HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses

By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.

The investigation of active HHV infections includes, among other markers, the detection of viral nucleic acids, typically by quantitative PCR (qPCR). In addition, the simultaneous detection of these pathogens has been shown to be beneficial, as their recognition may be difficult based on the clinical presentation alone (9)(10)(11)(12)(13)(14)(15).
While several multiplex qPCRs have been introduced for detection of HHVs (16)(17)(18)(19), none are designed to quantify them all. In addition, only few of the existing protocols distinguish between the closely related HHV-6A and HHV-6B, a distinction that may be crucial, as the former still lacks clear association to disease (20)(21)(22)(23).
In the present study, we developed a pan-herpes multiplex assay, HERQ-9, that quantifies and discriminates each of the HHVs using three separate triplex-qPCRs: the first amplifies herpes simplex viruses 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), the second EBV, HCMV, and KSHV, and the third HHV-6A, -6B, and -7. We validated our assay using prequantified reference materials and evaluated its performance with various clinical samples as well as solid tissue material.

RESULTS
In silico evaluation of amplicons, primers, and probes. The designed primers and probes were queried against all available sequences (full or partial genomes) in the NCBI database. The oligonucleotides showed perfect match for the different strains except for four sequences of HHV-6A (GenBank accession numbers KY316054.1, KT355575.1, KY316056.1, and KY316047.1) and two of HCMV (GenBank accession numbers KY490070.1 and KP745685.1) for which one to two mismatches were observed far from the 3= end.
We found no nonspecific binding to other viruses or human DNA except for the primers and probe of HSV-2, which also had complete homology to chimpanzee alpha-1 herpesvirus (GenBank accession number JQ360576.1).
In silico analysis of amplicons, primers, and probes revealed no relevant secondary structures, primer-dimers, or cross-dimers (see Fig. S1 and S2 in the supplemental material).
The multiplex assay detected all HHVs correctly from infected cell lines without cross-amplification of other HHVs, human DNA, or near-full-length or full-length genomes of parvovirus B19 (B19V) or the polyomaviruses BK virus (BKPyV), JC virus (JCPyV), and Merkel cell virus (MCPyV). All the no-template water controls remained negative throughout the PCR analyses.
The assay showed excellent short-term repeatability and long-term reproducibility in both singleplex and multiplex formats as well as with pMIXI to -III. The highest standard deviations in quantification cycle (C q ) values (intra-assay) and coefficients of variation between runs (interassay) were seen at the lowest template copies (Tables 1 and 2).
The method was linear in the range of 10 1 to 10 6 copies per l, and the qPCR efficiencies were between 95.9% and 103.8% in all the experiments.
The HHV plasmid dilutions spiked with 500 ng of human DNA (HaCaT cells) showed equal linearity to pure HHV plasmids ( Fig. 1; see also Fig. S4 in the supplemental material).
Analysis of clinical samples. We tested several types of clinical samples and compared the positive and negative agreements against reference methods. A summary of the results is presented in Table 4.
(ii) Mucocutaneous swabs. We tested 114 mucocutaneous swab samples previously investigated for HSV-1 and -2 or VZV at Turku University Hospital.
HERQ-9 identified correctly all the mucocutaneous swab samples that had tested positive by rapid viral culture for HSV-1 (n ϭ 35; median, 5.6 ϫ 10 7 ; range, 1.4 ϫ 10 5 to 8.2 ϫ 10 9 copies/ml of collection medium) and HSV-2 (n ϭ 30; median, 3.3 ϫ 10 7 ; range, 2.9 ϫ 10 5 to 3.5 ϫ 10 8 copies/ml of collection medium). In contrast, the 15 culture- negative controls showed no amplification for HSV-1 or HSV-2. However, two of these negative samples were positive instead for VZV, at 2.0 ϫ 10 7 and 3.2 ϫ 10 4 copies/ml of collection medium, and were confirmed to be VZV DNA positive with a control PCR (4,31). In addition, 5/5 HSV-1-positive and 4/4 HSV-2-positive DNA extracts previously tested by a reference PCR (4,31) were also positive by HERQ-9.  All the VZV samples positive (n ϭ 15) by enzyme immunoassay (EIA) were positive by the new assay, at a median quantity of 4.5 ϫ 10 7 copies/ml of collection medium (range, 8.0 ϫ 10 6 to 2.7 ϫ 10 9 ). On the other hand, among 10 VZV antigen-negative samples, 2 contained VZV DNA at 3.6 ϫ 10 6 and 4.7 ϫ 10 3 copies/ml of collection medium. Of these, the former was confirmed to be VZV DNA positive by the reference PCR. Incidentally, among the remaining eight samples negative for the VZV antigen, three showed positivity for HSV-1 instead, at loads of 7.7 ϫ 10 7 , 7.2 ϫ 10 7 , and 6.3 ϫ 10 1 copies/ml of collection medium. Only the two samples with the highest copy numbers were confirmed to be positive for HSV-1 DNA by the control PCR.
Moreover, we codetected other HHVs in these mucocutaneous swabs (Fig. 4C and  D). Of the HSV-1-positive samples, 20.9% were also positive for EBV DNA, 4.7% for The copy numbers of the other codetected HHVs (generally log 2 to log 3 copies/ml of collection medium) were always lower than those for HSV-1, HSV-2, or VZV. However, a few samples had log 5 to log 6 copies/ml of EBV DNA. Of all the mucocutaneous swab samples negative for HSV-1, HSV-2, and VZV (n ϭ 18), one (5.6%) tested positive for EBV DNA.
Coquantification of mixed high-and low-abundance targets. We tested uneven copies of whole HHV genomes in the same reaction (range, 4.5 ϫ 10 0 to 1.1 ϫ 10 6 copies/l) and found that all the viruses were correctly differentiated and accurately quantified by HERQ-9 (Pearson correlation coefficient [r] ϭ 0.996, P Ͻ 0.01). Higher coefficients of variation were seen at lower viral copy numbers (Table 5).

DISCUSSION
Our newly developed pan-herpes multiplex-qPCR assay, HERQ-9, stands out for its ability to differentiate and quantify the genomes of all nine human herpesviruses.
HERQ-9 was designed on three distinct triplex-qPCRs to meet, on the one hand, the clinical needs and, on the other, the technical constraints inherent to PCR multiplexing. Indeed, the capacity to codetect several targets is restricted by the spectral overlap of different fluorophores as well as the number of channels in the qPCR instrument (maximum of six) (32). Moreover, a greater number of targets can increase crossreactions between primers and probes, hampering assay performance.
HERQ-9 had good agreement with reference materials. The observed dissimilarities were likely to be related to the types and sensitivities of different methodologies (e.g., viral culture, EIA, and spectrophotometry), sample processing (e.g., DNA extraction methods), and the design of the PCR methods compared. Regarding the last item, the primer and probe design, amplicon size, target gene and its polymorphisms, reagents, and standards can all account for disagreements between qPCRs (37)(38)(39). In fact, these discrepancies have urged the introduction of WHO international standards for EBV, HCMV, and HHV-6B to increase the commutability between assays (39-42). However, this has had only relative value since, even after standardization, the interlaboratory variabilities continue to be high (up to 1.5 log 10 IU/ml, on average) (39,42).
Our findings emphasize the importance of multiplexing for comprehensive diagnosis and clinical management. Indeed, we identified additional HHVs in clinical samples that had been tested only for a single pathogen, encountering several unforeseen HSV-1 or VZV findings in mucocutaneous swabs, as well as EBV-HCMV coreactivations in plasma of immunodeficient patients. In addition, we codetected other HHVs in plasma in several combinations (EBV/HSV-1, EBV/HHV-6B, HCMV/HHV-6B, EBV/HCMV/ HSV-1, EBV/HCMV/HHV-6B, and EBV/HHV-6B/HSV-1). These coincidental discoveries, also noted by others (4,19,43), may have a significant impact on risk assessment and prognosis. Indeed, HHVs are thought to individually or synergistically contribute to viral syndromes (5,8,44), organ rejection (19), or the development of cancer (45,46). Moreover, we found other HHVs besides HSV-1, HSV-2, or VZV in mucocutaneous lesions (up to four in the same sample). The most common were EBV, HCMV, and HHV-7, whose low viral loads were likely to represent skin virome (47) or latency in  mobilized leukocytes (48). Yet in a few samples, EBV DNA levels approached those of HSV-1 or HSV-2, suggestive of in situ coreactivation or coinfection. Our detection of both EBV and HSV-1 in mucocutaneous lesions and plasma supports an interplay between these two viruses, as has been shown in vitro by Wu et al. (49). To the best of our knowledge, we are the first to report on HHV co-occurrences in classical herpetic lesions.
In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance for clinical management, the high sensitivity and specificity of this method will be of particular value for studies of the human virome generally dealing with minute quantities of persisting HHVs.
Uninfected HaCaT cells were used for human DNA spiking experiments. Prequantified reference material. All the reference materials are presented in Tables 3 and 4.
(i) Cell-free viral nucleocapsids. HSV-1 and HSV-2 nucleocapsids were isolated from pseudonymized dermal or mucosal lesion samples at the virus diagnostic unit of Turku University Hospital. The viruses were initially typed by a rapid viral culture immunoperoxidase assay (53) and confirmed by HSV type-specific gD (US6) gene-based PCR (54). For viral nucleocapsid DNA preparations, low-passagenumber stocks were generated in Vero cells (African green monkey kidney; ATCC), and the viral genomic DNA was prepared as described previously (55, 56) (see Text S2 in the supplemental material for a more detailed description). Two strains of HSV-1 (HSV-H1211 and HSV-H1215) and HSV-2 (HSV2-H12211 and HSV2-H1526) were prepared and the viral copies determined spectrophotometrically to be used as reference standards in dilutions of 1:10,000 and 1:100,000.
(iii) KSHV genome in bacterial artificial chromosome (BAC). KSHV-BAC16 DNA (a generous gift from Carolina Arias, University of California, Santa Barbara [UCSB], CA), derived from the KSHV strain of primary effusion lymphoma (PEL) cell line JSC-1, was purified from E. coli (60). The viral copy numbers were estimated spectrophotometrically. This reference was analyzed in dilutions of 1:10,000, 1:100,000, and 1:1,000,000 with the multiplex assay.
Clinical samples. All the clinical HHV samples were collected at the virus diagnostic unit of Turku University Hospital, and details are presented in Table 4. These included 80 mucocutaneous swab samples, of which 35 were positive for HSV-1 and 30 for HSV-2 by rapid viral culture immunoperoxidase assay (53), 25 mucocutaneous swab samples, of which 15 were positive for VZV by antigen enzyme immunoassay (61), 5 HSV-1 and 4 HSV-2 PCR-positive DNA extracts from mucocutaneous swab samples tested by a reference PCR (4,31), and 8 CSF samples, of which 2 were VZV positive by a control PCR (4,31).
In addition, 60 plasma samples were investigated, of which 17 had been studied only for EBV (GeneProof EBV PCR kit), 17 only for HCMV (GeneProof CMV PCR kit), and 26 for both. Of these, 13 and 16 samples were reported as EBV and HCMV positive (Ͼ200 copies/ml of plasma), respectively, while 9 and 5 had borderline copy numbers (50 to 200 copies/ml of plasma). Tonsillar tissues. Altogether, 35 mechanically homogenized tonsillar tissues were screened for the nine HHVs. The patients were 2 to 69 years of age (mean, 26), with eight Ͻ12 years (48). The viral loads were normalized per 10 6 cells, determined with the human single-copy gene RNase P qPCR (48).
Primers and hydrolysis probes. Primers and probes were designed for all HHVs, except for EBV (62). For each virus, several primer pairs were constructed in silico, in conserved genes (16,18,63,64). Degenerate primers were designed for HCMV to cover polymorphisms in the target area. Moreover, for HHV-6A, two probes were custom-designed to contain six locked nucleic acids (LNA) each (for shorter probe length) and a single-nucleotide difference for specific binding to different strains ( Table 6).
The tendency of primers and probes to form secondary structures, primer dimers, and cross-dimers was evaluated with Multiple Primer Analyzer (Thermo Fisher Scientific), while the propensity of the amplicons to form secondary structures was checked with the Mfold web server. A BLAST search (65) was performed to confirm primer binding to each of the virus strains in the nucleotide collection database (NCBI).
Primer candidates designed in silico were tested at a 200 nM concentration with plasmid dilutions (see "Plasmids" above), human DNA (HaCaT; 500 ng/reaction), and nuclease-free water in a SYBR green format (Maxima SYBR green qPCR master mix; Thermo Fisher Scientific), followed by melting-curve analysis. Primer pairs showing the highest efficiency and sensitivity with no primer dimer formation were chosen for further testing with the hydrolysis probes. Concentrations of the primer pairs were optimized empirically with a matrix of reactions ranging from 100 nM to 600 nM. The probes were tested in a 100 nM to 400 nM range. The final primer and probe concentrations are presented in Table 6.
Quantitative PCR protocol. Four commercial master mixes were pretested with HSV-1, HSV-2, and VZV plasmids and viral genomes, with special consideration given to the performance in the presence of human DNA and the coamplification of markedly low-and high-abundance targets. Consequently, TaqPath ProAmp multiplex master mix (Thermo Fisher Scientific) was chosen for the multiplex assay.
The qPCR thermal profile comprised initial denaturation at 95°C for 10 min followed by 45 cycles of 15 s at 95°C and 60 s at 60°C. The qPCRs contained 5 l of template, 2ϫ TaqPath ProAmp multiplex master mix, primers and probes (Table 6), and nuclease-free water in a final volume of 20 l. Water was used as negative control in all the qPCR runs. The samples were run in duplicate in AriaMx real-time PCR system (Agilent) and analyzed with Aria real-time PCR software (v.1.3) provided by the manufacturer. The adaptive fluorescence baseline, efficiency, slope, R 2 values, and intercept were calculated by the software. Background-based threshold was set for cycles 5 to 9 for the FAM and Texas Red dyes and 8 to 11 for the JOE dye.
Pretesting of primers in the SYBR green format consisted of the above-mentioned thermal profile, followed by a melting-curve analysis at 95°C for 60 s, 45°C for 30 s, and 95°C for 30 s. The melting-curve analysis was performed with a resolution of 0.5°C and soak time of 5 s. DNA extraction. DNAs from plasma, mucocutaneous swabs, CSF, and WHO international standards were extracted from 200 l of starting material with the QIAamp DNA blood minikit (Qiagen), DNAs from cells and virus-infected cell lines were extracted with the QIAamp DNA minikit (Qiagen), DNA from KSHV BAC was extracted with the NucleoBond Xtra Midi EF kit (Macherey-Nagel), and transformed plasmids were extracted with GeneJET plasmid miniprep kit (Thermo Fisher Scientific), according to the manufacturers' instructions. The final elution volumes were 100 l (with the exception of 50 l for plasma and 60 l for CSF samples). In every extraction, at least two negative controls (phosphate-buffered saline) were included.
Analytical sensitivity and specificity. The analytical sensitivities were determined in singleplex and multiplex formats using eight replicates of each HHV plasmid template in 50, 25, 15, 10, 5, 3, and 1 copy per reaction. The proportion of positive results was fit into a generalized linear model using probit link function (MATLAB v.R2018b) to approximate the limit of detection (LOD 95 ) for a given target.
The analytical specificities were evaluated by cross-testing (i) 10 7 copies of viral genomic DNA extracted from infected cell lysates and plasmid constructs of each HHV and (ii) plasmids containing near full-length or full-length genomes of polyomaviruses BKPyV, JCPyV, and MCPyV and parvovirus B19 genotype 1. In addition, 1,000 ng of cellular DNA extracted from HaCaT cells and 500 ng from human foreskin fibroblasts were tested for nonspecific amplification of human DNA.
Repeatability and reproducibility. The intra-assay and interassay variations were calculated using three separate qPCR runs using five replicates of HHV plasmids (10 6 to 10 1 copies/l) in the singleplex and multiplex formats, the latter both as single plasmids or in mixes pMIXI, pMIXII, and pMIXIII (10 6 to 10 1 copies/l). Two of the replicates were used to generate a standard curve, and three were marked as unknowns. The standard deviations of the C q values of the five replicates were used as a measure of intra-assay variation. A coefficient of variation calculated from the copy numbers of unknown replicates from three separate runs was used to estimate the interassay variation.
Ethics statement. The Ethics Committee of the Helsinki and Uusimaa Hospital District approved the collection of tonsils. Informed consent was obtained from all the donors or their parents prior to the surgery.

ACKNOWLEDGMENTS
We thank Leena-Maija Aaltonen for collecting the tonsil samples and Ritva Kajander and Huda Habib for help in the preparation of the viral DNA from HSV-1 and -2 nucleocapsids.