Differential Virus-Specific IFN-Gamma Producing T Cell Responses to Marek’s Disease Virus in Chickens With B19 and B21 MHC Haplotypes

Marek’s disease virus (MDV), the etiologic agent for Marek’s disease (MD), causes a deadly lymphoproliferative disease in chickens. Causes of the well-documented association between genetically defined lines of chicken and resistance to MD remain unknown. Here, the frequencies of IFN-gamma producing pp38 and MEQ-specific T cell responses were determined in line N (B21 haplotype; MD-resistant) and line P2a (B19 haplotype, MD-susceptible) chickens after infection with vaccine and/or virulent (RB1B) strains of MDV using both standard ex vivo and cultured chIFN-gamma ELISPOT assays. Notably, MDV infection of naïve and vaccinated MD-resistant chickens induced higher frequencies of IFN-gamma producing MDV-specific T cell responses using the cultured and ex vivo ELISPOT assay, respectively. Remarkably, vaccination did not induce or boost MEQ-specific effector T cells in the susceptible chickens, while it boosted both pp38-and MEQ-specific response in resistant line. Taken together, our results revealed that there is a direct association between the magnitude of T cell responses to pp38 and MEQ of MDV antigens and resistance to the disease.

First report of Serotype-1 Marek’s disease virus (MDV-1) with oncogenic form in backyard turkeys in Turkey: a molecular analysis study

Background Marek’s disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens. Results This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64–100% and 99.79–100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies. Conclusions These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

The Regulatory Microenvironment in Feathers of Chickens Infected with Very Virulent Marek’s Disease Virus

Vaccines against Marek’s disease can protect chickens against clinical disease; however, infected chickens continue to propagate the Marek’s disease virus (MDV) in feather follicles and can shed the virus into the environment. Therefore, the present study investigated if MDV could induce an immunoregulatory microenvironment in feathers of chickens and whether vaccines can overcome the immune evasive mechanisms of MDV. The results showed an abundance of CD4+CD25+ and CD4+ transforming growth factor-beta (TGF-β)+ T regulatory cells in the feathers of MDV-infected chickens at 21 days post-infection. In contrast, vaccinated chickens had a lower number of regulatory T cells. Furthermore, the expression of TGF-β and programmed cell death receptor (PD)-1 increased considerably in the feathers of Marek’s disease virus-infected chickens. The results of the present study raise the possibility of an immunoregulatory environment in the feather pulp of MDV-infected chickens, which may in turn favor replication of infectious MDV in this tissue. Exploring the evasive strategies employed by MDV will facilitate the development of control measures to prevent viral replication and transmission.

Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek’s Disease Virus Reduces Lymphoid Atrophy but Is Less Protective

Marek’s disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Hypoxia and HIF-1 Trigger Marek’s Disease Virus Reactivation in Lymphoma-Derived Latently Infected T Lymphocytes

Latency is a hallmark of herpesviruses, allowing them to persist into their host without virions production. Acute exposure to hypoxia (below 3% O 2 ) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gamma-herpesviruses (KSHV and EBV). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek’s disease virus (MDV), sharing biological properties with EBV and KSHV (notably oncogenic properties), into lymphocytes. Acute exposure to hypoxia (1% O 2 ) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia-response element consensus sequences on 119 open reading frames. RT-qPCR analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl 2 ) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor-1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. In such conditions, lytic cycle was detected in cells with a sustainable HIF-1α expression, but also in HIF-1α negative cells, indicating that MDV reactivation is mediated by HIF-1, in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors. IMPORTANCE Latent-to-lytic switch of herpesviruses (aka reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alpha-herpesvirus establishing latency in T-lymphocytes and causing lethal T-lymphomas in chickens. In vivo , a second lytic phase is observed during tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently-infected T-lymphocytes, like previously shown for EBV and KSHV in B-lymphocytes. In this study, we demonstrated that acute hypoxia (1% O2) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor (HIF-1) overexpression induces MDV reactivation to a similar extend than hypoxia after 24 hours. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genus.

Antibody profiles of avian leukosis virus subgroups A/B and J In layer flocks suspected to have Marek’s disease in Nigeria

Previous reports indicate high seroprevalence of avian leukosis virus (ALV) p72 antigen in layer flocks suspected to have Marek’s disease (MD) in Kaduna and Plateau States. However, the specific subgroups responsible for ALV infection in layers in the States are still unknown, hence the need for this study. Therefore, the objective of this study was to determine the antibody profiles of ALV subgroups A/B and J in layer flocks suspected to have MD in Kaduna and Plateau States. Sera from 7 and 16 layer flocks suspected to have MD in Kaduna and Plateau States respectively, were screened for the presence of antibodies to ALV subgroups A/B and J using IDEXX enzyme linked immunosorbent assay (ELISA) kits. Out of the seven layer flocks screened in Kaduna State, antibodies to ALV subgroup A/B was detected in six of the flocks (85.7%), while antibodies to ALV subgroup J was detected in only one flock (14.3%). Antibodies to both ALV subgroups A/B and J were detected in one flock (14.3%), which suggests co-infection of the two ALV subgroups. Out of the 16 flocks screened in Plateau State, antibodies to ALV subgroup A/B were detected in 15 flocks (93.8%), while antibodies to ALV subgroup J were detected in six flocks (37.5%). Antibodies to both ALV subgroups A/B and J were detected in five flocks (31.3%). The high detection of antibodies to ALV A/B suggests that ALV infection in layers is mostly due to ALV subgroup A or B in the study areas.

A Cell Culture System to Investigate Marek’s Disease Virus Integration into Host Chromosomes

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.