Epigenetic Silencing of MicroRNA-126 Promotes Cell Growth in Marek's Disease.

During latency, herpesvirus infection results in the establishment of a dormant state in which a restricted set of viral genes are expressed. Together with alterations of the viral genome, several host genes undergo epigenetic silencing during latency. These epigenetic dysregulations of cellular genes might be involved in the development of cancer. In this context, Gallid alphaherpesvirus 2 (GaHV-2), causing Marek's disease (MD) in susceptible chicken, was shown to impair the expression of several cellular microRNAs (miRNAs). We decided to focus on gga-miR-126, a host miRNA considered a tumor suppressor through signaling pathways controlling cell proliferation. Our objectives were to analyze the cause and the impact of miR-126 silencing during GaHV-2 infection. This cellular miRNA was found to be repressed at crucial steps of the viral infection. In order to determine whether miR-126 low expression level was associated with specific epigenetic signatures, DNA methylation patterns were established in the miR-126 gene promoter. Repression was associated with hypermethylation at a CpG island located in the miR-126 host gene epidermal growth factor like-7 (EGFL-7). A strategy was developed to conditionally overexpress miR-126 and control miRNAs in transformed CD4+ T cells propagated from Marek's disease (MD) lymphoma. This functional assay showed that miR-126 restoration specifically diminishes cell proliferation. We identified CT10 regulator of kinase (CRK), an adaptor protein dysregulated in several human malignancies, as a candidate target gene. Indeed, CRK protein levels were markedly reduced by the miR-126 restoration.

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. Our study provides further confirmation of the involvement of specific DNA methylation patterns in the regulation of vTR expression and vTR importance for virus-induced tumorigenesis.

SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

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.

Test selection for antibody detection according to the seroprevalence level of Schmallenberg virus in sheep.

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.

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.