Journal of Virological Methods, 38 (1992) 195-204 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00

VIRMET

195

01341

Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus Erik C. Stabell and Paul D. Olivo Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO (USA)

(Accepted

14 January

1992)

Summary A cell line which can be used in a simple, sensitive, and rapid histochemical assay was isolated for detection of herpes simplex virus (HSV). The cell line was derived by selection of G418 resistant colonies following co-transfection of baby hamster kidney cells with a plasmid which contains a G418 antibiotic resistance marker and a plasmid which contains the Escherichia coli LacZ gene placed behind an inducible HSV promoter. The promoter is from HSV-1 UL39 which encodes ICP6, the large subunit of ribonucleotide reductase (RRl). This promoter has a number of features which make it ideal for the detection of HSV. First, there is no constitutive expression from this promoter in uninfected cells. Second, activation of the promoter appears to be specific for HSV. Third, expression from this promoter occurs within hours after infection. Fourth, this promoter is strongly transactivated by the virion associated trans-activator protein VP16. As early as six hours after infection HSV-infected cells can be detected by histochemical staining for /3-galactosidase activity. Infected cells stain intensely blue whereas uninfected cells show no staining, and a single infected cell can easily be recognized in a microscopic field of uninfected cells. Both HSV-1 and HSV-2 are detected with this cell line, but after infection with human cytomegalovirus (HCMV), varicella zoster virus (VZV), adenovirus, and sindbis virus no blue cells were detected. Quantitation of HSV-1 stocks on this cell line by counting blue cell forming units (BFU) reveals that the number of BFU/ml closely approximates the number of plaque forming units (PFU)/ml as determined by plaque assays on the parent cell line. This cell line should Correspondence to: Paul D. Olivo, P.O. Box 8051, Washington Euclid Avenue, St. Louis, MO 63110, USA.

University

School of Medicine,

660 South

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provide a useful adjunct in the diagnostic detection of HSV in clinical specimens.

virology

laboratory

for the rapid

Herpes simplex virus; Diagnosis; /?-Galactosidase; Histochemistry _

Introduction Herpesviruses have become increasingly important causes of human morbidity and mortality (Whitley, 1990). Herpes simplex viruses types 1 and 2 (collectively HSV) in particular, infect a large number of individuals each year. Primary infection of immunocompetent patients with HSV usually leads to a mucocutaneous syndrome such as herpes labialis or herpes genitalis, the latter being one of the most common sexually transmitted diseases today. Following primary infection the virus persists in a latent state and patients can suffer from recurrent bouts of reactivation throughout their lifetime. In addition, HSV can cause visceral infections, the most serious of which are sightthreatening keratitis and life-threatening encephalitis. Furthermore, HSVrelated disease in immunocompromised patients such as newborns, leukemia patients, organ transplant recipients and AIDS patients has become an increasingly prevalent and difficult problem. There have been significant advances in the treatment of HSV infections in the past decade. Acyclovir has become the drug of choice for most HSV infections and it has had a dramatic effect on the outcome of infections in immunocompromised patients. Toxicity is usually relatively minimal and resistance has not been a significant clinical problem except in specialized circumstances such as AIDS patients treated for long periods. Advances in antiviral therapy have led to an expansion of the role of the diagnostic virology laboratory and the diagnosis of HSV infection has become one the more useful tests performed by diagnostic virology laboratories. Furthermore, effective therapy for HSV has stimulated the need for more sensitive, accurate, and rapid diagnostic tests. Tests that are useful for the diagnosis of HSV infections mostly involve the detection of viral antigens or intact infectious virus (McIntosh, 1990) since serological tests are of very limited value. Antigen detection assays offer the advantage of rapidity and specificity but can lack sensitivity (Kowalski and Gordon, 1989). Polymerase chain reaction (PCR) technology is a promising tool for the detection of HSV particularly in cerebrospinal fluid specimens, but it remains experimental (Puchhammer-Stock1 et al., 1990). The most reliable test involves inoculation of specimens onto tissue culture cells followed by detection of infectious virus by microscopically observing a characteristic cytopathic effect. Although HSV is a relatively easy virus to culture since it replicates on a wide variety of continuous cell lines, virus propagation in tissue culture can be slow and expensive. Recently, improved techniques have been

197

developed for the detection of viruses from clinical specimens. The shell vial technique, for instance, has greatly increased the sensitivity and the rapidity of HSV detection. When this method is combined with antigen detection by immunohistochemistry, HSV can be positively identified within 24 h in the majority of the cases (Gleaves et al., 1985; Ziegler et al., 1988). While this type of protocol is sensitive, specific and relatively rapid, it is relatively labor intensive and a signiticant number of specimens are not identified as positive until 48 h. We have developed a method for identifying HSV which we believe is as sensitive and specitic as currently used methods but which is much less labor intensive and more consistently rapid. Using this method, HSV can be detected in specimens within 612 h with a sensitivity and specificity equal to a standard plaque assay. Our method involves inoculation of specimens onto a cell line whose genome contains the Escherichia coli LacZ gene behind an inducible HSV promoter. The promoter, which has a number of features which make it ideal for this purpose (see ‘Discussion’), is from the HSV-1 geneUL39 which encodes ICP6, the large subunit of ribonucleotide reductase (RRl) (Goldstein and Weller, 1988). After infection, HSV-infected cells can be detected by histochemical staining of the cells for P-galactosidase activity. We found that HSV-infected cells stain intensely blue whereas uninfected cells show no staining, and a single infected cell can easily be recognized in a microscopic field of uninfected cells. Both HSV-1 and HSV-2 are detected with this c8ell line, but after infection with human cytomegalovirus (HCMV), varicella zoster virus (VZV), adenovirus, and sindbis virus, no blue cells were seen. Quantitation of HSV-1 stocks on this cell line by counting blue cell-forming units (BFU) in parallel with plaque assays on the parent cell line revealed that the number of BFU/ml closely approximated the number of PFU/ml. This cell line should provide a useful tool in the diagnostic virology laboratory for the rapid detection of HSV in clinical specimens.

Materials and Methods Cells and viruses Baby hamster kidney (BHK-21) cells were a gift of C. Hahn and C. Rice (Washington University, St. Louis, MO). They were propagated in MEM medium (Gibco BRL, Gaithesburg, MD) supplemented with 7% fetal calf serum (Gibco Co). BHKICP6LacZ lines were isolated by a liposomal transfection protocol (lipofection, Gibco, BRL, Gaithesburg, MD) followed by selection in medium containing G418 (Geneticin, Sigma, St. Louis, MO). Clones were isolated by trypsinization followed by plating at limiting dilution onto 96 well tissue culture dishes. G418 resistant cell lines were maintained in G418 at 400 pg/ml. Herpes simplex virus type one (KOS strain) was obtained from M. Challberg (NIH, Bethesda, MD). The Sindbis virus was obtained from

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C. Hahn and C. Rice, (Washington University, St. Louis, MO). The varicellazoster virus was obtained from L. Gelb (Washington University, St. Louis, MO). The other clinical isolates used in this study were obtained from G. Starch (St. Louis Children’s Hospital Diagnostic Virology Laboratory). The d120 mutant of HSV-1 containing a deletion in the ICP4 gene was a gift of N. DeLuca (Harvard University, Boston, MA) (DeLuca et al., 1985). Plasmids

pD6p was kindly provided by Dr. Sandra Weller (University of Connecticut). This plasmid contains the ICP6-LacZ fusion cassette (Goldstein and Weller, 1988) which was removed by BamHI digestion and subcloned into the BamHI site of the vector pUC18 to make pYBICP6LacZ. pMAMneo which contains the SV40 early promoter-neo resistance gene cassette was purchased commercially (Clontech, Palo Alto, CA). pMon3375 which contains the HSVlUL48 gene behind the MMTV LTR was a gift of Dr. Paul Hippenmeyer (Monsanto Corp.). Histochemical

staining

Cells were washed three times in phosphate buffered saline (PBS) pH 7.2 then fixed in 2% formaldehyde, 0.4% glutaraldehyde in PBS for 5 min at 4°C. After washing 2 times in PBS, ceils were incubated for 2 h at room temperature in staining solution (1 mg/ml X-gal (5-bromo-4-chloro-3 indolyl-P-Dgalactopyranoside), (Sigma, St. Louis, MO), 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM MgC12 in PBS. The staining solution was made up fresh before use with concentrated stock solutions of each reagent. The X-Gal stock solution was 40 mg/ml in N,N-dimethylformamide. The reaction was stopped by washing cells with PBS. Blue cells were visualized by light microscopy and stored in PBS at room temperature. b-Galactosidase

assay

The calorimetric assay for /?-galactosidase activity was done on whole cell lysates using o-nitrophenyl-/I-D-galactopyranoside (ONPG, Sigma, St. Louis, MO) as substrate in a standard assay (Maniatis, et al., 1990). Protein determinations of the lysates was done using a commercially available kit based on the Bradford method (Bio-Rad, Richmond, CA).

Results Preparation

of the BHKICP6LacZ

A 4.2 kb BamHI fragment

cell line

from plasmid pD6P was cloned into the BamHI

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420 428 434 440

HinD III Pst I Sal I BamH I

l

EcoR I 4592

Fig. 1. Schematic of plasmid used to make BHKICP6LacZ-5 cell line. A 4.2 kb fragment from pD6P was cloned into the BumHI site of the plasmid pUC8. This fragment contains the ICP6 promoter and 180 bp of the ICP6 open reading frame fused in-frame to the LacZ reading frame.

site of pUC18 to generate pYBICP6:LacZ as shown in Fig. 1. Baby hamster kidney cells were co-transfected with 0.5 pg of pMAMneo and 5 pg of pYBICP6:LacZ using lipofection (Gibco/BRL). 2 days after transfection the cells were placed in medium containing 1 mg/ml G418 for 1 wk at which time the G418 concentration was lowered to 400 pg/ml. The cells were maintained at this concentration for 2 wk with media changes every 4 days. Six G418 resistant clones were then isolated and analyzed. None of the clones revealed positive histochemical staining (i.e. blue staining) for P-galactosidase activity. However after infection with HSV type one (strain KOS) at high m.o.i. (> 10) most of the cells in the culture dish showed blue staining at 12 h. Several cell lines displayed up to lO--20% nonstaining cells even after high m.o.i infection and therefore, one of the cell lines (BHKICP6LacZ-5) which consistently displayed >95% blue cells 12 h after infection was chosen for further studies. Infection of BHKICP6LacZ-5

with HSV

BHKICP6LacZ-5 cells were infected with HSV-1 at various m.o.i. and histochemically stained for j?-galactosidase activity 12 h after infection. Fig. 2a

200 a

A b

B

Fig. 2. Histochemical staining of BHKICP6LacZ-5 cells infected with HSV-1. A. Macroscopic view of 1 cm’ wells of cells mock-infected (top row, well 1) or infected with increasing dilutions of a virus stock (row 1, well 2 through row 2, well 6). B. Microscopic view (40 x ) of row 2, well 4. The cells were fixed and stained as described in the ‘Materials and Methods’ section at 6 h after infection.

shows a macroscopic view of BHK-ICP6lacZ cells mock-infected (row 1, well 1) or infected with decreasing amounts of HSV-1 (row 1, well 2 through row 2, well 6). Microscopic examination of the wells which do not appear to stain blue macroscopically revealed individual blue cells among a predominance of unstained cells (Fig. 2b). We also titred the dilutions used in the experiment shown. in Fig. 2 in a standard plaque assay on the parental BHK cells. We found that the number of plaque-forming units (PFU)/ml (2.2 x 10’) approximately equaled the number of blue cell-forming units/ml (3.0 x 10’). The time-course of appearance of /I-galactosidase activity was next examined following infection with HSV-1 (Fig. 3). BHKICP6LacZ-5 cells were infected with HSV-1 at a high m.o.i. (10) and at various times after infected the cells were lysed and assayed for B-galactosidase activity by a calorimetric assay. Activity was first detected four hours after infection and peaked at 6 h after infection. When the same type of experiment was analyzed by histochemical staining similar results were found: a few blue staining cells could be seen at three hours after infection and >95% of the cells were blue after 6 h. After infection with a low m.o.i. (0.1) however, blue cells were not noted until 6 h after infection (data not shown).

201

I

2

3 Hours

4 after

5

6

7

0

infection

Fig. 3. Time-course of a calorimetric ~-gaiactosidase assay performed on @sates from HSV-infected BHKICP6LacZ-5 cells. Approximately lo6 cells were infected at an m.o.i. of 10 and at the indicated times whole cell lysates were made and submitted to a standard fi-galactosidase assay. The assay was stopped after 20 min and the 0D~zs measured. OD readings were normalized to amount of protein in the lysates.

The specificity of this assay for HSV was also examined by infecting BHKICP6LacZ-5 cells with clinical isolates of other viruses. Not unexpectedly, HSV-2 induced /?-galactosidase activity in a manner indistinguishable from HSV-1 (data not shown). However, inoculation of human cytomegalovirus (HCMV), varicella zoster virus (VZV), Adenovirus type 5 and a laboratory strain of Sindbis virus resulted in no blue staining cells at 12 h despite obvious cytopathic effects (except for the HCMV for which BHK cells are not permissive) (data not shown). We have not assayed beyond 12 h. The HSV-1 ICP6 promoter and its homolog in HSV-2 (ICPlO) contain a cisacting response element found in immediate-early genes which is important in virion mediated transcriptional transactivation and several groups have shown using transient transfection assays that the ICP6 and ICPIO promoters are responsive to the virion trans-activator protein VP16 (Goldstein and Weller, 1988; Wymer et al., 1989). We.therefore tested whether /?-galactosidase activity could be induced in our cell line by VP16 in the absence of HSV infection. We transfected BHKICP6LacZ-5 cells with plasmid pMon3375 which contains the VP16 gene (HSVUL48) behind the human cytomegalo%rus major immediate early promoter/enhancer element, a strong constitutive promoter. We histochemically stained the cells 24 h after transfection and no blue cells were seen. pMon3375 did however signi~cantly enhance expression from an ICP4 promoter in co-transfection assays (data not shown). It appears therefore that, in this particular cell line at least, integration of the ICP6 promoter into the chromosome makes it inaccessable to VP16 mediated transactivation in the absence of infection. We also tested whether non-infectious virus could induce &galactosidase activity in this cell line. We UV inactivated a stock of HSV-I

202

and found that both PFU and BFU were reduced greater than 10 OOO-fold after 2 min of UV exposure (data not shown). Therefore, under the conditions we used, both infectability and VP16 transactivateability seem to have been inactivated. Whether there are conditions which inactivate the virus but keep virion associated VP16 functionally intact is a subject of investigation. Another interesting characteristic of the ICP6 promoter is that its expression is independent of the essential immediate-early gene product ICP4 which is a major transactivator of many early HSV genes (DeLuca, et al., 1985). It is unclear why the gene encoding the large subunit of ribonucleotide reductase is expressed in the absence of ICP4. Nevertheless, we infected BHKICP6LacZ-5 cells with a host-range mutant virus which contains a deletion in the ICP4 gene (d120) (DeLuca et al., 1985). The cells were histochemically stained 8 h after infection and blue cells were seen. As shown for wild type virus, titration of d120 stocks on BHKICP6LacZ-5 cells revealed that BFU/ml correlated with PFU/ml determined on permissive cells (E5 cells) (data not shown). Therefore, this methodology can be used to detect and quantitate certain HSV mutants even though the BHKICP6LacZ cell line is non-permissive for such mutants.

Discussion A cell line which has potential use in a histochemical P-galactosidase assay to detect HSV was developed. The assay is simple and rapid, and based on limited studies thus far, it appears to be both sensitive and specific. The cell line was derived from BHK cells which were co-transfected with two plasmids: a plasmid that contains the E. coli neo gene behind the SV40 early promoter and a plasmid that contains the E. coli LacZ gene behind the HSV-1 UL39 promoter. After selecting for G418-resistant colonies we identified several cell lines which displayed B-galactosidase activity following infection with HSV- 1. One such cell line, BHKICP6LacZ-5, displayed no detectable B-galactosidase activity after mock infection and pronounced activity following infection with HSV. It was found that when virus stocks were titred on this cell line the number of blue staining cells seen following histochemical staining for /?galactosidase approximately equaled the titre of PFU. Therefore, a single blue cell corresponds to a single infectious virion of HSV. Both HSV-1 and HSV-2 are detected by this assay and thus the assay cannot distinguish between them. However, no induction of detectable /J-galactosidase activity was seen following infection with clinical isolates of two other Herpes viruses (HCMV and VZV), another DNA virus (Adenovirus), and a laboratory strain of an enveloped RNA virus (Sindbis virus). In order for this histochemical assay to be useful in a diagnostic virology laboratory, it must satisfy certain minimal criteria. It is important that there be no background staining in either uninfected cells or in cells infected with other viruses, particularly viruses which might be present in clinical specimens (nasopharyngeal swabs, cerebrospinal fluid, etc.) frequently assayed for the

203

presence of HSV. It is also important that it be highly sensitive, ideally as sensitive as a cytopathic assay. It must be more rapid than the cytopathic assay and ideally more rapid than the shell vial/immunoassay method. In addition it is important that it be a simple test to perform, in that it not be labor intensive and not require expensive equipment not normally found in a diagnostic laboratory. The histochemical assay using the cell line that we describe in this report clearly has the potential to fulfill all of these criteria. Studies using this system to detect HSV in clinical specimens are ongoing. The key aspect of the cell line which provides this potential is the nature of the particular promoter we have used to drive the LacZ gene. We used the promoter from the HSV-1 UL39 gene which encodes the large subunit of ribonucleotide reductase (ICP6 or RRl) (Goldstein and Weller, 1988). This promoter has a number of features which make it ideal for this purpose. Firstly, there is no constitutive expression from this promote in uninfected BHKICP6LacZ-5 cells, thus eliminating background staining. Secondly, activation of the promoter appears to be specific for HSV types 1 and 2. Thirdly, expression from this promoter occurs within hours after infection, making the test relatively rapid. Fourthly, this promoter is strongly transactivated by the virion-associated trans-activator protein VP1 6, as well as the immediate early gene product ICPO (Goldstein and Weller, 1988) thus increasing the sensitivity of the assay. Since the ICP6/ICPlO promoter has been shown to be transactivated by VP16 in the absence of HSV infection in transient transfection assays (Wymer et al., 1989) theoretically this assay can detect defective virion particles as long as they could enter the nucleus and transactivate the ICP6 promoter. Consistent with this the d120 mutant, which contains a deletion in the essential ICP4 gene, can induce expression of the ICP6:LacZ gene in the BHKICP6LacZ-5 cells despite the fact that they are not permissive for this mutant. However, we did not detect b-galactosidase activity following transfection of a plasmid containing the VP16 gene (UL48) behind a constitutive promoter. This result suggests that other events besides the presence of VP16 in the nucleus must occur during HSV infection of the BHKICP6LacZ-5 cells in order to allow accessability of the integrated ICP6 promoter to transactivation. Regardless, the good correlation between BFU titre and PFU titre which we observe suggests that from a practical standpoint only infectious virus is detected in this assay. This general methodology could theoretically be applied to many viruses. Recently a histochemical P-galactosidase assay was described for the detection and analysis of HIV-infected cells (Rocancourt et al., 1990). A HeLa-CD4+ cell line containing a recombinant HIV-l provirus with a 1acZ gene placed behind the HIV-l LTR was made. This cell line was shown to be very useful as a sensitive indicator of HIV-l, HIV-2, and SIV-infected cells and as a simple method to determine the dose response of antiviral drugs. In this system problems with uninfected cell P-galactosidase activity had to be circumvented by adding a nuclear localization signal to the /?-galactosidase. In the HSV

204

system described here no constitutive /I-galactosidase activity is detectable and the cytoplasmic location of the blue product following histochemical staining makes it easy to identify an individual infected cell. Application of this approach to the diagnosis of other Herpes viruses such as HCMV, is theoretically possible. The key question is whether a promoter with similar characteristics can be found. One advantage of this method is that the cell type used to make the cell line need not be fully permissive for the virus. In the case of HCMV, which is permissive only for diploid human cells (Smith, 1986) and is usually cultured on primary libroblast cultures, a wide number of susceptible but nonpermissive cells could be used to make the cell line used to detect the virus. Finally, in addition to its potential as a tool in virus detection assays, the BHKICP6LacZ-5 cell line we describe here could be useful in automated assays for screening for antiviral agents and for performing drug sensitivity testing. We are currently developing such assays.

References DeLuca, N.A., McCarthy, A.M. and Schaffer, P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J. Virol. 56, 558-570. Cleaves, C.A., Wilson, D.J., Wold, A.D. and Smith, T.F. (1985) Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 hours postinoculatidn. J. Clin. Microbial. 21, 29932. Goldstein, D.J. and Weller, S.K. (1988a) Herpes simplex virus type l-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 1acZ insertion mutant. J. Virol. 62, 196205. Goldstein, D.J. and Weller, S.K. (1988b) An ICP6: LacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J. Virol. 62, 2970-2977. Kowalski, R.P. and Gordon, Y.J. (1989) Evaluation of immunologic tests for the detection of ocular herpes simplex virus. Ophthal. 96, 1583-1586. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1990) Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory. McIntosh, K. (1990) Diagnostic Virology. Virology. Raven Press, New York. Puchhammer-Stockl, E., Popow-Kraupp, T., Heinz, F.X., Mandl, C.W. and Kunz, C. (1990) Establishment of PCR for the early diagnosis of herpes simplex encephalitis. J. Med. Virol. 32, 77-82. Rocancourt, D., Bonnerot, C., Jouin, H., Emerman, M. and Nicholas, J.-F. (1990) Activation of a /3-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells. J. Virol. 64, 2660-2668. Smith, J.D. (1986) Human cytomegalovirus: demonstration of permissive epithelial cells and nonpermissive fibroblastic cells in a survey of human cell lines. J. Virol. 60, 583-588. Whitley, R.J. (1990) Herpes simplex viruses. Virology. Raven Press, New York. Wymer, J.P., Chung, T.D., Chang, Y.-N., Hayward, G.S. and Aurelian, L. (1989) Identification of immediate-early-type c&response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2. J. Virol. 63, 2773-2784. Ziegler, T., Waris, M., Rautianen, M. and Arstila, P. (1988) Herpes simplex virus detection by macroscopic reading after overnight incubation and immunoperoxidase staining. J. Clin. Microbial. 26, 2013-2017.