detection of viruses by chemical and biological …...gardâ€”chemical and biological studies of...

Detection of Viruses by Chemical and Biological Means

SVENGARD

( Department of Virus Research, Karolinska Institutet Medicai School, Stockholm, Sweden )

The discovery of the polyoma virus ( 80 ) provides the strongest incentive yet for continuedserious efforts to find a human cancer virus. According to previous experience in the field of animal viruses, the polyoma virus should prove to beone member of a group of related viruses withdifferent species specificities, causing similar diseases. When attempts to isolate human viruseshave failed, we have reason to question the suitability of the materials and methods used. Thisattitude served as a basis for the present discussion of virus detection by biochemical and biological means. An attempt was made to envisagesituations in which a virus would not be readilydetected and to analyze the reasons for this,rather than summing up the various current technics for virus detection and their limitations. Forthis purpose the discussion was grouped underthree headings, corresponding to the knownstages in the developmental cycle of viruses:provirus, vegetative virus, and mature virus.1

I. PROVIRUSThe evidence of the existence of a provirus

stage is conclusive only for bacteriophages. Inthis case, however, prophage may be regarded asthe "natural" form of the virus and lysogeny as a

mechanism safeguarding the survival of bothhost and virus. For a complete review of the lysogeny problems reference is made to Lwoff(50 ). In this connection only some salient pointswill be summarized.

The fate of an infecting temperate phageseems to be determined at the moment of penetration or very soon thereafter. The factor deciding between the alternatives reduction to prophage or production of vegetative phage appearsto be the metabolic state of the host cell. Reduction does not automatically lead to establishmentof lysogeny; the prophage first has to become attached to and apparently interact with a specific

1 The term mature virus, referring only to properties ofthe virus particle itself, is preferred to infective virus withits implication of host-virus interaction.

intracellular site, to all appearances a specificchromosomal locus. By this interaction both therespective locus and the prophage seem to bestabilized.

The infection represented by the lysogenicstate seems, in most cases, to remain completelysilent, with no morphologic or metabolic characteristics to distinguish a lysogenic from an unin-fected cell. The two important features revealingthe true status of the lysogenic bacterium are ( a )"immunity," i.e., refractoriness to lysis by related

(incompatible) virulent phages, and (fo) susceptibility to spontaneous or sometimes also toartificial "induction."

The phenomenon called immunity suggeststhat a specific chromosomal locus is also involvedin infection with virulent phages. If a lysogenicbacterium is exposed to a related virulent phage,the latter is adsorbed and its nucleic acid injected. However, as long as the prophage blocksthe specific locus, no multiplication of virulentphage takes place, although its nucleic acid mayremain potentially active for considerable periods of time. If the prophage is activated by induction, synthesis of both the temperate and thevirulent phage is initiated.

In the situation as here described the geneticinformation contained in the prophage DNAseems not to find any demonstrable expression.This is not always the case, however, the mostnotable example being the toxogenic C. diph-teriae. The capacity of toxin production is invariably associated with lysogeny, and lysogeniza-tion by the temperate phage from a toxogenicbacterium invariably confers toxogenic capacityto a nontoxogenic variant ( 26 ). Thus, if the bacterial gene determining toxogenicity is not identical with the prophage DNA it must be linkedto it with an unusual firmness. It would seem amore plausible explanation that the same DNAmolecule which in a "resting" state controls the

synthesis of toxin, when activated is commanding synthesis of phage. A quantitative and qualitative analysis of the phage DNA might provide

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GARDâ€”Chemical and Biological Studies of Virimeli

an answer to this question. As another exampleof "phenotypic" prophage manifestation the mu-

coid growth of certain lysogenic enteric bacteriamay be mentioned.

Whether or not animal proviruses exist is amatter of discussion. The majority of the inap-parent, persistent infections with animal viruseshave been clearly shown to represent a carrierstate as principally distinct from virogeny. In atissue culture system a carrier state is establishedif the rate of growth of the cell population andthe rate of spread of infection are in balance.This can be artificially achieved with susceptiblecells and virulent vims by addition to the medium of neutralizing antibodies in the properconcentration, as first described by Ackermannand Kurtz (1). A similar equilibrium may existin a relatively resistant cell population even without antibodies in the medium ( 15, 60). In a typical carrier culture only a fraction of the total cellpopulation is infected at any given time. Persistence of infection depends upon extracellulartransfer of virus as shown by the facts that theculture can be "cured" by addition to the me

dium of antibody in high concentrations as wellas by cloning.

In some Rous virus-carrying tissue cultureseach individual cell may actually be infected( 83 ). However, since virus seems to be continuously released, the situation can hardly be analogous to lysogeny in bacteria. Most probably theintracellular virus is in a vegetative phasethroughout, although the rate of virus synthesisis sufficiently low not to interfere significantlywith the growth of the host cell. The same explanation seems to fit in the persistently infected,resistant cell line, described by Puck and Cie-ciura (60).

Persistent infections in the macro-organism areusually more difficult to analyze. Inapparent infections in higher animals, with one exception,seem to be associated with continuous production of antibodies, an indication that mature virus is released at least intermittently. The exception is LCM virus-carrying mice which, according to Traub (86), do not develop serologie immunity. As the infection in this case is initiatedin utero, Burnet (14) suggested that immunologie tolerance might be involved, which indeedseems to be the case (34).

A slow or intermittent release of mature virusis by no means inconsistent with the assumptionof virogeny. As a matter of fact the most characteristic property of a lysogenic culture is the continuous, spontaneous release of mature phage.Therefore, the demonstration in persistently in

fected tissue of small amounts of virus cannotserve to distinguish between carrier state andvirogeny. If, on the other hand, systematic attempts consistently fail to demonstrate the presence of mature virus during clinically silent periods, the assumption of a true virogenic stategains in probability. For such reasons the recurring herpes simplex and the swine influenza virus in the intermediate lungworm host (72) appear to be likely candidates, even if positive evidence is lacking.

The only definitely established provirus, theprophage, is of the DNA type and somehow associated with the genome of the host cell. A priori there is no obvious reason not to expect asimilar pattern of infection within other groupsof viruses as well, and then primarily among intranuclear DNA agents, i.e., a category to whichthe herpes virus belongs. No RNA viruses haveyet been found in a provirus stage, and the wholequestion must here be left wide open.

Detection of a provirus may be extremely difficult. The most promising approach should beattempts to induce formation of vegetative andmature virus, for instance, by application of ionizing irradiation or carcinogenic and mutagenicchemicals. Some prophages, however, have resisted all attempts at artificial induction. It is possible that systematic studies of such systems willreveal additional physical or chemical agentswith inducing capacity. Prolonged cultivation invitro to improve the chances of spontaneous induction should also be tried.

The only possibility to detect and identify aresting provirus would seem to be by chemicalmeans. In all probability provirus consists of nucleic acid only and can hardly be expected tocarry any immunological markers. Thus, the formidable task of the biochemist would be to singleout and identify as foreign a nucleic acid thatcan be expected to appear in the pool in a ratioof one molecule per cell. This will obviously notbe theoretically possible, unless the provirus hassome unique constituent.

Until recently nucleic acids were supposed tocontain no more than four bases: in DNA, ade-nine, guanine, cytosine, and thymine; in RNAthe same set, except that uracil replaces thymine.The structural specificity is presumably accounted for entirely by the sequential order ofthe base pairs or bases in the chain. Virus nucleicacids generally seem to have no particular chemical characteristics setting them apart from thoseof the host cell. However, in 1953 Wyatt andCohen (95 ) isolated from DNA of T-even phagesa previously unknown base, 5-hydroxymethyl-



730 Cancer Research Vol. 20, June, 1960

cytosine ( HMC ), so far not found anywhere elsein nature. These phages contain no cytosine,which is thus completely replaced by HMC. Asanother unique feature glucose enters into themolecule, probably in glucosidic linkage with themethyl group of HMC (76, 88). It might also bementioned that Tessman et al. (84, 85) recentlyreported an abnormally high irradiation sensitivity of the small phages S 13 and <j>X 174, whichwas interpreted as an indication of a single-stranded DNA instead of the double helix of theWatson-Crick model (89).

In recent years new bases, primarily methylated purines and pyrimidines, have been identified as minor constituents of nucleic acids (3, 18,48 ). The significance of these observations is stillobscure; sometimes the appearance of such basesor an increase in their concentration is observedunder abnormal metabolic conditions togetherwith deviations from the base ratios required bythe Watson-Crick theory. It would thus seemthat the chemical composition of nucleic acidsmay be less monotonous than hitherto assumed,which means that chemical analysis might become a practicable means of identification of individual nucleic acids.

II. VEGETATIVE VIRUSMethods devised for the detection of vegeta

tive virus will assume particular practical importance in cases of slowly growing viruses whererelatively small amounts of mature virus are released and extracellular virus is continually neutralized by specific antibodies or inactivated byother, nonspecific agents present in the tissues.This type of infection should be expected principally in species and tissues with a high degreeof resistance to the infectious agent, which, inturn, would mean that demonstration of smallamounts of mature virus by means of infectionexperiments would be almost impossible if noother more sensitive test systems were available.

A complete analysis of the vegetative phaseshould include both viral and nonviral substancesproduced in the course of the infection. To beconsidered are (a) virus nucleic acid or nucleo-protein, (b) one or more proteins forming integral parts of the mature virus particle, (c) specific byproducts of the virus synthesis, regularlyappearing as "soluble antigens," etc., but appar

ently not incorporated in the virus particle, and,finally (d) substances of a nonspecific nature.

A. NUCLEICAcrosSynthesis of virus DNA may take place in the

nucleus as seems to be true of papilloma, herpes,

adeno, and several insect viruses, or in the cytoplasm. So far only two groups of cytoplasmicDNA viruses are recognized, the pox viruses andthe capsular insect viruses. The members of thesegroups can be characterized as structurally complex viruses, probably more autonomous thanothers. Whether or not they should be classifiedas true viruses is debatable. As pointed out byBurnet (14) the replication pattern of the poxviruses resembles that of the psittacosis group.The fact that the fibroma-myxoma virus apparently belongs among the pox viruses indicatesthat cytoplasmic DNA viruses cannot be disregarded as possible tumor agents. However, thesize and morphological peculiarities to be expected from viruses of this category makes itseem unlikely that they should remain unnoticed and escape identification even if attemptsto demonstrate infectivity were unsuccessful. Forthis reason cytoplasmic DNA agents shall not bemade the subject of further deliberations in thepresent connection.

The intranuclear replication of animal DNAviruses shows some features resembling those observed in the synthesis of the T'-even phages:marginationâ€”"precipitation"â€”of the chromatin,appearance of a structureless "pool" of viralDNA, and subsequent "crystallization" with formation of spatially oriented virus "nuclei" (43,44, 57). To this writer's knowledge no attempts

have been made to clarify the processes leadingto the breakdown of the chromosomes of the hostcell. It would be of interest to know what chemical relations, if any, exist between such extremely radical alterations of the structure and the disturbances of the nuclear activity observed in malignant tumor cells.

Also, RNA virus replication seems to be possible both in the nucleus and in the cytoplasm. Intranuclear synthesis of fowl plague G-antigen(9) has been definitely established; electron micrographie evidence of formation of poliovirus"nuclei" in the nucleolar and perinucleolar region

has been presented (64). The most convincingevidence to date of cytoplasmic virus RNA synthesis is the recent report of crystalline structuresin the cytoplasm of cells infected with one insectvirus (94) and Coxsackie B 5 virus (56), respectively.

The problem of detection by chemical meansof vegetative virus nucleic acid is less formidablethan the task of finding a pro virus. At present,the success of such attempts will depend entirelyupon the presence of specific constituents, primarily purines and pyrimidines, possibly sugars.The base ratios determined on the pooled nucleic



GARDâ€”Chemical and Biological Studies of Viruses 731

acid extracted from a tissue represent, of course,averages. Supposedly such mixtures are made upof thousands of individual molecular species withquite possibly widely differing ratios. The virusparticles, on the other hand, contain only one orvery few molecules of nucleic acid, and the purified preparations, therefore, possess a chemicalhomogeneity entirely lacking in the first type ofpreparation. It is hardly justifiable to base conclusions concerning structural characteristics ona comparison of two such incongruous materials.For a further elucidation of this question methods will have to be developed by which a separation of individual nucleic acids in a mixtureis feasible. It does not seem entirely Utopian tohope that structural specificity might be made towork for such purposes. Even methods by whichthe degree of homogeneity could be estimatedwithout a complete separation of molecular species would be of great help.

It is usually assumed that the pure nucleic acidis devoid of antigenic activity, and immune seraprepared against whole virus are reported tohave no neutralizing effect on the infectivity ofphenol-extracted virus (30). However, in studies on poliovirus-infected cells with the fluorescent antibody technic Buckley (11) found a veryfaint but apparently specific fluorescence of thenucleolus and the perinucleolar region, i.e., thatpart of the nucleus which probably serves as thesite of virus RNA synthesis. The nucleus remained otherwise unstained, while the cytoplasmwas gradually filled with staining material. Thesignificance of this observation is somewhat obscure. Two interpretations are possible: the faintfluorescence of the nucleolus may indicate eitherthe presence of minute amounts of a specific protein or else that the RNA as such has a certainimmunologie specificity. A further study of thephenomenon appears justifiable.

The vegetative phase corresponds largely tothe "eclipse" in the cycle of infection, character

ized by a loss of infectivity which was reportednot to return until morphologically typical virusparticles reappeared during the maturationphase. In view of the now established fact thatthe pure nucleic acid of many viruses is by itselfinfective, the current explanation of the eclipsephenomenon is hardly satisfactory. Virus nucleicacid is being synthesized throughout the wholeperiod, and failure to demonstrate its presence ininfection experiments can apparently not be attributed solely to the disintegrated state of thevirus. As perhaps the most likely explanation ofthe failures it may be assumed that earlier methods used for extraction of nucleic acid from the

cells were unsuitable. In particular, it should beessential during preparation to protect the nucleic acid from tissue nucleases in order to prevent inactivation.

There is some evidence to indicate that theseassumptions might be correct. Wecker andSchÃ¤fer(91) isolated infectious EEE and WEERNA from infected cells by treatment with coldphenol, whereas extraction of purified virus withthe same technic yielded negative results.Wecker (90) later found that the virus proteinwas soluble in hot phenol and that treatment ofthe virus at temperatures of 40Â°-60Â°C. gave nu

cleic acids of an activity comparable to that previously found in RNA prepared from cells. Heconcluded that the cellular RNA most probablyrepresented vegetative virus. Phenol treatmentundoubtedly inactivates all tissue enzymes,which might account for the positive results.

There are thus good reasons to hope that detection of vegetative virus with biological methods will prove to have a more general applicability. The low yields of activity obtained with thephenol technic as it is now practiced are an obvious disadvantage. The reason may be partialinactivation in the process of extraction, or a lowavidity of the pure nucleic acid for the cell, orboth. However, improvements in the technics ofpreparation as well as infectivity testing canprobably be achieved. Eventually tests on purenucleic acid rather than mature virus may proveto offer great advantages. It was recently reported (52, 58 ) that poliovirus RNA could causeinfection in cells completely resistant to maturevirus. Infection was followed by one cycle of virus replication only. Apparently the mature virusreleased from the first infected cells was incapable of initiating a second cycle. Similar resultswere obtained with phage when the receptormechanism was sidetracked by simultaneous removal of the bacterial membrane (protoplastformation) and liberation of the phage DNAcore by osmotic shock (77). Even if self-propagating infections could not be established inthese experiments, the possibility by such meansto widen the host range of a virus must be considered as a valuable addition to the diagnosticarsenal.

B. VIRUSPROTEINSThe chemical properties of a number of plant

virus proteins have been studied in detail, including amino acid analyses and preliminary sequence determinations (46). In general the protein of the virus particle seems to be made up ofa number of identical subunits linked together




by relatively weak secondary bonds. Dissociationand reassociation of the subunits of rod-shapedplant viruses are easily achieved, whereas spherical viruses are more resistant. The number ofsubunits may vary considerably, from about 20to more than 2000 per virus particle, with molecular weights from 18,000 and upwards.

So far no exotic or unusual amino acids havebeen found in plant virus proteins. Although theamino acid composition is specific enough to distinguish between variants of the same virus, itdoes not show any peculiarities, setting virus proteins clearly apart from those of the host cell.Some enzyme-resistant proteins are supposed tohave a hemicyclic structure without free N-ter-minal groups (25 ).

The highly specialized proteins of phages shallbe mentioned only in passing. The anatomy ofthe bacterial cell calls for a particular functionaldifferentiation of the virus particle which canhardly be expected to be copied in any animalviruses.

In comparison, information on animal virusproteins is meager. Among the best studied arethe relatively complex myxoviruses. As shown byHoyle (37) the virus particle can be disruptedby treatment with ether with release of two distinct types of subunits : hemagglutinin and groupantigen. SchÃ¤ferand co-workers (9, 35, 66-68,92), studying fowl plague virus, describe thehemagglutinin as a spherical particle, 30 m/j. indiameter, and composed of protein and polysac-charide. It carries the hemagglutinating, en-zymic, and antigenic capacity (type specific Vantigen) of the intact virus. The group antigen(S-antigen, or G-antigen in SchÃ¤fer'sterminol

ogy) appears in the shape of 12-m^. particlesmade up of nucleoprotein. The hexagonal discsobtained by Valentine and Isaacs (87) by tryp-tic digestion of the virus represent presumablythe intact nucleoprotein subunit. In the virusparticle it is supposed to be arranged in one turnof a spiral; when liberated by enzyme treatmentthis structure collapses, forming the hexagonaldiscs; in the course of ether treatment it apparently breaks down into smaller fragments. Eachvirus particle is supposed to contain six hemagglutinin units in close packing, the nucleoproteinoccupying the central space between them andlipides filling out the rest. The lipides are presumably derived mainly from the host cell surface.

So far the chemical characteristics of animalvirus proteins have hardly been systematicallystudied. Ion exchange chromatography used forpurification purposes has revealed certain inter

esting adsorption-elution patterns (36), whichpresumably must be attributed to the proteincomponent. Present data do not permit any general conclusions and very little speculation beyond the fact that the technic itself looks promising.

The enzyme activity of certain virus proteins,notably those found in the myxovirus group,might appear to offer possibilities of detectionand identification of such proteins by purelychemical methods. However, even after manyyears of systematic studies the chemistry of influenza virus enzyme-substrate reactions is notsufficiently clarified to make a chemical identification feasible. The highly specific affinities tobe expected would also make a search for a substrate for an unknown virus an extremely haphazard undertaking. Less specific enzymes likethe ATPase reported to enter into some fowl leu-kosis viruses (54) cannot be distinguished fromnormal tissue components and would thus haveno diagnostic potentialities.

Therefore semibiologic methodsâ€”hemaggluti-nation-elution, hemadsorption, and immunologieanalysisâ€”are the most valuable of those at present available. The capacity of certain viruses orpurified proteins of agglutinating red cells isbased on the presence in the cell surface of a receptor substance of sufficient specificity. As anequivalent of this phenomenon the in vitro reaction between purified influenza virus and purified receptor substance leads to formation of aprecipitate (82). Poliovirus, with which no he-magglutination has been obtained, neverthelessseems to react specifically with a cellular lipo-protein (33), presumably lacking in erythro-cytes. This observation, further supported by theapparently specific capacity of susceptible cellsto adsorb virus (42), indicates that virus-receptor mechanisms may have to be expected in mostvirus-host cell systems. Detection by direct precipitation of virus and receptor substance requires a preparatory purification of both components which also have to be used in high concentrations. A possibility that might be worthtrying, when naturally agglutinable cells cannotbe found, is coating of native or tannic acid-treated red cells with receptor substance in anattempt to develop a more sensitive and less exacting test system. The receptors so far identified seem to belong in the class of surface lipo-glycoprotein antigens.

The simplest and by far most sensitive methodof detecting the protein component in the cell iswith the aid of specific antibodies, particularlyby means of Coons's fluorescent antibody technic.



GARDâ€”Chemicaland Biological Studies of Viruses 733

Applying specific antibodies against the G-anti-gen and the hemagglutinin, respectively, SchÃ¤ferand his group found that the former first appeared in the nucleus of cells infected with fowlplague virus, later probably migrating out intothe cytoplasm. The synthesis of the hemagglutinin seemed to be initiated later than the G-anti-gen, and it was found only in the cytoplasm. Thesynthesis of several other virus proteins has beenfollowed by the same technic (11, 49, 59, 93).Generally the amount of stainable material in thecells is remarkable. In the later stages of infection the entire cytoplasm appears fluorescent, anindication that much more specific protein is synthesized than is needed for incorporation in thecomparatively small amount of mature virus thatis eventually released ( representing a fraction ofabout 10~5 of the cell mass).

C. NONVIHAL,SPECIFICSUBSTANCESSome of the nonviral material produced in and

released from infected cells might simply be regarded as components of the virus particle, synthesized in excess of what is finally incorporatedinto the mature virus. These substances differ inno respect from the counterparts that can be extracted from the virus particles. The intracellularmyxovirus hemagglutinin and soluble antigensexemplify the principle.

Often, however, other nonviral substances appear, specific in the sense that they are foreign tothe host cell and are synthesized only in thecourse of a specific infection. A particularly interesting example is provided by the so-called incomplete influenza virus (51). The incompletevirus forms particles of a somewhat varying sizebut of the same order as that of the classical virus. They seem to contain normal hemagglutininand large amounts of lipides. According to Ada( 2 ) they may be free of nucleic acid or containreduced amounts of this constituent. In view ofthe fact that the mature particle is supposed tocontain one single RNA molecule, Ada's inter

pretation of the analytical data would imply thatonly a fraction of a molecule were incorporatedin the incomplete virus particle, which in turnwould indicate aberrations in the RNA synthesis.However, the results obtained by Ada could beequally well explained by the assumption thathis preparations represented mixtures of complete and incomplete virus, a likely explanationconsidering the technical difficulties associatedwith the preparative separation of the two. Incomplete virus has the capacity of interferingwith mature virus in a peculiar way. Thus, afterexposure of the cells to a mixture of mature and

a large proportion of incomplete virus at highmultiplicities, the amount of hemagglutinin released from the cells remains at normal levels butis accounted for mainly by incomplete virus. Theproportion of RNA-containing mature virus is reduced, sometimes to 10~6 of a normal yield. The

incomplete virus alone seems not to be able toincite any infection or induce the cells to production of hemagglutinin.

It is tempting to speculate on the mechanismunderlying these phenomena. The RNA contentof the virus particle corresponds, as already mentioned, to a single molecule of 2 X IO8 mol. wt.One molecule is supposed to carry sufficientcoded information for synthesis of one specificprotein only. If this is so, the myxovirus RNAcould provide the pattern for the synthesis of either the G-antigen protein or the hemagglutininbut not both. The information needed for production of one of the proteins would have to besupplied from some other source. Most likely, theprotein itself would serve this purpose. According to SchÃ¤ferthe complete G-antigen enters thecell, whereas the hemagglutinin is supposed tobe largely retained on the cell surface. However,it would be difficult to explain the interferencephenomenon without assuming penetration intothe cell of the interfering agent. It seems reasonable, therefore, to assume that the hemagglutininis capable of entering and that this is indeed asine qua non for the synthesis of virus.

According to this line of thought, then, at leastone of the six hemagglutinin subunits of the virusparticle would have to enter the cell togetherwith the nucleoprotein. The RNA of the latter ispresumably self-replicating, provides the information necessary for synthesis of the G-antigenprotein, and somehow activates the cell in such away that the hemagglutinin can serve as a primerfor the synthesis of the identical enzyme in thecytoplasm. If more than one hemagglutinin unitenters, presumably inducing an excessive hemagglutinin synthesis, the RNA synthesis would seemto be inhibited, and the end-product consistslargely of incomplete virus.

This phenomenon, infection ending in dissolution and destruction of the cell with release ofconsiderable amounts of a specific, noninfectivematerial (but none or only insignificant quantities of mature virus), resembles the productionof bactericines, lytic but noninfective substances,identical with or closely related to phage lysin(75). Both phenomena provide examples of situations in which the true nature of the processesas induced by a virus infection might be almostimpossible to assess.



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In the formation of incomplete virus the synthesis of the basic components of the virus maybe considered to proceed in a regular fashionâ€”unless Ada is right, in which case an aberrationof the nucleoprotein synthesis would have to bepostulated. The main abnormality, however, is inthe maturation process. To what extent this situation is replicated in other virus-host systems isat present not known. Obviously, the above reasoning is applicable only to more complex viruses.In the small viruses, containing a single protein,all information needed for the virus synthesis canbe carried by the nucleic acid. Nevertheless, material morphologically slightly resembling incomplete virus seems to appear regularly in polio-virus-infected cells. Schwerdt and Schaffer (70)reported the presence in purified virus preparations of a nucleic acid-free protein component.Immunologically, this component is distinct fromthe virus particles (47), in electron micrographsit looks like an empty sphere of approximately thesame size as the virus particle ( 69 ). The significance of these observations is obscure. The non-infective soluble antigen might represent a regular byproduct of the virus synthesis or the resultof an erratic synthesis.

Substances discussed in this section, being pro-teinaceous, are antigenic. For their detection inthe interior of the cell the fluorescent antibodytechnic should be particularly suitable. Cell extracts may preferably be analyzed with the aid ofany of the gel precipitation technics or immuno-electrophoresis.

D. NONSPECIFICSUBSTANCESThe phenomenon of interference has generally

been regarded as an expression of a competitionbetween viruses for certain key enzyme systemsin the cell. However, in a series of papers Isaacsand co-workers (12, 13, 38-40) have presentedevidence of an alternative mechanism of a presumably entirely different nature. Cells exposedto inactivated myxoviruses or to active virus oflow or moderate virulence were found to producea substance capable of conferring resistance toother cells against virulent strains of a variety ofrelated as well as unrelated viruses. This substance, presumably a protein, was called inter-feron.

Interferon does not interact with virus in vitro;it does not prevent adsorption of virus on thecell surface, but it does inhibit synthesis of(myxovirus) nucleoprotein. Resistance inducedby treatment of the cells with interferon requires12-24 hours to reach maximum levels. Induced

cells do not themselves release interferon, unlessthey are challenged with active virus. In the chickembryo chorioallantoic membrane infected withinfluenza virus, no interferon seems to be releasedas long as virus is produced at a high rate. Whenvirus synthesis has passed its peak, however, interferon appears in the allantoic fluid (39).

Isaacs has hazarded a guess that interferonmight represent an aberration of the normal virussynthesis at some intermediate stage and that thepresence of interferon in the cell would serve toredirect the synthesis to production of more interferon instead of the normal intermediate. Considering the proteinaceous nature of interferonthis could hardly explain the inhibition of nucleicacid synthesis that is apparently established. Asanother possibility it might be suggested thatinterferon has a place in a normal regulatorymechanism in the cell, serving the purpose ofcontrolling the nucleic acid synthesis. If so, interferon would be expected to show a certain degreeof species specificity, which indeed seems to bethe case (39).

Apparently interferon might become an important means of detection of infections withmoderate viruses in which histologie lesions areabsent and the amounts of virus or nonviral specific substances are below the threshold of de-monstrability. The chemistry of interferon is notyet known to such an extent as to permit a suggestion of chemical diagnostic methods. For thetime being only biological demonstration is possible, including determination of resistance ofinfected cells to challenge with selected indicatorviruses and induction of resistance with materialreleased from infected cells.

III. MATURE VIRUSThe mature virus would by definition possess

infectivity as its most characteristic marker. If,for various reasons to be discussed below, infection tests are unsuccessful, other methods of detection might be tried. In vitro demonstration bychemical or immunological methods wouldâ€”inmost casesâ€”require a concentration and partialpurification of the test material. Occasionally, asfound by Beard's group ( 20, 21, 55 ) with certain

strains of fowl leukosis virus, enormous amountsof the agent may appear free in the blood orother body fluids, from which it is easily obtainedsimply by centrifugation. More often virus willbe scarce and found only in tissues. Withoutmethods by which results of fractionation experiments can be evaluated, purification will be extremely difficult. Beyond a statement to the effect




that differential centrifugation probably has awider applicability than any other method tested,few general recommendations can be made.

Whatever the outcome of other attempts todemonstrate the existence of a virus, the crucialtest will always be infection experiments in volunteers, in test animals, or in tissue cultures. Provided a virus exists, infection tests may yet yieldnegative results for any one or more of the following reasons.

A. VIRUSCONCENTRATIONThe virus concentration in the test material

may be too low for detection by biological means.This again may be accounted for in several ways.

1. Only small quantities of virus may be released from the cells and at a low rate. In suchcases a concentration of the test material mightproduce positive results. It is, of course, important to select material of initially highest possiblevirus content.

2. The virus may be continuously inactivatedas it is released from the cell. A very labile virusmay simply undergo spontaneous inactivation;it may be sensitive to tissue enzymes; or it mayinteract with specific or nonspecific inhibitors,including antibodies. Accordingly, attention mustbe paid to the methods for preparation of inocula.Rapid preparation at low temperatures may beessential; the use of stabilizers may be tried, suchas cysteine, reducing the oxidation rate. It hasbeen shown that some viruses are inactivated byenzymes and that the enzyme sensitivity spectrum to a certain extent serves to characterizeindividual viruses (53). The effect of tissue enzymes on the yield of active virus seems not tohave been studied, however, and enzyme inhibitors have not been used in attempts to improveyields. Antibodies and inhibitors can be removedby perfusion, extensive washing, electrophoresis,etc., before the cells are disrupted and intracel-lular virus is set free.

B. VIRULENCEOFTHEVIRUSViruses of very low virulence may fail to pro

duce manifest infections. In principle, two different situations may be envisaged: the virus maybe regularly infective but the infection established remain inapparent; or the infection ratemay be low. The latter case will be discussed ina following section in connection with host resistance.

At least theoretically a state of virogeny mightaccount for an inapparent infection. As alreadymentioned, no unequivocal evidence of the exist

ence of this state in the field of animal virologyhas been presented. Since lack of evidence provesnothing, it would be unwise to disregard thepossibility, however. Detection of virogeny mightbe possible either by means of induction andidentification of subsequently appearing vegetative or mature virus, or it may express itself asa change in some particular property of the hostcell.

Artificial induction of prophage can beachieved with agents apparently affecting thechromosomal structures in a manner as yet unknown. According to Lwoff (50) induction ispossible only under certain conditions; the cellshave to be metabolically preconditioned. In somecases all attempts at artificial induction havefailed. What is known about provirus and its induction refers exclusively to DNA viruses, presumably attached to or incorporated in the chromosomes. It would be futile now to discuss thehypothetical case of an RNA provirus, its possiblepoint of attachment in the cell, and the mechanisms of its induction.

So far no successful attempts to induce cytologie manifestations in inapparently infected tissue cultures have been reported. The enhancedsusceptibility of irradiated cells reported by Puckand co-workers (61 ) obviously is a phenomenonof a different nature. Systematic studies of theeffects of conditioning and induction on suchcarrier cultures as those described by Puck andCieciura (60) and by Deinhardt et al. (15)would seem to be of great interest.

Even in host-prophage systems where attemptsat artificial induction have failed to produce results, spontaneous induction occurs. This wouldhave to be expected also of an animal provirus.Spontaneous induction is a comparatively rareevent even in inducible prophage systems. Evidence of an activation, therefore, can be expectedonly in a small fraction of a population of infected host cells. A recent report by Dmochowskiet al. ( 17 ) is of a certain interest in this connection. The authors describe virus-like structures inhuman leukemic tissues as well as in cells of suchorigin cultivated in vitro. Rounded particles of auniform size in a double-contoured membraneappeared either in cytoplasmic inclusions or ex-tracellularly. A characteristic feature was thesporadic occurrence of this phenomenon. In diseased tissues large areas showed no reaction atall of this type; in limited areas affected cellswere observed in perhaps one out of 30 sections.In tissue cultures 2-12 weeks of culturing and upto ten subcultures were needed to bring out the




phenomenon. This observation thus conformswith what could be expected from a virogeniccell population. Needless to say, many other interpretations are possible. In any event, thetechnic used was exactly what can be generallyrecommended for detection of a provirus, i.e.,prolonged cultivation of presumably infectedcells in vitro.

It was already pointed out that prophage atleast sometimes confers an easily demonstrablenew hereditary property to the host cell, e.g., tox-ogenic capacity. In cases of suspected virogeny itmay therefore be worth while to look for characteristics distinguishing supposedly infected fromnormal cells. Tumor biologists have of coursepaid much attention to the genetic constitutionof tumor cells and listed large numbers of characteristics, distinguishing them from normal tissuecells as well as one tumor from the other, thetendency being to regard each individual tumoras an equivalent of a separate biological species.Such an analysis includes all the innumerablegenes stemming from the parent cell from whichthe tumor took its origin, whereas in the presentconnection the interest should be centeredaround one single "gene" that would probably be

the same in a large proportion of the various tumors observed. In other words, we should lookfor common, not for distinguishing, characteristics of tumors. One such common feature of human cancers has indeed been reported. BjÃ¶rklund(7), using immune cytolysis, and Zilber (96),using active anaphylaxis as an aid in immunologie analysis of cancer cells, have found a common antigen in all tumors examined which seemsto be absent in normal tissues. According toBjÃ¶rklundet al. (8) the antigen is a phospholipo-protein localized to the cell surface.

Even with virus in the vegetative phase, continuously evolving into maturation and being released, an infection may remain inapparent, as inthe system described by Puck and Cieciura (60 ).In that case infected cells could be cloned andgive rise to infected populations. Whether or notan infected cell will be able to grow and divideprobably depends upon the rate at which virus isbeing synthesized. In Jacob's (41) elegant ex

periments on phage it was shown that virus synthesis had a priority over the synthesis of hostmaterial. In bacteria kept on a strict metabolicregimen only phage was synthesized, if the rateof assimilation was kept below a certain thresholdvalue. With assimilation in excess of what wasneeded for virus synthesis at maximum rates,metabolites and energy for synthesis of host material became available; a residual growth of the

host cell, at a rate proportionate to the rate ofexcess assimilation, was observed. Eagle (19)and others have also shown that certain metabolites may be indispensable for the production ofvirus. Together these observations suggest certain approaches to the study of inapparent infections in tissue cultures.

In a stable system the establishment of an in-apparent infection could be expected to causesome changes in growth and metabolism of thetissue. Deinhardt et al. (15) observed that theirNDV-carrying culture had a higher rate of respiration but a lower rate of growth than an un-infected culture of the same cell line. In thepresent writer's laboratory another system is cur

rently being studied where the only manifestation of a presumed virus infection is a consistentreduction in the metabolic rate of the tissue cultures.

As the "masked" state of infection probably is

a function of the ratio virus/host synthesis, itshould be possible to affect the manifestations ofinfection by selective nutritional regimentation.Thus, for unmasking, optimal conditions for virussynthesis should be established, while at thesame time the normal metabolic activity of thecell would have to be restricted. There are already many indications that conditions of nutrition of tissue cultures may have a decisive influence upon virus yields and incubation periods.Systematic studies of this problem on inappar-ently infected cultures should yield much valuable information.

C. HOSTRESISTANCEFailure to produce infection after transfer of

virus-containing material to a new host maysimply be a failure to infect, accounted for byresistance of the intended host organism or hostcell. In principle, host resistance may be eitherconstitutional or conditioned.

1. It is hardly to be doubted that resistanceand susceptibility to infection may be geneticallydetermined. This is true for the macro-organismas well as at the cellular level. As examples maybe mentioned that Gross's agent of mouse leuke

mia (31) goes in the inbred mouse lines C3Hand C57BL but not in DBA or the heterozygousSwiss, whereas the reverse is true of Friend's vi

rus ( 28 ). Hardly any two lines of HeLa cells areidentical with regard to susceptibility to viruses;Puck and Cieciura ( 60 ) described a method forselection of resistant lines. As illustrated by themouse leukemia viruses, resistance and susceptibility may be strictly specific and therefore largely unpredictable characters. For such reasons it




is important that a sufficient number of genetically distinct host organisms be used in tests forunknown viruses.

Resistance is seldom absolute; it can often beovercome by massive infection. Papilloma virusin domestic rabbits may serve as an illustration;one ID50 corresponds in this case to about 80million physical particles (5). Thus, in many situations the problem of detection can be reducedto that discussed on page 735 of this section: positive results might be obtained, if a sufficient virus concentration in the test material can be established.

As a particular type of constitutional resistancethe examples mentioned on page 731 should beremembered. In those systemsâ€”chick embryo orchick tissue culture cells/poliovirus RNA andbacterial protoplasts/osmotically shocked phageâ€”resistance depends upon the barriers againstpenetration of virus into the cell. If the processcan be shunted around these barriers, a manifestinfection might follow.

2. A naturally susceptible cell may be conditioned into resistance by interference with thereceptor mechanism. Thus, the lining of thechorioallantoic cavity of the chick embryo acquires a temporary resistance to influenza virusinfection after treatment with receptor destroyingenzyme or periodate ( 23, 81 ).

In most cases the mechanism of conditioning isless obvious. A possible effect of nutrional factorsupon the relative rates of synthesis of virus andhost material was discussed in a previous section.Similar mechanisms operating on the macro-organism level might be responsible for changesin resistance observed in connection with avitaminosis or amino acid deficiencies. In the presentconnection the effect of hormones is of specialinterest. Shwartzman (73, 74), studying polio-virus infection in the hamster, found that cortisone treatment enhanced the susceptibility of theanimals, whereas testosterone reduced it. In thiscase the hormones probably act on the brown fattissue. Cortisone stimulates the brown fat, thecells grow in size, and assimilation of lipides increases. Under these conditions virus multipliesin the tissue to high concentration levels. Testosterone has the opposite effect. The analogy to thewell known, less well understood conditioningeffect of hormonal stimulation in the development of the Bittner tumor in mice is apparent.

Another example of growth stimulation as aconditioning factor has been described by Friede-wald (27). Methylcholanthrene applied on therabbit epidermis acts as an irritant, producingproliferation and hyperkeratinization. A skin area

so treated may be found as much as 10,000 timesmore susceptible to papilloma virus infectionthan is normal skin.

In the bacteriophage field, lysogeny is one ofthe most common and most important causes ofresistance to infection. In metazoans immunityafter a previous exposure probably plays a greater role. However, inapparent infection can alsoproduce resistance through interference (cf. p.734). This possibility may be particularly important to keep in mind in a discussion of cancerand viruses. Apparently, some of the known tumor viruses are widely disseminated, almost ubiquitous; the animals seem to become infected verysoon after birth or maybe already in utero or inovo; they carry the infection in a clinically in-apparent form for considerable periods of time.If the etiology of human cancer is a virus (orviruses), the epidemiology of the disease canhardly be explained without assumption of asimilar near ubiquity of the causative agent.Therefore, inapparent infection might be expected to be of common occurrence, causingresistance of the macro-organism as well as ofcells in tissue culture.

In this connection a phenomenon, familiar tomost workers handling tissue cultures, assumesspecial significance. Trypsinized tissue usuallygrows rapidly during a period of about a weekafter explantation. Sooner or later growth slowsdown, and after three to five subcultures the cellpopulation remains stationary or decreases. Sometimes one or more islands of morphologicallychanged elements appear in the sheet of cells.This metamorphosis is usually associated withincreased growth capacity, and the cells now canbe established in permament culture. The morphology of such cells resembles that of established tumor cells; capacity of giving rise tomalignant tumors has been observed; and theimmunological character of such cells has beenfound indistinguishable from that of cancer cells.Obviously, such observations have to be evaluated with great caution. This is not the place foran exhaustive discussion of the various possibleinterpretations; it shall only be emphasized thatthe possibility of an induction of inapparent virusinfections should not be neglected. If that is theexplanation of the phenomenon, resistance because of inapparent infection might make detection of tumor viruses by means of tissue culturesdifficult, depending upon how widely virus is disseminated. Dmochowski et al. (17) reported thatthe agent found in human leukemic tissue produced suggestive changes in monkey but not inhuman tissue culture. Without access to more




detailed data it is difficult to evaluate this pieceof information; it may prove to be significant,however.

IV. SOME CHEMICAL AND BIOLOGICALCHARACTERISTICS OF KNOWN

TUMOR VIRUSESBy a rough classification four groups of tumor-

inducing viruses may be distinguished, producingpapillomas, leukosis, sarcomas, and canceroustumors, respectively. Each one of these groupscontains several members with different hostspecificities or immunological properties. It mustbe emphasized, however, that a classification atpresent can be only tentative, as the true interrelationships between the various tumor virusesare unknown.

Only two tumor viruses have been prepared ina sufficiently pure state to permit conclusionsconcerning their chemical characteristics. Rabbitpapilloma virus (10, 45, 65) contains DNA andprotein and is synthesized in the cell nucleus.One fowl leukosis virus (4 ) was found to containRNA; the site of replication is apparently in thecytoplasm. The fact that ATPase activity wasfound to be invariably associated with this virus(55) suggests that virus synthesis takes place inor in the immediate vicinity of mitochondria.

Most tumor viruses can be regarded as moderate. The incubation periods are often remarkably long, and manifestation of the infectionsometimes requires some conditioning stimulus.Rous sarcoma virus retains its moderate character in tissue cultures; it does modify the morphology of the host cell (83), but has so far notacquired cytocidal properties on subcultivation.Others, like avian lymphomatosis virus (24, 71)and polyoma virus (79), produce degenerativechanges in tissue cultures and may becomeadapted in the course of serial passages. Theviruses so far isolated have a narrow host rangein vivo. They may be less specific in tissue culture.

Polyoma virus is of special interest because ofits versatility. It propagates and produces symptoms in several breeds of mice and in hamsters.Large inocula produce degenerative lesions inmice, particularly in the kidneys; otherwise tumor formation is the main symptom, involvingmany different organs and tissues ( 78 ). The virusis antigenic, and both neutralizing and CF antibodies are produced (62, 63). It possesses he-magglutinating capacity (22). With fluorescentantibody, stainable material appears first in thenuclei of infected cells; later a migration to the

cytoplasm occurs, and the nuclei lose the capacityto fix antibody. At this stage the cells begin toshow signs of degeneration, and hemagglutininappears (32 ). The similarities with myxovirus infection are considerable. Early in the course ofinfection the cells develop resistance to super-infection with vesicular stomatitis virus, oftenused as an indicator virus in studies on interference.

Some tumor viruses appear as physical, apparently mature particles in the interior of the cellâ€”papilloma virus in the nucleus, the milk factor inthe cytoplasm ( 16 ). Avian leukosis virus is foundin cytoplasmic inclusions (6). No intracellularparticles are demonstrable in Rous virus-infectedcells; maturation in this case seems to take placeat the surface of the cell as in myxovirus infections (29).

It would appear from this brief summary thattumor viruses by no means form a uniform group.So far there are few indications of any characteristics setting them apart from other animal viruses. However, the behavior of Rous virus in tissueculture is somewhat unusual. The infection isapparent, the manifestation being a distinctchange in the morphology of the host cell. Virusseems to be continuously synthesized and released. Yet the viability of the host cell seems notto be impaired.

The strict host specificity of the mouse leukosisviruses is another unusual feature. Geneticallydetermined resistance to infection is usually notan all-or-none phenomenon; the term resistancehas to be applied in a relative sense. It is possiblethat future studies with improved technic willprove this to be true also of resistance to theagents causing leukemia. On the basis of presentevidence, however, the possibility of an "immunity" analogous to that found in lysogenic bac

teria should be seriously considered.

REFERENCES1. ACKERMANN,W. W., and KURTZ,H. Observations con

cerning a Persisting Infection of HeLa Cells withPoliomyelitis Virus. J. Exper. Med., 102:555-65,1955.

2. ADA, G. L. Ribonucleic Acid in Influenza Virus. In:C. E. W. WOLSTENHOLMEand E. C. P. MILLAR (eds.),The Nature of Viruses, pp. 104-15. Ciba FoundationSymposium. London: Churchill, 1957.

3. AMOS, H., and KORN, M. 5-Methyl Cytosine in theRNA of Escherichia coli. Biochem. et Biophys. acta29:444-45, 1958.

4. BEARD, J. W. Isolation and Identification of TumorViruses. Texas Rep. Biol. Med., 15:627-58, 1957.

5. . Bearing of Virus Mass on "Masking" and Laten

cy. In: D. L. WALKER,R. P. HANSON,and A. S. EVANS(eds.); Latency and Masking in Viral and RickettsialInfections, pp. 20-39. Minneapolis: Burgess, 1958.




6. . Viral Tumors in Cancer Research, in: M. POL- 24.LARD (ed.), Perspectives in Virology, pp. 197-230.New York: Wiley & Sons, 1959.

7. BJÃ–HKLUND,B., and BJÃ–RKLUND,V. Antigenicity ofPooled Human Malignant and Normal Tissues byCyto-immunological Technique: Presence of an In- 25.soluble, Heat-labile Tumor Antigen. Internat. Arch.Allergy appi. Immunol., 10:153-84, 1957.

8. BJÃ–RKLUND,B.; LUNDBLAD,G.; and BJÃ–RKLUND,V. 26.Antigenicity of Pooled Human Malignant and NormalTissues by Cyto-immunological Technique: II. Natureof Tumor Antigen. Internat. Arch. Allergy appi. Im- 27.munol., 12:241-61, 1958.

9. BREITENFELD,P. M., and SCHÃ„FER,W. The Formation of Fowl Plague Virus Antigens in Infected Cells,as Studied with Fluorescent Antibodies. Virology, 28.4:328-45, 1957.

10. BRYAN,W. R. and BEARD,J. W. Studies on Purification and Properties of Rabbit-Papilloma-Virus-Protein. 29.J. Nat. Cancer Inst., l :607-73, 1941.

11. BUCKLEY,S. M. Visualization of Poliomyelitis Virus byFluorescent Antibody. Arch. ges. Virusforsch., 6:388- 30.400, 1955.

12. BURKE,D. C., and ISAACS,A. Further Studies on Interferon. Brit. J. Exper. Path., 39:78-84, 1958. 31.

13. . Some Factors Affecting the Production of Interferon. Ibid., pp. 452-58.

14. BURNET, F. M. Principles of Animal Virology. NewYork: Academic Press, 1955. 32.

15. DEINHARDT,F.; HENLE, G.; BERGS,V. V.; and HENLE,W. Persistent Infection in Tissue Culture with Newcastle Disease and Mumps Viruses. In: D. WALKER,R. P. HANSON,and A. S. EVANS(eds.), Latency and 33.Masking in Viral and Rickettsial Infections, pp. 118-22. Minneapolis: Burgess, 1958.

16. DMOCHOWSKI,L., and GREY, C. E. Electron Micros- 34.copy of Tumors of Known and Suspected Viral Etiology. Texas Rep. Biol. & Med., 15:704-53, 1957.

17. DMOCHOWSKI,L.; GREY,C. E.; SYKES,J. A.; SHULLEN-BERCER,C. C.; and HOWE, C. D. Studies on humanleukemia. Proc. Soc. Exper. Biol. & Med., 101:686-90, 1959. 35.

18. DUNN, D. B., and SMITH, J. D. Occurrence of a NewBase in the Deoxyribonucleic Acid of a Strain of Bacterium coli. Nature, 175:336-37, 1955. 36.

19. EAGLE, H., and HABEL, K. The Nutritional Requirements for the Propagation of Poliomyelitis Virus by theHeLa Cell. J. Exper. Med., 104:271-87, 1956.

20. ECKERT,E. A.; SHARP, D. G.; BEARD,D.; and BEARD, 37.J. W. Variation in Infectivity and Virus-Particle Content of Individual Plasmas from Birds with Erythro-myeloblastic Leukosis. J. Nat. Cancer Inst., 1S':533- 38.

42, 1952.21. ECKERT, E. A.; SHARP, D. G.; MOMMAERTS,E. B-; 39.

REEVES,R. H.; BEARD,D.; and BEARD,J. W. Virus ofAvian Erythromyeloblastic Leukosis; Interrelations of 40.Plasma Particles, Infectivity, and Enzyme Dephos-phorylating Adenosine Triphosphate. J. Nat. CancerInst., 14:1039-53, 1954. 41.

22. EDDY,B. E.; ROWE,W. P.; HARTLEY,J. W.; STEWAHT,S. E., and HUEBNER,R. J. Hemagglutination with theSE Polyoma Virus. Virology, 6:290-91, 1958. 42.

23. FAZEKASDE ST. GROTH, S. Modification of Vims Receptors by Metaperiodate. I. The Properties of 104-treated Red-Cells. Austral. J. Exper. Biol., 27:65-81, 43.1949.

FONTES, A. K.; BURMESTER,B. R.; WALTER, W. G.;and ISELER,P. E. Growth in Tissue Culture of Cyto-pathogenic Agent from Strain of Virus Which Produces Avian Lymphomatosis. Proc. Soc. Exper. Biol. &Med., 97:854-57, 1958.FRAENKEL-CONHAT,H. and SINGER, B. The PeptideChains of Tobacco Mosaic Virus. J. Am. Chem. Soc.,76:180-83, 1954.FREEMAN,V. J. Studies on the Virulence of Bacterio-phage-infected Strains of Corynebacterium diph-theriae. J. Bact., 61:675-88, 1951.FRIEDEWALD,W. F. Cell State as Affecting Susceptibility to a Virus. Enhanced Effectiveness of the RabbitPapilloma Virus on Hyperplastic Epidermis. J. Exper.Med., 75:197-219, 1942.FRIEND,C. Cell-free Transmission in Adult Swiss Miceof a Disease Having the Character of a Leukemia.J. Exper. Med., 105:307-18, 1957.GAYLORD,W. H., JR. Virus-like Particles Associatedwith Rous Sarcoma as Seen in Sections of Tumor.Cancer Research, 15:80-83, 1955.GIERER, A., and SCHRAMM,G. Infectivity of Ribo-nucleic Acid from Tobacco Mosaic Virus. Nature,177:702-3, 1956.GROSS, L. "Spontaneous" Leukemia Developing in

C3H Mice Following inoculation, in Infancy, withAK-leukemic Extracts, or AK-embryos. Proc. Soc.Exper. Biol. & Med., 76:27-32, 1951.HENLE, G.; DEINHARDT,F.; and RODRIGUEZ,J. TheDevelopment of Polyoma Virus in Mouse EmbryoCells as Revealed by Fluorescent Antibody Staining.Virology, 8:388-91, 1959.HOLLAND,J. J.; MCLAREN,L. C.; and SYVERTON,J. T.A Structural Factor in Susceptibility of Primate Mammalian Cells to Poliovirus. Fed. Proc., 18:573, 1959.HOTCHIN,J. E. Some Aspects of Induced Latent Infection of Mice with the Virus of Lymphocytic Chorio-meningitis. in: D. WALKER,R. P. HANSON,and A. S.EVANS (eds.), Latency and Masking in Viral andRickettsial Infections, pp. 59-65. Minneapolis: Burgess, 1958.HOTZ, G., and SCHÃ„FER,W. Ultrahistologische StudieÃ¼berdie Vermehrung des Virus der klassischen GeflÃ¼gelpest.Ztschr. Naturforsch., 10b:l-5, 1955.HOYER, B. H.; BOLTON, E. T.; ORMSBEE, R. A.;LEBOUVIEH,G.; RITTER, D. B.; and LARSON, C. L.Mammalian Viruses and Rickettsiae. Science, 127:859-63, 1958.HOYLE, L. Structure of the Influenza Virus. The Relation between Biological Activity and Chemical Structure of Virus Fractions. J. Hyg., 50:229-45, 1952.ISAACS,A., and BURKE,D. C. Mode of Action of Interferon. Nature, 182:1073-74, 1958.

. Viral Interference and Interferon. Brit. M. Bull.,15:18.5-88, 1959.ISAACS,A., and LINDENMANN,J. Virus Interference.I. The Interferon. Proc. Roy. Soc. s.B, 147:258-67,1957.JACOB, F. Influence du rÃ©gimecarbone sur le dÃ©veloppement des bactÃ©riophages chez un pseudomonaspyocyanea. Ann. l'Inst. Pasteur, 82:578-602, 1952.

KAPLAN,A. S. Comparison of Susceptible and RÃ©sistant Cells to Infection with Poliomyelitis Virus. Ann.N.Y. Acad. Se., 61:830-38, 1955.KELLENBERGEH,E.; SÃ‰CHAUD,J.; and RYTER,A. Electron Microscopical Studies of Phage Multiplication.




IV. The Establishment of the DNA Pool of Vegetative 61.Phage and the Maturation of Phage Particles. Virology, 8:478-98, 1959. 62.

44. KJELLEN, L.; LAGERMALM, G.; SVEDMYR,A.; andTHORSSON,K. G. Crystalline-like Patterns in the Nucleiof Cells Infected with an Animal Virus. Nature, 175:505-6, 1955. 63.

45. KNIGHT, C. A. Amino Acids of the Shope PapillomaVirus. Proc. Soc. Exper. Biol. & Med., 75:843-46,1950.

46. . Chemistry of Some Virus Variants, in: E. BRODA 64.and W. FniscH-NiccEMEYER (eds.), Biochemistry ofViruses, pp. 80-84. London: Pergamon Press, 1959.

47. LEBOUVIER,G. L.; SCHWEHDT,C. E.; and SCHAFFEH,F. L. Specific Precipitates in Agar with Purified Polio- 65.virus. Virology, 4:590-93, 1957.

48. LrrTLEFiELD, J. W., and DUNN, D. B. The Occurrenceand Distribution of Thymine and Methylated Adenine 66.Bases in Ribonucleic Acids. Biochem. J., 68:8P, 1958.

49. Lru, C., and COFFIN, D. L. Studies on Canine Distemper Infection by Means of Fluorescein-labeledAntibody. 1. The Pathogenesis, Pathology and Diag- 67.nosis of the Disease in Experimentally Infected Ferrets. Virology, 3:115-31, 1957.

50. LWOFF, A. Lysogeny. Bact. Rev., 17:269-337, 1953.51. MAGNUS,P. VON. Propagation of the PR8 Strain of

Influenza A Virus in Chick Embryos. II. The Forma- 68.tion of Incomplete Virus following Inoculation ofLarge Doses of Seed Virus. Acta pathol. microbio!.Scandinav., 28:278-93, 1951.

52. MCLAREN,L. C.; HOLLAND,J. J.; and SYVEHTON,J. T. 69.Adsorption of Poliovirus to Cultivated Cells of Primateand Non-primate Origin. Fed. Proc., 18:585, 1959.

53. MERRILL,M. H. Effect of Purified Enzymes on Virusesand Gram-negative Bacteria. J. Exper. Med., 64:19- 70.28, 1936.

54. MOMMAERTS,E. B.; ECKERT,E. A.; BEARD,D.; SHARP,D. G.; and BEARD,J. W. Dephosphorylation of Ade- 71.nosine Triphosphate by Concentrates of the Virus ofAvian Erythromyeloblastic Leucosis. Proc. Soc. Exper.Biol. & Med., 79:450-55, 1952.

55. MOMMAERTS,E. B.; SHARP, D. G.; ECKERT, E. A.; 72.BEARD,D.; and BEARD,J. W. Virus of Avian Erythromyeloblastic Leukosis; Relation of Specific PlasmaParticles to Dephosphorylation of Adenosine Triphosphate. J. Nat. Cancer Inst., 14:1011-25, 1954. 73.

56. MORGAN,C.; HOWE, C.; and ROSE, H. M. Intracellu-lar Crystals of Coxsackie Virus Viewed in the ElectronMicroscope. Virology, 9:145-49, 1959.

57. MORGAN,C.; JONES, E. P.; HOLDEN, M.; and ROSE, 74.H. M. Intranuclear Crystals of Herpes Simplex VirusObserved with the Electron Microscope. Virology,5:568-71, 1958.

58. MOUNTAIN,J. M., and ALEXANDER,H. E. Study of 75.Infectivity of Ribonucleic Acid (RNA) from Type IPoliovirus in the Chick Embryo. Fed. Proc., 18:587,1959.

59. NOYES, W. F., and WATSON, B. K. Studies on the 76.Increase of Vaccine Virus in Cultured Human Cellsby Means of the Fluorescent Antibody Technique. 77.J. Exper. Med, 102:237-41, 1955.

60. PUCK,T. T., and CIECIURA,S. J. Studies on the Virus 78.Carrier State in Mammalian Cells. Jn: D. L. WALKER,R. P. HANSON,and A. S. EVANS (eds.), Latency andMasking in Viral and Rickettsial Infections, pp. 74-79.Minneapolis: Burgess, 1958. 79.

PUCK, T. T., and MARCUS,P. I. Action of X-Rays onMammalian Cells. J. Exper. Med., 103:653-66, 1956.ROWE, W. P.; HARTLEY, J. W.; BRODSKY,I.; andHUEBNER,R. J. Complement Fixation with a MouseTumor Virus (S.E. polyoma). Science, 128:1339-40,1958.ROWE, W. P.; HARTLEY, J. W.; ESTES, J. D.; andHUEBNER,R. J. Studies of Mouse Polyoma Virus Infection. I. Procedures for Quantitation and Detectionof Virus. J. Exper. Med., 109:379-91, 1959.RUSKA,H.; STUART,D. C., JR.; and WINSSER,J. Electron Microscopic Visualization of Intranuclear Virus-like Bodies in Epithelial Cells Infected with Poliomyelitis Virus. Arch. ges. Virusforsch., 6:379-87,1955.SCHACHMAN,H. K. Physical Chemical Studies onRabbit Papilloma Virus. J. Am. Chem. Soc., 73:4453-55, 1951.SCHÃ„FER,W., MUNK, K.; and MUSSGAY,M. Physikalisch-chemische und biologische Eigenschaften desf ereinigten "lÃ¶slichen Antigens" der klassischen Ge-

Ã¼gelpest.Ztschr. Naturforsch., llb:330-38, 1956.

SCHÃ„FER,W., and ZILLIG, W. Ãœberden Aufbau desVirus-Elementarteilchens der klassischen GeflÃ¼gelpest.Mitteilung I: Gewinnung, physikalisch-chemische undbiologische Eigenschaften einiger Spaltprodukte.Ztschr. Naturforsch., 9b:779-88, 1954.SCHÃ„FER,W.; ZILLIG, W.; and MUNK, K. Isolierungund Charakterisierung hÃ¤magglutinierender, nichtinfektiÃ¶ser Einheiten bei klassischer GeflÃ¼gelpest.Ztschr. Narurforsch., 9b:329-40, 1954.SCHWEHDT,C. E. The Properties of Purified Polio-viruses, in: E. BRODAand W. FRISCH-NIGGEMEYER(eds.), Biochemistry of Viruses, pp. 130-41. London:Pergamon Press, 1959.SCHWERDT,C. E., and SCHAFFER,F. L. Purificationof Poliomyelitis Viruses Propagated in Tissue Culture.Virology, 2:665-78, 1956.SHARPLESS,G. R.; DEFENDÃ•,V.; and Cox, H. R. Cultivation in Tissue Culture of the Virus of Avian Lym-phomatosis. Proc. Soc. Exper. Biol. & Med., 97:755-57,1958.SHOPE, R. E. The Swine Lungworm as a Reservoirand Intermediate Host for Swine Influenza Virus. II.The Transmission of Swine Influenza Virus by theSwine Lungworm. J. Exper. Med., 74:49-68, 1941.SHWARTZMAN,G. Enhancing Effect of Cortisone uponPoliomyelitis Infection (Strain MEF1) in Hamstersand Mice. Proc. Soc. Exper. Biol. & Med., 75:835-38,1950.SHWAHTZMAN,G.; ARONSON,M. A.; TEODORU,M. A.;and JAHIEL, R. Endocrinological Aspects of Pathogenesis of Experimental Poliomyelitis. Ann. N.Y. Acad.Sc., 61:869-76, 1955.SIMINOVITCH,L. Relation entre le dÃ©veloppementabortii du prophage chez Bacillus megatherium 91(1)et la synthÃ¨se de l'acide desoxyribonuclÃ©ique. Compt.

rend. Acad. Se., 233:1694-96, 1951.SINSHEIMER,R. L. Nucleotides from T2 r+ bacterio-phage. Science, 120:551-53, 1954.SPIZIZEN, J. Infection of Protoplast by Disrupted T2Virus. Proc. Nat. Acad. Sc., 43:694-701, 1957.STEWART,S. E., and EDDY,B. E. Properties of a Tumor-inducing Virus Recovered from Mouse Neoplasms. In: M. POLLARD(ed.), Perspectives in Virology, pp. 245-55. New York: Wiley & Sons, 1959.STEWART, S. E.; EDDY, B. E.; and BORCESE,N. G.




Neoplasms in Mice Inoculated with a Tumor AgentCarried in Tissue Culture. J. Nat. Cancer Inst., 20:1223-44, 1958.

80. STEWART, S. E.; EDDY, B. E.; GOCHENOUR,A. M.;BOHGESE,N. C.; and GHUBBS,G. E. The Induction ofNeoplasms with a Substance Released from MouseTumors by Tissue Culture. Virology, 3:380-400, 1957.

81. STONE, J. D. Prevention of Virus Infection with Enzyme of V. cholerae. I. Studies with Viruses of Mumps-Influenza Group in Chick Embryos. II. Studies withInfluenza Virus in Mice. Austral. J. Exper. Biol., 26:49-64, and 287-98,1948.

82. SVEDMYR,A. Studies on a Factor in Normal AllantoicFluid Inhibiting Influenza Virus Haemagglutination.Precipitation-dissolution Reaction in Mixtures of Active Virus and Inhibitor. Brit. J. Exper. Path., 30:237-47,1949.

83. TEMIN, H. M., and RUBIN, H. A Kinetic Study of Infection of Chick Embryo Cells in Vitro by Rous Sarcoma Virus. Virology, 8:209-22, 1959.

84. TESSMAN,I. Some Unusual Properties of the NucleicAcid in Bacteriophages S13 and 0X174. Virology, 7:263-75, 1959.

85. TESSMAN,I.; TESSMAN,E. S.; and STEHT, G. S. TheRelative Radiosensitivity of Bacteriophages S13 andT2. Virology, 4:209-15, 1957.

86. TRAUB,E. The Epidemiology of Lymphocytic Chorio-meningitis in White Mice. J. Exper. Med., 64:183-200, 1936.

87. VALENTINE,R. C., and ISAACS,A. The Structure of

Viruses of the Newcastle Disease-Mumps-Influenza(Myxovirus) Group. J. Gen. Microbio!., 16:680-85,1957.

88. VOLKIN,E. The Linkage of Glycose in Coliphage Nucleic Acids. J. Am. Chem. Soc., 76:5892-93, 1954.

89. WATSON,J. D., and CRICK,F. H. G. Molecular Structure of Nucleic Acids. Nature, 171: 737-38, 1953.

90. WECKER, E. The Extraction of Infectious Virus Nucleic Acids with Hot Phenol. Virology, 7:241-43,1959.

91. WECKER,E., and SCHÃ„FER,W. Eine infektiÃ¶se Komponente von RibonukleinsÃ¤ure-Charakter aus dem Virus der amerikanischen Pferde-Encephalomyelitis TypOst. Ztschr. Naturforsch., 12b:415-17, 1957.

92. . Studien mit 32P-markiertem Virus der klassischen GeflÃ¼gelpest. I. Mitteilung: UntersuchungenÃ¼berdas Verhalten des Virus beim Eindringen in dieWiertszelle. Ibid., pp. 483-92.

93. WELLEH, T. H., and COONS,A. H. Fluorescent Antibody Studies with Agents of Varicella and HerpesZoster Propagated in vitro. Proc. Soc. Exper. Biol. &Med., 86:789-94, 1954.

94. WILLIAMS, R. C., and SMITH, K. M. A CrystallizableInsect Virus. Nature, 179:119-20, 1957.

95. WYATT, G. R. and COHEN, S. S. The Bases of the Nucleic Acids of Some Bacterial and Animal Viruses: theOccurrence of 5-Hydroxymethylcytosine. Biochem. J.,55:774-82,1953.

96. ZILBER, L. A. Specific Tumor Antigens. Adv. CancerResearch, 5:291-329, 1958.



1960;20:728-741. Cancer Res Sven Gard Detection of Viruses by Chemical and Biological Means

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