Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay

Abstract

The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences between HHV-6A and HHV-6B has led to their recent reclassification as separate species. As it is now especially relevant to investigate each virus, our objectives were to first design a multiplex HHV-6A and HHV-6B ddPCR assay and then to investigate the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and patients with MS, a disease in which HHV-6 is thought to play a role. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus, which has important implications in clinical settings. Interestingly, upon comparing the saliva from MS patients and healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva; increased detection of HHV-6A in MS patients is consistent with other studies suggesting that this viral species (thought to be more neurotropic than HHV-6B) is more prevalent among MS patients compared to healthy donors. As the biology and disease associations between these two viral species differ, identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of each in health and disease.

Figure 1. HHV-6A and HHV-6B duplex ddPCR assay design and specificity validation.

(A) Primers were designed to amplify an 89 base pair region of U57, 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.

Figure 2. HHV-6 viruses detected in 57% healthy donor PBMC samples: 50% coinfection of HHV-6A and HHV-6B.

Representative ddPCR plots with corresponding housekeeping gene (RPP30) 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.

Figure 3. HHV-6 viruses detected in 30% healthy donor serum samples: 62% coinfection of HHV-6A and HHV-6B.

Representative ddPCR plots with corresponding housekeeping gene (RPP30) 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.

Figure 4. HHV-6 viral DNA detected in the serum may not reflect contamination from circulating blood cells.

(A) Of sixteen normal donors with matched PBMC and serum samples, 11 were positive in at least one compartment and nine were positive in both compartments. For these nine donors, the data are displayed ratio of the copies per well of serum to the copies per well of PBMC. The dashed line at 1 represents an equal amount of virus in each compartment, while points above the line contain more virus in the serum compared to PBMC. The point circled in red is donor 25611, with the highest ratio of virus detected in the serum compared to PBMC. (B) In the serum samples of these sixteen donors, there is no correlation between copies of HHV-6 and copies of a cellular housekeeping gene RPP30 (there are two copies of RPP30 per cell). The point corresponding to donor 25611 is again circled in red, showing that despite a high serum/PBMC viral ratio, the serum did not contain increased cellular material relative to the other samples.

Figure 5. Identification of potentially chromosomally integrated blood donors using duplex and triplex ddPCR assays.

(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 RPP30 (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.

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

Representative ddPCR plots with corresponding housekeeping gene (RPP30) 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.