LATENT VIRAL INFECTION OF CELLS IN TISSUE CULTURE BY JOHN P. BADER, PH.D., AND HERBERT R. MORGAN, M.D.

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1 Published Online: 1 February, 1961 Supp Inf: Dwnladed frm jem.rupress.rg n August 22, 218 LATENT VIRAL INFECTION OF CELLS IN TISSUE CULTURE VII. ROLE OF V~ATER-SOLUBLE VITAMINS IN PSITTACOSIS VIRUS PROPAGATION IN L CELLS* BY JOHN P. BADER, PH.D., AND HERBERT R. MORGAN, M.D. (Frm the Luis A. Wehle Virus Research Labratry f the Department f Bacterilgy, The University f Rchester Schl f Medicine and Dentistry, Rchester, New Yrk) (Received fr publicatin, Octber 1, 196) The multiplicatin cycle f psittacsis virus (6BC strain) in chick embry cells (1) r muse fibrblasts (L ceils) (2) is interrupted if these cells are expsed t a nutritinally deficient medium prir t infectin, and an experimental latent infectin is established. Several hurs after initial infectin, virus can n lnger be demnstrated in the cells, but the additin f a cmplete nutrient medium results in the reappearance f infectius virus. The imprtance f amin acids and vitamins fr the cmpletin f the infectius cycle was demnstrated thrugh the use f a synthetic medium (3), and subsequently the specific amin acid requirements fr the stimulatin f psittacsis virus grwth were defined (4). The specific vitamins required fr the prductin f psittacsis virus in L cells are examined in these experiments. Materials and Methds Virus.--The 6BC strain f psittacsis virus was maintained in this labratry by ylk sac passage, and virus suspensins were prepared frm infected chick embrys as previusly described (1). Fr certain studies, stck virus suspensins were prepared frm L cells infected with psittacsis virus and, in rder t dilute vitamins in the infecting inculum beynd an effective cncentratin, infected L ceils were maintained n a medium which was free frm the specific vitamin under examinatin. When cytpathic effects were first evident in these cultures, the fluids were remved and used as stck virus suspensins. A 1-8 dilutin f virus suspensin in Earle's balanced salt slutin (BSS) r in vitamin-deficient medium was emplyed as the infecting incuium. Virus Titratlns.--The single dilutin methd f Glub (5) was used t determine the amunt f virus in the tissue culture fluids..25 ml. f a 1 "I dilutin f the culture fluid was inculated int the ylk sac f each f a den 7-day-ld chick embrys and vires titers were expressed as the lg~lds. Test Media.--The basic cmplete synthetic medium (CM) emplyed, cntaining nly amin acids, water-sluble vitamins, glutamine, glucse, and inrganic salts, has been pre- * Supprted by a grant frm the Natinal Cancer Institute, Natinal Institutes Of Health, Public Health Service, and the Nelsn B. Delevan Virus Research Fund. 271

2 272 LATENT VIRAL INFECTION OF CELLS IN VITRO. VII viusly described (4) and prvides fr survival but nt multiplicatin f L cells. Each test medium was deficient in a single cmpund and/r its related derivative. L Cell Cultures.--Cultures f L cells were grwn in T-15 flasks 1 with a medium cntaining 2 per cent hrse serum,.2 per cent lactaibumin hydrlysate,.7 per cent yeast extract, and Earle's BSS (2). Depletin Prcedures.--Mter a unifrm layer f L cells had grwn ver the glass surface, the grwth medium was replaced with BSS; BSS was added again the fllwing day, and n the 3rd day, psittacsis virus was included in the replacement fluid t infect the cells. The fluids were changed with BSS after 24 hurs and 2 days after infectin the test media were added t the cultures and replaced each day fr 2 mre days. Virus titers f tissue culture fluids were determined daily. In certain experiments, grwth medium n the L cell cultures was replaced with a medium deficient in a single vitamin. The fluids were replaced n alternate days until the day f infectin. Cultures were infected with psittacsis virus when mrphlgical changes were nted in the cells which seemed t be a result f vitamin depletin r when a time cnsidered sufficient fr vitamin depletin had elapsed. One grup f depleted cells was maintained n the deficient medium after infectin, and the cmplete medium (CM) was added t anther grup. After infectin, the fluids were replaced daily until the cultures had disintegrated and these fluids were titrated fr infectius psittacsis virus. Cellular Activity.--The hydrgen in cncentratin f the test fluids cvering the cells was estimated clrimetrically utiliing phenl red in the medium in cmparisn with phenl red clr standards. Cell cultures were examined micrscpically each day and the mrphlgical appearances recrded. RESULTS Depletin ~th BSS.--Metablic depletin f L cells with BSS fr 2 days rendered the cells incapable f supprting a cmplete cycle f psittacsis virus grwth. The specific vitamin requirements fr the prpagatin f psittacsis virus in this system were examined by adding a synthetic medium deficient in a single vitamin t the infected L cells. Of the vitamins and vitamin cmbinatins which were examined, nly thiamine was a specific requirement in the medium fr psittacsis virus grwth (Table I). Media deficient in ther vitamins were fully capable f supprting the prductin f infectius virus in cells which had been depleted with BSS. The ph f the cmplete medium cvering infected cells was nt altered significantly ver the 24 hur interval between replacement f fluids, but was maintained at abut. The ph f mst f the vitamin-deficient media als remained cnstant, but culture fluids frm cells expsed t a thiamine-free medium were cnsistently mre acidic than cntrl fluids. The lack f stimulating activity f the thiamine-deficient medium fr psittacsis virus prductin was assciated with its failure t prduce recvery f the cells frm the mrphlgical changes assciated with the nutritinal depletin. When L cell cultures were maintained n BSS fr a ttal f 4 days, a decrease in cellular cytplasm was bserved micrscpically. The additin f i Obtained frm Kntes Glass C., Vineland, New Jersey.

3 J. P. BADER AND H. R. MORGAN 273 the cmplete medium t these cultures restred the cells t a mrphlgically healthy cnditin, usually within 48 hurs. Cells which were treated with a vitamin-deficient medium capable f stimulating virus grwth als shwed an increase in cytplasm cmparable t cells treated with the cmplete medium. Thiamine-deficient medium was capable f ellciting sme cytplasmic increase but the mrphlgical recvery f the cells was nt as cmplete as the recvery TABLE I Effect f Vitamin-Deficient Media n the Grwth f Psittacsis Virus Culture medium Virus grwth Recvery f cells fllwing BSS treatment ph f medium n 5th day after infecun Cmplete medium BSS Minus thiamine. " pantthenate. " insitl. " chline. " bitin. " ribflavin. " PABA* " flic acid. " PABA and flic acid. " niacin. " niacinamide.. " niacin and uiacinamide. " pyridxal. " pyridxine. " pyridxai and pyridxine L cells were treated with BSS fr 2 days prir t infectin with psittacsis virus and maintained fr 2 additinal days n BSS. Test media were added and culture fluids tested fr virus grwth. N virus was demnstrable in the cultures at the time f additin f test media. * Para-aminbenic acid. nted in cultures treated with cmplete medium r media deficient in ther vitamins. Depletin f Single Vitarains.--Since nly thiamine appeared t be essential fr psittacsis virus prductin in cells which had been treated with BSS, it seemed prbable that a mre extensive depletin perid fr ther vitamins wuld be necessary fr a fair estimate f the vitamins required fr virus prpagatin. L cells will nt survive lnger than 8 r 9 days when maintained n BSS alne but the recvery f nutritinally depleted cells after treatment with certain vitamin-deficient media indicated that L cells culd be maintained fr lnger perids n the vitamin-deficient media than n BSS alne. Therefre,

4 274 LATENT VIRAL INFECTION OF CELLS IN VITRO. VII attempts t deplete L cell cultures f specific vitamins were made by maintaining the cells n a medium deficient in the vitamin under examinatin until the cells were infected with psittacsis virus. In these studies the virus inculure was prepared frm L cells which were maintained fr a shrt perid f time n the vitamin-deficient medium, as described in Materials and Methds. A. Thiamine Depletin.--Depletin f thiamine frm L cells and the dependence f psittacsis virus prpagatin n this vitamin was demnstrated again after a 5 day treatment f L cells with a thiamine-deficient medium (Fig. 1). 6" iv' ILl :E 5- < "1r- I-, U) X~X e~e THIAMINE-DEFICIENT MEDIUM COMPLETE MEDIUM I.- a: 4-- :S J, CI l-i 3" ~- 2"..I (O.l -5 i DAYS FIG, 1. L cell cultures were maintained n thianine-deficient medium fr 5 days befre infectin with 6BC psittacsis virus. Mter 24 hurs cmplete medium was added t ne grup f cultures and thiamine-deficient medium was added t the ther grup. N virus was demnstrable in the fluids f infected cells which had been treated with thiamine-deficient medium, but virus prpagatin did ccur when cmplete medium was added after infectin. Fluids f cultures cntaining the thiamine-deficient medium were mre acidic than culture fluids frm the cntrls, and cytpathic changes which were typical f virus actin were seen in cultures cntaining cmplete medium befre any cytpathlgy was nted in the thiamine-deficient cultures. The disintegratin f L cell cultures cntaining the deficient medium was quite different frm that usually seen after virus infectin and culd prbably be attributed t the vitamin depletin. B. Pantthenate Depletin.--A few days after the initial expsure f L cells t pantthenate-deficient medium, the culture fluids became acidic, and it was

5 J. P. BADER AND H. R. MORGAN 275 necessary t replace fluids daily in rder t keep the ph within the range f the phenl red indicatr. The cultures were infected with psittacsis virus n the 12th day f pantthenate depletin. Cntrl cultures t which the cmplete medium was added after infectin did nt becme acidic, and the grwth f psittacsis virus in these cultures reached high levels within a few days. Cmplete cytpathic degeneratin ccurred by the 7th day after infectin in these 7- I,d Z a: 5' tli I- u~ n- >,,.,, ur) 4- I- I- _ Z 4...-I 2 ~ H (.1 X-X PANTOTHENATE-DEFICIENT MEDIUM e--e (~OMPLETE MEDIUM //CULTURE DISINTEGRATED -,2,' ;2,,,'6,8 DAYS FIG. 2. L cell cultures were maintained n pantthenate-deflcient medium fr 12 days befre infectin with psittacsis virus. After 24 hurs cmplete medium was added t ne grup 5 cultures and pantthenate-deficient medium was added t the ther grup. cultures. The rate f virus prductin in the pantthenate-deficient cultures was retarded and the yield f virus never reached the magnitude f the cntrl cultures (Fig. 2). Cytpathic effects were nt apparent in the deficient cultures until lng after the cntrl cultures had disintegrated, and virus cntinued t be prduced by the surviving cells. C. Niaci -Niacinamide Depletin.--Mter maintenance f cells fr 14 days n niacin-niacinamide-deficient medium the mrphlgy f the cells began t change frm the typical biplar cells t cells with multiple cytplasmic extensins. The number f cells exhibiting this change increased with cntinued niacin-niacinamide depletin, and after 18 days cnsiderable vaculiatin f

6 276 LATENT VIRAL INFECTION OF CELLS IN VITRO, VII cells was nted. On the 24th day f depletin, ~ f the cultures were infected with psittacsis virus. The remaining cultures were maintained fr 2 mre weeks n the deficient medium and infected at this time. The cultures infected at 24 days and treated with cmplete medium demnstrated a pattern f psittacsis grwth (Fig. 3) nt significantly different frm that usually fund in L cell cultures. Cytpathic effects were bserved after 6 days and the cultures had cmpletely disintegrated by the 9th day after infectin. Virus prductin ~ 7~ 6' N m 5' IM _ I-- I-- > to Q -1 2' ~t ne~ X~X NIACIN AND NIAOINAMtDEmDEFIGtENT MEDIUM e..-e COMPLETE MEDIUM //CULTURE DISINTEGRATED I'O DAYS FIG. 3. L cell cultures were maintained n niacin- and niacinamide-deficient medium fr 24 days befre infectin with 6BC psittacsis virus. After 24 hurs cmplete medium was added t ne grup f cultures and the deficient medium was added t the ther grup. was nt as rapid in cultures which were treated with niacin-niacinamidedeficient medium, and the peak titers were nt fund until 2 days after the cntrl cultures had reached maximum titers. Initial cytpathic effects were bserved in the cultures fed with cmplete medium 2 days befre the cultures n the deficient medium, and extensive degeneratin f the cultures was als delayed in the cultures with medium deficient in niacin and niacinamide. Similar results were nted in cultures which were infected n the 38th day after initiatin f niacin-niacinanfide depletin. Virus prductin was delayed in the deficient cultures and cmplete disintegratin f cells did nt ccur until 4 days after the cntrl cells had lysed. Sme cytlysis due t vitamin deple-

7 J. P. BADER AND ~. R. "M'ORGAN 277 tin alne was apparent at this time, since uninfected cultures which had been maintained n niacin-niacinamide-deficient medium had begun t disintegrate. D. Pyridxal-Pyr~txine Depletin.--Cultures which were maintained n a medium deficient in pyridxal and pyridxine shwed n significant mrphlgical changes ver the first 3 weeks, but after this perid the cells became slightly runded, and a few ceils began t disengage frm the glass surface. After 24 r 38 days f depletin the cultures were infected with psittacsis 5- q n- ne w t- i Ig > I-4 2 gig _ I: J, MEDIUM 1.1 [, I DAYS Fx. 4. L ceg cultures were maintained n pyridxal (pyridxine)-deficient medium fr 24 days befre infectin with 6BC psittacsis virus. After 24 hurs, cmplete medium was added t ne grup f cultures and the deficient medium was added t the thr grup. virus, and cultures which were maintained n pyrldxal-pyridxne-deficient medium were cmpared with cultures t which cmplete medium was added after infectin. Virus prductin after depletin fr 24 days was cnsiderably delayed in the cultures with the pyridxal-pyridxine-deficient medium (Fig. 4). Infectius virus did nt appear in these cultures until 2 days after it had appeared in the cntrls fed with cmplete medium. Cytpathic effects were extensive in the cntrl cultures by the 1th day after infectin, but in the deficient cultures there was nly a slw disintegratin f cells until the 19th day after infectin when lysis was practically cmplete. A simi]ar delay in the appearance f infectius virus was seen in the pyridxalpyridxine-deficient cultures which were infected n the 38th day after initiatin f the depletin prcedure. N differences in the cytpathic effects culd

8 278 LATENT VIRAL INFECTION OF CELLS IN VITRO. VII be demnstrated, hwever, because f the extensive cellular degeneratin which was nted during the experimental perid in bth infected and nn-infected vitamin-deficient cultures. E. Chline Depletin.--L cells were maintained n a chline-deficient medium fr 24 days prir t infectin. At this time the cells had changed nly slightly in their mrphlgical characteristics, althugh cells had begun t detach frm the glass surface. Cultures cntaining cmplete medium prduced higher levels 7" n~ l,ij I'- 6' 5. 4 in ) n... > a i.,,,i D 2' --I lu.p ji /) 3E J, J = X~X CHOLINE-DEFICIENT MEDIUM e~e COMPLETE MEDIUM //CULTURE DISINTEGRATED I 'CO.I i - :>4 DAYS FIG. 5. L cell cultures were maintained n chline-deficient medium fr 24 days befre infectin with 6BC psittacsis virus. Mter 24 hurs, cmplete medium was added t 1 grup f cultures and chline-deficient medium was added t the ther grup. f psittacsis virus than the cultures withut chline (Fig. 5). The large differences in titers culd nt be attributed t the lss f cells frm deficient cultures during this perid, but by the 6th day after infectin cellular disintegratin was extensive in bth deficient and cntrl cultures, and adequate determinatins f vitamin essentiality were nt pssible after this time. F. Bitin, Insitl, and Para-aminbenic Acid (PABA) Flic Acid Depletin.--Depletin f L cell cultures with a bitin-deficient, insitl-deficient, r PABA and flic acid-deficient medium had little effect n the capacity f the cells t prduce psittacsis virus. Cultures were maintained n bitindeficient medium fr 2 days befre infectin and subsequent viral titers f

9 J. P. BADER AND H. R. MORGAN 279 cultures cntaining the deficient medium were nt significantly different frm titers f cntrl cultures. Cultures were maintained fr 45 days n insitldeficient medium prir t infectin, but the prductin f psittacsis virus did nt appear t be affected, since cmplete medium cntrls and insitl-deficient cultures yielded similar amunts f virus. PABA and flic acid depletin f cultures fr as lng as 6 days had n apparent effect n the capacity f the cells t supprt the grwth f psittacsis virus. Virus prductin prceeded at similar rates in cntrl cultures and cultures cntaining the medium deficient in PABA and flic acid, and n differences were nted in the pattern f cytpathlgy exhibited by the test cultures. G. Ribflavin.--After the 12th day f depletin with a ribflavin-deficient medium, cells became enlarged and degenerative changes restricted the perid f experimentatin. When cultures were infected after 7 days' depletin, n significant differences in psittacsis prductin culd be demnstrated between the cultures cntaining ribflavin-deficient medium and thse cntaining cmplete medium. The relatively shrt depletin perid may nt be sufficient t lwer the effective cncentratin f ribflavin in the cells, and it is nt felt that a ribflavin requirement fr the grwth f psittacsis virus has been eliminated. DISCUSSION L cells which have been depleted f their nutritinal reserves by maintenance n BSS fr 48 hurs prir t infectin are incapable f supprting the grwth f psittacsis virus (2), and the specific amin acids which are required in a medium capable f supprting psittacsis virus prductin have been delineated (4). In this system the deletin f specific vitamins frm the cmplete synthetic medium had little effect n the ptency f the medium fr the supprt f virus grwth, with ne exceptin. Of the vitamins examined, nly thiamine was demnstrated t be an imprtant individual cnstituent f the medium. The acidity f the fluids frm L cell cultures which had been treated with the thiamine-deficient medium is indicative f thiamine deficiency in the cells, since this vitamin is knwn t be an imprtant factr in the metablism f a-ket acids and is prbably necessary fr the aerbic xidatin f carbhydrates. It has been bserved that cultures cntaining a minimum f free xygen are inhibited in their ability t supprt psittacsis virus prductin (6) and aerbic xidatin may be essential fr the synthesis f psittacsis virus. Cnsidering the ease with which L cells can be depleted f their essential free amin acid cnstituents (4), it is apparent that mst vitamins d nt diffuse readily frm the cell r are present in such cncentratin that many mre changes f maintenance media are necessary t dilute the vitamin t a cncentratin which is ineffective fr ptimal metablic activities. All f the vitamins examined have been shwn t exist in cmbined frms with ther metablic cmpunds, and these vitamins may be bund in L cells, rendering

10 28 LATENT VIRAL INFECTION OF CELLS IN VITRO. VII unlikely the depletin f the cells f a specific vitamin in such a shrt perid f time. The rapid depletin f thiamine frm L cells and the dependence f psittacsis virus prpagatin n this vitamin was further shwn by the failure f psittacsis virus t prpagate in cells which were maintained n a medium deficient nly in thiamine. L cells required much lnger incubatin perids with media deficient in ther vitamins befre any reductin in psittacsis virus grwth was demnstrable. The inability f cells t supprt high levels f psittacsis virus prductin after maintenance n a pantthenate-deficient medium may als be related t a dependence n the aerbic xidatin f carbhydrates and fats, since pantthenate is a cnstituent f cenyme A, an imprtant cmpund in the intermediary metablism f these substances. Darnell and Eagle (7) demnstrated that HeLa cells which had been depleted f pyridxine culd nt supprt the grwth f plivirus if glucse, glutamine, and salts were subsequently used as a maintenance medium. Hw= ever, if free amin acids were added the cells readily supprted plivirus grwth, and these authrs cncluded that the available free amin acid pl was depleted in vitamin Brdepleted cells. In the present studies, there is clearly indicated a dependence upn pyridxine which ges beynd that f depletin f the free amin acids, since these latter substances were regular cnstituents f the pyridxine-deficient medium. The failure f the present studies t demnstrate a grwth requirement f psittacsis virus fr flic acid is cnsistent with ther evidence which indicates that this virus is capable f bringing abut a synthesis f flic acid in infected cells. L cells apparently lack the capacity fr flic acid synthesis, since Eagle has demnstrated a requirement fr this vitamin in the grwth f L cells (8). Cln and Mulder (9) shwed that flic acid was present in purified preparatins f psittacsis virus, and Hltermann et al. (1) demnstrated an increase in the flic acid cntent f L cells infected with psittacsis virus. Als, the flic acid cntent f purified preparatins f anther member f the psittacsis grup, muse pneumnitis virus, was demnstrated t increase during in vitr incubatin (11). Eagle (8) was nt able t demnstrate a bitin r insitl requirement fr the grwth f L cells and n requirement fr these vitamins in the grwth f psittacsis virus in L cells culd be demnstrated in the present studies. Althugh a number f specific vitamin requirements have been demnstrated fr the maximum prductin f psittacsis virus in L cells, the exclusin f these vitamins, except thiamine, had little effect n the capacity f the medium fr stimulating the prductin f infectius virus frm a latent infectin. Als, extended depletin f L cells f specific vitamins did nt prduce a latent infectin in vitr as has been recently demnstrated with phenylalanine r isleucine depletin (6).

11 J. P. BADER AND H. R. MORGAN 281 SUMMARY A study f the metablic requirements fr the grwth f psittacsis virus in L ceils has been extended t the water-sluble vitamins. In a system in which a balanced salt slutin was used t deplete the ceils f their vitamin cnstituents, nly thiamine was essential fr psittacsis virus prductin. Extended depletin f ceils with media deficient in specific vitamins demnstrated that pantthenate, niacin (niacinamide), pyridxine (pyhdxal), and chline, in additin t thiamlne, were essential fr maximal grwth f psittacsis virus. N requirement fr bitin, insitl, flic acid, r ribflavin was demnstrated, althugh the pssibility f incmplete vitamin depletin f the ceils has nt been eliminated. In mst cases in which a specific vitamin requirement was shwn the decreased yield f virus was crrelated with a delay in the cytpathic effects prduced in the ceu cultures by psittacsis virus. We gratefully acknwledge the technical asdstance f Angel P. Andrese. BIBLIOGRAPHY 1. Mrgan, H. R., Latent viral infectin f ceils in tissue culture. I. Studies n latent infectin f chick embry tissues with psittacsis virus, J. Exp. Meg., 1956, 18, Mrgan, H. R., and Bader, J. P., Latent viral infectin f cells in tissue culture. IV. Latent infectin f L cells with psittacsis virus, J. Exp. Meg., 1957,16, Jhnsn, K. M., and Mrgan, H. R., Latent viral infectin f cells in tissue culture. II. Relatinship f cell nutritin t initiatin f grwth f psittacsis virus, J. Exp. Meg., 1956, 13, Bader, J. P., and Mrgan, H. R., Latent viral infectin f ceils in tissue culture. VI. Rle f amin acids, glutamine, and glucse in psittacsis virus prpagatin in L cells, J. Exp. Meg., 1958, 18, Glub, O. J., A single dilutin methd fr the estimatin f LD6 titers f the psittacsis-lgv grup f viruses in chick embrys, J. Immunl., 1948, 59, Bader, J. P., and Mrgan, H. R., unpublished bservatins. 7. Darnell, J. E., and Eagle, H., Nutritinal requirements fr plivirus synthesis in HeLa ceils, Fed. Prc., 1958, 17, Eagle, H., The minimum vitamin requirements f the L and HeLa cells in tissue culture, the prductin f specific vitamin deficiencies, and their cure, J. Exp. Meg., 1955, 1'2, Cln, J. I., and Mulder, J. W., Flic acid in purified preparatins f members f the psittacsis grup f micrrganisms, J. Infect. Dis., 1959, 18, Hltermann, O. A., Mergenhagen, S. E., and Mrgan, H. R., Factrs related t psittacsis virus (strain 6BC) grwth. V. Flic acid-like factr in infected ceils, Prc. Sc. Exp. Bil. and Meg., 1959, 13, Cln, J. I., Frmatin f citrvrum factr in purified preparatins f muse pneumnitis virus, Bact. Prc. 1959, 85.