DETECTION OF HHV-8 IN AUTOPSY SAMPLES FROM AIDS PATIENTS

Human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is the most recently identified human herpesvirus. A key question regarding HHV-8 is the location of infected cells within HHV-8 seropositive individuals. Outside of tumor tissues, HHV-8 viral proteins have been detected in saliva, circulating B cells, and semen of some, but not all HHV-8 seropositive individuals. HHV-8 is the causative agent of Kaposi's sarcoma (KS) and is associated with two other distinct proliferative disorders: primary effusion lymphoma and some forms of multicentric Castleman's disease. To better understand viral infection including the cellular targets of infection, we have begun a systematic screening of autopsy tissues from HHV-8 seropositive men who died with AIDS. Using immunohistochemistry (IHC), my goals were to determine reservoirs of HHV-8 infection and latency in organ tissues, determine the type of viral infection (lytic and/or latent) of each tissue type, and attempt to identify the infected cell type. In this report, using IHC, we document the presence of HHV-8 infected cells in several organs including kidney, lung, liver, and gastrointestinal tract samples from the Multicenter AIDS Cohort Study (MACS). Both lytic and latent infections have been detected and the infected cells appear to consist of both immune and non-immune cells. These results demonstrate the ability of HHV-8 to establish infections in various organs which may affect the pathogenesis of the virus in infected individuals. Kaposi's sarcoma is currently a major public health concern, as it is the most common malignancy found in individuals with AIDS and iatrogenic KS is a key concern in the field of solid-organ transplantation. This study will attempt to identify reservoirs of HHV-8 infection within the body in order to better understand the biology of HHV-8 in infected individuals, and the role HHV-8 plays in disease pathogenesis

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.</p

New perspectives on respiratory syncytial virus surveillance at the national level: lessons from the COVID-19 pandemic

EditorialLearning from the COVID-19 pandemic and considering the effects of this pandemic, we provide recommendations that can guide towards sustainable RSV surveillance with the potential to be integrated into the broader perspective of respiratory surveillance.info:eu-repo/semantics/publishedVersio

New perspectives on respiratory syncytial virus surveillance at the national level:lessons from the COVID-19 pandemic

Learning from the COVID-19 pandemic and considering the effects of this pandemic, we provide recommendations that can guide towards sustainable RSV surveillance with the potential to be integrated into the broader perspective of respiratory surveillance. https://bit.ly/40TsO0

Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection

Oligonucleotides used in PCR, site-directed mutagenesis, and ChIP assays.

<p>Oligonucleotides used in PCR, site-directed mutagenesis, and ChIP assays.</p

Role of HCV-induced cellular kinases on TGF-β1 promoter activation.

<p>A) Mock- and HCV-infected cells were transfected with 500 ng of phTG1 and phTG5 TGF-β1 promoter-luciferase reporter. At 36 h posttransfection cells were serum starved for 4 h and treated with inhibitors against p38 MAPK (SB203580, 10 µM), JNK (SP600125, 30 µM), PI3K (LY294002, 50 µM), Src (SU6656, 10 µM), JAK 2/3 (AG490, 100 µM), and MEK1/2 (UO126, 20 µM) for 12 h. Cellular lysates were subjected to dual-luciferase reporter assay. The values represent the means standard deviations of three independent experiments performed in triplicate. *denotes p<0.05 compared to mock cells. **denotes p<0.05 compared to HCV-infected mock-treated cells. B) Mock- and HCV-infected cells were treated with kinase inhibitors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056367#s2" target="_blank">Materials and Methods</a>. Total cellular RNA was extracted and TGF-β1 mRNA was analyzed using TGF-β1 specific primers and SYBR green fluorescent dye. The values represent the means standard deviations of three independent experiments performed in triplicate. *denotes p<0.05 compared to mock cells. **denotes p<0.05 compared to HCV-infected mock-treated cells. C) Mock- and HCV-infected cells were treated with kinase inhibitors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056367#s2" target="_blank">Materials and Methods</a>. Cells were subjected to growth inhibition assay using CytoToxONE homogenous membrane integrity assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056367#s2" target="_blank">Materials and Methods</a>.</p