Impact of viral telomeric repeat sequences on herpesvirus vector vaccine integration and persistence.

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.

In vivo imaging reveals novel replication sites of a highly oncogenic avian herpesvirus in chickens.

In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host.

Identification of Marek's Disease Virus VP22 Tegument Protein Domains Essential for Virus Cell-to-Cell Spread, Nuclear Localization, Histone Association and Cell-Cycle Arrest.

VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.

Keratinocytes derived from chicken embryonic stem cells support Marek's disease virus infection: a highly differentiated cell model to study viral replication and morphogenesis.

BACKGROUND: Marek's disease is a virus disease with worldwide distribution that causes major losses to poultry production. Vaccines against Marek's disease virus, an oncogenic alphaherpesvirus, reduce tumour formation but have no effect on virus shedding. Successful horizontal virus transmission is linked to the active viral replication in feather follicle epithelial cells of infected chickens, from which infectious viral particles are shed into the environment. The feather follicle epithelium is the sole tissue in which those infectious particles are produced and no in vitro cell-systems can support this highly efficient morphogenesis. We previously characterized embryonic stem-cell-derived keratinocytes, showing they display a marker-gene profile similar to skin keratinocytes, and therefore we tested their susceptibility to Marek's disease virus infection. FINDINGS: We show herein that keratinocytes derived from chicken embryonic stem-cells are fully permissive to the replication of either non-pathogenic or pathogenic Marek's disease viruses. All viruses replicated on all three keratinocyte lines and kinetics of viral production as well as viral loads were similar to those obtained on primary cells. Morphogenesis studies were conducted on infected keratinocytes and on corneocytes, showing that all types of capsids/virions were present inside the cells, but extracellular viruses were absent. CONCLUSIONS: The keratinocyte lines are the first epithelial cell-line showing ectodermal specific markers supporting Marek's disease virus replication. In this in vitro model the replication lead to the production of cell-associated viral progeny. Further work will be devoted to the study of relationship between 3D differentiation of keratinocytes and Marek's disease virus replication.

Derivation of keratinocytes from chicken embryonic stem cells: establishment and characterization of differentiated proliferative cell populations.

A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

A full UL13 open reading frame in Marek's disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that is highly contagious in poultry. Recombinant RB-1B (rRB-1B) reconstituted from an infectious genome cloned as a bacterial artificial chromosome (BAC) is unable to spread horizontally, quite in contrast to parental RB-1B. This finding suggests the presence of one or several mutations in cloned relative to parental viral DNA. Sequence analyses of the pRB-1B bacmid identified a one-nucleotide insertion in the UL13 orthologous gene that causes a frame-shift mutation and thereby results in a theoretical truncated UL13 protein (176 aa vs. 513 aa in parental RB-1B). UL13 genes are conserved among alphaherpesviruses and encode protein kinases. Using two-step "en passant" mutagenesis, we restored the UL13 ORF in pRB-1B. After transfection of UL13-positive pRB-1B DNA (pRB-1B*UL13), the resulting, repaired virus did not exhibit a difference in cell-to cell spread (measured by plaque sizes) and in UL13 transcripts in culture compared to parental rRB-1B virus. Although 89% of the chickens inoculated with rRB-1B*UL13 virus developed tumors in visceral organs, none of the contact birds did. MDV antigens were clearly expressed in the feather tips of rRB-1B infected chickens, suggesting that the UL13 gene mutation did not alter virus tropism of the feather follicle. The results indicate that the correction in UL13 gene alone is not sufficient to restore in vivo spreading capabilities of the rRB-1B virus, and that other region(s) of pRB-1B might be involved in the loss-of-function phenotype. This finding also shows for the first time that a full UL13 ORF is dispensable for MDV tumor formation and feather follicle tropism.