Temperature-induced reactivation of Marek's disease virus-transformed T cells ex vivo.

Marek's disease virus (MDV) establishes latency in chicken T lymphocytes that can lead to T cell transformation and cancer. Transformed Marek's disease chicken cell lines (MDCCs) can be expanded ex vivo and provide a valuable model to study latency, transformation, and reactivation. Here, we developed MDCCs from chickens infected with MDV that fluoresce during lytic replication and reactivation. Sodium butyrate treatment increased fluorescent protein expression as evidenced by fluorescent microscopy, flow cytometry, and western blotting; however, it caused significant apoptosis and necrosis. Treatment of MDCCs by decreasing the temperature resulted in robust MDV reactivation without significant induction of apoptosis and necrosis. Furthermore, MDV reactivation was significantly affected by the time in culture that can affect downstream reactivation analyses. In all, our data show that fluorescent protein expression during reactivation is a robust tool to examine viral replication in live cells ex vivo, and temperature treatment is an efficient technique to induce reactivation without punitive effects on cell viability seen with chemical treatment.

The Herpesviridae Conserved Multifunctional Infected-Cell Protein 27 (ICP27) Is Important but Not Required for Replication and Oncogenicity of Marek's Disease Alphaherpesvirus.

The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

Downregulation of the KLF4 transcription factor inhibits the proliferation and migration of canine mammary tumor cells.

Canine mammary tumor (CMT) is the most common neoplasm in female dogs, and over 50% of CMTs are diagnosed as malignant. Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and is associated with cell proliferation, differentiation, migration, and apoptosis. Although the role of KLF4 is still controversial in various human cancers, KLF4 has been identified as an oncogene in human breast cancer. Moreover, high KLF4 expression is correlated with an aggressive phenotype in CMT. Therefore, investigating the function of KLF4 may help better understand the pathogenesis of CMT. In this study, partial sequences of canine KLF4 and KLF4 expression were identified in various normal canine tissues, as well as CMT cells and Madin-Darby canine kidney (MDCK) cells. Kenpaullone, a small molecule inhibitor of KLF4, downregulated KLF4 expression in CMT cells and reduced CMT cell proliferation, migration, and colony formation in soft agar. Kenpaullone treatment induced S and G2/M phase arrest in CMT and MDCK cells, and induced death in CMT cells, but not in MDCK cells. It was concluded that KLF4 is expressed in various normal canine tissues, and downregulation of KLF4 inhibited CMT cell proliferation and migration, and induced cell death. The results of this study suggest that KLF4 may represent a suitable therapeutic target for CMT therapy.