Human Herpesvirus 6

DNA and RNA were isolated from clinical samples and Nthy-ori3-1 cells as described [27] (link). All RNA preparations were devoid of DNA, as assured by multiple DNase digestions and lack of amplification in PCR reactions where retrotranscription (RT) had been omitted [14] (link). HHV-6 DNA presence and load were analyzed by PCR and real time quantitative (qPCR) specific for the U94 and U42 genes [27] (link), and samples were considered positive when 1 µg of cell DNA harbored more than 100 copies of viral DNA [27] (link). Amplification of the house-keeping human RNase P gene was used as a control. All clinical samples were analyzed in a randomized and blinded fashion. In addition, 15/28 control and 21/34 HT FNAs, when there was enough material to repeat the analysis, were tested again in a randomized and blinded fashion at a distant time from the first analyses.
HHV-6 variant A or B identification was obtained by restriction enzyme digestion with HindIII enzyme of the U31 nested PCR amplification product, as reported previously [26] (link). Digestion products were then visualized on ethidium bromide stained agarose gel after electrophoresis migration.
Virus transcription was assessed by PCR or qPCR after retrotranscription (RT-PCR, RT-qPCR), determining the presence of lytic (U42, U22) or latent (U94 in the absence of U42) mRNAs, as previously reported [27] (link). The sensitivity of the used PCRs was similar for all genes, detecting as few as 100 copies of target sequence.
Cell fractions derived by immunomagnetic separation of FNAs were characterized by RT-PCR specific for leukocytes transcripts (respectively CD45, CD3), using serial dilutions of cDNA template, corresponding to amounts of total extracted RNA ranging from 100 ng to 1 pg. Primers and PCR conditions for CD3 and CD45 were previously reported [53] (link), [54] (link), and amplification reactions were carried out for 30 cycles. In each assay the cDNAs obtained from JJhan T cells or Nthy-ori3-1 thyroid cells were also included as positive and negative controls respectively. Amplification of the house-keeping β-actin gene was used as a control.

Genomic DNA of HHV in the aqueous humour and vitreous fluids was measured through the use of two independent PCR assays: (1) a qualitative multiplex PCR and (2) a quantitative real-time PCR. The result analysis for the PCR is shown in fig 1.
DNA was extracted from samples using an E21 virus minikit (Qiagen, Valencia, CA) installed on a Robotic workstation for automated purification of nucleic acids (BioRobot E21, Qiagen). The multiplex PCR was designed to qualitatively measure genomic DNA of eight human herpes viruses, that is, herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), Varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpes virus type 6 (HHV6), type 7 (HHV7) and type 8 (HHV8). The PCR was performed using a LightCycler (Roche, Switzerland). Primers and probes of HHV1–8 and the PCR conditions have been described previously.8 (link) Specific primers for the virus were used with Accuprime Taq (Invitrogen, Carlsbad, CA). Products were subjected to 40 cycles of PCR amplification. Hybridisation probes were then mixed with the PCR products. Subsequently, real-time PCR was performed only for the human herpes virus, with the genomic DNA detected by multiplex PCR (fig 1). The real-time PCR was performed using Amplitaq Gold and the Real-Time PCR 7300 system (ABI, Foster City, CA). The sequence of the HHV1–8 primers and probes are shown in table 1. The primers of the viruses and the PCR conditions have been described in previous reports.9 (link)^–13 (link) Our research group has also previously reported the primers of the sequences for VZV.14 (link) All of the products obtained were subjected to 45 cycles of PCR amplification. The value of viral copy number in the sample was considered to be significant, when more than 50 copies/tube (5×103 (link)/ml) were observed.

The reference sera were analyzed for antibodies against selected HHV antigens (Table 1) by species-specific Monoplex and Multiplex Serology, as described previously [19 (link)]. Briefly, HHV GST-tag fusion proteins were loaded onto glutathione casein-coated fluorescence-labelled polystyrene beads (COOH-beads xMAP Technology Microspheres, Luminex Corp. Austin, Texas, USA) by in situ affinity purification from lysate. Up to 100 bead sets are distinguishable by the Luminex flow cytometer via different ratios of two fluorescent dyes within the polystyrene microspheres. Loading each antigen onto a specific bead set enables simultaneous measurement of antibodies against different antigens within one reaction vessel.
Detection of bound primary antibodies from serum took place with a biotinlyated goat-α-human IgM/IgG/IgA secondary antibody (1:1000, #109-065-064, Jackson Immunoresearch, West Grove, PA, USA) and subsequent incubation with streptavidin-R-phycoerythrin (1:750, PE-Streptavidin Conjugate, MOSS Inc., Pasadena, CA, USA) as reporter dye. Median Fluorescence Intensities (MFI) from at least 100 detected beads per bead set (e.g. antigen) were calculated for each serum. Monoplex Serology was conducted for each HHV species-specific assay in an individual experiment only comprising the species-specific antigens and GST-tag antigen for background subtraction in dilutions 1:100 and 1:1000. Optimal serum dilution was 1:1000 with the exception of VZV (1:100). In addition, performance of HHV Monoplex Serology assays were assessed in multiplex format by combining them with various pathogen-specific Monoplex Serology assays (e.g. human herpes viruses 6–8, human polyomaviruses, human papillomaviruses, human hepatitis B and C viruses) into a Multiplex Serology panel.

Patients
This was a descriptive, observational, retrospective analysis using data from the Microbiology Laboratory of Mohammed VI University Hospital of Marrakech. This study covered a period of 34 months between March 2018 and December 2020 and included all patients (adults and children) hospitalized in different intensive care units of Mohammed VI University Hospital of Marrakech who underwent a multiplex FilmArray® meningitis/encephalitis panel (FA MEP) PCR on a cerebrospinal fluid (CSF) sample. The Mohammed VI University Hospital of Marrakech is the only tertiary hospital in the region that receives patients referred from the south of Morocco.
This study included all pediatric and adult patients with suspected severe acute meningeal infection for whom the clinician had ordered meningeal PCR for an infectious etiology. Cerebrospinal fluid was collected by lumbar or transfontanellar puncture [1 (link),2 (link)].
Methods
After receiving the samples, the culture was performed first with the cytobacteriological examination, followed by a multiplex PCR search for microbial agents. The culture was conducted for all samples, while the PCR was only performed based on microbiological criteria (high cytology, hypoglycorrhachia, and high CSF proteins) or at the request of the clinician.
The cytobacteriological and biochemical study of the CSF was performed according to the standard procedures of the French Society of Microbiology. Protein and glucose concentrations were measured by immunoturbidimetric assay using the Roche/Hitachi Cobas 311 system (Basel, Switzerland) with calibrators and internal controls supplied by Roche and according to the manufacturer’s recommendations.
FilmArray® Meningitis/Encephalitis Panel (FA MEP) ​​​​TestApproximately 200 μL of CSF was used for the BioFire® FilmArray® meningitis/encephalitis panel PCR panel according to the manufacturer’s recommendations. This multiplex real-time PCR assay consists of an automated nucleic acid assay based on acid extraction, reverse transcription, and nucleic acid amplification, with an average result turnaround time of 75 minutes per run and a handling time of less than five minutes. Transportation of samples from clinical services to the laboratory typically took about 15 minutes.
This PCR detects 14 pathogens: Cytomegalovirus, Enterovirus, Herpes simplex virus (HSV) 1/2, HHV-6, Parechovirus, Varicella-zoster virus, Cryptococcus neoformans/gattii, Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae.
Statistical analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 23.0 (IBM SPSS Statistics, Armonk, NY, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Statistical comparisons were performed using the chi-square test. A probability value (p) of less than 0.05 was considered statistically significant.

The present prospective, cross-sectional study was conducted in three centers in Israel: the Bnai Zion, Hillel Yaffe, and Schneider Children’s Medical Centers. Twenty-seven children aged 0.5–18 years with pseudotumor cerebri were prospectively studied. Ethical approval was obtained from each hospital, and all participants or caregivers provided written informed consent. All children were examined by a pediatric neurologist and ophthalmologist. The data collected included clinical presentation, imaging, treatment, ophthalmic findings, and CSF analysis. The CSF was taken as part of the PTC diagnosis at the time of measuring the opening pressure. Using the ALLPLEX^TM meningitis panel, real-time PCR was used to test for the presence of 12 viruses that are known to cause infections of the central nervous system: cytomegalovirus, Epstein–Barr virus, herpes simplex virus 1 and 2, human herpesvirus 6 and 7, varicella–zoster virus, adenovirus, parvovirus B19, human parechovirus, measles, and hepatitis E (https://www.seegene.com/assays/allplex_meningitis_panel_assays , accessed on 1 December 2022). Twenty-three pediatric CSF samples without PTC or known infection were also tested using the ALLPLEX^TM meningitis panel and served as controls. The samples were kept at −80 Celsius, and the PCR panel was performed at the end of the study. All precipitants were vaccinated against varicella–zoster virus but not against any other pathogen included in the study.

We have previously established stable PC-3 and DU 145 human PCa cell lines that express the wild-type KLF5, the Ac-KLF5-mimicking mutant KLF5^K369Q (KQ), the acetylation-deficient mutant KLF5^K369R (KR), and the pLHCX vector control [22 (link)]. PC-3 cells expressing different forms of KLF5, including PC-3-KLF5, PC-3-KQ, PC-3-KR, and PC-3-pLHCX, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Biological Industries, HAMEK, Israel) and 1% penicillin/streptomycin (100 U/mL, Biological Industries). DU 145 cells expressing different forms of KLF5 were cultured in the minimum Eagle’s medium (MEM) (Corning, New York, USA) with 10% FBS.
PC-3-KLF5, PC-3-KQ, and PC-3-KR cells were infected with lentiviruses expressing the luciferase (HBLV-LUC-PURO, Hanbio, Shanghai, China). Infected cells were selected for stable cell populations in a medium containing puromycin (2 μg/mL).
RAW264.7 macrophage cell line was obtained from the American Type Cell Culture (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified eagle medium (DMEM) (Biological Industries) supplemented with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂.