BRITISH MEDICAL JOURNALLONDON SATURDAY MAY 26 1962

DEVELOPMENT OF VIROLOGY AS AN INDEPENDENT SCIENCE*BY

GEORGE K. HIRST, M.D.The Puiblic Health Resea7rch fnstituite of the City of New York Inc.. New York 9, Neiv York, U.S.A.

I welcome this opportunity for acknowledging thehonour of being invited as a visitor to this greatUniversity and of being asked to give the first PfizerLectures in Virology. The experience of residing herefor an extended period of time, of enjoying the intel-lectual stimulation of my colleagues, and of working inthe laboratories of the Institute of Virology have allgiven me unprecedented pleasure. I wish to thank thePfizer company for their generosity and foresight inmaking this lectureship possible and I hope that this willbe the first of a long series.The evaluation of virology as a separate science is an

appropriate topic for the opening general lecture of aseries sponsored by the Institute of Virology and itnecessitates a brief historical review, with specialemphasis on those forces which in recent years havehelped to unify the several branches. In this review Ishall try to make it clear that virology can make itsfullest contribution to biology and medicine only if itis treated and studied as an independent discipline andis allowed to be fully responsive to its own internalneeds. In emphasizing the importance of breaking awayfrom the traditional link to medicine, I have animalvirology mainly in mind, and I am fully aware that thisshift runs counter to the instincts of many of mycolleagues.

History of VirologyThe history of virology is a complex story of the

independent development of several different branches,each involving a different group of hosts: floweringplants, vertebrates, insects, and bacteria. Thesebranches arose and developed separately and havebecome interdependent only recently. The overlongperiod of isolation was, in part, a consequence of thevery different hosts involved, but the separation of thebranches was widely supported by the virologists them-selves, partly because the prominent systemic symptomsof virus infection obscured a conception of commonevents at the cellular level.There was no serious attempt to integrate the findings

in the various virus fields until Luria's textbook onGeneral Virology appeared in 1953. Later texts onanimal virology (Viral and Rickettsial Infections ofMan, 1959; Principles of Animal Virology, 1960) treatthe general approach to virology in only the most super-ficial way. Even as late as 1956 a prominent animalvirologist, writing about virus multiplication, was movedto say that resemblances between T2 and influenza

Given on October 25, 1961, at the University of Glasgowas the first Pfizer Lectures in Virology. The genetic experimentsdiscussed were carried out in collaboration with Dr. R. W.Simpson, with financial assistance from the United States PublicHealth Service (Grant E-2034).

viruses were "superficial and inappropriate for seriousgeneralization." To-day it is more appropriate toemphasize that it is the differences between animal andbacterial viruses which are superficial, and the intentionin this lecture is to emphasize the importance ofgeneralization in this field.

Initially, virology was an offshoot of pathologicalbacteriology, and this is a tie which has affected itsentire development. Although virus diseases have beenknown for centuries, the first virus, tobacco mosaicvirus, was described in 1892 by lwanowski. It is notsurprising that neither he nor his contemporaries fullyunderstood the meaning of an infective particle passingthrough an earthenware filter, and even in later yearsIwanowski's fame as the father of virology was appre-ciated abroad more than at home, and it was not untilthe middle 'fifties that one of the many virologicalinstitutes in Moscow was named after him. The firstanimal virus (foot-and-mouth disease of cattle) wasdescribed six years later, in 1898. From this time onthe number of filterable agents increased and, under-standably, there was a gradual acceptance of the ideathat they formed a distinct group and were not merelybacteria which had difficult growth requirements.The rules for inclusion of an organism within the virus

group developed slowly and empirically. The firstcriterion, that of size, was a governing factor from thebeginning, and the criterion of filterability, thougharbitrary, was unexpectedly successful in delimiting thisgroup without much ambiguity. The fact of obligateintracellular parasitism, the high specificity for the hostcell, and the frequency of a necrotic response afterinfection were all features common to all types ofviruses from the beginning, but they were not generallyconsidered of sufficient weight to suggest strongly thatthere was any real unity in the group, except by accident.We shall leave further consideration of this problemuntil we have reviewed the history of the other branches.

Plant VirologyPlant virology has usually been a branch of plant

pathology, and great emphasis has always been placedon the discovery of new viruses, description of theirmode of spread, and investigation of prophylacticmeasures. Nearly all plant viruses produce undesirablpeffects in their hosts, and the preponderance of attentionhas been placed on those agents which cause aneconomic loss, as is illustrated by the fact that nearlyall of the viruses which have been described are parasiticon plants of commercial importance, such as tobacco,potatoes, peaches, spinach, and others. In spite of this

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1432 MAY 26, 1962 VIROLOGY AS AN INDEPENDENT SCIENCE

intensely practical orientation, plant virus research hasoften been productive of new concepts which have beeninfluential throughout all virology.Not only were plant viruses the first viruses to be

discovered and the first to be crystallized, but they werethe first to evoke an interference effect in the laboratory.They were the first viruses in which the details of particlestructure were clarified at a molecular level, the firstviruses with which infection was initiated by nucleic acidalone, and the first in which mutation was inducedin vitro. Cloning of plant viruses was accomplished bya plaque technique long before a similar method wasapplied to animal viruses.The scope of plant virology began to widen in the

early 1930's when the crystallization of tobacco mosaicvirus (T.M.V.) attracted the eye of the biochemist, sincefiere was an infective agent which could be obtained inpure form and in gramme quantities.The careful biochemical and biophysical work which

followed was most important in establishing a generaltype of structure for the most elementary typo of virusparticle. In the case of T.M.V. this consisted of anucleic acid helix embedded in a helix of a repzatingsingle type of protein. In the case of some other plantviruses the nucleic acid is packed in a roughly sphericalshape but is covered by prot-ein units in such a way thatpolyhedra of great regularity are formed. Both of thesestructures have since been found among animal viruses,by electron microscopy with the phosphotungstic-acidstaining technique. These striking morphological simi-larities provide the most satisfying evidence obtainedthus far for the fundamental unity of the virus world.The discovery of the infectivity of T.M.V. nucleic

acid was followed almost immediately by similardemonstrations with. animal viruses and, later, withbacterial agents. This has affirmed the importance ofthe nucleic acid core as the repository of all the essentialinformation for replication of the total virus. It is alsoapparent from T.M.V. work that one of the mainfunctions of the coat is to preserve the core in the extra-cellular state, and this is probably one of its functionsfor all viruses. Whether or not all virus coats play thesame part in regard to attachment is not entirely clearas yet.The interest in plant viruses continues from the

academic standpoint and has extended now so that onephase of T.M.V. work involves the general problem ofprotein synthesis. The induction of mutants in T.M.V.with nitrous acid was rapidly followed by similardemonstrations with poliomyelitis and Newcastle diseasevirus, as well as with phage.The academic approach to plant virology has been

most fruitful and it has been very important in creatinga fundamental picture of the virus particle. Its impacton animal virology in other respects has been less, andthis -may well be due to necessarily different methodo-logies. It requires a very large amount of virus toinfect a plant, and the reasons for this and the detailsof the infective process are poorly understood. Thereis no way of studying the infection at the cellular level.The lack of a usable tissue-culture system and thenecessity of working with, whole plants or whole leaveshave made it difficult to study in detail such variedphenomena as penetration, eclipse, mixed infection,recombination, and other reactions which are the centreof interest in other fields.

Animal VirologyNow let us consider the development of the science

of the vertebrate viruses or of veterinary and medicalvirology. At the outset animal viruses were lumpedwith pathogenic bacteria, and for a great many yearsmost animal virus research was carried out either inmedical schools or in organizations closely allied withthe health sciences. A large proportion of virologistshave been primarily trained in medicine, and often theyhave had no formal education in microbiology apartfrom that given in medical school. The general attitudehas been that virology, lacking a well-developed body oftechnique, was a discipline which could be easilymastered in a couple of years by a medical-schoolgraduate, provided he had no aversion for the subject.

Inevitably with this background, the attention ofinvestigators was focused on viruses as agents of disease.The pressure in pathology and medicine was for theisolation of new agents from diseases of unknownaetiology, and for efforts to cure or prevent diseases ofknown virus aetiology. Relatively little emphasis wasplaced on the virus particle itself or on virus infectionat a cellular level.For many years the progress of animal virology was

hampered by the lack of good techniques. Since virusesmay have very specific tropisms, the discovery of suit-able hosts always remained a central problem. Therabbit, dog, and monkey of the earlier years wererapidly replaced in favour by the mouse, especially whenthe efficacy of intracerebral injection was discovered.The introduction of each new host was accompanied bythe isolation of some new agents; sometimes these wereagents latent in the host. The discovery of the highsusceptibility of ferrets to influenza by a natural routecreated a wide but transient interest in unusual animalsas potential hosts for stubborn viruses.The next step was the widespread use of the chick

embryo. This was important because it marked a

departure from the concept that the laboratory hostshould provide a reasonable facsimile of the humaninfection. While the chick embryo had many advantages,such as broad susceptibility, it was still a very com-plicated host. Therefore the next step, the use of animalcells cultivated in vitro, was by far the most importantadvance, not only because it fostered a great increase inthe number of viruses cultivable but because iteliminated a great many of the complications of work-ing in the whole animal and fostered investigation ofinfection at a cellular level. Actually the techniques oftissue culture are quite old, and this source of hostmaterial might well have been developed much earlierif virologists had not been so preoccupied with modelsof the clinical disease.In general, the discovery of new agents was followed

by a fairly regular pattern of development: adaptationof the virus to new and convenient hosts, hopefullyaccompanied by high levels of virus production; estab-lishment and evaluation of clinical diagnostic tests, andthe application of such tests to the epidemiology of thedisease; and, finally, the study of disease prevention,which usually took several forms-vaccination withkilled virus, vaccination with live but attenuated virus,and environmental or vector control.The magnitude of the effort in applied virology has

thus always been much greater than that devoted to a

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long-range approach, and there seemed to be adequatereason for satisfaction with this emphasis because theapplication of rather empirical methods to public healthproblems has actually worked very well. Smallpox,urban yellow fever, and poliomyelitis are all goodexamples of disteases brought under control by thesemethods.

It is a sort of paradox that the most sophisticatedsolution to the prophylaxis of a virus disease was theuse of live attenuated virus for protection against small-pox, and was due to Jenner in the eighteenth century,long before the era of microbiology. Urban yellow feverwas well controlled by an attack on the vector, Aedesaegypti, long before the dispute over the aetiologicalagent had been settled and the virus isolated. Finally,there is the recent example of poliomyelitis, againstwhich both killed and attenuated viruses have been used.No new principles were involved and success awaitedthe modern tissue-culture era which provided cleanhigh-titre virus preparations and the possibility ofantibody titrations on a large scale without the useof monkeys.We shall momentarily leave animal virology at this

point, to return to it again later after considering thehistory of bacterial virology, which should provide betterperspective concerning the more recent history.

BacteriophageWe turn our attention now to a most important event

in the history of our science, the discovery of bacterialviruses or bacteriophage. This was due to a Britishbacteriologist, Twort, who in 1915 described a disease ofstaphylococci. Among the liypotheses he consideredwas that of a viral disease of bacteria. This paper,though provocative and perceptive, passed unnoticed,and Twort did not further pursue the matter, possiblybecause it was wartime.The same reaction was independently discovered by a

Canadian, d'Herelle, who was looking for a synergisticaction between a virus and a bacterium in dysenteryinfections. d'Herelle did nearly all of tlie earlydefinitive work on the subject and summarized it in abook, published in 1921 and entitled Bacteriophage;its Role int Immunity. At this time the arguments ragedover whether bacteriophage was an organism or whetherit was something preformed in the cell, and on this pointd'Herelle took what was eventually to be accepted as thecorrect view. He had. however, an overwhelmingconviction that bacteriophages were powerful therapeuticagents, and a large part of his and of the general effortin phage research between 1920 and 1930 was spenttesting phages against all sorts of diseases ranging fromdysentery to tuberculosis.

Meanwhile, however, the scope of phage research wasbroadened with the description of many new phagespecies, which affected a wide variety of bacteria, andthe generality of the phenomenon was amply docu-mented but the central problems were postponedthrough a diffusion of effort. In the early 'thirties anumber of able investigators studied phage for briefperiods, and often with interesting results, but none ofthem continued with the subject. It is of interest that,even by this time, very few investigators had madebacteriophage the subject of their life work.A turning-point came in 1936 when Delbruck and

Luria, in a joint effort, initiated some phage studies in

which the governing motivation was to understand thephenomenon of bacteriophage rather than to developpractical applications. This was by no means the firstsuch study, but it was the first which led directly toother similarly organized efforts on what proved to be arapidly expanding front.The results were frtitful, the developments were

rapid, and it is worth while trying to understand thereasons for the marked success of this approach. Thescientists who participated were well trained in avariety of disciplines and very few came directly frommedicine. There were physicists, biochemists, cyto-logists, geneticists, microbiologists, electron micro-scopists, and others. They had a high degree of internaldiscipline and by common consent they concentratedtheir attention on a small group of viruses with acommon host. The importance of accurate quantitativeresults was stressed. Infection was studied at thecellular level. Scientific results were commonlyexchanged before publication, and the published resultshave an accuracy and reproducibility which was oftenlacking from the earlier phage literature.The success of this joint effort was immediate,

especially in providing a number of organized andintegrated findings, which was in marked contrast to thepast, and the approach was so powerful that the dis-organized effort in this field was swept away. Beforelong the influence of phage work extended beyondvirology to biology in general, and to-day is of thegreatest importance as a tool in the study of replication.There is, unfortunately, no time to consider this non-viral phase and we shall confine our attention to thoseaspects of bacterial virology which influence animalvirology.We left animal virology on the threshold of a new

era. Within the past ten years new techniques have beenchanging the outlook of the animal virologist and thereare definite signs that this branch of the science isdeveloping along lines which break with the past. Anattempt will be made briefly to assess the influence ofone branch upon another at the methodological level, atthe phenomenological level, and in terms of generalscientific organization.

MethodologyMethodology, although it sounds inherently dull, can

be a crucial factor in the development of a science, andthe introduction of the plaque technique by d'Herellewas certainly a turning-point for virology. The idea,derived from bacteriology, for the first time placedvirology in a position to be studied quantitatively, in thesense in which that term is ordinarily used. Theevaluation and refinement of this method was one of thefirst tasks carried out after the inception of the modernphage school.

Prior to 1952 the methods of quantitating theinfectivity of animal viruses were really very rough,depending by and large on the death or survival ofwhole animals that had been given widely varyingamounts of virus. There is no doubt that these andsimilar limitations greatly curtailed the development ofthe art. Therefore the introduction of the plaquemethod into animal virology by Dulbecco was a first-rate achievement. This advance was made possible bythe recent introduction of tissue-culture techniques intovirology. It is interesting and significant that the

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method was introduced into animal virology by a

student of phage and not an animal virologist.

In nine years the plaque method has been adapted toa wide variety of animal viruses, utilizing a variety ofanimal cells. This often reduced the errors of titrationfrom several hundred to less than 15°%. It provided a

ready and precise way of obtaining pure line clones.Another major consequence of the use of the techniqueis the possibility of studying the events of infection at a

cellular level, devoid of all the complications arisingfrom using a whole animal. The study of the neutraliza-tion phenomenon is a good example.

Formerly there was no good way of allowing virusand antibody to react in a test-tube and then testing for,say, residual active virus without putting the wholemixture in an animal. After inoculation the investigatorhas little control and no knowledge of what goes on inthe animal determining death or survival; for example,further reaction between antigen and antibody, dis-sociation of antigen and antibody, or slightly delayedinfection with subsequent mobilization of the protectiveresources of the animal. These are all possible andeach could affect the titration. In the new way, theantigen and antibody react in the test-tube, and then,after brief contact with the cells, the excess antibodyand virus can be removed and the assay becomesrelatively straightforward and uncomplicated.The animal virologist now has the same basic tools as

the bacterial virologist, although the animal-virus tech-niques are much more difficult than the correspondingbacterial ones. The host cells will not grow on simplemedia, and they will not grow into a monolayer over-

night. The host cells do not tolerate handling very well,and the development of plaques often requires days.These technological difficulties are reasons for using thebacteriophage and its host as an experimental model.

PhenomenologyIn considering the different phases of the life-cycle of

a virus the same subdivisions are useful for all viruses-adsorption, penetration, multiplication, maturation, andrelease-and these categories can be conveniently usedfor comparing the phenomenology of bacterial versusanimal-virus infection. The adsorptive phase in eachcase requires electrolyte, and in both cases severaldifferent stages of adsorption may be demonstrable.The penetration of viruses into cells appears to be one

of the most varied functions among the major virusgroups. The plant cell requires injury, apparently of a

special sort, for infection to take place. For bacterio-phage to breach the tough bacterial wall a complicatedand specialized injection apparatus has developed, theexistence of which has given rise to misgivings about therelationship of bacterial to animal viruses. Where thisinjection mechanism exists the virus coat is clearly leftoutside the bacterial cell, and this has definitely posedthe query: Does the animal-virus coat get into the cell ?Where the problem of animal-virus penetration has beenconsidered at all, some form of phagocytosis or pino-cytosis has been proposed as a likely mechanism, inwhich case the engulfment of the whole particle seeimsvery probable. However, recent experiments with polio-virus (Joklik and Darnell, 1961) have suggested thatsome weakening of the coat may take place outside thecell. Clearly. experiments should be designed to deter-

mine the fate of the capsomeres with animal-viruspenetration.The absence of mature virus particles within the cell

immediately after infection was first conclusivelydemonstrated with bacteriophage, and the reasons forthis eclipse phase were quickly discerned as being dueto separation of constituent virus parts on penetrationand formation of new virus by assembly of severaldifferent constituents, possibly made in different partsof the cell.

This work inspired a number of experiments withanimal viruses in which the occurrence of an eclipsephase was studied. The results were fairly conclusivewith some viruses, leaving no doubt that, at least afterpenetration, a cell may contain no demonstrable maturevirus particles, and the assumption that reproduction canproceed from a stripped virus core has been bolsteredby experiments with naked viral ribonucleic acid(R.N.A.).

Maturation by assembly seems to be a hallmark ofvirus reproduction, and the mechanism of this assemblyprocess has been worked out with T2 and T4. Thenon-specificity of assembly gives rise to a phenomenoncalled "phenotypic mixing," where particles are madeup of parts arising from different parents. This non-specificity of assembly was also discovered withinfluenza viruses, but the interpretation of the results as

being due to phenotypic mixing came directly froniphage work.

Genetic recombination of viruses in mixedly infectedcells provides still another example of a phenomenonworked out with bacteriophage which stimulated similarthough necessarily much more restricted research withinfluenza and vaccinia. The same may be said formultiplicity reactivation and cross-reactivation, the saltrequirements for virus adsorption, and the phenomenonof cell-killing by viruses. In many of these cases thebehavioural similarities of the two different virus groupsare striking. In some other areas, such as the changesin cell metabolism brought about by infection, the resultswith mammalian cells are quite different from thosefound with infected bacteria. This is not surprising, andat least part of the difference is related to the very largesize of mammalian cells and the relatively small part ofthe cellular metabolism which the synthesis of virusrequires. It is also probably due to the inherentlydifferent type of metabolism in animal cells.

Latent Infections

Perhaps the most important effects of phage researchon general virology may occur in the future, and thismay be in the area of latent infections. Certainly themost fascinating part of the phage story concerns thenature of the lysogenic state. An invading virusbecoming an integral part of the host cell genome, a

latent virus replicating synchronously with the cell itself,and an invading virus altering the host cell phenotypewere all fascinating and unexpected findings whichprovide a concept of parasite and host as evolutionarypartners. The results suggest the possibility of a viralorigin of at least part of the host genome and, con-

versely, mutation in the host genome as a possible modeof origin of the virus. The question is: Do any of thesesame phenomena occur with cells of higher organisms ?The possibilities are intriguing from many points of

view. There are a great many viruses which remain

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latent in higher organisms, and at the moment themechanism of their maintaining a latent condition iscompletely unknown. "Latent" or chronic infectionsof mammalian cells in vitro have been described andstudied in detail in recent years, but in most of thesesystems it is doubtful if the mechanism of virus survivalis the same as in nature. Experiments in which both thehost cell and the virus are irradiated have suggested,with bacterial viruses, that some homologies existbetween virus genome and host genome. This hasinspired similar attempts with irradiation of animalviruses and their host cells, but the results are incon-clusive. However, experiments with tumour viruseshave shown that these agents may multiply within a cellwithout killing it, and in some cases it is possible, thoughunproved as yet, that new cell antigens are produced asthe result of infection (Habel, 1962).

It thus appears that no very definite case has beenmade out so far for anything corresponding to lysogenywith animal viruses. The important thing to note is thatthe bacterial work has stimulated a great deal of interestin the concept of this kind of parasitism at the geneticlevel, and the failure to confirm the similarities is prob-ably mainly an indication that the state of the art ofhandling this type of virus and host material is not yetsufficiently advanced to answer such questions. Burnetsuggested in 1931, on the basis of some experiments inlysogeny, that the lysogenic virus might be an integralpart of the host genome, and, while this surmise wasessentially correct, it required 20 years and the develop-ment of bacterial and virus genetics -to prove it.

General OrganizationAt the third level, that of general organization, animal

virology is still in an undeveloped state. However, it isalready apparent that it is becoming more removed fromits close ties to medicine and is beginning to developfrom within. This certainly augurs well for a fruitfuldevelopment, and it may be hoped that it may proceedby a logical progression rather than in the hit-or-missway characteristic of much medical research. Workersof very diverse training are moving into this area. Atthe moment a wide variety of animal viruses of academicinterest are under study, and it is conceivable that, withtime, the workers in this field may concentrate on a fewkey types, in a move comparable to the concentration onthe T coliphages.

Personal Experimental WorkBefore finishing, I should like briefly to present some

work being carried on currently in my laboratory withthe collaboration of Dr. Robert W. Simpson (Simpsonand Hirst, 1961). The excuse for including it here isthat it furnishes a concrete example of the inter-disciplinary forces that have been discussed. I mentionthis work with some diffidence, however, because it hasnot been developed very far and, at the present time,permits of no very far-reaching conclusions.For the genetic analysis of a species genetic interaction

between two members of the species is required. It wassurprising to hear, just a short time after the birth ofbacterial genetics, that genetic recombination takes placebetween two phage particles invading the same cell.Shortly thereafter Burnet described influenza recombina-tion. Phage genetics turned bacterial virology into oneof the central sciences of modern biology, while theC

influenza story which we are to follow has slowed to analmost imperceptible pace.The basic experiment, which is due to Burnet and his

collaborators, consists of crossing two related but anti-genically different influenza A strains by putting bothinto either mouse brain or the allantoic sac andexamining the viral progeny. These two strains may beconsidered to have M and W as antigenic markers. TheW strain was virulent for mice by the intracerebralroute while the M strain was not. After mixed infectionit was possible to sort out four virus types from theprogeny, the two parent viruses and two recombinanttypes, avirulent W and virulent M. A few other recom-binants were shown in similar fashion over a period ofyears, and there is no doubt that these results were dueto recombination of genetic material between influenzaviruses.

Fraser (1959) confirmed and elaborated on thisexperiment, as shown diagrammatically in Table I, bycrossing these same strains, isolating a number of Mstrains each time, testing them for virulence and back-crossing them with the virulent W strain. Fraser showed

TABLE I.-Recombination of Virulence(1) NWS++++ xM---Mostly M-, M+, very few M++

etc. (also NWS+++, NWS++, etc.)(2) M+(from reaction 1)xNWS++++->M+, M++, and a

few M+++, etc.(3) M++(from reaction 2)xNWS++++->M++, M+++,

and some M+ + + +(4) M+ + +(from reaction 3) xNWS++++ ->M+ + + andM++++A schematic representation of how Fraser (1959) obtained fully

virulent M virus (M++-+ -) from completely avirulent M virus(M-) by crossing M- with NWS+ + + + (maximum virulence).Some M progeny of each cross showed a moderately enchancedvirulence over the M parent type. By successive backcrossingswith NWS, fully virulent Melbourne (M++++) was obtained.

by this method that strain M went from avirulence tofull virulence by a series of small steps, and he thusconclusively demonstrated that this kind of virulence ismultigenic in origin and therefore an unsuitable markerfor beginning the analysis of a complicated system.

It will not be profitable to review all of the develop-ments which came out of this attack on the problem,some of them from our own laboratory. The maindefect of this approach was that it could not be madereally quantitative, and quantitation has always been anessential part of genetics. After a few years this workwas dropped and there have been virtually no publica-tions in this field for the past four or five years.At about this time we began looking for an R.N.A.

virus which could be handled quantitatively and wassuitable for recombination studies. Newcastle diseasevirus seemed like a good possibility and was studiedextensively in our laboratory by Granoff (1959). Inspite of good markers and a good quantitative assaysystem, no evidence of recombination could be found.The reasons for this failure have remained obscure.

Poliovirus, which also lends itself well to quantitativework, was tried and, actually, a low frequency ofrecombination was obtained in a cross of two mutants,each of which had become resistant to a specific plaque-forming inhibitor, one in a bovine and one in a horseserum. This type of approach is being further pursued,but progress has been slow and some of the difficultiesin obtaining suitable markers have been described byDulbecco (1961).

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Influenza, as mentioned above, gives readily demon-strable recombination, even in crude systems, but it hasbeen very difficult to develop quantitative methods. Aswe have already indicated, an essential step for goodquantitation for viruses is a plaque-forming system, andit seemed unfortunate that most strains of influenza Aeither do not kill the cells in monolayers in such a waythat plaques can be detected or else the efficiency ofplaque formation is so low as to make the techniquevirtually unusable.Most strains of influenza are rather strictly pneumo-

tropic in the mouse, but a mutant of the original humaninfluenza strain (WS) was isolated many years agowhich has the capacity of multiplying in the endothelialcells of the mouse, and, as a consequence, it can multiplyin the brain, where it is lethal. This strain (WSN),inaccurately described as being neurotropic, alsoproduces excellent sharply outlined plaques on chickfibroblast monolayers, and does so with a very highefficiency. As noted before, influenza strains in generaleither produce poor plaques at low efficiency on thismedium or fail to produce plaques at all.

Using this plaque-forming strain, we have developeda system in which plaque formation is used as a selectivemarker for those particles which have undergonerecombination. Selection is an essential feature ofrecombinational systems where the amount of nucleicacid per particle is so small as to make recombinationa very infrequent event. The fact that all recombinantclones are efficient plaque-formers of course enhancesthe possibility of working with them on a quantitativebasis.The method is carried out as follows: The so-called

neurotropic plaque-forming strain (WSN) (or arecombinant derivative of it) is irradiated by ultra-violet light until very little survives, and it is then usedto "infect" suspended chick fibroblasts along withanother active non-plaque-forming (or weak plaque-forming) strain. These cells, which have received atleast one of each type of particle, are then plated asinfective centres on fibroblast monolayers and overlaidwith agar.

If the suspended cells are infected with either of thesetwo types of virus singly, no plaques will be formed andthe controls are therefore negative. After combinedinfection, however, a number of clearly outlined plaquesappear. These plaques are readily transmissible inseries, and the virus in them is plentiful and plates withhigh efficiency. In the case of some crosses the newplaques are caused by viruses of the same serotype asthe non-plaque-forming parent.

Similar experiments have been carried out using anumber of influenza A strains, and we have beensuccessful in isolating plaque-forming recombinantsfrom a wide variety of them, including old laboratorystrains isolated from epidemics in the early 'thirties aswell as those obtained from Asian influenza. Theactual frequency of recombination is not known and isdifficult to determine from this type of approach. Allthat can be said is that the rate varies enormously withdifferent crosses and may bear some relationship to thesimilarities of the strains being crossed. Strains thatwere very dissimilar antigenically, such as types A andB, gave no recombination.

In order to see whether this system was truly selective,plaque-type recombinants were examined for other

characters. A cross was carried out between aMelbourne strain (isolated in 1934 and converted toplaque formation by recombination) and the prototypeof A2 or Asian influenza, isolated in Japan in 1957.These two strains are quite disparate in a number ofrespects, although both are influenza A. Eighty-sixplaque-forming recombinants were tested for fivemarkers, such as the capacity to infect, position in thereceptor gradient, and heat stability. A very high rateof recombination was found for these markers. Moresurprisingly, these five markers appeared to assortindependently, as indicated in Table II, which shows thata large number of the possible combinations of recom-binant characters was actually found. For at least twopassages these new characters proved to be stable.TABLE II.-Distribution of Markers Among Progeny of a CrossBetween Plaque-formting M (UV) and Non-plaque-forming

J Strains

Phenotypic Classification of Virus for:

Type RHeat SanAgglutination Rabbit No. of

-ype Hes- Spons of RDE- Serum Infec- clones

tance Elution treated Inhibi- tivity~~~~Cells tion

J progeny _ _ _ 3recombinant _ + 1for plaque + _ _ + 9formation + + + + 3

+ + + 10

+ + + 2+ + 16

+ 4+ + + 1+ + + 4+ + I+ + + + 6

+ + + + 7+ + + -19

Total recom-binants 42 77 65 12 33 86

86 J type recombinants were analysed for the five characters shown.+ Indicates that the character was from the M parent (recombinant);- indicates J parent type (non-recombinant).

From these preliminary results we feel that it may bepossible to analyse the genetic system of the influenza Aviruses in a systematic manner along classical lines. Ifso, it will be the first time that a genetic system basedsolely on inheritance through R.N.A. has been soexamined. Certainly, if the -genetics of influenza virusinheritance can be worked out it will strengthen ourknowledge of this- very interesting and importantorganism in such a way that we shall be much betterable to cope with the practical problems which influenzapresents in the form of its apparently great mutabilitywith widely swinging variations in virulence for man.On the other hand, this may be only a step in the direc-tion of analysing other animal viruses, which we are notprepared to work with at the present time.

ConclusionWe return now to our point of entry, and we have

seen enough, I hope, to realize that the nature ofvirology as a biological science cannot be expressed ina word or in a phrase, or perhaps not precisely at all.If we must characterize it further, we can say that it isnot a taxonomic science like ornithology because verylikely there may be no direct relationship between manyviruses in an evolutionary sense. To say that the tiesthat unite virology are methodological is certainly true,but only partly true, and one has the feeling that theunity must transcend mere methods. Perhaps cytologyprovides a closer analogy than any other biologicalscience. In any case, viruses as subcellular reproducingagents occupy a niche whose relationship to other

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MAY 26, 1962 VIROLOGY AS AN INDEPENDENT SCIENCE BmoH 1437JOURNAL~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

biological entities remains to be discovered. At thepresent time the outstanding similarities are morpho-logical and the evolutionary similarities or differencesremain to be worked out.

In closing, I should like once again to refer to theInstitute. It was obviously in the spirit of fostering abroad academic approach that the Institute of Virologywas founded here at the University of Glasgow, and, asI have tried to indicate, the creation of this organizationwith such adequate housing and equipment, but muchmore importantly with such able leadership, was a wiseand timely move and should be the basis for self-congratulation on the part of the University. It is

certain that the organization will grow with the times,and, as I have indicated, the times seem most propitious.

REFRENCESDulbecco, R. (1961). In Poliomyelitis: Proceedings of Fifth

International Polio Congress, 1960, p. 21. Lippincott, Phila-delphia and Montreal.

Fraser, K. B. (1959). Virology, 9, 168, 178, 191, 202.Granoff, A. (1959). Ibid., 9, 636, 649.Habel, K. (1962). J. exp. Med., 115, 181.Joklik, W. K., and Darnell, J. E., jun. (1961). Virology, 13, 439.Principles of Animal Virology (1960), edited by F. M. Burnet,

2nd ed. Academic Press, New York and London.Simpson, R. W., and Hirst, G. K. (1961). Virology, 15, 436.Viral and Rickettsial Infections of Man (1959), edited by T. M.

Rivers and F. L. Horsfall, jun., 3rd ed. Lippincott, Phila-delphia and Montreal.

SOURCES AND SEQUELAE OF SURGICAL SEPSIS*BY

JOHN LOEWENTHAL, M.S., F.R.C.S., F.R.A.C.S.Professor of Surgery, University of Sydney, Australia

The problem of infection in surgical wounds has alwaysbeen a contentious one. Lord Lister certainly found itso. From time immemorial the surgeon's attention has,naturally enough, been mainly focused on the dramaand challenge of operation, but in recent years he hasrecognized that the pre-operative preparation andimmediate post-operative care of the patient are sointegral a part of the operative procedure that theoperator himself must assume major responsibility forthem. But, in general, the surgeon has been disinclinedto concern himself seriously about the " occasional "infected wound. It is rarely lethal, it usually resolvesultimately, complications such as wound herniation maynot develop for ages and may then be treated bysomeone else, and, in any case, it is best handled by hisregistrar or house-surgeon.

Indisputable evidence exists in all parts of the worldthat infection of surgical wounds is occurring in eitherthe operating-theatre or wards, that the organism mainlyconcerned is the Staphylococcus aureus, that theorganisms are being born and bred in the hospitals, andthat the likelihood is that they will increase in bothfrequency and possibly virulence. The surgeon'sreaction to this has often been mostly one of blandindifference; some surgeons flatly deny that any of theirwounds ever become infected. Others admit thatsometimes they have a little trouble, but never anythinglike the horrors that are reported from the professorialunit where this problem is being investigated. But, ofcourse, you have to expect some rather odd things froma professorial unit.Our interest in this work started some four years ago

when we instituted a purely clinical survey of cleansurgical wounds. Some 764 of them were carefullyscrutinized. They were all uncontaminated woundswhich should have healed by first intention, but 8%of them showed clinical infection (B. P. Morgan, 1958,personal communication).

Findings of a SurveyMore recently a much more intensive survey was

undertaken to try to identify the source of the infection.Over a six-months period patients and staff were

*Based on a paper read in the Section of Surgery at theAnnual Meeting of the British Medical Asiociation, Auckland,1961.

systematically and intensively investigated clinically andbacteriologically. In brief, our findings (Rountree et al.,1960) were:

(1) In spite of poor and antiquated operating-theatrefacilities with inadequate ventilation there was littleevidence that wound infection was initiated in the theatres.During 25 operations 167 nasal swabs were taken from 67people. In 46% Staph. aureus was isolated. Of these 67people, 42% were carriers on at least one occasion. It isof interest that during the 25 operations the people in thetheatre comprised:

Surgeons, anaesthetists, visiting doctors .. .. 21Nursing sisters .1. .4. . .. 14Student nurses .. .. .. .. .. 16Medical students . .. .. .. 10Instrument and x-ray attendants .. .. 6

The "permanent" members of the surgical teamnumbered nine. Of these nine, six were permanent nasalcarriers. However, no patient was apparently infected inthe theatre with any of these strains, and it would seemthat few of these carriers were dispersers of their organisms,and possibly their strain was not highly virulent But thepotentialities of this situation are considerable underdifferent carrier circumstances.

(2) A total of 217 wounds were intimately examined."Clean" wounds were those in which no possible reasonfor contamination was present. A cross-infection rate of14% occurred in " clean " wounds and 68% in " dirty"wounds (Table I).

TABLE I.-Cross-infection-" Clean " and "Dirty" Wounds

Wound No. of Cases Uninfected Infected

Clean .. 198 171 27 (14%)Dirty .. 19 6 13 (68%)

Total.. 217 177 40 (18%)

(3) The site of the wound on the body or the sex of thepatient did not significantly affect the infection rate, norwas it influenced greatly by the surgeon. However, the typeof dressing did. Gauze pads which absorbed blood, serum,or sweat and required early post-operative changing wercassociated with a much higher incidence of wound infectionthan when a spray-on plastic seal (" healex " or " nobecu-tane") was used. While 61% of wounds were infectedunder a gauze pad, only 7% were when sealed (Table II).

(4) Of 40 wounds showing cross-infection, 35 (87%) wereinfected with Staph. aureus, either alone or with otherorganisms. The predominant staphylococcus present in 60%of wounds was phage type 47, which is resistant to penicillin,streptomycin, tetracyclines, and usually chloramphenicol and

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