Avian leukosis virus subgroup J-associated myelocytoma in a hobby chicken.

The avian leukosis viruses (ALVs) are a major group of retroviruses associated with neoplastic diseases in poultry. The ALV-J strain was identified as a cause of myelocytomas in broiler breeder and broiler chickens in the UK in the 1980s; however, following eradication of the virus,commercial broilers have remained free of infection since the early 2000s. A pet chicken was submitted to Animal and Plant Health Agency (APHA) in 2013 with a history of croaking respirations, abnormality of the left eye and apparent paralysis. Postmortem examination of the bird showed widespread tumour-like infiltration of many organs, including the pectoral muscles, internal organs, sternum and ribs. Histopathological examination of the affected tissues revealed myelocytoma formation typical of the lesions associated with ALV-J, and the virus was confirmed by PCR testing and sequencing. Virus was not detected in blood samples in the other five chickens remaining in the flock. The source of infection was not established. This was the first time ALV-J had been seen in the UK since its eradicationand the case highlights the importance of continued surveillance of backyard and hobby chickens to detect potential new and re-emerging disease threats, such as ALV-J, which may be of significance to the wider poultry population.

Early pathogenesis during infectious bursal disease in susceptible chickens is associated with changes in B cell genomic methylation and loss of genome integrity.

We propose a model by which an increase in the genomic modification, 5-hydroxymethylcytosine (5hmC), contributes to B cell death within the chicken bursa of Fabricus (BF) infected with infectious bursal disease virus (IBDV). Our findings indicate that, following an IBDV infection, Rhode Island Red (RIR) chickens have fewer surviving B cells and higher levels of 5hmC in the BF than the more resistant 15l line of birds. Elevated genomic 5hmC levels within the RIR BF are associated with markers of immune responses: infiltrating T cells and increased expression of CD40L, FasL and iNOS. Such changes correlate with genomic fragmentation and the presence of IBDV capsid protein, VP2. To explore the effects of CD40L, the immature B cell line, DT40, was exposed to recombinant chicken CD40L that resulted in changes in nuclear 5hmC distribution. Collectively, our observations suggest that T cell infiltration exacerbates early immunopathology within the BF during an IBDV infection contributing to B cell genomic instability and death to facilitate viral egress and immunosuppression.

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 CEBPß. 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.

Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus.

T-lymphocytes are central targets of Marek's disease, a major chicken disease induced by the oncogenic alphaherpesvirus Marek's disease virus (MDV). T-lymphocyte infection is also associated with immunosuppression and virus latency. To decipher viral morphogenesis in T-lymphocytes, we used the recombinant vRB-1B 47EGFP marker virus to generate a new lymphoblastoid cell line, 3867K, that exhibited typical properties of other MDV-transformed chicken cell lines in term of cell markers, reactivation rate and infectivity. Examination of reactivating EGFP-positive 3867K cells by transmission electron microscopy revealed the presence of most types of herpesvirus particles inside the cells but no extracellular ones. Quantification of virion types indicated only 5% cytoplasmic particles, with 0.5% being mature. This study demonstrated that MDV morphogenesis is complete upon reactivation in T-lymphocytes, albeit with poor efficiency, with a defect in the exit of virions from the nucleus and secondary envelopment, as occurs in infected fibroblasts.

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 Marek's 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.

Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.

A B-cell targeting virus disrupts potentially protective genomic methylation patterns in lymphoid tissue by increasing global 5-hydroxymethylcytosine levels.

The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius. We discovered that embryonic B-cells have high 5mC content while 5hmC decreases during bursa development. We propose that a high 5mC level protects from the mutagenic activity of the B-cell antibody diversifying enzyme activation induced deaminase (AID). In support of this view, AID mRNA increases significantly within the developing bursa from embryonic to post hatch stages while mRNAs that encode Tet family members 1 and 2 reduce over the same period. Moreover, our data revealed that infectious bursal disease virus (IBDV) disrupts this genomic methylation pattern causing a global increase in 5hmC levels in a mechanism that may involve increased Tet 1 and 2 mRNAs. To our knowledge this is the first time that a viral infection has been observed to cause global increases in genomic 5hmC within infected host tissues, underlining a mechanism that may involve the induction of B-cell genomic instability and cell death to facilitate viral egress.

An avian retrovirus uses canonical expression and processing mechanisms to generate viral microRNA.

To date, the vast majority of known virus-encoded microRNAs (miRNAs) are derived from polymerase II transcripts encoded by DNA viruses. A recent demonstration that the bovine leukemia virus, a retrovirus, uses RNA polymerase III to directly transcribe the pre-miRNA hairpins to generate viral miRNAs further supports the common notion that the canonical pathway of miRNA biogenesis does not exist commonly among RNA viruses. Here, we show that an exogenous virus-specific region, termed the E element or XSR, of avian leukosis virus subgroup J (ALV-J), a member of avian retrovirus, encodes a novel miRNA, designated E (XSR) miRNA, using the canonical miRNA biogenesis pathway. Detection of novel microRNA species derived from the E (XSR) element, a 148-nucleotide noncoding RNA with hairpin structure, showed that the E (XSR) element has the potential to function as a microRNA primary transcript, demonstrating a hitherto unknown function with possible roles in myeloid leukosis associated with ALV-J.

Identification of a neurovirulence factor from Marek's disease virus.

In addition to tumors, Marek's 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.

Relationship between levels of very virulent MDV in poultry dust and in feather tips from vaccinated chickens.

To assess the effect of various vaccine strains on replication and shedding of virulent Marek's 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 U(S)2 gene of MDV-1, in order to measure Md5 level per 10(4) 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.

Induction of lymphomas by inoculation of Marek's disease virus-derived lymphoblastoid cell lines: prevention by CVI988 vaccination.

Lymphoblastoid cell lines 265(L) and 990(O) are monoclonal lymphomas, derived respectively from liver and ovarian tumours, generated in inbred P-line (MHC B(19)/B(19)) chickens infected with RB-1B strain of Marek's disease virus (MDV) and pRB-1B5 BAC clone respectively. These were inoculated into inbred, MDV-susceptible, P-line chickens by intra-venous or intra-abdominal routes. Additional groups of birds were vaccinated using 1000 plaque-forming units of CVI988 vaccine 8 days prior to inoculation of the cell lines. Non-vaccinated birds developed visceral Marek's disease tumours with an increased rate 30 to 60 days post inoculation. Vaccination prevented tumour and disease development in challenged birds. TCRß repertoire analysis by spectratyping and sequencing of the inoculum was used to track tumour identity in primary tumours and tumour cell lines derived from inoculated birds. These data revealed that the tumours were a consequence of de novo virus infection and not metastasis and expansion of the inoculated tumour cells. Moreover, the data showed that the two MDV-derived cell lines were not transplantable even in syngeneic P-line birds. The data also demonstrated the application of spectratyping as a tool to track tumour identity in lymphoma transplantation studies.

Novel microRNAs encoded by duck enteritis virus.

Duck enteritis virus (DEV) is an important herpesvirus pathogen associated with acute, highly contagious lethal disease in waterfowls. Using a deep sequencing approach on RNA from infected chicken embryo fibroblast cultures, we identified several novel DEV-encoded micro (mi)RNAs. Unlike most mardivirus-encoded miRNAs, DEV-encoded miRNAs mapped mostly to the unique long region of the genome. The precursors of DEV miR-D18 and miR-D19 overlapped with each other, suggesting similarities to miRNA-offset RNAs, although only the DEV-miR-D18-3p was functional in reporter assays. Identification of these novel miRNAs will add to the growing list of virus-encoded miRNAs enabling the exploration of their roles in pathogenesis.

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαß+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRß repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.

Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130.

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

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 Marek's 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.

Pathogenicity of a very virulent strain of Marek's disease herpesvirus cloned as infectious bacterial artificial chromosomes.

Bacterial artificial chromosome (BAC) vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs) are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130) of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

MicroRNA-26a-mediated regulation of interleukin-2 expression in transformed avian lymphocyte lines.

BACKGROUND: Micro(mi)RNAs are a class of small non-coding RNAs that play critical roles in the induction of various cancers, including lymphomas induced by oncogenic viruses. While some of the miRNAs are oncogenic, miRNAs such as miR-26a are consistently downregulated in a number of cancers, demonstrating their potential tumor suppressor functions. Global miRNA expression profiles of a number of virus-transformed avian lymphoma cell lines have shown downregulation of gga-miR-26a expression, irrespective of molecular mechanisms of transformation or the viral aetiology. The neoplastic transformation of lymphocytes by many viruses accompanies high levels of proliferative responses, mostly mediated through cytokines such as IL-2. Chicken IL-2 can modulate T-cell proliferation and cytotoxicity in vitro and in vivo and dysregulation of IL-2 expression is observed in diseases such as leukaemia. RESULTS: The expression levels of gga-miR-26a in chicken lymphoma cells transformed by 3 distinct avian oncogenic viruses, viz Marek's disease virus (MDV), avian leukosis virus (ALV) and Reticuloendotheliosis virus (REV) were consistently downregulated compared to the levels in the normal lymphocytes. This downregulation of miR-26a regardless of the viral etiology and molecular mechanisms of transformation was consistent with the tumor suppressor role of this miRNA. Notwithstanding this well-established role in cancer, we demonstrate the additional role of this miRNA in directly targeting chicken IL-2 through reporter and biochemical assays. The downregulation of miR-26a can relieve the suppressive effect of this miRNA on IL-2 expression. CONCLUSIONS: We show that miR-26a is globally downregulated in a number of avian lymphoma cells irrespective of the mechanisms of transformation, reiterating the highly conserved tumor suppressor function of this miRNA. However, with the potential for directly targeting chicken IL-2, the downregulation of miR-26a in these tumor cells could relieve the inhibitory effect on IL-2 expression assisting in the proliferative features of the transformed lymphocyte lines.

Homodimerization of the Meq viral oncoprotein is necessary for induction of T-cell lymphoma by Marek's disease virus.

Marek's 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 Meq(BZIP/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 Meq(Hom/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.

Interaction of Marek's disease virus oncoprotein Meq with heat-shock protein 70 in lymphoid tumour cells.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces the rapid onset of T-cell lymphomas in poultry. The MDV-encoded oncoprotein Meq plays an important role in oncogenicity, as its deletion abolishes the ability of the virus to induce tumours. It has been shown previously that Meq oncogenicity is linked to its interaction with C-terminal binding protein 1 (CtBP), a property also shared by other virus-encoded oncoproteins such as adenovirus E1A and Epstein-Barr virus EBNA3A and -3C. Therefore, this study examined whether Meq also shares the properties of these viral oncoproteins in interacting with other binding partners such as heat-shock protein 70 (Hsp70), a molecular chaperone protein linked to multiple cellular functions including neoplastic transformation. Confocal microscopic analysis demonstrated that MDV infection induced nuclear accumulation of Hsp70 and its co-localization with Meq. Biochemical evidence of Meq-Hsp70 interaction was obtained by two-way immunoprecipitation with Meq- and Hsp70-specific antibodies. To demonstrate further the Meq-Hsp70 interaction in virus-induced lymphomas, recombinant MDV was generated expressing an N-terminal tandem affinity purification (TAP) tag-fused Meq by mutagenesis of the infectious BAC clone of the oncogenic MDV strain RB-1B. Demonstration of Hsp70 in the TAP-tag affinity purified Meq from tumours induced by the recombinant virus, using quadrupole time-of-flight tandem mass spectrometry analysis, further confirmed the Meq-Hsp70 interaction in the transformed lymphocytes. Given the well-documented evidence of the tumorigenic properties of Hsp70 and its interaction with a number of other known viral oncoproteins, demonstration of the interaction of Meq and Hsp70 is significant in MDV oncogenesis.

Novel microRNAs (miRNAs) encoded by herpesvirus of Turkeys: evidence of miRNA evolution by duplication.

Herpesviruses account for 134 out of the 140 virus-encoded microRNAs (miRNAs) known today. Here we report the identification of 11 novel miRNAs encoded by herpesvirus of turkey (HVT), a virus used as a live vaccine in poultry against the highly oncogenic Marek's disease virus type 1. Ten of these miRNAs were clustered together within the repeat long region of the viral genome, demonstrating some degree of positional conservation with other mardiviruses. Close sequence and phylogenetic relationships of some miRNAs in this cluster indicate evolution by duplication. HVT miRNAs represent the first example of virus-encoded miRNAs that show evolution by duplication.