Evaluation of the Revogene® GBS DS assay performance for the intrapartum screening of group B streptococcus in comparison with intrapartum culture.

Prevention of perinatal Group B Streptococcus (GBS) transmission is crucial in our effort to prevent Early-onset GBS disease. Here, we established the performance of the Revogene GBS DS assay for the detection of group B streptococcus on intrapartum vaginal samples in a laboratory environment using a prospective noninterventional study design. Intrapartum vaginal swabs were enriched using a selective culture method which served as study reference method. Overall, 119 patients were enrolled with an antenatal and intrapartum Group B Streptococcus colonization prevalence of 12.9% and 11.8%, respectively. Compared to intrapartum culture, the Revogene GBS DS assay had a sensitivity of 92.9% and a specificity of 99.1%, while the antenatal culture displayed a sensitivity 78.6% of and specificity of 96.2%. The Revogene GBS DS assay displayed an acceptable performance according to the European Group B Streptococcus consensus recommendations. Complementary studies in clinical practice are needed to confirm these findings.

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay.

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Evaluation of the Abbott RealTime quantitative CMV and EBV assays using the maxCycle protocol in a laboratory automation context.

Real-time PCR are often used for the diagnosis and monitoring of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections in susceptible populations. In this context, we evaluated the analytical performances of the Abbott RealTime CMV/EBV maxCycle protocol automated on the m2000 platform (Abbott). It was compared to our routinely-used procedure consisting of a NucleoMag® DNA extraction automated on a STARlet platform followed by manually processed CMV and EBV quantitative real-time PCR (Diagenode). In this study, we showed that both EBV assays exhibited a similar sensitivity but with a better precision for the EBV Abbott RealTime assay. For the CMV performances, the Abbott assay was more sensitive and more precise than our routine method. The use of WHO International Standards also indicated a slight underestimation of the viral loads (-0.25 log10 IU/mL and -0.21 log10 IU/mL for CMV and EBV assays respectively) while these were rather overestimated with the Starlet/Diagenode method (0.48 log10 IU/mL and 0.19 log10 IU/mL for CMV and EBV assays respectively). These trends were confirmed using relevant whole-blood clinical samples and external quality controls. The workflows were also compared and we highlighted a significant technician hands-on time reduction (-63%) using the Abbott CMV/EBV maxCycle automated protocol.

Absence of varicella zoster virus reactivation after infliximab administration for plaque psoriasis.

BACKGROUND: Herpes zoster (HZ) in patients receiving tumor necrosis factor (TNF) antagonists may be more severe and the incidence seems increased. The influence of TNF antagonists on varicella zoster virus (VZV) reactivation is unknown. OBJECTIVE: To prospectively search in a pilot study for VZV DNA in sequential blood samples before and after infliximab administration. SETTING: University medical center. SUBJECTS AND METHODS: Blood samples of six patients with longstanding and severe plaque psoriasis were taken on day 1 (before infliximab administration) and on days 2, 7, 21 and 42 for the determination of VZV viremia by ORF21 real-time polymerase chain reaction. Patients with varicella, HZ and normal subjects were included as controls. RESULTS: None of the six patients presented VZV viremia at any of the time points. High-load viremia was present during varicella, low-load viremia in some HZ patients and no viremia in the control patients. LIMITATIONS: Small number of patients. CONCLUSIONS: In this pilot study, infliximab did not reactivate VZV and did not induce subclinical VZV viremia.

ORF9p phosphorylation by ORF47p is crucial for the formation and egress of varicella-zoster virus viral particles.

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

Transfection of immortalized keratinocytes by low toxic poly(2-(dimethylamino)ethyl methacrylate)-based polymers.

Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.

How to monitor NF-kappaB activation after photodynamic therapy.

The nuclear factor-kappa B (NF-kappaB) is a multipotent factor involved in many cellular processes such as inflammation, immune response and embryonic development and it can be activated by a large number of stimuli. Consequently, this transcription factor plays a pivotal role in many natural processes but also in different pathologies. For several years, photodynamic therapy (PDT) has emerged as an attractive alternative approach for the treatment of different affections involving various forms of cancer and an increasing number of reports have highlighted the activation of the NF-kappaB following PDT treatment. Furthermore, it has been shown that the mechanism of activation of the NF-kappaB as well as its target genes depends on the nature of the photosensitizers and the cell type used. As this transcription factor is known to be a key regulator of the immune response but also controls cell survival and proliferation, it is important to assess its activation status and its impact on the target genes. In this review, we will present different techniques allowing identification of the activation status of this factor, from the degradation of its inhibitor in the cytoplasm to its ability to induce the expression of a reporter gene under the control of a target promoter. As a working model we will present results obtained from a 5-aminolevulinic acid-PDT treatment on cervix adenocarcinoma cells.

The varicella-zoster virus immediate-early 63 protein affects chromatin-controlled gene transcription in a cell-type dependent manner.

BACKGROUND: Varicella Zoster Virus Immediate Early 63 protein (IE63) has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. RESULTS: In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-kappaB dependent genes such as IL-8, ICAM-1, and IkappaBalpha, it modulates transcription of these genes upon TNFalpha induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. CONCLUSION: While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-kappaB dependent genes by the accelerated resynthesis of the inhibitor IkappaBalpha.

Varicella-zoster virus modulates NF-kappaB recruitment on selected cellular promoters.

Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-alpha was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-alpha; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-kappaB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-alpha; and (iv) VZV induces the nuclear accumulation of the NF-kappaB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-alpha by strongly reducing NF-kappaB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-kappaB binding and the expression of target genes. Targeting NF-kappaB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.

Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.

Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex.

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans-repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151-213 were shown to be essential to the trans-repressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors.

Regions of the varicella-zoster virus open reading frame 63 latency-associated protein important for replication in vitro are also critical for efficient establishment of latency.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

Phosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity.

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level.