ANTIMICROBIAL AGENTS AND CHmoTHEzRAPY, Dec. 1975, p. 643-450Copyright C 1975 American Society for Microbiology

Vol. 8, No. 6Printed in U.S.A.

Comparison of the Antiviral Effects of 5-Methoxymethyl-deoxyuridine with 5-Iododeoxyuridine, Cytosine Arabinoside,

and Adenine ArabinosideLORNE A. BABIUK,* BLAIR MELDRUM, V. SAGAR GUPTA, AND BARRY 1'. ROUSE

Departments of Microbiology* and Physiological Sciences, Western College of Veterinary Medicine,University of Saskatchewan, and Animal Pathology Division, Health ofAnimals Branch, Agriculture

Canada Saskatoon, Saskatchewan S7N OWO, Canada

Received for publication 12 August 1975

The antiviral activity of5-methoxymethyl-2'-deoxyuridine (MMUdR) was com-pared with that of 5-iodo-2'-deoxyuridine (IUdR), cytosine arabinoside (Ara-C),and adenine arabinoside (Ara-A). At concentrations of 2 to 4 ,ug/ml, MMUdR wasinhibitory to herpes simplex virus type 1, but concentrations as high as 128 ,ugIml were not inhibitory to three other herpesviruses tested (equine rhino-pneumonitis virus, murine cytomegalovirus, and feline rhinopneumonitis virus)or to vaccinia virus. The other nucleosides, in contrast, were inhibitory at similarconcentrations (1 to 8 ,ug/ml) against all viruses tested. The inhibition of HSV-1by MMUdR appeared to be the result of interference with virus replication ratherthan the result of drug toxicity to host cells. The drug was not toxic to host cellsat 100 times the antiviral concentrations, and pretreatment of host cells withhigh concentrations of MMUdR had no effect on subsequent virus replication.Combination of MMUdR with either IUdR, Ara-A, or Ara-C gave an enhancedantiviral effect, suggesting that the mechanism of action ofMMUdR is differentfrom that of the other three drugs. Antiviral indexes were calculated for eachcompound and were found to be >250, 80, 40, and 8 for MMUdR, IUdR, Ara-A,and Ara-C, respectively. These were defined as the minimum dose at whichtoxicity was observed microscopically divided by the dose which reduced plaquenumbers by 50%.

In the search for effective drugs for thetreatment of herpesviruses, considerable in-terest has been shown in the use of nucleosideanalogues (5, 6, 26, 27). 5-Iodo-2'-deoxyuridine(IUdR) (21), 9-f3-n-arabinofuranosyladenine(Ara-A) (6, 16), and 1-f3-n-arabinofuranosyl-cytosine (Ara-C) (25) have been found to beeffective to varying degrees in the treatmentof infections due to herpesviruses. However,their high toxicity (9, 13, 25), especially torapidly proliferating cells (18, 19), limits theusefulness of these nucleoside analogues in thetreatment of viral infections. Recently, an-other nucleoside derivative, 5-methoxymethyl-2'-deoxyuridine (MMUdR), was synthesized(3), and in preliminary experiments it showedantiviral activity against infectious bovine rhi-notracheitis virus (14). In this communication,we report the antiviral activity of MMUdRagainst other herpesviruses and vaccinia virus(VV), and compare its antiviral activity andtoxicity towards mammalian cells with IUdR,Ara-C, and Ara-A.

MATERIALS AND METHODS

Drugs. MMUdR was synthesized (3). Ara-C waskindly supplied by The Upjohn Co., Kalamazoo,Mich. IUdR and Ara-A were obtained from SigmaChemical Co., St. Louis, Mo., and PhanstiehlLaboratories, Waukegan, Ill., respectively. Thestructures of the drugs used in these studies areillustrated in Fig. 1.

Cell culture. The following cell lines used were:RK-13 (rabbit kidney), secondary feline embryo,HEP-2 (human epidermoid carcinoma no. 2), HeLA(human), and a secondary mouse embryo cell culture.The cells were cultured in Eagle minimal essentialmedium (GIBCO, Grand Island, N.Y.). Each liter ofmedium was supplemented with 2 mmol of glutamine(GIBCO no. 5034), 2.5 g of NaHCO3, 10 ml ofnonessential amino acids (GIBCO, no. 114), and 10%fetal calf serum (GIBCO, no. 614) for growth or 4%for maintenance. For the antiviral assays, mono-layers of confluent cell cultures were prepared byseeding 5 x 104 cells into each cup of a microtitertissue culture plate (96 cups per plate; no. 3040,Falcon Plastics, Oxnard, Calif.). Monolayers wereconfluent within 24 h. The cultures were incubatedat 37 C in a humidified C02 (5%) atmosphere.

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NH2

N<

HOH2C 109

HO

A

HO.2C ,

HO

NH2

N<N N)H

HOH2C 0

HO

lodo Deoxyuridine(lUdR)

Cytosine Arabinoside(ARA-C)

9-p-D-Arabinofuranosyl Adenine(ARA-A)

0

HN CH20CH3

N'"HOH2C >0--

HO

Methoxymethyl Deoxyuridine(MM UdR)

FIG. 1. Structure of nucleoside analogues.

Virusles. The viruses used were: herpes simplexvirus type 1 (HSV-1); murine cytomegalovirus;equine rhinopneumonitis virus strain KyD; felinerhinopneumonitis virus; and vaccinia virus. Murinecytomegalovirus was provided by G. Lussier (In-stitute of Microbiology and Hygiene, Montreal,P. Q.); equine rhinopneumonitis virus was re-ceived from F. C. Thomas (Animal Disease ResearchInstitute, Hull, P. Q.); vaccinia virus was procuredfrom the Department of Bacteriology, Universityof Saskatchewan; HSV-1 was isolated in this labora-tory from a human labial lesion; and feline rhino-pneumonitis virus was isolated from a cat withrespiratory disease. Stock viruses were prepared aspreviously described (22).Drug inhibition assays. Confluent cell mono-

layers were infected with one of a range of virusconcentrations (10 to 100 plaque-forming units/culture) and allowed to adsorb for 1 h at 37 C. Theunadsorbed virus was removed by washing withPuck solution, and compounds at different con-centrations (dissolved in minimal essential medium+ 4% fetal calf serum) were added. For plaqueassays, 2 U of neutralizing antibody specific toeach herpesvirus was included in the culture fluids.In each experiment, toxicity controls (containingtest compound and medium only), cell controls(containing medium only), and virus controls (con-taining virus and medium only) were also run

simultaneously. In combination antiviral chemo-therapy experiments, each compound was dissolvedat the appropriate concentration and both com-pounds were added together. The tissue cultureplates were incubated at 37 C for 72 h, the culturefluid was removed, and the monolayers were washedwith saline, fixed, stained with 1% gentian violetin 70% ethanol, and examined for virus plaques.The plaques were enumerated after magnificationof plates, using an overhead projector or a dis-secting microscope. The percentage of inhibition(calculated from reduction in the number ofplaques)at each concentration of the compound was de-termined. In experiments where the inhibition ofvirus yield was determined, antiserum was not in-cluded in the overlay. Culture fluid was harvesteddaily and titrated to determine the amount of viruspresent (21).

RESULTSComparison of antiviral potency ofMMUdR,

IUdR, Ara-C, and Ara-A against herpesvirusesand vaccinia virus. In an initial series ofexperiments, several ether derivatives of 5-hydroxymethyl-2'-deoxyuridine (for structuralfonnulae see reference 14) and 5-mercapto-methyl-2'-deoxyuridine were screened for anti-viral activity against HSV-1 in HEP-2 cell

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cultures. MMUdR was markedly inhibitory,but the other derivatives investigated werewithout anti-HSV-1 activity. Because of itsanti-HSV-1 activity, we decided to assess itsantiviral activity against other herpesviruses.The results of a comparison of the spectrumof antiviral activity of MMUdR with othernucleoside derivatives (IUdR, Ara-C, andAra-A) of known anti-herpesvirus activity areshown in Table 1.Whereas IUdR, Ara-A, and Ara-C were

markedly inhibitory against all herpesvirusestested as well as against vaccinia virus,MMUdR appeared selectively antiviral againstHSV-1 (Table 1). Thus concentrations of 2 to 4,ug/ml were inhibitory to HSV-1, but levels of128 ,ug/ml or greater were needed to inhibitfeline rhinopneumonitis and vaccinia viruses.MMUdR failed to show antiviral activityagainst murine cytomegalovirus and equinerhinopneumonitis virus even at 256 ,ug/ml(highest dose tested).

In comparing the anti-HSV-1 activity of thefour compounds on a molar basis, the spectrumof activity was IUdR > Ara-C > MMUdR> Ara-A, suggesting that IUdR may be thedrug of choice to treat HSV-1. However, acomparison of the antiviral indexes (Table 2)revealed that MMUdR may be preferable overIUdR because of its lower toxicity and greatermargin of safety. The antiviral index wasdefined as the minimum dose at which toxicitywas observed microscopically divided by thedose that reduced plaque numbers by 50%.

Effect of MMUdR on HSV-1 replication indifferent cell lines. The antiviral activity of

MMUdR against HSV-1 was investigated byusing different cell lines to determine whetherthe host cell affected the antiviral activityof MMUdR. Experiments with HeLa, Hep-2,and RK-13 cells showed a similar pattern ofantiviral activity in all three lines (Fig. 2). At10 ,ug/ml there was a delay in the appearanceof infectious virus, and virus titers at 72 hpostinfection (p.i.) were markedly (2 to 3 logs),but not completely, reduced. At higher con-centrations (25 or 50 ,ug/ml), infectious viruscould not be detected at 72 h p.i.; however,by 96 h p.i. low levels (4 to 5 logs lessthan controls) became detectable. At presentit is not known whether the virus producedafter 3 days was due to the emergence of

TABLE 2. Antiviral indexes ofMMUdR, Ara-A,Ara-C, and IUdR

Minimum Minimum ef- Antiviral in-Compound toxic dosea fective doseb

(/g/ml) (Ag/Iml) dx

MMUdR >500 2 >250Ara-A 160 d 4 40Ara-C 4 0.5 8IUdR 40C 0.5 80

a Concentration required to produce definite evi-dence of microscopic cytotoxicity in confluent cellmonolayers.

b Concentration required to cause 50% reductionin plaque numbers.

c Determined by dividing the toxic dose by theminimal effective antiviral dose.

d Data taken from Miller et al. (15)." Data taken from DeClerq and Shugar (7).

TABLE 1. Relative in vitro antiviralpotencyofMMUdR, IUdR, Ara-C, and Ara-A against herpesviruses andvaccinia virusa

Amt of virus Compounds (jug/ml)'IUdR Ara-A Ara-C MMUdR

HSV RK-13 10 0.5 4 0.5 2100 1 8 0.5-1 4

ERV RK-13 10 1 2-4 1 >256 e100 2 8 2 >256

MCMV Mouse 10 1-2 2 1 >256100 4 8 1 >256

FRV Feline 10 2 2-4 0.5 128100 4 8 1 256

VV RK-13 10 0.5 4 1-2 128100 0.5-1 4-8 2 256

a All compounds were added immediately after virus infection.b Abbreviations: HSV, herpes simplex virus; ERV, equine rhinopneumonitis virus; MCMV, murine

cytomegalovirus; FRV, feline rhinopneumonitis virus; VV, vaccinia virus.c PFU, Plaque-forming units.d Concentrations required to cause 50% reduction in plaque numbers.e Highest concentration tested.

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a

0

0

-aI-

0

z

0

0a

1 2 3 4

DAYS POSTINFECTION

FIG. 2. Effect ofMMUdR on replication ofHSV-1in various cell lines. Confluent monolayers ofHeLa,Hep-2, and RK-13 cells were infected at a multi-plicity of infection of 0.01 and simultaneously ex-posed to various concentrations of drug (U, withoutdrugs; A, 10 pglml; 0-----0, 25 pg/ml; andO O, 50 jig/ml). Supernatant fluids were har-vested daily and titrated to determine the amountof infectious virus produced.

drug-resistant mutants or to decreased intra-cellular concentrations of MMUdR as a resultof cellular metabolic activity convertingMMUdR to other inactive metabolic products.In the presence of 100 ,g of MMUdR per ml,infectious virus production was totally in-hibited (data not shown). MMUdR was devoidof toxicity to cells at this concentration (videinfra). These results suggest that MMUdR atlower concentrations (up to 50 ,ug/ml) hasvirostatic activity and at higher concentrations(100 ,ug/ml) has virocidal activity.

Effect of MMUdR on uninfected HEP-2cells. The effect of MMUdR (50 ,ug/ml) andAra-C (5 to 50 ,ug/ml) on the growth andplating density of HEP-2 cells is shown inTable 3. Neither cell growth nor plating densitywas affected by 50 Ag of MMUdR per ml, adose sufficient to completely prevent HSV-1

replication for 72 h. Furthermore, toxicity wasnot detectable by light microscopy and no de-crease in cell viability was apparent even incultures treated for 96 h with 500 pg ofMMUdR per ml (highest concentration tested;data not shown). These results are consistentwith the notion that in the concentrationrange of 2 to 50 Ag of MMUdR per ml, virusreplication is inhibited directly rather than theinhibition being the result of drug toxicity tohost cells.

In contrast to MMUdR, Ara-C was markedlymore toxic. There was a significant decreaseboth in cell numbers and plating efficiency(Table 3). By light microscopy, cell degenera-tion was evident at 72 h with as little as 4ug of Ara-C per ml.

Susceptibility of MMUdR-treated HEP-2cells to HSV-1 infection. The following ex-periments were undertaken to further supportthe hypothesis that the antiviral activity ofMMUdR was directed at virus replicationrather than acting by reducing virus replicationindirectly as a result of its effect on the hostcell. Confluent HEP-2 cells were treated withvarying concentrations of MMUdR for 48 h.After washing of the monolayers to removeresidual drug, the cells were infected withHSV-1. The results (Table 4) show that pre-treatment of monolayers with MMUdR had noeffect on subsequent HSV-1 replication. Plaquenumbers of treated and control cultures wereessentially identical at all intervals tested.

Inhibition of HSV replication by simul-taneous treatment with combinations of twoantiviral drugs. Therapeutic synergismachieved by the use of a combination of drugshas been of considerable value in the treatmentof bacterial and neoplastic disease (9-11, 22).The rationale for combination therapy is thathost toxicity is reduced because lower levelsofdrugs can be used. Since all of the nucleosideanalogues tested showed marked inhibitoryactivity against HSV-1, experiments were un-dertaken to determine the antiviral activity

TABLz 3. Effect ofMMUdR and Ara-C on normalcell (HEP-2) growth

Original no. No. of cells PercentCompound of cells after 3 days ofcontrolplated

Control 2.5 x 10f 1.3 x 10' 100MMUdR (50 2.5 x 10 1.3 x 10' 100

/Ag/Ml)Ara-C

50 sg 2.5 x 10' 0.14 x 10' 10.720jtg 2.5 x 10' 0.20 x 10' 155pg 2.5 x 10 0.38 x 10' 26

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TABLE 4. Susceptibility ofMMUdR-treated cells to HSV-1 replicationa

Infected 8 h after drug removal Infected 48 h after drug removalDrug concn (jLg/ml)

Day 2b Day 4 Day 2 Day 4

0 4 x105 6 x 105 4 x 103 5 x 10525 3.8 x 105 7 x 105 3.5 x 103 4.9 x 10550 5 x 105 5.5 x 105 4.1 x 103 5.8 x 105

a Confluent monolayers of Hep-2 cells were treated for 48 h with various concentrations ofMMUdR. Themonolayers were washed and replaced with drug-free minimal essential medium for 8 or 48 h beforeinfection with HSV-2.

b Supernatants were titrated for infectious virus on day indicated.

of various drug combinations as compared withthe antiviral activity ofdrugs used individually.The compounds, the concentrations used, andthe effect on virus plaque number are sum-marized in Fig. 3 and 4. The combination ofIUdR (0.5 ,ug/ml) and Ara-C (0.5 jig/ml) hada greater antiviral activity than did eithercompound alone (Fig. 3A). Enhanced antiviralactivity was also seen with the combinationof Ara-A (2 or 4 ,ug/ml) and IUdR (0.5 ,ug/ml)(Fig. 3B). In contrast, the antiviral activityremained essentially unchanged when Ara-A(4 p,g/ml) and Ara-C (0.5 ug/ml) were usedtogether (Fig. 3C). Combination of MMUdR(4 ,ug/ml) with IUdR, Ara-A, and Ara-C re-sulted in increased antiviral activity over theactivity of these compounds used individually(Fig. 4).

DISCUSSIONHerpesviruses are one of the groups of

viruses that have not been successfully con-trolled by conventional immunization schemes.Consequently, their control by other means,such as antiviral chemotherapy, has beensought. Various drugs such as IUdR (24),Ara-A (6, 16), Ara-C (25), and Ara-T (G. A.Gentry and J. F. Aswell, Fed. Proc. 34:3325,1975) are effective to varying degrees againstherpesviruses both in vitro and in vivo. In ourpresent communication we show that a newlyavailable drug, MMUdR, is highly efficaciousagainst HSV-1, and that the lack of toxicityby MMUdR may have advantages over theother nucleoside analogues for treatment ofHSV-1 infections in man. Our observation thatthe antiviral effects of MMUdR was directedonly against HSV-1 but not against three otherherpesviruses or vaccinia virus is at presentnot understood. One explanation for the selec-tive action of MMUdR could be that HSV-1codes for enzymes that are specifically inhibitedby this compound.

Although the nucleoside analogues in currentuse have shown some promise as antiviral

agents, their usefulness has been limited be-cause of their toxicity to host cells. Organswith rapidly dividing cells such as the gastro-intestinal tract, bone marrow, the fetus, andlymphoid tissue (4, 13, 18, 19, 25) are theusual sites where toxicity is manifest. Ourdata suggest that the antiviral effects ofMMUdR are selectively directed against virusreplication and not due to a drastic alterationof the physiological state of host cells. Thusit was shown that MMUdR had no effect onconfluent (greater than 500 ,ug/ml) or rapidlydividing cells (greater than 50 jig/ml) in vitro,concentrations far in excess of those inhibitoryto virus replication (2 ug/ml). Moreover, pre-treatment of cells with MMUdR had no effecton the cells' susceptibility to subsequent virusinfection.A calculation of the margin of safety of the

respective drugs indicated that MMUdR wasmuch safer to use than any other drug tested.Thus, a computation of the antiviral indexdetermined by dividing the toxic dose by theminimal effective antiviral dose gave values of250, 80, 40, and 8 for MMUdR, IUdR, Ara-A,and Ara-C, respectively. Recently, MMUdRhas also been shown not to have any im-munosuppressive effects on lymphoid functions(L. A. Babiuk and B. T. Rouse, submittedfor publication). These observations were con-sidered important since recovery from herpes-virus infection probably depends upon an intactlymphoid system (1, 2, 17, 22). Although theeffects of MMUdR on all organs have not beenfully investigated, preliminary acute toxicitystudies using up to 1,000 mg of MMUdR inadult Swiss mice did not result in mortalitynor were any gross lesions or hematologicalchanges observed at 1 week post-administra-tion (unpublished observations). For thesereasons MMUdR may prove to be a very usefulanti-herpesvirus drug, possibly without theseveral adverse side effects observed with othernucleoside analogues.

In addition to the problem of toxicity with

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Control Ara-C lUdR Ara-C+ Ara-A lUdR Ara-A+IUdR Ara-A Ara-C Ara-A+ItJdR Ara-C

TREATMENTFIG. 3. Effect of combining two anti-herpesvirus compounds on plaque formation by HSV-1. Confluent

monolayers ofRK-13 cells were infected with 100 plaque-forming units of virus and simultaneously treatedwith various compounds. (A) Ara-C (0.5 pg/mi), Ess.; IUdR (0.5 pg/mi), DFl~; combination of Ara-C +IUdR, both at 0.5 pg/mi, AfIEl . (B) Ara-A (4 pg/mi), ~; IUdR (0.5 psg/mi), @H; combination of Ara-A(2 pg/mi) and IUdR (0.5 pg/mi) (left histogram,[EDn) or Ara-A (4 pg/mi) and IUdR (0.5 p/,gmi) (righthistogram,IEJJ). (C) Ara-A (4 pg/mi), ; Ara-C (0.5 pg/mi), S3; combination ofAra-A (4 p/mi) andAra-C (0.5 pg/mi), aoo^

Control MMUdR Ara-C MMUdR+ lUdR MMUdR+ Ara-A MMUdR+Ara-C lUdR Ara-A

TREATMENTFIG. 4. Effect of combining MMUdR with three other nucleoside analogues on plaque formation by

HSV-1. Confluent monolayers ofRK-13 cells were infected with 100 plaque-forming units ofHSV-1 virus andsimultaneously treated with various compounds: MMUdR (2 pglml), E ; Ara-C (0.5 pg/mi), M; IUdR(0.5 pg/ml), UllX; Ara-A 4 (pg/ml), I; or combinations of these drugs at the concentration gives above.

antiviral compounds, another major limitationto their widespread use is the emergence ofdrug-resistant mutants. The observation thatin the presence of 25 to 50 ,ug/ml some virusreplication was detectable after 72 h suggestseither that the drug was gradually depleted

below its effective concentration or that drug-resistant mutants had arisen. Although theformer may be partially correct, we favor thelater hypothesis since we have been able toisolate MMUdR-resistant mutants from in-fected cell cultures treated with MMUdR (L. A.

aLUt^InLU -Ja.OX t-uZtnOLU U

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ANTIVIRAL EFFECTS OF NUCLEOSIDE ANALOGUES 649

Babiuk, V. S. Gupta, and B. T. Rouse, sub-mitted for publication). We feel that this ob-servation should not discount MMUdR as use-ful for treatment of herpesvirus infectionssince apart from the fact that resistant mu-

tants also arise in the presence of nucleosideanalogues (24), MMUdR mutants are stillsusceptible to the antiviral effects of the othernucleoside analogues (Babiuk et al., sub-mitted). Combination chemotherapy may beone way of avoiding both the problem oftoxicity and the emergence of drug-resistantmutants. Experiments are presently in prog-

ress to determine whether enhanced antiviraleffects occur between MMUdR and othernucleoside analogues in vivo as well as whethercombination chemotherapeutic regimens willprevent the emergence of drug-resistant mu-

tants.The nature of the antiviral activity of

MMUdR is in need of future definition. Thenucleoside IUdR is believed to interfere withvirus replication after its nucleotide is in-corporated into deoxyribonucleic acid (DNA)(20). The antiviral activity of Ara-C has beenattributed to its ability to impair DNA syn-

thesis of both the host cell and virus, probablyby an inhibition ofthe enzyme DNA polymeraseby the nucleotide arabinose cytidine 5'-tri-phosphate (20), whereas arabinose adenosine5'-triphosphate inhibits ribonucleotide reduc-tase (24). The combination chemotherapy ex-

periments reported by Fiala et al. (8), as wellas those discussed in this report, further sup-

port the hypothesis that these nucleosideanalogues elicit their effects by interfering atdifferent biochemical stages ofDNA replication.Thus enhanced antiviral activity was demon-strated with MMUdR in combination withAra-A, Ara-C, and IUdR. Studies are presentlyunderway to determine the effect of MMUdRon isolated enzymes and on nucleic acid andprotein metabolism of normal and infectedcells in an attempt to elicit the mechanism(s)whereby MMUdR exerts its antiviral activity.

ACKNOWLEDGMENTSThe enthusiastic technical assistance of Lynn Fran-

son, Lorraine Khachatourians, and Ken Zuk was ap-

preciated.This work was supported by the MRC of Canada

(MA 3136 and 5524) and Animal Pathology Division,Health of Animals Branch, Agriculture Canada.

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In I. Gililand and J. Francis (ed.), Scientific basis ofmedicine: annual reviews. The Althone Press, Lon-don.

2. Allison, A. C. 1974. Interactions of antibodies, comple-

ment components and various cell types in immunityagainst viruses and pyogenic bacteria. Transplant.Rev. 19:3-55.

3. Bubbar, G. L., and V. S. Gupta. 1970. Synthesis of5-substituted ether derivatives of 5-hydroxymethyl-deoxyuridine and their a-isomers. Can. J. Chem.48:3147-3153.

4. Buskirk, H. H., J. A. Crim, H. G. Petering, K.Merritt, and A. G. Johnson. 1965. Effect of uracilmustard and several anti-tumor drugs on the primaryantibody response in rate and mice. J. Natl. CancerInst. 34:747-758.

5. Buthala, D. A. 1964. Cell culture studies on antiviralagents. 1. Action of cytosine arabinoside and somecomparisons with 5-iodo-2'deoxyuridine. Proc. Soc.Exp. Biol. Med. 115:69-77.

6. Ch'ien, L. T., N. J. Cannon, L. J. Charamella, W. E.Dismukes, R. J. Whitley, R. A. Buchanan, and C. A.AMord, Jr. 1973. Effect of adenine arabinoside onsevere herpes virus hominis infections in man. J.Infect. Dis. 128:658-663.

7. DeClercq, E., and D. Shugar. 1975. Antiviral ac-tivity of 5-ethylpyrimidine deoxynucleosides. Bio-chem. Pharmacol. 24:1073-1078.

8. Fiala, M., A. W. Chow, K. Miyasaki, and L. B. Guze.1974. Susceptibility of herpes viruses to three nu-cleoside analogues and their combinations and en-hancement ofthe antiviral effect at acid pH. J. Infect.Dis. 129:82-85.

9. Gray, G. D., F. R. Nichol, M. M. Michalson, G. W.Camienar, D. T. Gish, R. C. Kelley, W. J. Wechter,T. E. Moxley, and G. E. Neil. 1972. Immuno-suppressive, antiviral and antitumor activities ofcytarabine derivatives. Biochem. Phamacol. 21:465-475.

10. Grindey, G. B., and C. A. Nichol. 1972. Interactionof drugs inhibiting different steps in the synthesisof DNA. Cancer Res. 32:527-531.

11. Hendersen, E. S., and R. J. Samaka. 1969. Evidencethat drugs in multiple combinations have materiallyadvanced the treatment of human malignancies.Cancer Res. 29:2272-2280.

12. Jawetz, E. 1968. The use of combinations of anti-microbial drugs. Annu. Rev. Pharmacol. 8:151-170.

13. Kurtz, S. M., R. A. Fisken, D. H. Kaump, and J. L.Schardein. 1969. Toxicity of 9-P-D-arabinofuranosyl-adenine in mice and rabbits, p. 180-189. Antimicrob.Agents Chemother. 1968.

14. Meldrum, J. B., V. S. Gupta, and J. R. Saunders.1974. Cell culture studies on the antiviral ac-tivity of ether derivatives of 5-hydroxymethyldeoxy-uridine. Antimicrob. Agents Chemother. 6:393-396.

15. Miller, F. A., G. J. Dixon, J. Ehrlich, B. J. Sloan,and I. W. McLean. Jr. 1969. Antiviral activity of9-p-D-arabinofuranosyladenine. I. Cell culture stud-ies, p. 136-147. Antimicrob. Agents Chemother.1968.

16. Momparler, R. L. 1974. A model for the chemotherapyof acute leukemia with 1-p-D-arabinofuranosylcyto-sine. Cancer Res. 34:1775-1787.

17. Notkins, A. L. 1974. Commentary: immune mechanismsby which the spread of viral infections is stopped.Cell Immunol. 11:478481.

18. Percy, D. H., and D. M. Albert. 1974. Developmentaldefects in mice and rats treated postnatally withcytosine arabinoside. Exp. Mol. Pathol. 21:381-397.

19. Percy, D. H., and D. M. Albert. 1974. Developmentaldefects in rats treated postnatally with 5-iododeoxy-uridine (IUdR). Teratology 9:275-286.

20. Prusoff, W. H., and B. Goz. 1973. Potential mech-anisms of action of antiviral agents. Fed. Proc.32:1679-1687.

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21. Rouse, B. T., and L. A. Babiuk. 1974. Host defensemechanisms against infectious bovine rhinotracheitisvirus: in vitro stimulation of sensitized lymphocytesby virus antigen. Infect. Immun. 10:681-687.

22. Rouse, B. T., and L. A. Babiuk. 1975. Host defensemechanisms against infectious bovine rhinotracheitisvirus. II. Inhibition of viral plaque formation byimmune peripheral blood lymphocytes. Cell Im-munol. 17:43-56.

23. Sartorelli, A. C. 1969. Some approaches to the thera-peutic exploitation of metabolic sites of vulnera-bility of neoplastic cells. Cancer Res. 29:2292-2299.

24. Schabel, F. M., Jr., and J. A. Montgomery. 1972.Purines and pyrimidines, p. 231-363. In D. J. Bauer

ANTIMICROB. AGENTS CREMOTHER.

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