Herpes Simplex Virus Type 1 Replication in a Gingival Cell Line R. D. FLETCHER, J. GUGGENHEIMER, D. SUAlINEY, and D. PLATT School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

Propagation of herpes simplex virus type 1 (HSV-1) in gingival cells is advantageous since these cells are more definitive than HeLa cells in demonstrating viral-induced cytopathic effects and are easily and economically maintained in the laboratory. They also provide a potential medium for studying oral tissue reactions to HSV-1 infection. HSV-1-induced cytopathic effects are cle(arly characterized following inoculation with viruts from standard laboratory cultures and human infections. Primary and recurrent herpes simplex virus type I (HSV-1) infections are observed most fre(quenitly in the oral regions.'-3 Therefore, it would seem appropriate to use cell culttures derived from one of the oral tissues to isolate and identify this virus. The availability of an epithelial-like cell line from human gingiva4 prompted a study to determine the effects of HSV-1 on these cells. Previously this gingival cell line has been used to successfully propagate rhinovirus and influenza virus type B.5,6 Poliovirus could not be replicated in gingival cells,7 probably because this virus cannot synthesize RNA. Materials and Methods The gingival cells used in this study were obtained from Smulow and Glickmana.4 They were described as epithelial-like cells from a 19-year-old healthy white female. Further evidence of their epithelial-like nature has been the demonstration of hemiThis paper was presented, in part, at the 52nd general session of the IADR in Atlanta, Ga, March 1974. This investigation was supported by Life Science, US Army Research Office, grant DAHC-19-72-G-007 and by Public Health Grant DE-00108-07. Received for publication July 15, 1974. Accepted for publication October 29, 1974. a Department of Periodontology, Tufts University, School of Dental Medicine, Boston, Mass.

desmosomes and tonofibrils in these cells.8 TFiese gingival cells are classified by definition as a cell line and they are characterized as lhaving a pleomorphic nucleus, no contact inhiibition, and a chromosome modal number of 57. There is good continuity between cells withi good cell clarity and distinct nuclei (Illustration, A). The passage number of these cells at the time of this investigation was approximately 644. The cells were grown in 16 X 125-mm screw cap tubes containing Earle's mediumb supplemented with calf serum (10%), penicillin (100 uinits/ml), streptomycin (100 ,lg/ml), ftungizone (25 ;tg/ml) and sodium bicarbonate (0.085%). Cell monolayers were maintained in a similar medium except the calf serum was reduced to 2%. Herpes simplex virus (number 539, MacIntyre) was obtained from the American Type Culture Collection. Additional HSV-1 was isolated from patients with typical herpetic oral lesions and identified by 5-iodo2'deoxyuridine inhibition, diethyl ether inhibition, sized as greater than 100 nm by filtration (Millipore) and neutralized by HSV- 1 antiserum. These virus stocks (approximately l07 TCID.50/ml) were stored in Earle's medium at -60 C. Before use they were thawed at 25 C and inoculated directly into the gingival cell cultures. Additional cells for histological study were also grown in Earle's medium in 60 X 15 mm roundculture dishes containing a coverslip. When the coverslip cell monolayer was confluent it was inoculated with virus (multiplicity of infection 4), incubated at 33, 35, or 37 C in a candle-jar, and following the appearance of the cytopathic effect, stained with hemab

Grand Island Biological Co., Grand Island, NY.

441 Downloaded from jdr.sagepub.com at PENNSYLVANIA STATE UNIV on March 13, 2015 For personal use only. No other uses without permission.

:_*.( ,s s _M.-

.E_

_^ :.

^

_,

...........

..

_

w

iEaCE

-E-

^ X|,!

AAV

A ti# ML

SEI* ...i ,,,,O

'a,v%\^-'-''* Bq' . . . . .

b~~~~~~~~~~~~~~~~~~~~~~~..

....Z..-

.. . i:EX:

AI

B

,+t

tfi

4W. 4

&.

AS :::

IV

.S

i

Md

I p

Gingival cell monolayers (mag x 400) infected with HISV-1. A, uninfected gingival cell monolayer; B, 24-hour HSV-1 infection shows giant cell formation; C, 48-hour HSV-1 infection shows nuclear pyknosis and tadpolelike giant cell vacuoles; D, cytoplasm loss, intense nuclear pyknosis, and cell monolayer destruction after 72 hours.

442 Downloaded from jdr.sagepub.com at PENNSYLVANIA STATE UNIV on March 13, 2015 For personal use only. No other uses without permission.

VOI1 54 NO. 3

HSV-1 REPLICATION IN GINGIVAL CELLS

toxylin and eosin. These cells also were observed for the presence or absence of mitosis. Periodically during viral replication, viral titers were obtained by the tube end-point method.9

Results Gingival cells at 24 hours after HSV-1 inoculation with either ATCC or virus isolates showed loss of cytoplasm, cellular rounding, some disruption of the cell sheet, giant cells, and cellular contraction (Illustration, B). At 48 hours after virus inoculation there was increased nuclear pyknosis, cytoplasmic condensation with vacuolization, and giant cell formation (Illustration, C). Seventy-two hours after inoculation the gingival cells were extensively pyknotic (markedly condensed chromatin) and most of the cytoplasm was lost. By this time in the HSV-1 infection cycle most of the cells had detached from the glass surface (Illustration, D). HSV-1 virus infection of gingival cells incubated at 35 C inhibited mitosis at two hours after infection; the duration of the eclipse phase was 5 hours and replication of the virions reached a peak at 18 hours. Incubation temperatures of 33 C and 37 C were not as ideal at 35 C for HSV-1 replication.

Discussion Gingival cells are maintained easily and economically in the laboratory and provide a suitable medium for the isolation of herpes group viruses. The cells have extremely distinct components that facilitate early detection of the viral cytopathic effects. They provide a potential medium for studying a variety of oral pathologic conditions relating to HSV infection. This could include the establishment of new cell cultures from patients with a history of HSV infections for subsequent isolation and propagation of this virus. They are also advantageous in that unlike HeLa cells, gingival cells do not readily contaminate other cell lines in the laboratory.'0 Presently, plaque studies are being conducted in these cells in an attempt to detect

443

possible differences between HSV-1 and HSV2 as well as the effect, if any, of other agents in clinical samples.

Conclusions Herpes simplex virus type 1 can be propagated in gingival cell line monolayers, thus providing a new medium for its isolation and identification. A variety of potential investigations are now foreseen since these cells are derived from the oral cavity, the site most frequently infected by primary and recurrent HSV-1 infections in man. References 1. DOWDLE, W.R.; NAHMIAS, A.J.; HARwELL,

R.W.; and PAULS, F.P.: Association of Antigenic Type of Herpes Virus Hominis with Site of Viral Recovery, J Immunol 99: 974980, 1967. 2. EJEREITO, P.M.; KIEFF, E.D.; and RoIzMAN, B.: Characterization of Herpes Simplex Virus Strains Differing in Their Effects on Social Behavior of Infected Cells, J Gen Virol 2: 357-364, 1968. 3. SCHNEWEISS, K.E., and NAHMIAS, A.J.: Antigens of Herpes Simplex Virus Type 1 and 2-Immunodiffusion and Inhibition Passive Hemagglutination Studies, Z Immunitaetsforsch 141: 471-487, 1971. 4. SMULOW, J.B., and GLICKMAN, I.: An Epi-

thelial-like Cell Line in Continuous Culture from Normal Adult Human Gingiva, Proc Soc Exp Biol Med 121: 1294-1296, 1966. 5. FLETCHER, R.D., and PLATr, D.: Growth Characteristics of Rhinoviruses in Human Gingival Cells, Arch Gesamte Virusforsch 25: 1-7, 1968. 6. FLETCHER, R.D.; JAYAVASU, C.; ZANER, L.; and PLArr, D.: Gingival Cell Propagation of Influenza Virus, abstracted, IADR Program and Abstracts of Papers, No. 409, 1972. 7. MILLIGAN, W.H., III, and FLETCHER, R.D.: Failure to Detect Poliovirus Replication in Human Gingival Cell Cultures, abstracted, IADR Program and Abstracts of Papers, No.

201, 1971. 8. LANGKAMP, H.: Unpublished data. 9. CUNNINGHAM, C.H.: A Laboratory Guide In V'irology, 6th ed, Minneapolis: Burgess Publishing Co., 1966, p 62. 10. CULLITON, B.J.: HeLa Cells: Contaminating Cultures Around the World, Science 184:

1058-1059, 1974.

Downloaded from jdr.sagepub.com at PENNSYLVANIA STATE UNIV on March 13, 2015 For personal use only. No other uses without permission.

Herpes simplex virus type 1 replication in a gingival cell line.

Herpes Simplex Virus Type 1 Replication in a Gingival Cell Line R. D. FLETCHER, J. GUGGENHEIMER, D. SUAlINEY, and D. PLATT School of Dental Medicine,...
227KB Sizes 0 Downloads 0 Views