GaHV-2 ICP22 protein is expressed from a bicistronic transcript regulated by three GaHV-2 microRNAs.

Herpesviruses have a lifecycle consisting of successive lytic, latent and reactivation phases. Only three infected cell proteins (ICPs) have been described for the oncogenic Marek's disease virus (or Gallid herpes virus 2, GaHV-2): ICP4, ICP22 and ICP27. We focus here on ICP22, confirming its cytoplasmic location and showing that ICP22 is expressed during productive phases of the lifecycle, via a bicistronic transcript encompassing the US10 gene. We also identified the unique promoter controlling ICP22 expression, and its core promoter, containing functional responsive elements including E-box, ETS-1 and GATA elements involved in ICP22 transactivation. ICP22 gene expression was weakly regulated by DNA methylation and activated by ICP4 or ICP27 proteins. We also investigated the function of GaHV-2 ICP22. We found that this protein repressed transcription from its own promoter and from those of IE ICP4 and ICP27, and the late gK promoter. Finally, we investigated posttranscriptional ICP22 regulation by GaHV-2 microRNAs. We found that mdv1-miR-M5-3p and -M1-5p downregulated ICP22 mRNA expression during latency, whereas, unexpectedly, mdv1-miR-M4-5p upregulated the expression of the protein ICP22, indicating a tight regulation of ICP22 expression by microRNAs.

Specific transcriptional and post-transcriptional regulation of the major immediate early ICP4 gene of GaHV-2 during the lytic, latent and reactivation phases.

Transcriptional and post-transcriptional mechanisms are involved in the switch between the lytic, latent and reactivation phases of the viral cycle in herpesviruses. During the productive phases, herpesvirus gene expression is characterized by a temporally regulated cascade of immediate early (IE), early (E) and late (L) genes. In alphaherpesviruses, the major product of the IE ICP4 gene is a transcriptional regulator that initiates the cascade of gene expression that is essential for viral replication. In this study, we redefine the infected cell protein 4 (ICP4) gene of the oncogenic Marek's disease virus (MDV or gallid herpesvirus 2) as a 9438 nt gene ended with four alternative poly(A) signals and controlled by two alternative promoters containing essentially ubiquitous functional response elements (GC, TATA and CCAAT boxes). The distal promoter is associated with ICP4 gene expression during the lytic and the latent phases, whereas the proximal promoter is associated with the expression of this gene during the reactivation phase. Both promoters are regulated by DNA methylation during the viral cycle and are hypermethylated during latency. Transcript analyses showed ICP4 to consist of three exons and two introns, the alternative splicing of which is associated with five predicted nested ICP4ORFs. We show that the ICP4 gene is highly and specifically regulated by transcriptional and post-transcriptional mechanisms during the three phases of the GaHV-2 viral cycle, with a clear difference in expression between the lytic phase and reactivation from latency in our model.

Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript.

Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model - the LAT long non-coding RNA (LAT lncRNA) of Marek's disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron - to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3' splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5'-tailed mirtron. We have thus identified the first 5'-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway.

Hyperediting by ADAR1 of a new herpesvirus lncRNA during the lytic phase of the oncogenic Marek's disease virus.

Marek's disease virus, or Gallid herpesvirus 2 (GaHV-2), is an avian alphaherpesvirus that induces T-cell lymphoma in chickens. During transcriptomic studies of the RL region of the genome, we characterized the 7.5 kbp gene of the ERL lncRNA (edited repeat-long, long non-coding RNA), which may act as a natural antisense transcript (NAT) of the major GaHV-2 oncogene meq and of two of the three miRNA clusters. During infections in vivo and in vitro, we detected hyperediting of the ERL lncRNA that appeared to be directly correlated with ADAR1 expression levels. The ERL lncRNA was expressed equally during the lytic and latent phases of infection and during viral reactivation, but its hyperediting increased only during the lytic infection of chicken embryo fibroblasts. We also showed that chicken ADAR1 expression was controlled by the JAK/STAT IFN-response pathway, through an inducible promoter containing IFN-stimulated response elements that were functional during stimulation with IFN-α or poly(I:C). Like the human and murine miR-155-5p, the chicken gga-miR-155-5p and the GaHV-2 analogue mdv1-miR-M4-5p deregulated this pathway by targeting and repressing expression of suppressor of cytokine signalling 1, leading to the upregulation of ADAR1. Finally, we hypothesized that the natural antisense transcript role of the ERL lncRNA could be disrupted by its hyperediting, particularly during viral lytic replication, and that the observed deregulation of the innate immune system by mdv1-miR-M4-5p might contribute to the viral cycle.

Oncogenic Marek's disease herpesvirus encodes an isoform of the conserved regulatory immediate early protein ICP27 generated by alternative promoter usage.

Herpesvirus gene expression is temporally regulated, with immediate early (IE), early (E) and late (L) genes. ICP27, which is involved in post-transcriptional regulation, is the only IE gene product conserved in all herpesviruses. We show here that the ICP27 transcript of the oncogenic Marek's disease virus shares the same polyadenylation signal as the bicistronic glycoprotein K-ICP27 transcript and is regulated by alternative promoter usage, with transcription from its own promoter (pICP27) or that of gK (pgK). The pgK can generate a spliced ICP27 transcript yielding an N-terminal-deleted ICP27 isoform (ICP27ΔN) that, like ICP27, co-localizes with the SR protein in infected cells, but with a diffuse nuclear distribution. The pICP27 includes functional responsive elements (REs) for SP1, AP1 and CREB, is essentially active during the lytic phase and leads to exclusive expression of the native form of ICP27. The alternative promoter, pgK, including active REs for GATA, P53 and CREB, preferentially generates the gK transcript during the lytic phase and the spliced ICP27 transcript (ICP27ΔN) during the latent phase. An analysis of the DNA methylation marks of each promoter showed that pgK was systematically demethylated, whereas pICP27 was methylated during latency and demethylated during the lytic stage. Thus, MDV ICP27 gene expression is dependent on alternative promoters, the usage of which is regulated by DNA methylation, which differs between viral stages.

Marek's disease herpesvirus: a model of vaccine-dependent adaptation.

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes a rapid-onset T-cell lymphoma in chickens. In order to preserve the viability of poultry industry, non sterilizing vaccines have been used since fifty years, preventing lymphoma development but leading to an imperfect control of MD. Vaccination has been accompanied with the increase in virulence of MDV forcing the development of new vaccine formulations. Several loci of MDV genome are variable and have evolved in link with virulence of MDV strains. It has been shown that MDV is in fact constituted by a dynamic population of genetic variants with a distribution linked to viral strain phenotype. In this context, we have shown that CVI988/Rispens vaccine, still the most efficient one against hypervirulent MDV strains, is composed of twenty variants, variable from one batch to another, evolving likely as RNA virus quasispecies.

Marek's disease: Genetic regulation of gallid herpesvirus 2 infection and latency.

Gallid herpesvirus-2 (GaHV-2) is an oncogenic α-herpesvirus that causes Marek's disease (MD), a T cell lymphosarcoma (lymphoma) of domestic fowl (chickens). The GaHV-2 genome integrates by homologous recombination into the host genome and, by modulating expression of viral and cellular genes, induces transformation of latently infected cells. MD is a unique model of viral oncogenesis. Mechanisms implicated in the regulation of viral and cellular genes during GaHV-2 infection operate at transcriptional, post-transcriptional and post-translational levels, with involvement of viral and cellular transcription factors, along with epigenetic modifications, alternative splicing, microRNAs and post-translational modifications of viral proteins. Meq, the major oncogenic protein of GaHV-2, is a viral transcription factor that modulates expression of viral genes, for example by binding to the viral bidirectional promoter of the pp38-pp24/1.8 kb mRNA, and also modulates expression of cellular genes, such as Bcl-2 and matrix metalloproteinase 3. GaHV-2 expresses viral telomerase RNA subunit (vTR), which forms a complex with the cellular telomerase reverse transcriptase (TERT), thus contributing to tumorigenesis, while vTR independent of telomerase activity is implicated in metastasis. Expression of a viral interleukin 8 homologue may contribute to lymphomagenesis. Inhibition of expression of the pro-apoptotic factors JARID2 and SMAD2 by viral microRNAs may promote the survival and proliferation of GaHV-2 latently infected cells, thus enhancing tumorigenesis, while inhibition of interleukin 18 by viral microRNAs may be involved in evasion of immune surveillance. Viral envelope glycoproteins derived from glycoprotein B (gp60 and gp49), as well as glycoprotein C, may also play a role in immune evasion.

Vaccine and oncogenic strains of gallid herpesvirus 2 contain specific subtype variations in the 5' region of the latency-associated transcript that evolve in vitro and in vivo.

Gallid herpesvirus 2 (GaHV-2) is the alphaherpesvirus responsible for Marek's disease (MD), a T-cell lymphoma of chickens. The virulence of the GaHV-2 field strain is steadily increasing, but MD is still controlled by the CVI988/Rispens vaccine. We tried to determine distinguishing traits of the CVI988/Rispens vaccine by focusing on the 5' end region of the latency-associated transcript (5'LAT). It includes a variable number of 60-bp tandem repeats depending on the GaHV-2 strain. By analyzing six batches of vaccine, we showed that CVI988/Rispens consisted of a population of 5'LAT molecular subtypes, all with deletions and lacking 60-bp tandem repeat motifs, with two major subtypes that probably constitute CVI988/Rispens markers. Serial passages in cell culture led to a substantial change in the frequency of CVI988/Rispens 5'LAT subtypes, with non-deleted subtypes harboring up to four 60-bp repeats emerging during the last few passages. Dynamic changes in the distribution of 5'LAT-deleted subtypes were also detected after infection of chickens. By contrast, the 5'LAT region of the oncogenic clonal RB-1B strain, which was investigated at every step from the isolation of the clonal bacmid RB-1B DNA to the isolation of the ovarian lymphoma cell line, consisted of non-deleted 5'LAT subtypes harboring at least two 60-bp repeats. Thus, vaccine and oncogenic GaHV-2 strains consist of specific populations of viral genomes that are constantly evolving in vivo and in vitro and providing potential markers for epidemiological surveys.

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures.

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

The oncogenic microRNA OncomiR-21 overexpressed during Marek's disease lymphomagenesis is transactivated by the viral oncoprotein Meq.

Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression.

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells.

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.

A p53-dependent promoter associated with polymorphic tandem repeats controls the expression of a viral transcript encoding clustered microRNAs.

The tumor suppressor protein p53 plays a role in cellular responses to cancer-initiating events by regulating progress through the cell cycle. Several recent studies have shown that p53 transactivates expression of the members of the proapoptotic microRNA-34 family, which are underexpressed in several cancers. We demonstrate here that the latency-associated cluster of microRNAs (miRNA) encoded by an oncogenic herpesvirus, gallid herpesvirus 2 (GaHV-2), is a direct target of p53. Robust transcriptional activity was induced in three avian cell lines by a sequence mapping 600 base pairs (bp) upstream of the cluster of miRNAs. We found transcription start sites for the pri-miRNA transcript at the 3' end of this transcription-inducing sequence. The promoter has no consensus core promoter element, but is organized into a variable number of tandem repeats of 60-bp harboring p53-responsive elements (RE). The minimal functional construct consists of two tandem repeats. Mutagenesis to change the sequence of the p53 RE abolished transcriptional activity, whereas p53 induction enhanced mature miRNA expression. The identification of a viral miRNA promoter regulated by p53 is biologically significant, because all avirulent GaHV-2 strains described to date lack the corresponding regulatory sequence, whereas all virulent, very virulent, and hypervirulent strains possess at least two tandem repeats harboring the p53 RE.

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo.

BACKGROUND: Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment. RESULTS: We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants. CONCLUSIONS: TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.

The chicken miR-150 targets the avian orthologue of the functional zebrafish MYB 3'UTR target site.

BACKGROUND: The c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3'UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different. RESULTS: We identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3'UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3'UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3'UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3'UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3'UTR. CONCLUSION: Our study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals.

Marek's disease virus microRNA designated Mdv1-pre-miR-M4 targets both cellular and viral genes.

Mdv1-miR-M4 is one of 25 microRNAs (miRNAs) expressed by Marek's disease virus (MDV-1), an oncogenic alphaherpesvirus that induces fatal T-cell lymphoma in chickens. Mdv1-miR-M4 was shown to be the second functional viral ortholog of miR-155, a cellular miRNA that plays a crucial role in several physiological and pathological processes in lymphocyte biology. In this study, we investigated a panel of putative mdv1-miR-M4 targets involved in gene networks affecting both cellular and viral life cycles. Using luciferase reporter assays, we showed that mdv1-miR-M4-5P and miR-155 efficiently targeted a common set of 3' untranslated regions (3'UTR) of six cellular genes (GPM6B, RREB1, c-Myb, MAP3K7IP2, PU.1 and C/EBP). In addition, we also investigated the interactions between mdv1-miR-M4-5P and mdv1-miR-M43P and viral mRNAs encoding UL28 and UL32 in both reporter and western blot assays. Mdv1-miR-M4 specifically inhibited the translation of these two viral proteins, which are involved in the cleavage/packaging of herpesvirus DNA.

Preliminary phylogenetic identification of virulent Chlamydophila pecorum strains.

Chlamydophila pecorum is an obligate intracellular bacterium associated with different pathological conditions in ruminants, swine and koala, which is also found in the intestine of asymptomatic animals. A multi-virulence locus sequence typing (MVLST) system was developed using 19 C. pecorum strains (8 pathogenic and 11 non-pathogenic intestinal strains) isolated from ruminants of different geographical origins. To evaluate the ability of MVLST to distinguish the pathogenic from the non-pathogenic strains of C. pecorum, the sequences of 12 genes were analysed: 6 potential virulence genes (ompA, incA, incB, incC, mip and copN), 5 housekeeping genes (recA, hemD, aroC, efp, gap), and the ORF663 gene encoding a hypothetical protein (HP) that includes a variant 15-nucleotides coding tandem repeat (CTR). MVLST provided high discriminatory power (100%) in allowing to distinguish 6 of 8 pathogenic strains in a single group, and overall more discriminatory than MLST targeting housekeeping genes. ompA was the most polymorphic gene and the phylogenetic tree based only on its sequence differentiated 4 groups with high bootstrap values. The number of CTRs (rich in serine, proline and lysine) in ORF663 detected in the pathogenic strains was generally lower than that found in the intestinal strains. MVLST appears to be a promising method for the differential identification of virulent C. pecorum strains, and the ompA, incA and ORF663 genes appear to be good molecular markers for further epidemiological investigation of C. pecorum.

Vaccination against Marek's disease reduces telomerase activity and viral gene transcription in peripheral blood leukocytes from challenged chickens.

We investigated whether telomerase activity and viral gene transcription were associated with protection against the RB-1B strain of Marek's disease virus (MDV) in chickens vaccinated with Rispens CVI988 or the herpes virus of turkey (HVT). Telomerase activity in peripheral blood leukocytes (PBLs) seemed to be an appropriate marker of lymphoma and levels of viral transcription were correlated with the virulence of MDV strains. Vaccinated protected birds had lower levels of telomerase activity and RB-1B viral gene transcription than unvaccinated chickens infected with RB-1B. The decrease in RB-1B viral transcription was more marked in chickens vaccinated with CVI988 than in those vaccinated with HVT. Indeed, RB-1B viral transcription was not detectable after 14 days post-challenge. In conclusion, telomerase activity and gene transcription in challenge MDV strains are potential new reliable criteria of protection in vaccinated chickens.

Variations in the H/ACA box sequence of viral telomerase RNA of isolates of CVI988 Rispens vaccine.

The use of the complete DNA sequence for the Marek's disease virus (MDV) serotype 1 vaccine strain CVI988 Rispens in comparative genomic studies with virulent strains of MDV has revealed the presence of a number of insertions, deletions and single-nucleotide polymorphisms. In this study, we investigated a SNP in the H/ACA box of the viral RNA subunit of telomerase (vTR). We sequenced vTR from four different batches of CVI988 vaccine originating from a single commercial company. The A-to-G mutation defining the SNP in the H/ACA box of CVI988 vTR was present in only some of the batches. Thus, although this mutation affects CVI988 vTR function, it is not shared by all CVI988 isolates and may be a stochastic rather than causative event in CVI988 attenuation.

Extraintestinal pathogenic Escherichia coli strains of avian and human origin: link between phylogenetic relationships and common virulence patterns.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of human and avian origin show similarities that suggest that the avian strains potentially have zoonotic properties. However, the phylogenetic relationships between avian and human ExPEC strains are poorly documented, so this possibility is difficult to assess. We used PCR-based phylotyping and multilocus sequence typing (MLST) to determine the phylogenetic relationships between 39 avian pathogenic E. coli (APEC) strains of serogroups O1, O2, O18, and O78 and 51 human ExPEC strains. We also compared the virulence genotype and pathogenicity for chickens of APEC strains and human ExPEC strains. Twenty-eight of the 30 APEC strains of serogroups O1, O2, and O18 were classified by MLST into the same subcluster (B2-1) of phylogenetic group B2, whereas the 9 APEC strains of serogroup O78 were in phylogenetic groups D (3 strains) and B1 (6 strains). Human ExPEC strains were closely related to APEC strains in each of these three subclusters. The 28 avian and 25 human strains belonging to phylogenetic subcluster B2-1 all expressed the K1 antigen and presented no significant differences concerning the presence of other virulence factors. Moreover, human strains of this phylogenetic subcluster were highly virulent for chicks, so no host specificity was identified. Thus, APEC strains of serotypes O1:K1, O2:K1, and O18:K1 belong to the same highly pathogenic clonal group as human E. coli strains of the same serotypes isolated from cases of neonatal meningitis, urinary tract infections, and septicemia. These APEC strains constitute a potential zoonotic risk.

Involvement of the oncoprotein c-Myc in viral telomerase RNA gene regulation during Marek's disease virus-induced lymphomagenesis.

Marek's disease virus (MDV) is an alphaherpesvirus that induces a highly malignant T-lymphoma in chickens. The viral genome encodes two identical copies of a viral telomerase RNA subunit (vTR) that exhibits 88% sequence identity to its chicken ortholog chTR. The minimal telomerase ribonucleoprotein complex consists of a protein subunit with reverse transcriptase activity (TERT) and an RNA subunit (TR). The active complex compensates for the progressive telomere shortening that occurs during mitosis and is involved in the cell immortalization process. We show here that the upregulation of telomerase activity is associated with an increase in vTR gene expression in chickens infected with the highly oncogenic MDV strain RB-1B. A comparative functional analysis of the viral and chicken TR promoters, based on luciferase reporter assays, revealed that the vTR promoter was up to threefold more efficient than the chTR promoter in avian cells. We demonstrated, by directed mutagenesis of the vTR promoter region, that the stronger transcriptional activity of the vTR promoter resulted largely from an E-box located two nucleotides downstream from the transcriptional start site of the vTR gene. Furthermore, transactivation assays and chromatin immunoprecipitation assays demonstrated the involvement of the c-Myc oncoprotein in the transcriptional regulation of vTR. Finally, an Ets binding site was specifically implicated in the transcriptional regulation of vTR in the MDV-transformed lymphoblastoid cell line MSB-1.