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Concept: Marek's disease

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Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek’s disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.

Concepts: Immune system, Infectious disease, Natural selection, Microbiology, Malaria, Vaccination, Smallpox, Marek's disease

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Marek’s disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a Treg signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system.

Concepts: Protein, Infection, B cell, T cell receptor, Culture, Herpesviridae, Chicken, Marek's disease

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Although it has become increasingly popular to keep backyard chickens in the United States, few studies have provided information about these flocks. An online survey of backyard chicken owners was conducted, advertised through Master Gardeners' websites, social platforms, and other sites. The survey had 56 questions about flock history, husbandry, health care, and owner attitudes and demographics. Surveys received (n = 1,487) came almost equally from urban, suburban, and rural areas. Most (71%) respondents owned fewer than 10 chickens and had kept chickens for less than 5 yr (70%). Major reasons for keeping chickens were as food for home use (95%), gardening partners (63%), pets (57%), or a combination of these. Rural respondents had larger flocks (P ≤ 0.001) and were more likely to keep chickens as a source of income or for show (P ≤ 0.001) than urban and suburban respondents. Owners thought that eggs/meat from their chickens were more nutritious (86%), safer to consume (84%), and tasted better (95%) than store-bought products, and also that the health and welfare of their chickens was better (95%) than on commercial farms. The majority (59%) indicated no flock health problems in the last 12 mo. However, there was a lack of awareness about some poultry health conditions. Many knew either little or nothing about exotic Newcastle or Marek’s disease, and most (61%) did not vaccinate against Marek’s. Respondents wanted to learn more about various flock management topics, especially how to detect (64%) and treat (66%) health problems. The Internet was the main source of information (87%) used by backyard flock owners, followed by books/magazines (62%) and feed stores (40%). Minimizing predation was the most cited challenge (49%), followed by providing adequate feed at low cost (28%), dealing with soil management (25%), and complying with zoning regulations (23%). The evidence obtained from this survey will help to determine what information and resources are needed to maintain good biosecurity and improve the health and welfare of backyard flocks.

Concepts: Public health, Health, United States, Chicken, Gross domestic product, Property, Marek's disease, Gardening

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Marek’s disease virus (MDV) is an alphaherpesvirus that causes fatal lymphomas in chickens and is used as a natural virus-host model for herpesvirus-induced tumorigenesis. MDV encodes a telomerase RNA subunit (vTR) that is crucial for efficient MDV-induced lymphoma formation; however, the mechanism is not completely understood. Similarly, Epstein Barr-virus (EBV) encodes two RNAs (EBER-1 and EBER-2) that are highly expressed in EBV-induced tumor cells, however their role in tumorigenesis remains unclear. Intriguingly, vTR and EBER-1 have interaction partners in common that are highly conserved in humans and chickens. Therefore, we investigated if EBER-1 and/or EBER-2 can complement the loss of vTR in MDV-induced tumor formation. We first deleted vTR (v∆vTR) and replaced it by either EBER-1 or EBER-2 in the very virulent RB-1B strain. Insertion of either EBER-1 or EBER-2 did not affect MDV replication and their expression levels were comparable to vTR in wild type virus. Intriguingly, EBER-2 restored tumor formation of MDV that lacks vTR. EBER-1 partially restored MDV oncogenicity, while tumor formation was severely impaired in chickens infected with v∆vTR. Our data provides the first evidence that EBERs possess tumor-promoting properties in vivo using this natural model for herpesvirus-tumorigenesis.

Concepts: DNA, Gene, Gene expression, Virus, Herpesviridae, Epstein-Barr virus, Herpesviruses, Marek's disease

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Protection against diseases caused by the avian viruses, Marek’s disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates. The Marek’s disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3-21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.

Concepts: Infectious disease, Vaccine, Vaccination, Bird, Herpesviridae, Chicken, Animal virology, Marek's disease

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Marek’s Disease Virus (MDV) is an alphaherpesvirus that infects chickens, transforms CD4+ T cells and causes deadly lymphomas. In addition, MDV induces immunosuppression early during infection by inducing cell death of the infected lymphocytes, and potentially due to activation of regulatory T (Treg)-cells. Furthermore, immunosuppression also occurs during the transformation phase of the disease; however, it is still unknown how the disease can suppress immune response prior or after lymphoma formation. Here, we demonstrated that chicken TGF-beta+ Treg cells are found in different lymphoid tissues, with the highest levels found in the gut-associated lymphoid tissue (cecal tonsil: CT), fostering an immune-privileged microenvironment exerted by TGF-beta. Surprisingly, significantly higher frequencies of TGF-beta+ Treg cells are found in the spleens of MDV-susceptible chicken lines compared to the resistant line, suggesting an association between TGF-beta+ Treg cells and host susceptibility to lymphoma formation. Experimental infection with a virulent MDV elevated the levels of TGF-beta+ Treg cells in the lungs as early as 4 days post infection, and during the transformation phase of the disease in the spleens. In contrast to TGF-beta+ Treg cells, the levels of CD4+CD25+ T cells remained unchanged during the infection and transformation phase of the disease. Furthermore, our results demonstrate that the induction of TGF-beta+ Treg cells is associated with pathogenesis of the disease, as the vaccine strain of MDV did not induce TGF-beta+ Treg cells. Similar to human haematopoietic malignant cells, MDV-induced lymphoma cells expressed high levels of TGF-beta but very low levels of TGF-beta receptor I and II genes. The results confirm that COX-2/ PGE2 pathway is involved in immunosuppression induced by MDV-lymphoma cells. Taken together, our results revealed a novel TGF-beta+ Treg subset in chickens that is activated during MDV infection and tumour formation.

Concepts: Immune system, White blood cell, Cancer, Infection, Lymphatic system, Chicken, Induced demand, Marek's disease

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Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek’s disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10-5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV.

Concepts: Gene, Genetics, Infectious disease, Natural selection, Evolution, Virus, Herpesviridae, Marek's disease

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Deubiquitinases (DUBs) are essential regulators of intracellular processes involving ubiquitin (Ub) modification. The human DUB ubiquitin-specific protease 1 (hUSP1) interacts with human USP-associated factor 1 (hUAF1), and helps to regulate processes such as DNA damage repair. Previously, we identified a chicken USP1 homologue (chUSP1) during an investigation into the properties of Marek’s disease virus (MDV). However, chUSP1’s deubiquitination activity, interaction with chUAF1, and substrate specificity remained unknown. In the present study, we expressed and purified both chUAF1 and chUSP1 with or without putative catalytic core mutations using the Bac-to-Bac system, before investigating their deubiquitination activity and kinetics using various substrates. chUSP1 was shown to interact with chUAF1 both in cellular assays in which the two proteins were co-expressed, and in in vitro assays using purified proteins. Heterodimerization with chUAF1 increased the deubiquitination activity of chUSP1 up to 54-fold compared with chUSP1 alone. The chUSP1 mutants C91S, H603A, and D758A reduced the deubiquitination activity of the chUSP1/chUAF1 complex by 10-, 7-, and 33-fold, respectively, while the C91A and H594A chUSP1 mutants eliminated deubiquitination activity of the chUSP1/chUAF1 complex completely. This suggests that C91 and H594, but not D758, are essential for chUSP1 deubiquitination activity, and that a nucleophilic group at position 91 is needed for the deubiquitination reaction. The chUSP1/chUAF1 complex was found to have distinct substrate preferences; efficient hydrolysis of Ub dimers with K11-, K48-, and K63-linkages was seen, with weaker hydrolysis observed with K6-, K27-, and K33-linkages and no hydrolysis seen with a K29-linkage. Furthermore, other Ub-like substrates were disfavored by the complex. No activity was seen with SUMO1-GST, SUMO2- and SUMO3-dimers, ISG15-Rho, FAT10-Rho, or Ufm1-Rho, and only weak activity was observed with NEDD8-Rho. Overall, the data presented here characterize the activity and substrate preferences of chUSP1, and thus may facilitate future studies on its in vivo role.

Concepts: Enzyme, Enzyme substrate, Product, DNA repair, In vivo, In vitro, Chicken, Marek's disease

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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 (DSB) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV lifecycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSB during lytic infection in vitro and in the PBMCs 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 poultry industry and is also used as a small-animal model for virus-induced tumor formation. DNA damage is not only 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 demonstrated that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells, however is reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induced DNA damage were defective in the induction of tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV-lymphomagenesis.

Concepts: DNA, Gene, Cancer, Infection, Tumor, Herpesviridae, Virology, Marek's disease

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We compared immunohistochemical (IHC) staining of tissue sections of liver, kidney, spleen, lung, proventriculus, sciatic nerve, bursa of Fabricius, brain, heart, intestine and skin; immunocytochemical (ICC) staining of peripheral blood samples and touch preparations of liver, spleen and kidney of laying hens naturally infected with Marek’s disease (MD) virus. We used one hundred and fifty 5-17-week-old commercial hens. IHC and ICC staining were performed using polymer-based techniques. IHC staining exhibited mostly free immunopositive reactions in tumor cells and in the cytoplasm of the parenchymal cells of liver, kidney, spleen and bursa of Fabricius. In the sciatic nerve, severe reactions were observed in the cytoplasm of plasma and MD cells in the lymphoproliferative areas. Pronounced staining was found in the lymphoid cells in the medulla of intrafollicular regions in the bursa of Fabricius. Although immunostaining was observed in the liver and spleen touch preparations, there was no staining in the kidneys and peripheral blood cell samples. The presence of virus in the tissue and peripheral blood samples and in touch preparations was compared immunohistochemically and immunocytochemically. IHC and ICC techniques were helpful for diagnosis of MD. Peripheral blood samples are inappropriate for field conditions and natural infections.

Concepts: Kidney, Cancer, Blood, Organ, Lymphatic system, Chicken, Hieronymus Fabricius, Marek's disease