Detection and typing of lymphotropic herpesviruses bymultiplex polymerase chain reaction
Francisco Pozo *, Antonio Tenorio
Ser6icio de Microbiologıa Diagnostica, Centro Nacional de Microbiologıa, Instituto de Salud Carlos III, Ctra. Pozuelo, KM2,Majadahonda, E-28220 Madrid, Spain
Received 27 August 1998; received in revised form 12 November 1998; accepted 16 November 1998
Human lymphotropic herpesviruses, includingEbstein–Barr virus (EBV), cytomegalovirus
(CMV), Human herpesvirus 6, variants A and B(HHV6-A, HHV6-B), human herpesvirus 7(HHV7), and the most recently discovered humanherpesvirus 8 (HHV8), formerly termed Kaposi’ssarcoma-associated herpesvirus (KSHV), are re-sponsible for a wide variety of human diseases,caused either by primary infection or by reactiva-tion in immunosuppressive conditions. Some of
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these herpesviral diseases are undoubtedly associ-ated with only one virus. In most clinical syn-dromes of herpesviral etiology, differentherpesviruses can nevertheless be involved, requir-ing differential diagnosis not only for epidemio-logical purposes, but also because treatment withspecific antiviral drugs is now available.
Until recently, the etiological diagnosis of her-pesviral infections relied solely on serological testsor virus isolation in cell culture for each of theseviruses. Over the last few years, the amplificationof viral DNA by polymerase chain reaction(PCR) has become one of the most widely usedtechniques in human diagnostic virology. ViralDNA from several lymphotropic herpesviruseshas been detected, often separately, in infectionsor reactivations in which the presence of morethan one of these viruses is a common finding,such as several neurological syndromes and otheropportunistic infections in human immunodefi-ciency virus (HIV)-infected patients (Cinque et al.,1996; Belec et al., 1997; Fabio et al., 1997; Gau-theret-Dejean et al., 1997; Portolani et al., 1997),opportunistic infections in transplant recipients(Wang et al., 1996; Chan et al., 1997; Osman etal., 1997; Moschettini et al., 1998), and infectiousmononucleosis syndrome (Gan et al., 1994; Ya-mamoto et al., 1995). Furthermore, the relativeimportance of lymphotropic herpesviruses is wor-thy of investigation in several other clinical set-tings, including lymphoproliferative disorders(Luppi and Torelli, 1996; Valente et al., 1996),multiple sclerosis (Sanders et al., 1996; Martin etal., 1997; Merelli et al., 1997), chronic fatiguesyndrome (Di Luca et al., 1995; Sairenji et al.,1995) hepatitis (Mason et al., 1996) or inflamma-tory bowel disease (Wakefield et al., 1992).
Multiplex PCR is a modification of the originalPCR procedure; it allows the simultaneous am-plification of DNA from different pathogens bythe use of several primers pairs in the same reac-tion. Some advantages of multiplex PCR methodsare reduced labor intensiveness, and the economyof both test sample and reagents compared tosingle analytical PCRs (Edwards and Gibbs,1994). Nevertheless, only a few multiplex PCRmethods for simultaneous detection of several hu-man lymphotropic herpesviruses have been de-
scribed (Rozenberg and Lebon, 1991; Tenorio etal., 1993; McElhinney et al., 1995; Casas et al.,1997; Kidd et al., 1998; Moschettini et al., 1998).
Based on a technique described previously(Tenorio et al., 1993), a sensitive multiplex nested-PCR method was developed that enables the de-tection and typing of all human lymphotropicherpesviruses described to date. An internal con-trol has been included to test for the presence ofDNA polymerase inhibitors or reaction failures.Prior to examining a wide variety of clinical spec-imens, sensitivity of the PCR method was esti-mated by testing dilutions of specific DNAplasmids. This technique could avoid the need totest clinical specimens separately for eachlymphotropic herpesvirus.
2. Materials and methods
2.1. Virus strains, plasmids and controls
Human embryonic lung fibroblast cells wereinfected with CMV (AD169 strain) and harvestedwhen a cytopathic effect greater than 75% wasdetected. HHV6-A (GS strain) was propagated inHSB-2 cells and harvested when the cytopathiceffect was at its peak. EBV was obtained from theEBV-containing P3HR-1 cell line. Cell suspen-sions of HHV6-B (Z29 strain)-infected Molt-3cells and HHV7 (SB strain)-infected cord bloodlymphocytes were used, both kindly provided byPhilip E. Pellet. DNA from a Kaposi’s sarcomaskin lesion of a HIV-infected patient was used assource of the HHV8 genome. Purified DNA ofpseudorabies herpesvirus (PrV) (NIA-3 strain)was kindly provided by E. Tabares. Finally,mock-infected human DNA was obtained frommonolayers of human embryonic lung fibroblasts.All of the infected and mock-infected cells and theKaposi’s sarcoma skin biopsy were lysed in abuffer (50 mM KCl, 10 mM Tris–HCl pH 8.3)containing 0.5% Tween 20 and 100 mg/mlproteinase K (Boehringer Mannheim). After incu-bation (56°C, 45 min), proteinase K was inacti-vated by heating at 96°C for 10 min. Tostandardize the PCR assay, extracted DNA was
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diluted tenfold in sterile deionized water, andaliquots were stored at −20°C.
2.2. Cloning of PCR products
To obtain well characterized positive controlsand to determine sensitivity of the multiplexPCR method, a clone containing the completetarget sequence for each herpesvirus was ob-tained. Each herpesviral DNA was amplified us-ing specific oligonucleotide primers for the firstamplification, and the product (194 bp) wascloned using the pGEM-T Vector System I(Promega, Madison, WI). Recombinant plasmidswere propagated in Epicurian Coli XL1-Blueelectroporation-competent cells (Stratagene, LaJolla, CA). Transformants were selected on LB/ampicillin/IPTG/X-Gal plates. Plasmid DNA was extracted andpurified using Wizard Plus SV Minipreps DNAPurification System (Promega). The plasmidconcentrations were estimated spectrophotomet-rically at OD260.
To determine the sensitivity of the PCR assay,stock preparations at 2×108 copies/ml were di-luted in 100-fold steps to 2×104, and thereafterin 10-fold steps to 2×10−2 copies/ml. To obtaina weak positive control (sensitivity control, SC),plasmids of each human lymphotropic her-pesvirus were mixed and adjusted to a dilutioncontaining 20 copies/ml, such that 100 copies ofeach herpesvirus were added to a single reactiontube. To include an internal control in each re-action tube, 100 copies of the cloned fragmentof PrV, a non-human herpesvirus, were addedto the lysis buffer used to extract DNA fromclinical specimens.
2.3. Nucleotide sequences
The 194 bp DNA fragment selected for am-plification was derived from the DNA poly-merase gene (Tenorio et al., 1993). Thecorresponding nucleotide sequences for each her-pesvirus were retrieved from the GenBank data-base, either from the complete sequence of thevirion genome, as in the case of EBV (accessionno. X00784, nucleotide positions 154499–
154306), CMV (X17403, 77919–77726), HHV6-A (X83413, 57386–57193), and HHV7 (U43400,52202–52009), or from the sequence of theDNA polymerase gene, as in the case of PrV(L24487, 2191–2384). In the initial stages of de-velopment, the DNA polymerase gene was onlypartially sequenced for HHV6-B (U63465) andHHV8 (U63467), at a region flanking, but notincluding, the 194 bp target fragment. Then,prior to design specific primers, cloning and se-quencing of this region for both viruses wasneeded. A consensus-primer pair (pol2f, 5%-GAYTTYSMHAGYYTVTAYCC-3% and pol3r,5%-ACNARNTCNACNCCYTT-3%), each chosenin two different highly conserved amino acidmotifs (Fig. 1), allowed amplification of a 722bp fragment, which was used for a second hemi-nested PCR round, using pol3r as reverseprimer and specific HHV6-B (SeqH6, 5%-CCAA-GATGCAGTCCGAGCAA-3%) or HHV8(SeqH8, 5%-TAGCGGGTCTCCTGCGGAG-3%)as forward primers. The PCR-amplified prod-ucts, containing the 194 bp target fragment,were cloned as above and sequenced using thestandard dideoxynucleotide method, making useof Sequenase 2.0 (USB, Cleveland, OH) and la-beling with [a-35S]dATP. To exclude mismatchesdue to polymerase mistakes, forward and reversestrands from five clones of each Z29 strain andHHV8 were sequenced. Nucleotide sequences ofthese 194 bp DNA polymerase fragments foreach virus have been submitted to the GenBankdatabase, with accession numbers AJ007662 andAJ007663. The HHV8 genome was derived froma Kaposi’s sarcoma skin lesion in which thepresence of herpesviral DNA from HSV1,HSV2, VZV, EBV, CMV and HHV6 in the skinbiopsy was discarded by the multiplex PCR as-say designed by Tenorio et al. (1993). Althoughthe presence of HHV7 DNA was not discarded,the nucleotide sequence obtained for HHV8 wasdifferent from that known for HHV7. Further-more, full concordance was observed betweenthe sequence obtained for HHV8 and two othernucleotide sequences that have recently beensubmitted to the GenBank database (U93872,nucleotide positions 13582–13775 and U75698,nucleotide positions 13601–13794).
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Fig. 1. Schematic representation of primer positions in the DNA polymerase gene of herpesviruses. (a) Primers used for HHV6-B and HHV8 sequencing. Amplificationwith a consensus primer pair, pol2f and pol3r, was followed by a second hemi-nested PCR round, using pol3r as reverse primer and specific HHV6-B (SeqH6) orHHV8 (SeqH8) as forward primers. The PCR-amplified products contained the 194 bp target fragment. (b1) Primers used for the first amplification round of themultiplex nested-PCR, delineating a 194 bp fragment. (b2) Primers used for the second amplification round. Forward primers were designed to render amplificationproducts of different sizes for PrV (140 bp) and for each of the human herpesviruses, HHV7 (122 bp), HHV8 (97 bp), CMV (78 bp), HHV6-A or HHV6-B (68 bp),and EBV (54 bp).
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2.4. Oligonucleotide primers for the multiplex PCR
Several highly conserved regions were identifiedin the DNA polymerase gene according to thealignment of nucleotide sequences generated bythe MACAW 1.01 program (Schuler et al., 1991).Forward and reverse primers of the first PCRround, delineating 194 bp, and reverse primers ofthe second PCR round, target three of thesehighly conserved regions (Fig. 1). Forwardprimers of the second PCR round were designedto render amplification products of different sizefor PrV (140 bp) and for each of the humanherpesviruses, HHV7 (122 bp), HHV8 (97 bp),CMV (78 bp), HHV6-A or HHV6-B (68 bp), andEBV (54 bp). Primer pairs specific for each virusare listed in Table 1.
Primers used for HHV6 detection are unable todiscriminate between the two recognized variants,HHV6-A and HHV6-B, since both variants were
taken into account when designing the corre-sponding primers. The primer set for HHV6 de-tection previously reported by Tenorio et al.(1993), which was derived from the HHV6-Asequence, was redesigned after sequencing HHV6-B (Z29 strain).
2.5. DNA extraction from clinical specimens
Viral DNA from 50 ml of clinical specimens wasextracted as previously described (Casas et al.,1995) by mixing with 200 ml of a guanidiniumthiocyanate lysis buffer, which has been slightlymodified to include 100 molecules of a cloned andpurified genome fragment of PrV-DNA as inter-nal control. Cold (−20°C) isopropyl alcohol wasadded to precipitate nucleic acids, which werepelleted by centrifugation (14 000×g, 10 min,4°C). The pellet was washed with 70% ethanoland dissolved in 10 ml of sterile double-distilledwater.
2.6. Multiplex nested-PCR assay
A 5-ml aliquot of the appropriate DNA samplewas amplified in a reaction mixture (total volume50 ml) containing 10 mM Tris–HCl (pH 8.3), 50mM KCl, 4 mM MgCl2, 200 mM of each de-oxynucleoside triphosphate, 10 pmol of each for-ward and reverse primer (Amersham PharmaciaBiotech, Sweden) for each herpesvirus, and 1.25 Uof Taq polymerase (Perkin-Elmer, Norwalk, CT).Amplifications were carried out on a PTC-200Peltier Thermal Cycler (MJ Research, Watertown,MA). An initial denaturation step at 94°C for 2min was followed by 30 cycles, consisting of 30 sat 94°C, 1 min at 53°C, and 30 s at 72°C. A finalextension step at 72°C was carried out for 5 min.
After the first amplification round, 1 ml of thereaction product was added to 49 ml of a secondreaction mixture consisting of 60 mM Tris–HCl(pH 8.5), 15 mM (NH4)2SO4, 2 mM MgCl2, 200mM of each deoxynucleoside triphosphate, 10pmol of each forward and reverse primer for eachherpesvirus, and 1.25 U of Taq polymerase. Am-plification was performed under the same condi-tions used for the first round, except that theannealing temperature was 47°C. A total of 10 ml
Table 1Primer sequences for detection and typing of humanlymphotrophic herpesviruses
a Negative control specimens.b Specificity control specimens.c Not done.
of each second round amplification product waselectrophoresed in 4% agarose (MS8, Hispanlab,Spain) in 0.5×TBE gel stained with ethidiumbromide (0.5 mg/ml).
To avoid false positive PCR results by carry-over contamination, aliquoting of clinical speci-mens, preparation of reagents, DNA extraction,first round amplification, and nested-PCR, werecarried out in safety cabinets located in separatelaboratories, all of them far away from the areawhere amplified products were analyzed. Eachcabinet was equiped with an independent set ofreagents, micropipette sets, sterile reagent tubesand filtered pipette tips. Positive results for clini-cal specimens were confirmed by testing a secondfresh aliquot.
2.7. Patients and clinical specimens
To assess suitability and clinical applicability ofthe multiplex PCR method, 35 well-characterizedspecimens, including Kaposi’s sarcoma skin le-sions, serum, cerebrospinal fluid, saliva and urinesamples were tested (Table 2). With the exceptionof saliva samples, which were obtained from fourhealthy volunteers, and Kaposi’s sarcoma skin
biopsies from three HIV-infected patients, all re-maining specimens were obtained from ourarchival collection (stored at −20°C). These spec-imens were selected according to a previous posi-tive result for any of the lymphotropicherpesviruses using different diagnostic tests.These specimens included eight serum samples,four from children with infectious mononucleosis,which had shown EBV-specific serologic findingscompatible with an acute primary EBV infection(VCA-IgM positive, VCA-IgG positive and anti-EBNA negative), and four from hepatic trans-plant recipients, positive for CMV by the pp65antigen test. Five urine samples were from con-genitally infected infants, all positive for CMVculture by shell-vial test. Finally, six cerebrospinalfluid samples were taken from three HIV-infectedpatients with neurological disorders and retinitis,two HIV-infected patients with cerebrallymphoma, and a patient with chroniclymphocytic meningitis, for which the presence ofCMV, EBV and HHV6 had been ascertainedpreviously by an alternative PCR method (Teno-rio et al., 1993).
Negative control specimens consisted of threeserum samples from children with clinical infec-
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Fig. 2. Agarose gel showing the specific bands obtained for each herpesvirus after two rounds of amplification. MW1, 1 Kb DNAladder; MW2, molecular weight ladder prepared in our laboratory with the expected band sizes.
tious mononucleosis, negative for EBV, CMV orHHV6 by serologic tests, and three urine samplesnegative for CMV in culture by shell-vial. Specifi-city control specimens consisted of a CSF samplefrom a child with enteroviral meningitis, positiveby cell culture, and two CSF samples from pa-tients with HSV and VZV encephalitis, diagnosedby DNA detection.
3. Results
The specific bands obtained for each her-pesvirus after two rounds of amplification, result-ing in DNA fragments of 140 bp for PrV, 122 bpfor HHV7, 97 bp for HHV8, 78 bp for CMV, 68bp for HHV6-A or HHV6-B and 54 bp for EBV,are shown in Fig. 2.
The sensitivity of the multiplex nested-PCR as-say was estimated by testing serial 10-fold dilu-tions of specific DNA plasmids containing theamplified products of each herpesvirus. The mini-mal amounts of herpesviral DNA detected afteragarose gel electrophoresis was equivalent to 10–100 molecules, depending on the virus tested. Thespecificity of the method was demonstrated bytesting DNA extracted from cells infected withHSV1, HSV2 and VZV. No specific amplificationof any of these viruses was observed.
Results obtained with clinical specimens areshown in Fig. 3. Specific HHV7-DNA was de-tected in all four saliva samples tested, and spe-cific HHV8-DNA was detected in all three
Kaposi’s sarcoma skin biopsies. Serum samplesrendered the expected products, i.e. a band corre-sponding to EBV for those from children withinfectious mononucleosis (samples 8–11), and aband corresponding to CMV for those from hep-atic transplant recipients (samples 12–15). Nobands were obtained for three sera included asnegative control specimens (samples 16–18). Spe-cific CMV-DNA sequences were also detected infive urine samples that had yielded a positiveresult for CMV culture by shell-vial (samples 19–23) and in CSF samples from HIV-infected pa-tients with neurological disorders and retinitis(samples 27–29). Two other CSF samples fromHIV-infected patients with cerebral lymphomarendered bands corresponding to EBV. Finally,specific HHV6-DNA was detected in a CSF sam-ple from a patient with chronic lymphocyticmeningitis. No bands were evident for CSF sam-ples from patients with enteroviral meningitis,HSV encephalitis and VZV encephalitis, whichwere included as specificity control specimens.
The internal control reactions were positive(140 bp bands) for all clinical specimens tested,with the exception of a single urine sample, whichshowed no amplification after the first PCR anal-ysis, probably due to the presence of DNA poly-merase inhibitors (sample 22). SpecificCMV-DNA was detected after dissolving the re-maining 5 ml resulting from the DNA extractionin 45 ml (dilution 1:10) of sterile double distilledwater.
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Fig. 3. Results of multiplex nested-PCR on clinical specimens. (a) Saliva samples from healthy adults showing a band correspondingto HHV7. (b) Kaposi’s sarcoma skin biopsies showing the specific band for HHV8. (c) Serum samples. Specimens 8–11 fromchildren with infectious mononucleosis syndrome, positive for EBV-DNA. Specimens 12–15 from hepatic transplant recipients,positive for CMV-DNA. Three negative control specimens (16–18) were also included. (d) Urine samples from congenitally infectedinfants (specimens 19–23), positive for CMV-DNA. Three negative control specimens (24–26) were also included. No amplificationof the internal control was initially detected for sample 22 (lane identified as 22*), which revealed presence of CMV-DNA after asecond PCR analysis dissolving the remaining 5 ml resulting from the DNA extraction in 45 ml (dilution 1:10) of steriledouble-distilled water. (e) Cerebrospinal fluid samples. Specimens 27–29 from HIV-infected patients with neurological disorders andretinitis, positive for CMV-DNA. Specimens 30 and 31 from HIV-infected patients with cerebral lymphoma, positive for EBV-DNA.Specimen 32 from a patient with chronic lymphocytic meningitis, positive for HHV6-DNA. Three specificity control specimens(33–35) were also included. MW2, molecular weight ladder prepared in our laboratory with the expected band sizes; SC, sensitivitycontrol containing 100 copies of each human lymphotropic herpesvirus.
4. Discussion
The shared clinical features of lymphotropicherpesviruses and the increasing use of the PCRmethods in their diagnosis led us to develop amultiplex nested-PCR assay for simultaneous de-tection of EBV, CMV, HHV6-A, HHV6-B,HHV7 and HHV8 in a single assay.
One of the main problems with multiplex PCRmethods is to make compatible the oligonucle-otide primers mixed in the PCR reaction. Includ-ing the primer pairs added to detect PrV, theinternal control, a total of 12 primers are used inthe first round of PCR, and 12 different primersin the second nested round. This set of primerswas chosen from a highly conserved region of theDNA polymerase gene of the herpesvirus group.
Most of these primers had been used previously inother multiplex PCR (Tenorio et al., 1993) andreverse transcription (RT)-PCR (Casas et al.,1997) methods, showing excellent compatibility.Other multiplex PCR methods for simultaneousdetection of several human lymphotropic her-pesviruses have been described, but these proto-cols combine only two or three different primersets (McElhinney et al., 1995; Kidd et al., 1998;Moschettini et al., 1998) or use consensus primers,followed by restriction enzyme analysis of am-plified products (Rozenberg and Lebon, 1991).
Each reaction component and all thermal cy-cling parameters were thoroughly standardized,such that a powerful multiplex PCR method wasachieved, capable of detecting between 10 and 100molecules of each lymphotropic herpesvirus, ac-
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cording to our tests using 10-fold dilutions ofspecific plasmids. Sterile deionised water and asuspension of human DNA were used as negativecontrols. The sensitivity of the technique forCMV detection was also evaluated by an externalinternational quality assessment program (ViralQuality Control—VQC—CMV-DNA proficiencystudy, 1997). VQC panels consisted of two ten-fold dilution series of a well-characterized positiveplasma and undiluted positive and negativeplasma samples. Our multiplex PCR method de-tected 43 particles of CMV/ml of plasma, and nofalse positive or negative results were observed.
The results of the multiplex PCR assay aftertesting clinical specimens confirmed expectations.Saliva samples were considered appropriate speci-mens for HHV7 detection, as this virus is a consti-tutive inhabitant of healthy adult saliva accordingto reported isolation rates, ranging from 75 to81% (Wyatt and Frenkel, 1992; Black et al., 1993;Hidaka et al., 1993); HHV7-DNA detection byPCR shows an even higher (96%) prevalence(Kidd et al., 1996). HHV8-DNA was found in allthree Kaposi’s sarcoma skin biopsies from HIV-infected patients, concurring with reported detec-tion rates, ranging from 90 to 100% (Chang et al.,1994; Huang et al., 1995; Moore and Chang,1995; Rady et al., 1995). DNA from EBV, CMVand HHV6 were also detected in CSF samplesfrom HIV-infected patients, in serum samplesfrom transplant recipients and children with infec-tious mononucleosis, and in urine samples frominfants with congenital CMV infection. Viral di-agnosis for all these specimens had been previ-ously established by serologic tests, shell-vialculture, pp65 antigenemia or an alternative PCRmethod.
An internal control, consisting of 100 moleculesof a cloned and purified genome fragment ofPrV-DNA, was included in the DNA extractionbuffer at the beginning of the process, to detectfalse negative results. These are often due to lossof nucleic acid during the DNA extraction stage,reaction failures, or the presence of DNA poly-merase inhibitors. When testing clinical speci-mens, all internal control reactions were positive,except for sample 22. This was a urine samplethat revealed the presence of CMV-DNA after
dissolving the remaining 5 ml from the DNAextraction in 45 ml of sterile double-distilled water,suggesting that the initial lack of amplificationwas due to the presence of reaction inhibitors.
In summary, it is considered that the multiplexPCR method described above has an extensivespectrum of potential application in clinical set-tings in which more than one lymphotropic her-pesvirus could be involved. EBV, CMV, HHV6,HHV7 and HHV8 could be detected and charac-terized using a single assay, avoiding the need totest clinical specimens with separate PCRmethods.
Acknowledgements
We thank Philip E. Pellet (Centers for DiseaseControl, Atlanta, GA) for providing cell suspen-sions of HHV6-B (Z29 strain)-infected Molt-3cells and HHV7 (SB strain)-infected cord bloodlymphocytes, J. Gonzalez and P. Herranz (Servi-cios de Medicina Interna y Dermatologıa, Hospi-tal La Paz, Madrid, Spain) for supplying Kaposi’ssarcoma skin biopsies, and E. Tabares (Facultadde Medicina, Universidad Autonoma, Madrid,Spain) for the gift of purified DNA of PrV (NIA-3 strain). F. Pozo is a predoctoral fellow fundedby the Fondo de Investigacion Sanitaria, Becas deAmpliacion de Estudios (BAE 96/5231 and BAE97/5098). This work was supported by grantsfrom the Fondo de Investigacion Sanitaria (FIS96/0304 and FIS 98/0229).
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