Marek’s disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset lymphoma in chickens. Marek’s disease (MD) is effectively controlled using vaccination; however, MDV continues to break through vaccinal immunity, due to the emergence of highly virulent field strains. Earlier studies revealed that deletion of the meq gene from MDV resulted in an attenuated virus that protects against MD in chickens challenged with highly virulent field strains. However, the meq deleted virus retains the ability to induce significant lymphoid organ atrophy. In a different study, we found that the deletion of the vIL8 gene resulted in the loss of lymphoid organ atrophy in inoculated chickens. Here, we describe the generation of a recombinant MDV from which both meq and vIL8 genes were deleted. In vitro studies revealed that the meq and vIL8 double deletion virus replicated at levels similar to the parental very virulent plus (vv+) virus. In addition, in vivo studies showed that the double deletion mutant virus (686BAC-ΔMeqΔvIL8) conferred protection comparable to CVI988, a commercial vaccine strain, when challenged with a vv+ MDV virus, and significantly reduced lymphoid organ atrophy, when compared to meq null virus, in chickens. In conclusion, our study describes the development of a safe and effective vaccine candidate for prevention of MD in chickens.

Marek’s disease virus (MDV) is an oncogenic avian alphaherpesvirus whose genome consists of unique long (UL) and short (US) regions that are flanked by inverted repeat regions. More than 100 open reading frames (ORFs) have been annotated in the MDV genome, and are involved in various aspects of MDV biology and pathogenesis. Within UL and US regions of MDV, there are several unique ORFs, some of which have recently been shown to be important for MDV replication and pathogenesis. In this review, we will summarize the current knowledge on these ORFs and compare their location in different MDV strains.

Historically, virus isolation has been the method of choice for conducting surveillance for avian influenza virus (AIV) in avian species. More recently, the primary screening method has become real-time reverse transcription-polymerase chain reaction (RRT-PCR). We wanted to determine how these two testing methods (virus isolation and RRT-PCR) affected AIV prevalence estimation, particularly in an understudied, low-prevalence region-the waterfowl wintering grounds along the Texas mid-Gulf Coast. Cloacal swabs were collected from hunter-harvested waterfowl and other wetland-associated game birds during four consecutive hunting seasons (2005-2006 through 2008-2009). Overall prevalence by RRT-PCR (5.9%, 6.5%, 11.2%, and 5.5%) was approximately an order of magnitude higher than prevalence by virus isolation (0.5%, 1.3%, 3.9%, and 0.7%) for the four hunting seasons, respectively. Apparent AIV prevalence by virus isolation conducted only on RRT-PCR-positive samples resulted in estimates nearly identical in magnitude to those derived from parallel testing (0.5% vs. 0.6%, 1.3% vs. 1.7%, and 3.9% vs. 4.0% for 2005-2006, 2006-2007, and 2007-2008, respectively). Unlike most reports of seasonal variation in AIV prevalence, we documented differences in prevalence estimates among months by RRT-PCR only during 2008-2009 and by virus isolation only during 2006-2007 and 2007-2008. Our data indicate that screening samples by RRT-PCR followed by virus isolation only on RRT-PCR-positive samples provides a reasonable means to generate prevalence estimates close to the true prevalence as determined by virus isolation. We also confirmed the low prevalence of AIV in waterfowl wintering grounds along the Texas mid-Gulf Coast and demonstrated little variation in prevalence among months during the four hunting seasons sampled.

Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek’s disease virus research.

Herpesvirus-encoded microRNAs (miRNAs) have been discovered in infected cells; however, lack of a suitable animal model has hampered functional analyses of viral miRNAs in vivo. Marek’s disease virus (MDV) (Gallid alphaherpesvirus 2, GaHV-2) genome contains 14 miRNA precursors, which encode 26 mature miRNAs, grouped into three clusters. In this study, the role of MDV-encoded cluster 3 miRNAs, also known as mdv1-miR-M8-M10, in pathogenesis was evaluated in chickens, the natural host of MDV. Our results show that deletion of cluster 3 miRNAs did not affect virus replication and plaque size in cell culture, but increased early cytolytic replication of MDV in chickens. We also observed that deletion of cluster 3 miRNAs resulted in significantly higher virus reactivation from peripheral blood lymphocytes. In addition, pathogenesis studies showed that deletion of cluster 3 miRNAs resulted in more severe atrophy of lymphoid organs and reduced mean death time, but did not affect the incidence of MDV-associated visceral tumors. We confirmed these results by generating a cluster 3 miRNA revertant virus in which the parental MDV phenotype was restored. To the best of our knowledge, our study provides the first evidence that MDV cluster 3 miRNAs play an important role in modulating MDV pathogenesis.

Marek’s disease (MD), characterized by the rapid onset of T-cell lymphomas in chickens, is caused by Mardivirus gallidalpha2, an oncogenic alphaherpesvirus commonly known as Marek’s disease virus (MDV). MDV encodes a bZIP protein, Meq, which contains a bZIP domain (basic DNA-binding and leucine zipper dimerization domain) at the amino terminus and a transcriptional regulatory domain at the carboxyl end. Meq can transform murine and chicken fibroblasts in vitro and is essential for tumor formation in chickens. Meq homodimerization and heterodimerization through its bZIP domain are involved in Meq-mediated transformation. However, the role of Meq DNA-binding and transcriptional regulatory domains in transformation has not been investigated. In this study, we constructed recombinant Md5 (very virulent MDV) viruses expressing chimeric Meq proteins generated by swapping the DNA-binding and transcriptional regulatory domains of Meq of Md5 and vaccine (CVI988/Rispens) strains. Our results show that these recombinant viruses, rMd5-Md5/CVI-Meq (Md5 DNA-binding domain and CVI transcriptional regulatory domain) and rMd5-CVI/Md5-Meq (CVI DNA-binding domain and Md5 transcriptional regulatory domain), replicated at levels similar to parental rMd5 in cell culture and chickens and could transmit efficiently among chickens. Interestingly, parental rMd5 and chimeric viruses exhibited distinct pathogenic phenotypes in chickens: rMd5 caused 100% mortality, a moderate level of tumor incidence in visceral organs and small visceral tumors; rMd5-Md5/CVI-Meq caused 100% mortality, a high level of tumor incidence in visceral organs, and very large visceral tumors; while rMd5-CVI/Md5-Meq caused an average of 37% mortality, rarely induced tumors in visceral organs, and the visceral tumors were small. In conclusion, our study suggests that the DNA-binding domain of Meq plays an essential role in transformation (tumor incidence), while the transcriptional regulatory domain of Meq influences the distribution and size of MDV-induced tumors.