Lydia Kgosana

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Mareks disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

A functional microRNA-155 ortholog encoded by the oncogenic Marek's disease virus.

ABSTRACT Kaposis sarcoma-associated herpesvirus-encoded microRNA (miRNA) MiR-K12-11 was recently shown to be a functional ortholog of miR-155, a miRNA that plays a major role in lymphoid malignancies and the modulation of immune responses. Here we show that miR-M4, encoded by the highly oncogenic Mareks disease virus of chickens, shares common targets with miR-155 and thus is also a functional ortholog of miR-155, the first one identified in an alphaherpesvirus. The observation that two distinct oncogenic herpesviruses associated with distinct types of lymphomas in different species encode functional miR-155 orthologs suggested the importance of this miRNA in regulatory pathways and the biology of lymphomagenesis.

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens

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 Mareks 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.

Homodimerization of the Meq Viral Oncoprotein Is Necessary for Induction of T-Cell Lymphoma by Marek's Disease Virus

ABSTRACT Mareks disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B MeqBZIP/BZIP) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B MeqHom/Hom). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

Targeting Marek's disease virus by RNA interference delivered from a herpesvirus vaccine.

Live attenuated herpesvirus vaccines such as herpesvirus of turkey (HVT) have been used since 1970 for the control of Mareks disease (MD), a highly infectious lymphoproliferative disease of poultry. Despite the success of these vaccines in reducing losses from the disease, Mareks disease virus (MDV) strains have shown a continuing increase in virulence, presumably due to the inability of the current vaccines in preventing MDV replication. The highly specific and effective nature of RNA interference (RNAi) makes this technology particularly attractive for new antiviral strategies. In order to exploit the power of RNAi-mediated suppression of MDV replication in vivo delivered through existing vaccines, we engineered recombinant HVT expressing short hairpin RNA (shRNA) against MDV genes gB and UL29. The levels of protection induced by the RNAi-expressing HVT against virulent virus challenge were similar to the parent pHVT3 virus. However, chickens vaccinated with recombinant HVT expressing shRNA showed moderate reduction of challenge virus replication in blood and feather samples. Delivery of RNAi-based gene silencing through live attenuated vaccines for reducing replication of pathogenic viruses is a novel approach for the control of infectious diseases.

Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene

ABSTRACT In this study, we have identified an internal ribosome entry site (IRES) from the highly infectious herpesvirus Mareks disease virus (MDV). The IRES was mapped to the intercistronic region (ICR) of a bicistronic mRNA that we cloned from the MDV-transformed CD4+ T-cell line MSB-1. The transcript is a member of a family of mRNAs expressed as immediate-early genes with two open reading frames (ORF). The first ORF encodes a 14-kDa polypeptide with two N-terminal splice variants, whereas the second ORF is contained entirely within a single exon and encodes a 12-kDa protein also known as RLORF9. We have shown that the ICR that separates the two ORFs functions as an IRES that controls the translation of RLORF9 when cap-dependent translation is inhibited. Deletion analysis revealed that there are two potential IRES elements within the ICR. Reverse genetic experiments with the oncogenic strain of MDV type 1 indicated that deletion of IRES-controlled RLORF9 does not significantly affect viral replication or MDV-induced mortality.

Relationship Between Levels of Very Virulent MDV in Poultry Dust and in Feather Tips from Vaccinated Chickens

SUMMARY.  To assess the effect of various vaccine strains on replication and shedding of virulent Mareks disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator. DNA was extracted from feather and dust samples and subjected to real-time q-PCR, targeting the US2 gene of MDV-1, in order to measure Md5 level per 104 feather tip cells or per microgram of dust. Accuracy of DNA extraction from dust and real-time q-PCR were validated by comparing either q-PCR cycle threshold values or the calculated MDV genome level; for use in q-PCR, DNA was extracted from serial dilutions of MDV-infected dust diluted with noninfected dust, or DNA from MDV-infected dust was diluted with DNA from noninfected dust. The results confirmed the accuracy and sensitivity of dust DNA extraction and subsequent q-PCR and showed that differences in virus levels between dust samples truly reflect differences in shedding. Vaccination delayed both replication of Md5 in feather tips and shedding of Md5. First detection of Md5 in feather tips always preceded or coincided with first detection in dust in each group. pCVI988 and HVT+SB-1 were the most efficient vaccines in reducing both replication and shedding of Md5. There was close correlation between mean virus level in feathers of each group and mean virus level in the dust shed by that group. This relationship was similar in each of the vaccinated groups, demonstrating that measurement of the virus in dust can be used to monitor accurately both the infection status of the chickens and environmental contamination by MDV. RESUMEN.  Relación entre los niveles del virus de Marek muy virulento en el polvo de instalaciones avícolas y en cañones de las plumas en pollos vacunados. Para evaluar el efecto de diferentes cepas de vacunas sobre la replicación y diseminación del virus muy virulento de la enfermedad de Marek en pollos infectados experimentalmente, se desarrollaron métodos cuantitativos de PCR (q-PCR) para determinar con precisión el ADN viral en los pollos infectados y en el entorno en el que se alojaron. Se alojaron cuatro grupos de 10 pollos por unidad de aislamiento, estas aves fueron vacunadas al primer día de edad con una de cuatro vacunas que incluyen los tres serotipos vacunales. Posteriormente fueron desafiados a los ocho días de edad con la cepa muy virulenta del virus de la enfermedad de Marek, Md5. En intervalos de tiempo regulares, se recolectaron cañones de plumas de cada pollo y también se recolectó el polvo del prefiltro del extractor de cada unidad de aislamiento. Se extrajo el ADN de los cañones de las plumas y de las muestras de polvo y se analizaron por PCR en tiempo real, para el gene US2 del virus de Marek 1, con el fin de medir el nivel de virus Md5 por 104 células de plumas o por microgramo de polvo. Se validó la precisión de la extracción de ADN del polvo y el método de PCR en tiempo real mediante la comparación de los ciclos umbrales del método de PCR cuantitativo o por los valores del genoma del virus de Marek. Para su uso en método de PCR cuantitativo, el ADN fue extraído de diluciones seriadas de polvo contaminado con el virus de Marek, diluido con polvo no contaminado, o también, el ADN extraído de polvo contaminado con el virus de Marek se diluyó con ADN de polvo no contaminado. Los resultados confirmaron la precisión y la sensibilidad de la extracción de ADN del polvo y el método de PCR cuantitativo subsecuente, y mostró que las diferencias en los niveles virales entre las muestras de polvo verdaderamente reflejan diferencias en la eliminación del virus. La vacunación retrasó la replicación de la cepa Md5 en los cañones de las plumas y la eliminación de esta cepa Md5. La primera detección de la cepa Md5 en los cañones de las plumas siempre precedió o coincidió con la primera detección de polvo en cada grupo. Las vacunas pCVI988 y HVT + SB-1 fueron las más eficaces en la reducción de la replicación y de la diseminación de la cepa Md5. Hubo una estrecha correlación entre el nivel promedio de virus en las plumas en cada grupo y el nivel promedio de virus en el polvo eliminado por ese grupo. Esta relación fue similar en cada uno de los grupos vacunados, lo que demuestra que la medición del virus en el polvo se puede utilizar para muestrear con precisión tanto el estado de infección de los pollos y la contaminación ambiental por el virus de Marek.

Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformation

The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transformation of primary chicken spleen and bone marrow cells. Rapid transformation of avian haematopoietic cells by v-rel occurs through a process of deregulation of multiple protein-encoding genes through its direct effect on their promoters. More recently, upregulation of oncogenic miR-155 and its precursor pre-miR-155 was demonstrated in both Rev-T-infected chicken embryo fibroblast cultures and Rev-T-induced B-cell lymphomas. Through electrophoresis mobility shift assay and reporter analysis on the gga-miR-155 promoter, we showed that the v-rel-induced miR-155 overexpression occurred by the direct binding to one of the putative NF-κB binding sites. Using the v-rel-induced transformation model on chicken embryonic splenocyte cultures, we could demonstrate a dynamic increase in miR-155 levels during the transformation. Transcriptome profiles of lymphoid cells transformed by v-rel showed upregulation of miR-155 accompanied by downregulation of a number of putative miR-155 targets such as Pu.1 and CEBPβ. We also showed that v-rel could rescue the suppression of miR-155 expression observed in Marek’s disease virus (MDV)-transformed cell lines, where its functional viral homologue MDV-miR-M4 is overexpressed. Demonstration of gene expression changes affecting major molecular pathways, including organismal injury and cancer in avian macrophages transfected with synthetic mature miR-155, underlines its potential direct role in transformation. Our study suggests that v-rel-induced transformation involves a complex set of events mediated by the direct activation of NF-κB targets, together with inhibitory effects on microRNA targets.

Identification of a Neurovirulence Factor from Marek's Disease Virus

SUMMARY.  In addition to tumors, Mareks disease (MD) virus (MDV) can induce a variety of syndromes linked to the central nervous system. In fact, early descriptions of MD suggested that it was a condition affecting mainly the nervous system. Cytokines and other immune-related genes have been suggested to play a crucial role in MDV-mediated neuropathology, but the mechanisms behind the viral-induced neurologic dysfunction are still poorly understood. In the present study we have used reverse genetic strategies to show that pp14 is not involved in the oncogenic phenotype of MDV1 and is not required for viral replication; however, we provide evidence indicating that the absence of pp14 expression is correlated with increased survival of MDV1-infected chickens, and that its expression is associated with enhanced viral neurovirulence. Our data identify for the first time pp14 as a neurovirulence factor from MDV1 and open the possibility to investigate the molecular mechanisms by which pp14 mediates the damage to the avian nervous system. RESUMEN.  Identificación de un factor de neurovirulencia del virus de la enfermedad de Marek. Además de tumores, el virus de la enfermedad de Marek (MDV) puede inducir una variedad de síndromes relacionados con el sistema nervioso central. De hecho, las primeras descripciones de la enfermedad de Marek sugirieron que era una enfermedad que afectaba principalmente el sistema nervioso. Se ha sugerido que las citocinas y otros genes relacionados con la inmunidad desempeñan un papel crucial en la neuropatología mediada por la enfermedad de Marek, pero los mecanismos que están implicados en la disfunción neurológica inducida por este virus todavía son poco conocidos. En el presente estudio se utilizaron estrategias de genética inversa para demostrar que el gene pp14 no está implicado en el fenotipo oncogénico del virus de Marek 1 y que no es necesario para la replicación viral. Sin embargo, se proporcionan pruebas que indican que la ausencia de la expresión de pp14 se correlaciona con aumento de la supervivencia de los pollos infectados con Marek, y que su expresión se asocia con un aumento en la neurovirulencia viral. Los presentes datos identifican por primera vez al producto pp14 como un factor de neurovirulencia del virus de Marek 1 y abren la posibilidad de investigar los mecanismos moleculares por los cuales el producto pp14 media los daños en el sistema nervioso aviar.

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

Marek’s disease virus (MDV), an alphaherpesvirus of poultry, causes Marek’s disease and is characterized by visceral CD4+TCRαβ+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus–host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.