(Accepted 28 May 1981) SUMMARY

Transcription

1 J. gen. Vrol. (1981), 56, Prnted n Great Brtan 259 Key words: acyclovr/bromovnyldeoxyurdne/herpes smplex vrus~latent Effects of Oral Treatment wth Acyclovr and Bromovnyldeoxyurdne on the Establshment and Mantenance of Latent Herpes Smplex Vrus nfecton n Mce By H. J. FELD ~* AND E. DE CLERCQ 2 ~Department of Pathology, Unversty of Cambrdge, Dvson of Vrology, Addenbrookes Hosptal, U.K. and 2Rega nsttute for Medcal Research, Katholeke Unverstet, Leuven, Belgum (Accepted 28 May 1981) SUMMARY Mce nfected wth herpes smplex vrus (HSV) were treated (separately) wth the nucleosde analogues acyclovr or bromovnyldeoxyurdne by ncorporatng the drugs n the drnkng water. Ths method of treatment was found to be effectve for both drugs and compared favourably wth ntrapertoneal njecton. Prompt treatment wth ether compound could prevent the establshment of latent nfectons but latent nfectons once establshed were ntractable usng prolonged courses of oral admnstraton. NTRODUCTON Several new nucleosde analogues have been developed whch are potent nhbtors of herpes smplex vrus (HSV) wth low toxcty n vvo. Two of these, acyclovr or 9-(2-hydroxyethoxymethyl)guanne (ACV) and E-5-(bromovnyl-2'-deoxyurdne) (BVDU) behave as analogues of thymdne and are selectvely phosphorylated by the HSV-specfed pyrmdne deoxynucleosde knase (thymdne knase or TK). Both drugs are then metabolzed wthn the nfected cells to ther respectve nucleosde trphosphates whch then nterfere wth HSV-specfed DNA polymerase and nhbt vrus replcaton (Elon et al., 1977; Fyfe et al., 1978; De Clercq et al., 1979a). Both ACV and BVDU have been shown to suppress acute HSV nfectons n mce, rabbts and gunea-pgs (De Clercq et al., 1979a; Feld et al., 1979; Schaeffer et al., 1977; Kaufman et al., 1978; Maudgal et al., 1980). Trals wth ACV and BVDU for the treatment of HSV and varcella-zoster nfectons n man are now underway. However, there are complex nteractons between HSV and the perpheral nervous system nvolvng latency and recurrent dsease. The effects of chemotherapy on these aspects of the dsease wll be very dffcult to determne n man and, therefore, the study of anmal models s partcularly valuable n developng a ratonal approach to chemotherapy. There have been several reports on the effects of ACV on latent nfectons n anmals but none to date wth BVDU. Our own prevous studes wth ACV showed that prompt treatment [twce daly doses ntrapertoneally (.p.) totallng 50 mg/kg/day from the tme of vrus noculaton for 10 days] prevented or at least reduced the establshment of latency (Feld et al., 1979). Smlar results have been reported by others (Klen et al., 1979; Park et al., 1979). n a dfferent study Pavan-Langston et al. (1979) showed that already-establshed latent nfectons n mce could be cured by 15 days systemc treatment wth ACV; however, Feld et al. (1979), Klen et al. (1979) and Blyth et al. (1980) all reported that latent nfectons once establshed were not reduced by subsequent treatment wth ACV /81/ $ SGM

2 260 H.J. FELD AND E. DE CLERCQ The systemc treatments descrbed n all the above studes employed repeated njectons of ACV and had the dsadvantage that the tssue half-lfe of ACV s relatvely short. Ths was borne out by the recent study of Boulter et at. (1980) who showed that ntravenous (.v.) dosng at 6-h ntervals was essental n order to prevent progresson of the hghly neurotropc herpes vrus B (whch also has a lower susceptblty to ACV) nfecton n rabbts. n our prevously descrbed treatment schedule (Feld et al., 1979) there was a 15 h gap between.p. doses (.e. 6 p.m. to 9 a.m.) and ths presumably led to perods of relatvely low drug levels n the tssue (Schaeffer et al., 1977). n order to treat a farly large number of mce over lengthy perods the drug was gven ad lb. n the drnkng water. Ths proved to be a successful method of admnstraton of both ACV and BVDU. Ths paper compares the effects of oral and.p. admnstraton of the two nucleosde analogues and descrbes ther effects on the establshment of latent nfectons n mce. METHODS Vrus nfecton. Acute HSV nfectons were produced n 4-week-old Balb/c mce by noculatng HSV type 1 stran SC16 (Hll et al., 1975) nto the skn of the left pnna usng a dose of 104 or 105 p.f.u, suspended n 0.02 ml Glasgow-modfed Eagle's medum. Latent nfectons became establshed n the cervcal dorsal root gangla relatng to the sensory nerve supply of the ear. Measurement of vrus nfectvty, nflammaton and detecton of latent nfectons. Vrus ttres were determned by plaque ttraton n BHK cells of dlutons of homogenzed ear tssus usng standard vrologcal technques. Three mce were sampled at each tme pont; the tssues were ttrated ndependently and the mean vrus ttre obtaned. As a measure of the severty of the dsease, nflammaton produced durng the nfecton was assessed by the determnaton of ear swellng. The ear thckness was measured usng an engneer's mcrometer screw gauge on groups of three to fve mce at each tme pont. The swellng was taken to be the thckness of the left mnus the rght (unnoculated) pnna. Ths technque has been characterzed n detal n an dentcal mouse model (Nash et al., 1980). For the detecton of latent nfectons, the 2nd, 3rd and 4th cervcal gangla were explanted nto 0.5 ml Glasgow-modfed Eagle's medum contanng 1% calf serum. The three gangla from each mouse were pooled. The cultures were ncubated for 6 days at 37 C, then homogenzed and ttrated usng BHK cells as above. Drugs. ACV was a gft from Dr G. B. Elon, Burroughs Wellcome Ltd., Research Trangle Park, North Carolna, U.S.A. BVDU was syntheszed by R. Busson and H. Vanderhaeghe (Rega nsttute, Leuven, Belgum). ACV was dssolved n water at ph 6-8 usng a concentraton of 1 mg/ml and the drug-contanng water was substtuted for the normal water supply. n the case of BVDU, the ph was adjusted to 3 usng HC. ACV s tasteless and mce drank t normally, but BVDU was found to be more palatable at the lower ph. Both drugs are stable at room temperature under these condtons. Naturally, the water ntake of mce vares wth envronmental condtons. However, observatons over several weeks ndcated a surprsngly constant rate of drnkng. The n vtro actvty of the two drugs was determned by a plaque reducton test n monolayer cultures of Vero and BHK cells as descrbed by Feld et al. (1980). RESULTS A dmnstraton ofa C V and B VD U by means of drnkng water The daly ntake of drnkng water by groups of mce was observed over a!0 day perod. The daly consumpton (approx. 3 ml/day/mouse) remaned surprsngly constant. There was no reducton n the ntake of water contanng 1 mg/ml ACV compared to contros but a slght dstaste for BVDU-contanng water was noted. The consumpton of ByDU was

3 HSV n mce: oral nucleosde therapy 261 (a) (b) t o t Tme after vrus noculaton (days) Fg. 1. Effects of.p. or oral treatment wth ACV on HSV nfecton n mce. Mce were noculated wth 104 p.f.u. HSV-1 SC16 nto the left ear pnna. (a) Vrus replcaton n the skn. (b) nflammaton: ear swellng s the thckness of the left mnus the rght (unnoeulated) ear. 0, Untreated; K], ACV (50 mg/kg/day.p. n two njectons daly); O, ACV (1 mg/ml n drnkng water). Treatment commenced at the tme of vrus noculaton and contnued for 10 days thereafter. mproved slghtly by acdfyng the water to ph 3. The average daly consumpton of the drugs was ACV mg/kg/day, BVDU (ph 5-8) 137 _+ 28 mg/kg/day and BVDU (ph 3) mg/kg/day. After 10 days contnuous treatment the mce showed no obvous sgns of toxcty n ether case and there was no dfference between the weght of treated mce and the untreated controls. An attempt was made to combne drnkng water treatment wth ACV wth an addtonal dose of a further 50 mg/kg/day gven.p. Ths combned dose appeared to exceed the toxc dose for ths drug and the mce ruffled and ded after 7 to 10 days treatment. n vtro senstvty to A C V and B VDU HSV stran SC 16 was tested n BHK, Vero and prmary mouse embryo fbroblast cells for senstvty to ACV and BVDU by a plaque reducton test. n all cases BVDU appeared to be more actve than ACV. However, whle ACV showed smlar actvty n BHK and mouse embryo fbroblast cells (EDs0 = 0.05 ~tg/ml) BVDU was found to be somewhat less actve n the latter cell type (BVDU EDs0 for BHK cells /tg/ml and for prmary mouse fbroblasts = 0.01 ~tg/ml). Comparson of oral wth.p. treatment of acute HSV nfecton Mce were noculated wth the type 1 stran of HSV, SC 6, nto the skn of the left ear. A vrus noculum of 104 p.f.u, was employed and treatment ether by ncorporatng 1 mg/ml ACV n drnkng water or by two daly.p. njectons totallng 50 mg/kg/day was commenced from the tme of noculaton. The effects of treatment were assessed by examnng the amount of vrus replcaton n the ear pnna and by measurng the nflammaton as judged by ncreased skn thckness. Fg. 1 shows that treatment wth ACV n the drnkng water reduced vrus ttres n the ear by 2 to 3 log10 wth a concomtant reducton n nflammaton. n ths experment the conventonal.p. dosng route was less effectve. The experment shown n Fg. 2 was smlar to that descrbed above except that BVDU was substtuted n the drnkng water and for.p. admnstraton a larger dose of BVDU was

4 262 H J. FELD AND E. DE CLERCQ -(a) (b) : 6-3 \ E x 3C ~2 > \ \ 2c 1( Tme after vrus noculaton (days) Fg. 2. Effects of.p. or oral treatment wth BVDU on HSV nfecton n mce. Mce were noculated as for Fg. 1. (a) Vrus replcaton n the skn. (b) nflammaton: ear swellng s the thckness of the left mnus the rght (unnoculated) ear. O, Untreated; S], BVDU (100 mg/ml/day.p. n two njectons daly); O, BVDU (1 mg/ml/day n drnkng water). Treatment commenced at the tme of vrus noculaton and contnued for 10 days thereafter. employed (100 mg/kg/day). n ths case treatment by the two dfferent routes was ndstngushable. However, t was noted that, whle vrus ttres were reduced by 1 to 2 logl0, the drug appeared to be less effectve than ACV n ether reducng vrus replcaton or nflammaton. However, t s recognzed that the lower toxcty of BVDU for mce would enable hgher doses of BVDU, e.g. 250 to 500 mg/kg/day to be nvestgated n future studes. A further experment was carred out to compare drectly BVDU wth ACV employng the drnkng water treatment. Ths confrmed that whle BVDU certanly nfluenced the course of the nfecton when gven n the drnkng water, n ths partcular model ACV was clearly more effectve (data not shown). Effects of oral treatment wth A C V or B VDU on the establshment of latent nfectons n mce Prevous studes usng.p. admnstraton of ACV have ndcated that, provded treatment s nsttuted wthn 24 h of nfecton and the doses of vrus employed are moderate, the establshment of latent nfectons n the sensory gangla related to the noculaton ste was reduced or prevented. The mce remanng from the above experments (Fg. 1 and 2) were later tested for evdence of latent nfectons. The treated mce had been gven ACV or BVDU n the drnkng water from the tme of noculaton for 10 days thereafter. The cervcal dorsal root gangla were explanted nto culture vessels 4 weeks after noculaton (18 days after treatment ceased). Subsequent culture of the explanted gangla and testng for the presence of nfectous vrus ndcated (Table 1) that both drugs were effectve n preventng the establshment of latent nfectons whch can be detected by ths n vtro culture technque. One out of 18 mce from the BVDU-treatment groups was found to be harbourng the nfecton n the cervcal gangla. All other treated anmals yelded negatve cultures. The ncdence of latent nfecton n the control, untreated groups were 100% and 55 % n two separate experments. Effects of oral treatment wth A C V and B VD U on establshed latent nfectons n mce Mce whch had recovered from the acute phase of an HSV nfecton n the ear followng an noculum of l0 s p.f.u, were treated wth ACV n the drnkng water commencng 1 month

5 HS V n mce: oral nueleosde therapy 263 Table 1. Effect of A C V and B VDU on the establshment of latent HS V* n mce TreatmentS" ACV (1 mg/ml n drnkng water) BVDU (1 mg/ml n drnkng water) None No. of mce postves h Expt. 1 Expt. 2 0/7 0/18 ND 1/18 (6%) 7/7 11/20 (55 %) * HSV- SC16 noculated nto left pnna (104 p.f.u./mouse). J" Treatment commenced from tme of noculaton and contnued for 10 days thereafter. $ Vrus solated by culture of dorsal root gangla for 6 days (2nd, 3rd and 4th cervcal gangla pooled from each mouse). ND, Not done. Table 2. Effects of ACV on establ&hed latent HSV* n m&e Tme after termnatng No. of mce Vrus ttre n postves$ Treatmentt treatment (weeks) postve/total tested (geometrc mean _+ S.D.) ACV 0 Control 8/8 5.4 _+ 0.6 (1 month) Treated 8/ Control 6/ (1 month) Treated 6/ Control 3/ (1 month) Treated 5/ Control 3/ (1 month) Treated 5/ ACV 4 Control ND (2 months) Treated 7/8 4.3 _+ 2.3 BVDU 2 Control 6/ ± 1.6 (1 month) Treated 4/ Control 7/ _+ 0.9 (1 month) Treated 6/ * Vrus: HSV SC 16 noculated nto left ear pnna (10 5 p.f.u./mouse). ~" Treatment: 1 mg/ml n drnkng water for 1 or 2 months startng 1 month after noculaton. ~c Reactvaton by culturng explanted cervcal dorsal root gangla (2nd, 3rd and 4th pooled from each mouse). ND, Not done. after noculaton; treatment contnued for 1 or 2 months. Snce t has been suggested that the perod whch elapses between the tme of termnatng treatment and explantng gangla nfluences the detecton of latency (D. Pavan-Langston, personal communcaton) mce were tested at varous tmes from 0 to 5 weeks after treatment ceased. A total of 28 treated mce were compared wth a smlar number of untreated controls. There was no sgnfcant dfference n the number of mce n whch latent nfecton was detected or n the ttres of vrus n the ganglon explant homogenates (Table 2). A further group of mce was tested after 2 months treatment but, agan, these mce showed no evdence of reduced latency. A small number of mce were treated wth oral BVDU agan for 1 month and agan ths produced no effect on the latent nfecton as determned by ths method of n vtro reactvaton (Table 2). DSCUSSON Oral admnstraton of the two nucleosde analogues under nvestgaton proved to be an effectve method of treatment whch compared favourably wth twce daly.p. njectons. The results of pharmoknetc studes of BVDU n mce followng oral treatment have been reported prevously (De Clercq et al., 1979 b) but no datawere obtaned n the present study on the serum or tssue levels obtaned durng the course of treatment. Oral admnstraton also suffers from the dsadvantage that the quantty and regularty of drug ntake are unknown. Despte ths uncertanty, n practce, oral therapy may stll be an extremely valuable method

6 264 H.J. FELD AND E. DE CLERCQ for treatng mce expermentally snce large numbers of anmals can be gven the drug for extended perods wthout the need for regular handlng of ndvdual mce. t was noted that BVDU was less effectve than ACV n controllng the acute phase of the nfecton. Ths was true when BVDU was gven orally or by.p. njecton (usng a larger dose than that employed for ACV). The reasons for ths are not yet clear snce the n vtro actvty of BVDU appears to be greater than that of ACV and ths was confrmed to be true for the partcular stran of HSV used n the present study. One possblty s that BVDU may be nactvated or elmnated more readly than ACV. However, the extremely low toxcty of BVDU for mce means that much hgher dose levels can be examned. Ths was not possble n the present study because of the lmted quantty of BVDU avalable. Both drugs were found to prevent the establshment of latent nfectons as detected by an n vtro reactvaton technque. Ths phenomenon has prevously been descrbed for ACV (Feld et al., 1979; Park et al, 1979) and for other drugs, e.g. phosphonoacetc acd (Klen et al., 1978). t s assumed that the falure to establsh latency results from reduced replcaton n the skn. Lower levels of vrus replcaton n the skn resultng from smaller vrus nocula n untreated mce were found to nduce latency but less reproducbly (Feld et al., 1979). A prevous study has also shown that drect noculaton of vrus nto the nervous system does result n the establshment of latent nfecton despte contnuous therapy (Feld et al., 1979). However, the effectve suppresson of cutaneous vrus replcaton usng ether ACV or BVDU may mean that both drugs wll have a role n suppresson of recurrent nfectons n man and may reduce the subsequent colonzaton of further neurons durng such recurrences. Fnally, oral treatment of mce by means of the drnkng water enabled the effects of long-term contnuous treatment on establshed latent nfectons to be nvestgated. Usng explant culture of dorsal root gangla to reactvate latent vrus n vtro, no reducton n the latent nfecton was observed followng treatment for up to 2 months. Smlar negatve results have been prevously reported by ourselves (Feld et al., 1979) usng daly.p. admnstraton of ACV. These data would be consstent wth the falure to demonstrate specfc vrus products ncludng TK n latently nfected tssue (Fong & Scrba, 1980). An earler report descrbed the detecton of TK n mouse dorsal root gangla usng a senstve technque (Yamamoto et al., 1977), although even n ths case the levels of TK became undetectable after 60 days although the mce remaned latently nfected. The falure to elmnate latent vrus would be consstent wth our own n vtro observatons on the effects of ncubatng explanted latently nfected gangla n the presence of nhbtory levels of ACV. These studes ndcated that n vtro reactvaton could be suppressed for several days n ths way, but when the drug was removed reactvaton occurred wth normal knetcs (J. Rajcan & H. J. Feld, unpublshed observatons). t mght be argued, however, that f vrus expresson occurs n neurons durng a recurrent nfecton, then the nfected cell mght become amenable to chemotherapy. t has been shown (Darby et al., 1980; Nshyama & Rapp, 1979; Furman et al., 1980) that the growth of TK-transformed cels whch express the HSV TK gene are nhbted by low concentratons of ACV (< 1 gg/ml). However, these are dvdng cels and would not necessarly reflect the stuaton n the neuron whch harbours the latent vrus, ths beng a non-dvdng cell. Whle the nteractons of effectve nucleosde analogues wth HSV durng reactvaton events n vtro reman to be elucdated, the clear ndcatons from the work descrbed n ths paper are that these drugs should only be used to suppress the actve phase of prmary or recurrent nfectons. Ths should not only amelorate the dsease but would be expected to reduce the establshment of latent nfectons n further neurons durng a recurrence and the decrease n vrus ttres would lessen the chance of auto-noculaton or transmsson. However, the attempted treatment of the nfecton durng the latent phase appears frutless and

7 HSV n mce: oral nucleosde therapy 265 wdespread use of the drugs n ths way may only ncrease the rsk of developng strans of HSV whch are resstant to the drugs concerned (Feld & Darby, 1980). H.J.F. s funded by a Programme Grant from the Medcal Research Councl of Great Brtan. We thank Mrs Elzabeth Lay for her expert techncal help and Mrs Mary Wrght for help wth the preparaton of the manuscrpt. REFERENCES BLYTH, W. A., HARBOUR, D. A. & HLL, T. J. (1980). Effect of acyclovr on recurrence of herpes smplex skn lesons n mce. Journal of General Vrology 48, BOULTER, E. A., THORNTON, B., BAUER, D. J. & BYE, A. (1980). Successful treatment of expermental B vrus (herpesvrus smae) nfecton wth acyclovr. Brtsh Medcal Journal 280, DARBY, G., LARDER, B. A., BASTOW, K. F. & FELD, H. J. (1980). Senstvty of vruses to phosphorylated 9(2-hydroxyethoxymethyl)guanne revealed n TK-transformed cells. Journal of General Vrology 48, DE CLERCQ, E., DESCAMPS, J., DE SOMER, P., BARR, P. J., JONES, A. S. & WALKER, R. T. (1979a). (E)- 5-(2-bromovnyl)-2'-deoxyurdne; a potent and selectve ant-herpes agent. Proceedngs of the Natonal Academy of Scences of the Unted States of Amerca 76, DE CLERCQ, E., DESCAMPS, J., DE SOMER, P., BARR, P. J., JONES, A. S. & WALKER, R. T. (1979 b). Pharmoknetcs of E-5-(2-bromovnyl)-2'-deoxyurdne n mce. Antmcrobal Agents and Chemotherapy 16, ELON, G. B., FURMAN, P. A., FYFE, J. A., DE MRANDA, P., BEAUCHAMP, L & SCHAEFFER, H. J. (1977). Selectve acton of an antherpetc agent, 9(2-hydroxyethoxymethyl)guanne. Proceedngs of the Natonal Academy of Scences of the Unted States of Amerca 74, FELD, H. J. & DARBY, G. K. (1980). Strateges of drug resstance n herpes smplex. Nature, London 286, 842. FELD, H. J., BELL, S. E., ELON, G. B., NASH, A. A. & WLDY, P. (1979). Effect of acycloguanosne treatment on acute and latent herpes smplex nfectons n mce. AntmcrobaAgents and Chemotherapy 15, FELD, H. J., DARBY, G. & WLDY, P. (1980). solaton and characterzaton of acyclovr-resstant mutants of herpes smplex vrus. Journal of General Vrology 49, FONG, B. S. & SCRBA, M. (1980). Use of [125ldeoxycytdne to detect herpes smplex vrus specfc thymdne knase n tssues of latently nfected gunea pgs. Journal of Vrology 34, FURMAN, P. A., McGURT, V. V., KELLER, P. M., FYFE, J. A. & ELON, G. B. (1980). nhbton by acyclovr of cell growth and DNA synthess of cells bochemcally transformed wth herpes vrus genetc nformaton. Vrology 102, FYFE, J. A., KELLER, P. M., FURMAN, P. A., MLLER, R. L. & ELON, G. B. (1978). Thymdne knase from herpes smplex vrus phosphorylates the new antvral compound, 9-(2-hydroxyethoxymethyl)guanne. Journal of Bologcal Chemstry 253, HLL, T. J., FELD, H. J. & BLYTH, W. A. (1975). Acute and recurrent nfecton wth herpes smplex n the mouse: a model for studyng latency and recurrent dsease. Journal of General Vrology 28, KAUFMAN, H. E., VARNELL, E. D., CENTFANTO, Y. M. & RHENSTROM, S. n. (1978). Effect of 9-(2- hydroxyethoxymethyl-guanne on herpesvrus-nduced keratts and rts n rabbts. Antmcrobal Agents and Chemotherapy 14, KLEN, R. J., FREDMAN-KEN, A. E. & YELLN, P. B. (1978). Orofacal herpes smplex vrus nfecton n harless mce: latent vrus n trgemnal gangla after topcal antvral treatment. nfecton and mmunty 20, KLEN, R. J., FREDMAN-KEN, A. E. & DE STEFANO, E. (1979). Latent herpes smplex vrus nfecton n sensory gangla of harless mce prevented by acycloguanosne. Antmerobal Agents and Chemotherapy 15, MAUDGAL, P. C., DE CLERCQ, E., DESCAMPS, J., MSSOTTEN, L., DE SOMER, P., BUSSON, R., VANDERHAEGHE, H., VERHELST, G., WALKER, R. T. & JONES, A. S. (1980). (E)-5-(2-bromovnyl)-2'-deoxyurdne n the treatment of expermental herpes smplex keratts. AntmerobalAgents and Chemotherapy 17, NASH, A. A., FELD, H. J. & QUARTEY-PAPAFO, R. (1980). Cell-medated mmunty n herpes smplex vrus-nfected mce: nducton, characterzaton and antvral effects of delayed type hypersenstvty. Journal of General Vrology 48, NSHYAMA, Y & RAPP, F. (1979). Antcellular effects of 9-(2-hydroxyethoxymethyl)guanne aganst herpes smplex vrus transformed cells. Journal of General Vrology 45, PARK, N. H., PAVAN-LANGSTON, D. & MCLEAN, S. L. (1979). Acyclovr n oral and ganglonc herpes smplex vrus nfectons. Journal of nfectous Dseases 140, PAVAN LANGSTON, D., PARK, N. H. & LASS, J. (1979). Herpetc ganglonc latency. Acyclovr and vdarabne therapy. Archves of Ophthalmology 97, SCHAEFFER, H. J., BEAUCHAMP, L., DE MRANDA, P., ELON, G. S., BAUER, n. J. & COLLNS, P. (1977). 9-(2-hydroxyethoxymethyl)guanne actvty aganst vruses of the herpes group. Nature, London 272, YAMAMOTO, H., WALZ, M. A. & NOTKNS, A. L. (1977). Vral-specfc thymdne knase n sensory gangla of mce nfected wth herpes smplex vrus. Vrology 76, (Receved 30 March 1981)