Susan J. Baigent

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.

Inhibition of Beta Interferon Transcription by Noncytopathogenic Bovine Viral Diarrhea Virus Is through an Interferon Regulatory Factor 3-Dependent Mechanism

ABSTRACT The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-α/β or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-β induction pathway have been analyzed. The transcription factor NF-κB was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-κB by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

ABSTRACT Mareks disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the US2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

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.

Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines

ABSTRACT Mareks disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.

Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Mareks disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Mareks disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Mareks disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

Comparative sequence analysis of a highly oncogenic but horizontal spread-defective clone of Marek's disease virus.

Marek’s disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ‘‘non-spreading phenotype’’ of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.

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.

Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.

We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Mareks disease virus in feather tips of chickens, and this has been used clinically to monitor a flocks response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.

Cloning of Gallid herpesvirus 3 (Marek's disease virus serotype-2) genome as infectious bacterial artificial chromosomes for analysis of viral gene functions

Mareks disease virus serotype 2 (Gallid herpesvirus 3) is a non-pathogenic alphaherpesvirus belonging to the Mardivirus genus, used widely in live vaccines against Mareks disease. Although the complete genome sequence of the MDV-2 strain HPRS-24 has been published, very little is known about the gene functions. As a first step for carrying out functional genomic analysis of MDV-2, the full-length genome of the MDV-2 vaccine strain SB-1 was cloned as an infectious bacterial artificial chromosome (BAC) clone pSB-1. Virus reconstituted from the pSB-1 clone showed morphological and growth characteristics in cell culture very similar to the parent virus. Generation of SB-1 constructs deleted in glycoprotein E and viruses expressing Citrine-UL35 fusion protein by the application of different BAC mutagenesis techniques demonstrated the amenability of the pSB-1 clone for reverse genetics approaches to identify molecular determinants associated with different biological features of this virus. The generation of replication-competent infectious clones of SB-1, together with those of CVI988 and herpesvirus of turkey strains described previously, completes the portfolio of generating infectious BAC clones of the MD vaccine strains belonging to all the three serotypes, paving the way for the application of reverse genetics for functional analysis of immunogenic determinants of these vaccines as well as for developing novel recombinant vectors.