siRNA inhibition of HCMV replication and virus progeny production during multiple rounds of virus replication at low multiplicity of infection.

<p>(A) MRC-5 cells were untreated or transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54B), UL97 (siUL97A), and UL122/123 (siUL122B) and infected with HCMV AD169 (0.001 pfu/cell) at 24 hours post siRNA transfection. Representative images of HCMV-induced CPE are shown at 7 days post infection in siRNA transfected cells. Scale bars represent 100 µm. (B) HCMV growth curves in response to siRNA treatment or no treatment with (C) viral load relative to no siRNA treatment. Data presented as mean of triplicate biological experiments ± SD. Significant differences between siRNA treated and untreated groups is given as *p<0.05 and #p<0.0001 and shown in (C).</p

siRNA efficacy at inhibiting HCMV protein expression during a single round of HCMV replication at high multiplicity of infection.

<p>MRC-5 cells were transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54A/B), UL97 (siUL97A/B), and UL122/123 (siUL122A/B) followed by infection with HCMV (1 pfu/cell) at 24 hours post transfection. (A) Cells were harvested 24, 48 and 72 hours post infection and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein expression measured using western blot. (B) Protein expression in UL54A/B, UL97A/B and UL122A/B siRNA treated and HCMV-infected cells relative to cells treated with scrambled siRNA (siSc) measured using densitometry. Results derived from two representative samples from triplicate biological experiments and densitometry results presented as mean ± SD.</p

Update on the management of gestational trophoblastic neoplasia

<p>MRC-5 cells were untreated or transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54B), UL97 (siUL97A), and UL122/123 (siUL122B) and infected with HCMV AD169 (0.001 pfu/cell) at 24 hours post siRNA transfection. Cells were harvested 1, 4 and 7 days post infection and HCMV immediate-early (IE1p72), early (pp65), and early-late (gB) protein expression measured using immunofluorescence and quantitative image analysis. Data presented relative to HCMV-infected untreated cells and represent mean of 10 fields of view ± SD from duplicate biological experiments.</p

siRNA inhibition of HCMV protein expression during multiple rounds of viral replication at low multiplicity of infection.

<p>MRC-5 cells were untreated or transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54B), UL97 (siUL97A), and UL122/123 (siUL122B) and infected with HCMV AD169 (0.001 pfu/cell) at 24 hours post siRNA transfection. (A) Cells were harvested 1, 4 and 7 days post infection and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein expression measured using western blot (B) Protein expression in HCMV-infected, siRNA treated cells relative to HCMV-infected, untreated cells measured using densitometry. Western blot images (A) are of one representative sample from triplicate experiments and densitometry data (B) presented as mean ± SD of triplicate biological experiments.</p

siRNA efficacy at inhibiting HCMV viral progeny production during a single round of HCMV replication at high multiplicity of infection.

<p>MRC-5 cells were transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54A/B), UL97 (siUL97A/B), and UL122/123 (siUL122A/B) followed by infection with HCMV (1 pfu/cell) at 24 hours post transfection. (A) Supernatant was harvested 72 hours post infection and viral titre measured using plaque assay. (B) Viral load in HCMV-infected cells treated with siUL54A/B, siUL97A/B and siUL122A/B relative to HCMV-infected cells treated with scrambled siRNA (siSc). Data presented as mean of triplicate biological experiments ± SD. Significant differences between HCMV siRNA and siSc treated groups is given as #p<0.0001.</p

siRNA efficacy at inhibiting plasmid-derived HCMV protein expression.

<p>293T cells were transfected with 50 µg of plasmid expressing the HCMV siRNA target transcript 24 hours post siRNA transfection. (A) Cells were harvested 72 hours post plasmid transfection and protein expression measured using western blot. (B) Protein expression in siRNA treated and plasmid transfected cells relative to cells treated with control scrambled siRNA (siSc) measured using densitometry. Results are from duplicate biological experiments and densitometry results presented as mean ± SD.</p

Amino acid sequence alignment of the HBV small S protein.

<p>The numbers on the right indicate the amino acid position. The common substitutions identified in this study are shown in closed boxes. The “a” epitope is shown in dashed box. Asterisks indicate known genotype- or subtype-related variability. ‘-’ Indicates stop codon. Local references were samples obtained from SEALS at POWH dating from January 2007 until March 2013 with a serological profile of HBsAg positive, anti-HBc positive and HBV DNA positive.</p

Nucleotide substitutions over time in the HBV S gene of Patients 1 and 3.

<p>N/A Not Available</p><p>Nucleotide substitutions over time in the HBV S gene of Patients 1 and 3.</p

Patient List with related serological and molecular test results.

<p>Patient List with related serological and molecular test results.</p

Limit of detection of designed PCR in amplifying the S gene of HBV DNA.

<p>The PCR was performed on serially diluted World Health Organization International Standard for Hepatitis B virus DNA with an initial viral concentration of 1x10⁶ IU/mL. Lane 1: 1000bp ladder; lane 2: DNA from HBV infected sample (positive control); lane 3: 20,000IU/mL, lane 4: 2,000IU/mL; lane 5: 200IU/mL, lane 6: 100IU/mL; lane 7: 50IU/mL; lane 8: 25IU/mL; lane 9: 12.5IU/mL; lane 10: 6.25 IU/mL; lane 11: 3.125IU/mL; lane 12: 1.5625IU/mL; lane 13: dH₂O (negative control).</p