Principle components analysis (PCA) of rodent communities grouped by mean SNV prevalence.

<p>Total number of individuals of each species was entered as components. Factor loadings plots were standardized to two factors. Figure 1.a. Factor loadings plot for low (0–5%) prevalence communities. Figure 1.b. Factor loadings plot for moderate (7–14%) prevalence communities. Figure 1.c. Factor loadings plot for high (>17%) prevalence communities. PEMA = deer mouse, PETR = pinyon mouse, DIOR = Ord's kangaroo rat, NELE = desert woodrat, REME = western harvest mouse, PEPA = Great Basin pocket mouse, ONLE = Northern Grasshopper mouse.</p

Summary of species diversity, prevalence of Sin Nombre virus and density of deer mice (Peromyscus maniculatus) at 16 sites in central Utah sampled 2003–2005.

<p>Site identifier includes geographic location (TJ = Tintic Junction; LS = Little Sahara; UL = Utah Lake) and the numeric code (3–19). Species diversity was estimated using Simpson's Index (D) for each site per season. Prevalence of SNV was estimated as the number of deer mice positive for SN viral antibodies divided by the total number of deer mice sampled at each site per season x 100%. Density of deer mice (no./ha) was estimated using program DISTANCE. Dashes indicate sites that were not sampled in a particular season.</p

Up-regulation of CD46 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for CD46 expression. Top- Percentage of CD46 positive cells for each MSC population. Bottom- MFI ratio for CD46 expression on each MSC population. MFI ratio  =  (Median Fluorescence Intensity for CD46/Median Fluorescence Intensity for isotype control). The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. 0(B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD46 and unfilled histograms are the staining with the corresponding isotype control.</p

FIX-ΔLUNA infected CD14+ cells fail to express lytic transcripts following IL6 induced differentiation.

<p>A) Diagram of the timeline of infection. Infected cells were collected at 1–20 dpi, IL6 was added at 10 dpi to induce cellular differentiation, after which two additional time points were collected. B) Expression of viral RNAs in FIX-WT, FIX-ΔLUNA and FIX-Rev infected CD14⁺ cells (MOI = 1). Total RNA was collected over a 20 d time course together with mock RNA. At 10 dpi CD14⁺ cells were differentiated with IL-6. Five and ten days post differentiation, RNA samples were collected (these samples were termed as 15 d-IL6 and 20 d-IL6). From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. C) CD14⁺ cells were infected at an MOI = 1 with FIX-WT, FIX-ΔLUNA or FIX-Rev. D–E) Protein was isolated from infected CD14⁺ cells at the indicated time points and subjected to western blot analysis. Blots were probed with either a rabbit monoclonal a-LUNA or a mouse monoclonal a-IE1 antibody along with a-Tubulin as a loading control. qRT-PCR analysis was used to assay gene expression of UL138, vIL10 and US28 at the indicated times post infection. Viral mRNA was normalized to actin. All samples were tested in triplicate. Abbreviations used: d, days post infection., Mo; mock. IL6 was added at 10 dpi.</p

Up-regulation of CD59 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for expression of CD59. Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD59, and unfilled histograms are the corresponding isotype control staining. The MFI ratio for each MSC cell line was obtained by performing the following calculation: MFI ratio  =  (Median Fluorescence Intensity for CD59/Median Fluorescence Intensity for isotype control). (B) MFI ratio for each MSC population is shown. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC.</p

FIX-ΔLUNA virus does not increase viral genome copy number after IL6 induced differentiation of CD14⁺ cells.

<p>Viral DNA was quantified by qPCR. A) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected HF cells over a 20 d time course. B) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected CD14⁺ cells at 1 dpi, 3 dpi, 5 dpi, 10 dpi, 20 dpi. Differentiation was induced in CD14⁺ cells at 10 dpi by the addition of IL6 and at 5 days (sample termed as 15dpIL6) or 10 days (samples termed as 20dpIL6) total DNA was collected. All samples were run in triplicate. Statistical analysis was done using a Student <i>t</i> test comparing FIX-WT vs. FIXΔLUNA or FIX-Rev vs. FIX-ΔLUNA. The * indicates a <i>P</i>-value<0.01, and the ** indicates a <i>P</i>-value<0.001.</p

Up-regulation of CD55 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed for CD55 surface expression by flow cytometry. Top- Percentage of CD55 positive cells for each MSC population. Bottom- MFI ratio for CD55 expression on different MSC populations. MFI ratio  =  Median Fluorescence Intensity for CD55/Median Fluorescence Intensity for isotype control. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. (B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to each MSC cell population stained with antibody against CD55 and unfilled histograms correspond to isotype staining.</p

Primers for BAC mutagenesis and RT-PCR.

a<p>F: Forward Primer; R: Reverse Primer.</p

Effect of overexpression of HCMV US2 protein on MSC in a complement-mediated cytotoxicity assay.

<p>(A) The results depict representative data from a flow cytometry-based complement-mediated cytotoxicity assay from at least 3 independent experiments. The assay was performed in the presence of human IgM and rabbit serum complement (experimental) or in the presence of human IgM and absence of the rabbit serum complement (control). MSC stained with calcein were counted as viable, cells stained with Ethidium homodimer-1 were counted as dead, and cells doubly positive were counted as having membrane damage. Doubly negative cells were excluded from the analysis. A live/dead control (top) was used in order to set up the quadrant gates for alive, dead, and membrane-damaged populations and to set parameters for compensation between the 2 fluorescent channels. The experimental assay provided the percentage of dead cells under experimental conditions. The control assay provided the percentage of spontaneously dead cells. The same conditions were applied for non-transduced MSC and MSC-US2. (B) The percentage of cytotoxicity for each cell line was calculated as follows: (percent of dead cells under experimental conditions – percent of spontaneously dead cells)/(100 – spontaneously dead)*100. The percentage of experimental or spontaneously dead cells was determined by calculating the percentage of cells that were positive for Ethidium homodimer-1, and negative for calcein. The results depict the mean ± SEM of three independent experiments. * indicates p<0.05.</p

HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14⁺ Cells

<div><p>Human cytomegalovirus (HCMV) is a member of the <em>Herpesviridae</em> family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors <em>in vivo</em>. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.</p> </div