Journal: PLoS ONE

Article Title: Exploitation of Herpesviral Transactivation Allows Quantitative Reporter Gene-Based Assessment of Virus Entry and Neutralization

doi: 10.1371/journal.pone.0014532

Figure Lengend Snippet: ( A ) Human MRC-5 cells, transiently transfected with the pTA-Control plasmid (left panel) or pTA-GAS (right panel), were split into aliquots and seeded. Cells were infected with 1 PFU/cell HCMV-AD169, HCMV-Towne, HCMV-TB40/E (black bars) or left uninfected (white bars). After 24 h the cells were treated for additional 5 h with 500 U/ml human IFN-γ. Cells were lysed and luciferase activity was measured. The experiment was performed in triplicate and the arithmetic mean with the standard deviation is shown. The significance was tested with the Student's t-test: * p<0.05 and ** p<0.01. ( B ) ARPE-19, CV-1, Vero and Jurkat cells were transfected with 2.5 µg pTA-Control plasmid using the Lonza nucleotransfection system. Cells were split so that parallel measurements and infections originated from the same transfection. 20 h post transfection cells were left uninfected (mock; open bars) infected with 3 PFU/cell HCMV-AD169, HSV-1 F strain (black bars) or UV-inactivated virus (hatched bar) for additional 20 h. Cells were lysed and luciferase activity was measured. ( C ) MRC-5 cells were infected with 3 PFU/cell HCMV-AD169 or UV-inactivated HCMV-AD169. After the indicated time cells were lysed and total RNA or protein was prepared. Protein lysates were subjected to native sodium-deoxycholate-PAGE for the analysis of IRF-3 dimerization or to SDS-PAGE for the analysis of pp72-IE1 and β-actin protein amount. Proteins were transferred to filters and probed with the indicated antibodies. IFN-β and GAPDH mRNA was assessed by semi-quantitative RT-PCR from total RNA as. For GAPDH two log 10 dilutions were used as template to confirm measurement in the linear amplification range.

Article Snippet: Purified stocks of HCMV strains AD169 , Towne (ATCC VR-977), the BAC-derived HB5 and the BAC-derived endotheliotropic strain TB40/E were used.

Techniques: Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, Standard Deviation, SDS Page, Quantitative RT-PCR, Amplification

Journal: Herpesviridae

Article Title: Human cytomegalovirus induces apoptosis in neural stem/progenitor cells derived from induced pluripotent stem cells by generating mitochondrial dysfunction and endoplasmic reticulum stress

doi: 10.1186/2042-4280-4-2

Figure Lengend Snippet: Analysis on the expression of viral and cellular gene products in NSPC/iPSCs. (A) Morphological changes of Towne-infected NSPC/iPSCs were observed under the inverted microscope before infection (a) , 2 dpi (b) , 5 dpi (c) , and 7 dpi (d) . (B) RT-PCR analysis of HCMV-encoding gene expression. Total RNAs isolated from NSPC/iPSCs harvested before (−) HCMV infection or at 1, 2, 3, 5, and 7 dpi with HCMV Towne strain were subjected to RT-PCR assays. GAPDH gene expression was assayed for the control. (C) The kinetics of mRNA expression for IE1, UL89, and UL136 in Towne-infected NSPC/iPSCs was examined by real-time quantitative RT-PCR assay. The mRNA expression was normalized to that of G6PDH gene. Real-time PCR data was analyzed by the 2-ΔΔCT method. The fold induction was calculated as the ratio of mRNA levels detected at each time point to that detected at 1 dpi. The y-axis represents fold induction of IE1 and UL136 mRNA (left y-axis) and UL89 mRNA (right y-axis). (D) Immunoblot analysis of HCMV protein expression in HCMV-infected NSPC/iPSCs. Whole-cell lysates of NSPC/iPSCs harvested before (−) HCMV infection or at 1, 2, 5, and 7 dpi with HCMV Towne strain were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against IE1/IE2, pp65, gB, and α-tubulin. (E) RT-PCR analysis of pluripotency and neural differentiation marker gene expression in HCMV-infected NSPC/iPSCs. (F) Immunoblot analysis of neural differentiation marker protein expression in HCMV-infected NSPC/iPSCs. Whole-cell lysates of NSPC/iPSCs were analyzed by immunoblotting with antibodies against Musashi-1, Pax6, and Nestin. (G) hTERT-BJ1 cells inoculated with culture supernatant collected from mock-infected NSPC/iPSCs (upper panel) or Towne HCMV-infected NSPC/iPSCs (lower panel) at 8 dpi were subjected to immunofluorescence test with anti-IE1/IE2 antibody (green). Nuclei were stained with DAPI. Representative results from two independent experiments are shown.

Article Snippet: HCMV laboratory strain Towne (ATCC VR-977) was propagated in hTERT-BJ1 cells.

Techniques: Expressing, Infection, Inverted Microscopy, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot, SDS Page, Marker, Immunofluorescence, Staining

Journal: Virology Journal

Article Title: Evaluation of three Polymerase chain reaction tests targeting morphological transforming region II, UL-83 gene and glycoprotein O gene for the detection of Human Cytomegalovirus genome in clinical specimens of immunocompromised patients in Chennai, India

doi: 10.1186/1743-422X-3-20

Figure Lengend Snippet: Results of the three PCRs for the diagnosis of suspected CMV infections in comparison to pp65 antigenemia (gold standard) a .

Article Snippet: The PCRs for the aforementioned regions were already standardized in our laboratory using cultures of CMV AD-169 strain (ATCC VR-538). pp65 antigenemia assay is a rapid, reliable and superior to both rapid shell vial assay and conventional test tube culture in the detection of HCMV in the clinical specimens from immunocompromised patients indicating active HCMV disease.

Techniques:

Journal: Bioinformation

Article Title: Functional characterization of novel mutations in UL54 of ganciclovir resistant HCMV strain using structural analysis

doi:

Figure Lengend Snippet: The amplified products of region IV of the specimens along with others specimens detected positive to HCMV. Agarose Gel Electrophoretogram of PCR amplified product targeting Human cytomegalovirus UL54 gene of Region IV. Round II applied on Clinical Specimens. Some of the specimens positive for the region IV along with sample 2256 is shown. Lanes NC2: Negative Control - II round NC1: Negative Control - I round PC: Positive Control DNA extract from Human cytomegalovirus AD 169 MWM: Molecular weight marker 100 bp Ladder. Specimens positive for UL54 partial region: 2526, 4474, 4869, 1650, 4069

Article Snippet: DNA extracted from HCMV AD 169 (ATCC VR 538) was used as a positive control.

Techniques: Amplification, Agarose Gel Electrophoresis, Negative Control, Positive Control, Molecular Weight, Marker