Archives of Virology
Archives of Virology 54, 367--372 (1977)
© by Springer-Verlag 1977
The Effect of Ultraviolet Liflht on Primary Herpes Simplex Virus Infection in the Mouse By I). A. HARBOUR, T. J. HILL, and W. A. BLYTH Department of Bacteriology, University of Bristol, Medical School, Bristol, England With 2 Figures Accepted April 5, 1977
Summary The effect of irradiation with UV light on herpes simplex virus infection in the mouse ear was investigated. Irradiation 2 days before infection had no effect on the titre of virus in the skin 3 days after infection. In ears irradiated 24 hours or immediately before infection virus titres measured 3 days after infection were depressed. Irradiation 2 days after infection enhanced virus titres. The dose of irradiation used did not directly inactivate herpes simplex virus in the ear. The results are discussed with reference to theories of herpes simplex reactivation.
Introduction One theory of herpes simplex reactivation (3, 9) proposes that a stimulus in the dorsal root ganglion leads to breakdown of latent infection and the production of infectious virus. The virus then travels via peripheral nerves to the skin where a lesion is produced. Recently however it was proposed that the reactivating stimulus acts on virus in the skin rather than in the ganglion--the "skin trigger" theory (6). B y this theory, after a reactivating stimulus, altered conditions in the skin allow the virus to multiply sufficiently to produce a lesion, either by enhanced virus replication or by temporary suppression of local defence mechanisms. I t is commonly observed that ultraviolet (UV) light can act as a stimulus to recurrent herpes simplex (11, 2). In an effort to throw light on the mechanism of this reactivation we report some effects of irradiation with UV light on primary ttSV infection in the mouse ear.
Materials and Methods Cell Cultures
VERO cells were obtained from Gibco Biocult Ltd. and maintained by serial subculture in medium 199 (~VVellcome reagents Ltd.) supplemented with 5 per cent
368
D.A.
I-lag~o~R, T. J. I-IILL, and W. A. BLYT~I:
,(-irradiated foetal calf serum (Gibeo Biocult Ltd.), penicillin and streptomycin, 100 units/ml, and aerosporin, 50 units/ml. For virus assay monolayers of cells were grown in 16 m m diameter wells in plastic dishes (Multidish, Linbro ChemieM Co. Inc.). Maintenance medium used after infection was similar to that described above but contained 2 per cent serum and 0.75 per cent methyl cellulose.
Herpes Simplex Virus ( H S V ) One stock suspension of the 2nd passage in Vero cells of HSV1 strain SC16 (7) was used throughout the study.
In/eetion o/ Mice Four week old male Swiss white mice from a closed colony were anaesthetized by intraperitoneM sodium pentobarbitone and the right ears were inoculated intradermally with 200 P F U of virus in 20 B1. The inocutum was titrated after use to confirm the dose given.
Irradiation With U V Light Mice were anaesthetized and placed 22 cm vertically below a Hanovia model 1 UV lamp. The right ears were irradiated usually for 100 seconds which was about twice the 50 per cent erythemogenie dose. Control mice were anaesthetized but not irradiated.
Assay o/ In/ectious Virus 2'rein the Mouse Ear Mice were killed and the skin was scraped from both sides of the right ear with a scalpel blade. The serapings were ground in 0.4 ml of maintenance medium in a glass tissue grinder and serial 10 fold dilutions were inoculated in duplicate Oll to Vero cell monolayers; each well received 50 ~l. After 1 hour adsorption at 35 ° C the cultures were washed with PBSA and 1 ml of maintenance medium was added to each well. After 2 days incubation at 35 ° C cultures were fixed and stained so that plaques could be counted.
Results I n a series of e x p e r i m e n t s the titre of H S V in the ears of mice after i r r a d i a t i o n w i t h U V light was c o m p a r e d with t h a t of u n - i r r a d i a t e d animals. Groups usually of 15 animals were i n o c u l a t e d w i t h 2 × 102 P F U H S V s u b c u t a n e o u s l y in t h e r i g h t pinna. E i t h e r i m m e d i a t e l y before infection (within half an hour), or one or more days before or after infection a group of animals was i r r a d i a t e d w i t h U V l i g h t ; control groups were a n a e s t h e t i z e d at t h e same t i m e b u t n o t irradiated. The animals were killed either 3 days after infection or 3 days after i r r a d i a t i o n (when this was l a t e r t h a n infection) a n d H S V in the ear tissue was t i t r a t e d . T h e 3 d a y i n t e r v a l b e t w e e n i r r a d i a t i o n a n d sampling was chosen because in l a t e n t l y infected animals H S V was isolated from t h e ear m o s t often 3 days after i r r a d i a t i o n (2). H S V was isolated from 90 per cent of u n - i r r a d i a t e d mice t e s t e d 3 days after infection. T h e r e a f t e r t h e p r o p o r t i o n fell u n t i l after 9 days only 10 per cent of the ears c o n t a i n e d infectious virus (Table 1, Fig. 1). Titres of virus from these control mice were a b o u t 103 P F U / e a r on t h e t h i r d f o u r t h a n d fifth days after infection, t h e r e a f t e r t h e y fell (Fig. 1). I n i r r a d i a t e d mice even at the p e a k of the infection virus was usually isolated from a smaller p r o p o r t i o n of the ears t h a n in control animals (Table 1). I n a d d i t i o n virus disappeared more q u i c k l y from the ears of mice i r r a d i a t e d 3 or 4 days after inoculation. H S V was isolated f r o m only 2 of 27 ears (7 per cent) i r r a d i a t e d 4 days after infection (therefore sampled 7 days after infection) whereas in similar control animals I t S V was isolated from 17 of 30 ears (57 per cent).
UV Light and P r i m a r y HSV Infection
369
4.0
3.5
3.0
"T
z.s
IO0 9O
2.o
80
"~ 70
60
z~
E o
g so "E 40 30
2o 0
a 2
3 4 5 6 Days after infection
7
8
2
9
10
0
o) of HSV in un-irradiated ears. Bars represent Fig. 1. Incidence (A) and titres (o 4-1 standard deviation
Table 1. Isolation o] H S V / t o m the ears o] mice, either un-irradiated or irradiated with U V light
Days after infection 3
4
5
6
7
9
10
Not irradiated
No.@ve/total ~o
1t8/132 90
22/30 73
77/100 20/27 77 74
17/30 57
1/8 12
0/11 0
Irradiated
No.~-ve/total ~o
82/132 62
22/29 76
44/85 52
11/26 42
2/27 7
0/4 0
0/11 0
@2
~-3
@4
+6
+7
D a y of irradiation (infection= d a y 0)
-- 2, -- 1 or 0 + 1
I n 8 of 9 e x p e r i m e n t s where i r r a d i a t i o n p r e c e d e d infection (and s a m p l e s were t a k e n 3 d a y s a f t e r infection) t h e m e a n t i t r e of H S V in ears of i r r a d i a t e d a n i m a l s was lower t h a n in control a n i m a l s (Fig. 2). I r r a d i a t i o n 2 d a y s before infection h a d v i r t u M l y no effect on these titres. H o w e v e r when mice were i r r a d i a t e d e i t h e r 1 d a y or i m m e d i a t e l y before in~ect4on t i t r e s were decreased in 6 of 7 e x p e r i m e n t s . T h e decreases v a r i e d from 8 t o 25-fold a n d all were significant (p < 0 . 0 5 ) b y WmTE'S r a n k i n g t e s t (12). I n t h e s e v e n t h e x p e r i m e n t t h e i r r a d i a t e d g r o u p h a d a 4-fold higher m e a n t i t r e t h a n t h e control group, a difference t h a t was n o t significant (p > 0.05).
370
D.A.
HAI~BOUI~, T. J. HILI~, and ~A T. A. BLYTII:
By contrast, when animals were irradiated one or more days after infection mean titres of HSV in groups of irradiated mice were always higher, by about 2--7 fold, than those of control groups (Fig. 2). The difference was statistically significant (p
Archives of Virology 54, 367--372 (1977)
© by Springer-Verlag 1977
The Effect of Ultraviolet Liflht on Primary Herpes Simplex Virus Infection in the Mouse By I). A. HARBOUR, T. J. HILL, and W. A. BLYTH Department of Bacteriology, University of Bristol, Medical School, Bristol, England With 2 Figures Accepted April 5, 1977
Summary The effect of irradiation with UV light on herpes simplex virus infection in the mouse ear was investigated. Irradiation 2 days before infection had no effect on the titre of virus in the skin 3 days after infection. In ears irradiated 24 hours or immediately before infection virus titres measured 3 days after infection were depressed. Irradiation 2 days after infection enhanced virus titres. The dose of irradiation used did not directly inactivate herpes simplex virus in the ear. The results are discussed with reference to theories of herpes simplex reactivation.
Introduction One theory of herpes simplex reactivation (3, 9) proposes that a stimulus in the dorsal root ganglion leads to breakdown of latent infection and the production of infectious virus. The virus then travels via peripheral nerves to the skin where a lesion is produced. Recently however it was proposed that the reactivating stimulus acts on virus in the skin rather than in the ganglion--the "skin trigger" theory (6). B y this theory, after a reactivating stimulus, altered conditions in the skin allow the virus to multiply sufficiently to produce a lesion, either by enhanced virus replication or by temporary suppression of local defence mechanisms. I t is commonly observed that ultraviolet (UV) light can act as a stimulus to recurrent herpes simplex (11, 2). In an effort to throw light on the mechanism of this reactivation we report some effects of irradiation with UV light on primary ttSV infection in the mouse ear.
Materials and Methods Cell Cultures
VERO cells were obtained from Gibco Biocult Ltd. and maintained by serial subculture in medium 199 (~VVellcome reagents Ltd.) supplemented with 5 per cent
368
D.A.
I-lag~o~R, T. J. I-IILL, and W. A. BLYT~I:
,(-irradiated foetal calf serum (Gibeo Biocult Ltd.), penicillin and streptomycin, 100 units/ml, and aerosporin, 50 units/ml. For virus assay monolayers of cells were grown in 16 m m diameter wells in plastic dishes (Multidish, Linbro ChemieM Co. Inc.). Maintenance medium used after infection was similar to that described above but contained 2 per cent serum and 0.75 per cent methyl cellulose.
Herpes Simplex Virus ( H S V ) One stock suspension of the 2nd passage in Vero cells of HSV1 strain SC16 (7) was used throughout the study.
In/eetion o/ Mice Four week old male Swiss white mice from a closed colony were anaesthetized by intraperitoneM sodium pentobarbitone and the right ears were inoculated intradermally with 200 P F U of virus in 20 B1. The inocutum was titrated after use to confirm the dose given.
Irradiation With U V Light Mice were anaesthetized and placed 22 cm vertically below a Hanovia model 1 UV lamp. The right ears were irradiated usually for 100 seconds which was about twice the 50 per cent erythemogenie dose. Control mice were anaesthetized but not irradiated.
Assay o/ In/ectious Virus 2'rein the Mouse Ear Mice were killed and the skin was scraped from both sides of the right ear with a scalpel blade. The serapings were ground in 0.4 ml of maintenance medium in a glass tissue grinder and serial 10 fold dilutions were inoculated in duplicate Oll to Vero cell monolayers; each well received 50 ~l. After 1 hour adsorption at 35 ° C the cultures were washed with PBSA and 1 ml of maintenance medium was added to each well. After 2 days incubation at 35 ° C cultures were fixed and stained so that plaques could be counted.
Results I n a series of e x p e r i m e n t s the titre of H S V in the ears of mice after i r r a d i a t i o n w i t h U V light was c o m p a r e d with t h a t of u n - i r r a d i a t e d animals. Groups usually of 15 animals were i n o c u l a t e d w i t h 2 × 102 P F U H S V s u b c u t a n e o u s l y in t h e r i g h t pinna. E i t h e r i m m e d i a t e l y before infection (within half an hour), or one or more days before or after infection a group of animals was i r r a d i a t e d w i t h U V l i g h t ; control groups were a n a e s t h e t i z e d at t h e same t i m e b u t n o t irradiated. The animals were killed either 3 days after infection or 3 days after i r r a d i a t i o n (when this was l a t e r t h a n infection) a n d H S V in the ear tissue was t i t r a t e d . T h e 3 d a y i n t e r v a l b e t w e e n i r r a d i a t i o n a n d sampling was chosen because in l a t e n t l y infected animals H S V was isolated from t h e ear m o s t often 3 days after i r r a d i a t i o n (2). H S V was isolated from 90 per cent of u n - i r r a d i a t e d mice t e s t e d 3 days after infection. T h e r e a f t e r t h e p r o p o r t i o n fell u n t i l after 9 days only 10 per cent of the ears c o n t a i n e d infectious virus (Table 1, Fig. 1). Titres of virus from these control mice were a b o u t 103 P F U / e a r on t h e t h i r d f o u r t h a n d fifth days after infection, t h e r e a f t e r t h e y fell (Fig. 1). I n i r r a d i a t e d mice even at the p e a k of the infection virus was usually isolated from a smaller p r o p o r t i o n of the ears t h a n in control animals (Table 1). I n a d d i t i o n virus disappeared more q u i c k l y from the ears of mice i r r a d i a t e d 3 or 4 days after inoculation. H S V was isolated f r o m only 2 of 27 ears (7 per cent) i r r a d i a t e d 4 days after infection (therefore sampled 7 days after infection) whereas in similar control animals I t S V was isolated from 17 of 30 ears (57 per cent).
UV Light and P r i m a r y HSV Infection
369
4.0
3.5
3.0
"T
z.s
IO0 9O
2.o
80
"~ 70
60
z~
E o
g so "E 40 30
2o 0
a 2
3 4 5 6 Days after infection
7
8
2
9
10
0
o) of HSV in un-irradiated ears. Bars represent Fig. 1. Incidence (A) and titres (o 4-1 standard deviation
Table 1. Isolation o] H S V / t o m the ears o] mice, either un-irradiated or irradiated with U V light
Days after infection 3
4
5
6
7
9
10
Not irradiated
No.@ve/total ~o
1t8/132 90
22/30 73
77/100 20/27 77 74
17/30 57
1/8 12
0/11 0
Irradiated
No.~-ve/total ~o
82/132 62
22/29 76
44/85 52
11/26 42
2/27 7
0/4 0
0/11 0
@2
~-3
@4
+6
+7
D a y of irradiation (infection= d a y 0)
-- 2, -- 1 or 0 + 1
I n 8 of 9 e x p e r i m e n t s where i r r a d i a t i o n p r e c e d e d infection (and s a m p l e s were t a k e n 3 d a y s a f t e r infection) t h e m e a n t i t r e of H S V in ears of i r r a d i a t e d a n i m a l s was lower t h a n in control a n i m a l s (Fig. 2). I r r a d i a t i o n 2 d a y s before infection h a d v i r t u M l y no effect on these titres. H o w e v e r when mice were i r r a d i a t e d e i t h e r 1 d a y or i m m e d i a t e l y before in~ect4on t i t r e s were decreased in 6 of 7 e x p e r i m e n t s . T h e decreases v a r i e d from 8 t o 25-fold a n d all were significant (p < 0 . 0 5 ) b y WmTE'S r a n k i n g t e s t (12). I n t h e s e v e n t h e x p e r i m e n t t h e i r r a d i a t e d g r o u p h a d a 4-fold higher m e a n t i t r e t h a n t h e control group, a difference t h a t was n o t significant (p > 0.05).
370
D.A.
HAI~BOUI~, T. J. HILI~, and ~A T. A. BLYTII:
By contrast, when animals were irradiated one or more days after infection mean titres of HSV in groups of irradiated mice were always higher, by about 2--7 fold, than those of control groups (Fig. 2). The difference was statistically significant (p
