HHV-6 viruses detected in 57% healthy donor PBMC samples: 50% coinfection of HHV-6A and HHV-6B.

<p>Representative ddPCR plots with corresponding housekeeping gene (<i>RPP30</i>) as insets shown in A-C. (A) No positivity detected. (B) Only HHV-6B positivity (green droplets, lower right quadrant) detected. (C) Coinfection of HHV-6A and HHV-6B (blue droplets in upper left and green droplets in lower right quadrants, respectively) detected. (D) Group analysis of 46 healthy donor PBMC samples, with a mean (solid line) of 455 total HHV-6 copies/10⁶ cells. Each circle represents a donor. Closed circles represent detection of both HHV-6A and HHV-6B, while open circles represent detection of only HHV-6B. (E) The amount of HHV-6A and HHV-6B in the coinfected healthy donors (closed circles in D). The ratio of 6A/6B copies/10⁶ PBMC ranged from 0.01–0.25.</p

HHV-6A and HHV-6B duplex ddPCR assay design and specificity validation.

<p>(A) Primers were designed to amplify an 89 base pair region of <i>U57</i>, encoding the major capsid protein of HHV-6. The shared forward and reverse primers (in bold) amplify both HHV-6A and HHV-6B, while the probes are specific for each virus with a three base pair mismatch. The HHV-6A probe sequence is in blue, while the HHV-6B probe sequence is in green. (B) The probes distinguish HHV-6A and HHV-6B viral DNA with high specificity. The HHV-6A FAM-labeled probe binds HHV-6A DNA (blue droplets in left plot) but not HHV-6B DNA. Likewise, the HHV-6B VIC-labeled probe binds HHV-6B DNA (green droplets in right plot), but not HHV-6A DNA.</p

Identification of potentially chromosomally integrated blood donors using duplex and triplex ddPCR assays.

<p>(A) In a duplex reaction, donor 27867 was calculated to have 1.02 copies HHV-6A per cell. (B) In a duplex reaction, donor 28319 was calculated to have 0.98 copies HHV-6B per cell. The corresponding plots for the housekeeping gene <i>RPP30</i> (insets) were used to quantify the number of cells. (C) Triplex ddPCR of PBMC DNA from potentially chromosomally integrated donors. All three primer/probe sets (HHV-6A, HHV-6B and RPP30) can be assayed in a single well, using fluorescence intensities to distinguish between the droplet populations (labeled). In a triplex reaction, donor 27867 was calculated to have 1.09 copies HHV-6A per cell. (D) In a triplex reaction, donor 28319 was calculated to have 1.04 copies HHV-6B per cell.</p

Primer/probe sequences.

<p>Primer/probe sequences.</p

HHV-6 viruses detected in 30% healthy donor serum samples: 62% coinfection of HHV-6A and HHV-6B.

<p>Representative ddPCR plots with corresponding housekeeping gene (<i>RPP30</i>) as insets shown in A-C. (A) No positivity detected. (B) Only HHV-6B positivity (green droplets, lower right quadrant) detected. (C) Coinfection of HHV-6A and HHV-6B (blue droplets in upper left and green droplets in lower right quadrants, respectively) detected. (D) Group analysis of serum from 43 healthy donors, with a mean (solid line) of 2,069 total HHV-6 copies/ml. Each circle represents a donor. Open circles represent the detection of only HHV-6B, and closed circles represent the detection of both HHV-6A and HHV-6B. (E) The amount of HHV-6A and HHV-6B in the coinfected healthy donors (closed circles in D). The ratio of 6A/6B copies/ml serum ranged from 0.1 to 0.66.</p

Increased HHV-6A and HHV-6B coinfection in MS patient saliva samples.

<p>Representative ddPCR plots with corresponding housekeeping gene (<i>RPP30</i>) as insets shown in A-C. (A) No positivity detected. (B) Only HHV-6B positivity (green droplets, lower right quadrant) detected. (C) Coinfection of HHV-6A and HHV-6B detected (blue droplets in upper left and green droplets in lower right quadrants, respectively). (D) Group analysis of total HHV-6 copies/ml in saliva of healthy donors (n = 39) and MS patients (n = 59). Lines represent the mean total viral copies: 1.38×10⁴ copies/ml for healthy donors and 1.82×10⁴ copies/ml for MS patients. Each symbol represents a donor. Open symbols represent the detection of only HHV-6B, and closed symbols represent the detection of both HHV-6A and HHV-6B. (E) The proportion of donors with HHV-6A and HHV-6B coinfection (dark shading) in saliva is significantly increased in MS patients compared to healthy donors (p = 0.04, Χ² test). (F) The ratio of 6A/6B copies/ml saliva ranged from 0.005–0.6 in healthy donors and from 0.04 to 4.0 in MS patients.</p

<b>Table 3.</b> Summary of HHV-6 detection by ddPCR HHV-6 positive samples.

a<p>p = 0.04 (χ²).</p

Multi compartment analysis of ten normal donors.

a<p>ND  =  not detected.</p>b<p>Coinfected: 580 copies/ml HHV-6A, 580 copies/ml HHV-6B.</p>c<p>Coinfected: 92 copies/10⁶ cells HHV-6A, 369 copies/10⁶ cells HHV-6B.</p

Detection of CD4⁺CD25⁺ T cells in CSF of chronic virus infection and/or neuroinflammatory diseases.

<p>(A) Comparison of frequency of CD4⁺CD25⁺ T cells in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (B) Comparison of frequency of CD4⁺CD25⁺ T cells in peripheral blood using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (C) Comparison of frequency of FoxP3 in peripheral blood CD4⁺CD25⁺ T cells using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (D) Comparison of frequency of CTLA-4 in peripheral blood CD4⁺CD25⁺ T cells using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean.</p

Detection of B cells in CSF of chronic virus infection and/or neuroinflammatory diseases.

<p>(A) Comparison of frequencies of B cells (left) and B cell/monocyte ratio (right) in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (B) Detection of ASCs subset in B cells of CSF. (Left) Representative dot plots of IgD and CD27 staining in CSF CD19⁺ B cells of a ND and a HAM/TSP patient. IgD^- CD27⁺⁺ subsets (red rectangles) represent ASCs. (C) Comparison of frequencies of ASCs (left) and absolute number of ASCs (right) in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean.</p