Br. J. exp. Path. (1976) 57, 460
REPLICATION OF TYPE 2 HERPES SIMPLEX VIRUS IN HUMAN ENDOCERVICAL TISSUE IN ORGAN CULTURE J. BIRCH*, C. G4. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN From the Departments of Obstetrics and Gynaecology, Virology and Anatomy, University of Birmingham
Received for publication AMarch 18, 1976
Summary.-The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 370 and in serumsupplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a " non-human " herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions, and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ.
THE BEHAVIOUR of type 1 herpes simplex virus in tissue maintained in organ culture has been widely investigated in human fetal skin (Bang and Niven, 1958), human fetal trachea (Hoorn, 1964, 1966), rabbit adult oviduct (Barski, Cornfert and Wallace, 1959), and rabbit adult trachea (Hoorn, 1964). However, there is little information concerning the behaviour of type 2 herpes simplex virus. Balduzzi, Nasello and Amstey (1972) reported histological changes in herpes virus-infected cervical tissue fragments maintained for up to 3 weeks in culture, but did not investigate virus replication in quantitative terms. In recent years a considerable body of research has investigated type 2 herpes simplex virus. The virus is the aetiological agent of a relatively common venereal disease herpes genitalis and there is evidence that the virus is involved in the phenomenon of latency (Stevens and Cook, 1973). Of great interest, there * Present address: Department of Obstetrics Birmingham 15.
is evidence associating previous herpetic infection with pre-invasive and invasive carcinoma of the uterine cervix (Naib, 1966; Rawls, Tomkins and Melnick, 1969; Nahmias et al., 1970; Skinner, Thouless and Jordan, 1971). Type 2 herpes simplex virus is capable of in vitro cell transformation (Duff and Rapp, 1971; McNab, 1974; Skinner, 1976), and it is tempting to invoke such evidence towards incrimination of the virus in cervical carcinogenesis. By establishing a model organ culture system using human endocervical explants (Fink et al., 1973) we have attempted to study the replication of type 2 herpes simplex virus in tissue in which the architectural integrity is maintained. The organ culture technique has been extended to investigate the replication of type 2 herpes simplex virus in the explants, which may provide the basis for further investigation of cell transformation in contiguous human tissue. and Gynaecology, Birmingham Maternity Hospital,
VIRUS REPLICATION IN CULTURED CERVIX MATERIALS AND METHODS
Organ culture Collection of material.-Clinical information on the patients from whom tissue was obtained is shown in Table I. With the exception of one patient (78), who was recently divorced, all were married subjects. Endocervical tissue was obtained from uteri removed for non-malignant pathology. The tissue was cut from areas high in the endocervical canal in an attempt to select tissue which was free from metaplastic change. The specimens were transported in sterile Eagle's basal medium at 40. Technique of organ culture.-The organ culture system was based on the method of Trowell (1959) as adapted for human cervical organ culture by Fink et al. (1973). The tissue was maintained in Eagle's basal medium containing sodium penicillin (15 ,ug/ml) and streptomycin sulphate (15 ,ug/ml) in a humid atmosphere of 95% 02 and 5% CO2. Unless otherwise stated tissue was cultured at 370 using serum-free medium. Medium was changed every 3 days. Control tissue was selected by removing every fourth explant from the block of endocervical tissue. This tissue provided an indication of the amount and type of epithelium placed into culture. Routine histological sections were cut at 5 ju and stained with haematoxylin and eosin. Virus strains Strains HF, the HFEM derivative of the Rockefeller strain HF (Wildy, 1955), and strain 3345, a penile isolate, isolated at the Venereal Disease Clinic of the General Hospital, Birmingham, were used as type 1 and type 2 strains respectively (Geder and Skinner, 197 1; Plummer et al., 1974). The " Dekking " strain of pseudorabies virus was also used (Dekking, 1967). Antisera preparation Rabbits were given 7 i.m. injections of 200 mg virus antigen in Freund's incomplete adjuvant at 3-weekly intervals and thereafter given booster injections at 3-monthly intervals (Watson et al., 1966). Virus techniques
Infection of explants.-Explants were infected by suspension in virus-containing medium, with gentle agitation, for 2-5 h at 250. The explants were then washed thoroughly with fresh medium and residual unadsorbed virus was inactivated by suspension of the explants in immune serum (diluted 10-1 in medium) for 30 min at 37°. Following this procedure, the explants were thoroughly washed in media to remove the immune serum. To exclude undetected overt or latent
461
herpetic infection of the cervix, as a routine control uninfected explants were assayed for infectious virus before being placed into culture, and following culture for 3 days under the normal experimental conditions. As8ay of viru8 infectivity in the explants. Explants were placed individually in bijou bottles containing sterile distilled water (1 ml) and stored at -70°. Just prior to titration, the tissue was thawed and disrupted ultrasonically in a Megason bath. Histological examination of the tissue following sonication revealed complete destruction of the epithelial cell elements, with rather less marked stromal destruction (Fig. 1). Virus infectivity was titrated on BHK-21 cells (Macpherson and Stoker, 1962) by the plaque method of Russell (1962).
Replication errors between explants from one or more patients Mean values were compared by analysis of variance. Variances were compared by Snedecor's " F " test. The word " significant " is restricted to the statistical measure of P t< 0-05. The scatter in virus titres between replicate tissue from the same patient varied with the particular experimental conditions and tended to increase with the duration of the culture. The scatter in the values between tissues from different patients under the same experimental conditions was generally greater, although the difference did not reach significant levels. On this account and in view of the limited number of explants obtainable from any one patient, for certain purposes, data from experiments including one or more patients were evaluated as a whole.
Neutralization tests Kinetic neutralization tests were carried out as described by Skinner, Thouless and Jordan (1971). The neutralization constants (k-value) were calculated according to the formula: k
=
2-3 log V0 ctVt
where c = volume of antisera in test mixture divided by the total volume of test
mixture, t = time in minutes, V0 = initial virus concentration, Vt = virus concentration at time t.
RESULTS
Effect of the initial concentration of inoculum Tissues from 14 patients were used (Table I). Explants were exposed to
462
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
.r " .:z-..
FIG. 1.-(a) Cervical tissue following sonication. The columnar cells have detached into the lumen of the gland. Stromal disintegration is less marked. x 102. (b) Tissue before sonication. x 102. FIG. 6.-Cervical tissue 16 h following infection with type 2 herpes -implex virus. Columnar cells show gross vacuolation and destruction. x 160.
463
VIRUS REPLICATION IN CULTURED CERVIX
TABLE I.-Code for Patients from whom Tissue was Obtained Patient number 64 65 67 68 70 75 76 77 78 82 83 84 85 88 89 90 91 94 95 96 97
Age 52 25 40 25 30 43 52 49 43 46 36 36 30 42 31 29 40 49 31 31 32
Social class -
4 4 4 3 4 4 4 2 4 3 4 2 3 2 3
Parity 4 2+0 2 4+0 3
Blood group
0+
Serum neutralization rate constant (k-value) , Type 2 Type 1 0-16 0-12
A+
A+
0-67 0-0
0-16 0-14
0
0+
6 2+0
0+
3 -33 1 -87 0-19 73 -3 1 -80 2 -41 2-19
1 -45 0-78
3+0 3+0 3+0 3+0 1+3
1+0 3+1
2+0
9+0 1+0 2+0
1+0 3+0
varying multiplicities of virus infection and cultured for up to 7 days. Only with multiplicities of infection of greater than 108 plaque-forming units (pfu) per explant was there residual input or " background " virus infectivity following antisera treatment. The time of maximum virus replication varied according to the multiplicity of infection (Fig. 2); at 106-108 pfu per explant maximum infectivity occurred at 3 days, whereas with 104-105 pfu per explant infectivity reached a plateau level between 3 and 6 days of culture. At the lowest multiplicities (102-104 pfu per explant) virus replication was just detectable by the third day of culture, but had increased one thousand-fold by 6 days of culture. At very high multiplicities (viz., > 108 pfu per explant), while residual virus infectivity was not completely eliminated, there was clear evidence of virus replication by 16 h following infection. The proportion of explants infected was also related to the multiplicity of infection, reaching by 6 days of culture 100% at high multiplicities of infection, and falling to less than 50% at low
B+ A+
0+
A+ 0+ AB+ 0+ A+
A+ A+ O+ O+ O+ B+
0
2 -00
1 -07 2 -38 0-50 0-21 0-87
0-06
0
0-02 2-14 2 -60 4-73 3 -25 3-14
multiplicities of 102-104pfu (Table II).
0-12 0-69 1 -09 0 -57 0
0-72
per
explant
Influence of temperature on virus replication Explants (patient 88) were infected with 5 ml of virus suspension containing 5-6 x 104 pfu per explant and cultured for 3, 6 and 7 days at 320 and 37°. Total virus replication (the summated titres over the entire period of culture) was significantly decreased at the lower temperature (P < 0-01, and Table III).
Influence of serum on virus replication Explants from 2 patients (82 and 83) with 3-7 x 103 pfu of virus The explants were randomly arranged into 2 groups, one cultured in medium supplemented with 10% (v/v) calf serum, and the other cultured in serum-free medium. In serum-supplemented medium there was approximately a hundred-fold increase in virus replication (Fig. 3) and the proportion of successful infections was increased from 41 % in serum-free medium to 73% in serumsupplemented medium (Table II).
were infected per explant.
464
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
DAYS OF CULTURE FIG. 2.-Replication of type 2 herpes simplex virus following infection of the tissue on Day 0 with: > 108pfu/explant(EZ L0); 106-108 pfu/explant (* );104-105 A); 102-104pfu/explant pfu/explant ( ( .. ). Tissue was cultured in serumfree medium at 370. Mean + s.e. mean were determined using number of explants shown in Table II.
DAYS OF CULTURE FIG. 3.-Replication (mean ± s.e. mean) of type 2 herpes simplex virus cultured in the 0) presence (A---A) and absence ( of 10% v/v calf serum. Number of explants per determination and initial inoculum of virus are shown in Table II.
gated; for this purpose relatively low input multiplicities of 1.8 x 104pfu per explant were used. The cultures were in serum-supplemented Influence of organ culture age on virus maintained medium as described. A previously replication proportion of the explants harvested at The replication of virus in explants each time point were retained for histothroughout 13 days of culture was investi- logical examination. The bulk of virus TABLE II.-Proportion of Explants* showing Virus Replication during Culture Time of harvest (days) Experimental conditions Type 2 herpes simplex virus > 108 pfu/explant 106-108 pfu/explant
Patient code
,1
2
3
5
6
95, 97 64, 70 76, 78 75, 78 76, 78
47/47 24/25
9/9 25/25
8/8 30/30
11/15
8/8 7/7
A
7
104-105 pfu/explant 12/16 4/4 17/19 102-104 pfu/explant 1/11 7/15 Type 2 herpes simplex virus 4/5 3/5 With serum 4/5 82, 83 3 - 7 x 103 pfu/explant 2/3 1/5 4/9 Without serum j 82, 83 Type 1 herpes simplex virus 2/2 6/6 3/6 3 x 106 pfu/explant 84, 85 Pseudo-rabies virus 2/2 6/6 2 x 107 pfu/explant 89 * Numerator denotes the number of explants showing evidence of virus replication. Denominator denotes the number of explants exposed to virus inoculum.
VIRUS REPLICATION IN CULTURED CERVIX
465
Ix
uL
itJ 0.
O12i3
4
5 6 DAYS OF CUL
FIG. 4. Replication (± s.e. mean) of type 2 herpes simplex virus in tissue cultured for up to 12 days. Tissue from one patient was exposed to 6 - 8 x 104 pfu/explant on Day 0 and cultured for 12 days (A---A); tissue from a second patient was exposed to 1-8 x 104 pfu/explant on Day 0 and cultured for 9 days (0 *). The mean number of explants per point was 5. Infected explants were cultured at 370 in medium supplemented with 10% v/v calf serum.
replication occurred by 3 days, reaching maximum levels at 8 days, and was still detectable at similar levels by 13 days of culture (Fig. 4). Histological examination (vide infra) revealed good architectural preservation after this prolonged period of culture. As it was not apparent from the foregoing experiment whether the explants had synthesized new virus at 13 days of culture, or whether infectivity represented stable infective virus particles synthesized at an earlier time in the culture period (viz. 3 days of culture), explants were infected de novo with 1 x 106 pfu per explant at 4-day intervals from establish-
ment of the culture. In addition to evaluation of the relative involvement of the stromal and columnar epithelial cells in the continued support of virus replication, " standard " explants were bisected into " stromal " and predominantly epithelial explants. A number of standard explants were included for control purposes. Both " stromal " and " epithelial" explants could be infected de novo up to 12 days of culture with virus infectivity detected at 16 days of culture. Virus or replication equalled control " standard" levels in predominantly epithelial explants, but was significantly
TABLE III.-Replication of Type 2 Herpes Simplex Virus in Endocervical Tissue Cultured at 32° and 370 ± s.e. mean
Proportion of explants infected
3-2±2*7x 104 1 5±08x 104 4.8±29x 104
5/5 5/7 4/4
2 8±1*8x 103 6.4±38x 102 1*3±09x 104
5/5 3/4 3/3
Mean virus titre
Time of harvest (days) 37° culture 3 6 7 320 culture 3 6 7
,~ ~ ~ -k
466 J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
TABLE IV.- Virus Replication (±s.e. mean) in Epithelial and Stromal Explants Infected at Various Time Intervals following Establishment of Culture. Explants were Infected with 5 x 106 pfu of Type 2 Herpes Simplex Virus and Harvested 4 days after Infection. Each Mean Value was Derived from 5 Replicate Infected Explants. There was only One Standard Explant at Each Time Point Day of infection of explant
r--
Standard* Epithelial Stromal
0 2 1 x 104
8*3±2*61x 104 3 6±2 2x 104
4 2-4x 106 2*0+0*3x106 4-4+1 2x 104
8 1 9x 105 5 3A3 4x 105 7 1±43x 104
* " Standard " explant refers to tissue prepared in the normal epithelial and stromal elements.
decreased in " stromal " explants with the exception of stromal explants infected de novo at time zero (Table IV).
Influence of immune status of explant donor Serum was obtained from every patient whose tissue was used in our experiments. The neutralization rate constants for each serum against type 1 and type 2 herpes simplex virus are shown in Tables I and II, from which it is apparent that virus was capable of replication in tissue from both seropositive and sero-negative patients. Replication of other herpes viruses Explants (patients 84, 85 and 89) were infected with type 1 herpes simplex virus and pseudo-rabies virus using initial inocula of 13 x 106 and 241 x 107 pfu per explant respectively. There was replication of both types of virus (Fig. 5), although the titres were lower than those in explants infected with type 2 herpes simplex virus under similar experimental conditions. The proportion of successfully infected explants was in general agreement with results obtained with type 2 herpes simplex virus (Table II).
Histological appearances Cytopathological changes were identified in columnar epithelium by 16 h of infection: viz., cell vacuolation with gross cell destruction (Fig. 6). By 3 days of
way
12 3 6x 105 1-2±10x 106 2-2±2-Ox 103
containing both
infection there was evidence of further cell damage, margination of nuclear chromatin, and multinucleate giant cell formation (Fig. 7). At this time these features were less evident in the stroma, where they were generally confined to stromal tissue immediately adjacent to the infected columnar cells; in the deeper stromal layers cytopathic changes were
DAYS OF CULTURE FIG. 5.-Comparison of replication (h s.e. mean) of type 1 herpes simplex virus (A --A); type 2 herpes simplex virus (0 *) and pseudo-rabies virus in
explants
cultured
in
serum-
free medium at 37°. Data on number of patients and explants used, and initial inoculum of virus are given in Table II.
VIRUS REPLICATION IN CULTURED CERVIX
467
FIG. 7.-Columnar cells showing margination of nuclear chromatin and multinucleate giant cell formation. x 410. FIG. 8. Blood vessel following infection of the explant with type 2 herpes simplex virus. A multinucleate giant cell is apparent in the lumen. Other cells show evidence of infection; viz., margination of nuclear chromatin. x 410.
468
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
.. .
FiG. 9. Infected (A) and control (B) fibroblasts following culture for 12 days. Infected nuclei have a " spotty " appearance due to nuclear chromatin clumping. x 1024. FiG. 10. Infected cervical tissue following 12 days culture. In one gland there is gross cellular destruction (A) with spread to surrounding stromal tissue. In other areas there is normal columnar cell architecture (B). x 256.
VIRUS REPLICATION IN CULTURED CERVIX
almost entirely localized in the endothelial cells of the lymphatics and vascular system (Fig. 8). At later times in culture (viz., 12 days), cytopathic changes had extended to the deeper stromal layers and now involved fibroblastic cells which developed a " spotty " appearance as a result of chromatin margination (Fig. 9). However, even at this late stage there were areas of well maintained columnar and stromal cells with no evidence of viral cytopathic effect (Fig. 10). Similar cytological appearances were evident in explants infected with type 1 herpes simplex virus and swine pseudo-rabies virus. DISCUSSION
This study has provided quantitative evidence of replication of type 2 herpes simplex virus in cultured endocervix. In the initial studies with serum-free medium, high inocula of virus were used, which not only led to more rapid virus replication, with increased virus titre, but also increased the proportions of infected explants (Fig. 2 and Table II), thus providing a more reproducible experimental system. This was possible as residual virus inoculum was effectively eliminated by treatment of the explants at time zero with immune sera. The effects of both temperature (Table III) and serum supplementation (Fig. 3) were investigated. Although type 2 herpes simplex virus is reported to be relatively thermolabile (Plummer, Waner and Bawling, 1968; Figueroa and Rawls, 1969), and Farnham and Newton (1959) reported higher virus titres in type 1 infected cells cultured at 320, in our experimental system virus titres were significantly depressed in explants maintained at the lower temperature. This may have been due to poorer maintenance of the tissue at this temperature. Addition of serum to the culture medium increased the rate of virus replication, the virus titres and the proportion of infected explants (Fig. 3, Table II). This was probably related in part at least to
469
improved maintenance of the tissue in serum-supplemented medium as judged by histology of tissue cultured for 15 days. It was interesting that, while there were quite marked differences in virus replication with serum-deprived and serumsupplemented tissue by 3 days of culture, no histological differences with respect to tissue preservation were apparent. This suggests that virus replication may provide a quantitative and sensitive index of tissue viability. Virus replication was not detected until 16 h following infection. This contrasts with studies of herpes simplex virus replication in continuous hamster kidney cell lines where, for example, virus replication was detected within 8 h following infection (Watson, Wildy and Russell, 1964). However, in our explants there may be relatively few susceptible cells, and a proportion of these may be masked by mucus secretion or cell debris. Thus there may be few cells engaged in initial virus replication and, in conjunction with virus inactivation on account of thermolability and possibly interferon or interferon-like substances in the explant, our failure to detect virus infectivity during the early hours of culture may not be surprising. This is in part substantiated by the long delay (3 days) before appearance of infectious virus particles in explants treated with very low input multiplicities of (e.g.) 5 x 102 pfu per explant. This may model the venereal transmission of type 2 herpes simplex virus in human subjects where a (presumably) low inoculum of infectious virus is transmitted. Consistent with our findings, clinical features are not usually apparent until 3-7 days following venereal exposure (Rawls et al., 1971). It is also possible that the delay in appearance of infectious virus represents a genuine, but extended, " eclipse phase " (Watson ,Wildy and Russell, 1964) where the virus has penetrated the cells of the explant but has not yet been assembled into new infectious virus particles. A prolonged eclipse phase might be attribut-
470 J. BIRCH, C. G. FINK, G. R. 1B. SKINNER, G. H. THOMAS AND J. A. JORDAN
able to the particular explant cell involved in virus replication, or indeed to the particular conditions of organ culture. These hypotheses might be discriminated by numerical investigation of explant cells in the early stages of infection for evidence of early virus antigen production. The influence of organ culture " age on virus replication was investigated under optimal conditions of culture. Infectious virus was detectable until at least 13 days of culture (Fig. 4). It was not clear from this experiment whether new virus synthesis was complete by 3 days of culture, or whether virus produced by this time was merely " surviving " until 13 days, stabilized by the highly proteinaceous intracellular environment of the endocervical cells. However, it was apparent that these explants were capable of de novo virus synthesis even following 14 days of culture (Table IV). Virus replication was evident in tissue from sero-negative and sero-positive patients, with no apparent association between virus titres and the serological status of the donor subject; thus tissue from sero-negative patients was not " inherently immune " to herpes simplex virus infection and tissue from seropositive patients was not protected from virus infection by local neutralizing antibody. Type 1 herpes simplex virus, an uncommon associate of genital herpes virus infection (Dowdle et al., 1968) also replicated in cultured cervix, although the level of infectivity was lower than with type 2 herpes simplex virus (Fig. 5). The well established association between site of infection and virus type cannot therefore be wholly explained by " tropism " and raises the possibility that the virus type might be modified by multiple in vivo passage. This seems unlikely as although there is evidence of intermediate mutant or recombinant laboratory herpes simplex virus strains (Ejercito, Kieff and Roizman, 1968; Timbury and Subak-Sharpe, 1973), there are no reports to our knowledge of naturally occurring oral or genital herpes
isolates which demonstrate clear-cut " intermediate " serological behaviour. It was interesting that pseudo-rabies virus, a virus normally associated with infection of pigs, replicated, albeit rather poorly, in the tissue explants. Characteristic cytopathic effects of type 1 and type 2 herpes simplex virus and pseudo-rabies virus were noted in both columnar and stromal cells, and resembled the histological appearance of an in vivo infection of the cervix by type 2 herpes simplex virus (Naib et al., 1973). Moreover, both epithelial and stromal elements of the explants supported infection of type 2 herpes simplex virus although titres in the columnar cells were generally higher (Table IV). This might be expected in view of the natural history of herpes virus infection, which is usually transmitted by direct cutaneous contact and will frequently find a foothold in the cells of the squamo-columnar junction. In these experiments a model system permitting investigation of type 2 herpes simplex infection of the uterine cervix has been established. This will allow identification of virus antigens produced in human endocervical tissue, which in turn may suggest which type specific antibodies, if any, may be represented in human sera, thus offering a more precise serological method of detecting previous herpetic infection in the human subject. This is of great importance in view of sero-epidemiological evidence relating type 2 herpes simplex virus and carcinoma of the cervix. In addition it may be possible to investigate the phenomenon of type 2 herpes simplex virus-induced cell transformation in the appropriate cells (in contiguity rather than in isolated cells of a laboratory " cell line ") of the appropriate host. This would, therefore, seem a useful compromise between in vitro cellular studies and in vivo or human experimentation which is clearly unacceptable. We thank the medical staff of the Birmingham and Midland Hospital for Women for kindly providing tissue; we are
VIRUS REPLICATION IN CULTURED CERVIX 471 Epithelia for the Study of Respiratory Viruses. indebted to Mrs D. Blackmore and Mr C. Acta path. microbiol. scand., 183, 1. H. Hubble for providing technical assist- MACPHERSON, I. & STOKER, M. (1962) Polyoma ance, and to Miss J. M. Allen and Mrs G. Transformation of Hamster Cell Clones-an Investigation of Genetic Factors Affecting Cell A. Powell for their invaluable assistance Competence. Virology, 16, 147. with photography. We are grateful to McNAB, J. C. M. (1974) Transformation of Rat Embryo Cells of Temperature Sensitive Mutants Professor H. C. McLaren, Department of of Herpes Simplex Virus. J. gen. Virol., 24, 143. Obstetrics and Gynaecology, and Pro- NAHMIAS, A. J., JOSEY, W. E., NAIB, Z. M., LUCE, fessor P. Wildy, Department of Virology, C. F. & GUEST, B. (1970) Antibodies to Herpesvirus Hominis Types 1 and 2 in Humans. II. University of Birmingham for much Women with Cervical Cancer. Am. J. Epidem., valuable advice and criticism. This work
91, 547. NAIB, Z. M. (1966) Exfoliative Cytology of Viral Cervical-vaginitis. Acta cytol., 10, 126. Campaign. NAIB, Z. M., NAHMIAS, A. J., JOSEY, W. E. & ZAKI, S. A. (1973) Relation of Cytohistopathology of Genital Herpesvirus Infection to Cervical Anaplasia. Cancer Res., 33, 1452. PLUMMER, G., WANER, J. L. & BAWLING, C. P. (1968) REFERENCES Comparative Studies of Type 1 and Type 2 BALDUZZI, P. C., NASELLO, M. A. & AMSTEY, M. S. Herpes Simplex Viruses. Br. J. exp. Path., 49, (1972) Experimental Infection of Human Cervix 202. by Herpes Virus Type 2 in Organ Culture. PLUMMER, G., GOODHEART, C. R., MIYAGI, M., Cancer Res., 32, 243. SKINNER, G. R. B., THOULESS, M. E. & WILDY, P. BANG, F. B. & NIVEN, J. S. F. (1958) A Study of (1974) Herpes Simplex Viruses: Discrimination of Infection in Organised Tissue Cultures. Br. J. Types and Correlation between Different Characexp. Path., 39, 317. teristics. Virology, 60, 206. BARSKI, G., CORNFERT, F. & WALLACE, R. E. (1959) RAWLS, W. E., TOMPKINS, W. A. E. & MELNICK, J. Response of Ciliated Epithelium of Different L. (1969) The Association of Herpes II Virus and Histological Origin to Virus Infections in vitro. Carcinoma of the Uterine Cervix. Am. J. Proc. Soc. exp. Biol. Med., 100, 407. Epidem., 89, 547. DEKKINC, F. (1967) described by WATSON, D. H., RAWLS, W. E., GARDNER, H. L., FLANDERS, R. W., WILDY, P., HARVEY, B. A. M. & SHEDDEN, W. I. LOWRY, S. P., KAUFMAN, R. H. & MELNICK, J. L. H. (1967) Serological Relationships among (1971) Genital Herpes in Two Social Groups. Viruses of the Herpes Group. J. gen. Virol., 1, Am. J. Obstet. Gyn., 10, 682. 139. RUSSELL, W. C. (1962) A Sensitive and Precise DOWDLE, W., NAHMIAS, A. J., HARWELL, R. & Plaque Assay for Herpes Virus. Nature, Lond., PAULS, F. (1968) Association of Antigenic Type of 195, 1028. Herpes Virus Hominis with Site of Viral Recovery. SKINNER, G. R. B., THOULESS, M. E. & JORDAN, J. A. J. Immunol., 99, 974. (1971) Antibodies to Type 1 and Type 2 Herpes DUFF, R. & RAPP, F. (1971) Properties of Hamster Virus in Women with Abnormal Cervical Cytology. Embryo Fibroblasts Transformed in vitro after J. Obstet. Gynaec. Br. Commonuw., 78, 1031. Exposure to UV-irradiated Herpes Simplex Virus SKINNER, G. R. B. (1976) Transformation of Primary Type 2. J. Virol., 8, 469. Hamster Fibroblasts by Type 2 Herpes Simplex EJERCITO, P. M., KIEFF, E. D. & ROIZMAN, B. (1968) Virus; Evidence for a Hit and Run Mechanism. Characterisation of Herpes Simplex Virus Br. J. exp. Path. (In press.) Strains Differing in their Effects on Social STEVENS, J. G. & COOK, M. L. (1973) Latent InfecBehaviour of Infected Cells. J. gen. Virol., 2, 357. tions Induced by Herpes Simplex Viruses. FARNHAM, A. E. & NEWTON, A. A. (1959) The Effect Cancer Res., 33, 1399. of Some Environmental Factors on Herpesvirus TIMBURY, M. C. & SUBAK-SHARPE, J. H. (1973) Growth in He-la Cells. Virology, 7, 449. Genetic Interactions between Temperature FIGUEROA, M. E. & RAWLS, W. E. (1969) Biological Sensitive Mutants of Types 1 and 2 Herpes Markers for Differentiation of Herpes Virus Simplex Viruses. J. gen. Virol., 18, 347. Strains of Oral and Genital Origin. J. gen. Virol., TRAWELL, 0. A. (1959) The Culture of Mature Organs 4, 259. in a Synthetic Medium. Expl Cell Res., 16, 119. FINK, C. G., THOMAS, G. H., ALLEN, J. M. & JORDAN', WATSON, D. H., WILDY, P. & RUSSELL, W. C. (1964) J. A. (1973) Metaplasia in Endocervical Tissue Quantitative Electron Microscope Studies on the Maintained in Organ Culture-an Experimental Growth of Herpes Virus using the Technique of Model. J. Obstet. Gynaec. Br. Commonw., 80, 75. Negative Staining and Ultramicrotomy. GEDER, L. & SKINNER, G. R. B. (1971) DifferenVirology, 24, 523. tiation Between Type 1 and Type 2 Strains of WATSON, D. H., SHEDDEN, W. I. H., ELLIOT, A., Herpes Simplex Virus by an Indirect ImmunoTETSIJKA, T., WILDY, P., BOURGAUX-RAMOISY, D. fluorescence Technique. J. gen. Virol., 12, 1 79. & GOLD, E. (1966) Virus Specific Antigens in HoORN, B. (1964) Respiratory Viruses in Model Mammalian Cells Infected with Herpes Simplex Experiments. Transactions of the 15th Congress Virus. Immunology, 11, 399. of the Scandinavian Oto-Larynyological Society, WILDY, P. (1955) Recombination with Herpes 1963. Acta oto-lar., 188, Suppl., 138. Simplex Virus. J. gen. Microbiol., 13, 346. HOORN, B. (1966) Organ Cultures of Ciliated
was supported by the Cancer Research
.q I
REPLICATION OF TYPE 2 HERPES SIMPLEX VIRUS IN HUMAN ENDOCERVICAL TISSUE IN ORGAN CULTURE J. BIRCH*, C. G4. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN From the Departments of Obstetrics and Gynaecology, Virology and Anatomy, University of Birmingham
Received for publication AMarch 18, 1976
Summary.-The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 370 and in serumsupplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a " non-human " herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions, and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ.
THE BEHAVIOUR of type 1 herpes simplex virus in tissue maintained in organ culture has been widely investigated in human fetal skin (Bang and Niven, 1958), human fetal trachea (Hoorn, 1964, 1966), rabbit adult oviduct (Barski, Cornfert and Wallace, 1959), and rabbit adult trachea (Hoorn, 1964). However, there is little information concerning the behaviour of type 2 herpes simplex virus. Balduzzi, Nasello and Amstey (1972) reported histological changes in herpes virus-infected cervical tissue fragments maintained for up to 3 weeks in culture, but did not investigate virus replication in quantitative terms. In recent years a considerable body of research has investigated type 2 herpes simplex virus. The virus is the aetiological agent of a relatively common venereal disease herpes genitalis and there is evidence that the virus is involved in the phenomenon of latency (Stevens and Cook, 1973). Of great interest, there * Present address: Department of Obstetrics Birmingham 15.
is evidence associating previous herpetic infection with pre-invasive and invasive carcinoma of the uterine cervix (Naib, 1966; Rawls, Tomkins and Melnick, 1969; Nahmias et al., 1970; Skinner, Thouless and Jordan, 1971). Type 2 herpes simplex virus is capable of in vitro cell transformation (Duff and Rapp, 1971; McNab, 1974; Skinner, 1976), and it is tempting to invoke such evidence towards incrimination of the virus in cervical carcinogenesis. By establishing a model organ culture system using human endocervical explants (Fink et al., 1973) we have attempted to study the replication of type 2 herpes simplex virus in tissue in which the architectural integrity is maintained. The organ culture technique has been extended to investigate the replication of type 2 herpes simplex virus in the explants, which may provide the basis for further investigation of cell transformation in contiguous human tissue. and Gynaecology, Birmingham Maternity Hospital,
VIRUS REPLICATION IN CULTURED CERVIX MATERIALS AND METHODS
Organ culture Collection of material.-Clinical information on the patients from whom tissue was obtained is shown in Table I. With the exception of one patient (78), who was recently divorced, all were married subjects. Endocervical tissue was obtained from uteri removed for non-malignant pathology. The tissue was cut from areas high in the endocervical canal in an attempt to select tissue which was free from metaplastic change. The specimens were transported in sterile Eagle's basal medium at 40. Technique of organ culture.-The organ culture system was based on the method of Trowell (1959) as adapted for human cervical organ culture by Fink et al. (1973). The tissue was maintained in Eagle's basal medium containing sodium penicillin (15 ,ug/ml) and streptomycin sulphate (15 ,ug/ml) in a humid atmosphere of 95% 02 and 5% CO2. Unless otherwise stated tissue was cultured at 370 using serum-free medium. Medium was changed every 3 days. Control tissue was selected by removing every fourth explant from the block of endocervical tissue. This tissue provided an indication of the amount and type of epithelium placed into culture. Routine histological sections were cut at 5 ju and stained with haematoxylin and eosin. Virus strains Strains HF, the HFEM derivative of the Rockefeller strain HF (Wildy, 1955), and strain 3345, a penile isolate, isolated at the Venereal Disease Clinic of the General Hospital, Birmingham, were used as type 1 and type 2 strains respectively (Geder and Skinner, 197 1; Plummer et al., 1974). The " Dekking " strain of pseudorabies virus was also used (Dekking, 1967). Antisera preparation Rabbits were given 7 i.m. injections of 200 mg virus antigen in Freund's incomplete adjuvant at 3-weekly intervals and thereafter given booster injections at 3-monthly intervals (Watson et al., 1966). Virus techniques
Infection of explants.-Explants were infected by suspension in virus-containing medium, with gentle agitation, for 2-5 h at 250. The explants were then washed thoroughly with fresh medium and residual unadsorbed virus was inactivated by suspension of the explants in immune serum (diluted 10-1 in medium) for 30 min at 37°. Following this procedure, the explants were thoroughly washed in media to remove the immune serum. To exclude undetected overt or latent
461
herpetic infection of the cervix, as a routine control uninfected explants were assayed for infectious virus before being placed into culture, and following culture for 3 days under the normal experimental conditions. As8ay of viru8 infectivity in the explants. Explants were placed individually in bijou bottles containing sterile distilled water (1 ml) and stored at -70°. Just prior to titration, the tissue was thawed and disrupted ultrasonically in a Megason bath. Histological examination of the tissue following sonication revealed complete destruction of the epithelial cell elements, with rather less marked stromal destruction (Fig. 1). Virus infectivity was titrated on BHK-21 cells (Macpherson and Stoker, 1962) by the plaque method of Russell (1962).
Replication errors between explants from one or more patients Mean values were compared by analysis of variance. Variances were compared by Snedecor's " F " test. The word " significant " is restricted to the statistical measure of P t< 0-05. The scatter in virus titres between replicate tissue from the same patient varied with the particular experimental conditions and tended to increase with the duration of the culture. The scatter in the values between tissues from different patients under the same experimental conditions was generally greater, although the difference did not reach significant levels. On this account and in view of the limited number of explants obtainable from any one patient, for certain purposes, data from experiments including one or more patients were evaluated as a whole.
Neutralization tests Kinetic neutralization tests were carried out as described by Skinner, Thouless and Jordan (1971). The neutralization constants (k-value) were calculated according to the formula: k
=
2-3 log V0 ctVt
where c = volume of antisera in test mixture divided by the total volume of test
mixture, t = time in minutes, V0 = initial virus concentration, Vt = virus concentration at time t.
RESULTS
Effect of the initial concentration of inoculum Tissues from 14 patients were used (Table I). Explants were exposed to
462
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
.r " .:z-..
FIG. 1.-(a) Cervical tissue following sonication. The columnar cells have detached into the lumen of the gland. Stromal disintegration is less marked. x 102. (b) Tissue before sonication. x 102. FIG. 6.-Cervical tissue 16 h following infection with type 2 herpes -implex virus. Columnar cells show gross vacuolation and destruction. x 160.
463
VIRUS REPLICATION IN CULTURED CERVIX
TABLE I.-Code for Patients from whom Tissue was Obtained Patient number 64 65 67 68 70 75 76 77 78 82 83 84 85 88 89 90 91 94 95 96 97
Age 52 25 40 25 30 43 52 49 43 46 36 36 30 42 31 29 40 49 31 31 32
Social class -
4 4 4 3 4 4 4 2 4 3 4 2 3 2 3
Parity 4 2+0 2 4+0 3
Blood group
0+
Serum neutralization rate constant (k-value) , Type 2 Type 1 0-16 0-12
A+
A+
0-67 0-0
0-16 0-14
0
0+
6 2+0
0+
3 -33 1 -87 0-19 73 -3 1 -80 2 -41 2-19
1 -45 0-78
3+0 3+0 3+0 3+0 1+3
1+0 3+1
2+0
9+0 1+0 2+0
1+0 3+0
varying multiplicities of virus infection and cultured for up to 7 days. Only with multiplicities of infection of greater than 108 plaque-forming units (pfu) per explant was there residual input or " background " virus infectivity following antisera treatment. The time of maximum virus replication varied according to the multiplicity of infection (Fig. 2); at 106-108 pfu per explant maximum infectivity occurred at 3 days, whereas with 104-105 pfu per explant infectivity reached a plateau level between 3 and 6 days of culture. At the lowest multiplicities (102-104 pfu per explant) virus replication was just detectable by the third day of culture, but had increased one thousand-fold by 6 days of culture. At very high multiplicities (viz., > 108 pfu per explant), while residual virus infectivity was not completely eliminated, there was clear evidence of virus replication by 16 h following infection. The proportion of explants infected was also related to the multiplicity of infection, reaching by 6 days of culture 100% at high multiplicities of infection, and falling to less than 50% at low
B+ A+
0+
A+ 0+ AB+ 0+ A+
A+ A+ O+ O+ O+ B+
0
2 -00
1 -07 2 -38 0-50 0-21 0-87
0-06
0
0-02 2-14 2 -60 4-73 3 -25 3-14
multiplicities of 102-104pfu (Table II).
0-12 0-69 1 -09 0 -57 0
0-72
per
explant
Influence of temperature on virus replication Explants (patient 88) were infected with 5 ml of virus suspension containing 5-6 x 104 pfu per explant and cultured for 3, 6 and 7 days at 320 and 37°. Total virus replication (the summated titres over the entire period of culture) was significantly decreased at the lower temperature (P < 0-01, and Table III).
Influence of serum on virus replication Explants from 2 patients (82 and 83) with 3-7 x 103 pfu of virus The explants were randomly arranged into 2 groups, one cultured in medium supplemented with 10% (v/v) calf serum, and the other cultured in serum-free medium. In serum-supplemented medium there was approximately a hundred-fold increase in virus replication (Fig. 3) and the proportion of successful infections was increased from 41 % in serum-free medium to 73% in serumsupplemented medium (Table II).
were infected per explant.
464
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
DAYS OF CULTURE FIG. 2.-Replication of type 2 herpes simplex virus following infection of the tissue on Day 0 with: > 108pfu/explant(EZ L0); 106-108 pfu/explant (* );104-105 A); 102-104pfu/explant pfu/explant ( ( .. ). Tissue was cultured in serumfree medium at 370. Mean + s.e. mean were determined using number of explants shown in Table II.
DAYS OF CULTURE FIG. 3.-Replication (mean ± s.e. mean) of type 2 herpes simplex virus cultured in the 0) presence (A---A) and absence ( of 10% v/v calf serum. Number of explants per determination and initial inoculum of virus are shown in Table II.
gated; for this purpose relatively low input multiplicities of 1.8 x 104pfu per explant were used. The cultures were in serum-supplemented Influence of organ culture age on virus maintained medium as described. A previously replication proportion of the explants harvested at The replication of virus in explants each time point were retained for histothroughout 13 days of culture was investi- logical examination. The bulk of virus TABLE II.-Proportion of Explants* showing Virus Replication during Culture Time of harvest (days) Experimental conditions Type 2 herpes simplex virus > 108 pfu/explant 106-108 pfu/explant
Patient code
,1
2
3
5
6
95, 97 64, 70 76, 78 75, 78 76, 78
47/47 24/25
9/9 25/25
8/8 30/30
11/15
8/8 7/7
A
7
104-105 pfu/explant 12/16 4/4 17/19 102-104 pfu/explant 1/11 7/15 Type 2 herpes simplex virus 4/5 3/5 With serum 4/5 82, 83 3 - 7 x 103 pfu/explant 2/3 1/5 4/9 Without serum j 82, 83 Type 1 herpes simplex virus 2/2 6/6 3/6 3 x 106 pfu/explant 84, 85 Pseudo-rabies virus 2/2 6/6 2 x 107 pfu/explant 89 * Numerator denotes the number of explants showing evidence of virus replication. Denominator denotes the number of explants exposed to virus inoculum.
VIRUS REPLICATION IN CULTURED CERVIX
465
Ix
uL
itJ 0.
O12i3
4
5 6 DAYS OF CUL
FIG. 4. Replication (± s.e. mean) of type 2 herpes simplex virus in tissue cultured for up to 12 days. Tissue from one patient was exposed to 6 - 8 x 104 pfu/explant on Day 0 and cultured for 12 days (A---A); tissue from a second patient was exposed to 1-8 x 104 pfu/explant on Day 0 and cultured for 9 days (0 *). The mean number of explants per point was 5. Infected explants were cultured at 370 in medium supplemented with 10% v/v calf serum.
replication occurred by 3 days, reaching maximum levels at 8 days, and was still detectable at similar levels by 13 days of culture (Fig. 4). Histological examination (vide infra) revealed good architectural preservation after this prolonged period of culture. As it was not apparent from the foregoing experiment whether the explants had synthesized new virus at 13 days of culture, or whether infectivity represented stable infective virus particles synthesized at an earlier time in the culture period (viz. 3 days of culture), explants were infected de novo with 1 x 106 pfu per explant at 4-day intervals from establish-
ment of the culture. In addition to evaluation of the relative involvement of the stromal and columnar epithelial cells in the continued support of virus replication, " standard " explants were bisected into " stromal " and predominantly epithelial explants. A number of standard explants were included for control purposes. Both " stromal " and " epithelial" explants could be infected de novo up to 12 days of culture with virus infectivity detected at 16 days of culture. Virus or replication equalled control " standard" levels in predominantly epithelial explants, but was significantly
TABLE III.-Replication of Type 2 Herpes Simplex Virus in Endocervical Tissue Cultured at 32° and 370 ± s.e. mean
Proportion of explants infected
3-2±2*7x 104 1 5±08x 104 4.8±29x 104
5/5 5/7 4/4
2 8±1*8x 103 6.4±38x 102 1*3±09x 104
5/5 3/4 3/3
Mean virus titre
Time of harvest (days) 37° culture 3 6 7 320 culture 3 6 7
,~ ~ ~ -k
466 J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
TABLE IV.- Virus Replication (±s.e. mean) in Epithelial and Stromal Explants Infected at Various Time Intervals following Establishment of Culture. Explants were Infected with 5 x 106 pfu of Type 2 Herpes Simplex Virus and Harvested 4 days after Infection. Each Mean Value was Derived from 5 Replicate Infected Explants. There was only One Standard Explant at Each Time Point Day of infection of explant
r--
Standard* Epithelial Stromal
0 2 1 x 104
8*3±2*61x 104 3 6±2 2x 104
4 2-4x 106 2*0+0*3x106 4-4+1 2x 104
8 1 9x 105 5 3A3 4x 105 7 1±43x 104
* " Standard " explant refers to tissue prepared in the normal epithelial and stromal elements.
decreased in " stromal " explants with the exception of stromal explants infected de novo at time zero (Table IV).
Influence of immune status of explant donor Serum was obtained from every patient whose tissue was used in our experiments. The neutralization rate constants for each serum against type 1 and type 2 herpes simplex virus are shown in Tables I and II, from which it is apparent that virus was capable of replication in tissue from both seropositive and sero-negative patients. Replication of other herpes viruses Explants (patients 84, 85 and 89) were infected with type 1 herpes simplex virus and pseudo-rabies virus using initial inocula of 13 x 106 and 241 x 107 pfu per explant respectively. There was replication of both types of virus (Fig. 5), although the titres were lower than those in explants infected with type 2 herpes simplex virus under similar experimental conditions. The proportion of successfully infected explants was in general agreement with results obtained with type 2 herpes simplex virus (Table II).
Histological appearances Cytopathological changes were identified in columnar epithelium by 16 h of infection: viz., cell vacuolation with gross cell destruction (Fig. 6). By 3 days of
way
12 3 6x 105 1-2±10x 106 2-2±2-Ox 103
containing both
infection there was evidence of further cell damage, margination of nuclear chromatin, and multinucleate giant cell formation (Fig. 7). At this time these features were less evident in the stroma, where they were generally confined to stromal tissue immediately adjacent to the infected columnar cells; in the deeper stromal layers cytopathic changes were
DAYS OF CULTURE FIG. 5.-Comparison of replication (h s.e. mean) of type 1 herpes simplex virus (A --A); type 2 herpes simplex virus (0 *) and pseudo-rabies virus in
explants
cultured
in
serum-
free medium at 37°. Data on number of patients and explants used, and initial inoculum of virus are given in Table II.
VIRUS REPLICATION IN CULTURED CERVIX
467
FIG. 7.-Columnar cells showing margination of nuclear chromatin and multinucleate giant cell formation. x 410. FIG. 8. Blood vessel following infection of the explant with type 2 herpes simplex virus. A multinucleate giant cell is apparent in the lumen. Other cells show evidence of infection; viz., margination of nuclear chromatin. x 410.
468
J. BIRCH, C. G. FINK, G. R. B. SKINNER, G. H. THOMAS AND J. A. JORDAN
.. .
FiG. 9. Infected (A) and control (B) fibroblasts following culture for 12 days. Infected nuclei have a " spotty " appearance due to nuclear chromatin clumping. x 1024. FiG. 10. Infected cervical tissue following 12 days culture. In one gland there is gross cellular destruction (A) with spread to surrounding stromal tissue. In other areas there is normal columnar cell architecture (B). x 256.
VIRUS REPLICATION IN CULTURED CERVIX
almost entirely localized in the endothelial cells of the lymphatics and vascular system (Fig. 8). At later times in culture (viz., 12 days), cytopathic changes had extended to the deeper stromal layers and now involved fibroblastic cells which developed a " spotty " appearance as a result of chromatin margination (Fig. 9). However, even at this late stage there were areas of well maintained columnar and stromal cells with no evidence of viral cytopathic effect (Fig. 10). Similar cytological appearances were evident in explants infected with type 1 herpes simplex virus and swine pseudo-rabies virus. DISCUSSION
This study has provided quantitative evidence of replication of type 2 herpes simplex virus in cultured endocervix. In the initial studies with serum-free medium, high inocula of virus were used, which not only led to more rapid virus replication, with increased virus titre, but also increased the proportions of infected explants (Fig. 2 and Table II), thus providing a more reproducible experimental system. This was possible as residual virus inoculum was effectively eliminated by treatment of the explants at time zero with immune sera. The effects of both temperature (Table III) and serum supplementation (Fig. 3) were investigated. Although type 2 herpes simplex virus is reported to be relatively thermolabile (Plummer, Waner and Bawling, 1968; Figueroa and Rawls, 1969), and Farnham and Newton (1959) reported higher virus titres in type 1 infected cells cultured at 320, in our experimental system virus titres were significantly depressed in explants maintained at the lower temperature. This may have been due to poorer maintenance of the tissue at this temperature. Addition of serum to the culture medium increased the rate of virus replication, the virus titres and the proportion of infected explants (Fig. 3, Table II). This was probably related in part at least to
469
improved maintenance of the tissue in serum-supplemented medium as judged by histology of tissue cultured for 15 days. It was interesting that, while there were quite marked differences in virus replication with serum-deprived and serumsupplemented tissue by 3 days of culture, no histological differences with respect to tissue preservation were apparent. This suggests that virus replication may provide a quantitative and sensitive index of tissue viability. Virus replication was not detected until 16 h following infection. This contrasts with studies of herpes simplex virus replication in continuous hamster kidney cell lines where, for example, virus replication was detected within 8 h following infection (Watson, Wildy and Russell, 1964). However, in our explants there may be relatively few susceptible cells, and a proportion of these may be masked by mucus secretion or cell debris. Thus there may be few cells engaged in initial virus replication and, in conjunction with virus inactivation on account of thermolability and possibly interferon or interferon-like substances in the explant, our failure to detect virus infectivity during the early hours of culture may not be surprising. This is in part substantiated by the long delay (3 days) before appearance of infectious virus particles in explants treated with very low input multiplicities of (e.g.) 5 x 102 pfu per explant. This may model the venereal transmission of type 2 herpes simplex virus in human subjects where a (presumably) low inoculum of infectious virus is transmitted. Consistent with our findings, clinical features are not usually apparent until 3-7 days following venereal exposure (Rawls et al., 1971). It is also possible that the delay in appearance of infectious virus represents a genuine, but extended, " eclipse phase " (Watson ,Wildy and Russell, 1964) where the virus has penetrated the cells of the explant but has not yet been assembled into new infectious virus particles. A prolonged eclipse phase might be attribut-
470 J. BIRCH, C. G. FINK, G. R. 1B. SKINNER, G. H. THOMAS AND J. A. JORDAN
able to the particular explant cell involved in virus replication, or indeed to the particular conditions of organ culture. These hypotheses might be discriminated by numerical investigation of explant cells in the early stages of infection for evidence of early virus antigen production. The influence of organ culture " age on virus replication was investigated under optimal conditions of culture. Infectious virus was detectable until at least 13 days of culture (Fig. 4). It was not clear from this experiment whether new virus synthesis was complete by 3 days of culture, or whether virus produced by this time was merely " surviving " until 13 days, stabilized by the highly proteinaceous intracellular environment of the endocervical cells. However, it was apparent that these explants were capable of de novo virus synthesis even following 14 days of culture (Table IV). Virus replication was evident in tissue from sero-negative and sero-positive patients, with no apparent association between virus titres and the serological status of the donor subject; thus tissue from sero-negative patients was not " inherently immune " to herpes simplex virus infection and tissue from seropositive patients was not protected from virus infection by local neutralizing antibody. Type 1 herpes simplex virus, an uncommon associate of genital herpes virus infection (Dowdle et al., 1968) also replicated in cultured cervix, although the level of infectivity was lower than with type 2 herpes simplex virus (Fig. 5). The well established association between site of infection and virus type cannot therefore be wholly explained by " tropism " and raises the possibility that the virus type might be modified by multiple in vivo passage. This seems unlikely as although there is evidence of intermediate mutant or recombinant laboratory herpes simplex virus strains (Ejercito, Kieff and Roizman, 1968; Timbury and Subak-Sharpe, 1973), there are no reports to our knowledge of naturally occurring oral or genital herpes
isolates which demonstrate clear-cut " intermediate " serological behaviour. It was interesting that pseudo-rabies virus, a virus normally associated with infection of pigs, replicated, albeit rather poorly, in the tissue explants. Characteristic cytopathic effects of type 1 and type 2 herpes simplex virus and pseudo-rabies virus were noted in both columnar and stromal cells, and resembled the histological appearance of an in vivo infection of the cervix by type 2 herpes simplex virus (Naib et al., 1973). Moreover, both epithelial and stromal elements of the explants supported infection of type 2 herpes simplex virus although titres in the columnar cells were generally higher (Table IV). This might be expected in view of the natural history of herpes virus infection, which is usually transmitted by direct cutaneous contact and will frequently find a foothold in the cells of the squamo-columnar junction. In these experiments a model system permitting investigation of type 2 herpes simplex infection of the uterine cervix has been established. This will allow identification of virus antigens produced in human endocervical tissue, which in turn may suggest which type specific antibodies, if any, may be represented in human sera, thus offering a more precise serological method of detecting previous herpetic infection in the human subject. This is of great importance in view of sero-epidemiological evidence relating type 2 herpes simplex virus and carcinoma of the cervix. In addition it may be possible to investigate the phenomenon of type 2 herpes simplex virus-induced cell transformation in the appropriate cells (in contiguity rather than in isolated cells of a laboratory " cell line ") of the appropriate host. This would, therefore, seem a useful compromise between in vitro cellular studies and in vivo or human experimentation which is clearly unacceptable. We thank the medical staff of the Birmingham and Midland Hospital for Women for kindly providing tissue; we are
VIRUS REPLICATION IN CULTURED CERVIX 471 Epithelia for the Study of Respiratory Viruses. indebted to Mrs D. Blackmore and Mr C. Acta path. microbiol. scand., 183, 1. H. Hubble for providing technical assist- MACPHERSON, I. & STOKER, M. (1962) Polyoma ance, and to Miss J. M. Allen and Mrs G. Transformation of Hamster Cell Clones-an Investigation of Genetic Factors Affecting Cell A. Powell for their invaluable assistance Competence. Virology, 16, 147. with photography. We are grateful to McNAB, J. C. M. (1974) Transformation of Rat Embryo Cells of Temperature Sensitive Mutants Professor H. C. McLaren, Department of of Herpes Simplex Virus. J. gen. Virol., 24, 143. Obstetrics and Gynaecology, and Pro- NAHMIAS, A. J., JOSEY, W. E., NAIB, Z. M., LUCE, fessor P. Wildy, Department of Virology, C. F. & GUEST, B. (1970) Antibodies to Herpesvirus Hominis Types 1 and 2 in Humans. II. University of Birmingham for much Women with Cervical Cancer. Am. J. Epidem., valuable advice and criticism. This work
91, 547. NAIB, Z. M. (1966) Exfoliative Cytology of Viral Cervical-vaginitis. Acta cytol., 10, 126. Campaign. NAIB, Z. M., NAHMIAS, A. J., JOSEY, W. E. & ZAKI, S. A. (1973) Relation of Cytohistopathology of Genital Herpesvirus Infection to Cervical Anaplasia. Cancer Res., 33, 1452. PLUMMER, G., WANER, J. L. & BAWLING, C. P. (1968) REFERENCES Comparative Studies of Type 1 and Type 2 BALDUZZI, P. C., NASELLO, M. A. & AMSTEY, M. S. Herpes Simplex Viruses. Br. J. exp. Path., 49, (1972) Experimental Infection of Human Cervix 202. by Herpes Virus Type 2 in Organ Culture. PLUMMER, G., GOODHEART, C. R., MIYAGI, M., Cancer Res., 32, 243. SKINNER, G. R. B., THOULESS, M. E. & WILDY, P. BANG, F. B. & NIVEN, J. S. F. (1958) A Study of (1974) Herpes Simplex Viruses: Discrimination of Infection in Organised Tissue Cultures. Br. J. Types and Correlation between Different Characexp. Path., 39, 317. teristics. Virology, 60, 206. BARSKI, G., CORNFERT, F. & WALLACE, R. E. (1959) RAWLS, W. E., TOMPKINS, W. A. E. & MELNICK, J. Response of Ciliated Epithelium of Different L. (1969) The Association of Herpes II Virus and Histological Origin to Virus Infections in vitro. Carcinoma of the Uterine Cervix. Am. J. Proc. Soc. exp. Biol. Med., 100, 407. Epidem., 89, 547. DEKKINC, F. (1967) described by WATSON, D. H., RAWLS, W. E., GARDNER, H. L., FLANDERS, R. W., WILDY, P., HARVEY, B. A. M. & SHEDDEN, W. I. LOWRY, S. P., KAUFMAN, R. H. & MELNICK, J. L. H. (1967) Serological Relationships among (1971) Genital Herpes in Two Social Groups. Viruses of the Herpes Group. J. gen. Virol., 1, Am. J. Obstet. Gyn., 10, 682. 139. RUSSELL, W. C. (1962) A Sensitive and Precise DOWDLE, W., NAHMIAS, A. J., HARWELL, R. & Plaque Assay for Herpes Virus. Nature, Lond., PAULS, F. (1968) Association of Antigenic Type of 195, 1028. Herpes Virus Hominis with Site of Viral Recovery. SKINNER, G. R. B., THOULESS, M. E. & JORDAN, J. A. J. Immunol., 99, 974. (1971) Antibodies to Type 1 and Type 2 Herpes DUFF, R. & RAPP, F. (1971) Properties of Hamster Virus in Women with Abnormal Cervical Cytology. Embryo Fibroblasts Transformed in vitro after J. Obstet. Gynaec. Br. Commonuw., 78, 1031. Exposure to UV-irradiated Herpes Simplex Virus SKINNER, G. R. B. (1976) Transformation of Primary Type 2. J. Virol., 8, 469. Hamster Fibroblasts by Type 2 Herpes Simplex EJERCITO, P. M., KIEFF, E. D. & ROIZMAN, B. (1968) Virus; Evidence for a Hit and Run Mechanism. Characterisation of Herpes Simplex Virus Br. J. exp. Path. (In press.) Strains Differing in their Effects on Social STEVENS, J. G. & COOK, M. L. (1973) Latent InfecBehaviour of Infected Cells. J. gen. Virol., 2, 357. tions Induced by Herpes Simplex Viruses. FARNHAM, A. E. & NEWTON, A. A. (1959) The Effect Cancer Res., 33, 1399. of Some Environmental Factors on Herpesvirus TIMBURY, M. C. & SUBAK-SHARPE, J. H. (1973) Growth in He-la Cells. Virology, 7, 449. Genetic Interactions between Temperature FIGUEROA, M. E. & RAWLS, W. E. (1969) Biological Sensitive Mutants of Types 1 and 2 Herpes Markers for Differentiation of Herpes Virus Simplex Viruses. J. gen. Virol., 18, 347. Strains of Oral and Genital Origin. J. gen. Virol., TRAWELL, 0. A. (1959) The Culture of Mature Organs 4, 259. in a Synthetic Medium. Expl Cell Res., 16, 119. FINK, C. G., THOMAS, G. H., ALLEN, J. M. & JORDAN', WATSON, D. H., WILDY, P. & RUSSELL, W. C. (1964) J. A. (1973) Metaplasia in Endocervical Tissue Quantitative Electron Microscope Studies on the Maintained in Organ Culture-an Experimental Growth of Herpes Virus using the Technique of Model. J. Obstet. Gynaec. Br. Commonw., 80, 75. Negative Staining and Ultramicrotomy. GEDER, L. & SKINNER, G. R. B. (1971) DifferenVirology, 24, 523. tiation Between Type 1 and Type 2 Strains of WATSON, D. H., SHEDDEN, W. I. H., ELLIOT, A., Herpes Simplex Virus by an Indirect ImmunoTETSIJKA, T., WILDY, P., BOURGAUX-RAMOISY, D. fluorescence Technique. J. gen. Virol., 12, 1 79. & GOLD, E. (1966) Virus Specific Antigens in HoORN, B. (1964) Respiratory Viruses in Model Mammalian Cells Infected with Herpes Simplex Experiments. Transactions of the 15th Congress Virus. Immunology, 11, 399. of the Scandinavian Oto-Larynyological Society, WILDY, P. (1955) Recombination with Herpes 1963. Acta oto-lar., 188, Suppl., 138. Simplex Virus. J. gen. Microbiol., 13, 346. HOORN, B. (1966) Organ Cultures of Ciliated
was supported by the Cancer Research
.q I
