replication. Taken together, these data not only hold promise in identifying the biochemical molecules utilized by MDV replication in host cells, but also provides a new insight into understanding MDV-host interactions.

Inhibition of Marek's Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro.

Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.

Serum amyloid A regulates TLR2/4-mediated IFN-β signaling pathway against Marek's disease virus

Serum amyloid A (SAA), an acute response phase protein (APP), is crucial for the innate immune response during pathogenic microorganisms' invasion. Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that activates multiple innate immune molecules, including SAA, in the host during infection. However, the pathway through which SAA participates in MDV-induced host innate immunity remains unknown. The present study aimed to elucidate the pathway through which SAA exerts its anti-MDV function. We observed that MDV infection in vivo and in vitro significantly elevated SAA expression. Furthermore, through SAA overexpression and knockdown experiments, we demonstrated that SAA could inhibit MDV replication. Subsequently, we found that SAA activated Toll-Like Receptor 2/4 (TLR2/4) -mediated Interferon Beta (IFN-β) promoter activity and IFN regulatory factor 7 (IRF7) promoter activity. During MDV infection, SAA enhanced TLR2/4-mediated IFN-β signal transduction and messenger RNAs (mRNAs) expression of type I IFN (IFN-I) and interferon-stimulated genes (ISGs). Finally, TLR2/4 inhibitor OxPAPC inhibits the anti-MDV activity of SAA. These results demonstrated that SAA inhibits MDV replication and enhancing TLR2/4-mediated IFN-β signal transduction to promote IFNs and ISGs expression. This finding is the first to demonstrate the signaling pathway by which SAA exerts its anti-MDV function. It also provides new insights into the control of oncogenic herpesviruses from the perspective of acute response phase proteins.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

Marek's Disease Virus and Reticuloendotheliosis Virus Coinfection Enhances Viral Replication and Alters Cellular Protein Profiles.

Coinfection with Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) causes synergistic pathogenic effects and serious losses to the poultry industry. However, whether there is a synergism between the two viruses in viral replication and the roles of host factors in regulating MDV and REV coinfection remains elusive. In this study, we found that MDV and REV coinfection increased viral replication in coinfected cells as compared to a single infection in a limited period. Further, we explore the host cell responses to MDV and REV coinfection using tandem mass tag (TMT) peptide labeling coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS). Compared with MDV/REV-infected cells, 38 proteins increased (fold change > 1.2) and 60 decreased (fold change < 0.83) their abundance in MDV and REV coinfected cells. Differentially accumulated proteins (DAPs) were involved in important biological processes involved in the immune system process, cell adhesion and migration, cellular processes, and multicellular organismal systems. STRING analysis found that IRF7, MX1, TIMP3, and AKT1 may be associated with MDV and REV synergistic replication in chicken embryo fibroblasts (CEFs). Western blotting analysis showed that the selected DAPs were identical to the quantitative proteomics data. Taken together, we verified that MDV and REV can synergistically replicate in coinfected cells and revealed the host molecules involved in it. However, the synergistic pathogenesis of MDV and REV needs to be further studied.

Marek's disease virus encoded miR-M6 and miR-M10 are dispensable for virus replication and pathogenesis in chickens

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides long non-coding RNAs, and virus-encoded miRNAs play an important role in pathogenesis. Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes immunosuppression and tumors in its natural host, chicken. In the MDV genome, 14 miRNA precursors and 26 mature miRNAs were identified, thus MDV has been used as a model to study the function of viral miRNAs in vivo. Recently, a cluster of miRNAs encoded by MDV, Cluster 3 miRNAs (miR-M8-M10), has been shown to restrict early cytolytic replication and pathogenesis of MDV. In this study, we further analyzed the role of miR-M6 and miR-M10, members of cluster miR-M8-M10, in MDV replication and pathogenicity. We found that, compared to parental MDV, deletion of miR-M6−5p significantly enhanced the replication of MDV in cell culture, but not in chickens. The replication of miR-M6−5p deletion MDV was restored once the deleted sequences were re-inserted. Our results also showed that deletion of miR-M10−5p did not affect the replication of MDV in vitro and in vivo. In addition, our animal study results showed that deletion of miR-M6−5p or miR-M10−5p did not alter the pathogenesis of MDV. In conclusion, our study shows that both miR-M6 and miR-M10 are dispensable for MDV replication and pathogenesis in chickens, while also suggests a repressive role of miR-M6 in MDV replication in cell culture.

DEAD/DEAH-box helicase 5 is hijacked by an avian oncogenic herpesvirus to inhibit interferon beta production and promote viral replication

DEAD-box helicase 5 (DDX5) plays a significant role in tumorigenesis and regulates viral replication of several viruses. An avian oncogenic herpesvirus, Marek's disease virus (MDV), is widely known to cause immunosuppression and lymphoma in chickens. However, the underlying mechanisms of how DDX5 plays a role in viral replication remain unclear. In this study, we show that MDV inhibits the production of interferon beta (IFN-β) in chicken embryo fibroblasts (CEFs) by increasing the expression level and promoting the nuclear aggregation of DDX5. We further reveal how DDX5 down-regulates melanoma differentiation-associated gene 5/toll-like receptor 3 signaling through the fundamental transcription factor, interferon regulatory factor 1. MDV replication is suppressed, and the production of IFN-β is promoted in the DDX5 absented CEFs. Taken together, our investigations demonstrate that MDV inhibits IFN-β production by targeting DDX5-mediated signaling to facilitate viral replication, which offers a novel insight into the mechanism by which an avian oncogenic herpesvirus replicates in chicken cells.

Artesunate derivative TF27 inhibits replication and pathogenesis of an oncogenic avian alphaherpesvirus

Nucleoside analogues have been the cornerstone of clinical treatment of herpesvirus infections since the 1970s. However, severe side effects and emergence of drug resistant viruses raise the need for alternative treatment options. We recently investigated the broad and strong antiherpesviral activity of the optimized artesunate derivative TF27 in vitro. TF27 efficiently inhibited replication of the highly oncogenic Marek's disease virus (MDV), a virus that infects chickens, causes deadly lymphomas and threatens poultry populations worldwide. In this study, we used this natural virus-host model for herpesvirus-induced cancer by infecting chickens with MDV, and evaluated the protective efficacy of TF27 and the nucleoside analogue valganciclovir (VGCV) on virus replication and tumorigenesis. We could demonstrate that both drugs reduced viral load in the blood and prevented tumor development in a large portion of the animals. Antiviral treatment also had a positive impact on body weight gain, while no negative compound-associated side effects were observed. This research provides the first evidence that the artesunate derivative TF27 and VGCV can be used in avian species and that they inhibit MDV replication and tumorigenesis. In addition, our study paves the way for promising approaches in future antiherpesviral drug development.

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.

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

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

The significance of the individual Meq-clustered miRNAs of Marek's disease virus in oncogenesis.

Marek's disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXΔmiR-M2, GXΔmiR-M3, GXΔmiR-M5, GXΔmiR-M9 and GXΔmiR-M12 was reduced from 100 % to 18 %, 30 %, 48 %, 24 % and 14 %, coupled with gross tumour incidence reduction from 28 % to 8 %, 4 %, 12 %, 8 % and 0 %, respectively. Our data confirm that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.

Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek's disease viruses in vivo

Marek's disease (MD) is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV). MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1), therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H+S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses.

Role of the short telomeric repeat region in Marek's disease virus replication, genomic integration, and lymphomagenesis.

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. 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 influences the core gut microbiome of the chicken during the early and late phases of viral replication.

Marek's disease (MD) is an important neoplastic disease of chickens caused by the Marek's disease virus (MDV), an oncogenic alphaherpesvirus. In this study, dysbiosis induced by MDV on the core gut flora of chicken was assessed using next generation sequence (NGS) analysis. Total fecal and cecum-derived samples from individual birds were used to estimate the influence of MDV infection on the gut microbiome of chicken. Our analysis shows that MDV infection alters the core gut flora in the total fecal samples relatively early after infection (2-7 days) and in the late phase of viral infection (28-35 days) in cecal samples, corresponding well with the life cycle of MDV. Principle component analyses of total fecal and cecal samples showed clustering at the early and late time points, respectively. The genus Lactobacillus was exclusively present in the infected samples in both total fecal and cecal bird samples. The community colonization of core gut flora was altered by viral infection, which manifested in the enrichment of several genera during the early and late phases of MDV replication. The results suggest a relationship between viral infection and microbial composition of the intestinal tract that may influence inflammation and immunosuppression of T and B cells in the host.

Marek's Disease Virus Morphogenesis

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

Visualization of Marek’s disease virus in vitro using enhanced green fluorescent protein fused with US10

Marek's disease virus (MDV) is an highly cell-associated avian alphaherpesvirus. Although viral replication is supported in chicken embryo fibroblasts (CEF) or duck embryo fibroblasts, identification of MDV-infected cells is quite cumbersome especially during the early stages of virus replication when plaques can be difficult to recognize. To visualize MDV replication in infected cells and characterize MDV US10 in vitro, rMd5-US10-EGFP, a recombinant MDV, was generated that expresses enhanced green fluorescent protein (EGFP) as a tagged protein fused with US10 at the C-terminal end. The expression of US10-EGFP was detected in infected CEF using fluorescent microscopy and the expression intensity was quantified using flow cytometry analysis. In addition, confocal microscopic analysis provided information on subcellular localization of US10-EGFP in virus-infected cells. In conclusion, rMd5-US10-EGFP virus can be used to help monitor virus activity in vitro.

Marek's disease viral interleukin-8 promotes lymphoma formation through targeted recruitment of B cells and CD4+ CD25+ T cells.

Marek's disease virus (MDV) is a cell-associated and highly oncogenic alphaherpesvirus that infects chickens. During lytic and latent MDV infection, a CXC chemokine termed viral interleukin-8 (vIL-8) is expressed. Deletion of the entire vIL-8 open reading frame (ORF) was shown to severely impair disease progression and tumor development; however, it was unclear whether this phenotype was due to loss of secreted vIL-8 or of splice variants that fuse exons II and III of vIL-8 to certain upstream open reading frames, including the viral oncoprotein Meq. To specifically examine the role of secreted vIL-8 in MDV pathogenesis, we constructed a recombinant virus, vΔMetvIL-8, in which we deleted the native start codon from the signal peptide encoding exon I. This mutant lacked secreted vIL-8 but did not affect Meq-vIL-8 splice variants. Loss of secreted vIL-8 resulted in highly reduced disease and tumor incidence in animals infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals by the natural route, infection and lymphomagenesis in contact animals were severely impaired. In vitro assays showed that purified recombinant vIL-8 efficiently binds to and induces chemotaxis of B cells, which are the main target for lytic MDV replication, and also interacts with CD4(+) CD25(+) T cells, known targets of MDV transformation. Our data provide evidence that vIL-8 attracts B and CD4(+) CD25(+) T cells to recruit targets for both lytic and latent infection.

Allele-specific expression analysis reveals CD79B has a cis-acting regulatory element that responds to Marek's disease virus infection in chickens

Marek's disease (MD) is a T cell lymphoma disease of domestic chickens induced by the Marek's disease virus (MDV), a highly infectious and naturally oncogenic alphaherpesvirus. Enhancing genetic resistance to MD in poultry is an attractive method to augment MD vaccines, which protect against MD but do not prevent MDV replication and horizontal spread. Previous work integrating QTL scans, transcript profiling, and MDV-chicken protein-protein interaction screens revealed 3 MD resistance genes; however, a major challenge continues to be the identification of the other contributing genes. To aid in this search, we screened for allele-specific expression (ASE) in response to MDV infection, a simple and novel method for identifying polymorphic cis-acting regulatory elements, which may contain strong candidate genes with specific alleles that confer MD genetic resistance. In this initial study, we focused on immunoglobulin β (CD79B) because it plays a critical role in the immune response and, more important, is transcriptionally coupled with growth hormone (GH1), one of the previously identified MD resistance genes. Using a coding SNP in CD79B and pyrosequencing to track the relative expression of each allele, we monitored ASE in uninfected and MDV-infected F(1) progeny from reciprocal intermatings of highly inbred chicken lines 6(3) (MD resistant) and 7(2) (MD susceptible). Upon screening 3 tissues (bursa, thymus, and spleen) at 5 time points (1, 4, 7, 11, and 15 d postinfection), we observed that MDV infection alters the CD79B allelic ratios in bursa and thymus tissues at 4 and 15 d postinfection in both mating directions. Our results suggest that CD79B has a cis-acting regulatory element that responds to MDV infection and probably cooperates with GH1 in conferring genetic resistance to MD. This result helps validates the use of ASE screens to identify specific candidate genes for complex traits such as genetic resistance to MD.