Activation of MAPK/c-Fos induced responses in oral epithelial cells is specific to Candida albicans and Candida dubliniensis hyphae

Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae

Correction: Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected.

[This corrects the article DOI: 10.1371/journal.pone.0098077.]

HIV-1 entry via the endocytic pathway results in productive viral infection in epithelial cells.

<p>Two fold serial dilutions of VSV-G pseudotyped HIV-1 (MOI 1 –0.125) were added to TR146, FaDu, A431 and TZM-bl control cells. Infection is measured 16–24 h later by flow cytometry as percentage of GFP expression. The effect of AZT (500 mM) on GFP expression was also measured at the highest virus inoculum. Error bars show standard error from the mean. Data are representative of three independent experiments</p

Basal HIV-1 receptor surface expression in resting epithelial cells.

<p>TR146, FaDu, A431, TZM-bl and NP2-R5 and –X4 expressing cells were examined for surface expression of CD4, CCR5, CXCR4, DC-SIGN, GalCer and HSPG's by flow cytometry using monoclonal primary antibodies specific to each receptor with a FITC-labeled secondary antibody. HSPG's were not analyzed in NP2-R5 or -X4 expressing cells. Data are presented as percentage of cells expressing each receptor in a minimum of three independent experiments. Bars indicate ± standard error of the mean. *** = <i>P</i><0.001, ** = <i>P</i><0.01, * = <i>P</i><0.05.</p

Detection of integrated HIV-1 genome in epithelial cells by qPCR.

a<p>C8166 cells express CXCR4 and were used for X4 viral infections only.</p>b<p>NP2-R5 cells express CCR5 and were used for R5 viral infections only.</p><p>+, Integrated HIV-1 product detected (cycle threshold detection in brackets).</p><p>ND, Integrated HIV-1 product not detected.</p><p>Real-time PCR assay to detect integrated HIV-DNA using HIV-1 LTR and human Alu-specific primers and a U5 specific probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098077#pone.0098077-Mbisa1" target="_blank">[92]</a>. TR146, FaDu, A431 and PM-1 (control) cells were exposed to YU2 (R5) or LAI (X4) virus for 48 h at 37°C. DNA was extracted, digested with DpnI to degrade any plasmid DNA contaminant and analysed by real-time PCR.</p

Basal HIV-1 receptor mRNA expression in resting epithelial cells.

<p>TR146, FaDu, A431 and TZM-bl cells were examined for mRNA expression of CD4, CCR5, CXCR4, DC-SIGN and the HSPG's syndecan-1 and -4 by quantitative RT-PCR. Data are presented as mRNA transcripts (arbitrary units) normalized to β-actin in a minimum of three independent experiments. PBMCs showed significantly higher expression of CD4, CCR5 and CXCR4 than oral (TR146 and FaDu) or vaginal (A431) cell lines. A431 cells show significantly higher expression of SDC-1, whilst FaDu and A431 show significantly higher expression of SDC-4 than TR146. Bars indicate ± standard deviation from the mean. *** = <i>P</i><0.001, * = <i>P</i><0.05.</p

Robust and sensitive amplicon-based whole-genome sequencing assay of respiratory syncytial virus subtype A and B

ABSTRACTPrevention of respiratory syncytial virus (RSV) infection is now a global health priority, with a long-acting monoclonal antibody and two RSV vaccines recently licenced for clinical use. Most licenced and candidate interventions target the RSV fusion (RSV-F) protein. New interventions may be associated with the spread of mutations, reducing susceptibility to antibody neutralization in RSV-F. There is a need for ongoing longitudinal global surveillance of circulating RSV strains. To achieve this large-scale genomic surveillance, a reliable, high-throughput RSV sequencing assay is required. Here we report an improved high-throughput RSV whole-genome sequencing (WGS) assay performed directly on clinical samples without additional enrichment, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. Using upper respiratory tract (URT) RSV-positive clinical samples obtained from a sentinel network of primary care providers and from hospital patients (29.7% and 70.2%, respectively; n = 1,037), collected over the period 2019 to 2023, this assay had a threshold of approximately 4 × 103 to 8 × 103 copies/mL (RSV-B and RSV-A sub-types, respectively) as the lowest amount of virus needed in the sample to achieve >96% of whole-genome coverage at a high-quality level. Using a Ct value of 31 as an empirical cut-off, the overall assay success rate of obtaining >90% genome coverage at a read depth minimum of 20 was 96.83% for clinical specimens successfully sequenced from a total of 1,071. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national programs for the surveillance of RSV genomic variation.IMPORTANCEIn this paper, we report an improved high-throughput respiratory syncytial virus (RSV) whole-genome sequencing (WGS) assay performed directly on clinical samples, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national and global programs for the surveillance of RSV genomic variation. The quality of sequence produced is essential for preparedness for new interventions in monitoring antigenic escape, where a single point mutation might lead to a reduction in antibody binding effectiveness and neutralizing activity, or indeed in the monitoring of retaining susceptibility to neutralization by existing and new interventions

Primer sets detecting gene expression of HIV-1 associated receptors.

<p>Primer sets detecting gene expression of HIV-1 associated receptors.</p

Post-integration HIV-1 mRNA transcription and <i>de novo</i> viral protein production in epithelial cells (MOI: 0.2).

<p>(A) Detection of spliced HIV-1 <i>tat</i> mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.</p

Transfer of captured HIV-1 from epithelial cells to permissive cells via transcytosis.

<p>TR146, FaDu and A431 were grown on polycarbonate transwell membranes and incubated with R5 (YU2) and LAI (X4) virus for 4 h. Following extensive washing the transwells were placed in a separate plate that overlaid a confluent monolayer of TZM-bl cells and incubated for a further 48 h at 37°C. TZM-bl cells were fixed, stained for β-galactosidase expression with X-Gal stain, and counted using light microscope at 100x magnification. Resulting replicate colony counts were averaged and analyzed by two factor (cell line and virus tropism) ANOVA and post hoc Fisher PLSD tests. Colony counts through A431 monolayers were significantly greater than those through TR146 and FaDu (<i>P</i><0.05). Colony counts resulting from exposure of epithelium to X4 and R5 were significantly greater (<i>P</i><0.05) than control wells with no virus exposure, but were not significantly different from each other. Data are representative of three independent experiments. * <i>P</i><0.05.</p