JOURNAL OF BACrERIOLOGY, July, 1966 Vol. 92, No. 1Copyright © 1966 American Society for Microbiology Printed in U.S.A.

Effects of Pancreatin on the Growth of ReovirusCRAIG WALLIS, JOSEPH L. MELNICK, AND FRED RAPP

Department of Virology and Epidemiology, Baylor University College of Medicine, Houston, Texas

Received for publication 31 March 1966

ABSTRACTWALLIS, CRAIG (Baylor University College of Medicine, Houston, Tex.), JOSEPH

L. MELNICK, AND FRED RAPP. Effects of pancreatin on the growth of reovirus. J.Bacteriol. 92:155-160. 1966.-The influence of pancreatin and other proteolyticenzymes on the growth, plaque formation, and antigenicity of reovirus was studied.Single-cycle yields of virus in the presence of enzyme were not increased, but multi-ple-cycle yields of virus were greatly enhanced. Immunofluorescence studies demon-strated that the transmission of reovirus from cell to cell is more rapid in the presenceof the enzyme. These findings led to the development of a rapid plaque assay systemfor reovirus, a virus which has previously been difficult to assay by theplaque method. In the recommended procedure, pancreatin is incorporated into theagar overlay. Monkeys immunized with enzyme-treated reovirus yielded higher anti-body titers than animals receiving the same amount of untreated virus.

The low plaquing efficiency of reoviruses hasbeen attributed to inhibitors in the agar overlay(9). However, since the proteolytic enzymes havebeen shown to enhance the infectivity of reovirus(2, 6), a proteinaceous covering on the virus maybe the responsible factor in suppressing cell-to-cell transmission under agar. This protein mayalso explain the long period required to growreovirus in cells bathed in fluid medium. Previ-ous electron microscope studies (5, 7) have ac-tually revealed the virus to be embedded in amatrix as it is formed in the course of its replica-tion cycle. The present study is concerned with theeffect of proteolytic enzymes on the growth,plaque formation, and antigenicity of reovirus,aspects which have not been previously investi-gated.

MATERIALS AND METHODS

Monkey kidney (MK) cells. Kidneys from imma-ture rhesus monkeys were trypsinized, grown inM-H medium, and maintained in M-E medium, asdescribed in detail elsewhere (4).

Viruses. Type 1 reoviruses, strains 716 and Lang,were grown in MK cultures maintained with M-Emedium.

Virus assays. The viruses were assayed by plaque-counting methods, with results expressed as plaque-forming units (PFU). Overlay medium consisted ofEarle's salt solution, 0.4% NaHCO3, 0.1% skimmilk, 1:60,000 neutral red, and 1.5% agar (Difco).The preparation of the reagents for use in the overlaymedium has been described (8).

Pancreatin. Buffered pancreatin tablets were eachdissolved in 50 ml of distilled water, as described by

the manufacturer (Oxoid, Ltd., distributed by ColabLaboratories, Inc., Chicago Heights, IHl.), and filteredthrough a 0.220-,u Millipore filter. This solution wasfrozen as undiluted stock until used, at which timeit was diluted as indicated in the text below. Thisenzyme preparation gave reproducible results fromlot to lot, whereas purified forms of pancreatin fromother manufacturers tested at different levels did notgive reproducible results. Other enzymes used in thisstudy were obtained from Nutritional BiochemicalsCorp., Cleveland, Ohio, and were either two or threetimes crystalline (except for lipase, which was reagentgrade).

Immunofluorescence technique. Cells were grownon round 15-mm cover slips in petri dishes incubatedin an atmosphere of 5% CO2 at 37 C. When mono-layers had formed, the cover slips were drained ofgrowth medium, transferred to dry petri dishes, andinoculated with 0.1 ml of virus per cover slip. Thevirus was allowed to adsorb for 1 hr at 37 C, andthen the cover slips were flooded with M-E medium,some with and some without pancreatin diluted 150-fold. Cells harvested for immunofluorescent stainingwere washed three times with warm tris(hydroxy-methyl)aminomethane (Tris) buffer (pH 7.4), air-dried, and fixed for 10 min in acetone. Viral antigenwas detected by reacting the cells first with reovirusantiserum prepared in monkeys, and then with horseglobulin that had been prepared against normal mon-key serum globulin and labeled with fluoresceinisothiocyanate. Detailed fractionation and labelingmethods have been described (3).

REsuLTsEffect ofpancreatin in overlay medium on plaque

formation. The two reovirus strains were selected155

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WALLIS, MELNICK, AND RAPP

because they were known to differ in their plaque-forming ability. Without special treatment, theLang -strain fails to produce plaques, whereas the716 strain produces tiny plaques about 1 to 2 mmin diameter in about 5 days. In the present ex-periments, 0.1 ml of each strain in serial 10-folddilutions was inoculated onto drained cultures (in1-oz bottles containing 106 cells), and after an ad-sorption period of 2 hr at 37 C, representativecultures were overlaid with medium containingthe pancreatin stocks that had been diluted 30 to120 times. The results are shown in Table 1.By the 3rd day after inoculation, the 716 strain

had not produced plaques under the usual overlay;however, the virus titer in cultures containing1: 30 pancreatin in the overlay was 106 5 PFU/ml,and the diameter of plaques ranged from 2 to 5mm. By the 6th day, the virus in the control cul-tures had attained a titer of 106 5 with 1- to 2-mmplaques as compared with 107-7 under the pan-creatin-containing overlay, in which the plaquesaveraged 6 to 8 mm. Figure 1 illustrates the dif-ferences in plaque size found between control andpancreatin overlays on the 3rd day.The Lang strain gave similar results. However,

no plaques were visible in the pancreatin-freecultures even on the 6th day, whereas the titerunder pancreatin-containing overlays was 1065PFU/ml with an average plaque diameter of 2to 3 mm.

Trypsin and chymotrypsin were also tested, butthe toxicity of these enzymes for MK cells pre-cluded their routine use. At enzyme concentra-tions where plaque enhancement occurred, read-ings could only be made up to the 3rd day, sincecell integrity was lost thereafter.

Effect of pancreatin on reovirus during growthcycles under fluid medium. These experimentswere designed to test the effect of the enzyme on

the growth of the virus in single and multiplecycles of replication.

Strain 716 was inoculated at a multiplicity of 1PFU per cell onto drained 1-oz cultures, contain-ing 106 cells. After an adsorption period at 37 Cfor 2 hr, the cultures were repeatedly washed toremove unadsorbed virus. Some of the cultureswere then held in M-E medium at 37 C. Duplicatecultures were maintained in the same mediumcontaining pancreatin diluted 150 times, which iswell beyond the toxic concentration of enzyme forthe cells. Representative cultures were frozen atdifferent intervals, beginning at 2 hr and endingat 16 hr after the addition of virus. Harvests were

assayed by thawing cultures, centrifuging themlightly for a few minutes, and plating the clarifiedsupernatant fluids under pancreatin overlays. Inthese experiments on the single growth cycle, no

differences between the pancreatin-treated andenzyme-free cultures were detected. Newlyformed virus was first detected after 10 hr in bothenzyme-free and control pancreatin-treated cul-tures; both stocks were similar in titer (106PFU/ml). At 12 hr, titers of 106.5 and 1066 (con-trol and pancreatin) were obtained, and at 16 hr107-5 and 107-3, respectively.

Multiple-cycle experiments were performed insimilar fashion to those above. The input of viruswas 10 PFU per culture of 106 cells, and harvestswere collected at 2 hr through 6 days. The resultsof a typical experiment are illustrated in Fig. 2.Cytopathic effects (CPE) were first evident in thepancreatin-containing cultures on the 2nd day,at which time 10% of the cells were involved.Infected pancreatin-free cultures showed similardegrees of CPE only on the 4th day.

Virus harvested 24 hr after inoculation con-

tained 103.0 PFU/ml in the infected control cul-tures and 103.4 PFU/ml in cultures maintained

TABLE 1. Effect of pancreatin on plaque titers and diameters of type 1 reoviruses

Plaque titer (PFU/ml) Plaque size (mm)b

Overlay" Strain 716 Lang Strain 716 Lang

3rd day 6th day 3rd day 6th day 3rd day 6th day 3rd day 6th day

No added enzyme. <2.0 6.5 <2.0 <2.0 0 1-2 0 0Pancreatin added

1:30.6.5 7.7 <2.0 6.5 2-4 6-8 0 2-31:60. 6.2 6.9 <2.0 4.1 1-2 4-5 0 1-21:120.<2.0 6.4 <2.0 <2.0 0 1-3 0 0

a Viruses were inoculated onto drained MK cultures at various dilutions and, after 2 hr of adsorp-tion at 37 C, cultures were overlaid with control medium (see Materials and Methods) or medium con-taining pancreatin at dilutions indicated. Dilutions of 1:15 caused slight toxic effects on cells, andhigher concentrations destroyed them.

b About 100 plaques were counted to determine average diameter.

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VOL. 92, 1966 EFFECTS OF PANCREATIN ON GROWTH OF REOVIRUS

with pancreatin. However the 2nd-day harvestswere significantly different. Infected control cul-tures manifested titers of 103 7, whereas the cul-tures containing pancreatin increased to 106 °. Themaximal titer of 107-7 PFU/mI was reached inpancreatin cultures on the 4th day, as comparedwith only 106 4PFU/ml, which was not reachedin the infected control cultures until the 6th day.The effect of the enzyme on the production of

viral antigen was also studied by immunofluores-cence. Different dilutions of reovirus were ad-sorbed to monolayers on cover slips. After anadsorption period of 1 hr at 37 C, the cultureswere rinsed repeatedly, and a series of cover slipswere maintained in M-E medium. A duplicateseries was made with medium containing 1:150pancreatin. Cover slips were fixed at differenttimes, reacted with antireovirus antiserum, andexamined for fluorescence. The results of this ex-periment are shown in Table 2. In the pancreatin-containing cultures, the number of infected cellsgreatly exceeded the number of positive cells inthe enzyme-free cultures. Thus, at an inoculumof 10-4, on day 2 only 1 % of the cultures without

FIG. 1. Effect of pancreatin on plaque formationof reovirus (716 strain). Photographed 3rd day afterinoculation. Left culture: inoculated with 20 to 40PFU of reovirus; overlay made with pancreatin-freemedium. Right culture: same inoculum, but pancreatin(1:60) included in overlay medium.

8

.01~~~~~~

Z_,a PANCREATIN /

c / CONTROL

/0

3 /I0 2 3 4 5 6

DAY OF VIRUS HARVEST

FIG. 2. Effect of pancreatin on multiple-cycleyields of reovirus. The 716 strain was inoculated intodrained cultures with an input of about 10 PFU per

culture. Cultures were maintained with M-E medium,and M-E medium containing the enzyme (1:150).

TABLE 2. Fluorescence studies with reovirus(strain 716)

Percentage of cells exhibiting fluorescence

Virus Cultures without added Cultures with addedinoculum pancreatin pancreatin

Day I Day 2 Day 3 Day I Day 2 Day 3

10-2 25 90 Ua 50 U Ulo-- 1 25 90 25 U U104 <0. 1 1 25 1 50 U0_6 0 <0.1 1 0 25 U

a Unsatisfactory because of advanced cyto-pathic effects.

added enzyme were positive, in contrast to 50%of the cells in the pancreatin-treated cultures. Inthe presence of pancreatin, cells showed such ad-vanced cytopathic effects 2 days after inoculationof high doses of virus and 3 days after inoculationof even the low doses that immunofluorescencereadings were not possible.

Effect of pancreatin on adsorption of reovirus.When reovirus is treated with trypsin or otherproteolytic enzymes and then plated, the infec-tivity of the sample is increased over untreatedsamples (2, 6), suggesting that the enzyme actsby removing inhibitors from the virus surface.However, from the results of the multiple-cycleyields shown above, pancreatin might have actedby enhancing virus adsorption. This was testedexperimentally.

Cultures were drained of medium and inocu-lated with 0.1 ml containing 5,000 PFU of reo-

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virus (716 strain), determined on the basis ofprevious titrations with the usual overlay. Dupli-cate drained cultures were inoculated with virussuspended in medium containing 1:10 pancreatin.After adsorption periods up to 40 min, 1 ml ofTris buffer was added to the cultures, and unad-sorbed virus was recovered after rotary agitationof the diluent over the monolayer. The amount ofvirus recovered was the same, regardless ofwhether pancreatin had been present during ad-sorption.Another experiment was performed with cells

pretreated with pancreatin. After 2 hr of treat-ment at 37 C, the cell layer was repeatedly rinsedto remove residual pancreatin, and cultures werethen inoculated with 10 to 20 PFU of virus con-tained in a 0.1-ml volume of M-E medium. Cul-tures pretreated with pancreatin manifested noenhancement of plaque counts.

Penetration of virus. Since there is no enhancedadsorption of virus which could be detected inthe presence of enzyme, the possibility of morerapid penetration of the virus into cells was in-vestigated. A typical experiment follows. Reo-virus was diluted to contain 100 PFU/0.1 ml in(i) 1:10 pancreatin and (ii) pancreatin-free dilu-ent. These suspensions were used to inoculate alarge number of drained cultures. After adsorp-tion periods ranging from 15 to 480 min, cultureswere washed repeatedly to remove unadsorbedvirus, and they were then treated with a concen-tration of reovirus antiserum capable of neu-tralizing 10,000 PFU, to neutralize the virus thathad adsorbed but had not yet penetrated the cell.After 1 hr of incubation at 37 C, cultures werewashed to remove free antibody. The cultureswere then provided with maintenance medium,and were immediately frozen and thawed. Theclarified supernatant fluids of these harvestswere assayed to compare the amount of cell-associated virus beyond the reach of antibody butwhich had not yet been "eclipsed." The infectedcultures with or without pancreatin yielded har-vests that had insignificant differences in theirvirus content. Thus, the effects of pancreatin areapparently not due to enhanced cell penetration.

Effects ofpancreatin on harvests obtained at dif-ferent stages in the growth cycle. The followingexperiment was performed to determine when inthe stage of virus growth the enzyme was effective.Reovirus grown in single-cycle yields (describedabove) in M-E medium was used as the source ofvirus. Different harvests (taken from 2 to 14 hrpostinfection) were diluted 10-fold in (i) Hankssalt solution and (ii) 1 :10 pancreatin in salt solu-tion. Samples were held at 37 C for 1 hr and thenassayed. The 10-hr harvest was the first harvest

showing newly formed virus, and it was activated30-fold by enzyme treatment. The 12-hr harvestwas enhanced 25-fold by pancreatin treatment,and the 14-hr harvest, 40-fold. Thus, virus formedat different times during the single cycle of growthcould be enhanced to approximately the samedegree.The following experiment was performed to de-

termine whether the inhibitor was bound to reo-virus during the intracellular replication phase oronly after virus release.

Reovirus (716 strain) was inoculated ontodrained cultures at an input of 1 PFU/cell. After2 hr of adsorption at 37 C, the cells were washedrepeatedly, and then M-E maintenance mediumwas added. After 24 hr, the fluid phase was har-vested, and the cells were washed and harvestedseparately. Virus was released from the cells byfreezing and thawing. The fluid and cell-associ-ated samples were then diluted 10-fold in (i)1:10 pancreatin or (ii) Hanks salt solution. Bothsamples were activated to the same degree (25-and 30-fold, respectively), which indicates thatintracellular and extracellular virus are equallysensitive to the action of the enzyme.

In some experiments, a small input of 10 PFUper culture was used, and multiple-cycle harvestswere collected after 4 days of incubation. Inconfirmation of the results of Spendlove andSchaffer (6), the extracellular virus in the lateharvest could not be activated by pancreatin,whereas the late intracellular virus was readilyactivated 25-fold.

Effect ofenzymes on the antigenicity ofreovirus.In view of the enhanced infectivity describedabove, viral antigenicity was also tested in ani-mals inoculated with enzyme-treated virus. Theantigenicity of reovirus in 2 M MgC12 was alsotested, as this salt has also been shown to enhanceinfectivity (9). Reovirus (716 strain) was dilutedwith an equal volume of (i) Hanks salt solution,(ii) stock pancreatin, (iii) 0.1% chymotrypsin,(iv) 0.1% trypsin, (v) 0.1 % papain, (vi) 0.1%lipase, and (vii) 4 M MgCl2. The samples in theenzymes were held at 37 C for 1 hr, and repre-sentative samples in MgCl2 were heated at 50 Cfor 15 min. All samples were then assayed forvirus, and in addition 2-ml portions were injectedintramuscularly into young rhesus monkeys. Pre-inoculation sera were taken from all the monkeys,and none was found to neutralize reovirus. After10 days, all animals were bled and given a boosterinjection of the same inoculum used initially; asecond bleeding was made 10 days thereafter.The results of the antibody tests are shown in Ta-ble 3.Assay of antigens used for injection of the mon-

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EFFECTS OF PANCREATIN ON GROWTH OF REOVIRUS

TABLE 3. Antigenicity of reovirus treated with enzymes or MgC12

Serum titersb

Reovirus treatmenta Logso titer, Multiplicity of Monkey no. - _ - ____________PFU/ml activation Ist bleeding, 2nd bleeding,Preinjection 10th day' 20th day

Hanks salt solution........... 6.0 2702 <10 40 802709 <10 <10 802710 <10 20 160

Pancreatin (IX stock) ........ 7.5 30X 2703 <10 320 6402704 <10 80 320

Trypsin, 0.1% ................ 6.8 6X 2705 <10 160 320Chymotrypsin, 0.1% .......... 7.5 30X 2706 <10 80 640Papain, 0.1% ................. 7.0 lox 2707 <10 80 320Lipase, 0.1% .................. 6.5 3X 2708 <10 <10 80MgCI2, 2 MHeated at 50 C, 15 min. 7.0 lox 2711 <10 80Unheated ................... 6.1 2712 <10 <10 40

a Undiluted reovirus (strain 716) was mixed with an equal volume of diluents indicated.b Values are 90% plaque-reduction end points.c A second injection of each material was given on the 10th day.d Monkey died after first bleeding.

keys showed an activation multiplicity of 30 timesfor reovirus treated with pancreatin, 6 times iftreated with trypsin, 30 times with chymotrypsin,3 times with lipase, 10 times with papain, and 10times for the sample heated in MgCI2. The anti-body response in monkeys was consistently higherif the animals received treated samples thatshowed enhanced infectivity. The three monkeysinjected with untreated virus having a titer of 106PFU/ml yielded neutralizing titers of <1:10 to1:40 at the first postinjection bleeding and 1:80to 1:160 at the second bleeding. The monkeyinoculated with pancreatin-treated virus (30-foldactivation) had titers of 1:80 and 1:320 (firstbleeding) and 1:320 and 1:640 (second bleed-ing); trypsin-treated virus (sixfold activation)elicited titers of 1:160 and 1:320; chymotrypsin(30 times activation), 1:80 and 1:640; papain(10 times activation), 1:80 and 1:320; lipase(three times activation), <1:10 and 1:80. Thesample heated in MgC12 (10 times activation) in-duced a serum titer of 1:80 at the first bleeding.Prior to the second bleeding, this monkey died,but not due to viral infection. The monkey im-munized with virus treated with unheated MgC12(which did not increase viral infectivity) had alow antibody response similar to the animals re-ceiving untreated virus.

DiscussIoNThe addition of pancreatin to the agar overlay

results in the development of large reovirusplaques in rhesus kidney cells within 3 days afterinoculation. In the presence of enzyme, the cell-

to-cell transmission of the virus is greatly facili-tated, and during multiple cycles of viral replica-tion the yield of infectious virus is higher than incultures maintained in the absence of enzyme.Enhancement of reovirus infectivity after expo-sure to either MgCJ2 (9) or to proteolytic enzyme(2, 6) had been noted previously.Spendlove and Schaffer (6) suggested that the

enzymatic enhancement of reovirus infectivitywas due to proteolytic action upon a virus-associ-ated substrate. The present study indicates thatpancreatin does not appear to enhance the ad-sorption of reovirus to the monkey cells, but thatcell-to-cell transmission is enhanced in the pres-ence of the enzyme, as shown by increased plaquesize and the more rapid spread of viral antigensin the presence of pancreatin. The pancreatin maybe activating noninfectious virus. One possiblemechanism of activation might be the removalfrom the virus surface of inhibitors that interferewith penetration or uncoating of the virus. Experi-ments carried out in this study did not indicatethat pancreatin enhanced reovirus penetrationinto cells more rapidly than untreated virus, norwas there any evidence of an earlier eclipse phasewith pancreatin than in its absence.The possibility exists that the enzyme is de-

stroying interferon. However, Spendlove andSchaffer (6) showed that pelleted virus (presuma-bly free from interferon) could still be activated byenzymes. Furthermore, in the present study wefound that virus stocks diluted 10,000-fold, wellbeyond the range of interferon activity, were stillactivated by treatment with pancreatin.

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Reovirus treated with enzymes was a morepotent antigen in monkeys than untreated virus.The increased antibody response may be a reflec-tion of the higher infectivity of the treated virus.

Experiments on virus harvested after multiplecycles of growth showed that intracellular viruswas activated by pancreatin to the same degree asthe extracellular virus obtained from a single cycleof growth. However, the extracellular virus ob-tained after multiple (but not single) cycles ofgrowth showed little or no activation by the en-zyme. This confirms the finding of Spendlove andSchaffer (6), who had shown that late fluid har-vests were not amenable to activation with en-zymes. However, by replacing the culture fluidswith fresh nutrient just before release of virus intothe medium, they obtained enzyme-sensitive virus.It seems to us that extracellular virus may be al-ready enhanced by the proteolytic enzymes pro-duced by the cells in the culture, and thus the ef-fects of exogenously added enzymes would not bedetectable. Production of proteolytic enzymes byMK cultures has been described by Baron andBarnett (1).

Although many of the observations reported inthis study cannot be explained at the present time,the fact that treatment of reovirus with pancreatinis followed by the enhancement of reovirus in-fectivity and the formation of large and reproduci-ble plaques within 3 days after the inoculationof monkey cells should prove useful for thoseworking with these agents.

ACKNOWLEDGMENTSThis study was supported by Public Health Service

grant Al 05382 from the National Institute of Allergyand Infectious Diseases.

The excellent technical assistance of Fred Morales,Joycelyn Powell, Ira Wimberly, and Matilde Olive isgratefully acknowledged.

LITERATURE CITED1. BARNETT, E. V., AND S. BARON. 1959. An activator

of plasminogen produced by cell cultures. Proc.Soc. Exptl. Biol. Med. 101:308-311.

2. LERNER, A. M., J. D. CHERRY, AND M. FINLAND.1963. Hemagglutination with reoviruses. Virol-ogy 19 :58-65.

3. MELNICK, J. L., S. E. STINEBAUGH, AND F. RAPP.1964. Incomplete simian papovavirus SV40.Formation of non-infectious viral antigen in thepresence of fluorouracil. J. Exptl. Med. 119:313-326.

4. MELNICK, J. L., H. A. WENNER, AND L. ROSEN.1964. Enteroviruses, p. 194-242. In E. H.Lennette [ed.], Diagnostic procedures for viraland rickettsial diseases, 3rd ed. AmericanPublic Health Association, Inc., New York.

5. RHIM, J. S., L. E. JORDAN, AND H. D. MAYOR.1962. Cytochemical, fluorescent-antibody andelectron microscopic studies on the growthof reovirus (ECHO 10) in tissue culture. Virology17:342-355.

6. SPENDLOVE, R. S., AND F. L. SCHAFFER. 1965.Enzymatic enhancement of infectivity of reo-virus. J. Bacteriol. 89:597-602.

7. TOURNIER, P., AND M. PLISSIER. 1960. Le developpe-ment intracellulaire du REOvirus observeau microscope electronique. Presse Med. 68:683-688.

8. WALLIS, C., M. BIANCHI, AND J. L. MELNICK.1963. Factors influencing enterovirus and reo-virus growth and plaque formation. TexasRept. Biol. Med. 20:693-702.

9. WALLIS, C., K. 0. SMITH, AND J. L. MELNICK.1964. Reovirus activation by heating and inacti-vation by cooling in MgCl2 solutions. Virology22 :608-619.

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