Beverly Barton Rogers,* Stephen L.Josephson,t and Solida K. Mak*From the Departments ofPathology, Women and Infants'Hospital and Rhode Island Hospital, t Rhode IslandHospital, and the Brown University Program in Medicine,Providence, Rhode Island
The polymerase chain reaction (PCR) was used toamplify herpes simplex virus DNA using a single setofprimers that amplify both herpes simplex virus I(HSVI) andH (HSVII). The viruses can be differenti-ated by a single restriction enzyme cleavage. Virusfrom dilutions ofHSV-infectedA549 cell suspensionswere amplified and the infectivity endpoints of cellculture were compared with the PCRJ and with an-other direct detection method, the enzyme-linkedimmunosorbent assay (ELISA). The PCR was capableof detecting virus at a 10-4 dilution for both HSVIand MMII, when the corresponding TCID50 endpointswere lo-59 and 10 -57 respectively. The EUSA de-tected virus only doum to the 10' dilution The am-plification procedure showed the greatest sensitivitywhen an initial protease digestion was followed byfiltration ThePCR may have use in detection ofHSVin clinical situations in which a rapid result is de-sirable. (AmJ Pathol 1991, 139:1-6)
The detection of herpes simplex virus (HSV) has impor-tant implications for patient care, particularly for the preg-nant patient, who may pass the infection to the fetus inutero or at the time of delivery.' Virus isolation in cell cul-ture has been the method of choice for detecting HSV.However, in instances in which rapid diagnosis is desir-able, such as the detection of HSV before delivery, cellculture diagnosis may not be timely.2 Other methodssuch as immunofluorescence and enzyme-linked immu-nosorbent assay (ELISA) have been used in place of cul-
ture to detect HSV directly in clinical specimens, therebyresulting in a more rapid diagnosis.3'4 However thesemethods lack the sensitivity of cell culture isolation.5'6Consequently it has been necessary to consider alterna-tive approaches.
The polymerase chain reaction (PCR), an efficient andrapid DNA amplification procedure, has been applied tothe detection of a wide variety of viruses, includingherpes simplex virus.7-'O All of the studies involving HSVhave used primers to amplify small fragments of the HSVgenome, requiring hybridization for confirmation of thePCR product and differentiation of HSVI and HSVII. How-ever this additional step of hybridization increases theassay time by 1 to 2 days. We have designed primersthat amplify HSVI and HSVII simultaneously and allowdifferentiation of the two viruses using restriction enzymecleavage without the necessity for hybridization. This re-sults in a diagnosis in 8 to 10 hours from the time ofreceipt of the specimen. We have determined the detec-tion limits of the PCR procedure for HSV and have testeda number of DNA viruses to confirm the specificity of theprimers. Furthermore we compared the PCR results todirect antigen detection.
Materials and Methods
Cell CultureAs a preliminary step, nine representative stock isolatesof HSVI (n = 4) and HSVII (n = 5) were grown in HumanEmbryonic Lung (HEL) cell monolayers until 95% cyto-pathic effect (CPE) was observed. Undiluted superna-tants from these cultures then were tested for the pres-ence of HSV using PCR followed by endonuclease cleav-age and hybridization. In addition, one isolate each of
Accepted for publication April 17,1991.Address reprint requests to Beverly B. Rogers, MD, Department of
Pathology, Women and Infants' Hospital, 101 Dudley St., Providence, RI02905.
1
2 Rogers, Josephson, and MakAJPJuly 1991, Vol. 139, No. 1
cytomegalovirus, adenovirus, and varicella-zoster viruswere evaluated using the same methodology. The cyto-megalovirus and varicella were grown in HEL cells andthe adenovirus in HEp-2 cells.
Detection Limits
The detection limits of the PCR procedure described be-low were determined by testing dilutions of HSV-infectedcell suspensions. Briefly, cell culture tubes containingA549 cell monolayers heavily infected (more than 95%CPE) with either HSVI or HSVII were harvested by replac-ing the cell culture medium with 1 ml of phosphate-buffered saline (PBS), dislodging the adherent cells witha scraper, and disrupting cell clumps with a pasteur pi-pette. Serial 1 0-fold dilutions (10- 1 to 1 -8) of the result-ing HSVI- and HSVII-infected cell suspensions were pre-pared in PBS. Each dilution was inoculated into A549 cellculture tubes (five tubes per dilution) to determine theinfectivity endpoint. The TCID50 (tissue culture infectivedose) for the HSVI and HSVII suspensions was calcu-lated by the standard Karber method.1 1 All of the dilutionswere assayed for the presence of HSV using PCR fol-lowed by endonuclease cleavage and hybridization. Fur-thermore the dilutions were assayed for the presence ofHSV using a commercially available HSV antigen captureELISA. The results were compared to the TCID5Q.
HSV Antigen Capture ELISA
The HSV antigen capture ELISA from Ortho Diagnostics(Raritan, NJ) was used to detect HSVI and HSVII antigendirectly in samples. The test was done according to themanufacturer's instructions.
Polymerase Chain Reaction
The PCR was done using primers that have homology toboth HSVI and HSVII and span a 476-nucleotide (nt) seg-ment in the DNA polymerase gene. The upstream primersequence is 5'-CAGTACGGCCCCGAGTTCGTGA-3'and the downstream sequence is 5'-GTAGATGGT-GCGGGTGATGTT-3'. The PCR was done according toprocedures previously published.12 Briefly, reactionstook place in a total volume of 100 microliters (,ul) con-taining 50 mmol/l (millimolar) NaCI, 10 mmol/l TRIS-CI,pH8.3, 1.5 mmol/l MgC12, and 100 ,umol/l (micromolar)each dNTP. The dNTPs were purchased from CetusCorp. (Cetus, Emeryville, CA) and the remainder of thereagents were combined into a PCR buffer in our labo-ratory. After adding the DNA template (see below), the
test samples were overlaid with mineral oil and boiled for5 minutes to destroy nuclease activity and to lyse intactcells. The tubes were allowed to cool for a few minutesbefore adding 0.5 ,ul of Taq DNA polymerase (Cetus). ADNA Thermal Cycler was used (Perkin Elmer-Cetus, Nor-walk, CT) to amplify the specimens. An initial 950C soakfor 5 minutes was followed by 35 cycles of 940C for 1.5minutes; 650C for 2 minutes; and 720C for 2 minutes. Anadditional 2 seconds was added to the 720C extensionstep with each cycle. This results in optimal extensiontime for the polymerase to act as the amount of templateincreases. We found that the additional time was neces-sary to obtain optimal amplification.
The amplified products were electrophoresed in a 4%agarose gel (1% Ultrapure, Bio-Rad, Richmond, CA, and3% NuSieve, FMC BioProducts, Rockland, ME) and aSouthern blot was prepared using 0.2 ,um Nytran and1OX sodium chloride sodium citrate according to stan-dard methods (Schleicher and Schuell, Keene, NY). Hy-bridization was performed using oligonucleotide probesspecific for either HSVI or HSVII and labeled with32PddATP using a 3' end-labeling kit (Boehringer-Mannheim, Indianapolis, IN). The sequences of the inter-nal oligonucleotide probes are 5'- AGGCCGTCTTGAAG-GACAA-3' for HSVII and 5'-TACTGCATACAGGAT-TCCC-3' for HSVI. Hybridization took place overnight atTm -50C (51°C for HSVI and 530C for HSVII) and washeswere carried out at the Tm (560C for HSVI and 580C forHSVII). Autoradiography was done for approximately 2hours using DuPont Intensifying Screens (Hoffman Es-tates, IL) at - 700C.
Fragments seen using ethidium bromide-stained gelswere cleaved with Avail restriction enzyme (InternationalBiotechnologies, Inc., New Haven, CT) by placing 10 ,ulof the PCR product into a microfuge tube and adding 2 ,ulof 1 X buffer, 1 ,ul of the enzyme, and 7 ,ul of water. Thereis a single Avall site in the amplified sequence of HSVIIthat cleaves the 476-nt product into 87- and 389-nt frag-ments. An additional Avall site is present in the amplifiedfragment of HSVI, resulting in fragments of 87, 183, and206 nt. This allows differentiation of HSVI and HSVII witha single restriction enzyme cleavage.
The diluted cell suspensions used in the determina-tion of PCR detection limits were processed in two sep-arate ways for the PCR. A 250-p1 aliquot was placed intoa 500-,lI microfuge tube and spun in a fixed-speed mi-crocentrifuge with a fixed-angle rotor at 16,000g for 10minutes. The supernatant was aspirated from the pelletand the pellet retained to do the PCR directly. An addi-tional 250 ,ul of each dilution of the cell suspensions wasdigested with 500 ,ug/ml (12.5 pl1 of a 1 0-mg/ml solution)proteinase K (Boehringer-Mannheim) at 550C for 1 hourand transferred to a microcentrifuge tube containing a0.22-,um low-binding Durapore microporous membrane
Polymerase Chain Reaction and Herpes Virus 3AJP julu 1991, Vol. 139, No. I
(Millipore Corp., New Bedford, MA). The samples werecentrifuged at 2000g until all the liquid had passedthrough the filter (approximately 5 minutes). The filtratewas transferred to a microcentrifuge tube with a 100,000Nominal Molecular Weight Limit, polysulfone ultrafiltration(UF) membrane (Millipore Corp.) and centrifuged at2000g until only 70 [LI remained above the filter (approx-imately 15 minutes). Two hundred fifty microliters of TE(10 mmol/l TRIS, pH 8.0,1.0 mM mmol/l ethylenediaminetetra acetic acid) was added and centrifugation was con-tinued until only 70 ,u remained above the filter. Forty-fivemicroliters of the remaining 70 RI (called the filtered sam-ple) was taken to do the PCR.
The superinfected stock virus cultures used to evalu-ate the specificity of the primers were not filtered or cen-trifuged before amplification. Because these culturescontained high titers of virus, we used 5 RI directly in thePCR mixture.
Results
As expected, PCR amplification of genomic DNA presentin nine HSV stock cultures yielded a 476-nt product. Sub-sequent cleavage of the PCR products with Avall endo-nuclease resulted in fragments consistent with eitherHSVI or HSVII (Figure 1). This methodology correctlyidentified four isolates as HSVI and five as HSVII. Furtherconfirmation of viral typing as determined by restrictionenzyme cleavage was obtained when the PCR productswere hybridized with HSV-type specific probes (data notshown).
When other DNA viruses, including adenovirus, cyto-megalovirus, and varicella-zoster virus, were subjected toPCR amplification with the HSV primers, specific DNAproducts were not detected in ethidium bromide-stainedgels. Hybridization also did not detect nonspecific ampli-fication of these viruses.
To optimize conditions and determine the detectionlimits of the PCR procedure, serial 10-fold dilutions ofHSVI and HSVII cell suspensions each with a known levelof infectivity (TCID50) were evaluated by PCR using twodifferent processing methods. In one case, a portion ofeach dilution was centrifuged at 16,000g. After removingthe supernatant, the pellets were tested in the PCR. Al-ternatively an identical portion of each dilution was di-gested with proteinase K and then filtered several timesto remove debris larger than 0.2 ,um and low-molecular-weight components (less than 100,000 MW).The remaining filtered material was tested in the PCR.With the sediments obtained by centrifugation alone, wedetected HSVI and HSVII down to the 10-3 dilution byusing direct gel analysis and restriction enzyme cleavage(Figure 2a and Table 1). When the hybridization step was
-476-389
-206-183
-87
Figure 1. 7he etbidium bromide-stained gel shows 476-nt ampli-fiedproductsfor HSVI and HSVII in lanes labeled I and II, respec-tivelv. Restriction enzyme cleavage uwith AvaI gives 389- and 87-ntfragments for HSVII (lane IIC) and 206-, 183-, and 87-nt frag-mentsfor HSVI (lane IC). Mlolecular-uweight size markers (lane M)are a HaeIII digest ofPhiX174 andgive bands of 1350, 1080, 870,603, 310, 270, 230, 194, and 118 nt top to bottom.
performed, the virus also was detected in the 10-4 dilu-tion (Figure 2b) using a 2-hour radiograph exposure.However it should be noted that we did not detect virus inthe 100 and the 10 -1 dilutions by analysis of the pelletspun from the 250 ,ul. When analyzing the filtered spec-imens, HSV was detected in all dilutions down to 10-4 byboth direct gel analysis and hybridization after a 2-hourexposure of the radiograph. In the case of the antigencapture ELISA, the detection endpoint for HSVI and HS-VII was 10-1. Virus antigen was not detected at higherdilutions (10-2 to 10 -8). The infectivity titers of the undi-luted HSVI and HSVII cell suspensions were 105-9TCID,J0.2 ml and 105 7 TCID5J0.2 ml, respectively.
Discussion
Herpes simplex virus infection of the fetus or neonateresulting from exposure to the virus at the time of deliverycan cause a severe, disseminated infection and evendeath.13 Studies have shown that 1.4% of women with ahistory of genital herpes infection will shed the virus at thetime of delivery.2 If infection is suspected, HSV is routinely
ivi II li (") i IC Ivi
4 Rogers, Josephson, and MakAJPJu4l 1991, Vol. 139, No. 1
Sediment
10 10-1 10 :T 10i 10
Sediment
CO 3 Filtered C-)c_:>aJ ---- ------------------ ---- ,-
:7 :s- ino in in- in.- in. in n L
Filtered
z~ E* 10, 10 10 10....I loml ,l O
Figure 2. a: Ethidium bromide-stained gel ofHSVI amplifiedproductsffrom representative di-lutions ofvirusfrom 1(01 to 10 The amplifiedproducts are present onl4' in the 10'2 and10-3 dilutions in specimens amplifiedffrom thecell pellet (sediment), but amplified productsare seen in dilutions 10° to 10-4 when ana-lvzing thefiltered specimens treated u'ith an ini-tial proteinase K step (filtered). b: The hybrid-ization for HSVI shous positive resultsfor dilu-tions 10-2 to 10-4for the sediment and 10j to10- 4 for the filtered specimens. Films were ex-posed at - 70°Cfor 2 hours.B
cultured during the last week of pregnancy. However theresults may not accurately predict the presence or ab-sence of virus at the time of delivery due to the typicalshort period of asymptomatic shedding. Consequentlythe most reliable results are obtained when the patient istested near the time of delivery.We have designed a PCR procedure that can provide
a rapid diagnosis of HSV within 1 day, which could beused in the situation described above. Polymerase chain
reaction amplification of herpes DNA followed by Avallrestriction endonuclease cleavage of PCR products canbe used to detect and identify HSVI and HSVII usingdirect gel analysis, without the need for time-consuminghybridization. As with most PCR procedures used to de-tect viral genome, the possibility of identifying defective ordead virus exists, and the significance of a positive cul-ture in a clinical setting remains to be determined.
While developing the PCR procedure for the detection
Table 1. Dilution ofDetectable Virus Versus TCID50
CellCulture
TCID50
10-5910-57
HSVIHSVII
PCR
Sediment
Direct
10-310-3
Filtered
Hyb
10-410-4
Direct
10-410-4
Hyb
10-410-4
Direct, direct gel analysis.Hyb, hybridization result.
of HSV, we observed that the method by which speci-mens were processed for PCR testing affected the sen-sitivity of the test. Suspensions of cells infected with HSVIand HSVII, instead of viral suspensions alone, were usedto evaluate different processing methods. This approachwas taken because clinical specimens, normally col-lected with a sterile swab, could be expected to containprimarily cellular material.
Optimal PCR results were achieved using proteinaseK and a multistep filtration processing method rather thancentrifugation and analysis of a pellet alone. We wereunable to detect virus in the 10° and 10-1 dilutions byanalysis of the pellet, whereas virus was easily amplifiedfrom these dilutions after filtration. The most logical expla-nation for this result is that inhibition of the PCR occurredwith the 1 0° and 10-1 dilutions. Because we were usingdilutions of cells containing virus, the higher dilutions of1 O° and 101- would not only have greater amounts ofvirus but also greater numbers of cells. This would resultin an increased burden of nucleases or cellular inhibitorsthat could affect the PCR. It is known that inhibitors to thePCR exist14'15 and these inhibitors can be detected bydoing an additional amplification using a set of primersthat amplify a part of the human genome as a control. It isalso possible, but less likely, that manipulation of the pel-let when analyzing the cell pellet after centrifugation maybe responsible for the lack of detection of HSV in the 1 0°and 10-1 dilutions. As seen here, purification of speci-mens, although adding 2 hours to the procedure, may benecessary to ensure uniform results.
The PCR detected virus at a 10-4 dilution for bothHSVI and HSVII, while the corresponding TCID,0 end-points were 10-59 and 10-57, respectively. The abilityof cell culture to detect a lower amount of virus than thePCR has been described before.14 Although our PCRprocedure was between 10 and 100 times less sensitivethan culture, it was 1000 times more sensitive than theELISA. This difference in the sensitivity of PCR relative toculture was confirmed when the experiment was re-peated. We achieved an increase in the sensitivity of thePCR procedure by exposing the radiograph for 24 hoursinstead of 2 hours. Virus was detected in the 10-5 dilutionafter a 24-hour exposure of the radiograph obtained fromanalysis of the filtered specimens. However we have de-cided to limit the exposure time to approximately 2 hoursto adapt this technology to the clinical setting.
The ability of a PCR procedure to amplify the targetDNA depends on the primers chosen, the form andquantity of the template, the size of the fragment, the tem-perature for annealing of the primers, and the number ofcycles chosen. We do not see significant nonspecific am-plification by our primers, and they do not have significantcomplementarity with each other, indicating that there isno loss in efficiency of amplification due to 'primer-dimer'
formation. Our annealing temperature and the number ofcycles in the PCR were optimized in earlier experiments,and it was noted that a 2-second extension at 72°C witheach cycle gave best results. While it may be possible toamplify more efficiently a fragment shorter than 476 nt, wechose this length because it was necessary to pick anarea of total homology in the DNA polymerase gene forHSVI and HSVII that included restriction enzyme sites toallow viral typing. The primers selected had these char-acteristics. It may be possible to use nested primers toincrease the sensitivity, but this would add another day tothe procedure.
The large amount of homology present in the DNApolymerase gene allowed us to pick primers that recog-nize both HSVI and HSVII. However the potential forcross-reactivity of the primers to other DNA viruses exists.To address this, we analyzed varicella-zoster virus, cyto-megalovirus, and adenovirus and did not demonstratecross-reactivity of the primers with these viruses. Also toensure the fidelity of the sequences to which the primersanneal in the DNA polymerase gene, we amplified a totalof nine isolates (four HSVI and five HSVII) and obtainedamplification in each case.We designed a PCR procedure for the amplification of
HSVI and HSVII using a single set of primers. The ampli-fication product can be confirmed and the viruses typedby a single restriction enzyme cleavage. We also deter-mined a method of purification of the DNA before PCRprocessing that may ensure the reliability of the PCR.
Acknowledgments
The authors thank Mary Ellen Sullivan, Linda Covill, Priscilla Mc-Dermott, Judy Thibaudeau, Dana Chiolfi, Jacqueline Costello,Janet Gardner, and Fatima Muriel for technical assistance andDr. Don B. Singer for continued support and careful review of themanuscript.
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