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.

Host-dependence of in vitro reassortment dynamics among the Sathuperi and Shamonda Simbuviruses.

Orthobunyaviruses are arboviruses (Arthropod Borne Virus) and possess multipartite genomes made up of three negative RNAs corresponding to the small (S), medium (M) and large (L) segments. Reassortment and recombination are evolutionary driving forces of such segmented viruses and lead to the emergence of new strains and species. Retrospective studies based on phylogenetical analysis are able to evaluate these mechanisms at the end of the selection process but fail to address the dynamics of emergence. This issue was addressed using two Orthobunyaviruses infecting ruminants and belonging to the Simbu serogroup: the Sathuperi virus (SATV) and the Shamonda virus (SHAV). Both viruses were associated with abortion, stillbirth and congenital malformations occurring after transplacental transmission and were suspected to spread together in different ruminant and insect populations. This study showed that different viruses related to SHAV and SATV are spreading simultaneously in ruminants and equids of the Sub-Saharan region. Their reassortment and recombination potential was evaluated in mammalian and in insect contexts. A method was set up to determine the genomic background of any clonal progeny viruses isolated after in vitro coinfections assays. All the reassortment combinations were generated in both contexts while no recombinant virus was isolated. Progeny virus populations revealed a high level of reassortment in mammalian cells and a much lower level in insect cells. In vitro selection pressure that mimicked the host switching (insect-mammal) revealed that the best adapted reassortant virus was connected with an advantageous replicative fitness and with the presence of a specific segment.

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.

Modelling the evolution of Schmallenberg virus seroprevalence in a sheep flock after natural infection.

Modelling the long-term seroprevalence evolution against Schmallenberg virus (SBV) is of first interest to plan vaccination strategies and to predict viral resurgence. The objectives of this study were first to estimate the duration of colostral immunity and the persistence of active immunity in a sheep flock that encountered two episodes of natural SBV infection and then to model the evolution of SBV seroprevalence by considering immune status as well as zootechnical variables. The entire sheep flock of the University of Namur composed by around 400 ewes producing 600 lambs a year and characterized by an annual 24.0% renewal rate was used for this study from January 2012 until December 2016. Antibody titers were estimated by performing Virus Neutralization Test (VNT) from blood and colostrum samples collected in lambs and adult sheep. Colostral antibodies against SBV of lambs born to seropositive ewes were detectable during four months. A significant increase of anti-SBV antibody titers was observed in lambs' serum as well as in ewes' colostrum between the time of first viral episode (2011) and the time of SBV reemergence (2012) suggesting a booster effect of viral resurgence on immune status. In naturally SBV infected adult sheep, the active immunity was estimated to last at least four years. These results combined with flock management data allowed to develop a mathematical model to predict the evolution of SBV seroprevalence at a herd scale. The accuracy of this model was assessed by VNT experiment performed at the end of the study. By applying this model to the sheep flock of the University of Namur, it was estimated that an annual 24.0% renewal rate led to total seronegativity, and so high susceptibility to viral resurgence, in 50 months after time of last natural infection. The third SBV episode detected in this research sheep flock within the expected time demonstrated huge within-flock susceptibility.

S segment variability during the two first years of the spread of Schmallenberg virus.

A large sheep flock screened over a two-year period showed active spreading of Schmallenberg virus (SBV) during the summers of 2011 and 2012. Transplacental infections were observed during the two associated lambing periods (the winters of 2012 and 2013). Analysis of small (S) segment sequences of 38 SBV-positive samples, collected during periods of viral spreading and lambing revealed intra-herd sequences diversity and sub-consensus variability occurring after transplacental infections. In comparison with the nucleoprotein (N), which appeared to be conserved, the non-structural protein (NSs) showed the highest level of variability at the time of viral emergence and over the two-year analysis period.

Schmallenberg virus infection of ruminants: challenges and opportunities for veterinarians.

In 2011, European ruminant flocks were infected by Schmallenberg virus (SBV) leading to transient disease in adult cattle but abortions and congenital deformities in calves, lambs, and goat kids. SBV belonging to the Simbu serogroup (family Bunyaviridae and genus Orthobunyavirus) was first discovered in the same region where bluetongue virus serotype 8 (BTV-8) emerged 5 years before. Both viruses are transmitted by biting midges (Culicoides spp.) and share several similarities. This paper describes the current knowledge of temporal and geographical spread, molecular virology, transmission and susceptible species, clinical signs, diagnosis, prevention and control, impact on ruminant health, and productivity of SBV infection in Europe, and compares SBV infection with BTV-8 infection in ruminants.

Characterization of messenger RNA termini in Schmallenberg virus and related Simbuviruses.

Schmallenberg virus (SBV) is an emerging arbovirus infecting ruminants in Europe. SBV belongs to the Bunyaviridae family within the Simbu serogroup. Its genome comprises three segments, small (S), medium (M) and large (L), that together encode six proteins and contain NTRs. NTRs are involved in initiation and termination of transcription and in genome packaging. This study explored the 3' mRNA termini of SBV and related Simbuviruses. In addition, the 5' termini of SBV messenger RNA (mRNA) were characterized. For the three SBV segments, cap-snatching was found to initiate mRNA transcription both in vivo and in vitro. The presence of extraneous nucleotides between host RNA leaders and the viral termini fits with the previously described prime-and-realign theory. At the 3' termini, common features were identified for SBV and related Simbuviruses. However, different patterns were observed for the termini of the three segments from the same virus type.

Schmallenberg virus among female lambs, Belgium, 2012.

Reemergence of Schmallenberg virus (SBV) occurred among lambs (n = 50) in a sheep flock in Belgium between mid-July and mid-October 2012. Bimonthly assessment by quantitative reverse transcription PCR and seroneutralization demonstrated that 100% of lambs were infected. Viremia duration may be longer in naturally infected than in experimentally infected animals.

In vivo and in vitro identification of a hypervariable region in Schmallenberg virus.

Detected for the first time in 2011, Schmallenberg virus (SBV) is an orthobunyavirus of the Simbu serogroup that caused a large outbreak in European ruminants. In a tight time frame, data have been obtained on SBV epidemiology and the clinical pictures associated with this new viral infection, but little information is available on the molecular biology of SBV. In this study, SBV sequence variability was characterized from the central nervous system of two stillborn lambs in a naturally infected herd. A hypervariable region (HVR) was detected in the N-terminal region of the SBV Gc glycoprotein through sequencing and analysis of the two full-length genomes representative of intra-herd SBV dissemination. In vitro growth assays coupled with full-length genome sequencing were performed on the two isolates after successive cellular passages, showing an in vitro adaptation of SBV and mutation accumulation inside the HVR in the absence of immune selective pressure.

The impact of naturally-occurring, trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep.

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the 'over-wintering' of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008. The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n=4); (2) fetal malformations suggestive of BTV infection (n=10); (3) positive PCR results from red blood cells in-lambs (n=7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n=9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.

Interspecific recombination between two ruminant alphaherpesviruses, bovine herpesviruses 1 and 5.

Homologous recombination between different species of alphaherpesviruses has been described between herpes simplex viruses 1 and 2 but has not yet been observed between other alphaherpesviruses. In the present study we chose to assess to what extent in vitro recombination can occur between members of a well-defined group of closely related viruses such as ruminant alphaherpesviruses. At 24 h after infection of epithelial bovine kidney cells with a double-deleted mutant of bovine herpesvirus 1 (BoHV-1) (containing green fluorescent protein and red fluorescent protein genes) and different ruminant alphaherpesviruses, four types of progeny viruses were detected and distinguished according to their phenotype. Frequent recombination events between identical or different strains of BoHV-1 were observed (up to 30%), whereas only two BoHV-1/BoHV-5 recombinants were identified, and no recombinants between BoHV-1 and less closely related caprine and cervine herpesviruses were detected. Restriction analysis of the genomes of the two BoHV-1/BoHV-5 recombinants showed different genetic backgrounds. One possessed a restriction pattern close to BoHV-1, whereas the other one was close to BoHV-5. This exhaustive analysis of each combination of coinfection in a unique situation of five closely related alphaherpesviruses revealed the importance of a high degree of genetic relatedness and similar parental virus growth kinetics for successful interspecific recombination.