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Induction of herpes simplex virus lesions on rabbit lip mucosa
Oral pathology American
Academy
of Oral
Pathology
Donald Kerr, Editor
Induction of herpes simplex virus lesions on rabbit lip mucosa James W. Griffin, D.D.S., Atlanta, DEPARTMENT
Oh” PATHOU)GY,
EMORY
Ga. UNIVERSITY
SCHOOL
OF
DRNTISTRY
T
he induction, with herpes simplex virus (HSV) , of an experimental lesion on the palatal mucosa of the ra,bbit has been reported by Rizzo and Ashe,l and Force and associates*have demonstrated production of skin lesions by the administration of HSV in conjunction with hyaluronidase. The present investigation was undertaken to provide a simpler experimental model which would more closely resemble human lesions and produce recurrent lesions as experienced by human beings. MATERIALS
AND
METHODS
Original isolations of virus used in the study were obtained by rupturing a clinical recurrent vesicular lesion from a human subject and collecting vesicular lluid in a Pasteur pipette. Vesicular fluid was diluted with 0.2 ml. Earle’s -Medium 199 supplemented with 10 per cent calf serum. The diluted virus was immediately inoculated into tubes containing 1 x 1@ HEp-2 cells. The HEp-2 cells had been initiated in culture with Earle’s Medium 199 supplemented with 10 per cent calf serum, and after 24 hours of culture a maintenance medium containing 3 per cent calf serum was substituted. The virus was allowed to replicate until a 75 to 100 per cent cytopathogenic effect (CPE) developed. On occasion, diluted virus was frozen at -65’ C. prior to initial passage until cells became available for inocula.tion. Subsequent to initial passageand development of CPE, infected1 cells and supernatant virus were directly frozen at -65’ C. for future passage of the virus isolate. Thirteen isolations were obtained in this manner and designated by an arbitrary letter. Virus was prepared for inoculation of rabbits by passage in HEp-2 cells, as previously described, with inocula of 0.1 to .2 ml. of virus stock sufficient to This
study
was
supported
by
General
Research
Support
Grant
1 SOL
FR,-5308-04.
765
produce 100 per cent CPE in a %4 hour culture and frozen a,t -@j* (:. prior to use. Titration of virus for rabbit inoculation was perf’ormed on prima,ry rabbit kidney cultures as described by Ship and asso&tetes” and by Ashe and Sclierp.4 The average titer of rabbit inocula was IOi PFU per 0.1 ml. virus stock with 100 per cent 24 hour CPE at previous passage. Three albino female ra,bbits, each weighing approximately 2 kilograms at the time of initial inoculation, were used for the experimental model (Rabbits 1, 2, and 3). Two ad,ditional rabbits were initia.ted into this study subsequent to immunization with a single isolate of HSV and intracardial blood sampling for a separate study (Rabbits 4 a.nd 5). The a.nimals wrre restrained in a conventional restraining box. Immediately thawed HSV isolates were inoculated into the maxillary right vermilion border of the lip with a multipuncture technique, using 0.1 ml. of virus preparation as described above. Rabbit 2 received only one isolate of virus at the different time intervals, and the other four animals received va.rious isolates (Table I). Animals were exa.mined twice daily for 4 to 6 days for development of lesions. Lesions that developed were random tested by means of cytologic material obtained with a small curette, spread on a glass slide, and air dried for fluorescent antibody (F’A) examination. Direct immunofluorescent staining was employed, utilizing rabbit immune sera Iabeled with fluorescein isothioeyanate and purified by DEA%I column elution.4t 5 Biopsy specimens of lesions were also taken immediately a.nd fresh frozen without fixation for cryostat sectioning for both hematoxylin-e&n and fluorescent antibody staining. Sections for FA staining were air dried. Blocking re-
Fig. on April
1. Cluster 18, 1966.
of vermilion
lip
vesicular
lesions
48 hours
after
inoculation
of Rabbit
2
Volume Number
Inch&ion
23 6
of herpes
siwlplex
virus
lesio,ts
767
a&ions were employed with FA staining by reacting tissue specimen with unlabeled whole HSV immune sera prior to staining with labeled immune sera. Random re-isolation of virus from rabbit lesions was casried out in the same manner as for initial isolation of human lesion virus. RESULTS The results of lesion development are presented in Table I. The right vermilion border of the lip would become edematous a few hours after inocula5tion and initial signs of a developing lesion consisting of a very small (1 mm. or less) vesicle would be present within 18 hours to 24 hours. The lesion would progress in 48 to 72 hours to maximum size of 3 mm. Between 48 and 96 hours iafter ‘inoculation the vesicle would rupture, leaving a shallow ulcer. Occarsionally multiple vesicular lesions would appear in a cluster arrangement (Fig. 1). All aaimals developed lesions following t.heir initial exposure to HSV lip inoculation. Frozen sections of biopsy specimens stained with hematoxylin and eosin revealed intraepitheliad vesicle formation with cells exhibiting a cytopathogenic effect of the virus. As previously described by other autho,rs,l a marked eosinophilia was present in the inflammatory infiltrate. FA sta.ining of air-dried cytologic and biopsy frozen sections gave positive immunofluorescent reaction@-lo in all instances in which an apparent clinical lesion was present. Blocking procedures completely inhibited a positive fluorescent response. The repeated exa,mination of animals during an uninoculated period did not reveal a spontaneous lesion. The virus was successfully re-isolated in five out of six random attempts with re-inoculation into HEp-2 cells. DISCUSSION The experimental formation of a true vesicular lesion on rabbit lip offers an experimental model which is more accessible and more easily
mucosa
induced
Table I HSV Date
Rabbit
4/16/65 4/28/65 5/12/65 5/26/65 6/ 9/65 6/23/65 g/24/65 l/11/66 3/ 9/66 3/22/66 4/18/66 5,’ 2/66 *Biopsy
1
1
TC ‘NJ TC TC TC TC TC TC
K (+I* K C-1 K (+) s (+I TC (+)*
or
K C-j K C-1 TC C-1 B (+) A (+) TC (-) cytologic
Rabbit
inocwlwm 8
(+)* C-1 C-1 C--J (-) (-) C-1 (+j*
TC (+I TC C-1 TC (+)* fluorescent
antibody
1
and
reaction
Rabbit
3
TC (+) K (+)* R (-) TC (-) t-3 C-1 K (+) TC (-) TC (+I TC (-) B (+) A (+I* TC (+) verification
(-t- or -) 1
Rabbit -.584: ‘g g i
4
.g gi A (+I* A (+I TC (+) B (+I A (+I* TC (+I of
HSV
cytopathogenic
)
Rabbit a8v2 ‘g g E ‘B .f: 6% s (+)* s C-t) TC (+) B (+> A (+) TC (+)* effect.
5
exudate. Fig. 8. Byncytial cell CPE in vesicle inflammatory x440; redwed l/s.) of biops, y frozen section. Magnification,
Fig. (HSV-1E
(Hematoxylin
and eosin stain
Volume Number
23 6
Induction
of her.pes simplex
virus
lesions
769
than previously reported experimental lesions. Animals were not exposed to factors occasionally related to recurrent human lesions (ultraviolet radiation, trauma, emotiona. stress, etc.). The ideal experimental model would be an aaima1 that would develop spontaneous recurrent lesions. From Table I, it can be seen that induced lesions tended to occur without regard to, antibody, particularly in Rabbits 4 and 5 which had been hyperpreimmunized with HSV isolate for collection of serum to be used in a separate study. This aspect correlates with the apparent lack of antibody significance in recurrent HSV infection in man. Inoculation with varioas isolates produced .more lesions tha,n repeated inoculation with the same isolate. There is a variation’in FA experiments between five of the isolates used as inocula (manuscript in prepa,ration), but there was no correlation to positive lesion production with variation of HSV antigen, Experiments on minimum virus dose for lesion production and effects of antibody titer are now in progress, as well as experiments on et.iologic factors for spontant?wus development of lesions. the
The author experiments
is most reported
grateful in this
to Mrs. article.
Katie
W. Carpenter
for
her
technical
assistance
in
REFERENCES
1. 2. 3. 4. 5. 6. 7. Y. !). 10. ll.
Rizzo, A. A., and Ashe, W. K.: Arch. Oral Biol. 9: 713-724, 1964. Force, E. E., Stewart, R. C., and Haff, R. F.: Virology 23: 363-369, 1964. Ship, I. I., Ashe, W. K., and Scherp, H. W.: Arch. Oral Biol. 3: 117-124, 1961. Ashe, W. K., and Scherp, H. W.: J. Immunol. 91: 658-665, 1963. Levy, H. B., and Sober, H.: Proe. Sot. Exper. Bid. & Med. 103: 250-252, 1960. Riggs, J. L., Loh, P. C., and Eveland, W. C.: Proc. Sot. Exper. Biol. & Med. 106: 658, 1961. Weller, T. H., and Coons, A. H.: Proc. Sot. Exper. Biol. & Med. 86: 789-794, 1954. Lebrun, J., and Crouse, A.: Virology 2: 496-650, 1956. Biegeleisen, J. F., Scott, L. V., and Lewis, V.: Science 129: 640-641, 1959. Griffin, J. W.: ORAL SURG., ORAL MED. & ORAL PATH. 16: 945-952, 1963. Griffin, J. W.: ORAL SURG., ORAL MED. & ORAL PATH. 19: 209-213, 1965.
655
Academy
of Oral
Pathology
Donald Kerr, Editor
Induction of herpes simplex virus lesions on rabbit lip mucosa James W. Griffin, D.D.S., Atlanta, DEPARTMENT
Oh” PATHOU)GY,
EMORY
Ga. UNIVERSITY
SCHOOL
OF
DRNTISTRY
T
he induction, with herpes simplex virus (HSV) , of an experimental lesion on the palatal mucosa of the ra,bbit has been reported by Rizzo and Ashe,l and Force and associates*have demonstrated production of skin lesions by the administration of HSV in conjunction with hyaluronidase. The present investigation was undertaken to provide a simpler experimental model which would more closely resemble human lesions and produce recurrent lesions as experienced by human beings. MATERIALS
AND
METHODS
Original isolations of virus used in the study were obtained by rupturing a clinical recurrent vesicular lesion from a human subject and collecting vesicular lluid in a Pasteur pipette. Vesicular fluid was diluted with 0.2 ml. Earle’s -Medium 199 supplemented with 10 per cent calf serum. The diluted virus was immediately inoculated into tubes containing 1 x 1@ HEp-2 cells. The HEp-2 cells had been initiated in culture with Earle’s Medium 199 supplemented with 10 per cent calf serum, and after 24 hours of culture a maintenance medium containing 3 per cent calf serum was substituted. The virus was allowed to replicate until a 75 to 100 per cent cytopathogenic effect (CPE) developed. On occasion, diluted virus was frozen at -65’ C. prior to initial passage until cells became available for inocula.tion. Subsequent to initial passageand development of CPE, infected1 cells and supernatant virus were directly frozen at -65’ C. for future passage of the virus isolate. Thirteen isolations were obtained in this manner and designated by an arbitrary letter. Virus was prepared for inoculation of rabbits by passage in HEp-2 cells, as previously described, with inocula of 0.1 to .2 ml. of virus stock sufficient to This
study
was
supported
by
General
Research
Support
Grant
1 SOL
FR,-5308-04.
765
produce 100 per cent CPE in a %4 hour culture and frozen a,t -@j* (:. prior to use. Titration of virus for rabbit inoculation was perf’ormed on prima,ry rabbit kidney cultures as described by Ship and asso&tetes” and by Ashe and Sclierp.4 The average titer of rabbit inocula was IOi PFU per 0.1 ml. virus stock with 100 per cent 24 hour CPE at previous passage. Three albino female ra,bbits, each weighing approximately 2 kilograms at the time of initial inoculation, were used for the experimental model (Rabbits 1, 2, and 3). Two ad,ditional rabbits were initia.ted into this study subsequent to immunization with a single isolate of HSV and intracardial blood sampling for a separate study (Rabbits 4 a.nd 5). The a.nimals wrre restrained in a conventional restraining box. Immediately thawed HSV isolates were inoculated into the maxillary right vermilion border of the lip with a multipuncture technique, using 0.1 ml. of virus preparation as described above. Rabbit 2 received only one isolate of virus at the different time intervals, and the other four animals received va.rious isolates (Table I). Animals were exa.mined twice daily for 4 to 6 days for development of lesions. Lesions that developed were random tested by means of cytologic material obtained with a small curette, spread on a glass slide, and air dried for fluorescent antibody (F’A) examination. Direct immunofluorescent staining was employed, utilizing rabbit immune sera Iabeled with fluorescein isothioeyanate and purified by DEA%I column elution.4t 5 Biopsy specimens of lesions were also taken immediately a.nd fresh frozen without fixation for cryostat sectioning for both hematoxylin-e&n and fluorescent antibody staining. Sections for FA staining were air dried. Blocking re-
Fig. on April
1. Cluster 18, 1966.
of vermilion
lip
vesicular
lesions
48 hours
after
inoculation
of Rabbit
2
Volume Number
Inch&ion
23 6
of herpes
siwlplex
virus
lesio,ts
767
a&ions were employed with FA staining by reacting tissue specimen with unlabeled whole HSV immune sera prior to staining with labeled immune sera. Random re-isolation of virus from rabbit lesions was casried out in the same manner as for initial isolation of human lesion virus. RESULTS The results of lesion development are presented in Table I. The right vermilion border of the lip would become edematous a few hours after inocula5tion and initial signs of a developing lesion consisting of a very small (1 mm. or less) vesicle would be present within 18 hours to 24 hours. The lesion would progress in 48 to 72 hours to maximum size of 3 mm. Between 48 and 96 hours iafter ‘inoculation the vesicle would rupture, leaving a shallow ulcer. Occarsionally multiple vesicular lesions would appear in a cluster arrangement (Fig. 1). All aaimals developed lesions following t.heir initial exposure to HSV lip inoculation. Frozen sections of biopsy specimens stained with hematoxylin and eosin revealed intraepitheliad vesicle formation with cells exhibiting a cytopathogenic effect of the virus. As previously described by other autho,rs,l a marked eosinophilia was present in the inflammatory infiltrate. FA sta.ining of air-dried cytologic and biopsy frozen sections gave positive immunofluorescent reaction@-lo in all instances in which an apparent clinical lesion was present. Blocking procedures completely inhibited a positive fluorescent response. The repeated exa,mination of animals during an uninoculated period did not reveal a spontaneous lesion. The virus was successfully re-isolated in five out of six random attempts with re-inoculation into HEp-2 cells. DISCUSSION The experimental formation of a true vesicular lesion on rabbit lip offers an experimental model which is more accessible and more easily
mucosa
induced
Table I HSV Date
Rabbit
4/16/65 4/28/65 5/12/65 5/26/65 6/ 9/65 6/23/65 g/24/65 l/11/66 3/ 9/66 3/22/66 4/18/66 5,’ 2/66 *Biopsy
1
1
TC ‘NJ TC TC TC TC TC TC
K (+I* K C-1 K (+) s (+I TC (+)*
or
K C-j K C-1 TC C-1 B (+) A (+) TC (-) cytologic
Rabbit
inocwlwm 8
(+)* C-1 C-1 C--J (-) (-) C-1 (+j*
TC (+I TC C-1 TC (+)* fluorescent
antibody
1
and
reaction
Rabbit
3
TC (+) K (+)* R (-) TC (-) t-3 C-1 K (+) TC (-) TC (+I TC (-) B (+) A (+I* TC (+) verification
(-t- or -) 1
Rabbit -.584: ‘g g i
4
.g gi A (+I* A (+I TC (+) B (+I A (+I* TC (+I of
HSV
cytopathogenic
)
Rabbit a8v2 ‘g g E ‘B .f: 6% s (+)* s C-t) TC (+) B (+> A (+) TC (+)* effect.
5
exudate. Fig. 8. Byncytial cell CPE in vesicle inflammatory x440; redwed l/s.) of biops, y frozen section. Magnification,
Fig. (HSV-1E
(Hematoxylin
and eosin stain
Volume Number
23 6
Induction
of her.pes simplex
virus
lesions
769
than previously reported experimental lesions. Animals were not exposed to factors occasionally related to recurrent human lesions (ultraviolet radiation, trauma, emotiona. stress, etc.). The ideal experimental model would be an aaima1 that would develop spontaneous recurrent lesions. From Table I, it can be seen that induced lesions tended to occur without regard to, antibody, particularly in Rabbits 4 and 5 which had been hyperpreimmunized with HSV isolate for collection of serum to be used in a separate study. This aspect correlates with the apparent lack of antibody significance in recurrent HSV infection in man. Inoculation with varioas isolates produced .more lesions tha,n repeated inoculation with the same isolate. There is a variation’in FA experiments between five of the isolates used as inocula (manuscript in prepa,ration), but there was no correlation to positive lesion production with variation of HSV antigen, Experiments on minimum virus dose for lesion production and effects of antibody titer are now in progress, as well as experiments on et.iologic factors for spontant?wus development of lesions. the
The author experiments
is most reported
grateful in this
to Mrs. article.
Katie
W. Carpenter
for
her
technical
assistance
in
REFERENCES
1. 2. 3. 4. 5. 6. 7. Y. !). 10. ll.
Rizzo, A. A., and Ashe, W. K.: Arch. Oral Biol. 9: 713-724, 1964. Force, E. E., Stewart, R. C., and Haff, R. F.: Virology 23: 363-369, 1964. Ship, I. I., Ashe, W. K., and Scherp, H. W.: Arch. Oral Biol. 3: 117-124, 1961. Ashe, W. K., and Scherp, H. W.: J. Immunol. 91: 658-665, 1963. Levy, H. B., and Sober, H.: Proe. Sot. Exper. Bid. & Med. 103: 250-252, 1960. Riggs, J. L., Loh, P. C., and Eveland, W. C.: Proc. Sot. Exper. Biol. & Med. 106: 658, 1961. Weller, T. H., and Coons, A. H.: Proc. Sot. Exper. Biol. & Med. 86: 789-794, 1954. Lebrun, J., and Crouse, A.: Virology 2: 496-650, 1956. Biegeleisen, J. F., Scott, L. V., and Lewis, V.: Science 129: 640-641, 1959. Griffin, J. W.: ORAL SURG., ORAL MED. & ORAL PATH. 16: 945-952, 1963. Griffin, J. W.: ORAL SURG., ORAL MED. & ORAL PATH. 19: 209-213, 1965.
655