INFECTION AND IMMUNITY, Sept. 1972, p. 348-354Copyright @ 1972 American Society for Microbiology

Vol. 6, No. 3Prinited in U.S.A.

Identification of a Canine Adenovirus (InfectiousCanine Hepatitis Virus) Inhibitor in Dog Liver

Extracts as ArginaseL. E. CARMICHAEL

Veteriniary Virul.s Research Institatte, New York State Veteriniary College, Cornlell University,Ithaca, New York 14850

Received for publication 15 May 1972

Extracts of canine liver inhibited growth of infectious canine hepatitis (ICH)virus, a canine adenovirus. Purified extracts from mammalian, but not avian, livertissue contained the inhibitor, and evidence is presented that the inhibitory factor isthe enzyme arginase (arginine ureohydrolase). This study further emphasized theneed for arginine in adenovirus growth and may explain some of the difficulties inisolating small amounts of ICH virus from suspensions of liver.

During studies on pathogenesis of infectiouscanine hepatitis (ICH) virus in this laboratoryover a period of several years, two unexplainedobservations have been made. (i) The degree ofillness and mortality rates of dogs inoculated withsuspensions of infective liver generally were lowerand variability in clinical response appearedgreater than those of susceptible dogs inoculatedby the same routes with comparable doses of viru-lent ICH virus propagated in dog kidney cell(DKC) cultures. (ii) When titrations of liver tissuesuspensions (usually 10 to 20%-o) taken from dogsat various stages of illness were performed, cyto-pathic effects (CPE) in DKC cultures generallyoccurred earlier, and were more pronounced, atdilutions greater than 1:10, especially whenamounts of virus in this organ were low. Theseobservations were given little attention until re-cently, when Lee (13) considered possible reasonsfor failures to isolate ICH virus from liver tissuesamples obtained from guinea pigs after paren-teral inoculation with large viral doses. This ani-mal has been reported to be susceptible to thisvirus (24). The possibility of a viral inhibitor inguinea pig tissues then was considered, and asearch for inhibitory activity in triturated guineapig liver revealed a heat-labile proteinaceous sub-stance that reduced ICH viral CPE in DKC cul-tures greater than 1,000-fold (13). Viral growthwas inhibited, but to a somewhat lesser extent.Inhibitory activity also was found in homogenatepreparations and in ammonium sulfate extracts ofnormal liver but not from other organs obtainedfrom various animals (dog, cat, rabbit, pig). Al-though the mode of action had not been deter-

mined, it was found that the viral inhibitory factordid not act extracellularly, for viral neutralizationand interference with absorption to DKC couldnot be demonstrated. The liver inhibitor wasfound active against a variety of other deoxyribo-nucleic acid (DNA) viruses, but it had very littleeffect on growth or CPE produced by selectedribonucleic acid viruses.

In further studies on the mode of viral inhibi-tion by liver extracts (LE) prepared from normaldogs, it occurred to us that arginase might be in-volved. This enzyme is found almost exclusively inthe liver of mammals, and its substrate, L-arginine,has been demonstrated as an essential amino acidfor replication of several DNA viruses, includingmembers of the adenovirus (8, 20), herpesvirus(3, 11), and papovavirus (10, 23) groups. A reo-virus (14) also has been reported to require argi-nine for synthesis of infectious virus. As early as1963, adenovirus replication was shown to havean absolute requirement for arginine; without itthere was no net viral synthesis (20). This impor-tant observation emphasized the necessity for uti-lizing cell cultures for adenovirus growth that arefree from arginine-utilizing microorganisms. Stud-ies on the mechanism of arginine dependence foradenovirus replication have been reported re-cently (8).

In this report, data are presented that indicatearginase as the principal canine adenovirus inhib-itor found in extracts from liver tissue.

MATERIALS AND METHODSCells. Tube cultures of DK cells were prepared ac-

cording to standard procedures. Inoculation mediumwas Earle saline solution that contained 5% heated

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ARGINASE AS CANINE ADENOVIRUS INHIBITOR

lamb serum, 0.5%`7O lactalbumin hydrolysate, penicillin,streptomycin, and amphotericin B.

Virus. Cornell-I strain ICH virus was used in all ex-periments. Stock virus used had been passaged 59 to61 times in primary DKC cultures. Repeated tests formycoplasmal contamination of virus stock were nega-tive.

Preparation of LE. Extracts from canine, bovine,chicken, and pheasant liver were prepared in threeways. (i) Crude extracts of pieces of liver that had beenwashed in several changes of cold isoosmotic KCI(0.9%) were homogenized as a 20 or 25%, suspensionin a blender (Omnimixer, Sorvall). Homogenates wereclarified by low-speed centrifugation (2,000 rev/min,International PR2 centrifuge) for 15 min, and thesupernatant fluids then were collected and centrifugedfor 30 min at 28,000 rev/min in the no. 30 rotor of amodel L ultracentrifuge (Beckman). Such extractswere termed crude LE. (ii) Crude LE was further frac-tionated by treatment with saturated ammonium sul-fate solution to attain final saturation of 65%. Theprecipitate material that formed was stirred for 1 hrand, after centrifugation, was dissolved in approxi-mately 0.25% the original volume in distilled water.The dissolved precipitate then was dialyzed against0.15 M phosphate-buffered saline (PBS), pH 7.2, or0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydro-chloride buffer (Sigma 121), pH 7.0, until free of am-monium ions as determined with Nessler's reagent.(iii) Canine liver homogenate preparations (50%70) wereextracted repeatedly with cold acetone and an acetone-dried liver powder was made. This material was frac-tionated in six steps exactly as described by Bach et al.(2) for purification of arginase to the precrystallizationstage (fraction VI). Tests for viral inhibitory activityin concert with tests for arginase activity, noted below,were carried out at each stage of purification. FractionVI was used for characterization studies.

Virus titrations. Titrations for viral infectivity weredone in tube cultures by standard procedures, employ-ing 10-fold virus dilutions and inoculums of 0.1 ml pertube. Two or three tubes were inoculated per dilution.Inoculated cultures were observed daily, and endpoints were expressed as median tissue culture infectivedose (TCID5o) per 0.1 ml inoculum. End point calcu-lations were done by the Karber method.

Analytical methods. Arginase was assayed accordingto the method described in the Worthington EnzymeManual (Worthington Biochemical Corp., Freehold,N.J., 1972), using as standard enzyme bovine liver ar-ginase (arginine ureohydrolase, Worthington) thatcontained approximately 20 units/mg. Prior to assay,LE fractions were dialyzed against 0.1 M Tris-hydro-chloride buffer, pH 7.0, to avoid precipitate formationwith the manganese maleate buffer employed in thereaction system. Protein determination was by the bi-uret method, or by measurement with the Folin phenolreagent (15). Acrylamide gel electrophoresis was doneby the method described by Davis and Ornstein (6),using 10-cm columns of 7.5% polyacrylamide gel(Eastman reagents) and 0.2-ml amounts of test samplematerial containing approximately 0.1 mg of proteindissolved in 20% sucrose. Electrophoresis was carriedout at 5 mA per gel column until the tracking dye (bro-

mophenol blue) had reached the end of the column,usually after 2.5 or 3 hr. Gel columns were removedfrom the glass tubing and stained with 0.05%Coomassie brilliant blue (R250) in 10%c trichloroaceticacid for 3 hr, followed by repeated washes with 7%acetic acid until clarified. In some instances, slices ofunstained gel that corresponded to replicate samplesthat had been fixed and stained were eluted into 0.1 NiTris-hydrochloride buffer, pH 7.0, and tested for argi-nase and ICH virus inhibitory activities. Sedimenta-tion behavior of LE fraction VI was examined by thesucrose gradient centrifugation technique described byMartin and Ames (16). By this method, utilizing twocharacterized enzymes as markers (alkaline phospha-tase and beef liver catalase), the sedimentation coeffi-cients of these enzymes could be compared. Their mo-lecular weights then could be roughly estimated.Linear sucrose (5 to 20%) gradients in PBS were pre-pared in 5-ml Lusteroid tubes and stored at 4 C for 4to 5 hr before use. Samples (0.2 ml per gradient) of anenzyme mixture in PBS were layered on each gradient.Each mixture contained (per 0.2 ml) 0.1 mg of alkalinephosphatase (Worthington), 0.4 mg of dog liver frac-tion VI (arginase activity), and 0.1 mg of beef livercatalase (Sigma, St. Louis, Mo.). The SW39 rotor(Beckman, Palo Alto, Calif) was run at 37,500rev/min for 11 hr at approximately 5 C. At the end ofthe runs, 10-drop fractions were collected, using a de-vice similar to that described by Martin and Ames(16). Fractions were made up to 0.5-ml volumes with0.01 M Tris-hydrochloride buffer, pH 7.0, and storedat -20 C until analyzed for enzyme activities and ICHviral inhibition.

RESULTSViral inhibition by LE preparations. Results of

tests for viral inhibition by crude LE from variousspecies are presented in Table 1. Both crude LEand ammonium sulfate precipitated (ASP) frac-tions from canine and bovine tissues inhibitedICH virus. However, similar materials preparedfrom livers of a pheasant and a chicken failed toaffect ICH viral growth in DKC cultures. Viralgrowth studies utilizing LE fraction IV obtainedduring the purification of arginase from dog liverindicated that viral replication indeed was inhib-ited and that the effect of LE preparations was notmerely inhibition of viral CPE. A typical growthexperiment where the initial ICH virus inoculumwas 103.2 TCID50 and 0.1 ml of a 1:10 dilutionof LE was added per culture is shown in Fig. 1.The LE fraction used (fraction IV, 2.5 mg of pro-tein/ml) had been shown in preliminary tests toinhibit growth of 500 TCID50 ICH virus at a dilu-tion of 1:128 and arginase activity (,umoles ofurea per mg of protein per 30 min) was 46,700.

Relationship between LE-inhibitor and arginase.In Table 2, ICH viral inhibitory titers are shownin relation to arginase activity. The viral inhibi-tory activity was not related to the initial proteinconcentration of LE preparations, but it increased

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CARMICHAEL

TABLE 1. Inihibition of infectious virus hepatitis (ICH) viruis by liver extract (LE) preparationis

Donor animal Age

Dog 12 wk

Dog

Dog

Calf

Pheasant

Chicken

1 hr

1 day

5 mo

poult

4 mo

LE preparation

20%o crudeASPb

ASP

ASP

20%0 crudeASP

25% crudeASP

25% crudeASP

Protein (mg/ml)

26024

13

20

30018

470128

700300

ICH virus titerLogio virus inhibition

Control

6.56.8

5.8

5.8

6.06.0

5.85.8

6.26.2

+LEa

1.5 -5.02.5 -4.3

1.8

2.0

1.01.8

5.86.1

6.06.2

-4.0

-3.8

-5.0-4.2

0

+0.3

-0.20

a LE added in 0.1-ml amounts to dog kidney cell cultures 2 hr before virus titrations were done.I ASP means 65%-saturated ammonium sulfate precipitate material, dialyzed against phosphate-

buffered saline, pH 7.2 to 7.4, until free of ammonium.

61 <~~~~~~~<53RSNLY

_

2-- VVIPUS LE (rnctc' E

20 30 4r 5C 6C 7C

TIME rs.

FIG. 1. Effect of caninte LE (fractiont IV) ont growthofICH virus in DK cell cultures. LE was added to DKcultures 2 hr before virus inioculum.

markedly with increased arginase specific activity.Those fractions that contained the enzyme alwaysinhibited viral growth. In contrast, those fractionslacking arginase activity failed to inhibit ICH viralmultiplication. Of significance was the finding thatliver fractions from fowl (pheasant, chicken) nei-ther inhibited ICH virus, nor did they have detect-ible arginase activity. It is known that avianspecies (uric acid excretors) have negligible argi-nase in their tissues; what little arginase occurs ispresent in the kidney (7).

Further evidence for arginase as the ICH viralinhibitor was obtained with ASP fractions, as wellas with the more highly purified dog LE (fraction

TABLE 2. Infectious canliine hepatitis viral-inihibitoryand arginase activities of various liver extract

(LE) preparations

Protein ViralbPreparation (m )inhibitory Arginasebmgm titera

Crude LE (dog) 260 1:16 630ASPC (dog) 24 1:40 580ASP (neonatal dog) 13 1:16 420LE (fraction VI, 0.7 1:400 73,500dog)

Crude LE (chicken) 700 <1:2 0ASP (chicken) 300 < 1:2 0Crude LE (pheas- 470 <1:2 0

ant)ASP (pheasant) 128 <1:2 0

a Titer indicates dilution of LE (0.1-ml inocu-lum) that caused 90% inhibition of 300 to 500median tissue culture infective dose of infectiouscanine hepatitis virus.

b Arginase activity expressed as ,1moles of urealiberated per mg of protein per 30 min.

c ASP means precipitate fraction after am-monium sulfate (65% saturation) treatment ofcrude LE. Precipitate was dialyzed against phos-phate-buffered saline until free of ammonium.

VI) subjected to rate-zonal sucrose (5 to 20%)density-gradient centrifugation. Figure 2 com-pares arginase activity and presence or absence ofICH viral inhibitor of 1-ml fractions collectedafter centrifugation of 1.0 mg of protein (fractionVI) for 34 hr at 24,000 rev/min in the SW25 rotor.

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ARGINASE AS CANINE ADENOVIRUS INHIBITOR

Again, viial inhibitory material was found only inthose fractions that had arginase activity.

Effect of arginase on ICH viral growth. From aninitial dilution of 100 ,ug/ml, beef liver arginasewas diluted in twofold steps in cell culture mediumand added (2-ml samples) to a series ofDKC cul-tures. After 2 hr at 36 C, virus titrations were donein arginase-treated and in untreated DKC cul-tures. Even under these suboptimal conditions forarginase action, viral inhibitory activity was found(Table 3). For optimal arginase activity, manga-nese and pH 9.2 is required. In this study, no at-tempt was made to employ arginine-depletedmedia, or media supplemented with ingredientsthat enhance the activity of this enzyme. The op-timal pH for arginase is unsatisfactory for main-tenance of cell cultures.

Reversibility of viral inhibition by arginine.Three sets of DKC cultures were treated as fol-lows. Set I (virus control) was inoculated with

ICHVInhibition

ill. Canine LE Fraction S

651/e Ammonium Sulfate ppt.

i

I.4:: '''------$-d2 6 10 14 18 22 26 30

Fraction No. ( 1 ml. samples )

FIG. 2. Arginase and ICH viral inhibitory activitiesof canine liver extract preparations after sucrose gra-dient (5 to 20°%1) centrifugation.

104 5 TCID50 ICH virus. Set II (inhibitor control)was treated with 0.1 ml of a 1:10 dilution of dogLE (fraction VI) plus virus. Set III was the sameas Set II, but each culture was supplemented with10 mg of arginine-HCl adjusted with 0.1 N NaOHto pH 7.0. At intervals following virus inocula-tion, duplicate cultures were frozen and thawedthree times and then titrated for viral infectivity.Results (Fig. 3) indicate the reversibility of theLE viral inhibitor by arginine.

Characterization of the canine liver ICH viralinhibitor. The viral inhibitor was precipitated by60 to 65%, but not by 50% saturated ammoniumsulfate. Full inhibitory activity also was retainedby precipitation with 37.5% cold acetone. It with-stood heating for 5 min at 56 C, but was inacti-vated by boiling for 1 min or by heating for 5 minat 70 C. Polyacrylamide gel electrophoresis offraction VI revealed a principal, heavy band thatcorresponded to a similar one obtained when ap-proximately the same quantity of beef liver argi-nase was subjected to electrophoresis (Fig. 4). Inboth preparations there were two additional veryfaint bands that did not correspond. Although itwas possible to elute a portion of the original ar-ginase activity from corresponding areas of a pairof unstained gel columns, viral inhibitory activitycould not be recovered in detectible amounts.By determining the relative sedimentation rates

of dog LE (fraction VI) ultracentrifuged togetherwith beef liver catalase (molecular weight 225,000)and calf intestine alkaline phosphatase (molecularweight approximately 100,000), the S20,w could beroughly estimated. Replicate trials gave similarresults (Fig. 5), indicating, by this method, thatthe molecular weight of the arginase viral inhibi-tor was approximately 120,000 with an S20,W valueof 5.3.

TABLE 3. Effect of arginiasea oni inifectious caninehepatitis virus titers

Arginase effect

Arginase (pg) Expt. 1 Expt. 2per DKC

culturebTiter Titer

Virus titer change Virus titer change(logio) (logiao)

None 5.5 6.512.5 5.5 0 6.5 025 4.1 -1.4 5.5 - 1.050 4.1 -1.4 4.5 -2.0100 3.5 -2.0 4.5 -2.0

a Beef liver arginase (Worthington, Freehold,N.J.). Activity approximately 20 units/mg ofprotein.

I DKC, Dog kidney cell.

5t.

C 4 .

3 r

2

'-7! ''/~~

7*Virus onlyx

Virus . LE ( Fraction VI)Arginine HCI

vP,I.. -rVirus(Fraction VI )

C,,,."

10 20 30 40 50

TIME: Chrs'

FIG. 3. Reversion ofcanine LE (fraction VI) inlhibi-tion ofICH virus by arginine hydrochloride.

zC\lo

.90

0 JU .5

.3n

i .16

.4-

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CARMICHAEL INFECT. IMMUNITY

ICH VIRALInhibition

Iz Beef Liver Catalase

(m wt. 225,000)

II - Dog Liver Fraction X

m =Alkaline Fhosphatosem wt 100,000)

FIG. 4. Polyacrylamide gel electrophoresis patternsof 0.1-mg samples of: (1) partially purified canine LE,fraction IV; (2) purified canine LE, fractioni VI; (3)beef liver arginase (Worthinigton). Electrophoresis fromtop (cathode) to bottom (anode) in 7.5% gel. Photo-graph is print of Coomassie brillianit blue-stainied gelreproduced from color tranisparency.

DISCUSSIONA great variety of substances and conditions

have been described that are capable of interferingwith viral growth in laboratory hosts. Of the vari-ous nonspecific (nonimmunoglobulin) inhibitorsreported active against viruses, excluding inter-feron, sulfated polysaccharides (heparin, sub-stances present in commercial agar) have beenmost extensively studied (22). This class of com-pounds acts extracellularly, preventing absorptionof virus to cells. Similarly, nonspecific, heat-stableinhibitors of various adenoviruses, including ICHvirus, have been found in normal bovine, horse,rabbit, and swine serums. These inhibitors werefound in the globulin fraction of serum, but werenot specific antibody, although they formed non-dissociable complexes with various adenoviruses(5, 9). In the present study, there was no evidencethat virus was inactivated extracellularly. The pro-

<, 5) 1V 15 20 25 30 35 40

Fraction No. (10 drops)

45 403 345 238 23 173 115 81 0

Distance (mm) From Meniscus

FIG. 5. Sedimentation patterns of beef liver catalase(I), dog liver fractioni VI (II), and calf intestine alkalinephosphatase (III). Enzyme activities are relative. Alka-line phosphatase anid arginase (LE fraction VI) activi-ties were determined by methods described in theWorthingtonz Enzyme Manual (1972); calalase was as-

sayed by microtitration of twofold dilutions of eachfraction. Addition of I drop of5% H202 to each dilu-tionI cup (Disposo-tray, Linibro, New Haven, Conti.)caused vigorous bubbling wheni catalase was presenit.

teinaceous inhibitory substance from liver re-ported active against certain DNA viruses, includ-ing ICH and canine herpesvirus, was found inextracts from liver of various normal mammalsand acted intracellularly where it was postulatedthat it interfered with viral synthesis in an un-known manner (13).Data introduced in the present study extend the

observations of Lee (13) and indicate thatthe principal inhibitor of ICH viral growth foundin liver extracts is arginase. Although definitivestudies of the nutritive requirements for adeno-virus synthesis are not available, certain aminoacids such as arginine (8, 23) and methionine (18)have been shown essential for viral growth.Adenoviruses contain an internal nucleoproteincomponent which is approximately 23 moles per-cent arginine (12, 21), and it has been suggestedthat this amino acid may be an important factorin limiting synthesis of members of this viralgroup, as well as others. Recent studies by Everittet al. (8) have confirmed the necessity of argininefor adenovirus type 2 synthesis. They showed thatboth cell and viral synthesis was severely impairedby complete arginine deprivation, which was re-versible by addition of arginine. It was hypothe-sized that arginine deficiency in adenovirus in-fection interfered with synthesis of host- orvirus-derived proteins made early in the infectiouscycle. In the studies reported here, the mechanismof inhibition of ICH viral formation by liver ex-

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ARGINASE AS CANINE ADENOVIRUS INHIBITOR

tracts was not investigated, for experiments werewere done under conditions considered adequatefor cell growth, but not optimal for arginase ac-tivity. Nevertheless, there was strong correlationbetween the activity of this enzyme and the ICHviral inhibitor. Of significance was the absenceof both inhibitor and arginase activities fromextracts of liver from avian species. Fowl livercontains less than 0.01 % arginase activity thanthat of mouse liver (7). In most mammals, thisenzyme is found essentially in liver tissue only,with very small amounts (less than 0.5%,,) inthe kidney and testis (7). Few other enzymesthat may be of significance as regards participa-tion in inhibiting viral synthesis are distributedin this manner. Lee (13) did not detect inhibitoryactivity in extracts of various tissues, other thanthe liver. The methods employed for purificationof the LE inhibitor, i.e., ASP and acetone ex-traction, probably would eliminate nonspecificlipid inhibitors, such as that isolated from brainby Burnstein et al. (4) and found active againstmeasles virus. However, we did not test forpresence of lipids in LE preparations.Ackenhusen (1) reported on an inhibitor of

viral and cellular DNA synthesis obtained froman extract of hamster liver. The inhibitor, whichinterfered with synthesis of Celo virus (an avianadenovirus), had an estimated molecular weightof 31,000. It appears that the hamster LE in-hibitor differs from the one reported in thisstudy, since the molecular weight of the inhibitorin canine liver was approximately 120,000. Thismolecular weight estimation could vary as muchas 25%, however, for variances of 10 to 25%may occur, depending on the enzyme standardsused (16). In any case, the dog LE that had viralinhibitory and arginase activities had a molecularweight greater than 100,000 and less than 150,000.The molecular weight of beef liver arginase hasbeen estimated as 138,000 and the enzyme existsin multimolecular forms (7).

It would be of interest to investigate arginaseactivity of extracts prepared from cell culturesderived from neoplastic tissues, for an inhibitoryproteinaceous substance active against herpessimplex virus (an arginine-requiring virus) re-cently was found in extracts of Burkitt'slymphoma cells (19). We have found that thecanine LE was equally effective in interferingwith growth of canine herpesvirus (13, unpub-lished results).A biological role for the viral inhibitor in

liver is undetermined. However, presence ofreleased arginase may account for difficultiesin isolating ICH virus from triturated samplesof pathological liver material, especially if viraltiters of this organ were low and inoculums con-

sisted of 20%o liver suspensions. This stud alsoyfurther emphasizes the requirement for argininein ICH viral growth. Methionine, phenylalanine,tryptophane, threonine, leucine, valine, andhistidine all have been reported as required forherpes simplex virus growth in L cells (17).Failures to find ICH or canine herpes viral in-hibitory activity in fractions of crude LE ob-tained after rate zonal centrifugation in 5 to20%7, sucrose gradients, other than in those witharginase activity, suggests that the inhibitionwas not a complex mixture of enzymes activeagainst various other amino acids.

ACKNOWLEDGMENTS

This study was supported in part by the John M. Olin Foun-dation and The Whitehall Foundation.

I thank Beverly S. Medic and Walter Pronsky for excellenttechnical assistance.

LITERATURE CITED

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2. Bach, S. J., R. A. Hawkins, and D. Swaine. 1963. A shortmethod for the purification cf arginase from ox liver. Bio-chem. J. 89:265-267.

3. Becker, Y., U. Olshevsky, and J. Levitt. 1967. The role ofarginine in the replication of herpes simplex virus. J. Gen.Virol. 1:471-478.

4. Burnstein, T., L. J. Swango, and D. P. Byington. 1971. Non-specific btain inhibitor against measles virus. Arch.Gesamte Virusforsch. 34:396-399.

5. Carmichael, L. E., G. F. Atkinson, and F. D. Barnes. 1963.Conditions influencing virus-neutralization tests for infec-tious canine hepatitis antibody. Cornell Vet. 53:369-388.

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7. Dixon, M., and E. C. Webb. 1964. Enzymes, p. 638-643.Academic Press Inc., New York.

8. Everitt, E., B. Sundquist, and L. Philipson. 1971. Mechanismof the arginine requirement for adenovirus synthesis I. Syn-thesis of structural proteins. J. Virol. 8:742-753.

9. Gold, E., and H. S. Ginsberg. 1962. An inhibitor of adeno-viruses in ox serum. J. Immunol. 88:513-518.

10. Goldblum, N., Z. Ravid, and Y. Becker. 1968. Effect of with-drawal of arginine and other amino acids on the synthesisof tumour and viral antigens of SV40 virus. J. Gen. Virol.3:143-146.

11. Inglis, V. B. M. 1968. Requirement of arginine for the repli-cation of herpes virus. J. Gen. Virol. 3:9-17.

12. Laver, W. G. 1970. Isolation of an arginine-rich protein fromparticles of adenovirus type 2. Virology 41:488-500.

13. Lee, H. 1972. Inhibition of certain deoxyribonucleic acidviruses by liver extracts. Infect. Immunity, in press.

14. Loh, P. E, and H. K. Oie. 1969. Role of lysine in the replica-tion of reovirus. I. Synthesis of complete and empty virions.J. Virol. 4:890-895.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurements with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

16. Martin, R. C, and B. N. Ames. 1961. A method for deter-mining the sedimentation behavior of enzymes: applicationto protein mixtures. J. Bicl. Chem. 236:1372-1379.

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17. Pelmont, J., and H. R. Morgan. 1959. Facteurs nutritifs in-fluencant la croissance du virus de l'herpes dans la souchede cellules L de Earle. Ann. Inst. Pasteur (Paris). 96:448-453.

18. Pett, D. M., and H. S. Ginsberg. 1971. Methionine require-ment for multiplication of type 5 adenovirus. Bacteriol.Proc. 71:212.

19. Rabson, A. S., S. A. Tyrrell, and F. Y. Legallais. 1971. Aninhibitor of Herpesvirus hominis in extiacts of cultures ofBurkitt's lymphoma cells. Proc. Soc. Exp. Biol. Med. 137:264-267.

20. Rouse, H. C., V. H. Bonifas, and R. W. Schlesinger. 1963.Dependence of adenovirus replication on arginine and in-

INFECT. IMMUNITY

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22. Takemoto, K. K., and P. Fabisch. 1963. Influence of acidpolysaccharides on plaque fcrmation by influenza A2 andB viruses. Proc. Soc. Exp. Biol. Med. 114:811-814.

23. Winters, A. L., and R. A. Consigli. 1971. Effects of argininedeprivation on polyoma virus infection of mouse embryocultures. J. Gen. Virol. 10:118-194.

24. Wright, N. G. 1965. Experimental infectious canine hepatitis.I. The pathology of the disease in guinea-pigs. J. Comp.Pathol. 75:449-453.

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