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<0.05) by WHITE'S ranking test (12) in only 1 of the 9 experiments. However, the fact that in all 9 experiments titres in the irradiated mice were increased could not be evaluated by this test. Therefore the data were subjected to analysis of variance (10) which showed a significant difference (0.05 > p > 0.01) overall, between the mean titres in all the irradiated mice and all non-irradiated mice. The analysis of variance technique was judged to be an appropriate method since the variance within each of the 18 groups of mice was sho~rn to be homoseedastie b y BARTLETT'Stest (1). D a y s a f t e r i n f e c t i o n when samples t a k e n 3

3

3

4

5

6

.1,0

~0.8 *0.6

o

*0,4

8

o o o

*0-2 0

o

-0'2

o

-0.4 -0.6

P > 0.05

o

P < 0.05

•

-0'8 -1.0

4b

-1,2 -1.4 -1,6

-2

-1

Day

of irradiation

0 (infection

.I

+2

*3

=day 0)

Fig. 2. Comparison of titres of HSV in irradiated and un-irradiated ears * lg geometric mean titre in irradiated ea,rs minus lg geometric mean titre in un-irradiated ears

The finding that in ~nimals irradiated 3 - 4 days after infection the ear became free from virus earlier than in control animals was surprising in view of the fact that in those irradiated 1 or 2 days after infection titres of HSV were higher than in controls. The possibility that this earlier recovery from infection resulted from inactivation of virus in the tissue by UV light was therefore tested. Groups of animals were infected with 2 × 102 P F U ttSV; 3 days later they were irradiated with various doses of UV light. The mice were killed within 10 minutes of being irradiated and the infectious HSV in the tissue of the right pinna was titrated (Table 2). Virus was isolated from 26 of the 30 control mice (87 per cent) and from 98 of the 104 irradiated mice (94 per cent). Irradiation for up to 100 sec (the time used in all previous experiments) made no difference to the titre of ttSV in the ear tissue, but when longer times were used titres were decreased; with 200 sec irradiation the decrease was about tenfold.

UV Light and Primary HSV Infection

371

Table 2. Titres o / H S V in ears immediately aJter irradiation with U V light~ Time (sec) of irradiation 0

50

100

t50

200

No.@ve/total

11/15

14/15

11/15

--

9/15

Mean lg titre s,d.

2.77 0.88

2,92 0.87

2,97 1.26

--

1.91 1.16

No. ÷ veltotal

15/15

14/15

14/15

12/14

14/15

Mean lg titre s.d.

3.51 1.02

3.68 0.74

3.19 0.55

2.71 1.06

2.57 1.08

Experiment 1

Experiment 2

Ears were irradiated 3 days after infection; Samples were taken within 5 minutes of irradiation

Discussion The effects of irradiation with UV light on primary HSV infection are clearly complex and influenced by the timing of virus inoculation and irradiation. There was a decrease in virus growth when the skin was irradiated before the virus was inoculated and this parallels observations made in vitro with CV-1 cells (4). Irradiation of these cells before infection with tISV1 decreased virus DNA synthesis and production of infectious virus. This probably resulted directly from UV irradiation damage to the cell DNA which could also cause the decreased growth now reported. However in the skin the effects m a y be complicated by the inflammation that follows irradiation (8). In tissue already infiltrated by neutrophils and macrophages removal of virus would be facilitated, and this would accentuate the direct effect of irradiation damage to cellular DNA. These mechanisms could also underly the early disappearance of virus from ears irradiated 3 4 days after inoculation. Virus was not inactivated directly b y the dose of UV hght used, but it was when this was increased. In contrast to the decreased virus titres when irradiation preceded infection and the shortening of the infection by irradiation 3 - - 4 days after inoculation, virus titres were increased by irradiation 1--3 days after inoculation of virus. Though not so pronounced as the previous decreases, the increase in titres occurred in all of nine experiments, a result that was statistically significant (p < 0.05) when tested by analysis of variance. This result suggests either that the factors acting against the virus were absent during this period (which seems unlikely) or that their effects were overcome b y other processes which favoured virus multiplication. I t could be, t h a t once established, virus infection is stimulated by some of the physiological agents induced by UV light, for instance prostaglandins (5). One of these, prostaglandin E2, produces a significant increase in the size of HSV plaques in Vero cells (Harbour, Blyth & Hill, unpublished observations) and m a y be involved in reactivation of ItSV infection in mouse skin (2). How is the present work related to the reactivation of latent herpes simplex by UV light in humans (11) and mice (2)? Two theories of the mechanism of herpes reactivation have been discussed elsewhere (6). By the "ganglion trigger" theory a stimulus such as UV light which acts first on the skin must thereafter

372

D . A . HARBOUI~ et al. : UV Light and P r i m a r y HSV Infection

a c t i v a t e virus in t h e ganglion. The virus m u s t t h e n t r a v e l to t h e skin to i n i t i a t e infection a n d would arrive t - - 2 d a y s after i r r a d i a t i o n (6). D u r i n g t h e e a r l y p a r t of this period, b y e x t r a p o l a t i o n from t h e p r e s e n t results, conditions in t h e skin w o u l d be less f a v o u r a b l e t h a n usual for infection. B y c o n t r a s t t h e " s k i n t r i g g e r " t h e o r y (6) suggests t h a t subclinical foci of infection f r e q u e n t l y occur in t h e skin. If these were p r e s e n t before a stimulus occurred, the s i t u a t i o n would be similar to t h a t in the p r e s e n t e x p e r i m e n t s in which virus was i n o c u l a t e d 2 d a y s before the skin was i r r a d i a t e d . Results of these exp e r i m e n t s suggest t h a t conditions would t h e n be p a r t i c u l a r l y f a v o u r a b l e for the infection so t h a t t h e r e would be an increased chance t h a t clinical lesions w o u l d develop. R e a c t i v a t i o n of herpes lesions b y U V light would therefore be m o r e easily e x p l a i n e d b y t h e " s k i n t r i g g e r " t h e o r y t h a n b y t h e " g a n g l i o n t r i g g e r " theory. Aeknowledgements We are grateful to Miss Anne Dempsey and Mr. Peter Standing for their skilful technical assistance, and to Dr. A. J. Hedges for his advice on the statistical analysis of the results. This work was supported by grants from the Medical Research Council.

Re~erenees 1. BAt~TLETT, M. S. : Some examples of statistical methods of research in agriculture and applied biology. Suppl. to J. Royal Star. Soc. 4, I37--170 (t937). 2. BLY~H, W. A., HILL, T. J., FIELD, H. J., HA~BO~, D. A. : Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement

3. 4. 5. 6. 7.

of prosta-

glandins. J. gen. ViroI. 33, 547--550 (1976). COOK, M. L., STEVENS, J. G. : Pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection. Infect. Immun. 7, 272 --288 (1973). COPPE¥, J., NOCENTINI, S. : Herpes virus and viral DNA synthesis in ultraviolet light-irradiated cells. J. gen. Viroh 32, 1--15 (1976). GREAVES, M. W., SONDERGAA~D, J. : Pharmacologic agents released in ultraviolet inflammation studied b y continuous skin perfusion. J. invest. Dermatol. 54, 365 --367 (1970). HILL, T. J., BLYTH, ~V. A. : A n alternative theory of herpes simplex recurrence and a possible role for prostaglandins. Lancet i, 397--399 (1976). HILL, T. J., FIELD, H. J., BL'YTH, W. A. : Acute and recurrent infection with herpes

simplex virus in the mouse: a model for studying latency and recurrent disease. J. gen. Viroh 28, 341--353 (1975). 8. LOGAN, G., WILHELM, D. L.: The inflammatory reaction in ultraviolet injury. Brit. J. exp. Pathol. 47, 286--299 (1966). 9. MERIGAN, T. C.: Host defenses against viral diseases. New Eng. J. Med. 290, 323

--329 (1974). 10. SNEDECOP~, G. W. : Statistical methods applied to experiments in agriculture and biology, Chapter 11. Ames, Iowa: Iowa State College Press 1950. t l . WHEELERS, C. E. : Pathogenesis of recurrent herpes simplex infections. J. invest. Dermatol. 65, 341--346 (t975). 12. V~THITE,C. : The use of ranks in a test. of significance for comparing two treatments. Biometrics 8, 33--41 (1952). Authors' address: Dr. D. A. tIAgBOVa, Department of Bacteriology, University of Bristol Medical School, University Walk, Bristol BS 8 1TD, England. Received F e b r u a r y 7, 1977