MDV Infection Research Articles

Use of nucleic acids amplification methods in Marek's disease diagnosis and pathogenesis studies.

Marek's disease (MD) is a lymphoproliferative disease of chickens with strong economic impact on poultry industry. Although successful vaccination has enabled control of the disease, outbreaks occur in commercial flocks, resulting in substantial economic losses. Together with vaccination, accurate and fast diagnosis of MD remain the most important tools for its efficient control. MD diagnosis currently relies mainly on the identification of its causative agent, Marek's disease virus type 1 (MDV-1). Nucleic acid amplification techniques have been successfully applied for identification of MDV DNA in field samples and also for studies of virus-host interactions. In this review we want to summarize recent advances in the development of standard and quantitative PCR techniques and their use in rapid MD diagnosis, including differentiation of pathogenic and vaccine MD viruses. PCR protocols have served as a useful tool for clarification of processes associated with MDV infection in chickens, such as virus spread and release, and effect of vaccine virus on progress of MD. Here, we also describe a novel multi-species qPCR methodology for identification and quantification of MDV DNA, enabling its detection in various bird species that are the most susceptible to MDV infection. Keywords: Marek's disease; MDV; diagnosis; nucleic acid detection; duplex quantitative PCR.

Antiviral effect of baicalin on Marek\u2019s disease virus in CEF cells

BackgroundBaicalin, the main metabolic component of Scutellaria baicalensis Georgi, has various pharmacological properties including anti-inflammatory, anti-oxidant, anti-apoptotic, anti-bactericidal and anti-viral. The purpose of this study was to investigate the anti-Marek’s disease virus (MDV) activities of baicalin in CEF cells.ResultsHere, we showed that baicalin could inhibit viral mRNA, protein levels and overall plaque formation in a time-dependent manner. We also found that baicalin could consistently inhibit MDV replication and directly affect the virus infectivity. Moreover, baicalin treatment has no effect on expression level of antiviral cytokine and inflammatory cytokines in MDV infected CEFs.ConclusionsThese results demonstrate that baicalin could be a potential drug against MDV infection.

Milk vetch dwarf virus infection in the Solanaceae and Caricaceae families in Southeast Asia

AbstractMilk vetch dwarf virus (MDV) is an important member of the genus Nanovirus and is transmitted by the aphid Aphis craccivora. MDV has multiple single‐stranded DNA genome components, each approximately 1 kb, and two or three alpha‐satellite molecules. It mainly infects plants of the legume family Fabaceae. Recently, papaya (Carica papaya) collected in Yesan, South Korea, displaying symptoms of leaf yellowing and dwarfism, was identified as a new host for MDV. To examine the geographical distribution of MDV, papaya samples with symptoms were harvested in South Korea, Vietnam, and Taiwan in August 2018, along with tomato and pepper samples grown in adjacent fields in Vietnam. The results revealed the presence of MDV not only in papaya but also in pepper and tomato. This MDV infection in members of the Solanaceae family was confirmed by Southern blot hybridization performed using a PCR product of segment S as a probe. Based on sequence analysis of three MDV components (M, S, and C3), we verified the presence of three different isolates of MDV in these three countries and homology between sequences of isolates from papaya and from members of the Solanaceae from Vietnam. Taken together, our results clearly demonstrate MDV infection in Vietnam and Taiwan for the first time and confirm that MDV can infect economically important pepper and tomato.

Potential Role of Macrophage Migration Inhibitory Factor in the Pathogenesis of Marek's Disease.

IntroductionMarek’s disease virus (MDV) can cause malignant T-cell lymphomas and immunosuppression in chickens. Macrophage migration inhibitory factor (MIF) not only plays a critical role in inhibiting T-cell responses, but also contributes to multiple aspects of tumour progression. The aim of this study was to reveal the potential role of MIF in the pathogenesis of MDV infection.Material and MethodsMIF gene expression levels were measured by using real-time PCR. Expression was assayed at different times in chicken embryo fibroblast (CEF) cells and tissue samples of SPF chickens infected with different MDV strains and fold change was calculated by the 2–△△CT method.ResultsThe expression of MIF was significantly downregulated (p < 0.05 and FC > 2) in CEF cells infected with the very virulent MDV RB1B strain at 48 h post infection (hpi) and in the skin and spleen at 14 days post infection (dpi). The reduction of MIF expression was also found in CEF cells infected by reticuloendotheliosis virus (REV), avian leukosis virus subgroup J (ALV-J), and MDV vaccine strain CVI988 or in HD11 cells stimulated with TLR2, 3, 4, and 7 ligands. Interestingly, MIF expression decreased continuously from 7 to 28 dpi in the thymus after RB1B virus infection while it increased after CVI988 virus infection. Upregulated expression of MIF was found in CEF infected with RB1B at 96 hpi and in the spleen and skin at 21 and 28 dpi.ConclusionThe present study revealed the different expression pattern of MIF in response to MDV infection and indicated that MIF level may be associated with MDV pathogenesis.

Co-infection of vvMDV with multiple subgroups of avian leukosis viruses in indigenous chicken flocks in China

BackgroundIn China, although the ALV eradication program and the MD vaccination strategy greatly reduce the disease burdens caused by the infection of ALV and MDV, the frequent emergence of novel ALV-K or vvMDV in the vaccinated chicken flock challenges the current control strategies for both diseases.ResultsIn Guangdong Province, an indigenous chicken flock was infected with neoplastic disease. Hematoxylin–eosin staining of the tissues showed the typical characteristics of MDV and classical ALV infection. The PCR and sequencing data demonstrated that the identified MDV was clustered into a very virulent MDV strain endemic in domestic chickens in China. Moreover, subgroups ALV-A and ALV-K were efficiently recovered from two samples. The full genome sequence revealed that the ALV-K isolate was phylogenetically close to the ALV TW3593 isolate from Taiwan Province.ConclusionsA co-infection of vvMDV with multiple ALV subgroups emerged in a chicken flock with neoplastic disease in Guangdong Province. The co-infection with different subgroups of ALV with vvMDV in one chicken flock poses the risk for the emergence of novel ALVs and heavily burdens the control strategy for MDV.

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.

Integrated analysis of lncRNA and mRNA repertoires in Marek\u2019s disease infected spleens identifies genes relevant to resistance

BackgroundMarek’s disease virus (MDV) is an oncogenic herpesvirus that can cause T-cell lymphomas in chicken. Long noncoding RNA (lncRNA) is strongly associated with various cancers and many other diseases. In chickens, lncRNAs have not been comprehensively identified. Here, we profiled mRNA and lncRNA repertoires in three groups of spleens from MDV-infected and non-infected chickens, including seven tumorous spleens (TS) from MDV-infected chickens, five spleens from the survivors (SS) without lesions after MDV infection, and five spleens from noninfected chickens (NS), to explore the underlying mechanism of host resistance in Marek’s disease (MD).ResultsBy using a precise lncRNA identification pipeline, we identified 1315 putative lncRNAs and 1166 known lncRNAs in spleen tissue. Genomic features of putative lncRNAs were characterized. Differentially expressed (DE) mRNAs, putative lncRNAs, and known lncRNAs were profiled among three groups. We found that several specific intergroup differentially expressed genes were involved in important biological processes and pathways, including B cell activation and the Wnt signaling pathway; some of these genes were also found to be the hub genes in the co-expression network analyzed by WGCNA. Network analysis depicted both intergenic correlation and correlation between genes and MD traits. Five DE lncRNAs including MSTRG.360.1, MSTRG.6725.1, MSTRG.6754.1, MSTRG.15539.1, and MSTRG.7747.5 strongly correlated with MD-resistant candidate genes, such as IGF-I, CTLA4, HDAC9, SWAP70, CD72, JCHAIN, CXCL12, and CD8B, suggesting that lncRNAs may affect MD resistance and tumorigenesis in chicken spleens through their target genes.ConclusionsOur results provide both transcriptomic and epigenetic insights on MD resistance and its pathological mechanism. The comprehensive lncRNA and mRNA transcriptomes in MDV-infected chicken spleens were profiled. Co-expression analysis identified integrated lncRNA-mRNA and gene-gene interaction networks, implying that hub genes or lncRNAs exert critical influence on MD resistance and tumorigenesis.

Inhibition of DNA-Sensing Pathway by Marek's Disease Virus VP23 Protein through Suppression of Interferon Regulatory Factor 7 Activation.

The type I interferon (IFN) response is the first line of host innate immune defense against viral infection; however, viruses have developed multiple strategies to antagonize host IFN responses for efficient infection and replication. Here, we report that Marek's disease virus (MDV), an oncogenic herpesvirus, encodes VP23 protein as a novel immune modulator to block the beta interferon (IFN-β) activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) in chicken fibroblasts and macrophages. VP23 overexpression markedly reduces viral DNA-triggered IFN-β production and promotes viral replication, while knockdown of VP23 during MDV infection enhances the IFN-β response and suppresses viral replication. VP23 selectively inhibits IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) activation. Furthermore, we found that VP23 interacts with IRF7 and blocks its binding to TANK-binding kinase 1 (TBK1), thereby inhibiting IRF7 phosphorylation and nuclear translocation, resulting in reduced IFN-β production. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.IMPORTANCE Despite widespread vaccination, Marek's disease (MD) continues to pose major challenges for the poultry industry worldwide. MDV causes immunosuppression and deadly lymphomas in chickens, suggesting that this virus has developed a successful immune evasion strategy. However, little is known regarding the initiation and modulation of the host innate immune response during MDV infection. This study demonstrates that the cGAS-STING DNA-sensing pathway is critical for the induction of the IFN-β response against MDV infection in chicken fibroblasts and macrophages. An MDV protein, VP23, was found to efficiently inhibit the cGAS-STING pathway. VP23 selectively inhibits IRF7 but not NF-κB activation. VP23 interacts with IRF7 and blocks its binding to TBK1, thereby suppressing IRF7 activation and resulting in inhibition of the DNA-sensing pathway. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.

Induction of DNA Damages upon Marek's Disease Virus Infection: Implication in Viral Replication and Pathogenesis

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis.IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

Isolation and Identification of Very Virulent Strains of Marek’s Disease Virus from MDVVaccinated Flocks in Egypt

Marek’s disease is still a major problem among Egyptian poultry flocks, despite the intensive vaccination programs against the disease. This study was conducted to investigate the prevalence of MDV infection among forty-four flocks of breeders and layers with ages ranged from 4-20 months. Feather follicle specimens were collected during 2012-2015 from 44 vaccinated chicken flocks showing emaciation and visceral tumors. The samples were tested by PCR using three pairs of primers. Nineteen flocks were positive for MDV using primers targeting the UL 19 gene with a percentage of 43.2%. Three flocks were shown to be positive by primers targeting the meq and 132 bp tandem repeat genes with a percentage of 6.8%. Inoculation of duck embryo fibroblast (DEF) and chick embryo fibroblast (CEF) showed CPE in the form of plaques formation within 5-14 days post inoculation. Sequencing of meq and 132 tandem repeat genes of the 3 samples revealed that the isolated strains exhibited 99% homology with the very virulent European, Chinese, American, Indian and Egyptian MDV isolates. In conclusion, although the availability of MDV vaccines especially HVT vaccine which is used in the examined flocks in the present study, the disease was recorded. Thus, indicating that HVT vaccines are unable to protect completely against more virulent strains. Therefore, there is a need to develop a new strategy and types of vaccination to be able to protect against new strains of virus.

A novel method for genome-wide profiling of dynamic host-pathogen interactions using 3\u2032 end enriched RNA-seq

Marek’s disease is a contagious lymphoproliferative disease of chickens and typical model of viral oncogenesis. Mapping changes or different states over the course of infection for both host and pathogen would provide important insights into dynamic host-pathogen interactions. Here we introduced 3′ end enriched RNA-seq as a novel method to study host-pathogen interactions in chicken embryo fibroblasts cells challenged with Marek’s disease virus. The method allowed accurate profiling of gene expression and alternative polyadenylation sites for host and pathogen simultaneously. We totally identified 476 differentially expressed genes and 437 APA switching genes in host, including switching in tandem 3′ UTRs and switching between coding region and 3′ UTR. Most of these genes were related to innate immunity, apoptosis and metabolism, but two sets of genes overlapped a little, suggesting two complementary mechanisms in gene regulation during MDV infection. In summary, our results provided a relatively comprehensive insight into dynamic host-pathogen interactions in regulation of gene transcription during infection of Marek’s disease virus and suggested that 3′ end enriched RNA-seq was a promising method to investigate global host-pathogen interactions.

Evaluation of factors influencing the development of late Marek’s disease virus-induced immunosuppression: virus pathotype and host sex

ABSTRACTMarek’s disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.

Differences in CD8αα and cecal microbiome community during proliferation and late cytolytic phases of Marek's disease virus infection are associated with genetic resistance to Marek's disease.

Marek's disease (MD) is an important neoplastic disease of chickens caused by Marek ': s disease virus (MDV), a highly oncogenic alphaherpesvirus. In this study using two chicken lines, one resistant and another susceptible to MD, splenic T cells and cecal microbiome were profiled to gain a better understanding of primary differences in these lines. The percent of splenic CD4+ T cells were similar regardless of MDV challenge status in both bird lines. In contrast, CD8αα profiles were different (P < 0.005) between chicken lines under naïve status and under MDV challenge, suggesting that CD8αα T cells play a key role in mediating MDV infection. Microbiome composition was different between naïve resistant (Blautia spp.) and susceptible birds (Streptococcus spp.) (P < 0.05) during initial colonization. With MDV challenge, both chicken lines showed lower numbers of beneficial Faecalibacterium spp. and increased number of Lactobacillus spp. Metabolic profiles between naïve chicken types were similar but with MDV challenge, there were differences in metabolism in both chicken lines, with amino acid metabolism impacted in resistant birds and lipid metabolism in susceptible birds. These results provide insights into immune response and potential interplay with the microbiome during infection with an oncogenic virus.

Severe necrotic dermatitis in the combs of line 63 chickens infected with Marek’s disease virus

ABSTRACTMarek’s disease virus (MDV), the aetiological agent of Mareks' disease (MD), is a highly cell-associated oncogenic α-herpesvirus that replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. We investigated the possible effect of MDV infection on the exacerbation of necrotic dermatitis in the combs of MD-susceptible (72) and MD-resistant (63) chicken lines at 21 days post infection. MDV-infected birds of line 63 are relatively resistant to tumour development but exhibit an unusual necrosis of combs, wattles, and footpads that is intensified when infected with MDV. Chickens from line 72, on the other hand, are highly susceptible to MDV infection and tumour development. Real-Time PCR analysis revealed that IL-6, IL-8, IL-12, IL-18, iNOS, and IFNγ were all up regulated in the comb tissues of MDV-infected susceptible line 72 with no visible necrotic damage. With the exception of IL-8 and iNOS, the expression of all the other tested genes was barely detected in the necrotic combs of the resistant line 63. Real-Time PCR analysis revealed the MDV meq oncogene transcripts in the spleen tissues of both infected lines but in the comb tissues of only the susceptible line 72. A significant infiltration of macrophages and lymphocytes was detected in the comb tissues of both resistant and susceptible lines. Histopathological analysis also showed thinning and erosion of epidermis and inflammation, lympho-plasmocytic infiltration, heterophilic, and histocytic cellulitis within the connective tissues of the necrotic combs. Gram stain of the sectioned frozen comb samples exposed the presence of Gram-positive micrococcus.

Effects of sodium tanshinone IIA sulfonate against Marek's disease virus in experimentally infected chickens

Chickens experimentally infected with Marek's disease virus J-1 strain were used to evaluate the anti-Marek's disease virus (MDV) activity of sodium tanshinone IIA sulfonate (STS) in vivo. Chickens in same group were kept in one pen and control group chickens were housed in negative pressure isolator. Chickens were treated with different dose of STS or ABOB for 21 consecutive days. Peripheral T lymphocyte proliferation, expression level of IFN-γ and IL-10 in serum, and MDV load in spleen were determined. The results showed that the treatments with STS and ABOB could significantly increase stimulating index (SI) of peripheral T lymphocytes while the SI is dropping due to the MDV infection, SI of chickens in STS prevention groups were significantly higher than that in STS treatment group and ABOB group (P<0.05 or P<0.01); IFN-γ and IL-10 level of chickens in STS groups were higher than that in other groups (P<0.05 or P<0.01). The results of qPCR demonstrated that STS could inhibit the virus replication in spleen of chickens infected with MDV. These findings indicated that STS can be potentially applied as an anti-MDV drug and set a solid basis for further investigating the antiviral mechanisms of STS.

DNMT gene expression and methylome in Marek’s disease resistant and susceptible chickens prior to and following infection by MDV

Marek’s disease (MD) is characterized as a T cell lymphoma induced by a cell-associated α-herpesvirus, Marek’s disease virus type 1 (MDV1). As with many viral infectious diseases, DNA methylation variations were observed in the progression of MD; these variations are thought to play an important role in host-virus interactions. We observed that DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) were differentially expressed in chicken MD-resistant line 63 and MD-susceptible line 72 at 21 d after MDV infection. To better understand the role of methylation variation induced by MDV infection in both chicken lines, we mapped the genome-wide DNA methylation profiles in each line using Methyl-MAPS (methylation mapping analysis by paired-end sequencing). Collectively, the data sets collected in this study provide a more comprehensive picture of the chicken methylome. Overall, methylation levels were reduced in chickens from the resistant line 63 after MDV infection. We identified 11,512 infection-induced differential methylation regions (iDMRs). The number of iDMRs was larger in line 72 than in line 63, and most of iDMRs found in line 63 were overlapped with the iDMRs found in line 72. We further showed that in vitro methylation levels were associated with MDV replication, and found that MDV propagation in the infected cells was restricted by pharmacological inhibition of DNA methylation. Our results suggest that DNA methylation in the host may be associated with disease resistance or susceptibility. The methylation variations induced by viral infection may consequentially change the host transcriptome and result in diverse disease outcomes.

Expression Pattern of Genes of RLR-Mediated Antiviral Pathway in Different-Breed Chicken Response to Marek’s Disease Virus Infection

It has been known that the chicken's resistance to disease was affected by chicken's genetic background. And RLR-mediated antiviral pathway plays an important role in detection of viral RNA. However, little is known about the interaction of genetic background with RLR-mediated antiviral pathway in chicken against MDV infection. In this study, we adopted economic line-AA broilers and native Erlang mountainous chickens for being infected with MDV. Upon infection with MDV, the expression of MDA-5 was upregulated in two-breed chickens at 4, 7, and 21 d.p.i. It is indicated that MDA-5 might be involved in detecting MDV in chicken. Interestingly, the expression of IRF-3 and IFN-β genes was decreased in spleen and thymus of broilers at 21 d.p.i, but it was upregulated in immune tissues of Erlang mountainous chickens. And the genome load of MDV in spleen of broiler is significantly higher than that in Erlang mountainous chickens. Meanwhile, we observed that the death of broiler mainly also occurred in this phase. Collectively, these present results demonstrated that the expression patters of IRF-3 and IFN-β genes in chicken against MDV infection might be affected by the genetic background which sequently influence the resistance of chicken response to MDV.

Down-regulation of promoter methylation level of CD4 gene after MDV infection in MD-susceptible chicken line

BackgroundMarek’s disease virus (MDV) is an oncovirus that induces lymphoid tumors in susceptible chickens, and may affect the epigenetic stability of the CD4 gene. The purpose of this study was to find the effect of MDV infection on DNA methylation status of the CD4 gene differed between MD-resistant (L63) and –susceptible (L72) chicken lines.MethodsChickens from each line were divided into two groups with one group infected by MDV and the other group as uninfected controls. Then, promoter DNA methylation levels of the CD4 gene were measured by Pyrosequencing; and gene expression analysis was performed by quantitative PCR.ResultsPromoter methylation of the CD4 gene was found to be down-regulated in L72 chickens only after MDV infection. The methylation down-regulation of the CD4 promoter is negatively correlated with up-regulation of CD4 gene expression in the L72 spleen at 21 dpi.ConclusionsThe methylation fluctuation and mRNA expression change of CD4 gene induced by MDV infection suggested a unique epigenetic mechanism existed in MD-susceptible chickens.

Proteomic analysis of host responses to Marek's disease virus infection in spleens of genetically resistant and susceptible chickens

Resistance to Marek's disease (MD) in chickens is genetically regulated and there are lines of chickens with differential susceptibility or resistance to this disease. The present study was designed to study comparative changes in the spleen proteomes of MD-susceptible B19 and MD-resistant B21 chickens in response to MDV infection. Spleen proteomes were examined at 4, 7, 14 and 21 days post-infection (d.p.i.) using two-dimensional gel electrophoresis and subsequently the protein spots were identified by one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). On average, there were 520 ± 27 distinct protein spots on each gel and 1.6 ± 0.7% of the spots differed quantitatively in their expression ( p ≤ 0.05 and fold change ≥2) between infected B19 and B21 chickens. There was one spot at 4 d.p.i. and three spots each at the rest of the time points, which had a qualitative difference in expression. Most of the differentially expressed proteins at 4 and 7 d.p.i. displayed increased expression in B21 chickens; conversely the differentially expressed proteins at 14 and 21 d.p.i. showed an increase in expression in B19 chickens. The differentially expressed proteins identified in the present study included antioxidants, molecular chaperones, proteins involved in the formation of cytoskeleton, protein degradation and antigen presentation, signal transduction, protein translation and elongation, RNA processing and cell proliferation. These findings shed light on some of the underlying processes of genetic resistance or susceptibility to MD.

Early Replication in Pulmonary B Cells After Infection with Marek's Disease Herpesvirus by the Respiratory Route

Abstract Natural infection with Marek's disease virus occurs through the respiratory mucosa after chickens inhale dander shed from infected chickens. The early events in the lung following exposure to the feather and squamous epithelial cell debris containing the viral particles remain unclear. In order to elucidate the virological and immunological consequences of MDV infection for the respiratory tract, chickens were infected by intratracheal administration of infective dander. Differences between susceptible and resistant chickens were immediately apparent, with delayed viral replication and earlier onset of interferon (IFN)-gamma production in the latter. CD4(+) and CD8(+) T cells surrounded infected cells in the lung. Although viral replication was evident in macrophages, pulmonary B cells were the main target cell type in susceptible chickens following intratracheal infection with MDV. In accordance, depletion of B cells curtailed viremia and substantially affected pathogenesis in susceptible chickens. Together the data described here demonstrate the role of pulmonary B cells as the primary and predominant target cells and their importance for MDV pathogenesis.