Herpes simplex virus type 1 and type 2 infection in human orai mucosa in culture

Y. Yura, H. Iga, Y Kondo, K. Harada, T. Yanagawa, H. Yoshida and M. Sato Second Department ol Oral and Maxillolacial Surgery, Tokushima University School ol Dentistry, Tokushima, Japan

Yura Y, Iga H, Kondo Y, Harada K, Yanagawa T, Yoshida H, Sato M: Herpes simplex virus type 1 and type 2 infection in human oral mucosa in culture. J Oral Pathol Med 1991; 20: 68-73. To examine the sensitivity of human oral mucosa to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection, human gingival mucosa explants were infected with either HSV-1 or HSV-2 in vitro and the expression of virus specific antigen was examined by the immunofluorescent antibody technique. HSV-2 antigen was found in the basement membrane, basal cell layer and lower prickle cell layer. This finding was consistent with the HSV-1 infection. Electron microscopic study revealed the presence of nucleocapsids and enveloped virus particles in the basal cells of HSV-2-infected organ cultures. These findings indicate that human gingival mucosa is sensitive to infection with HSV-2, as well as HSV-1, and that the virus may replicate in the undifferentiated epithelial cells of mucosal epithelium.

Herpes simplex virus type 1 (HSV-1) is most frequently isolated from the oral cavity, whereas herpes simplex virus type 2 (HSV-2) is more commonly isolated from genitalia (1). However, recent studies have indicated an increasing number of genital lesions with HSV-1, e,g,, 15-37% of primary genital herpes simplex virus (HSV) infection is now due to HSV-1 (2, 3). Since HSV-1 was found to be excreted in saliva of asymptomatic individuals (4-7), the most likely method of HSV-1 transmission is by orogenital contact. Saliva may be the mediator of this transmission. Persistence of HSV-2 shedding from asymptomatic women after recovery from genital herpes infection was reported (8, 9), suggesting that HSV-2 from genitalia can be also transmitted by orogenital contact. However, HSV-2 infection of the oral cavity seems to be a very rare disease even among a population actively engaging in oral sex (10-12). In one study, none of 55 isolates from the oral cavity was HSV-2 (12), The reason why HSV-2 can be rarely isolated from the oral cavity has not been clarified. Evidence for HSV type specific receptors on cellular plasma membranes was presented (13), indicating that HSV-1 and HSV-2 may exhibit a difference in the affinity to a specific cell or tissue. Moreover, it is proposed that the female genital tract offers a more permissive environment for HSV-2 than for HSV-1

(14). Thus, it is also possible that the oral cavity is more sensitive to infection with HSV-1 than with HSV-2, though the possibility has not been investigated. Organ culture systems have been developed to study the pathogenesis of HSV in a natural host (15,16). Recently, we have established an organ culture system with human gingival mucosa explants (17). Using this organ culture system, the present study was conducted to determine whether human oral mucosa is sensitive to infection with HSV-2 as well as with HSV-1. Material and methods Tissue culture - Normal gingival tissues were obtained during the removal or fenestration of impacted incisional, premolar and molar teeth. For organ culture, gingival tissues were obtained from 7 men and 8 women ranging in age from 9-38 yr. To prepare single-cell suspensions, gingival tissues were obtained from four patients including 2 tnen and 2 woman patients with ages ranging from 9-15 yr. These patients had no history of recurrent herpes labialis and there were no ulcerative lesions in their oral cavity when operations were carried out. Their serologic status, e.g., HSV seropositive or seronegative, was not investigated. A gingival tissue was washed in Dulbecco's phosphate buffered saline (D-PBS) and divided into three

Key words: herpes simplex virus, type-1 and type-2, oral mucosa, human: organ culture; viral antigen. Yoshiaki Yura, Second Department of Oral and Maxillofacial Surgery, Tokushima tjniversity School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770, Japan. Accepted for publication August 29, 1990.

fragments. Approximate size of one fragment was 3 mm. To remove blood from tissue, fragments were placed in 60 mm dishes, covered with 5 ml of Eagle's minimal essential medium (MEM) and incubated for 1 h with intermittent shaking. MEM was exchanged for three times. Thereafter, fragments were infected with HSV and then subjected to organ culture. The gingival fragments were placed on a nitrocellulose membrane supported with a metal mesh in a 60 mm organ culture dish (Falcon Plastic Co., CA. U.S.A.) and cultured in 2 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 300 ng/ml ascorbic acid, 1 ng/ml hydrocortisone, 100 units/ ml penicillin and 100 ^ig/ml streptomycin in the presence of 5% CO, in an incubator at 35 C. After 1 or 3 days, the cultured mucosal fragments were embedded in embedding medium for frozen tissue specimens (O.T.C. compound; Miles Laboratories, IA. U.S.A.) and stored at -80°C. To prepare singlecell suspensions, the mucosal tissues were treated with 0.25% (W/V) trypsin in DMEM at 4 C for 4-6 h. Thereafter, the epithelium was separated from connective tissue with a fine forceps and dissociated by pipetting and then passed through a metal mesh, followed by washing twice with DMEM. After infection with HSV, cells were cultured in DMEM containing 10% FBS, 100

Herpes virtis infection in organ culttire 69 units/ml penicillin and 100 ng/ml streptomycin. Cell viability was tested by the trypan blue exclusion test. Under the present experimental condition, 85-90% of cells were viable. The African green monkey kidney cell line Vero was cultured in MEM supplemented with 5% newborn calf,serum and 2 tnM L-glutamine, Virus and virus assay - HSV-1 laboratory strain HF (18), HSV-2 laboratory strain U'W-268 (19) and clinical isolates were used in the present study. The types of clinical isolates were determined by immunofluorescent antibody staining based on reactivity with a monoclonal antibody against HSV-1 or HSV-2 (Syva Company, CA. U.S.A.) and/or by restriction endonuclease analysis of viral DNAs (20). Each virus was grown in Vero cell tnonolayers. The infected cells were disrupted by 3 cycles of freezing and thawing. After centrifugation at 500 x g for 10 min, the supernatant was stored at - 80°C. Both types of HSV were assayed by plaque-forming ability on confluent Vero cell monolayers. Plaques formed after 3 days were stained with 0.5% (W/V) crystal violet. Infection of gingival mucosa explants and mucosal cells with HSV - For HSV infection, mucosal fragments washed in D-PBS and MEM were placed in a 24well tissue culture plate (Falcon Plastic Co.), covered with 1.5 ml of tnedium containing 1.5x10' plaque fonning units (PFU) of HSV-1 or HSV-2 and incubated at 35^C for 2 h. During this adsorption period, the plate was rotated intermittently. Thereafter, fragments were washed in 10 ml of MEM for three times to remove unadsorbed viruses and cultured in organ culture dishes as described above. Cells dissociated from gingival mucosa were pelleted by centrifugation at 500 xg for 10 min. The pellet of 10-^ cells was suspended in 1 ml of medium containing 2x10'' PFU of HSV-1 or HSV-2. After adsorption period of 2 h, cells were washed with D-PBS by two cycles of centrifugation and suspension.

Table 1. Clinical isolates of HSV-1 and HSV-2 Patient Ym Im Kj Fr Ys Ki Kn

Age (Y) 49 35 7 31 28 32 25

ND = not determined.

Sex

Disease

F M

Gingivostomalitis Herpetic whitlow Gingivostomatitis Genital herpes Genital herpes Skin herpes Skin herpes

M F F M M

Thereafter, cells were pelleted again, susElectron microscopy - For transmispended in 1 ml of medium, seeded in a sion electron microscopy, the HSV-in24-well tissue culture plate at a density of fected gingival rnucosa explants were I X IO'/well and cultured at 35'C. fixed in 2.5% glutaraldehyde buffered Immunofluorescent antihody staining with 0,1 M phosphate (pH 7.3) contain- Four micrometer thick frozen sections ing 5 mM calcium phosphate for 2 h. frorn the mucosa fragments were fixed After washing in phosphate buffer, they in acetone at 4'C for 10 min and air- were postfixed with 2% osmium tetroxdried. The fixed sections were incubated ide for 1 h and dehydrated step by step with 50 |il of fluorescein isothiocyanate with ethanol, followed by embedding in (FITC)-conjugated anti-HSV-1 anti- Epon 812 resin, Ultrathin sections were body or FITC-conjugated anti-HSV-2 cut on a LKB ultramicrotome and antibody (Syva). After incubation at stained with uranyl acetate and lead hy37°C for 30 min, the sections were droxide. The final preparations were washed with D-PBS for 30 min, mount- observed under a Hitachi electron mied with buffered glycerol (pH 9.4) and croscope, model H-500. examined under a Nikon fluorescent microscope (Nikon Co., Tokyo Japan). Cell suspension from the gingival muco- Results sa were placed on glass slides (coated HSV-1 and HSV-2 infection in gingiva with 0.1% (W/V) oil-resistant synthetic mucosa explants - Clinical isolates used rubber (Neoprene; Ohken-Shoji Co., in this study are shown in Table 1, Three Tokyo, Japan) to faciliate firm attach- strains of HSV-1 were isolated from pament of the cells), air-dried and fixed in tients with pritnary herpetic gingivostocold acetone for 10 min. Coexpression matitis and one was from a genital of keratin and HSV antigen in these lesion. One of HSV-2 strains was recovcells was investigated by double-anti- ered from a genital lesion and the other body staining. The cells fixed in acetone two strains from skin vesicles. The type were incubated with rabbit antikeratin of these viruses was detennined on the antibody (diluted 1:50. Dakopatts, Co- basis of reactivity with monoclonal antipenhagen, Denmark) and mouse anti- bodies against HSV-1 and HSV-2 and/ HSV-1 antibody (diluted 1:50) prepared or by restriction endonuclease analysis in this laboratory as described previous- of viral DNAs. Two mucosal explants ly (17). The antikeratin antibody (Da- were obtained from each of 7 patients kopatts Code No. A575) used was (3 men and 4 women) and one frotrt 8 shown to stain all of the cell layers of patients (4 men and 4 women). Total 22 human epidermis (21). After an over- mucosal explant satnples were used to night incubation at rootn tetrtperature, determine whether human oral tnucosa samples were washed with D-PBS for can be infected with HSV-2 as well as 30 min and incubated for a further 1 HSV-1. Each excised tissue was further h at 37°C with tetramethylrhodamine divided into three fragtnents. Two of isothiocyanate-conjugated swine anti- thetn were infected with HSV and the rabbit immunoglobulin (diluted 1:40. other part of the specimen was mockDakopatts) and FITC-conjugated swine infected and the expression of a HSV anti-mouse immunoglobulin (diluted specific antigen was examined by imtnu1:40. Dakopatts) with a subsequent nofluorescent antibody staining. If viral washing. Samples were examined under antigen was detected in one or both of a Nikon fluorescent microscope using the HSV-infected fragments, the mucothe appropriate filter systems for FITC sal explant satnple was considered to and tetramethylrhodatnine isothiocya- be HSV antigenpositive. Since in our nate. previous study HSV antigen appeared in HSV-1-infected gingival mucosa explants within 3 days post infection (17), the HSV-1- or HSV-2-infected fragments were cultured for 1 to 3 days Antibody at first visit followed by iinmunolluorescent anti(complement-fixing test) Type body staining. A representative result of immunoHuorescent antibody staining is < x4 1 shown in Fig. 1. In a gingival fragment < x4 1 < x4 1 infected with HSV-2 strain Kn, viral an< x4 I tigen was demonstrated predominantly ND 2 in the basetnent membrane, basal cell ND 2 layer and lower prickle cell layer, where2 ND as there were no antigenpositive cells in

70

YURA ct al.

Fig. I. Immunofluorograph of HSV-l (A)- or HSV-2 (B)-infected human gingival mueosa fragment. Expression of HSV antigen is found in basement membrane, basal eell layer and lower priekle eell layer. Arrow heads indicate the epithelialmesenehymal junction x 170.

either the keratinized cell layer or the granular cell layer (Fig. IB). In general, subepithelial connective tissue did not express HSV-2 antigen, but in a few cells viral antigen was observed beneath the basement tnembrane. Likewise, HSV-1 antigen was demonstrated in cell layers composed of undifferentiated epithelial cells of the mucosal fragment infected with HSV-l strain Ym (Fig. IA). Table 2 summarizes the results of the data obtained by immunofluorescent antibody staining. Typespecific viral antigen was demonstrated in all of the .samples (12/12) infected with HSV-1 strains and in 7 out of 8 samples infected with HSV2 strains 3 days post infection. There was no difference between HSV-1 and HSV-2 in the presence site of viral antigen. Viral antigen was not demonstrated in the mock-infected fragment obtained from each explant sample using anti-HSV-1 or HSV-2 anfibody. Moreover, the HSV-infected fragments

were not stained with normal mouse serum (Dakopatts) and FITC-conjugated swine anti-mouse immunoglobulin. HSV-1 and HSV-2 infection in tnucosal cells - To determine the proportion of cells which are sensitive to HSV infection, cells were dissociated from gingival epithelium and infected with HSV-1 or HSV-2 at a multiplicity (MOI) of 20 PFU/cell. In this condition, it was assumed that all of the cells would come into contact with infectious viruses. Four separate experiments were conducted using cell preparations from 4 patients. Since only one mucosal tissue was obtained from each ofthe 3 patients and the number of cells available from one patient was limited, 2 or 3 HSV strains were tested in experiment I, 3 and 4. In experiment 2, mucosal tissues were obtained from two locations ofthe patient. Thus, in this case, the number of the cells was sufficient for the study of 2 HSV-1 strains and 4 HSV-2 strains.

Table 2. Expression of HSV antigen in the human gingival mucosa explant.s Strains of HSV Type 1

Type 2

Days after infection

HF

Ym

Im

Fr

UW-268

I 3

I/I* 2/2

ND 6/6

ND 1/1

ND 3/3

ND 2'2

Ys

Kn

0/1 2/3

ND 3/3

ND = not determined. *Number of explant samples expressing HSV antigen/number of explant samples tested.

As experiments were perfortned in duplicate, the percentage ofantigen-posive cells, shown in Table 3, indicates the tnean of 2 determinations. A satnple infected with strain Itn showed the lowest percentage of antigenpositive cells, whereas those infected with strain UW268 showed higher values, but if compared in each experitnent, the values in samples infected with HSV-1 were sitnilar to those in the samples infected with HSV-2. For example, in experitnent 3, the percentage of HSV-1 atitigcnpositive cells was 10.8 and that of HSV-2 antigenpositive cells was 11.9. The proportions of antigenpositive cells throughout the experitnents in those infected with HSV-1 or HSV-2 strains were 6.3 + 3.6% and 6.4 + 2.8%, respectively. Viral antigen was not demonstrated in the uninfected cells obtained from each individual with HSV typespecific antibodies. Moreover, HSV-1 or HSV-2-inrected cells were not stained with the combination of normal mouse serutn and FITC-cotijugated swine antitnouse itntnunoglobulin. The cells of experiment 2 were infected with HSV-1 strain Ym and tested for the presence of keratin and HSV antigen by doubleantibody staining. This experiment had two objectives, first, to demonstrate that the tnajority of the cells prepared were epithelial cells but not fibroblasts which were derived from subepithelial connective tissue and could be included during the preparation of cells, and, second, to confirtn that the cells expressing HSV antigen were epithelial cells. As a result, approximately 95% of the cells were found to be keratinpositive and HSV antigen was observed in polygonal cells containing keratin (Fig. 2). Electron microscopic ohscrvalion of the HSV-2-infecled gingival mucosa fragments ~ HSV-2-infected mucosa fragments were subjected to electron microscopic study 3 days after infection. The structures of basement metnbrane, epithelial cell layers and subepithelial connective tissue were preserved. A nutnber of nucleocapsids with and without a dense core were observed in the nuclei of the basal cells, which contained bundles of tonofilament, desmosomes and hemidesmosomes (Fig. 3A and B). Enveloped virus particles were also present in the vacuoles of the cytoplasm. An interesting finding was that HSV nuclocapsids were demonstrated in the nucleus of a fibroblast etnbedded in the collagen fibers of the connective tissue beneath the basetnent membrane (Fig. 3C), though no evidence of HSV

Herpes virus infcclion in organ eullure Table 3. Expression of HSV antigen in cells dissociated from gingival mucosa Strains; of HSV Experiment number 1* 2 3 4

Type 1

Type 2

HF

Im

Ki

Fr

UW-268

Ys

Ki

Kn

3.4** ND

ND 3.3 ND ND

ND ND 10.8 ND

ND 4.4 ND 9.4

ND 3.8 11.9 6.9

ND 5.2 ND ND

ND 3.9 ND 9.1

5.7 4.7 ND ND

ND ND

ND = not determined. *Each experiment was performed with eells derived ironi one individual. **Percentage of HSV antigen-positive eells.

infectioti was observed in other fibfoblasts examined.

Discussion Human gingival tnucosa explants were infected with HSV-1 or HSV-2 in vitro and the expression of HSV antigen was examined. The present study detnonstrated that human gingival mucosa can be infected with HSV-1 and HSV-2 in a sitnilar manner. It is well established that most cell lines are pertnissive for the replication of HSV. In addition. RONES el al.

(22)

and EBBHSEN el al. (23) have shown that hutrtan gingival epithelial and fibroblast cells grown in vitro support HSV tnultiplication. However, cells grown in vitro and cell lines derived from tutnors or mesoblastic tissue have lost their nortrtal

anatotnical relationships and no longer express all of the characteristics of differentiated cells. In this regard, organ cultures are composed of cells at various stages of differentiation and preserve their original structure, providing a proper in vitro systetii to study the interactions between HSV and human oral mucosa. When gingival mucosa explants were tested for the sensitivity to various strains of HSV-1 or HSV-2, all of the samples except for one allowed the expression of viral antigen 3 days after infection. Moreover, it was found that HSV antigen was only expressed in cells of the basal cell layer, the lower prickle cell layer and in basement rnembrane, but not in more differentiated epithelial cells. This suggests that the replication of HSV may be dependent on the stage of epithelial differentiation. However, the failure of expression of

Fig. 2. Double label inimunonuorograph showing simultaneous presence of keratin (A) and HSV antigen (B) in cells infected with HSV-1. Arrow heads indicate same cell, x 380.

71

viral antigen in differentiated epithelial cells of upper cell layers tiiay be due to the inability of these cells to come into contat with infectious viruses. To examine this possibility and to know what percentage of epithelial cells can be infected, cell suspensions were prepared from gingiva explants and tested for the sensitivity to HSV infection. Consequently, even if cells were infected with a large nutnber of infectious viruses, HSV antigen was only allowed to express in a .small proportion (3.3-11.9%) of the cells infected with either HSV-1 or HSV-2. Although the number of cultures infected with each HSV strain was small, there was no difference between the two groups infected with HSV-1 or HSV-2 strains in the proportion of HSV antigen-positive cells. Thus, it is considered that a part of the cells, e.g., differentiated epithelial cells in the upper cell layers of the epithelium, may be not so pertnissive as undifferentiated basal cells and that a similar proportion of cells will express typespecific viral antigen. The finding in the study by doubleantibody staining suggests that most of the cells used are epithelial cells containing keration and the contatnination by fibroblasts is minimal. Since this polyclonal antikeratin antibody stains various types of keratin (21), it is unknown whether the presence of a specific type of keratin is correlated with the sensitivity of the cells to HSV-1 or HSV-2. Although studies with immunofluorescent antibody staining demonstrated the appearance of HSV antigen in undifferentiated mucosal epithelial cells, it was not detertnined whether HSV could replicate in these antigenpositive cells, resulting in the production of virus particles. It is possible that epithelial cells permit the expression of a specific genome of HSV. To address this question, HSV-2-infected gingiva fragments were examined by electron rnicroscope. Nucleocapsids and enveloped virus particles were detected in the basal cells. In addition, nucleocapsids were detected in a fibroblast located near the basement membrane. Since HSV antigenpositive cells, though infrequently, were observed in the connective tissue (Fig. 1), the fibroblast containing nucleocapsids may correspond to a cell expressing \iral antigen. At present it is unknov\n what percentage of fibroblasts can be infected with HSV, but HSV multiplication in the fibroblasts of organ cultures may be an uncommon event because there were no virus particles in other fibroblasts examined.

72

YuRA el al.

repeatedly sampled, 27% or more of individuals with antibody were found to be excreting HSV-1 at some period during the interval studied (4, 7). On the other hand, HSV-2 shedding has been observed in 10-14% of asymptomatic women after recovery from genital infection (8, 9). Since the incidence of HSV-1 infection is higher than that of HSV-2 infection, it is likely that HSV-1 shedding will occur more frequently than HSV-2. The duration of each shedding and the amount of virus may be more important for the transmission of HSV by orogenital contact and the establishment of infection at the inoculated sites. Alternatively, the low frequency of HSV-2 infection in the oral cavity may be partially explained by the fact that infeetion by HSV-1 usually occurs earlier in life than that by HSV-2, i.e., previous oral HSV-) infection may protect against reinfection by HSV-2.

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