WS Moc 6/2475 HANDBUCH VIRUSFORSCHUNG HERAUSGEGEBEN VON PROF. Da. R. DOERR unp PROF. Dr. C. HALLAUER BASEL BERN ERSTE HALFTE DIE ENTWICKLUNG DER VIRUSFORSCHUNG UND IHRE PROBLEMATIK - MORPHOLOGIE DER VIRUSARTEN - DIE ZUCHTUNG DER VIRUSARTEN AUSSERHALB IHRER WIRTE - BIOCHEMISTRY AND BIOPHYSICS OF VIRUSES BEARBEITET VON F. M. BURNET-MELBOUBNE - R. DOERR-BASEL + W. J. ELFORD-LONDON G. M. FINDLAY-LONDON - M. HAITINGER-WIEN - C. HALLAUER-BERN M. KAISER-WIEN - W. M. STANLEY-PRINCETON MIT 71 ZUM TEIL FARBIGEN ABBILDUNGEN IM TEXT WIEN VERLAG VON JULIUS SPRINGER 1938 Inhaltsverzeichnis. XI Seite I. The technique of chorioallantoic membrane inoculation.............. 420 1. The anatomy of the extraembryonic membranes of the chick embryo 420 Histology of the normal chorioallantois ...........-. 2 ees eee eee 421 2. Technical details of the method of inoculation used in the Haiti Tnstitnte 2. ee ee ee ee tenet ete teen ene 422 3. Other methods of inoculation ...... 06... cee ee ee eee 424 Ii. Virus lesions of the chorioallantois and embryo ............- geceeee 424 1. The criteria necessary to establish that a virus is being propagated on the chorioallantois .... 6.0... cece eee ene n enna 424 2. Nonspecific lesions of the chorioallantois ........ 06... ee ese ee eens 425 3. Specific chorioallantois lesions. .... 01... 66sec eee ee tenes 426 The histological development of focal lesions 426 — The macro- scopic appearence of virus foci 43] — Conditions modifying the nature of the membrane lesions 432. 4. Effects of virus infection on the embryo0.....-.. 6... eee e eee eee ees 434 HI. The main applications of the method in virus research .........---- 435 1. Titration of viruses on the chorioallantois.........0. 60 c eee eee 435 a) Titration of viruses rapidly lethal for the embryo............. 435 b) The “pock-counting” method of virus titration in the chorio- allantois 0.0... cee et eee eee teeta tee eens 436 2. Immunological applications ©... 0... cece cee ee ee eee ete ean eee 438 a) Titration of virus-neutralizing antibodies on the egg membrane 438 b) The use of egg-grown virus as immunizing agent ............. 439 c) Egg membrane virus as antigen in complement fixation tests ... 441 IV. Summary of the viruses wich have been propagated on the chorioallantois 441 1. Viruses pathogenic for man....... 6. 6c eee eee eee eee ee eee 442 2. Viruses pathogenic for other mammals..........--.+-.0eeeeeeeee 442 3. Viruses pathogenic for birds .......... 02... eee eee eee teens 443 4, Viruses which have failed to grow on the chorioallantois ......... 443 Vierter Abschnitt. Biochemistry and Biophysics of viruses. References 0... ccc cc ccc ccc eee ee ee eee eee e eee e eee UE By W.M. STaxLey, ROCKEFELLER Institute for Medical Research, Princeton, N.J. With 20 illustrations. J. Inactivation of viruses by different agents ............. 00.000 c eevee eee 447 Introduction 0.0.00... 0c cee eee ene tree ten enneee 447° Effect of enzymes .. 2... eee een net ne nares 448 Effect of chemical reagents... 0.0... cece eee eee teen teens 449 Effect of physical agents. 0.0... cc cece ener rene tenet tenes 452 Bibliography... .... ccc eee ete tenet nee e eens 454 _ II. Concentration and purification of viruses ......... 00. eee eens 458 Introduction 2... 0. cee cece ee ce eee eee ene teeta ete n een eneee 458 : The elementary bodies of vaccinia and of other viruses...........-. 460 The fowl tumor viruses 6.0... cece cee eee eee eee neat eee nae 463 a Chemical methods ........ 0. ccc cee cece rene eee e een etnnnene 463 ‘ Physical methods... 2... ce. cece cece teeter e eee e estates 465 j Tobacco mosaic Virus.......-..0eee eee nee een teen een nes 467 “ Early work 2.0.0.0. 0 02 ccc cence cee e been e nner ene eeeee 467 Use of safranine and Jeadacetate...... 0. ccc ee ete e ener e erence 468 XIT Seite Vinson and PETRE’s procedure .....-.. 6c reece reer en eee 470 Other methods ......... 2.2 cee eee e eee ee erent ete nees 471 Isolation of crystalline tobacco mosaic virus protein ....-.-.-+--- 472 Modified isolation procedure .......-. 2s eee c eee e eee cree erences 407 Foot-and-mouth disease Virus. ..... 0.2 eee e ence nec e nee e cnet 478 Poliomyelitis Virus... 60... cee eee eee ener eee ennrtes 480 Viruses isolated by ultracentrifugation ....-..--.ee eee ee eee eee tee 481 Chemical versus ultracentrifugal methods .........-+. 0s ere ees 481 The centrifuges... 0... ccc eee eee eee n ene 482 Tobacco ring spot VITUS... 6.6... eee eee ee teeter ents 485- Latent Mosaic VITUS ... 6. ee eee eee eee ete eees 486 Severe etch ViIruS..... 22.00 cece eee eee tenner tees 487 Cucumber mosaic VITUS 02.6.6... cee ee eet eee neat enens 487 Shope rabbit papilloma virus .... 6.6.1 eee e ee eee ents 488 Equine encephalitis virus..... 66... sec c ete eee eee er eens 488 Bacteriophages . 1.6... cee cee eee nett e ene teen ene 489 The transformation agent of the pneumococeus .......---+- +e eee 491 Bibliography... ccc cece eee ee eee eee ee teen aes 492 Ill. Chemical and physical properties of viruses .........--- 622 e reese eens 498 Tntrodnction oo... eee eee teense ene n tenes 498 Tobacco mosaic Virus protein. ... 6 ee eee tt eet 500 Virus activity 0... eee tenet ene e tees 500 Protein-free virus preparations ....-.. see ete eee ee eee tenes 502 Analysis and general properties ......6-- ++ essere eee eee etn 502 Effect of Enzymes .......... 0c eee teeter tees 505 Crystallization and solubility experiments ......-.-+2+- +e essere 506 X-ray diffraction pattern... ..... cece eee eee ete eens 507 Absorption spectrum... 0... eee eee eee cece nee tenet eee nnes 508 Inactivation by ultraviolet light and X-rays ...-.-----++-2++5-55 509 Effect of drying.........2 6. eee eee tere eens 510 Visible mesomorphie fibres ©... 0.6.6 e cee e eee teen eee 511 Double refraction of flow ......6 606 c eee eee ee eee eens 51L Sedimentation constants ....... 65. eee cece ee ee eee eters 514 Viscosity, molecular weight, shape, diffusion constant etc. .....--- 516 Virus protein from different hosts 2.01... + sss esse eee eres 517 Virus protein of different strains... 000-666. sees eee erect 519 Correlation of virus activity with protein...........25s ree ree 52) Reactivation of inactive virus protein .........--+- heen eee eee 524 Tobacco ring spot virus protein 1.2.2.6... eee eee eee teeters 526 Latent mosaic of potato virus protein. .... 6... eee eee eee eres 528 Bushy stunt virus protein... 2.6.0.6. ee cece eect eee tenes 530 Cucumber mosaic virus protein .... 6.6. eee ee eee ees 531 Shope papilloma virus protein ........- 6+ eee eee eter eee erties 532 Vaccinia elementary bodies. ..... 5.0... eee tee eee eee nents 533 Analysis and general properties ...... 66.6.6 e eee teens 533 Soluble antigens......... 6.0. c erence eect teers 534 Mode of formation of soluble antigens..........5.50 +r eee reece 535 Nature of the elementary body..... 0.6... e eee e eee enter eres 536 Norrurop’s bacteriophage protein ..-... cee eee eee eee tenets 537 General properties and size....- 1... ee eee nee eens 537 Correlation of activity with protein ....- 66 ee eee eee tenes 539 Bibliography... cece eee e eee eee eee eee nee eee ene ees 540 Inhaltsverzeichnis. Vierter Abschnitt. Biochemistry and biophysics of viruses. . by W. M. Srantey, Princeton, New Jersey, U.S.A. (The Rockefeller Institute for Medical Research.) I. Inactivation of viruses by different agents. Introduction. Studies on the effect of different chemical and physical agents on the activity of viruses were in progress even before viruses were recognized as a separate group of infectious entities and have been continued to the present time. During the earlier work two objectives were sought, one the preparation of immunizing antigens and the other the elucidation of the nature of viruses. These have continued to remain as objectives and recently a third has been added; during the past few years studies on the effect of different agents on viruses have been made with a view towards establishing conditions and reagents that could be used in the purification and concentration of viruses. Considerable difficulty has been encountered, not only during the progress of the studies but also in the interpretation of the results that were obtained. Much of this has been due to the great variation in the physical and chemical properties of the different viruses and to an apparent variation in the properties of the same virus in different preparations. The latter appears to have been due to the presence of varying amounts of extraneous material in the different virus preparations. For a great many years the presence of extraneous material made it impossible to be certain that any given physical or chemical property was one of the virus itself. This point is discussed at somewhat greater length in the third section of this chapter. Recently the effect of enzymes on viruses has been studied in an effort to learn something of their nature. However, until very recently only crude enzyme preparations containing a mixture of materials were available, and there is con- siderable doubt concerning the significance of results obtained with such pre- parations, for Prrrz’s work indicates that the inactivation of some viruses was due to extraneous material rather than to the enzymes. The isolation within the past few years of several enzymes in crystalline and apparently pure form has made it possible to study more accurately the effect of enzymes on viruses. Although a vast amount of work has been done in attempting to achieve the three objectives mentioned above, no effort will be made to consider all the work in this section, because the general situation has been considerably altered within the last few years by the isolation of several viruses in apparently pure form. It is obvious that studies on the nature of viruses and on the preparation of immuniz- ing antigens should be made with such purified preparations of virus rather The transformation agent of the pneumococcus. 491 active protein isolated by fractional precipitation with ammonium sulphate bet- ween 0,2 and 0,4 saturation at px 7. Norruror was able to isolate about 50 mg. of protein, representing about 25 per cent of the activity in the starting material, . from 200 liters of lysed culture. The detailed procedure for an average experi- ment is shown in table 3. It may be seen that the protein nitrogen could be reduced from about 4000 mg. to about 40 mg. without any great loss of phage activity, but that further purification was accompanied by loss of activity. Norturop found the preparations to become increasingly unstable as purifi- cation proceeded and encountered considerable difficulty in achieving final puri- fication. However, he has subjected the purified phage protein to extensive studies in which the activity and protein have been correlated by several diffe- rent procedures, and has secured good evidence not only that the phage activity is a property of the protein but that the phage protein is essentially pure. The phage activity was found to be greater than that of any preparation previously reported, for only 10-6 gm. protein nitrogen of purified phage was found suffi- cient to cause lysis. The physical and chemical .properties of the phage protein will be considered in the ‘next section. The transformation agent of the pneumococcus. In 1928 Grirrrru found that he could transform one specific S type of pneu- mococcus into another specific S type through the intermediate stage of the R form. He effected the transformation by injecting mice with nonvirulent R forms, together with large amounts of heat-killed S pneumococci of a type other than that of the organisms from which the R cells were derived. Living virulent S organisms of the same type as the heat-killed S forms were then recovered from the animals. These results were confirmed by NeEvrELD and LevrntHaL and by Dawson. Later Dawson and Sra demonstrated that the transformation in type could be accomplished in vitro by inoculating small amounts of R organisms derived from 8 organisms of one type into blood broth containing anti-R serum and heat- killed § cells of the other type, or more strikingly by the use of an extract of the cells of several times frozen type-specific pneumococci. The latter finding was confirmed by ALLoway, who found that cell-free, heated and filtered extracts of one type of S pneumococci could be used to induce the conversion of R forms derived from another § type into the same type as that of the cells used to prepare the extract. It is obvious that there is a factor which may be obtained from any one of the S type of organisms that is normally absent from R. type cells, but that when added to such cells induces their conversion into the same type of S orga- nisms from which the factor was derived, with the very important result that more of the factor is produced in the induced S cells. This phenomenon is virus-like, and it is because of this and the fact that it may become important from the standpoint of the chemistry of viruses that a discussion is included here. The various type-specific pneumococci may be regarded as cells infected with different “virus” strains and only the R organisms as healthy. The R organisms may be converted into any one of what we refer to as type-specific organisms by “‘in- fection” with any one of the different “viruses”. By appropriate treatment it is again possible to free the pneumococci of “virus” and secure the healthy R type. It is ofinterest, therefore, to examine the nature of this factor or “virus”. The type- specificity of the pneumococcus is determined by its capsular polysaccharide, hence it might be assumed that the type of soluble specific substance or poly- saccharide isolated by HEIDELBERGER and AvERY or the acetyl derivative isolated by Avery and GorBEL from pneumococcus type 1 might be responsible 3la* 492 W.M. STANLEY: Biochemistry and biophysics of viruses. for this conversion. However, Dawson and S1A found that the specific capsular polysaccharide in chemically pure form would not induce the transformation in - type. It seems probable, therefore, that, if the polysaccharide plays a role in the transformation, it does so only when in combination with some other substance. Alloway, in attempting to purify the active agent, found that considerable in- active material could be removed by dissolving heat-killed S organisms with sodium desoxycholate, precipitating with cold alcohol, and extracting the pre- cipitate with salt solution. The extract was then heated to 60° C., centrifuged, the supernatant liquid filtered through charcoal, and again precipitated with alcohol. The precipitate was dissolved in water and centrifuged to give a colorless, water- clear supernatant liquid containing practically all of the original activity. No chemical tests were made on these purified preparations, hence nothing is known about the nature of the active agent. It is to be hoped that the study of this agent will be continued because of its virus-like nature. 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Extracts containing virus plus greater or smaller amounts of extraneous material were subjected to the action of different chemical and physical agents, to filtration through membranes of known porosity, and more recently to centrifugation in known fields of force. These studies, some of which are described in the two preceding sections, were very valuable from the stand- point of serving to increase our general knowledge of viruses. Frequently, however, they did not yield information concerning the chemical and physical properties of the viruses themselves, despite the fact that the results were usually so inter- preted, for the viruses were always accompanied by extraneous material, the effect of which it was impossible to evaluate. In some virus preparations the inert extraneous material probably comprised over 99 per cent of the solids, whereas in others it probably comprised less than 20 per cent of the solids. The nature and amount of extraneous matter varied with the host from which the virus extract was prepared. The presence of this extraneous material was either a real or a potential source of interference in the establishment of the true properties of a given virus, and before viruses were concentrated and purified it was practically impossible to be certain that a given property was really characteristic of a given virus. For example, the thermal inactivation point of tobacco mosaic virus is usually given as 93° C., because the virus in freshly expressed juice from diseased Turkish tobacco plants is usually inactivated on heating to 93° C. for 10 minutes. However, this point varies from sample to sample, depending upon the concentration of virus and upon the host from which the virus was obtained, for these two factors affect the relationship between virus and extraneous material. It has been impossible to determine the thermal inactivation point of the virus itself in such preparations because of the effect of the extra- neous matter. When this extraneous matter is removed and the tobacco mosaic virus is obtained in the form of crystalline virus protein, the thermal inacti- vation pointis found to be, not 93° C., but about 75° C., and the point remains the same regardless of the source of the virus. The filtration experiments on the virus of latent mosaic of potato may be given as another example of the erroneous impressions that may result from work with unpurified preparations of virus. These filtration results indicated that the particle size of the latent mosaic virus is 75-112 my, or about 3 or 4 times that of tobacco mosaic virus. This virus has recently been concentrated and obtained in the form of a homogeneous purified virus protein having a sedimentation constant of 113, a value that is only about 60 per cent that of tobacco mosaic virus protein. 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