FTIR Spectroscopic Method for Detection of Cells Infectedwith Herpes Viruses

AHMAD SALMAN,1 VITALY ERUKHIMOVITCH,2 MARINA TALYSHINSKY,2 MAHMOUD HULEIHIL,3

MAHMOUD HULEIHEL2

1 Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

2 The Institute for Applied Biosciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

3 Department of Solar Energy and Environmental Physics, Blaustein Institute for Desert Research, Ben-Gurion Universityof the Negev, Beer-Sheva 84105, Israel

Received 9 November 2001; revised 16 February 2002; accepted 16 February 2002Published online 17 July 2002 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.10171

ABSTRACT: Microscopic FTIR spectroscopy was used to investigate the spectral differencesbetween normal cells in culture and cells infected with various members of the herpesfamily of viruses [Herpes simplex (HSV) and Varicella zoster (VZV)]. The main objective ofthis study is to evaluate the possibility of developing microscopic FTIR spectroscopy as asensitive assay for the detection of herpetic infections at their early stages. The advantageof this method over conventional FTIR spectroscopy is that it facilitates inspection ofrestricted regions of tissue. Our results showed significant and consistent differencesbetween all normal and HSV or VZV infected cells that were tested. Detectable andsignificant spectral differences between normal and infected cells are seen as early as 24 hpostinfection, but the damage of the cells (cytopathic effect), caused by the infecting virus,can be seen by optical microscope observations at only 3 days postinfection. An impressiveincrease in the levels of vital cellular metabolites was seen in the herpes virus infected cellscompared to normal cells. It seems that this spectral behavior is unique for infection withherpes viruses, because when these cells were infected with other viruses from differentfamilies like retroviruses, a considerable decrease in the levels of vital cellular metaboliteswas seen in infected cells compared to normal cells. Cluster analysis performed on FTIRmass chromatography yielded 100% accuracy in classifying control uninfected and VZV orHSV infected cells. Our data strongly support the possibility of developing FTIR microscopyas a diagnostic method for early detection of herpetic infections. © 2002 Wiley Periodicals, Inc.Biopolymers (Biospectroscopy) 67: 406–412, 2002

Keywords: spectroscopy; herpes viruses; FTIR microscopy; Varicella zoster virus;retroviruses

INTRODUCTION

Herpes family viruses are involved in various se-vere infections (disorders) in animals and hu-

mans. This family of viruses includes severalmembers like Herpes simplex types 1 and 2(HSV-1, HSV-2) and Varicella zoster (VZV) vi-ruses, which are involved in various infections ofthe skin, eyes, genital system, lungs, and brain.In most cases, it is difficult to distinguish (differ-entiate) between herpetic infection and other pos-sible infections caused by other agents such asbacteria and fungi or irritations of the body, par-

Correspondence to: M. Huleihel (mahmoudh@bgumail.bgu.ac.il).Biopolymers (Biospectroscopy), Vol. 67, 406–412 (2002)© 2002 Wiley Periodicals, Inc.

406

ticularly at the early stages of the infection. Theroutinely used assays for the detection of herpesviruses are cell culture and immunoassays,1,2

which are time consuming and expensive.Scientists have already begun exploring the

application of FTIR spectroscopy in biomedi-cine,3–6 particularly for detecting and monitoringcharacteristic changes in molecular compositionsand structures that accompany cellular changeslike a transformation from a normal to a cancer-ous state.7–11 Several features of IR techniquesindicate that FTIR may be applied as an accurateand sensitive method for the diagnosis and studyof different diseases: IR, having a longer wave-length than UV and visible radiation, penetratesto a greater depth and is absorbed with less scat-tering by the tissue; many of the vibration bandsin the IR region are well resolved, and thereforesubtle changes in the molecular structure may bemonitored during development of the dis-ease.12–15

In our previous studies we used FTIR micros-copy for the detection and characterization of ma-lignant cells. FTIR microscopy enables us to focussolely on specific relevant regions of a tissue orcell culture section and hence to avoid undesir-able signals from unsuitable regions. Our dataproved FTIR microscopy as a good, sensitive, andreliable method for the detection and character-ization of malignant cells transformed by retrovi-ruses.10,16

In the present study we examined the FTIRspectra of normal cells in culture and cells in-fected with either HSV-1 or VZV using FTIR mi-croscopy. Our results showed significant and in-teresting spectral differences between infectedand uninfected cells. The contents of the vitalmetabolites were significantly higher in the virusinfected cells than in the control cells.

MATERIAL AND METHODS

Cells and Viruses

Monkey kidney (Vero), human fibroblast (HF),and rabbit fibroblast (RF) cells were grown at37°C in RPMI medium supplemented with 10%new born calf serum (NBCS) and the antibioticspenicillin, streptomycin, and neomycin.

HSV-1 and VZV stocks were used for infectingthe cells.

Cell Infection and Estimation of Viral Infection

Monolayers of cells grown in 9 cm2 tissue cultureplates were incubated with HSV or VZV at 37°Cfor 2 h at various multiplicities of infection (moi)in RPMI medium containing 2% NBCS. The un-absorbed virus particles were removed, fresh me-dium containing 2% NBCS was added, and themonolayers were incubated at 37°C. At varioustimes postinfection the infected cells were exam-ined by FTIR microscopy and for the appearanceof the cytopathic effect (CPE), defined as areas ofcomplete destruction of cells or of morphologicallymodified cells in the inspected fields, using a lightinverted microscope. The CPE was expressed as apercentage of the damaged cells in the inspectionfield.

Examination of Apoptosis

Apoptotic cells show high activity of the enzymecaspase 3. In order to test for this enzyme activ-ity, our cell extracts were mixed with a substratefor the enzyme, which was a peptide bound top-nitroaniline (CalBioChem Co.). The optical den-sity (OD) at 420 nm was tested and read in aspectrophotometer (high activity of caspase 3gives high OD values at 420 nm).

Preparation of Slides

Because ordinary glass slides exhibit strong ab-sorption in the wavelength range of interest to us,we used zinc sellenide crystals, which are highlytransparent to IR radiation. Normal cells frompassages 3–5 or cells infected with herpes viruseswere picked up from the tissue culture platesafter treatment with trypsin (0.25%) for 1 min.The cells were pelted by centrifugation at 1000rpm for 5 min. Each pellet was washed twice withsaline and resuspended in 100 �L of saline. Thenumber of cells was counted with a hematocytom-eter, and all tested samples were pelted again andresuspended in an appropriate volume of saline togive a concentration of 1000 cells/�L. A 1 �L dropof each sample was placed on a certain area on thesellenide crystal, air dried, and examined byFTIR microscopy. The radius of the 1 �L drop wasabout 1 mm.

FTIR Spectra

FTIR measurements were performed in transmis-sion mode with a liquid nitrogen cooled FTIR

FTIR FOR HERPES CELL DETECTION 407

microscope (Bruker IR Scope II) with an MCTdetector coupled to an FTIR spectrometer (Equi-nox model 55/S, Bruker; OPUS software). Thespectra were obtained in the wavenumber rangeof 600–4000 cm�1 in the mid-IR region. A spec-trum was taken as an average of 128 scans toincrease the signal to noise ratio, and the spectralresolution was at 4 cm�1. The aperture used inthis study is 100 �m. We found that this apertureis the best for a good signal to noise ratio on oneside and a limited preferable region on the otherside. The spectra were normalized to amide I afterbaseline correction. For each cell type, we took atleast five different measurements at various sites.

Statistical Analysis

The spectral differences in the 1000–1480 cm�1

region between uninfected and infected cells werestatistically assessed by t test.

Cluster Analysis

Cluster analysis is an unsupervised techniquethat examines the interpoint distances betweenall the samples and it represents the informationin the form of a 2-dimensional plot, known asdendrogram. The dendrogram presents the datafrom high-dimensional row spaces in a form thatfacilitates the use of human pattern recognitionabilities. To generate a dendrogram, cluster anal-ysis methods form clusters of samples based ontheir nearness in row space. Cluster analysis wasperformed with good quality spectra having highsignal to noise ratios. The total number of spectraused in each analysis was 150, including normaland infected samples. The Ward’s minimum vari-ance method was used for the cluster analysis,which was provided in the OPUS software.

RESULTS AND DISCUSSION

CPE of Infected Cells with Herpes Viruses

Infected cells with herpes viruses are damagedafter several rounds of replication of the infectingvirus [3–4 days postinfection (p.i.)]. The progenyof the infecting virus reinfect neighboring unin-fected cells. The damaged cells represent the CPEof these viruses.

Vero cells grown on plastic dishes in RPMImedium with 2% NBCS appear as flat cells [Fig.1(a)]. At day 1 or 2 after infection with the herpes

viruses, the infected cells are morphologicallyidentical to normal uninfected cells [Fig. 1(b)].Moreover, these infected cells lose their flat shapeand become round at 3–4 days p.i. [Fig. 1(c,d)].

Cell Apoptosis

Uninfected and infected cells with 1 moi of herpesviruses (HSV-1 and VZV) were examined forapoptosis at various times postinfection. Our re-sults (Fig. 2) show an increase in caspase 3 activ-ity as a function of the time of infection. Thehighest activity values were obtained at 3 daysp.i. These results indicate that infected cells withherpes viruses started the apoptosis process asearly as 24 h p.i.

Comparison of IR Spectra for Normal and HSV orVZV Infected Cells

Cells from different mammalian species (Vero,HF, and RaF) were infected with HSV-1 or VZV.At various times postinfection the cells were ex-amined by FTIR microscopy.

An analysis of the FTIR spectra of the cellsattributed the dominant bands at 1655 and1546 cm�1 to protein amide I and II bands,respectively.17,18 The shoulder at about 1730cm�1 was attributed to lipid CAO stretching vi-brations.17,19 The band at 1465 cm�1 was as-

Figure 1. (a) Uninfected Vero cells, (b) HSV-1 in-fected Vero cells at 24 or 48 h p.i., (c) HSV-1 infectedVero cells at 72 h p.i., and (d) VZV infected Vero cells at72 h p.i.

408 SALMAN ET AL.

signed to the CH2 bending mode of the cell lipids.The bands at 1454 and 1397 cm�1 were due to theasymmetric and symmetric CH3 bending modes ofmethyl groups and branched methyl groups ofproteins and lipids, respectively.11,20 From infor-mation obtained from previous studies,15,17,18,21

we assigned the remaining IR bands as follows:the peaks at 1237 and 1082 cm�1 were attributedto PO2

� asymmetric and symmetric stretching vi-brations, respectively, in nucleic acids. The peakat 1064 cm�1 resulted from the overlap of severalbands, including absorption due to the vibrationalmodes of OCH2OH and the COO stretching vi-bration coupled to the COO bending mode of cellcarbohydrates.

Our results showed that the FTIR spectra ofcells infected with herpes viruses differed mark-edly from those of normal cells taken from thedifferent mammalian species. In general, the dif-ference between the spectra was evident in thehigher intensities of absorbance of the cells in-fected with HSV-1 or VZV (Figs. 3–5).

The level of amide II was significantly higherin all infected cells compared to the normal cells.

In addition, we found that the phosphate con-centrations in the infected cells were much higherthan those in control uninfected cells as can beseen in Figure 6 and Table I. The amount ofphosphates presented in Table I was calculated asthe integrated areas in the 950–1350 cm�1 re-gion.

The ratio of the absorbance at 1121 cm�1 tothat at 1020 cm�1 provides a measure of the cel-lular RNA/DNA ratio,23 whereas the ratio of theabsorbance intensity at 1045 cm�1 to that at 1545cm�1 gives an estimate of the carbohydrate con-centrations.24 Our findings (Table I) showedlower values for the RNA/DNA ratio and highervalues for the carbohydrates for the infected cells(with either HSV or VZV) versus those for normalcells. These results could be explained by thelower metabolic activity of the infected cells com-pared to the normal cells.

Figure 2. Cell apoptosis. The caspase 3 activity of (�)control uninfected Vero cells or cells infected with ei-ther (F) HSV-1 or (Œ) VZV was tested at various timespostinfection.

Figure 3. FTIR microspectroscopy of uninfected Verocells and HSV-1 infected cells at 24 or 72 h p.i.

Figure 4. FTIR microspectroscopy of uninfected Verocells and VZV infected cells at 24 or 72 h p.i.

FTIR FOR HERPES CELL DETECTION 409

IR Spectra at Various Times Postinfection withHerpes Viruses

Vero cells were infected with 0.5 moi/cell of eitherHSV-1 or VZV and examined by FTIR microscopyat various times after infection. Our data showhigher spectral discrepancies between normaland infected cells at 3 days p.i. compared to the 1day p.i. in cells infected with either HSV-1 (Fig. 3)or VZV (Fig. 4). However, the spectral discrepan-cies between normal and cells infected with eitherof these viruses at 1 day p.i. were statisticallysignificant as assessed by a t test (p � 0.001) andenough for detection of infected cells. It is impor-tant to point out that at 24 or 48 h p.i. with herpesviruses, the infected cells are morphologicallyidentical to normal uninfected cells [Fig. 1(a,b)].These results correlate with our results showingan increase in caspase 3 activity, which indicatethe presence of apoptotic cells,24 as early as 24 hp.i. with herpes viruses (Fig. 2). Thus, it seemsthat FTIR microscopy can be used as a sensitivemethod for the early diagnosis of herpetic infec-tions.

In addition, normal uninfected and HSV orVZV infected cells were classified by cluster anal-ysis, which is considered as one of the simplestand fastest procedures, that was adopted to clas-sify the FTIR spectra. Cluster analysis was per-formed for different segments of the spectra toobtain the best grouping results. The best resultswere obtained in the 950–1350 cm�1 region. Ta-ble II shows the results of our cluster analysis ofthe FTIR spectra of normal uninfected cells (Veroand HF cells) and cells infected with either HSVor VZV. It can be clearly seen that this classifica-tion method provided 100% accuracy in differen-

tiating between normal uninfected cells (in bothkinds of cells Vero and HF) and cells infected witheither HSV or VZV. Also, using this assay wecould distinguish between cells infected with HSVand others infected with VZV with a significantaccuracy of about 85–92%.

More detailed studies are required to gain in-sights into specificity and the development of invivo mode applications in the future.

Comparison of Spectra of Herpes Virus InfectedCells with Retrovirus Infected Cells

In order to confirm whether the spectral changesobtained as a result of the infection of cells withherpes viruses were unique to this family of vi-ruses and not general for all viral infections, wecompared them with spectra of other viral infec-tions. As a first step we chose one of the retroviralfamily members, murine sarcoma virus (MuSV),which is capable of transforming normal cells tomalignant cells in culture.

The FTIR spectra of normal RF cells were com-pared to those of RF cells infected with eitherHSV-1 or MuSV at 3 days p.i. Our data (Fig. 7)showed that the FTIR spectra of MuSV infectedcells differed markedly from those infected withHSV-1. The most significant differences were inthe 1000–1500 cm�1 region. The bands in thisregion represent PO2

� symmetric and asymmetricstretching vibrations, a CH2 bending mode of thecell lipids, and symmetric and asymmetric CH3

Figure 6. FTIR microspectroscopy in the 1200–1400cm�1 region of the various normal and HSV-1 or VZVinfected cells: (�) HF, (E) Vero, (‚) HF HSV-1, (Œ) HFVZV, (�) Vero HSV, and (�) Vero VZV cells.

Figure 5. FTIR microspectroscopy of uninfected HFcells and either HSV-1 or VZV infected cells at 72 h p.i.

410 SALMAN ET AL.

bending modes. The metabolic turnover is re-flected in the phosphate levels, which are madeup from contributions from energy producers,such as ATP and GTP, and other biomolecularcomponents, including phospholipids, nucleic ac-ids (DNA and RNA), and phosphorylated pro-teins. In this study the intensity of the absor-bance due to these PO2

� vibrations was muchhigher for HSV infected cells compared to MuSVinfected cells (Fig. 7).

In addition, it is clear that in all tested casesthe absorbance due to PO2

� vibrations were higherfor HSV infected cells compared to normal cellswhereas in the case of MuSV infected cells theabsorbance was lower than normal cells (Fig. 7).These results are in agreement with our previousdata16 showing that NIH/3T3 cells (mouse fibro-blasts) transformed by MuSV display lower IR

absorbance due to PO2� vibrations compared to

normal cells.The spectral differences between cells infected

with herpes viruses or retroviruses are mostlikely related to their different activity. Retrovi-ruses are involved in the transformation of nor-mal to malignant cells whereas herpes viruses arelytic, which cause the death of the infected cellsseveral days postinfection (4–5 days p.i.). Thispossibility is supported by previous findings25

showing IR spectral differences between cells atvarious phases (S, G1, or G2 phases) of theirreplication cycle.

The spectral results obtained in this study arealso in agreement with our results demonstratingsignificant activity of caspase 3, which is associ-ated with apoptosis of cells, as early as 24 h p.i.with herpes viruses (Fig. 2), although the retro-virus infected cells did not show any activity ofcaspase 3 even several days postinfection (datanot shown). For more understanding of thesespectral discrepancies and variations, furtherspectral examinations of cells infected with otherviruses from each group of viruses (lytic or retro-

Figure 7. FTIR microspectroscopy of uninfected RFcells and either HSV-1 or MuSV infected cells at 3 daysp.i.

Table I. Phosphates, Carbohydrates and RNA/DNA Levels for Normal and VeroCells Infected with HSV or VZV at 3 Days Postinfection

Normal Vero Cells

Vero Cells Infected With

HSV VZV

Phosphate levels 117.9 � 4.7 201.9 � 7.3 236.9 � 15.0RNA/DNA ratio 1.38 � 0.05 1.30 � 0.02 1.26 � 0.03Carbohydrate levels 29.3 � 1.47 38.08 � 1.53 47.08 � 1.88

Table II. Results of Cluster Analysis for Normaland Infected Vero and HF Cells

NormalVero Cells

VeroInfected

Cells with

HSV VZV

Normal Vero cells 100% 0% 0%Vero cells infected with

HSV 0% 85% 8%VZV 0% 15% 92%

NormalHF Cells

HF InfectedCells with

HSV VZV

Normal HF cells 100% 0% 0%HF infected cells with

HSV 0% 88% 10%VZV 0% 12% 90%

FTIR FOR HERPES CELL DETECTION 411

viruses) and cells treated with agents inducingapoptosis like phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate26 are being done in our labo-ratory.

CONCLUSIONS

FTIR microspectroscopy proved to be a usefultechnique in distinguishing normal from herpesviruses infected cells at early stages of infection.The phosphate levels and other metabolite con-centrations, such as proteins and carbohydrates,were higher in the herpes virus infected cells.Therefore, it seems certainly worthwhile to con-tinue with the development of FTIR microscopyfor the purpose of herpes viruses infection diag-nosis.

REFERENCES

1. Megdam, M. M.; Todd, D.; Al-Abosi, M. Microbio-spectroscopy 2001, 105, 111–118.

2. Markoulatos, P.; Fountoucidou, P.; Marinakis, G.;Krikelis, V.; Spyrou, N.; Vamvakopoulos, N.; Mon-cany, M. L. J Clin Lab Anal 1997, 11, 146–153.

3. Cohenford, M.; Godwin, T. A.; Cahn, F.; Bhandare,P.; Caputo, T. A.; Rigas, B. Gynecol Oncol 1997, 66,59–65.

4. Wong, P. T.; Wong, R. K.; Caputo, T. A.; Godwin,T. A.; Rigas, B. Proc Natl Acad Sci USA 1991, 88,10988–10992.

5. Jackson, M.; Kim, K.; Tetteh, J.; Mansfield, J. R.;Dolenko, B.; Somorjai, R. L.; Orr, F. W.; Watson,P. H.; Mantsch, H. H. SPIE 1998, 3257, 24–34.

6. Mantsch, H.; Chapman, D. Infrared Spectroscopyof Biomolecules; Wiley: New York, 1996.

7. Diem, M.; Boydston-White, S.; Chiriboga, L. ApplSpectrosc 1999, 53, 148–161.

8. Mordechai, S.; Argov, S.; Salman, A.; Cohen, B.;Ramesh, J.; Erukhimovitch, V.; Goldstein, J.;Sinelnikov, I. SPIE 2000, 4129, 231–241.

9. Franck, P.; Nabet, P.; Dousset, B. Cell Mol Biol1998, 44, 273–275.

10. Huleihel, M.; Talyshinsky, M.; Erukhimovitch, V.Spectrosc Int J 2001, 15, 57–64.

11. Wong, P.; Goldstein, S.; Grekin, R.; Godwin, A.;Pivik, C.; Rigas, B. Cancer Res 1993, 53, 762–765.

12. Yazdi, H. M.; Bertrand, M. A.; Wong, P. T. ActaCytol 1996, 40, 664–668.

13. Benedetti, E.; Bramanti, E.; Rossi, I. Appl Spec-trosc 1997, 51, 792–797.

14. Chiriboga, L.; Xie, P.; Zarou, D.; Zakim, D.; Diem,M. Cell Mol Biol 1998, 44, 219–229.

15. Yang, D.; Castro, D.; El-Sayed, I.; El-Sayed, M.;Saxton, R.; Nancy, Y. SPIE 1995, 2389, 543–550.

16. Huleihel, M.; Salman, A.; Erukhimovitch, V.; Ja-gannathan, R.; Hammody, Z.; Mordechai, S. J Bio-phys Biochem Methods 2001.

17. Wong, P. T.; Wong, R. K.; Caputo, T.; Godwin, T. A.;Rigas, B. Proc Natl Acad Sci USA 1992, 88, 10988–10992.

18. Krupnik, E.; Jackson, M.; Bird, R. P.; Smith, I. C.;Mantsch, H. H. SPIE 1998, 3257, 307–310.

19. Binding, U.; Wasche, W.; Liebold, K.; Winter, H.;Gross, U. M.; Frege, P.; Muller, G. SPIE 1998,3568, 38–45.

20. Rigas, B.; Morgello, S.; Goldman, I. S.; Wong,P. T. T. Proc Natl Acad Sci USA 1990, 87, 8140–8144.

21. Wong, P.; Lacelle, S.; Yazdi, H. Appl Spectrosc1993, 47, 1830–1836.

22. Andrus, P. G.; Strickland, R. D. Biospectroscopy1998, 4, 37–46.

23. Parker, F. S. Application of Infrared Spectroscopyin Biochemistry, Biology and Medicine; Plenum:New York, 1971.

24. Demeester, N.; Baier, G.; Enzinger, C.; Goethals,M.; Vandekerckhove, J.; Rosseneu, M.; Labeur, C.Mol Membr Biol 2000, 17, 219–228.

25. Boydston-White, S.; Gopen, T.; Houser, S.; Bargon-etti, J.; Diem, M. Biospectroscopy 1999, 5, 219–227.

26. Torgeman, A.; Ben-Aroya, Z.; Huleihel, M.; Gruns-pan, A.; Zelin, E.; Hallak, M.; Lochelt, M.; Flugel,R. M.; Wolfson, M.; Kedar, I.; Aboud, M. Exp CellRes, submitted.

412 SALMAN ET AL.