11 research outputs found
Modelling the evolution of Schmallenberg virus seroprevalence in a sheep flock after natural infection.
Role of DNA Methylation and CpG Sites in the Viral Telomerase RNA Promoter during Gallid Herpesvirus 2 Pathogenesis
Gallid herpesvirus type 2 (GaHV-2) is an oncogenic alphaherpesvirus that induces malignant T-cell lymphoma in chicken. GaHV-2 encodes a viral telomerase RNA subunit (vTR) that plays a crucial role in virus-induced tumorigenesis, enhances telomerase activity, and possesses functions independent of the telomerase complex. vTR is driven by a robust viral promoter, highly expressed in virus-infected cells, and regulated by two c-Myc response elements (c-Myc REs). The regulatory mechanisms involved in controlling vTR and other genes during viral replication and latency remain poorly understood but are crucial to understanding this oncogenic herpesvirus. Therefore, we investigated DNA methylation patterns of CpG dinucleotides found in the vTR promoter and measured the impact of methylation on telomerase activity. We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within c-Myc REs showed specific changes in methylation after in vitro reactivation and in infected animals over time. Promoter reporter assays indicated that one of the c-Myc REs is involved in regulating vTR transcription, and that methylation strongly influenced vTR promoter activity. To study the importance of the CpG sites found in c-Myc REs in virus induced tumorigenesis, we generated recombinant virus containing mutations in CpG sites of c-Myc REs together with the revertant virus by two-step Red-mediated mutagenesis. Introduced mutations in the vTR promoter did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication of the mutant virus in infected chickens was severely impaired, and tumor formation completely abrogated. Our data provides an in-depth characterization of c-Myc oncoprotein REs and the involvement of DNA methylation in transcriptional regulation of vTR.
IMPORTANCE Previous studies demonstrated that telomerase RNAs possess functions that promote tumor development independent of the telomerase complex. vTR is a herpesvirus-encoded telomerase RNA subunit that plays a crucial role in virus-induced tumorigenesis and is expressed by a robust viral promoter that is highly regulated by the c-Myc oncoprotein binding to the E-boxes. Here, we demonstrated that the DNA methylation patterns in the functional c-Myc response elements of the vTR promoter change upon reactivation from latency, and that demethylation strongly increases telomerase activity in virus-infected cells. Moreover, the introduction of mutation in the CpG dinucleotides of the c-Myc binding sites resulted in decreased vTR expression and complete abrogation of tumor formation. ation patterns in the regulation of vTR expression and vTR importance for virus induced tumorigenesis
Test selection for antibody detection according to the seroprevalence level of Schmallenberg virus in sheep
<div><p>Schmallenberg virus (SBV), initially identified in Germany in 2011, spread rapidly throughout Europe causing significant economic losses in ruminant livestock. The ability to correctly detect emerging and re-emerging diseases such as SBV with reliable tests is of high importance. Firstly, we tested diagnostic performance, specificity, and sensitivity of three different assays used in SBV antibody detection using control sheep samples of determined status. After obtaining the results from the control samples, we assessed the potential of the assays to detect previously infected animals in field situations. The samples were investigated using IDEXX Schmallenberg virus Antibody Test Kit, ID Screen Schmallenberg virus Competition Multi-species ELISA and Serum Neutralisation Test (SNT). Analysis of control samples revealed that SNT was the most suitable test, which was therefore used to calculate concordance and test performance for the two other ELISA tests. To evaluate whether different assay performances had an impact under field conditions, sheep samples from two different contexts were tested: the emergence of SBV in Ireland and the re-emergence of SBV in Belgium. Comparing the results obtained from different assays to the non-reference standard assay SNT, we showed considerable differences in estimates of their sensitivity to detect SBV antibodies and to measure seroprevalence of the sheep flocks. Finally, a calculation of the number of randomly selected animals that needs to be screened from a finite flock, showed that SNT and ID.Vet are the most suitable to detect an introduction of the disease in low seroprevalence situations. The IDEXX ELISA test was only able to detect SBV antibodies in a higher seroprevalence context, which is not optimal for monitoring freedom of disease and surveillance studies.</p></div
Differences in seroprevalence estimates from samples obtained after Schmallenberg virus (SBV) infection in two areas of Ireland (I) and in Belgium (II) using two ELISA assays and the Serum Neutralisation Test (SNT) with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).
<p>Differences in seroprevalence estimates from samples obtained after Schmallenberg virus (SBV) infection in two areas of Ireland (I) and in Belgium (II) using two ELISA assays and the Serum Neutralisation Test (SNT) with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).</p
Diagnostic performances of three serological tests used to detect Schmallenberg virus antibodies from samples of known status with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).
<p>Diagnostic performances of three serological tests used to detect Schmallenberg virus antibodies from samples of known status with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).</p
Diagnostic differences between ELISA tests used for samples collected in Ireland (April 2013) and in Belgium (September 2016) with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).
<p>Diagnostic differences between ELISA tests used for samples collected in Ireland (April 2013) and in Belgium (September 2016) with inclusion of suspect/doubtful in the negative results (a) and in the positive results (b).</p
The theoretical fraction of random animals required to be tested from finite sheep flock (n = 200) in order to demonstrate that the population is SBV infected.
<p>Calculations with sensitivities and specificities (Se/Sp) obtained from total control testing from two scenarios: <b>(A)</b> all suspect/doubtful reads were included in positive results, <b>(B)</b> all suspect/doubtful reads were included in negative results. The results for the given theoretical prevalence are shown for Serum Neutralisation Test (SNT), ID.Vet test and IDEXX test. A minimum number of positive animals needed to be recognised from all tested animals in order to conclude that the population is SBV infected is indicated in square brackets for each test. NA—an error if the calculations could not be achieved within the limits of the population and/or maximum sample size.</p