a sm eae! Po por ee iz aS é ee ne err, thas mie ee E #OS5Te00 TOED 0 M0 IOHM/18lN THE VIRUSES Volume 3 ANIMAL VIRUSES | aie DA 4% 7 Mas { i ip i i ; i OS | a { 1 we 1 * ‘ : 1 ; . Pi { { i uy i * i i i * - a i 1 y ° a - 1 1 1 \ i 5 5 a u i t i . i iss > 2 - J ‘ i ai 2 Bs ; 4 c t i i i rv 4 a i 2 i = J} y i i = 1 ts if 7 ¥ 7 i i x 1] t : ! i - . - \ i i = THE VIRUSES Biochemical, Biological, and Biophysical Properties Edited by F. M. BURNET W. M. STANLEY The Walter and Eliza Hall Virus Laboratory Institute of Medical Research University of California Melbourne, Australia Berkeley, California Volume 3 ANIMAL VIRUSES 1959 ACADEMIC PRESS - NEW YORK - LONDON Academic Press Ine. 111 Fifth Avenue New York 3, New York U.K. Edition, Published by Academic Press Inc. (London) Ltd. 40 Pall Mall London, S.W.1 COPYRIGHT © 1959 By ACADEMIC PRESS INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS Library of Congress Catalog Card Number : 59-7923 PRINTED IN GREAT BRITAIN AT THE UNIVERSITY PRESS ABERDEEN Contributors to Volume 3 F. M. Burnet, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia 8S. G. ANDERSON, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia ALFRED GOTTSCHALK, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia F. B. Bane, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland ' Auick Isaacs, National Institute for Medical Research, London, England R. WALTER SCHLESINGER, Saint Louis University School of Medicine, Saint Louis, Missouri Frank L. Horsratu, Jr., The Rockefeller Institute for Medical Research, New York, New York Frank FENNER and Joun Cairns, Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia T. Francis, Jr., University of Michigan, Ann Arbor, Michigan Howarp B. ANpDERvoNT, National Cancer Institute, National Institutes of Health, Bethesda, Maryland KennetH M. Smiru, Agricultural Research Council, Virus Research Unit, Cambridge, England I. Animal Viruses: A Comparative Survey sy F. M. Burnet....... ie Demnitrons of Animal MVinusegsa2 oo states cee See ede nos See II. A Basis for the Classification of Animal Viruses............ ge HE SILLACOBIS ALOU ers him ota meres tek. Meee eS Colne oe ee Ne oat SORA ee Se as ke ey roe ee Ace en ee a are eats eR cee Se Peres vIRWS 52 or fein ati shee Sete ots ete ae tert haa Me VY ONMRUS Sr wsiectinnah. tin aay omesb eee aati ae he Sauer Nolo ete SUNGENOVINUSES = £5 Gh cone. ait ache etotas he econ tae IAGGEVIRIISES eae lice ene ke Da ate en ere eae MB ILEROVIRUSOS ere sos NA sane a eee Ble Bara Qe ers Other Groups: a= mes Rea ae eee cee aie Semen iM TheiComparative Pictures... = 2. sek oon ee, ne ee ae ee eee iV Evolution’ of the: Animal Viruses... 2. 2.5.2.6. ise asc eee V. Is There Any Functional Unity among the Animal Viruses? mod ddonw> PRuCRET EMC OG oe eye tae ART ey PIE Ce Ee II. The Initiation of Infection by Animal Viruses spy F. M. Burnet . RL CLEREM COS Oro Aion eee rote tee ted wofears teehee II. Hemagglutination by Animal Viruses By S. G. ANDERSON....... Ee Phe My covikUseGLOrps. 2. seh etis ciyer bese aie hegreicre eee SpLISLOTICAL c/s ta tirte csc eae & A ES aces cies eee . Enzymes with Receptor-Destroying Properties.......... emdlinG@icabor VATUS! cones meer oe nae mete oe aa ee cle ae » Imbibitors/of Memacelutimation: -).4..0..e-e 2 = oe «1 oe os . Mechanism of Hemagglutination.. «.5....5.....+5....+. AQAA OW > II. Particulate Hemagglutinin with No Eluting Enzyme........ A. Arthropod-borne Encephalitides...................... So FHMPSLONILIISES! cxc cok a Ve Ee ane ete OM Ae Fit et minds eas III. Hemagglutinin Separate from the Virus Particle............ Ave SILLACOSIS LOUD Ay, Jou, aid vaerte ete ee eee eine aaah ea BPE ORWARUSES 655th tes eta R re ha eee ey caer oie wre, EPC LELEMCOS Tee Pree Ree are re Men Ah ak 76935 ANTAHNTOOOMKO FP WO Re Re — © vii CONTENTS IV. Chemistry of Virus Receptors By ALFRED GOTTSCHALK.......... 51 References 0.5.3: Seaineenag cian ee Ge meena ees see 59 V. The Morphological Approach py F. B. Banea................... 63 T.. Introduchon.. 704.062 aes es ie eins eo See ee eee 64 TT. (Normal ‘Cell. .a4 22h Seceeuaa ce eee ect eee ee eee 65 A, PinOCytOsis..c0... 00 2 agwawwie oc ders cree a eee ana 67 Bi Mitochond ta: ones 5 asim ee © els mates el eine eee 67 Cm Nueleuss 232 suscvsaare. s acianaiceetsoe 2 ten eee 67 Di Secretion): i 5.2. See ee Scie eee ieee oe 67 Be Osmotic Changes: 2... dic vets seria es beck cia ee 68 III. Pathological Aspects of Cell Morphology................... 68 ix... Cell SUElaces) si Was sen Sond Oca iain ie ee 69 By -Osmoticvimbalanee, 2cc-' om: osicwia cersatrg 2, oer cee 69 Cos Mitochomeria satis cs ctaa ca epe ee met eens hace eee 70 Di Paranuclear Hypertrophy sj<.0 1 oe sne eee te 70 H:! Hy pertrophied or Giant Cells). 32. isos arctan on ote 70 FR. Amino Acid Deficiencies. 72-5 cencisencec onan see a G. Cytoplasmic and Nuclear “Degenerative” Changes....... 71 IV. Bacterial and Rickettsial Infections.....................-- 72 V. Psittacosis Group Viruses (Miyagawanella)............... oo ee Ay Wisswe Culture Stages 2 215)600/< 2.6 a5 aio o oe See 72 B. Hlementary Bodies... 2. asec. sstie ie ics eee) s) ete 73 C: “Fluorescent Antibody Stamine. 5.020525 fans eee 75 Wal Poxvirus oe Seesaw crs wees osiea sites Gis: os Ree ears Speman 15 (Neb VACCINI Ata. a etuae Saad Bets caret Aeon e sae 75 Be TRO WIPORY are sic "heise arb ocho: Bo ene nee Danae ae eae (ee) OC. = Shope Hibromain. 5 ..tn545 nestles see cheer Womens 80 WIT: Herpeswarus’) kocsis. 2 etna ee Bee a hse 80 A. “intranuclear Inclusion).)... 5 ce...) oe os ee eee ee 80 B, Sistochenmis try: 2.3. 50 cacao ie eee tiene ote tener rere 81 C:, Hlectron: Microscopy... cisa5s-8 os eee eee Iee 81 D.. Herpes Birra ssuwis i. ocicas aie stein s carn asters a center 81 K.. Fluorescent’ Antibody Stammg.). 05.22.0220 sone eee 82 WILT. Myxoviruses.s5.0: 00 25s dimes ants ae sone eee 83 AS. Entroductions 02% ob tau tae en nets ate te rok nee eae 83 By Filament: Formation: 52.05 one cc. esas arene 85 C; lhivang Cellsi 2 s3.ccuni caste Wiens Aoi aes eee Spee 86 DD: BixedTissies # dies. ctcaie otione ¥ ciatnae ue Reon eee 86 Ei: Electron: Microscopy-.«. « Mammary Canceriof Mices 2. os-ace cheese eee a. eewkemua of Mice ts oie oc bee eee seer eee Heo OW > Conclusion eves eo erste ae ee ee References ic. o os Se ne XIII. The Insect Viruses sy Kenneru M. Smiru...................-- ir lub III. IV. NG Wille Introdnetion\.. 2 22 coset desay, dem ste mele ieee 2. Influence of Environmental Conditions .............cceeeeeeeeeeees Jo influence of species of Red Cells. 5.028. soaes = oe eae ode tne se sieclens 4, Effect on Hemagglutination of Physicai and Chemical Agents... ...... 5. Effect on Red Cells of Physical and Chemical Agents................ 6. Increase in Titer of Hemagglutinin at 37°C. in the Presence of Ceils.... RED eIEIOM 4 Siaieie oc cere iais tie ols eiane ays anuto wel ocsilete S wintehoto ts cid aetna Giuince.apesiele epetee tele ieeeroduction: of sha hilized, Cells. 2226 cla tails sxeaeie crelaters wleletale oldar ope Denbroperties/or Stabilized (Celis. yjscs = eis crickets seo clals/aimiersis ohelersleveyayar siaieks D. Enzymes with Receptor-Destroying Properties...............++-2-2-- 1, The Receptor-Destroying Enzyme (RDE) of Vibrio cholerae........... Bmp GHYC GOED VITUS. 5 rane cove cia, siete Oe rch cla) etuexereacias 6 cymapsteielarelener io. shatels octane eeinhibitors OL HemagelutiMatlOli.,. « | bo me HC C alkali “ yc C-CO.H (CHOH), SNH. Oz COOH A 2 (CHOH), PYRUVIC Ne CHOH ACID PYRROLE-2CARBOXYLIC TETROSE CH,OH ACID D-GLUCOS AMINE Fie. 1. Degradation of N-acetylneuraminic acid to pyrrole-2-carboxylic acid. exists predominantly in the pyranose form and that ON-diacetylneur- aminic acid has the O-acetyl group attached to C, (Blix e¢ al., 1956; Klenk e¢ al., 1956). Siahe acid, the group name for ON-diacetylneuraminic acid, N-acetyl- neuraminic acid, N-glycolylmeuraminic acid, and other acylated neuraminic acids (for nomenclature see Blix, et al., 1957), is an intrinsic component of all hemagglutinin inhibitory mucoproteins (Odin, 1952, 1957; Gottschalk, 1955a, 1956a; Werner and Blix, 1956). NANA has been obtained crystalline from UM, serum mucoprotein, from the inhibitory mucoproteins present in meconium, ovarian cyst fluids, human cervical mucus, human milk, and from ovomucin; diacetylneuraminic acid from BSM (Klenk et al., 1955; Bohm et al., 1957; Zilliken et al., 1955; Odin, 1955; Blix, 1936). As product of enzymatic cleavage (virus or vibrio enzyme) NANA has been obtained with BSM, UM, and serum and meconium mucoproteins as substrates (Faillard, 1957; Bohm et al., 1957; Zilliken et al., 1957). Moreover, Klenk and Lempfrid (1957) were able to split off NANA from human erythrocytes with RDE and to crystallize 56 A. GOTTSCHALK the compound. This contribution proved beyond doubt the previously postul- ated chemical analogy between cellular receptors and soluble inhibitory muco- proteins, an analogy also reflected in the reduction of the net negative surface charge of both human erythrocytes and inhibitory mucoproteins upon RDE action (Hanig, 1948; Ada and Stone, 1950; Pye, 1955). It is of interest to note that the sialic acid of all potent hemagglutinin inhibitors is NANA. Thus, lipid- free extracts of equine erythrocytes (stromata), known to contain N-glycolyl- neuraminic acid, are devoid of inhibitory power, whereas the corresponding extracts of human erythrocytes, contaming NANA, inhibit virus hemagelu- tination (Yamakawa, 1956). This observation may have some bearing on the fact that equine red cells, in contrast to human red cells, are not readily agglu- tinated by influenza virus (Clark and Nagler, 1943). Another example is BSM. This mucoprotein has the highest sialic acid content known so far (17 %), of which at least 80 °% is terminal (Gottschalk, 1956b). Yet, its virus hemagglut- inin inhibitory power is very limited. BSM efficiently inhibits only influenza PR8 indicator virus; LEE indicator virus is inhibited very slightly, and eight other influenza virus strains are not inhibited at all. When living PR8 or LEE virus was allowed to act on BSM, no significant change in the net negative charge of the muco-protein was observed (Curtain and Pye, 1955). According to Blix (1958) the sialic acids of BSM are a mixture of ON-diacetylneur- aminic acid, N-acetyl-O-diacetylIneuraminic acid and N-glycolylmeuraminic acid. With regard to the position and linkage of sialic acid in inhibitory mucoproteins, it was shown by Gottschalk (1956b) that in BSM sialic acid occupies a terminal position and is joined through the potential keto group glycosidically to the adjacent unit. RDE released 64 °% of the total sialic acid present in BSM (Gottschalk, 1955b, 1956b). Heimer and Meyer (1956) and Faillard (1957) arrived at the same results. With UM as substrate, both the influenza virus enzyme and RDE split off 50-60 % of the total sialic acid present, and sialic acid only (Klenk e¢ al., 1955; Faillard, 1957). The over-all structure of the soluble receptors was early recognized (Gottschalk, 1952) as that of conjugated proteins, containing as prosthetic groups relatively small sized polysaccharides or oligosaccharides. In the case of UM, the prosthetic group, detachable by alkali, consists of galactose, mannose, fucose, glucosamine, galactosamine, and NANA in the molar ratios 6:3:1:6:2:3. The molecular weight of the prosthetic group is of the order of 12,300 (assuming acetylation of the amino sugars). Since the molecular weight of UM is 7 x 10® and its carbohydrate content about 21.5% (calculated as anhydro sugar), approximately 120 individual prosthetic groups are assumed to be attached to the protein core (Gottschalk, 1958). BSM contains 11.4 % N-acetylgalactosamine and 17 °{ ON-diacetylneura- minic acid, i.e., about equimolar quantities of the two components; in addition, CHEMISTRY OF VIRUS RECEPTORS Dit small amounts of galactose (0.7 4), mannose (0.2 °4), fucose (0.7 °%), and N-acetylglucosamine (1.0%) are present (Gottschalk and Ada, 1956; Heimer and Meyer, 1956). By very gentle alkali treatment, Gottschalk (1957c) has detached a disaccharide from BSM. This reducing disaccharide was recently obtamed in an analytically pure state; its formula is C,,H;,0,,N, and it consists of NANA linked ketosidically to C, of N-acetylgalacto- samine (NAGal), as shown in Fig. 2. The viral enzyme and RDE split the disaccharide into NANA and N-acetylgalactosamine. (Gottschalk and Graham, H NHAc HCOH | ECan ()CHOH 6-«-D-N-Acetyineuraminyl-N-acetylgalactosamine Fic. 2. 1958). By this action the enzyme is characterized as an O-glycosidase (ketosidase). Since both the viral enzyme and RDE invariably release from their substrates a terminal, ketosidically lmked, acylated neuraminic acid, the enzyme has been termed “neuraminidase” (Gottschalk, 1957b). From the evidence presented it would appear that the soluble influenza virus receptors (hemagglutinin inhibitors) are conjugated proteins with oligosaccharides as prosthetic groups. The size of the oligosaccharide may vary with the type of mucoprotein, and so may vary the number of prosthetic groups. Acetylated neuraminic acid residues are terminal units of the oligosaccharides; these terminal units are joined through a neuraminidase- susceptible glycosidic linkage to an adjacent sugar residue. There is every 58 A. GOTTSCHALK reason to believe that the cellular receptors are built up in a similar way. Both the terminal neuraminic acid of the prosthetic group and the polypeptide core providing the framework for an orderly spatial arrangement of the prosthetic groups are essential for attracting and binding the influenza virus particle. Enzymatic removal from the prosthetic group of the terminal acetylated neuraminic acid deprives the receptor of its binding power. Breakdown of the protein framework has the same effect. Thus, it has long been known that trypsin inactivates inhibitory mucoproteins (Burnet e¢ al., 1947; Hirst, 1948; Gottschalk and Lind, 1949a). The oligosaccharide on its own does not inhibit hemagglutination by indicator virus, as was shown with the disaccheride (Fig. 2) and with sialyl-lactose. This trisaccharide, which is present in milk, consists of diacetylneuraminic acid and lactose (Kuhn and Brossmer, 1956a). In this case, neuraminic acid is linked ketosidically to C, of galactose, the ketosidic bond being of the «-type (Kuhn and Brossmer, 1958; Gottschalk, 1957b). The trisaccharide is susceptible to virus and V. cholerae neuraminidase. Just as different mucoproteins offer different structural situations to one and the same virus, different strains of influenza virus present different sur- face structures to one and the same receptor. Differences in reactivity of the various influenza virus strains with one receptor, the red cell receptor, were first observed by Burnet ef al. (1946). They found that red cells from which one strain of virus had become eluted were still agglutinable by certain other strains. Graded decrease by RDE of the number of intact receptors available at the red cell surface rendered in a definite order one virus strain after the other unable to agglutinate the increasingly impaired red cell (receptor gradient; for details see page 29). The physicochemical expression of this stepwise decrease in intact receptors available or, as we know now, of the stepwise loss of sialic acid residues is the gradual decrease in the net negative charge of the red blood cells. Thus, the electrophoretic mobility of human erythrocytes is reduced from the normal value of — 1.30 x 10~® cm.?/see.-1/ volt! to a value characteristic for each virus strain of the gradient, the lowest value of — 0.37 x 10~° cm.?/sec.-!/volt being attained with swine influenza virus (Ada and Stone, 1950). RDE action on human erythrocytes reduces their electrophoretic mobility to — 0.17 x 107° cm.?/sec.—4/volt=}, i.e., by 87%, indicating that the net negative surface charge of human erythrocytes is due nearly exclusively to the dissociated carboxyl group of sialic acid (pK’ = 2.60 + 0.05 at 20°C. and 0.05). The receptor gradient probably reflects the degree of steric hindrance to the close fit between the complementary combining groups of virus and receptor, exerted by the surface structure of the individual virus strains. In a similar fashion Burnet (1955) has invoked differences in “‘accessibility”’ of receptors to account for the gradient. Since the surface structures of the virus receptors, whether CHEMISTRY OF VIRUS RECEPTORS 59 cellular or soluble receptors, vary, it might be expected and was substantiated experimentally (Stone, 1949b; Burnet, 1949) that the position of the virus strains at the gradient varies with the type of receptor taken as reference substance. If the phenomenon of hemagglutination by viruses (see page 21) is inter- preted to mean the existence of specific receptor groupings at the red cell surface for the virus concerned, then many other receptor substances must exist. 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Virology 3, 464. Chapter V The Morphological Approach F. B. Bane Johns Hopkins School of Hygiene and Public Heaith, Baltimore, Md. Pee NAG POM CHLOE 6 cis) siatase siete! oys Sieve. o) 616 5/8) are aves ovals hci wi bide atayeievelordia Sco /piove aie Gere 64 Miners ETA AUK CU Ney aca wee ps Sa yay ee donee none tke: ohal Ss wiawelle case terest micros detainee ees 65 od BITRE OA SA EW LO gO a aR a i gee te 67 JE}; RArieotel evoya ts ba!) Rete arte ob eer tne 9 em eae nel ce Sole aR Laveen ae rea 67 SP PEPIN CCI OGIR staan ire etite ate eta le (ors een. o 8 eoys etait anit Sie aye omer Ne ste aeons Meets 67 PP SESCETCLION Hs toe cetenere aie ate o fettiere aioe ein ts ete eR bene Sam ee Ge oie 67 BP ONTRO LIC CHANVER 5 or1s c/o Warsnciasarsistailaran eee carol 5) sare a eteee aerate ae ies 68 iii. Pathological Aspects of Cell Morphology. ...:.........ccscccccevcsenses 68 PSP CMU SLEL ACER noc isa tcuettveds ave eerste Cae ace aiRieie cleats aietseierdiotetow Sem iow eee 69 Eye Omit cle “Lied DAIAHCO\ oh 3c ole state arelsuass aioeGPe wigs ateoe mein tacts mee ote eure ene 69 MP PeV UO FAO IAULTESL 3 aha, 20, salar gece at slong ao Geigy diet sishanchete atoeomra ae acetneiessas aime arate 70 Dy haranaclear Ey PerbrOopuy~ aceaelaie a ala aamicte weve iat tela 80 IES erat USOC MLCRIAR ETSY fo tess Fl de cos oasis incre SimVe7s ck alepan Siege ce x late Re tehes Oe I 81 Wee leCERORT MICEOSCO PY, fact rcle «ever uge ois craic waren a a Rilaiods Sia Rie ono ersbemas eee 81 HD MPEV OT CSOs raptadetans heterehsaake Weer are on by Seals SOSA rato DRM Ge Rie CRUE 8] HS blnorescent Antibody Sram 231.10 is adi.) ions eres ooo eos desis onien 82 eal ieee iy KOWACUISOS o/c¥ ie / Suenos testo abo oh ove w ie jeune Eola Rua oA eedeeere One 83 EES eeeloHl aIVIOT oH OEHAA.DIOU ws ats ic Staite ts. Nabe sito ey8s cere: auc cuhd oeiott ose ytala nia ordtaas of 85 ep itiyseIAE Ol Raretentorete ays ate came ene sos SRI te 5 os Sow ee ee ais Ui Se le 86 IDS d BO Co 3 UM WIEST econ oe Aga a Re eae eR an ean aa 86 me LEC ERO MICEORCOY Fie aiken stots cares es tS eagle Shae Re chow’ s bw wlan ee 87 64. F. B. BANG f, Fluorescent Antibody Staining). .< -.7..15 <0. -<)scma. eee oe 87 dM PBS £ sd fos os 3 eee os eee ee oe ek clae ci Shek een 88 2) Maa eN a 3S aps. 5 5 Bsn eae @ eres means Sshenstisus “oesyaieh sop aistae eaten eee 88 3. Bowl Plague. cys « conus te Reeve ae te ore ore oem etna 2. he ee 88 Gs dIncomplete. Varuse- 5. nos os. ccets atals pected scala loro each stats) aie hee eee 89 TEX; Aen OVINUISOS aie. tic 5 eS ee hod aayeres leks wele oiero Crmlenanae sliproteve tors) a) ier cee eee 89 A. Cytochemistry: d22.. (5c. fol os 5 aks alaveae: Obes ee cdeveare era iets eee eda 89 Be Mlectron Microscopy... +. 2 bocce Se. oki = selec eee cies 90 X. Poliomyelitis and Other Neurotropic Viruses.................00sseeeeeee 91 A, Nerve Cellais oo oa se-ccars dacies eg acee ge osu eae Lb ausie arn 91 LsClassie Windings.< 0.0.6. os. 2a sins oe ool ose ee a 91 2.9 Tissue: Culture: sco. ow neal eos aiiiom < 9000). Fie. 12. Contiguous thick section showing the same crystal which is strongly colored by the Feulgen reaction. (Courtesy Dr. Councilman Morgan, from J. Biophys. Biochem. Cyt. 3, 1, 1957) (Magnification: « 3700). Fic. 13. Electron micrograph showing partial destruction of rat fibroblast by virus of eastern equine encephalomyelitis in tissue, culture (From Bang and Gey, 1952) (Magni- fication: x 20,000). Fic. 14. Tissue culture of human sarcoma cells by phase microscopy. (Courtesy Stoler and Gey, 1953.) Fic. 15. Tissue culture of cells from same line (AF) showing initial stages of cell destruction with poliomyelitis virus. (Courtesy Stoler and Gey, 1953.) THE MORPHOLOGICAL APPROACH oi same general thickness as the virus particles themselves, would contain different proportions of the virus particles, dependent upon the amount of spherical virus caught in the particular section. Low and Pinnock (1956) have further analyzed these appearances and rule out other arrays as explain- ing the pictures obtained by Morgan et al. (1956b). Recent proposals by Valentine and Hopper (1957) that the true shape of the virus is polygonal (as seen in dried specimens), and that the shape is dependent upon the method of preparation (Tousimis and Hilleman, 1957) may have relevance to this characteristic crystallme pattern. Bloch et al. (1957) studied intranuclear lesions from the same nuclei in Feulgen-stained and in electron microscope preparations. By studying alternate thin and thick sections of Hela cells infected with types 3, 4, and 7, the intranuclear Feulgen-positive masses were positively identified with the ordered arrays of virus particles (Figs. 11, 12). It was proposed that the virus particles developed from a Feulgen- negative matrix. It was further suggested that viral and host DNA may be differentiated by their reaction to a Feulgen azure-stainmg method. Finally, type 5 virus infection of another cell type (Hep 2) has been shown to contain an unusual crystalline protein within the infected nuclei. This new crystal- line lattice consists of much smaller units and the complete inclusion is Feulgen-negative. Tt is clear from all of these studies that an exciting beginning to the study of intracellular lesions with this virus has been made. The dynamic aspects will need not only sequential studies, but living cells and stained preparations must be analyzed with fluorescent antibody. X. POLIOMYELITIS AND OTHER NEUROTROPIC VIRUSES A. Nerve Cells 1. Classic Findings Until the relatively recent evidence that a variety of tissue culture cells was susceptible to poliomyelitis (Enders, 1954), attention had centered on the lesions of the central nervous system and preeminently on the anterior horn cell. Although the pathology has been fully reviewed (Bodian, 1948; Howe, 1952), the main points will be recapitulated in comparison with lesions produced in the tissue culture system. The progressive changes begin with an initial massing of chromatin, continue with chromatolysis, which progresses even while mitochondria remain intact (McCann, 1918), and proceed eventu- ally to complete necrosis of the cell and subsequent variable neuronophagia. Of particular interest now are some samples of a motor nerve cell lesion which was occasionally present after the acute stage of paralysis (Bodian, 1948). This lesion comprised a large eosinophilic mass, sometimes with the nucleus 92 F. B. BANG “apparently verging on extrusion from the cytoplasm.” In addition to actual destruction of the nucleus in the terminal stage of the infection, small eosinophilic intranuclear inclusions were sporadically found. Electron microscope studies of cells of the central nervous system infected with poliomyelitis have not been reported. However, the identification of the Nissl substance which is first destroyed by virus as endoplasmic reticulum (Palay and Palade, 1955) and the presence of quantities of this material at the tips of the dendrites adjoining axons (Palay, 1956) would suggest that the virus is able to parasitize susceptible material throughout the cytoplasm of these specialized cells. 2. Tissue Culture Living nerve cells have been studied in relation to the effects of the virus upon them in tissue culture. In all three types of virus (Hogue et al., 1955, 1958), the first identifiable lesion was contraction and disintegration of the extended dendrites. Contraction began at the tips of these long processes and progressed until a process had withdrawn, at times leaving a bulbous extrusion. Later the cell became granular, lost its surface film, and assumed the character of a loose mass of granules surrounding the nucleus. Serial photographs demonstrate these changes. Although pinocytosis has been seen to occur at the tips of the axons in nerve cells in tissue culture (Hughes, 1953), the role of this event in susceptibility to virus has not been considered. B. Epithelial Cells and Frbroblasts There have been many careful studies of the effect of poliomyelitis virus on tissue culture cells other than nerve cells. These include phase (Stoler and Gey, 1953; Barski et al., 1955; Riessig et al., 1956; Klone, 1955a; b) and bright field (Harding et al., 1956) studies on living cells (Figs. 14, 15). There have also been several sequential studies of such changes in which cells were stained and fixed in correlation with the time of virus release (Riessig et al., 1956; Dunnebacke, 1956a). 1. Morphological Changes Observed in Living Cells The sequence of changes has been studied in individual cells by phase microscopy. Kléne (1955a) followed the effect of virus on monkey kidney cells which showed no lesions 23 hours after infection. These retracted from surrounding cells at 25} hours, the nucleolus lost form, and protoplasmic extrusions appeared. Mitochondria remained normal through all of this activity. At 26} hours the cellular contraction continued, and the sharply defined, irregular cell extrusions progressed. By 274 hours the extrusions developed a series of fine extensions and branches, the cell nucleus was only obscurely visible, and vacuoles appeared in the cytoplasm. In the course of a THE MORPHOLOGICAL APPROACH 93 few hours, therefore, changes in the nucleus and cytoplasm appeared almost simultaneously. In comparing these findings with the data on fixed and stained cells, it is important to remember that only extremely careful cyto- logical preparation would preserve intact the irregular peripheral spread of endoplasm so that it could be identified in fixed and stained material. Barski et al. (1955) recorded the changes produced in large fibroblasts derived from tonsils in motion pictures taken by phase microscopy and described the development of large eosinophilic paranuclear masses. These occurred with both types 1 and 2 in cultures kept at 30 to 31°C. for 20 hours after the addition of a virus. The cell remained motile, and filamentous mito- chondria persisted. Later, a series of bubbling extrusions developed, and the authors suggest that this is the mechanism of virus release. Very similar, if not identical, paranuclear lesions have been described in anterior horn cells by Bodian (1948); it is difficult to differentiate this lesion from the giant centrosphere described by Lewis (1920) in degenerating mesenchyme and in tumor cells. It may represent an abnormal accumulation of normal cellular material, rather than a specific virus “inclusion.” The material has been shown to be Feulgen-negative (Harding et al., 1956). 2. Relation to Virus Release The exact relation of these cell changes to the time of virus release has not been settled. In studies on isolated individual cells, Lwoff et al. (1955) showed that about one hour before the beginning of virus release into the medium the cell started to contract, and a hyaline zone developed at the periphery of the cell. At the time of virus release this zone became vacuolized and then disintegrated, by which time virus release ceased. Riessig et al. (1956) have followed the changes in cultures of monkey kidney cells during one phase of virus multiplication. Most of the preparations utilized hematoxylin, eosin, and Feulgen stains. Osmium fixation was not used. Some of the living cells were studied by phase microscopy. Although gross changes were not apparent in the infected test tubes until 8 hours after infection, the stained preparations showed a patchy disappearance of the chromatin network of the nucleus and development of an eosinophilic cyto- plasmic mass by 5 hours, with the persistence of the nucleolus. The formation of intracellular virus began at about 4 hours, and extracellular virus increased from 5 hours on. The authors suggest that the changes in the appearance of the cell seen with phase microscopy do not begin until 1 or 2 hours after intracellular virus increases. In studies on monkey kidney, HeLa, and human fetal cells, Dunnebacke (1956a,b) found that virus release occurred several hours after nuclear pyknosis, contraction of cytoplasm, and rounding of the cell. In contrast, human amniotic cells released virus much later, and the lesion in these cells 94 F. B. BANG as contrasted with monkey kidney, started with a disappearance of the nucleolus (Dunnebacke 1956b, 1957). An interesting attempt to segregate the process of viral synthesis from the changes seen in the cell used the metabolic inhibitor, fluorphenylalanine (Ackerman et al., 1954). Apparently, once the virus infection was initiated, although actual production of infectious virus was inhibited, cell changes proceeded. The changes described are mainly terminal, however, and a more detailed comparison of the viral growth and the cell changes in the same cultures is needed. 3. Other Findings An interesting attempt was made by Kléne (1955b) to determine whether infection of monkey kidney cells had any direct effect on mitotic activity. No increase or decrease in mitosis rate was observed, but certain findings suggested that cells might be infected before mitosis set in, and that division would then proceed with eventual death of both daughter cells. By following individual cells for a number of hours, he was able to show that both daughter cells were destroyed at similar but not identical times. In one case, a cell was followed through to an abnormal telephase, from which the cell did not recover but fell apart. In an electron microscope study of “virus-like bodies” in the nuclei of epithelial cells infected with type 1 poliomyelitis virus, Ruska e¢ al. (1956) have reported on the occurrence of accumulation of particles of about 26 mp In size, in close association with remnants of nucleoli. The occurrence of these particles, which in themselves are not highly distinctive, needs to be corre- lated with the appearance of infectious virus and the sequence of cellular changes. The localization of poliomyelitis antigen by fluorescent antibody (Buckley, 1956) shows that type 1 may be found both in the nucleus and the cytoplasm and may be found in peripheral blebs of the cell as it breaks down. In the early stages specific fluorescence was diffuse or granular. A summary of concurrent findings on the effects of poliomyelitis virus on living cells in tissue culture would include: (1) Peripheral contraction of the thin endoplasmic spread, which leaves ghosts of branched material behind to simulate irregular extrusions; (2) disappearance of the nucleolus; (3) forma- tion of a large, paranuclear mass. It seems clear that the morphological steps which lead to final death and disintegration of the infected cell cannot as yet be set in proper sequence. The cells vary in their response to the virus so that, as yet, early cytoplasmic changes have not been differentiated from nuclear destruction. Undoubtedly, part of the difficulty is that different cells react in different ways to the same virus (Dunnebacke, 1957). Thus, morphological evidence does not tell us THE MORPHOLOGICAL APPROACH 95 whether virus may be multiplying in the cytoplasm or the nucleus. The mito- chondria, however, seem both in the nerve cell and tissue culture to remain unaffected until fairly late in the destruction. C. Encephalitis Although there have been relatively few cytological studies of the effect of the arbor (arthropod-borne) viruses on cells, these do illustrate certain general problems and so will be discussed here. The virus of eastern equine encephalomyelitis produced varying degrees of gross destruction of cells in tissue culture, dependent upon the cell strain used (Bang and Gey, 1952; Bang et al., 1957). Using a tissue culture method of preparing cells for electron microscopy, the cytological aspects of these infections were studied. It was shown (Bang and Gey, 1952) that piecemeal destruction of the cell occurred. Individual fibrillar processes (Fig. 13), normal for the rat fibroblast, were destroyed by the virus before any effect on the rest of the cell was apparent. In other cells, the virus was found on the very edge of the cell, sometimes in the presence of destruction, sometimes without any change. Subsequent quantitative studies by Dulbecco of virus release from cells showed that this virus is gradually released from cells over a period of some hours. This picture of varying or limited amounts of destruction is completely changed when the same virus is studied in primary explants of chick em- bryos, either in roller tube cultures (Bang, 1955a) or in slide cultures (Bang and Gey, 1949). Here, the destruction was shown to be accompanied by an almost complete replacement by the virus. A somewhat related virus (Egypt 101) has been studied by the fluorescent antibody technique not only in mouse brain but also in cultures of human epidermoid carcinoma (Noyes, 1955). The antigen was found 24 hours after infection in the cytoplasm, but there was no nuclear staining. There was some concentration of the antigen in the paranuclear area at first, but later it was found distributed throughout the cytoplasm. XI. Tumor VIRUSES Three fundamental questions may be raised concerning tumors induced by viruses: 1. Are cells which are infected by neoplastic viruses morphologically different from cells infected with other viruses? Specifically, are there differ- ences in the localization of the virus, the alterations in ultrastructure in the cell, the mitotic abnormalities, and the mechanisms of virus multiplication and virus release? 2. Is there any direct relationship between the presence of morphologically identifiable virus and the malignant nature of the cell? Are there reliable 96 ; F. B. BANG controls, is there morphological evidence of lysogeny and/or latency, and is the continued presence of virus demonstrable throughout the existence of the tumor? 3. Is the specificity ascribed to some tumor viruses reflected in analogous specific effects in tissue culture? This question remains unsettled, even with respect to the well-studied Rous chicken tumor virus. Although the answers to these questions cannot yet be conclusive, it is encouraging that it 1s now feasible to ask them, and that the implements for finding the answers are available. The five selected types of tumors will emphasize the variety of cellular reactions to the viruses. This variety of reactions should not, how- ever, obscure the fact that data on the above questions are not complete for any one virus-host cell system relationship. A. Rous Chicken Sarcoma Although this tumor was proved to be of viral etiology almost 50 years ago (Rous, 1911), there is a present need to understand the effect of this virus on living cells of established strains. Many early observations on living cells have been obscured by controversy over the nature of the tumor cell, whether it is macrophage or fibroblast. It is ticklish today to describe with assurance the effect of the virus on macrophages and fibroblasts, for it has been pro- posed both that the virus could convert macrophages into fibroblasts (Carrel and Ebeling, 1926), and that under other virus-induced conditions fibro- blasts could become phagocytic (Fischer and Laser, 1927). Some light may have been shed on the enigma by the study of Sanford et al. (1952), who showed that fibroblasts grown in horse sera would support growth of the virus for six months, while macrophages failed to support it after a few days. There is no record of a comparable experiment using chicken sera. Borel (1926) showed that cultures of the Rous tumor consisted of two types of cell: round basophilic cells and fusiform fibroblasts. The macrophages often yielded multinucleated plasmodia which at times reached a diameter of 600 ». The fusiform cells were occasionally equally large. Both types of cells showed an accumulation of eosinophilic material in the cytoplasm corresponding to the eosinophilic paranuclear mass seen in the tumor cells in the animal. Hypertrophied nuclei and an accumulation of fat droplets were characteristic, as was an extraordinarily lavish network of mitochondria. 1. Effect on Cells in Tissue Culture The most detailed study of these cells as cultivated in vitro is that of Doljanski and Tenenbaum (1942; Tenenbaum and Doljanski, 1943). They documented and fully illustrated the “distinctive syndrome of severe cell disease.”’ Although their work was carried out on an “18-year old strain of the tumor,” it was maintained with the regular addition of fragments of THE MORPHOLOGICAL APPROACH 97 normal tissue. Thus, the cell types themselves cannot be evaluated. In spite of this factor, the variety of lesions and the detailed photographic accounting of them are unsurpassed. These authors not only confirmed Borel’s findings but described lesions of the nuclei in the basophilic cells in which an aggre- gation of the chromatin was seen within a decidedly granular nucleus. Some of the nucleoli assumed peculiar shapes or were pushed to one side by eosino- philic masses negative to Feulgen stain. Occasional nuclei were packed with eosinophilic inclusions. The marking-off of the central region of the cyto- plasm was accompanied by the formation of intracellular crystals. Atypical mitoses were common as was giant cell formation, and chromosome lag during mitoses was illustrated. A phase microscopic study (Lo et al., 1955) of living cultured fibroblasts in non-chicken media and inoculated with the Rous virus showed that normal fibroblasts were transformed into abnormal cells which developed typical paranuclear inclusions and fatty accumulations. Giant cells formed again with great networks of normal appearing mitochondria. 2. Electron Microscopy Since the first cautious description of ‘“‘small bodies, the size of that esti- mated for the transmitting tumor agents,” having the appearance of ex- traneous entities within chicken tumor cells in tissue culture (Claude et al., 1947), there has been a series of descriptions of these particles both in tissue cultures (Bernhard ef al., 1953; Epstein, 1956) and in sections of tumors (Gaylord, 1955; Bernhard et al., 1956a). The identification of these particles as the tumor agent is as yet incom- plete, but several of the criteria (Bang, 1955b) for such identification are gradually being fulfilled. The particles from the first were recognized as having an internal area of high density and a peripheral area of slight density. Recent pictures show an external membrane and a thinner internal membrane between the dense central portion and the external membrane (Bernhard e¢ al., 1956a). Thus, there is a fairly characteristic arrangement or grouping of the particles and an internal morphology for the individual particles. It is much more difficult, however, to make a clear statement relat- ing these characteristic particles to the infectiousness of the material. Epstein (1956) was able to show a correlation between the percentage of cells showing these particles in vacuoles and the infectiousness of the extract. Whether these round and vacuolated cells obtained from ascites-like passages of the Rous tumor are indeed the tumor cells or are infected macrophages like those originally described by Borel is not pertinent to the identification of the particles. However, it is clear that there is a correlation between (a) the cells which contain particles and (b) the infectiousness of the suspension; and thus, indirectly with the particles. A more direct comparison of the VOr. ie / 98 F. B. BANG number of particles seen in sections of tumors and the infectiousness of the tumor extract has recently been completed (Hagenau ef al., 1958). In this study the more infectious tumors showed the greatest number of character- istic particles, and the pellets obtained from highly infectious extracts showed many more particles than those of low infectiousness. It is clear that a beginning in the identification has been made. However, the immuno- logical tests, purification, spray droplet correlations, etc., which may con- clusively establish these particles as the agents of infection have not been completed. Confirmation is especially important. A small percentage of normal chick embryo tissue cultures yield preparations with large numbers of identical particles (Bang, 1954; Gey and Bang, 1951). Further complications are added when related tumors, such as the Murray- Begg endothelioma (Rouiller et al., 1956) and leukemias (Benedetti et al., 1956), are studied for both of these have similar particles. In the latter, 3 of 24 spleens from “normal” chickens showed the same particles. The morpho- logical difficulties have then emphasized the fact that most stocks of chickens acquire antibodies to these agents (Andrewes, 1939; Bang and Haley, 1958), and that uncontaminated control animals are difficult to obtain. It is tempt- ing to explain these contradictions by supposing that virus tumor cells carry the virus in a latent lysogenic state, and that the infectious and visible form of the virus is important in producing what may well be secondary patho- logical changes in infected cells, whereas the really malignant cell does not necessarily carry the virus in this form. The recognition that the disturbed cell is one in which host cell and virus are not in balance, whereas the trans- missible tumor cell is in balance with its virus would support this idea (Bang, 1955a). 3. Localization of Virus In all of the above studies on the Rous sarcoma the viruses have been found predominantly at the surface of the cell or within vacuoles which are in a way not in contact with the cell. It is, therefore, not easy to perceive the method of formation of the complete virus. In a few cases particles have aggregated within vesicles which could well be altered mitochondria. The possibility that the characteristic virus particle, which is assumed to be the infectious unit, represents only a terminal phase of the partnership which is the tumor cell, focuses particular interest on the morphology of the tumor cell. Marly observations on this are presented in the beginning of this chapter. Recent electron microscope studies of the “fibroblast-like tumor cells of Rous sarcomata”’ (Epstein, 1957) show that the ultrastructure of these cells is similar to that of other cells and fails to bring out any qualitative morphological difference between normal fibroblasts and the tumor cells. Attention was, however, directed upon the tightly packed piles of smooth THE MORPHOLOGICAL APPROACH 99 cisternae. These were found in two separate areas of the tumor cell, whereas they are normally found in only one area. These may represent part of the story of the hypertrophied paranuclear area. B. Mammary Tumors of Mice Although this tumor may now be listed among those associated with viruses, the biological data establishing this pomt are more complex than those available for the chicken tumors. The milk agent operates within a more restricted set of conditions (sufficient hormonal stimulus and a particular genetic background are essential) and the tumor itself has an extremely delayed incubation period. Secondly, the spontaneous mammary tumors which occur in low incidence in mice in the absence of “‘the milk factor’ nevertheless have, in many cases, a histological structure indistinguishable from milk factor tumors (Dunn, 1953). In the continuing search for the nature of the cancer cell attention has repeatedly focused on variations in neoplastic cell structure. Nevertheless, the behavior of a group of cells has remained more diagnostic than the appearance of any one cell. For this reason, the recent study of Foulds (1956) is of particular value in orienta- tion for a detailed study of individual cells. The restricted portion of tissue which can be studied in the electron microscope field has so far prevented adequate correlation of ultrastructure with the distribution of the developing malignancy. 1. “Inelusion’’ and Virus Particles Kosmophilic cytoplasmic inclusions were described by Guerin in 1955 in several lines of mammary tumors. Almost at the same time, studies with the higher resolution of electron microscopy (Bang’and Andervont, 1953; Bang et al., 1956a,b; Bernhard et al., 1956b; Dmochowski, 1954) showed that masses of intracytoplasmic particles thought to be virus could be commonly found in mammary tumor cells. Direct comparison of the cytoplasmic in- clusions (Bernhard et al., 1956b) and the eosinophilic inclusions showed that a large part, if not the entire mass, is made up of such particles. The first description of these particles is without question accreditable to Porter and Thomson (1948), who found clusters of these particles with an average out- side diameter of 130 mu and a dense center averaging 75 my in epithelial cells grown in tissue cultures from spontaneous mammary tumors. They were found in three of the six tumors studied in this way. It is likely that these “virus-like bodies” actually were at the surface of the cell, although several clusters which appear to be within the osmium-digested cell are also shown. Sections of infected cells, however, have the great advantage of affording accurate localization of the particles. Remarkably similar pictures were 100 F. B. BANG obtained by three separate groups of investigators (Bang, 1955b). Two types of particles were found and may be interpreted as representing two phases of development. One type was seen in paranuclear clusters of spherical (oval as compressed in sectioning) particles, having an outside diameter of about 65 to 70 mu. The particles were also scattered throughout the cytoplasm and sometimes encircled small vesicles like a series of beads. They did not appear to have any specific relation to intact mitochondria, but it is possible that some of these small vesicles were remnants of degenerating mitochondria. Pictures suggestive of this have been published (Bang et al., 1956a,b). Bernhard e¢ al., (1956b) has shown that this type of particle is actually double-layered, with an internal diameter of about 38-50 my and an external, sometimes less dense layer of about 10 my. 2. Morphological Evidence of Virus Release The second group of particles is larger, was found either within vesicles, on the surface of the cell, or attached to the microvilli of the cell. The possibility that they are formed by the ejection of the smaller particle either from microvilli (Bang, 1955a; Bang et al., 1956a,b; Bernhard et al., 1956b) or from the free surfaces of the cell follows the pattern of virus release in other cell infections (Figs. 16 and 17). Their presence as dense particles surrounded by less dense “cytoplasm,” yet still within the cell, might result from the projec- tion of finger-like extrusions of the host cell cytoplasm into an intracellular vesicle which was the residuum of a collapsed mitochondrial structure. There is, however, no evidence of selective localization in or near mitochondria. Serial sections have shown that they are released all along the free surface of a cell and that they may also project by a series of microvilli into fluid pockets between cells (Bang et al., 1956a,b). The particles may be readily distinguished from the larger, more homogeneous milk particles or droplets the secretion of which entails a continuous breakdown of the cell surface (Bang et al., 1956a,b; Bernhard e¢ al., 1956b). Except for one preliminary, unconfirmed report (Kinosita et al., 1953), there is no record of their presence in the nucleus of the host cell. Since identical particles have been found by three different groups in a number of mammary milk factor tumors and rarely, if ever, in normal cells, and since their characteristic shape, arrangement, and apparent release from the cell surface follow those of other viruses, they are the most promising candidates for identification as the virus of mammary tumors. However, present biological knowledge of the virus is limited to factors known to be transmissible by milk and possibly by seminal fluid. Again, there is agree- ment (Bang et al., 1956a,b; Bernhard et al., 1956b; Dmochowski et al., 1955) that identical particles are found in mammary tumors which lack the milk factor. Hither these particles do not represent the milk factor or they may Fig. 16. Vesicle between two mammary tumor cells. Clump of early particles in right lower area of cytoplasm. Microvilli with virus particles within them projecting into vesicle. (From Bang et al., 1956a) (Magnification: x 18,000). Fie. 17. Collection of virus particles in cytoplasm of mammary tumor of mice. Two projecting microvilli are seen in uprer left area. (Courtesy Dr. W. Bernhard: Bernhard, Guerin, and Oberling, 1956b.) Fia. 18. Tissue culture preparation of frog adenocarcinoma with cytoplastic inclusion and lobulated nucleus. (Courtesy Dr. W. Duryee.) Fic. 19. Thin section of frog adenocarcinoma showing mature virus particles within cytoplasmic vesicles. Complex structure of mature virus particles apparent. (Courtesy Dr. D. Fawcett, 1956.) THE MORPHOLOGICAL APPROACH 101 just look the same and may be equally essential to the mammary cell but spread from mouse to mouse by a route other than the milk. Further com- plexity was created when Fawcett and Wilson (1955) found somewhat larger particles in hepatoma cells from the mice (milk factor C; H mice) which commonly develop mammary tumors. Most of the work reviewed here on the mammary tumor agent has been concerned with the comparative morpho- logy of tumor cells and normal cells. There is sucha dearth of knowledge about living cells that Lasfargues’s (1957a) cultures of adult normal and malignant mouse mammary cells are of particular interest. His observation of particles resembiing those seen in the sections but found at the surface of the tissue culture cells (Lasfargues, 1957b) adds to the potential importance of this method of study. C. Warts (Human Papillomas) The viral etiology of these persistent benign growths has been indicated for a number of years (van Rooyen and Rhodes, 1948). In any common clinical sample of warts a few may be observed to differ from their neigh- bors at the same sites in that they have a smooth margin, less keratinization, and a surrounding erythematous inflammatory halo (Bunting e¢ al., 1952). Water extracts of such warts were shown to have masses of small, round, uniform particles which were often in a crystalline array (Straus e¢ al., 1949). Bunting showed that these apparent virus particles were closely packed within the nuclei of affected cells and in such crowded conditions measured about 38 mp in diameter. In the cells of the Malpighian layer of the neo- plastic epidermis, the particles were in the nucleus and did not occur in the prominent eosinophilic intranuclear inclusions or in the cytoplasm which contained characteristic discrete dark masses. In cells forming the lower stratum corneum of the wart, at a time when the nucleus was no longer recognizable, the particles occupied almost the entire cell (Bunting, 1953a). Bunting (1953b) later suggested that “apparently as the inclusion body becomes larger, it becomes granular and then becomes ‘converted’ into virus particles. These then increase in number and fill the nucleus. At first they are not in an array, but when the number is so great as to distend the nucleus when they are tightly packed then the array appears.” Investigation of these papillomas was suspended upon Bunting’s untimely death, but the similarity of these lesions with the changes wrought by some strains of adenovirus is striking. D. Frog Adenocarcinoma Since the incidence of spontaneous carcinoma of the kidney of frogs may be greatly increased by the injection of various filtrates from such tumors, 102 F. B. BANG and since intranuclear inclusions occur in these tumor cells (Lucké, 1939), it is generally accepted that this tumor is caused by a filterable agent. Since investigation of the agent is still preliminary, this discussion will be limited to the cytological changes which differentiate the tumor cells from normal. The recent much-needed tissue culture studies of Duryeé (1956) have brought us much closer to realizing a method by which a known number of virus particles may be placed on a known number of cells so that the morpho- logical events involved in a one-step growth curve may be followed. The changes, which he has labeled as precancerous, may then eventually be quite precisely determined. Under less exact conditions, Duryeé (1956) has studied the following changes produced by this virus in living cells: (1) Large irregular nucleoli, which often showed pulsations. (2) Large cytoplasmic inclusions, which were thought to originate in the nucleus, and were seen passively to extrude through the nuclear membrane. (3) Giant cells with 3 to 55 nuclei per cell. In addition to these direct observations, it was determined by means of a microdissector that the intranuclear inclusions were semisolid, gelatinous masses (Fig. 18). An electron microscope study of these tumors by Fawcett (1956) has revealed numerous “‘virus particles’ throughout these cells in about one-third of the tumors examined. These are hollow spheres (90-100 my) with a thick capsule and a dense inner body (35-40 mu). These particles were found in the cytoplasm, occasionally in the nucleus, and in the microvilli, where they were presumably being extruded into the extracellular spaces (Fig. 19). The intranuclear inclusion bodies which are described above were found to be largely made up of hollow spherical vesicles with a thin limiting mem- brane. These are thought to be “immature virus particles.” A few of these contain a dense inner body like the “mature” cytoplasmic particles. Bundles of dense filaments and vacuoles, which were also found in the infected cells, were not explained. A sequence of development of the virus is suggested, but, again, is completely dependent upon hypothetical order of events. In summarizing the distribution of virus particles in the different virus tumors we have included data on the Shope fibroma, even though it is re- viewed in another section. It is evident that the tumor viruses, like other viruses, are found throughout the cell. Moreover, there is nothing distinctive about the type of cell pathology produced by them. This lends credence to the idea that these tumor cells in which virus is found and changes are produced are the cells which are out of balance with their parasitic virus. Thus, the balanced state, which indeed may be one analogous to that of the lysogenic bacteria, has not as yet been technically distinguishable from the normal cell. THE MORPHOLOGICAL APPROACH 103 XII. Discussion AND CONCLUSIONS In summarizing the experimental data on virus infections no attempt will be made to systematize contemporary knowledge of individual points but rather to collate some of the ideas and trends which are reorienting and even reorganizing research in this area. 1. It is axiomatic that a morphological study of virus infections involves the interaction of virus and cell. In this context the pathology of the cell may be expressed as virus action, cell reaction, and subsequent interaction until the cell overcomes or is destroyed by the virus. Given, then, the pathological state, the first necessity is identification of the virus, and for this there is no set of rules. Criteria for the identification of viruses in the electron microscope have been tabulated and discussed elsewhere (Bang, 1955b). These criteria have been generally fulfilled in the case of the poxviruses and perhaps in several others. Viral antigen may now be identified within the cell by the fluorescent antibody technique (Coons, 1957), and the application of this technique will no doubt be greatly expanded. The determination of that part of cell pathology which is a reaction to the virus is more difficult. Similar changes which are known to occur under various unfavorable conditions have perhaps been taken too little into account. An excellent example is the hypertrophied paranuclear area in infection with poliomyelitis, which is simulated if not duplicated in degenerating mesenchyme and in a variety of tumor cells. 2. The pathology of the cell now implies, not only the reaction of the entire cell, but also lesions of parts of the cell independent of apparent simultaneous change elsewhere within it. The adenoviruses are identified as masses of virus particles inside the nuclei of otherwise intact cells. The mitochondria in infections with poliomyelitis or Rous sarcoma virus remain intact until late in the extensive cellular changes which entail the accumula- tion of paranuclear material and, in poliomyelitis, the onset of nuclear de- struction. Great growth of abnormal microvilli, which seem to have virus withm them, develop at the surface of a cell infected with influenza or New- castle disease virus, and yet the mitochondria are normal even at high re- solutions in the electron microscope. Thus, we are beginning to get away from the term “inclusion,” and we have avoided it here except when used in an established framework. It would be more accurate today to speak of lesions ° within the cell and to describe them as virus, virus by-products, or reactions on the part of the cell to the virus. 3. The ways in which a cell can react to a virus are probably limited, and the same reactive processes may be induced under a variety of conditions. The microvilh, which are normal extrusions from epithelial cells, seem to contain and, in some cases, to release virus in vaccinia, influenza, Newcastle disease, and frog adenocarcinoma. Even rickettsia may be extruded from the 104 F. B. BANG cell by means of long fibrils. The intracellular fluid vacuoles, which develop in tissues infected with the larger viruses, may be considered as a way of separating parasite from host cytoplasm. The loss of osmotic competence of the cell is reflected in ballooning of the internal membrane system (endo- plasmic reticulum) and the development of fluid vacuoles between the nucleus and the cytoplasm. 4. A series of electron microscope studies, particularly with the myxo- viruses, has focused interest on the changes occurring at the cell surface. From this we have inferred that virus release, and perhaps maximum virus activity, occurs here. These data tell us nothing about where the various parts of the virus are synthesized. Indeed, interpretation of some morpho- logical data on herpes has suggested that the early virus forms are made in the nucleus and that they acquire more complete clothing as they progress from nucleus to cytoplasm to the surface of the cell. The conclusiveness of a given interpretation may be hazardous, as witness the fact that two strains of the same virus may produce different cellular lesions (Bang, 1953b; Bankowski and Hyde, 1957), contingent upon their respective virulence. Furthermore, the same virus may produce different cellular lesions in dif- ferent cell types. This latter circumstance has been nicely shown in the infec- tion of rabbit fibroblasts and epithelium by the virus of myxoma by Chap- ronniere (1957). These may be compared with macrophages (Maral, 1957). It is also apparent in the effect of poliomyelitis on different cell types (Dunnebacke, 1956a,b). 5. There is a great need for correlative studies of one virus on one cell system by different methods, with concomitant correlations of the amount of virus released with the various cellular lesions as they develop. Presum- ably, this may best be done by simultaneous infections of all cells with a relatively high multiplicity of virus infectious units. Appropriate reference has been made in the text to the few cases where this has been done. More studies which correlate the fluorescent antibody technique with other methods are needed. Coffin and Liu (1957) were able by this technique to identify the specific intracellular localization of distemper virus and to separate this from other cellular lesions. , 6. Tumor viruses have not been shown to differ from other viruses in their localization in the cell or in their intracellular lesions. Virus is found in the cytoplasm, in the microvilli, and within the nucleus in varying degrees. Its association with mitochondria is unsettled, possibly because most of the viral studies have concentrated on the obviously diseased cell, a factor which would unerringly select the cell in which virus and host were out of balance and virus was overcoming host cell. No significant morphological data on the balanced malignant cell produced by virus infection is available. THE MORPHOLOGICAL APPROACH 105 TABLE II LocATION oF VIRUS AND CELLULAR LESION In Some Virus Tumors Virus Location in cell Predominant lesion Rous chicken sarcoma Surface, occasional intra- Paranuclear hypertrophy cytoplasmic vesicle Mammary tumor of Cytoplasmic vesicles, Large cytoplasmic mice microvilli “inclusion’’ in some strains Shope fibroma Cytoplasmic Cytoplasmic granular masses, nucleolar changes Human warts Nucleus, entire cell when Kosinophilic intranuclear keratinization takes place inclusion Frog adenccarcinoma Cytoplasm, nucleus, Intranuclear inclusion bodies microvilli in about 1/3 of tumors REFERENCES Ackerman, W. W., Rabson, A., and Kurtz, H. (1954). J. Exptl. Med. 100, 437. Adams, W. R., and Prince, A. M. (1957). J. Exptl. Med. 106, 617. Algire, G. (1957). Federation Proc. 16, 601. Amies, C. R. (1938). J. Pathol. Bacteriol. 47, 205. Andrewes, C. H. (1939). J. Pathol. Bacteriol. 48, 225. Andrewes, C. H., Bang, F. B., and Burnet, F. M. (1955). Virology 1, 176. 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Pathol. Bacteriol. 68, 159. Wolman, M. (1955). Haperientia 11, 22. Wolman, M., and Behar, A. (1952). J. Infectious Diseases 91, 63. Wyckoff, R. W. G. (1951). Proc. Natl. Acad. Sci. U.S. 37, 565. Wyckoff, R. W. G. (1953). J. Immunol. 70, 187. Wyckoff, R. W. G., Croissant, O., and Lepine, P. (1956). Ann. inst. Pasteur 90, 18. Chapter VI Biological Aspects of Intracellular Stages of Virus Growth AuickK Isaacs National Institute for Medical Research, London, England MRLTE POCUUIC EIQ IN ccc eltre Gait es ieithe rate ae ene lapel aay ohm a8 ey Feb fatale, Shas woes we Aelia 111 T,. Ging dd aes Nore es Meo Tae Aloe Sen a aise ea minim etorer ieee 112 A. The Amount of Infective Virus Recoverable during the Lag Period...... 113 ( LESee rely) dic ecg pane el an Sewer adore: tober Obi aes ce eee mean 113 Easily Amiri 16USeSicys S Sicha S:4 Mola wigtsat ein esiwictelersuahe cs Povete isin Selon aes 114 ete MeCN Sized. V INIISES ay) i 0h Alois S sige. Bass Sem use aeus, 5 Reiss Qareie ele A 115 PREM eA SGTy WECLISCS nic Sreseisin dyoie Rate Barnes Sue asia Scie aera hnestaie ee era 116 B. Attempts to Demonstrate Virus during the Lag Period by Its Antigenic JER OS ght ee ioaen Aer stiri reer Dairies aie MEA a cep iam rua era 120 C. Tke Significance of Virus Recoverable during the Lag Period............ 122 III. Development within Infected Cells of Antigens Associated with Virus Multi- dex rte Re rks Mee ae a AC Ou eee came t TOMO deo army hmemar er net 126 A. The 30 S Complement-Fixing (Soluble) Antigen of Influenza and Related ATER ITE SS Fe ac ae Pa a re Seeger vee aan A eae gare A penn tenis genioiey= tay. 8 126 pel bertemag py labmimOl Ny xOVIRUSOS!. «eis alee css e vs weiss oot oats 129 C. Cell-Associated Antigens of Other Viruses... 2.0... ..5 cece eens eee enes 132 IV. Dynamics of the Development of Infective Virus .........--.----+-++++++> 133 Pe ANORN Elin VRS) oa te isos? 4m Jose ee mi Sree om iatakta cr vig ety vet 133 Be Western. Equine: bncephalitig: VPUBs..o.:~2 <)< a a ea 2 a0 aim ales cisigi aye se 134 Soplnehisie za ONV RUB a ahs © erode rare kl ofan. 0 Sire eae asks Mie orate. ay sispeue Sena ONAL 135 IDS Neweastie Disease \Virus.Of Wows, .< co. s7-a:ce <3 sls © oo cies ee ess 136 Pip oR es SIMI p le ke VITUS. Asia's yatia7 once kie a aos Satie eo nate a eel Saige se a 137 EY recut ITS ar Ui <, Wiha cactciepe ache ea oe eee Saoirse eis 7 137 Wo the Release of Virus from Infected Cellet 5. ccs o c.aiee cece vee sess ee ees 138 A. Western Equine Encephalitis and Poliomyelitis Viruses................ 138 1B), Nbc) ES elena oacacdiintn tre BrsToiaici a AC aad as tea G rlcerr ciple iron toe eer 139 C2 Herpes Sunplex and Vacemia) Viruses’. << o..2 250. ose ee tem eet ee ee 141 D. Mechanism of Release of Virus and Virus Filaments .................. 142 bm inicoMm plete, VarUs Ss uia-ctere ee cise sie etatets see bec ele Ave. Sandie sed oper eleuetons ayers as 143 ee Properticn' Ol imcomplote: VITUB\.. <0). ci. co iees ace ae ele aie Se Ase hse = 144 ieee roducton ol ineomplove: VAEUS c.ch acta cys cios arco oem sins sei > see & 146 C. Partial Cycle of Virus Multiplication Produced by Incomplete Virus...... 149 D. Incomplete Viruses Other Than Influenza.......... 22.0... e cece eee eee 150 Whale (CETEG TES TCOT Siete, - aie erent Magee ee Caen ON are ee eae 151 PEST EN CERT OREO Gh enh. tae oc als HARE Sek none ERIS el pas aelolne Sas ss 153 I. IntTRoDUCTION The information which we have at the moment of how animal viruses multiply within cells is insufficient to allow us to build up a connected picture lll 112 ALICK ISAACS of the main processes involved. Present ideas have been largely shaped by work on bacteriophages, and, according to inclination, workers have tried to point to resemblances or differences between animal and bacterial viruses. At the moment, the most significant recent observations on virus multiplica- tion are probably those of Hershey and Chase (1952) on bacteriophages and Gierer and Schramm (1956) and Fraenkel-Conrat (1956) on tobacco mosaic virus; these studies make it clear that infection can be initiated by virus nucleic acid preparations from which the bulk of the virus protein is absent. Recently Colter et al. (1957) and Wecker and Schafer (1957a) have described similar experiments with animal viruses and the results suggest tentatively that infection could be produced by “nucleic acid extracts” of encephalomyo- carditis (Mengo) and eastern equine encephalitis viruses (see also Section VII). Apart from these findings, our ideas on how animal viruses multiply are to a large extent governed by our interpretation of experiments on the eclipse phase and on virus recombination. Virus recombination is the theme of Chapter IJ; in this chapter the eclipse phase will be considered first, because of its importance to our concepts of virus multiplication. Burnet (1955) defines viruses as microorganisms less than 0.4 in diameter which can multiply only within living cells of a susceptible host, and which undergo conversion into a noninfective form as a necessary step in their multi- plication. It is in this sense that the term “eclipse phase’”’ will be used in this chapter. Burnet pointed out that in the great majority of animal virus types it was not possible, on the basis of evidence then available, to conclude that an eclipse phase was present or absent. In the following section the experi- mental evidence bearing on the existence of an eclipse phase for a number of different animal viruses is reviewed. II. Tae Eciipsr PHASE One of the striking findings about bacteriophage multiplication is that within a short time of the initiation of infection, no infective virus can be detected in disintegrated bacteria and this eclipse phase persists until halfway through the lag period.* Many similar investigations have been carried out with animal viruses, but while in all cases the amount of virus which could be recovered in the lag period represented only a fraction of the amount taken up by the cells, in only one case was the situation strictly analogous to that found with bacteriophages, i.e., western equine encephalitis virus (Rubin et al., 1955) in which no virus was recovered from cells during the lag period. In all other cases some infective virus could be recovered throughout the lag period, ranging from an extremely small fraction to quite * For the present purpose the term “‘lag period” is used to indicate the time between the initiation of infection and the appearance of newly formed virus. BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 113 a large proportion of the virus taken up. Since most studies with animal viruses have not been carried out with isolated cells, two interpretations of these results are possible. In the first, 1t 1s assumed that the virus which is detectable during the lag period is the parent of the new virus produced, and the virus which is not detectable has been destroyed by the cells and plays no further role in multiplication. In the second interpretation it is thought that the virus which is detectable during the lag period is adventitious, while the virus which has become undetectable has entered the eclipse phase on the road to producing new virus. It is also possible that the process is not uniform throughout animal viruses, although it would obviously be preferable to try to fit the experimental results into a single theoretical framework. We may therefore consider separately the following questions. 1. How much of the infectious virus taken up by the cells is recoverable during the lag period? 2. What happens to other viral properties, e.g., antigenic properties, during the lag period? 3. What is the significance of the infective virus which is detectable during the lag period? Thereafter, we may try to arrive at an assessment of the idea of an eclipse phase as a general phenomenon among animal viruses and as an essential stage in the multiplication process. It is perhaps unfortunate that more work on the multiplication of animal viruses has been carried out on influenza and related viruses than on any others, but an attempt will be made to balance observations on influenza viruses with the findings in regard to smaller viruses, e.g., poliomyelitis and the encephalitis viruses and larger viruses, such as vaccinia and herpes simplex. A. Amount of Infective Virus Recoverable during the Lag Period 1. Bacteriophages As a standard with which to compare animal viruses, the experiments of Doermann (1952) on the eclipse phase of bacteriophages should be quoted. Doermann studied coliform bacteria infected with T4 phage. At intervals after infection the bacteria were lysed by applying a large dose of heterolo- gous phage along with cyanide to stop further phage synthesis, a treatment which was shown to liberate as much phage during the terminal stages of intracellular development as occurs naturally. When bacteria were infected at a multiplicity of 1, lysis at 10} minutes after infection revealed less than 0.01 °% of the final yield, or less than 1 phage particle in 80 bacteria. Clearly, therefore, the eclipse phase in its strictest sense implies that cells which are known to be infected with virus and which would have produced new virus if they had been left, show no infective virus during the lag period. VOL. l11.—8 114 ALICK ISAACS There are many difficulties in applying these techniques directly to the study of animal viruses, where, until recently, 1t has not been possible to infect uniform populations of isolated suspended cells. The advantages enjoyed by workers with bacteriophages are that the infectivity of their virus is usually stable at 37°C.; that adsorption of phages to their host cells is usually efficient; that the assay of infectivity is simple and has a relatively low error; that it is possible to assay without difficulty the number of infected cells; that the bacterial cells can be effectively isolated from one another to prevent spread of virus from cell to cell; and that in the cases which have been studied most there is a one-to-one ratio between infectivity and total virus particles present, as determined by electron microscopic counts (Luria e¢ al., 1951). In some or all of these respects workers with animal viruses have been at a great disadvantage. Most studies of this kind have been carried out with whole animals, tissues such as the chick chorioallantoic membrane, or populations of cells cultured in vitro under conditions where it is not known how many cells are supporting virus multiplication. In addition, many animal viruses are inactivated at 37°C. at a rate which appreciably affects the interpretation of the results of virus absorption studies, and often the absorp- tion itself is not very efficient. In titrations carried out by methods other than pock- or plaque counting techniques the error of the titrations is usually high (Dulbecco, 1955); finally, with all animal viruses which have been adequately studied so far the minimal infective dose corresponds to at least 5 to 10 virus particles counted by electron microscopy. These difficulties should make for caution, and perhaps a little sympathy, in the interpretation of studies on the eclipse phase of animal viruses. 2. Small Animal Viruses Rubin et al. (1955) infected a monolayer of chick embryo cells with a high multiplicity of western equine encephalitis virus. After allowing 30 minutes at 37°C. for virus adsorption, the monolayer was washed and trypsinized and the suspended cells washed and diluted greatly to prevent reinfection of cells. This was taken as zero time and the diluted cells were then incubated at 37°C. in buffer. At intervals, samples were removed and assayed for the total number of infected cells by direct plating on a cell monolayer. At the same time, the intracellular virus content was measured by plating out cells which had been washed to remove superficially adsorbed virus and disintegrated by ultrasonic vibration (a procedure which was shown to have no detectable effect on virus infectivity). The sample at time “zero” was found to contain 1.3 x 10* infected cells per milliliter, but 2.9 ml. of an aliquot, when disinte- grated, revealed no intracellular infective virus. This corresponds to a recovery of less than 1 virus particle in about 40,000 infected cells. BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 115 Sanders (1953) found that when mice were inoculated in the tongue with the GD VII strain of encephalomyocarditis (EMC) virus, spread of virus occurred along the nerve to the hypoglossal nucleus. If this nucleus was removed 48 hours after inoculating the tongue, a suspension prepared from frozen and thawed nuclei contained no detectable infective virus, whereas suspensions prepared by incubating intact nuclei i vitro at 37°C. developed 3000 LD,, of virus per milligram of tissue. In these experiments it was not possible to say how much virus was initially present in the intact nuclei before incubation. Recently, Sanders et al. (1958) have studied the growth of EMC virus in agitated cultures of Krebs 2 mouse carcinoma cells under conditions where the cells remain separate, the virus being titrated by a plaque technique. At a multiplicity of infection of 0.1, about 95% of the virus is adsorbed to the cells within 15 minutes. During the lag period the amount of virus recoverable by breaking up the cells with glass beads was 104 plaque-forming units per milliliter, whereas the number of infective centers, determined by direct plating of intact cells, was 10° per milliliter. Hence, the recovery of virus during the lag period was 1 °% of the number of infective centers. As far as has been investigated, therefore, small animal viruses behave like bacteriophages in showing a low recovery of infective viruses during the lag period. 3. Medium-Sized Viruses Hoyle (1948) noted that during the lag period there was a low recovery of influenza virus from an infected chick chorioallantoic membrane. Henle (1949) carried out much more detailed experiments on similar lines; he found that after injecting various doses of influenza virus into the chick allantoic cavity about 30 °% of the virus infectivity of the inoculum could be recovered in the allantoic fluid during the lag period. Henle interpreted this to mean that 70 °% of the inoculum was taken up by the cells, but in view of later studies, which showed that the infectivity of influenza virus is reduced during incuba- tion at 37°C. at an appreciable rate (Horsfall, 1954; Paucker and Henle, 1955a), conclusions based on the amount of the more stable virus hemagglutinin taken up by the cells are probably more reliable. Cairns and Edney (1952) reported that over a wide range of virus dosage about 50% of influenza virus hemagglutinin was taken up by the allantoic cells during a 43 hour incubation period. Hoyle and Frisch-Niggemeyer (1955) arrived at a very similar figure for virus labeled with P*? by studying the residual radioactivity in the allantoic fluid after allowing a 14 hour period of absorption. Horsfall (1954), on the other hand, found an exponential decline in the titer of unadsorbed virus with time. When Henle’s (1949) figures are calculated on the conservative basis that 50 % of the seed virus is taken up by the cells, it 116 ALICK ISAACS appears that only 3.2%, on the average, of the amount of infective virus taken up could be recovered from the membranes during the lag period. Although a number of different techniques of extraction were used, the recovery of virus was not improved. Schafer and Munk (1952) inoculated very large doses (10°— 10!° LD.,) of fowl plague virus into de-embryonated eggs, washed out the inoculum with buffer 30-105 minutes afterwards, and then measured the infectivity of homogenized membranes. During the lag period, the infective titer was 107° to 10-°. This represents 0.01 °% of the virus inoculum, but it 1s not known how much of the inoculum was actually taken up by the cells. Granoff (1955) inoculated 107-4 LD,) of Newcastle disease virus (NDV) into the allantoic cavity of chick embryos and calculated that 80% of the seed was adsorbed after 2 hours (this figure would be an overestimate if a significant degree of virus inactivation occurs during 2 hours’ incubation at 37°C.). However, less than 1 °4 of the inoculated virus could be detected in ground membrane extracts at this time. More recently, Rubin et al. (1957) studied the growth of NDV in monolayers of chick embryo lung epithelium. The technique was similar to that used by Rubin et al. (1955) for western equine encephalitis virus. During the lag period, the number of virus infective doses recoverable by freezing and thawing the cells was about 1 % of the number of cell yielders (or infective centers). This proportion remained constant throughout the lag period. Rubin (1955) studied the development of the Rous sarcoma virus in suspensions of tumor cells. By inoculating cells onto the chick chorioallantoic membrane, it was shown that the number of cells capable of initiating pocks was about 1/8 (range of 1/1.9 to 1/17 in different experiments) of the total number in the suspensions. These cells release virus at a slow rate during incubation at 37°C., but when the cells were frozen and thawed three times before incubation (a procedure which of itself did not reduce virus infectivity) the amount of virus released corresponded to one infective dose for 250 cells. On the average, therefore, the recovery of virus during the lag period was about 3 virus particles for every 100 infected cells. The recoveries found for influenza and Rous sarcoma viruses during the lag period are slightly higher than the level of about 1 % (or less) of the virus taken up, which was found for the remaining viruses. However, at least for influenza viruses, this estimate is probably too high, as will be described in Section IT, C. 4. Larger Viruses A number of recent studies bear on the recovery of vaccinia and herpes simplex viruses from infected chorioallantoic membranes during the lag period, and, in general, the recoveries found have been higher than those BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 117 described for small and medium-sized viruses. Briody and Stannard (1951) and Crawford and Sanders (1952), working with vaccinia virus grown, respectively, on the chick chorion and in the rabbit skin in vitro, described a decrease in the number of infective particles that could be detected in the lag period, but the uptake of virus by the cells was not measured, so that the actual extent of the decrease is not known. Anderson (1954) inoculated eggs with 250 infective doses of vaccinia virus on the chorion and measured the amount of virus that could be recovered from ground membranes after various periods of incubation. The number of infective doses recoverable at 30 minutes was 70; at 1 hour, 350; this gradually declined to 18 infective doses at 9 hours, followed by a steep increase. This suggests that the virus was gradually adsorbed to the membrane over the first hour and that, as the virus entered the cells, there was a decrease in the amount of recoverable virus to about 7 % of that moculated; from the titers obtained at 1 and 2 hours, it appears that most of the virus inoculated was taken up by the cells. Metcalf (1955) described a slightly greater decline in titer following inocula- tion of 7 x 104 infective doses, but in this experiment the uptake of virus was not measured. By contrast, others who have worked with vaccinia virus report much higher recoveries during the lag period. The interpretation of Maitland and Tobin’s (1956) results is complicated, however, by what they call the “enhancement effect.”” They found that when a vaccinia elementary body suspension was prepared from rabbit skin by differential centrifugation and inoculated on the chorion, the amount of virus recoverable from the liquid on top of the membrane immediately after inoculation was considerably higher than the apparent titer of the inoculum (5 to 22 times in some experiments). This is presumably some sort of disaggregation effect; after this apparent rise in titer the virus entered the membrane. Thereafter the total virus concentration decreased, until at 2 to 4 hours it was 20 to 80 % of the peak titer (or 2 to 4 times the amount apparently inoculated) and most of this virus was recoverable from the membrane itself. Maitland and Magrath (1957) studied this decline in titer in more detail, using the same virus grown in chorioallantoic membrane in vitro. When pieces of chorioallantoic membrane were incubated with virus for 10 minutes, about 20 to 40 % of the inoculum became attached to the membrane. However, about three-quarters of this virus could be removed by washing, and the remainder constituted the baseline. Following incubation of washed pieces of membrane there was a slow decrease in the amount of virus recoverable. At 8 to 12 hours, the minimal titer was reached when about 20 to 50 °% of the baseline value was recoverable. A similar result was obtained with minced chick embryo cells. Trypsinized cells were left in contact with vaccinia virus for 10 minutes at 37°C., incubated in vitro, and titrated at intervals along with their suspending medium after disintegrating the cells mechanically. The base-line titration 118 ALICK ISAACS showed | infective dose of virus for every 25 to 50 cells, and between 50 and 90 % of this amount was recoverable throughout the lag period. It seems important to repeat these experiments with seed virus which does not show the enhancement effect. Overman and Tamm (1957) incubated very large doses of virus (18 x 10° infective doses) with pieces of chorioallantoic membrane 7n vitro. The amount of virus taken up by the cells was not meas- ured but there was only a slight decline, roughly 2-fold, between the third and eighth hours of incubation in the amount of virus recoverable from ground membrane pieces. Shaffer and Enders (1939) noted that 14 hours after moculating 120 infective doses of herpes simplex virus on the chick chorioallantoic membrane, only 10 doses could be recovered from ground membranes. However, further experiments with antiserum made it appear that only a small proportion of the virus had been taken up by the membrane in that time. Scott et al. (1953) carried out similar experiments but with a much larger inoculum, 8 x 10? infective doses. The recovery of virus from ground membranes after 1, 4, and 6 hours’ incubation was usually less than 10 infective doses, but the uptake of virus during this time was not measured. In further experiments of the same kind, Modi and Tobin (1954) obtained a recovery of 1 to 10 °% of the inoculum in the interval 1 to 6 hours after inoculation. In this case the inoculum was between 2.10 and 5.108 infective doses, but the amount absorbed is not known. Wildy (1954) carried out a series of most ingenious experiments to investigate the question of a possible eclipse phase for this virus. He inoculated eggs with 100 infective units of virus and at different time intervals eggs were rotated through 120°. The effect of this was that the bubble of air forming the artificial air sac was carried around to a new area of the membrane, taking along with it any virus which had not previeusly been fixed to the membrane. In this way it was found that the total amount of imoculated virus was partitioned between the two areas, 90 % of the moculum being fixed to the first area in 90 minutes. The total amount of virus which could be recovered from membranes and overlying fluids declined gradually and was about 33 % of the original amount after 4 to 6 hours. In this experiment the inoculum was suspended in broth containing gelatin; after the period of incubation at 37°C., the eggs were chilled in order to solidify the gelatin and thus facilitate harvesting of all the virus in the overlying fluid. Since the gelatin was found experimentally to delay fixation of virus by the membrane, it is probable that part of the 33 °% of the virus recovered between 4 and 6 hours represented virus not yet fixed to the membrane. Gostling and Bedson (1956) infected trypsinized chick embryo cell suspensions by contact with herpes virus overnight at 4°C. Under these conditions the uptake of virus was found to be only about 1 %, or less, of the moculum. When such infected cell suspensions were incubated at 37°C., the level of infective virus recoverable from the cells BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 119 declined between the third and sixth hours to about 10% of the level initially present. Gostling (1956) later showed that no further virus was recovered from such cells by extracting the nuclei with 6 °/, sodium chloride solution. Yoshino and Taniguchi (1956) have investigated in detail the question of an eclipse phase for herpes simplex virus. The virus was applied to a glass cover slip, which was then inverted onto the dropped chorioallantoic membrane. Under these conditions and over quite a large range in inoculum size, about 80% of the inoculum was apparently absorbed by the cells within 30 minutes and 20 % remained unabsorbed for some hours. When extracts of chorioallantoic membrane were prepared, the residual unabsorbed virus became included with the virus present in the membrane, so it was necessary to carry out elaborate washing of the membrane to remove super- ficially absorbed virus. These experiments are discussed in Section C, which deals with the significance of the virus recovered during the lag period. Recently Stoker and Ross (1958) studied the growth of herpes simplex virus in sheets of HeLa cells. Infection was initiated at high multiplicity of exposure and most of the extracellular virus was then removed by washing and treatment with antiserum. The proportion of intact infected cells was determined by titrating cell suspensions on the chick chorioallantoic mem- brane during the lag period; in different experiments it varied between 2 and 88 % of the total number of cells. After disintegration in distilled water in a microblendor, cell debris gave a significantly lower yield of infective virus than intact cells, the recovery measured in this way being about 10 %. (Neither the method of disintegration nor the cell debris reduced the infectivity of free virus.) Thus the amount of virus detected in the lag period corresponded to about one-tenth of the number of intact infected cells found to be present. Therefore, the majority of cells which would ultimately yield virus did not reveal any infective virus when disintegrated during the lag period. The very large “viruses,” or Chlamydozoaceae, have some interest in connection with the question of an eclipse phase, since they occupy a position intermediate between smaller viruses and rickettsiae. Girardi e¢ al. (1952) inoculated chick embryos by the allantoic route with about 2 x 10° infective doses of meningopneumonitis virus and measured the content of virus in extracts of chorioallantoic membrane after different intervals of incubation. There was an increase in titer in the membranes as virus was taken up and this was followed by a slow and constant decline over a period of 20 hours. At that time the titer in the membranes was about 1 % of that initially present. Morgan (1956) has studied the growth of psittacosis virus in chick embryo mince which had been starved of essential metabolites. After inoculating 10%.° infective doses (LD, 9) of virus, the infectivity was rapidly lost and after 6 hours’ incubation in starved cells titration of ground cells did not reveal any virus. In this case the recovery of virus was less than 0.03 °% of that inoculated. 120 ALICK ISAACS However, inspite of the absence of demonstrable virus, addition of beef embryo extract caused an immediate and rapid production of large amounts of virus. A variation of these techniques occurs in the experiments of Girardi et al. (1952) on the recovery of meningopneumonitis virus during the lag period, and similar methods were used later by Sanders (1953), Anderson (1954), and Wildy (1954) in studies with encephalomyocarditis, vaccinia virus, and herpes viruses. The principle of the method is that a piece of tissue is removed during the lag period, part is homogenized and tested directly for its virus content, while an aliquot is cultured i vitro and then tested for its viral content. The object of the technique is to refine the experiments on recovery of virus during the lag period by removing a piece of tissue and stopping further virus absorption or spread of virus from neighboring cells or tissues. The method has given recoveries of virus in the homogenized tissue as compared with the cultured tissue of roughly 1 °% for meningopneumonitis, 7 % for vaccinia (Anderson, 1954) and 10 °% for herpes simplex (Wildy, 1954). It is difficult to summarize these varied studies on the recovery of different viruses during the lag period. In many, there is not sufficient information to allow us to estimate what proportion of the virus absorbed by the cells became undetectable, but in those studies which allow us to estimate the recovery, it is clear that, as a general rule, a large proportion of the virus absorbed loses its infectivity shortly after entering the cells. Taking the re- covery of less than 1 °% found for bacteriophages as a standard, and making a few assumptions about the experimental data, it appears that losses in infectivity of about this order of magnitude, or slightly less, have been described for western equine encephalitis, encephalomyocarditis, influenza, fowl plague, Newcastle disease, Rous sarcoma, meningopneumonitis, and psittacosis viruses. For herpes simplex the recoveries reported by Gostling and Bedson and by Wildy may be about 10-20 °; for vaccinia virus, recoveries of between 7 and 90 °% have been reported by different investigators. Leaving vaccinia and herpes aside temporarily, these large losses in virus infectivity might be thought of as due to breakdown of the virus after entering the cells or to the fact that the infectivity of animal viruses is often a very labile property which might be unable to withstand the coarse methods of extraction needed to prepare infected cell suspensions. ff the latter explanation were correct, it might be possible to show the presence of intact virus during the lag period by testing for a more stable viral property, such as its antigenic behavior. B. Attempts to Demonstrate Virus during the Lag Period by Its Antigenic Properties Most attempts to detect virus during the lag period by its antigenic behavior have been carried out with the influenza virus hemagglutinin. BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 121 Hoyle (1948) described a typical experiment in which an egg was inoculated with 1024 hemagglutinating units of virus. After 3 hours’ incubation, only about 10% of the hemagglutinin remained in the allantoic fluid and pre- sumably the rest had been taken up by the cells. However, when the membrane was ground with sand and incubated for 8 hours to allow elution of virus from cells, no hemagglutinin could be detected; this is less than 0.5 % of the amount that might have been expected to be present. Similar findings are recorded by Henle and Henle (1949), i.e., no hemagglutinins were recoverable from membranes in the first 2-3 hours after the use of very large inocula. But in this study no attempt was made to remove normal inhibitors of agglutina- tion in the membrane; this factor could, in theory, influence the recovery of hemagglutinin from membrane extracts, depending on the dosage and strain of virus used. However, Isaacs and Edney (1950) showed that the use of RDE (receptor-destroying enzyme of Vibrio cholerae) to inactivate chorioallantoic membrane inhibitor did not increase the recovery of hemagglutinin. In their experiments, less than 1° of the hemagglutinin taken up by the cells could be recovered in membranes which were ground and treated with RDE. They also tested membranes which had absorbed large doses of influenza virus for evidence of virus enzymatic (neuraminidase) activity during the lag period. Chorioallantoic membranes which had absorbed between 400 and 700 agglu- tinating doses of virus, each, were incubated for short periods at 37°C. in saline to allow elution of superficially absorbed virus. They were then ground and aliquots were incubated at 37 and 0°C, for 18 hours and the agglutinating inhibitory titer of the membranes measured. If any virus enzyme was present it should inactivate the inhibitor of agglutination during incubation at 37°C. but not at 0°C. In fact, no such enzymatic activity was demonstrated, whereas control membranes incubated with 100 agglutinating doses added after grinding had their agglutinating inhibitory titer reduced by more than 96 ° under the same conditions. The fact that during the lag period neither the hemagglutinating nor enzymatic activities of the virus could be detected suggested that a fundamental change in the majority of the virus particles occurred after they entered the cells. Confirmation of this idea was obtained in similar experiments with heat-imactivated virus. Very large doses of heated virus (roughly 5000—10,000 agglutinating doses of virus per egg) were absorbed by chorioallantoic membranes, which were then ground and treated with RDE. Such extracts showed no evidence of viral hemagglutinin, or of antigenic activity as determined in vitro by antibody-combining activity, or in vivo by antigenicity in mice. By contrast, heated virus incubated with chorioallantoic membrane extract in vitro, and then treated with RDE, showed high activity by all three tests. Tamm and Tyrrell (1954) studied the recovery of influenza virus hemag- glutinin after 1 hour’s incubation with pieces of chorioallantoic membrane 122 ALICK ISAACS in vitro. After homogenizing, the membranes were treated with RDE and 3.6-9.4 °%, of the virus which had been absorbed was recovered. Hoyle and Frisch-Niggemeyer (1955) studied the radioactivity and hemagglutinin content of chorioallantoic membranes which had absorbed 480 agglutinating doses of P*? labeled influenza virus during a period of 1-2 hours’ incubation. In extracts of the membrane, prepared by freezing and thawing and treatment with RDE, there was no demonstrable hemagglutinin, although a titer of 140 would have been expected on the basis of the radioactivity. The fact that the radioactivity was not due to hemagglutinin masked by combination with membrane inhibitor was shown by centrifuging the extract at 100,000g for 3 hours, when the greater part of the radioactivity remained in the super- natant. Hoyle and Frisch-Niggemeyer also observed that a high proportion of the phosphorus label could be found in the “residual membrane,” 1.e., the sedimentable brei from the original membrane extract. This fraction was not apparently tested for its viral content, but Henle (1949) had shown earlier that when extracts of infected chorioallantoic membrane were pre- pared during the lag period and centrifuged lightly (2000 r.p.m. for 20 minutes) the bulk of the virus infectivity remained in the supernatant. Isaacs and Lindenmann (1957) found that when heated influenza virus was absorbed by chorioallantoic membranes in vitro and then the membranes were washed, the interfering activity of the virus could not be detected in extracts of the membrane before incubation. If such membranes were incubated for 3 to 6 hours at 37°C. before extraction, interfering activity was found in the membranes and was later secreted ito the surrounding fluid, but this interfering activity was not due to the heated virus but to a product of the cell-virus interaction, which they called interferon. These examples show that during the lag period of influenza virus growth there is a low recovery of hemagglutinin and of virus enzymatic activity which closely parallels the low recovery of infective virus. The finding with heated influenza virus that hemagglutinin, viral antigenic activity, and interfering activity were lost suggests a fundamental alteration in the viral particle on entering the cell; this point is discussed later. It is unfortunate that only influenza viruses have been studied for their antigenic behavior dur- ing the lag period, but the problem is technically more difficult with other viruses. So far as they go, these results add emphasis to the low recoveries of infective virus during the lag period. C. The Significance of Virus Recoverable during the Lag Period In most studies described above small amounts of virus could be extracted from infected cells during the lag period, and we can now consider the significance of this recoverable virus. The most important consideration in relation to the question of an eclipse phase is whether the recoverable virus BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 123 is situated in an extracellular or an intracellular position at the time the cell extract is prepared for virus assay. The experimental method most commonly used to answer this question has been to find the effect of treating the cells with viral antiserum on the amount of virus recoverable after disrupting the cells. There is, however, a possible objection to this technique, that antibody might first combine with extracellularly situated virus, and, if bivalent, later combine also with intracellular virus when the cells are disrupted. Efficient removal of the antibody by washing is therefore an essential part of the technique, and if it is shown that treating control cells with antiserum after infection has been initiated does not reduce the final yield of virus from these cells, this can be taken as evidence that the antibody has not had much influence on the titer of intracellular virus. Two other types of experiment have also been carried out to test the localization of recoverable virus, i.e., treating the cells with RDE im the case of influenzal infection, and repeated washing of cells to see what is the effect on the amount of virus recoverable during the lag period. , Andrewes (1930) first showed that the growth of herpes simplex virus in tissue culture was not inhibited by viral antiserum inoculated after the initiation of infection. The effect of antiserum used in this way on the recovery of virus during the lag period has been studied by a number of workers. Henle and Henle (1949) noted that antibody given 30 minutes after a large inoculum of influenza virus (10°LD,;,) in the chick allantoic cavity reduced the recovery of virus in the membranes during the lag period by 3 logy, without reducing the eventual yield of infective virus in control eggs similarly moculated. Apparently, too, antibody was not fixed by uninoculated mem- branes. On the other hand, antibody reduced the yield of virus after a small inoculum, suggesting that some multivalent antibody combined with superficially absorbed virus and was then carried over into the tissue on extraction. It is best, therefore, as Henle and Henle point out, to accept the findings as giving qualitative evidence that a substantial part of the recover- able virus is superficially attached to the cells, without attempting to interpret the findings on a strictly quantitative basis. In essentially similar experiments, Schafer and Munk (1952) found that the recovery of fowl plague virus from the membrane was reduced by 3-4 log,, by treatment with antiserum; Tamm and Tyrrell (1954) and Ackermann and associates (1955) found a 1 log,, reduction with influenza virus grown in pieces of chorioallantoic membrane im vitro; and Ishida and Ackermann (1956), while investigating the effect of temperature on the fixation of virus to cells, noted that immune serum treatment caused a large reduction in the recovery of influenza virus after adsorption to membranes at 3°C. However, the latter workers also obtained, in the same way as Henle and Henle (1949) had done, results which suggested that in their experiments some serum becomes bound to the 124 ALICK ISAACS cells in association with superficial virus and may later mix with intracellular virus on grinding the cells. Gostling and Bedson (1956) found that chick embryo cell suspensions incubated with herpes simplex virus overnight at 4°C. and then treated with diluted antiserum overnight at 4°C. contained throughout the lag period about one-tenth the amount of virus of control cells in which the antiserum treatment was omitted. In these experiments evidence was shown that there was insufficient antiserum present to affect the results of the infectivity titrations and the findings are attributed to neutraliza- tion of superficially adsorbed extracellular virus. As mentioned earlier, the level of virus extractable from the cells then declined to one-tenth of the initial level, but this fraction of the initially absorbed virus was not neutralized by treatment with antiserum in their experiments. In the experiments of Yoshino and Taniguchi (1956) an antiserum in a 1/100 dilution was used to treat chorioallantoic membranes one-half hour after moculating herpes simplex virus by the cover slip technique mentioned above. The use of anti- serum was combined with an elaborate wash-drying procedure involving numerous washings for each membrane in buffered saline with careful drying on filter paper between washes. With this method about 0.0015 to 0.004 °{ of the inoculum was recoverable during the lag period compared with about a 20 % recovery in membranes not treated in this way. These workers also attempted to remove superficially adsorbed virus by experiments involving washing the membrane in situ, followed by sponging with gauze after each wash. After 5 washings with sponging between each, the amount of recoverable virus was about 1 °% of that present before washing; this degree of washing apparently did not harm the cells too much, as judged by the yield of virus from similar membranes incubated at 35°C. However, with 6 washings, involving sponging witb 3 changes of gauze between each washing, the yield of virus was drastically reduced following incubation at 35°C. Such a traumatic procedure might easily damage cells so that intracellular virus could be washed away. These experiments display novel methods of trying to answer this problem and it would be interesting to see them applied to vaccinia virus. That tissues which had adsorbed influenza virus when repeatedly washed (15 times) in buffer continued to release virus into the washings was demon- strated by Ackermann et al. (1955), and similar findings were noted after 12 washings of cells infected with herpes simplex virus (Gostling and Bedson, 1956). One method used to remove superficially adsorbed influenza virus was to treat the intact chorioallantoic membranes with large doses of RDE, followed by thorough washing before grinding (Isaacs and Edney, 1950). With the use of RDE the recovery of infective virus was reduced 10-fold, i.e., 0.04 °% of the seed compared with 0.6 % in membranes which had not been washed with RDE. Insufficient RDE was present to interfere with the assay of infectivity. BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 125 These experiments making use of antiserum, repeated washing, and RDE have not always given unequivocal answers but, in general, it seems that in nearly all the cases investigated most of the virus recoverable from cells during the lag period is extracellular and can be removed by one or other of these methods. Nevertheless, it has always been found too that a very small fraction of the virus taken up by the cells can be detected in the lag period and is not completely removed by antiserum, washing, or RDE. This virus is therefore assumed to be intracellular, and, depending on our point of view, we may regard it (in experiments in which isolated cell suspensions were not used) as the parent of the new virus yield which will remain infective through- out the lag period, or as virus which is about to enter into an eclipse period, or as adventitious virus of no significance in the multiplication cycle. Quantita- tive considerations, however, make the first explanation untenable for many of the above viruses. Thus, if we assume as average figures for influenza and fowl plague viruses that 0.1% of the virus taken up can be detected as intracellular virus during the lag period, we are obliged to conclude that only 1 out of every 1000 infective virus particles taken up by the cell survives to multiply, while the remaining 999 perish. But there is good evidence from electron microscopic counts and indirect counting methods that infection with these viruses can be initiated by 10 virus particles (Isaacs, 1957). When we consider, too, the results of Hoyle and Frisch-Niggemeyer (1955) on labeled influenza virus, it seems justifiable to conclude that on entering the cells the majority of influenza virus particles become changed into material which is either smaller or less dense than the original virus and which lacks infectivity and many of the other properties of the virus; and that this change may well be analogous to the eclipse phase of bacteriophages and cannot be explained other than as a stage in the life cycle of the virus. At the moment we cannot say what is the significance of virus which is not neutralizable by serum but is recovered during the lag period. With other viruses, the recovery during the lag period is almost as low as that found for bacteriophages, but one cannot yet be certain that this is a true eclipse. The findings with herpes simplex virus are conflicting. Wildy (1954), Yoshino and Taniguchi (1956), and Stoker and Ross (1958) favor the idea of an eclipse phase while Gostling and Bedson (1956) think it is not proved; the same conclusion was reached by Maitland and co-workers for vaccinia virus. Indeed, Maitland and Magrath (1957) report that a decline in infectivity of vaccinia virus paralleling that which occurred in experiments with intact chorioallantoic membrane was found when the virus was adsorbed to membranes which had been heated at 56°C. for 20 minutes, a procedure which destroyed the ability of the membranes to support virus multiplication. The evidence cited for vaccinia and herpes simplex viruses seems therefore to be insufficient at the moment to decide definitely in favor of or against an 126 ALICK ISAACS eclipse phase, although the findings of Stoker and Ross (1958) are in favor of an eclipse phase for herpes virus. More decisive evidence, particularly for vaccinia virus, will be provided by experiments along the lines of Rubin et al. (1955) with isolated cells, or if recovery of virus in the lag period is found to be low compared with the “plating efficiency,” 1.e., the ratio of infective virus count to total virus particle count. Nevertheless, the trend of the findings favors the idea of the eclipse phase as a general phenomenon among animal viruses. Morgan’s (1956) experiments with psittacosis virus are very difficult to interpret except on the assumption that the virus is in a noninfective phase in the starved cell and that it resumes its development when the cell is supplied with certain essential metabolites. The studies by Dulbecco and Vogt (1955) and Dulbecco (1957) on the sensi- tivity of poliomyelitis virus in infected cells to ultraviolet irradiation (based on the work of Luria and Latarjet (1947) on bacteriophages) also imply a change of state of the virus on entering the cells; from the evidence on influenza viruses, a change of state to a noninfective phase seems to be the fate of the majority of infecting virus particles. Ill. DEVELOPMENT WITHIN INFECTED CELLS OF ANTIGENS ASSOCIATED wiItH Virus MULTIPLICATION The development of fully mature virus in infected cells may be preceded or accompanied by the development of associated viral antigens. These antigens have excited interest and speculation as to the possibility that some of them might be building blocks which are later assembled to make mature virus. Unfortunately, although there are many published studies of the development of associated viral antigens, attempts to inculpate them as viral precursors have been carried out mainly with imfluenza and related viruses, with which, therefore, this section is mainly concerned. A. The 30 S Complement-Fixing (Soluble) Antigen of Influenza and Related Viruses During the growth of influenza and related viruses there develops in infected cells an antigen detectable by the complement fixation test, of much smaller particle size than the mature virus particle (Hoyle and Fairbrother, 1937). This antigen has a sedimentation constant of 30 8, compared with about 700 S for the virus particle, and is often called the mfluenza soluble antigen or S, as opposed to the viral antigen or V. The viral antigen has a greater serological specificity than the soluble antigen, and influenza A viruses with widely divergent antigenic characters are said to have a common soluble antigen. However, observations on complement fixation with the BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 127 soluble antigen have usually been made with sera from adults convalescent from influenza, although such sera are unsuitable for tests with viral antigen on account of their broad reactivity with different strains of influenza A virus. In order to measure specific viral antigens most workers use sera of con- valescent or immunized animals which have not had previous experience with infiuenza virus and therefore respond to infection or immunization in a more specific way. When sera from infants infected (presumably for the first time) with influenza virus were tested with soluble antigens prepared from different influenza A strains, the soluble antigens showed some strain specificity (Grist, 1957). There is therefore a real difficulty in defining the influenza soluble antigen precisely. It is normally taken to mean an antigen of sedimentation constant 30 8, present in infected tissues, and showing the broad serological reactivity of the virus serotype. It must be emphasized however, that the term soluble antigen may have been used by different workers to describe different things; this is especially true when studies with human convalescent serum are compared with investigations using animal sera reacting with soluble antigen. Hoyle (1948) found that in cells infected with influenza virus, soluble antigen could be detected at the end of the eclipse period and before the formation of mature virus. Subsequently, Hoyle (1950) showed that, on treating virus elementary bodies with ether, soluble antigen was liberated, and he therefore suggested that the soluble antigen represented the essential form in which the virus multiplied within the cell. This is an attractive theory which attempts to draw an analogy between the influenza soluble antigen and the nucleic acid of bacteriophages and tobacco mosaic virus. At the moment, the evidence concerning this theory is as follows: 1. Soluble antigen is detectable in tissues within 3 hours of inoculating influenza virus, whereas fully infective virus is not formed until the fourth to the fifth hour (Henle and Henle, 1949). However, while this finding has been generally confirmed, there is one reservation which should be made. In experiments on the time of appearance of soluble antigen, large virus inocula are required to produce measurable yields of soluble antigen, and under these conditions multiple infection of cells occurs and incomplete virus is formed (see Section VI). Hence, to overcome this objection, the rise of viral infecti- vity must be studied after the use of small inocula; this means that the development of soluble antigen and infective virus have not been observed under comparable conditions. There are a number of known instances with other viruses in which newly formed virus has been shown to appear earlier with large inocula, e.g., Dulbecco and Vogt (1954), so it is possible that the earlier appearance of the influenza soluble antigen may be more apparent than real. Recently, Ledinko and associates (1957) have described a rise in infectivity 24 to 23 hours after infection of chick embryo lung cells with 128 ALICK ISAACS influenza virus when large doses of RDE were present in the medium. Hemagglutinin appeared at the same time but soluble antigen was not studied in this system. Hoyle first thought that the increase in soluble antigen in the cells occurred at a logarithmic rate but later (Hoyle, 1953) he agreed that a linear increase fitted the experimental observations better. 2. Hoyle (1950) interpreted his findings on the effect of ether treatment of influenza virus particles as showing that when the particles were disrupted they liberated soluble antigen which had been enclosed within the intact particle. Fulton (1953) interpreted the same findings as indicating that ether treatment of the virus had degraded the viral antigens and blunted their serological specificity in an analogous way to the effect shown by ether treatment of rickettsiae (Fulton and Begg, 1946). These differences in interpretation emphasize the difficulty of identifying soluble antigen derived from infected tissues with the antigen liberated by ether treatment of virus elementary bodies. In favor of Hoyle’s interpretation are the findings of Lief and Henle (1956b) that with a standardized technique of ether extraction, a constant amount of soluble antigen was liberated from a given dose of influenza virus; in addition, less soluble antigen was liberated from incomplete virus than from standard virus. This finding is further discussed in Section VI, A, but the implication is that the amount of soluble antigen which can be liberated is closely bound up with the infectivity of the virus, a finding which would not be expected on Fulton’s hypothesis. 3. Evidence has been produced that the soluble antigen is nucleoprotein or is closely bound to nucleoprotein. Hoyle (1952) showed that the serological activity of the soluble antigen was greatly reduced by treatment with trypsin, but only slightly reduced by prolonged incubation with ribonuclease, although the ribonuclease could have been contaminated with small amounts of protease. Hoyle also found that the soluble antigen was precipitated by lanthanum acetate. More convincing evidence for the presence of ribonucleic acid in the influenza soluble antigen was provided by Ada and Perry (1954). They prepared extracts by differential centrifugation of infected chick embryo lungs from which the soluble antigen was precipitated by addition of immune mouse antiserum. Such precipitates were fractionated by a modified Schmidt-Thannhauser method and were found to contain roughly 4% of RNA and 0.5 % DNA. Later, Ada (1957) prepared influenza soluble antigen from infected chorioallantoic membranes and compared it with virus soluble antigens prepared by ether extraction of the virus. The two antigens were found to have comparable complement-fixing activities per milligram of dry weight, but while both showed the presence of ribonucleic acid, the total amount of acid and the proportions of nucleotides differed greatly in the two preparations. On the other hand, Schafer (1957) found that fowl plague virus BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 129 soluble antigens, prepared in the same way from infected tissues and from ether extracts of the virus, were very similar to one another in their behavior with antisera, nucleic acid content, and ultraviolet absorption spectrum. 4. Wiener and associates (1946) showed that soluble antigen could be liberated from influenza virus by ultrasonic vibration and this was confirmed by Lief and Henle (1956a). However, Lief and Henle found that after the ultrasonic treatment there was always more antigen which could be extracted subsequently with ether, and they interpreted this to mean that some soluble antigen was loosely adsorbed to the virus surface and some was present in the virus particles themselves. In summary, while the evidence is incomplete, it seems likely that the soluble antigen is a nucleoprotein which is produced in infected cells before mature virus appears, and that some of it becomes incorporated in the virus particles. While this statement may be an oversimplification of the events which occur and leaves many points unexplained, it suggests further useful lines of investigation. B. The Hemagglutinin of Myxoviruses In parallel with studies of the appearance of influenza soluble antigen in infected cells, the development of viral hemagglutinin (or V antigen) has been intensively investigated. However, hemagglutination may be caused by the virus elementary bodies, by particles which are either smaller or less dense than the elementary bodies (see below), or by small fragments produced, e.g. by ether treatment of elementary bodies—all these particles showing similar serological specificity. Unfortunately, in most studies the particle size of the hemagglutinin is not mentioned. This means that a great deal of useful information on the development of viral hemagglutinin in infected cells cannot yet be applied to the problem of which antigens might qualify as possible viral precursors. At this point it is necessary to anticipate some of the discussion in Section VI by considering the concept of incomplete viruses. Von Magnus (1946) showed that on serial passage of large inocula of influenza virus in the allantoic cavity incomplete virus was liberated into the allantoic fluid. The incomplete virus showed normal hemagglutinating and serological activity, but had a very lowinfectivity relative to that of virus similarly passaged at high dilution. There is still controversy as to whether incomplete virus represents virus which was prematurely liberated from the cells before it had acquired full infectivity, i.e., its normal development was interrupted; or whether it should be considered as malformed virus produced by overloaded cells. In the present discussion the term incomplete virus will be used strictly for the virus spontaneously liberated from cells when the experimental technique of von Magnus is applied. This limitation is necessary because there are two VOL. I1I—9 130 ALICK ISAACS other types of condition under which hemagglutinin of low relative infectivity is produced, but in both cases the hemagglutinm remains intracellular and is not normally liberated from the cells. The noninfective hemagglutinin produced in these other conditions shows some significant differences from the incomplete virus of von Magnus and therefore will not be called incomplete virus here. The first example was described by Granoff and associates (1950). They prepared extracts of chorioallantoic membranes infected with NDV and found that after high-speed centrifugation (20,000 r.p.m. for 20 minutes) the supernatant showed serologically specific hemag- glutinin of low relative infectivity. Virus from the allantoic fluid did not behave in this way after high-speed centrifugation. Granoff (1955) later confirmed these results with NDV and found that the same phenomenon could be demonstrated with the PR8 strain of influenza virus A. He called this hemagglutinin “S”’ for small, although of course its size was not defined by the centrifugation results, and it might be merely less dense than the elementary bodies. By passage experiments it was shown that the “S” influenzal hemagglutinin did not reproduce incomplete virus on passage, thus differentiating it from the incomplete virus prepared by the technique of von Magnus. Schafer and Munk (1952) have also reported finding hemagglutinin of low relative infectivity in the supernatant after high-speed centrifugation of membranes infected with fowl plague virus, and Schafer (1957) has shown the appearance of this hemagglutinin on electron microscopy (Schéfer calls these incomplete forms). This membrane hemagglutinin consists of balloon- like particles of variable size measuring 50-550 my in diameter in the flattened state. It seems, therefore, that in NDV, fowl plague, and influenza infection there develops in infected membranes hemagglutinin of low relative infectivity, sedimenting less readily than the virus elementary bodies; this hemagglutinin is not normally secreted into the allantoic fluid. By contrast, the same membrane extracts also contam hemagglutinm which shows the infectivity and sedimentation behavior characteristic of mature elementary bodies; and this is normally secreted into the allantoic fluid. It seems possible that the so-called “‘S’? hemagglutinin is one of the virus building blocks; further studies of its appearance in the tissues relative to that of mature elementary bodies might help to decide this point. The second situation in which hemagglutinin of low relative infectivity is produced in cells is when there is a partial cycle of influenza virus multi- plication, as was found in the mouse brain (Schlesinger, 1950), the chick chorion (Fulton and Isaacs, 1953), and Hela cells (Henle e¢ al., 1955). In these three sites, infection by strains of influenza virus which have not been specially adapted to these particular cells results in the formation of soluble antigen and hemagglutinin in readily detectable amounts, but fully infective virus is formed to only an insignificant degree. In these three sites, too, the BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 131 hemagglutinin remains intracellular, as in the case of the “‘S’’ hemagglutinin. Also, the morphological appearances described by Werner and Schlesinger (1954) for influenza hemagglutinin from mouse brain are very reminiscent of the tissue forms described by Schafer for fowl plague; in unpublished observa- tions made in collaboration with R. C. Valentine, it was noted that viral hemagglutinin from infected Hela cells showed the same morphological appearance as the forms described by Schafer (1957). On the other hand, incomplete virus obtained by the method of von Magnus and present in allantoic fluid appears on electron microscopy (Donald and Isaacs, 1954¢; Pye et al., 1956) to be much less pleomorphic than the tissue form of the hemagglutinin, and von Magnus (1954) states that incomplete and standard virus do not show any pronounced differences in size and shape on electron microscopy. It is tempting therefore, to suggest that in the cells of these three sites there is a lack of some factor required for completing the virus multiplica- tion cycle and that viral building blocks accumulate in the cells. There is, however, no firm experimental evidence which would let us identify any viral- associated antigen as a virus precursor. Henle et al. (1956) studied the ratio of infectivity : hemagglutinin titer (I/HA ratio) in the membranes and fluid media of de-embryonated eggs infected with influenza virus. With seeds of different varieties, 1.e., diluted or undiluted standard seeds and seeds prepared by serial passage of large inocula to produce incomplete virus (see Section VI), it was regularly found that the I/HA ratio in the membranes was about 1.5 log less than that of the liberated progeny. In any 2-hour liberation period about ten times the amount of infective virus was shed into the medium as was present in the membrane, whereas only about one-quarter of the hemagglutinin was released in the same time. In addition, V or viral antigen, measured by complement fixation and distinct from the viral hemagglutinin, could be shown to be present in the tissues but not in the fluid in any 2-hour period. Henle e¢ al. also found that on adding potassium cyanide the infectivity and hemagglutinin titers in the membranes decreased sharply and rose again, together, as the cyanide was removed. They concluded that the hemagglutinin in the membrane was noninfective at first and acquired infectivity later in its development, the production of noninfective hemagglutinin and conversion to mature virus forming a dynamic process. The noninfective hemagglutinin in these experi- ments may be the same as the “S” hemagglutinin of Granoff et al. and it seems important to investigate this possibility. In many studies, the times of first appearance of hemagglutinin, soluble antigen, and infective virus have been compared. As discussed earlier, it is difficult to compare strictly the time of appearance of mature virus with that of the other two antigens since they are not normally measured under identical conditions, although it appears that infective virus develops after 132 ALICK ISAACS the other two antigens (Henle e¢ al., 1954). As between soluble antigen and hemagglutinin, Hoyle (1948) found that soluble antigen appeared first, while Fulton (1949) found that the two appeared simultaneously. It is important in making this comparison to be sure that the inhibitor of viral hemagglutina- tion present in susceptible cells is completely inactivated; when this precau- tion was taken there was no difference between the time at which hemagglu- tinins and soluble antigen could be first detected (Liu and Henle, 1951; Burnet and Lind, 1954). It seems that the evidence that the soluble antigen appears before the hemagglutinin in infected cells is not definite. C. Cell-Associated Antigens of Other Viruses In almost all viruses which have been investigated, small particle antigens, detectable by complement fixation and usually called soluble antigens, have been found associated with viral multiplication. It has generally been found that these antigens remain intracellular, that they are distinctly smaller than the virus elementary bodies, and that they have less serological specificity than the elementary bodies. There are at least two distinct cell-associated antigens of vaccinia virus, the soluble LS antigen (Craigie, 1932), which is a protein, and the hemagglu- tinin (Nagler, 1942), a lipid-prote complex. During the growth of the virus these antigens increase in amount parallel to the increase of viral infectivity (Metcalf, 1955; Maitland and Tobin, 1956) and there is no evidence of their appearing before mature virus or of their being used up to produce new virus. A soluble antigen is produced during the growth of herpes simplex virus (Hayward, 1949) but there is no evidence for its preceding the appearance of infective virus (Scott et al., 1953; Modi and Tobin, 1954). Van den Ende and co-workers (1957) studied the soluble (12 my) antigen which develops in the brains of suckling mice infected with rabies virus. The antigen appeared after the rise in titer of infective virus and apparently continued to increase in amount after the maximal infective titer was reached. Differences in sensi- tivity of infectivity and complement fixation titrations affect the mterpreta- tion of all these results but there is no support for the idea that soluble antigens in general appear in cells before mature virus. Among the smaller viruses, poliomyelitis (Selzer and Polson, 1954), African horse sickness (Polson and Madsen, 1954), and foot-and-mouth disease (Bradish et al., 1952) viruses have all been shown to produce soluble complement-fixing antigens of small particle size; Polson has drawn attention to the fact that the estimated particle size for the soluble antigens of many different viruses is about 12 my. Apart from this, there is no evidence to suggest whether the soluble antigens of different viruses have a similar function or are produced by analogous processes. There is, however, an interesting BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 133 hint derived from studies of virus-infected cells stained with fluorescin- labeled antibody. Liu (1955) showed that fluorescence was first detectable in the nuclei of infected cells and that this nuclear fluorescence was caused by the presence of the soluble antigen; a similar observation has recently been made for fowl plague virus by Franklin (1957). Also, early nuclear fluorescence was found for herpes simplex virus (Lebrun, 1956), but it is not known whether the early fluorescence is due to elementary bodies or to the soluble antigen. If this point were investigated for a number of viruses it might give an important hint about the development and function of soluble antigens (see also Section VII). IV. Dynamics OF THE DEVELOPMENT OF INFECTIVE VIRUS In this section an attempt will be made to summarize studies on dynamic aspects of the development of fully infective virus in infected cells. Ideally such studies are carried out along the lines of the “one-step growth curves” of bacteriophages, 1.e., an attempt is made to limit experimentally the growth of virus to a single cycle by preventing spread of the newly produced virus to neighboring cells. In practice, studies with many viruses have not yet reached this ideal. Nevertheless, an attempt will be made to analyze for a number of animal viruses the rate at which infective virus appears, the yield of virus (measured as infective doses) per cell and the duration of the growth cycle. oo A. Poliomyelitis Virus Dulbecco and Vogt (1955) carried out one-step growth curves of polio- myelitis virus of all three types in suspensions of isolated monkey kidney cells. The technique was the same as that which they used with western equine encephalitis virus (see Section IV, B). The cells are incubated with virus in buffer to allow adsorption of virus. The cells are then washed and diluted greatly in nutrient medium so as to minimize readsorption of released virus. They found that after a lag period of about 4 hours there was a sharp rise in the titer of extracellular infective virus, which increased for 2-3 hours and then tailed off. During the period of rapid rise in infectivity the increase occurred at a nearly exponential rate. The yield of virus was roughly 100 infective doses per infected cell for all three types. Lwoff et al. (1955) studied the kinetics of virus release from single cells. They used type 1 poliomyelitis virus grown in monkey kidney cells and deposited in drops of paraffin oil after they were infected. The lag period was 53-7 hours, a longer period than found for suspended cells cultures, perhaps because of the difficulties of the technique of culturing individual cells. Once virus liberation started, the virus was re- leased extremely rapidly and 100-200 infective doses per cell were liberated 134 ALICK ISAACS from the cells within about half an hour. Again the rapid liberation may have been a reflection of the culture conditions. Howes and Melnick (1957) have also studied the growth of type 1 poliovirus in monkey kidney cell monolayers and they ensured that a single cycle of growth was observed by infecting their cells at a multiplicity of 4 or more. They found that after a lag period of 3 hours there was a rapid and, at first, exponential increase in the amount of cell-associated virus with the produc- tion of approximately 100 plaque-forming units of virus per infected cell. At about 54 hours, after the initiation of infection, 50 % of the total virus yield had been produced. The yield of extracellular virus lagged behind the cell-associated virus by about 1 hour. B. Western Equine Encephalitis Virus Dulbecco and Vogt (1954) used a dilution technique to make one-step growth curves of western equine encephalitis virus grown in chick embryo cell suspensions. The lag period was about 2 hours with a multiplicity of 4, and 34 hours with a multiplicity of 0.15. Once virus release commenced there was an exponential rise in the titer of extracellular virus for about 1} hours, followed by a slowing, with the maximal yield at 6-8 hours. The yield of virus was approximately 100-200 infective particles per cell, but conditions of cultivation were probably not optimal, since a yield of 200-1000 infective particles per cell was found when the virus was grown in a cell monolayer. With a high multiplicity of infection the yield of virus per cell was consis- tently higher than at low multiplicity, suggesting that more than one virus particle per cell can take part in the growth process. This last finding is an extremely interesting one, and should be tested in other virus systems with simultaneous study of the intracellular (or cell-associated) virus and the extracellular virus. Rubin e¢ al. (1955) studied the growth of this virus in a similar system but with particular attention to the rate at which intracellular or cell-associated virus developed. Monolayers were infected at high multiplicity and after a 30-minute absorption period the cells were washed and trypsinized and the suspended cells diluted greatly. After a lag period of about 1} hours the cell- associated virus, measured after rupturing the cells by ultrasonic vibration, was found to increase exponentially for a period of nearly 3 hours, the virus doubling in amount every 15 minutes. As described earlier, no virus could be detected in these cells at zero time, 1.e., the time at which the cell dilution was made. Rubin et al. calculated that the average maximum number of cell- associated infective virus particles per cell at the end of the period of exponen- tial rise was only 4 to 10, although each cell had spontaneously released 100 infective particles, i.e., the virus is very rapidly released when formed (this BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 135 point is further discussed in Section V). This suggests that the exponential rise in the amount of intracellular infective virus reflects a logarithmic rate of increase of one virus precursor, whose rate of production limits the rate of production of mature infective virus forms. C. Influenza Virus Henle et al. (1947) carried out one-step growth curves of influenza A and B viruses in the allantoic cavity of the chick embryo, using a large dose of ultraviolet irradiated heterologous virus as an interfering agent, to prevent the occurrence of a second cycle of virus growth. The validity of this tech- nique depends on whether the irradiated virus can be used to prevent a second cycle of virus growth without inhibiting the first cycle. In their paper, Henle et al. (1947) thought that the irradiated virus interrupted the readsorp- tion of virus released in the first cycle, but, since the irradiated virus was given 1 hour after the live virus, it may have also induced some interference in the cells initially infected. Hence the appearance of a step in the growth curve described by Henle et al. might be to some extent artificial, and indeed, in later work, Henle and associates (1954) showed that virus continued to be released from infected cells over a period of 30 hours or more. With this limitation, the results of Henle and his co-workers show that for the PR8 strain of influenza A, after a lag period of 6 hours, new virus was released into the allantoic cavity within the next 2 hours with an average value of 63 ID;, produced per ID,, of virus adsorbed. The corresponding figures for the Lee strain of influenza B were 36 ID,, produced per ID;, adsorbed after a lag period of 9 hours. Henle and Rosenberg (1949) later extended these findings to other strains of influenza A and B and found that, in general, the influenza B viruses showed a longer lag period and a lower viral yield than the A strains. However, if in fact the viral interference is induced at a constant time after moculating the irradiated virus, the apparently lower yield may simply reflect a slower rate of growth of the B viruses and it is known that the yield of some influenza B strains per egg is not significantly less than for A viruses. Cairns (1952) studied the release of influenza A virus hemagglutinin into the allantoic cavity and used the V. cholerae enzyme, RDH, to prevent readsorption of newly released virus. Viral hemagglutinin was first liberated after 5 hours but the 50 % liberation time, i.e., the time at which half the ultimate first-cycle yield of virus had been liberated, appeared to be about 8 hours. Cairns also measured the amount of virus liberated in each half-hour period (“differential response’) in this system and found that periods of peak liberation of virus occurred at 74-9 hours, 14-144 hours, and about 19 hours. He interpreted these findings in terms of cycles of virus growth, of which the first cycle lasted longer than succeeding cycles. However, it was later found that infected cells continue to liberate influenza virus over long periods of time 136 ALICK ISAACS (Henle et al., 1954). In addition, Schlesinger and Karr (1956) observed that in a similar system, when cells were infected at a high multiplicity, i.e., when presumably all cells were infected initially, periodic increases and decreases occur in the amount of liberated virus, associated with stepwise breakdown and partial restoration of the inhibitor of viral hemagglutination present in the chorioallantoic membrane. These periodic increases and decreases appear to correspond in time to the cycles observed by Cairns (1952). Recently, Cairns (1957) has shown that there is asynchrony in the initiation of infection by influenza virus, with great variation in the time before hemagglutinin appears in the allantoic fluid after infection with very small inocula. This finding makes it even more difficult to disentangle the different cycles which together make up the normal growth curve. Tyrrell (1955) found that when influenza virus was grown in tissue cultures of chick embryo lung the average yield of virus was 650 hemagglutinating particles per cell. In this technique one hemagglutinating particle corresponds roughly to one infective dose and 10 virus particles as counted in the electron microscope (Tyrrell and Valentine, 1957), 1.e., the yield was about 6500 virus particles per cell. The period of liberation observed was 72 hours. Recently, Ledinko et al. (1957) studied the growth of influenza virus in trypsinized suspensions of chick embryo lung cells, adding RDE after the initiation of infection in order to restrict the growth of virus to a single cycle. After a lag period of 24-23 hours there was an exponential rise in the titer of extracellular infective virus for about 2 hours, with a rate of increase of roughly 10-fold per hour. D. Newcastle Disease Virus of Fowls (NDV) Rubin et al. (1957) carried out one-step growth curve studies of NDV grown in monolayers of chick embryo lung epithelium, the cells being infected at a multiplicity of 2 infective doses per cell. The lag period, as measured by the first appearance of new virus, was 3-4 hours but the average latent period, defined as the time when the yield of virus corresponded to one infective particle per cell, was 5-6 hours. In studying the development of cell- associated viruses, the cells were treated with antiserum before disintegrating them by three cycles of freezing and thawing. The antiserum treatment was necessary to remove virus superficially adsorbed to the cells and to give a clearer picture of the development of intracellular virus. Intracellular virus was found to increase at a nearly exponential rate, with an approximately 30-fold increase between the 4th and 6th hours before the increase tailed off. The curve of extracellular virus increased more steeply and 24 hours after infection there were roughly 1000 infective doses of virus released per infected cell. About 50 °% of the final yield of virus was produced at 8-10 hours after the initiation of infection. BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 137 In selecting studies of viral growth to quote in this section an attempt was made to choose experiments in which either a one-step growth curve was carried out or some other method was used for restricting the viral growth to a single cycle of multiplication. For herpes simplex and vaccinia viruses, however, this has not been possible and values quoted for the rate of growth and yield of these viruses are subject to the qualification that growth of virus is accompanied by spread of newly formed virus to fresh cells with secondary and later cycles of virus growth. E. Herpes Simplex Virus Scott et al. (1953) studied the growth of herpes virus on the chick chorion. After a lag period of 6 hours the virus increased exponentially until about 16-18 hours, when a plateau was reached; this was followed by another slow rise from 24 to 48 hours. The smaller the moculum the longer was the lag period but the rate of viral mcrease was independent of the size of the inoculum, being about one log,, every 2 hours. During the period of rise of infectivity in the membranes there was a parallel rise of infectivity in fluid washings (1.e., extra-cellular virus), but the titer in the washings was about 1 log,» less than in the membrane. At 24—48 hours the titer in the washings was about the same as that in the membranes. The results quoted by Wildy (1954) follow closely those of Scott et al. After a lag period of about 6-8 hours there was an exponential increase of cell-associated virus with an increase of about 3 log,) over a period of about 10 hours; again the extracellular virus lagged considerably behind the cell-associated virus. The presence of “‘steps’’ in the growth curve, as suggested by Wildy, is not very clearly defined. F. Vaccinia Virus Briody and Stannard (1951) measured growth curves of vaccinia virus on the chick chorioallantoic membrane. They thought that steplike increases in virus activity occurred at 8 and 16 hours after infection, but the findings were not consistent or in line with those of other workers. Anderson (1954), working with the same system, noted a steady decrease in the amount of recoverable virus up to the 9th hour. Thereafter, the amount of virus in the membrane increased exponentially at a rate of about one log,, every 4 hours. Overman and Tamm (1957) studied the development of vaccinia virus grown in pieces of chick chorioallantoic membrane in vitro. After a lag period of about 8 hours the virus increased at a nearly exponential rate until the 48th hour. However, in this system the growth of virus was much slower than when virus was grown in vivo and the increase was only about 2 log,, over the 40-hour period. The maximal virus yield in the membranes occurred at 3 days. The titer of virus in the medium was always much less than that in the 138 ALICK ISAACS membrane at the same time, the proportion being about 1 % at 3 days; even at 7 days it was less than 10 %. Maitland and Magrath (1957) have also studied the growth of this virus in pieces of chorioallantoic membrane suspended in vitro, as well as in minced chick embryo and chick embryo cell suspensions. The increase in infectivity was variable from one experiment to another but tended to be more nearly exponential than linear. In reviewing studies of growth curves for such different viruses grown under varying cultural conditions it is surprising to find so many similarities. After a lag period, all the viruses studied have shown an exponential increase in titer for part of their growth cycle. Also, there is a suggestion that those viruses which have a longer lag period have a slower rate of increase of viral infectivity. It is surprising, too, how frequently a rate of increase of infectivity of the order of about one log,, per hour has been reported, i.e., for polio- myelitis (Dulbecco and Vogt, 1955), western equine encephalitis (Rubin et al., 1955), influenza (Henle et al., 1947; Ledinko e¢ al., 1957), and Newcastle disease virus (Rubin et al., 1957). It is interesting to note that all these viruses contain ribonucleic acid (RNA); on the other hand, herpes simplex and vaccinia, probably both DNA viruses, seem to have a longer lag period and a slower rate of growth. It is worth speculating whether the rate of growth of these different viruses may depend on the rate of synthesis of the different nucleic acids in infected cells. V. THe RELEASE OF VIRUS FROM INFECTED CELLS it is striking in comparing growth curves for different animal viruses to note great differences in the ratio of extracellular to cell-associated virus titer during the period of exponential increase of virus, e.g., for western equine encephalitis virus the extracellular virus titer was ten times greater than the cell-associated virus titer, whereas for vaccinia virus the extracellular virus was only 1/100 of the titer of cell-associated virus. These differences clearly reflect major differences in the rate at which fully infective virus is released from cells after it is formed. In this section the rate of release of different viruses 1s compared in order to gain some insight into the mechanism by which virus is berated from infected cells. A. Western Equine Encephalitis and Poliomyelitis Viruses In studying the release of western equine encephalitis virus from individual cells, Dulbecco and Vogt (1954) found that by 4 hours, 60 % of cells had released small amounts of virus, i.e., were low yielders, whereas by 7 hours, only 11 % of the cells were low yielders, the other cells now giving high yields of virus. This finding implies that cells were continuing to release virus over the period 4—7 hours, 1.e., individual cells release virus over a long period BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 139 of time. Rubin et al. (1955) tried to measure the release time for this virus. They assumed that the rate of increase of extracellular virus is at any instant proportional to the concentration of the intracellular virus present at that instant and to a velocity constant. This can be expressed as de/dt = K,B(t), where K, represents the probability per unit time that an intracellular virus particle will be released. (It is, however, theoretically possible that K, might alter during the growth cycle, if for example, the release of virus was accelera- ted as cell damage increased.) Therefore, 1/K, is the average time for virus to be released once it has become infective, and it can be calculated either mathe- matically or graphically from the experimental data on the rate of increase of intracellular and extracellular virus. For western equine encephalitis virus growing in chick embryo monolayers the average release time was calculated to be just under one minute. Lwoff et al. (1955) noted an extremely rapid release of poliomyelitis virus from individual monkey kidney cells, practically all the virus being released within half an hour. On the other hand, Howes and Melnick (1957) concluded that Type I poliomyelitis virus was slowly released from monkey kidney monolayers. This is based on the findings that the increase in extracellular virus began about one hour after the increase in cell-associated virus and that the free virus was always much lower in titer. Even after 11 hours’ growth, less than 20 % of the total virus was free. These findings are very different as regards extracellular virus from those of Dulbecco and Vogt (1955), who measured the rate of increase of extracellular (but not cell-associated) virus in the same cells. The only obvious difference between the two sets of experi- ments is that Dulbecco and Vogt used a one-step growth curve technique in order to minimize adsorption of newly released virus to fresh cells, whereas Howes and Melnick assayed samples from the monolayers directly, where presumably newly released virus might be rapidly adsorbed to fresh cells. Possibly, therefore, rapid release of virus is a more accurate picture for poliomyelitis virus, but direct comparison of the two techniques might help to decide this point. B. Myzxoviruses Cairns (1952) studied the release of influenza virus from eggs in- fected with virus at limiting infective dilution, where presumably a single allantoic cell was infected initially. He found that virus liberation occurred over a period of 3 hours. The technique was not suitable for measuring further liberation of virus since additional cycles of infection could not be prevented. However, Henle et al. (1954) inoculated de-embryonated eggs with 10° ID;, of influenza virus and measured the differential (hourly) release of virus. 140 ALICK ISAACS RDE was given with each hourly washing in order to restrict virus liberation to a single cycle, a finding which they demonstrated experimentally. It was found that virus liberation occurred at a constant rate for 36 hours, so that the virus must leave the cells by a process which does not destroy the cells. Cairns and Mason (1953) measured the amount of influenza hemagglutinin in chorioallantoic membrane and allantoic fluid at hourly intervals after infection with very large doses of virus and found that between the 4th and 8th hours the membrane titers were consistent with a constant release period of about one hour. The experiments were then repeated but with large doses of RDE given 2} hours after the virus. No hemagglutinin was detectable in the membrane until 7 hours after infection and thereafter the titer was about one-tenth of that in the fluid. The effect of the RDE was therefore to elute newly released virus from the surface of the cells, and Cairns and Mason calculated that the liberation time, excluding the time during which virus can be released by the action of RDH, is less than 2 minutes. The most obvious explanation of these findings is that once it is formed the viral hemagglutinin is rapidly liberated from the cells, but that it remains adsorbed to the cell surface, from which it is released within about one hour, presumably by its own enzymatic action. Support for this theory comes from the work of Ackermann and Maassab (1954). They found that the release of influenza virus from chick chorioallantoic membrane 7n vitro was inhibited by «-amino- p-rmethoxyphenylmethane sulfonic acid (AMPS), and that this effect was reversible by RDE given after the virus. When concurrent titrations of virus in fluid and membrane were carried out, it could be shown that the effect of the AMPS was to delay by many hours the liberation of virus from the cells. Ackermann and Maassab interpret these results as indicating that normally the viral enzyme functions in liberating virus from the cells and that AMPS inhibits the enzymatic activity of the virus, the action of AMPS being in turn reversible by RDE. Rubin et al. (1957) calculated the release time for Newcastle disease virus grown in chick embryo lung epithelium, using the same assumptions and formula as these workers had adopted in their work on western equine encephalitis virus. The average release time for NDV was found to be 80 minutes. However, by treating the cells with NDV antiserum before disrupt- ing them, it was found that a great deal of cell-associated virus could be neutralized during the period of exponential rise, although antiserum treat- ment had little effect when carried out during the lag period. This suggests that during the period of exponential rise in virus titer most of the cell- associated virus is superficially adsorbed to the cell surface; when the release period was recalculated to take this into account, it was found that only a few minutes were required for newly matured virus particles to reach the cell BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 141 surface. The results are strikingly similar to those obtained by Cairns and Mason for influenza virus. C. Herpes Simplex and Vaccinia Viruses In their studies of herpes simplex virus, both Scott e¢ al. (1953) and Wildy (1954) noted that the titer of released virus was about one log,, less than that in the cells during the period of exponential increase in virus titer. Similarly, for vaccinia virus, Overman and Tamm (1957) found that during the period of exponential increase less than 1 °% of the virus in the membranes was released into the medium. It is clear, therefore, that herpesvirus and vaccinia viruses, once formed, are only slowly released from the cells. Calculation of the release time would not be meaningful, however, since pock-producing viruses such as these probably spread from cell to cell without being liberated into the medium. Andrewes (1930) showed that herpesvirus could continue to grow in tissue culture in the presence of immune serum, which, however, prevented the initiation of infection and Black and Melnick (1955) noticed that herpes B virus grown in monkey kidney cells was able to form plaques even without an agar overlay. Primary plaques could not be prevented from developing by immune serum given shortly after the virus but the serum prevented the appearance of further plaques. By contrast, poliomyelitis virus grown in the same cells did not form plaques and immune serum could avert the spread of the virus even when infection was well under way. From what is known of influenza virus, it behaves very like poliomyelitis virus in this respect. We have, therefore, two contrasting modes of virus release and spread. In the first, exemplified by poliomyelitis, virus is rapidly released from cells once it is formed and spreads to new cells mainly by invasion via the medium. In the second, exemplified by B virus, virus is slowly released from cells once it is formed and can spread to neighboring cells by direct cell-to-cell spread. Cell-to-cell spread presumably occurs with varicella virus too, since Weller (1953) found that passage could be effected only by cell extracts and not by the fluid phase. For four different adenoviruses, Ginsberg (1957) has also provided evidence of slow release of virus from these cells. Once more, it is interesting to note that the division of animal viruses into those which are released rapidly from cells and those which are released more slowly corres- ponds (so far as it is known) to viruses which contain RNA and those which contain DNA. A possible division in terms of size alone is ruled out by the fact that the adenovirus, a DNA virus, is slightly smaller than influenza virus. At the moment, there is little guide to the possible significance of this observation, and, indeed, it is difficult to be sure of its accuracy, since the different viruses have not been studied under comparable conditions. However, this may be an interesting generalization and it seems important to see how far it is applicable among the animal viruses. 142 ALICK ISAACS D. Mechanism of Release of Virus and Virus Filaments During the multiplication of bacteriophages release of virus occurs when the bacteria lyse. The results quoted in the previous three sections show that for most animal viruses virus release can occur without cell lysis. Nevertheless, there is a range of behavior from a virus such as western equine encephalitis virus, which is very rapidly released from cells within a minute or two of its maturation, to a virus like that of varicella in which release is minimal under ordinary conditions of culture, and spread mostly occurs directly from one cell to its neighbors. Further details of the mechanism of virus release are best investigated by morphological studies; these are reviewed in Chapter 5. In passing, however, it may be noted that there is some morphological evidence to justify the biological findings on virus release for different viruses. Thus, Lwoff et al. (1955) studied the appearance of single cells by phase-contrast microscopy in parallel with biological observations on the release of polio- myelitis virus. A hyaline zone appeared at the periphery of the cell just before virus release occurred and underwent a pronounced vacuolization during the time of release. This suggests that release of virus occurs through lysis of part of the cell and that there was extensive cell disruption by the time virus release ended. On the other hand, Robinow (1950) found that vaccinia virus was excreted from cells along narrow filaments of cytoplasm protruding from the cells. The virus appeared to pass to the tip of these stalks and then to leave the cell with only minimal damage to the cell surface. There is a further contrast in a virus like adenovirus, which Morgan et al. (1956a) demonstrated in their beautiful electron micrographs to be almost exclusively intranuclear, and in influenza virus particles, which could be found just beneath the cell surface only (Morgan et al., 1956b). Presumably, influenza virus particles, in contrast with adenoviruses, are finally assembled just beneath the cell surface and are excreted from the cell soon after they are formed. From the biological point of view, one of the most interesting phenomena bearing on the question of formation and release of virus particles is the occurrence of filamentous forms of virus. Since they were first described by Mosley and Wyckoff (1946) filaments have been found associated with almost all members of the myxovirus group, but they are most characteristic of recently isolated strains of influenza virus A (Chu et al., 1949). The evidence obtained by Donald and Isaacs (1945b) from particle counts and infectivity titrations and on the effect of ultrasonic vibrations suggested that filaments were infectious, and that they could be fragmented into many hemagglutina- ting segments but without increasing the number of infective units. Burnet (1956) found that filaments could be ruptured by suspending them in water without reducing significantly the infectivity of the preparation; he suggested that infectivity was limited to the sphere frequently found at the tip of the filaments. Burnet also noted that filaments had some of the physicochemical BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 143 characters of the cell surface although their serological behavior is that of virus, since they are specifically agglutinated by viral antiserum. Valentine and Isaacs (1957) observed in an electron microscopic study that, on digestion with acid and trypsin, filaments largely disappeared, whereas trypsin- resistant nucleoprotein rings remained after similar treatment of virus spheres; Ada and Perry (1958) and Burke e¢ al. (1958) found that filamentous preparations of influenza virus contained much less ribonucleic acid than the corresponding spherical forms. Taken together, these results point to filaments having an infective “warhead”’ (Lindenmann, 1957) containing nucleic acid, and a long tail containing noninfective viral hemagglutinin. Morphological studies of filaments in the course of formation and cut in ultrathin sections show that the interior of the filament is continuous with the cytoplasm of the cell (Morgan et al., 1956b), and that the surface of the filament is continuous with the surface of the cell (Bang and Isaacs, 1957). The conclusion suggested by these findings is that viral spheres forming near the cell surface may drag out long filaments of the cell cytoplasm behind them; on the basis of Cairns and Mason’s work this process may occur during the time that the virus is trying to free itself by its enzymatic action from mucoprotein present at the cell surface. At some point, the filaments would become nipped off at their base and so be formed of variable length. If this is so, one must conclude that a large part of the cytoplasm near the surface of the infected cell has acquired the specific hemagglutinating and serological behavior of virus elementary bodies (see Section VII). It seems, too, that highly filamentous strains of virus cause little damage to the cell surface. On the other hand, as the virus becomes better adapted to a tissue, filaments become much less common and it may be that the process of adaptation is accompanied by an increasing ability of the virus to damage the cell surface, thus preventing the formation of filaments. In this connection, Bang (1955) has illustrated the different degrees of cell destruction produced by virulent and avirulent forms of virus. Finally, one cannot but be struck by the almost haphazard way in which influenza virus filaments and spheres appear to be assembled together and released from the cells, a picture which contrasts strikingly with the regular array of adenovirus particles demonstrated by Morgan et al. (1956a) within the cell nucleus. VI. IncoMPLETE VIRUS In the previous sections of this chapter different intracellular stages in the normal cycle of viral development have been described. This final section is devoted to the abnormal development of virus which occurs under certain conditions, particularly when cells are heavily infected with virus, and which results in the formation of what von Magnus called incomplete virus. There 144 ALICK ISAACS are still two possibilities with regard to incomplete virus. It may be thought of as deformed virus, produced as a result of a faulty cycle of multiplication, or it may be immature virus prematurely released from cells before its” development had been completed. At the moment, it seems to be logically impossible to distinguish between these two possibilities, and it may be best to regard incomplete virus simply as the product of an abnormal cycle of virus development without specifying the abnormality further. Unfortunately, most of the work on incomplete virus has been carried out with influenza viruses, although recently there have been hints that the same phenomena may apply to other viruses. The formation of incomplete virus is such an interesting phenomenon that search among other viruses seems well worth- while. A. Properties of Incomplete Virus Incomplete influenza virus, as defined by von Magnus (1946), and prepared by serial passages of undiluted allantoic fluid virus, has a very low infectivity relative to its agglutination titer. In comparison with standard influenza virus, incomplete virus may have a ratio of infectivity/hemagglutinin titer (I/HA ratio) of 101 to 10~® of that expected. The hemagglutinating behavior of incomplete virus does not differ from that of standard virus as determined by the number of virus particles (seen on electron microscopy) per agglutinat- ing dose (Werner and Schlesinger 1954; Donald and Isaacs 1954a). Hence, the characteristic property of incomplete virus is its low infectivity per virus particle. In addition to its low infectivity, incomplete virus is also reported to have a low toxicity when injected intracerebrally into mice (Bernkopf, 1950). However, it appears to be a good interfering agent, as shown by its ability after inoculation into the chick embryo to suppress hemagglutinin production by a large dose of standard influenza virus given 18 hours after the incomplete virus (von Magnus, 1954). These findings are much more significant than earlier results reported by von Magnus (1951a) of ““autointerference’’ induced by incomplete virus inoculated intranasally into mice, since the interpretation of autointerference in mice is complicated by the fact that the incomplete virus would provide an antigenic stimulus which might mimic interference. This possibility can be inferred from further experiments by von Magnus (1951a) from which it is clear that incomplete and standard influenza virus showed very similar antigenic behavior, both in im vitro hemagglutination inhibition tests and when formolized and tested for their antigenicity in mice. In the latter case, the antigenicity of the two preparations was proportional to their agglutination titer (i.e., particle count) and not to their infectivity. This finding implies that incomplete and standard virus may have a similar surface structure; further evidence for this came from experiments indicating BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 145 that both viruses showed similar behavior on adsorption to, and subsequent elution from, chick red cells. In addition, Svedmyr (1949) found similar enzymatic activity of both forms in their ability to destroy the inhibitor present in normal allantoic fluid. From present information, therefore, incomplete and standard virus have a similar surface structure and the difference between them is a more fundamental one. One difference which was noted early in a careful comparison of standard and incomplete virus was in their sedimentation behavior in the high-speed centrifuge (Gard et al., 1952). Standard virus consisted for the most part of rather homogeneous particles with a sedimentation constant of 747 A. In highly incomplete virus preparations the 747 S component was replaced by a very inhomogeneous slower sedimenting component of sedimentation constant varying from 430 to 675 S. As mentioned earlier, there was not sufficient morphological difference between particles of standard and incom- plete virus seen in electron micrographs to account for the different sedimenta- tion behavior, and a difference in particle density appeared to be the most likely explanation. Support for this came from a study by Uhler and Gard (1954), who found that incomplete virus had a higher lipid content than standard virus. From a preliminary analysis of the lipid content of prepara- tions showing varying degrees of incompleteness, it was concluded that the findings could not be explained by postulating virus of low and high lipid content, i.e., there was some variability in the lipid content among different virus particles. These findings might, therefore, account for the unusual sedi- mentation behavior of incomplete virus, but, at the moment, there is no explanation for the difference in lipid content in terms of mechanisms of viral synthesis, nor is it clear what is the influence, if any, of the lipid content of the virus particle on its infectivity. More pertinent to the problem of the infectivity of incomplete virus is the work of Ada and Perry (1956) on the nucleic acid content of preparations of influenza virus showing varying degrees of incompleteness. In a most important study, these workers showed that when the infectivity of different preparations (expressed as log infectivity titer/agglutination titer) was plotted against its nucleic acid content a linear relation was found. There was no simple proportionality between infectivity and nucleic acid content and a hundred-fold decrease in infectivity was accompanied by a drop in RNA content from about 1% to 0.5%. It is interesting to note that Lief and Henle (1956b) found a very similar ratio between the amount of “soluble antigen” (see Section III, A) which could be extracted from incomplete virus by ether treatment and the 1/HA ratio of the virus used, Le., there appears to be a close relationship between the soluble antigen content and the nucleic acid content of the virus. The proportions of the nucleotide bases in the study by Ada and Perry were not significantly different in incomplete and VOL. 11I—10 146 ALICK ISAACS standard virus. These findings imply that preparations of virus showing varying degrees of incompleteness are heterogeneous with regard to the total nucleic acid content of their individual virus particles. In view of the known significance of nucleic acids in the infectivity of bacteriophages and tobacco mosaic virus, it seems reasonable to conclude from Ada and Perry’s findings that the probability of a virus particle being able to initiate infection may be a function of its nucleic acid content. This conclusion gains further signifi- cance when we consider the mode of production of incomplete virus. B. Production of Incomplete Virus The all-important factor in producing incomplete virus seems to be that large doses of seed virus are essential. As is discussed below, seeds obtained after two, three, or more serial passages of undiluted virus are more effective in producing incomplete virus than seed which has not been passaged in this way. Nevertheless, the same fact holds with all these seeds, 1.e., if they are passaged in undiluted form, further incomplete virus is produced, whereas if they are passaged diluted 1/100 or more, standard virus is once more produced (von Magnus, 1951c). It is natural to suppose that the critical factor in producing incomplete virus is the multiplicity of infection. This poimt was first discussed by von Magnus (1951c) who found that about 10? agglutinating doses of virus, or more than 10° virus particles (Donald and Isaacs, 1954a) was the mmimum amount which would produce incomplete virus. Recent counts of the number of surface allantoic cells in 10- and 11-day eggs are between 1.8 x 107 (Tyrrell e¢ al., 1954) and 3 to 4 x 10? cells (Cairns and Fazekas de St. Groth, 1957); this would correspond to a multiplicity of between about 10 and 100 virus particles per cell. Since incomplete virus is not produced when seeds are diluted 1/100 or more, these figures support the idea that multiple infection of cells is an important factor in the production of incomplete virus. On the other hand, Cairns and Edney (1952) concluded from their results that incomplete virus was produced when only 1 % of the cells was infected initially. However, their calculations were based on the assump- tion that a multiplicity of 1 occurred when 10?-? agglutinating doses of virus were taken up by the allantoic cells; more recent techniques of counting virus particles and allantoic cells would suggest a much higher figure for the multiplicity after this inoculum. From Cairns and Edney’s experimental findings, incomplete virus production is first detectable when between 10 and 100 agglutinating doses of virus are taken up (10° to 10° virus particles) and the corresponding figure given by Horsfall (1954) is 3 x 107 “hemagglutina- ting’ particles, which is equivalent to 3 x 108 virus particles (Tyrrell and Valentine, 1957). There is, therefore, nothing in these estimates to refute the idea that multiple infection of cells is a critical factor in producing incomplete BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 147 virus. Since a low nucleic acid content appears to be the characteristic deficiency of incomplete virus, it seems that multiple infection may strain the cells’ ability to synthesize viral nucleic acid, a hypothesis which might be tested experimentally. The second factor which von Magnus (1951b) stressed was that the degree of incompleteness increased with serial passage. In a most detailed series of growth curves he studied the effect of serial passages of PR8 virus in the undiluted form. In the standard virus used to initiate the passages the ratio of infectivity : hemagglutinin titer (I/HA ratio) was about 10°. First passage virus had an I/HA ratio of about 10°; second passage, about 10%; third passage, between 10! and 10?; and fourth passage, between 1 and 10. Since no attempt was made to wash out the inoculum, the infectivity of the residual inoculum may have accounted for a proportion of the infectivity found, so that the I /HA ratios might have been even lower had the moculum been removed before the new yield of virus was liberated. This may explain the finding that, in the de-embryonated egg, where the inoculum is removed, there is more incom- plete virus produced in a single passage (Bernkopf, 1950), and the same is true in the intact egg (Cairns and Edney, 1952). Another finding which von Magnus (1951b) noted was that while the I/HA ratio declined on passage, the yield of hemagglutinin was not greatly affected until the fourth passage, when it was one-tenth (or less) of that produced in standard passages. As a result, when a fifth passage was made with the same volume of infected fluid, the number of particles present was considerably less than in earlier passages and, as would be expected on the hypothesis that multiplicity of infection was a critical factor, the I/HA ratio of fifth passage material again rose sharply. The low yield of hemagglutinin in fourth passage material is presumably a manifestation of viral interference, but since interference requires many hours to become established (Fazekas de St. Groth et al., 1952), it is only apparent in these experiments when the amount of infective virus in the inoculum is very small. Experimentally, von Magnus (1954) has demonstrated that a large inoculum of incomplete virus suppressed hemagglutinin production by a large dose of challenge virus, provided a few hours’ interval was allowed before the challenge virus was inoculated; an 18-hour interval gave the highest degree of interference. The serial passage experiments thus disclose a complex situation. As a result of multiple infection of cells with standard virus particles, the progeny has a low I/HA ratio and a low nucleic acid content, and on serial passages this process is continued. Experimental addition of about 10° ID,, of standard virus along with incomplete virus did not signifi- cantly influence the yield from the incomplete virus alone; large doses of standard virus led to a slightly greater yield of virus, but with a lower 1/HA ratio than when the same dose of standard virus alone was given (von Magnus, 1951c). Hence, when the incomplete virus particles infect a cell at the 148 ALICK ISAACS same time as standard virus particles, there is a sharing of the available nucleic acid among the progeny. If the incomplete virus is given sufficiently long before the standard virus, there is time for viral interference to become established and a diminished yield of total virus results. The suggestion that in multiple infection of cells the nucleic acid may be shared among the viral progeny is intended as an analogy to ideas of viral recombination. Another method of producing incomplete virus is to use as seed standard virus which has had its infectivity greatly reduced by incubation for a few days at 37 or 22°C. (Henle, 1953; Horsfall, 1954; Paucker and Henle, 1955a). In an experiment described by Paucker and Henle (1955a), a preparation of virus with an I/HA ratio of 10%-1 was incubated at 37°C. for 5 days, when its infectivity had dropped more than 100,000-fold, the I/HA ratio being 10°-°. When this was used undiluted as seed the progeny had an I/HA ratio of 10?-3; seed diluted 1/10 gave progeny with an I/HA ratio of 10?-°, while seed diluted 1/100 gave progeny with an I/HA ratio of 10°-4. These findings show that hemagglutinating virus of low infectivity is produced by seed virus which has been inactivated at 37°C.; there is a striking resemblance to the production of incomplete virus by von Magnus’s method in that only concentrated inocula produce this effect, while on dilution of the seed 1/100 standard virus is produced. One difference is that when incomplete virus is prepared by von Magnus’s method, passage of incomplete virus results in progeny with a lower I/HA ratio, whereas inoculation of virus inactivated at 37°C. leads to progeny with a slightly higher I/HA ratio than the seed. This finding argues against the possibility that incomplete virus formation in von Magnus’s experiments is caused by accumulation of virus spontaneously inactivated at 37°C., as does the fact that the incomplete virus released in the first 2 hours of the growth cycle has a low I/HA ratio, similar to that formed later in the cycle (Finter et al., 1955). At the moment, there is no evidence on the nucleic acid content of progeny from virus inactivated at 37°C.; this would be an interesting point to investigate. If the nucleic acid were low, the results might be explained by postulating that during prolonged incubation at 37°C. the virus nucleic acid becomes sufficiently damaged to prevent the normal synthesis of viral nucleic acid, but, provided multiple infection of cells occurs, it is still able to take part in the synthesis of a lower quota of nucleic acid with a resulting yield of incomplete virus. The findmg by Fazekas de St. Groth and Graham (1955) that, in eggs treated with metaperiodate, influenza virus growth results in the production of hemagglutinating virus of low infectivity may be due to the action of aldehydes on the virus rather than to an action on the cells, since a similar effect could be demonstrated on virus tn vitro (Liu et al., 1956; Schlesinger and Karr, 1956). Fazekas de St. Groth and Graham (1954) also investigated production of incomplete virus by different strains of influenza virus, using BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 149 the technique of serial passages (von Magnus, 1951b). They found that different strains varied in the ease with which they could be induced to form incomplete virus. When the strains were arranged in order of ease, the only biological activity with which the order corresponded was the time taken for effective entry of virus into the cell. At the moment, the significance of this interesting observation is not known. C. Partial Cycle of Virus Development Produced by Incomplete Virus In earlier work on incomplete virus it was assumed that the incomplete virus was noninfective, but more recently it has become clear that it can initiate partial cycles of virusdevelopment. Burnetet al. (1954, 1955) carried out some very interesting studies of the growth of incomplete virus in de-embryo- nated eggs. The technique used was to inoculate different doses of incomplete virus, remove the seed by washing, and treat the membranes with RDE in order to obtain a single cycle of growth. The fluids were then harvested at 7 hours and the yield of hemagglutinin titrated; in addition, the amount of hemagglutinin produced between 7 and 22 hours was measured. Over a given range of virus dosage the yield of hemagglutinin at 7 hours was proportional to the amount of virus taken up; this applied to both complete and incomplete virus. This finding shows that in both cases a single cycle of virus production was being observed. However, the yield from incomplete virus was much greater than would have been expected from the infectivity of the inoculum; in one experiment, quoted by Burnet e¢ al. (1954), the yield from an incomplete virus preparation with a I/HA ratio of 10~*-1 of the complete virus used gave a yield of one-sixth of the complete virus at all virus dosages tested. Since the yield per particle was independent of the virus dosage and on the basis of calculations of the multiplicity of infection the results could not be explained by multiplicity reactivation. Burnet et al. conclude that a consider- able proportion of the incomplete virus in the seed is able to undergo a single incomplete cycle of multiplication to produce viral hemagglutinin, but that this hemagglutinin is unable to continue to produce full infection. Paucker and Henle (1955b) have also found that virus heated at 37°C. and rendered noninfective was still able to produce hemagglutinin in a single cycle. They suggested that the live virus particles which remained in the seed after inactivation at 37°C. were able to initiate a single cycle of virus growth, but were prevented from initiating a second cycle by viral interference induced by the inactivated virus in the seed; as mentioned earlier (Section VI, B), this interference would not be established immediately and hence there would be no inhibition until the first cycle of virus growth had been completed. In the experiments of Burnet et al. (1954) the restriction to a single cycle of virus production at high virus doses may also be due to a late induction of inter- ference. 150 | ALICK ISAACS Beale and Finter (1956) studied the ability of preparations of incomplete virus to produce soluble antigen in the chorioallantoic membrane after inoculation into the allantoic cavity. They measured the appearance of soluble antigen 6 hours after inoculation and compared the amount found with that produced by corresponding amounts of standard virus when similarly inoculated. It was found that preparations of incomplete virus produced much more soluble antigen in this test than would have been expected on the basis of their infectivity, although less was produced than by the same number of standard virus particles (assessed by the hemagglutinin titer). In these respects the findings are strictly analogous to those of Burnet et al. (1954) on the production of viral hemagglutinin by incomplete virus. Furthermore, as a parallel to the findings of Paucker and Henle (1955b) on the production of hemagglutinin by virus inactivated at 37°C., Isaacs and Fulton (1953) found that virus inactivated by heating at 56°C. for 8 minutes produced signifi- cantly more soluble antigen when grown on the chick chorion than would have been expected from the infectivity of the inoculum. Beale (1954) has also found that the virus present in the chorioallantoic membrane 43 hours after inoculating a large dose of influenza virus has a low I/HA ratio, and that this, too, when examined by the 6-hour soluble antigen test, produces more soluble antigen than would be expected on the basis of its imfectivity. Presumably, this is another manifestation of the behavior of incomplete virus, in this case present in the chorioallantoic cells 45 hours after inoculation and before being liberated. However, a precursor to fully infective virus might be present in such membrane extracts and it would be interesting to test the behavior of the ““S” hemagglutinin in experiments of this kind. Incomplete influenza virus is not wholly noninfective, therefore, but is capable of initiating a partial cycle of virus multiplication, though not a complete cycle. In addition, virus which has been inactivated by heating at 37°C., or 56°C. for a short period, behaves in a way which is superficially similar to incomplete virus in this respect. It appears that virus which lacks its normal complement of nucleic acid, although unable to complete the virus multiplication cycle, may nevertheless initiate the synthesis of some viral constituents. D. Incomplete Viruses Other Than Influenza Although the formation of incomplete viruses is an important phenomenon to understand in itself and also because of the light it throws on virus multi- plication processes in general, almost all the work on the subject has had to be carried out with influenza viruses. Even such closely related viruses as NDV and fowl plague apparently do not give rise to incomplete virus when passaged by similar techniques to those used by von Magnus for influenza, BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 151 and outside this group of viruses there are very few published accounts of attempts to prepare incomplete virus forms. This seems a pity, since the phenomenon is most interesting and information on the conditions under which it is applicable to other viruses would be worth having. Mims (1956) described some indirect evidence that Rift Valley fever virus could be induced to show incomplete virus forms. Mims noted that when large inocula of virus were passaged serially in mice low infective titers resulted, whereas high titer virus was produced by small inocula. The low- infective virus was shown to be capable of interfering with the growth of infective virus by prolonging the incubation period and reducing the peak infectivity titer reached. However, these effects could be explained in alternative ways and, in particular, there is a possibility that if the seed population of virus particles is heterogeneous in respect of virulence, large doses of virus may act as an antigenic stimulant and produce an apparently similar effect (cf. Schlesinger, 1949). Sanders (1957) has shown that encephalomyocarditis virus can be titrated in three different ways. The plaque-forming titer measures the infectivity of a preparation in a plaque titration with ascites tumor cells of mice. The same cells can be used to measure a second property of the virus, its cell- killing ability; this depends on the fact that aqueous eosin stains dead cells and the virus can be titrated indirectly by this method by calculation from the number of unstained cells (the calculation is based on the Poisson distribution). Finally, the virus can be titrated by its hemagglutinin titer, and there is good evidence that all three titrations measure properties of the virus particles themselves. If virus is mcubated for more than 1 hour at 37°C., the plaque- forming titer is reduced more than is the titer by the other methods, and the same is true of virus freshly released from cells after infection with high multiplicities of virus. This phenomenon requires further investigation, but the results of these experiments seem to resemble closely those of von Magnus for influenza virus. Recently, Cooper and Bellett (1957) have produced some evidence that an autointerference phenomenon observed with vesicular stomatitis virus grown in chick embryonic cells in vitro is due to the produc- tion of incomplete forms of virus. The incomplete, or T, forms of virus are produced on serial undiluted passages, but not with diluted passages, and only when cells are mixedly infected with T and live virus particles; also the T forms are spontaneously liberated from cells, as in the case of influenza virus. VII. Conciusions Throughout this chapter, the attitude adopted has been that although research on the multiplication of bacteriophages has provided the greatest stimulus to the investigation of similar problems in animal viruses, in all 152 ALICK ISAACS consideration of work on animal viruses the results should be judged on their own merits. Clearly, there are great differences between the multiplication of animal and bacterial viruses, particularly in the way they penetrate and are released from their host cells. Nevertheless, such differences are quite overshadowed by the many strong resemblances found in their methods of multiplication. More particularly, in plant, bacterial, and animal viruses more and more evidence is accumulating to show that the key role in multi- plication is played by the viral nucleic acid. Some recent work from Tiibingen on animal viruses adds emphasis to this conclusion. Wecker and Schafer (1957b) examined chick embryonic cells during the lag period, Le., 30-180 minutes after infection with P%?-labeled fowl plague virus. They found that in extracts of these cells a high proportion of the radioactivity was not sedimented under conditions when the virus elementary bodies would have been sedimented. From such extracts a proportion of the radioactivity could be precipitated by antiserum to the soluble antigen and part of the remainder was shown to consist of viral nucleic acid by the fact that it was hydrolyzed by ribonuclease. It seems, therefore, that after the virus particles enter the cells many of them are broken down to give soluble antigen (a ribonucleo- protein) and free nucleic acid, with phospholipid in addition. The further course of infection in a similar system was studied by Breitenfeld and Schafer (1957) by means of antibody, labeled with fluorescin isocyanate, to both soluble antigen and hemagglutinin. This technique of localizing antigens within cells was preferred to that of homogenization and differential centri- fugation, since it was found that by the latter method viral constituents were nonspecifically adsorbed to the various cell components. They found that soluble antigen was first detected at 3 hours after infection and was localized to the cell nuclei (cf. Liu, 1955). From 5 hours the fluorescence diffused slowly out of the nucleus in the absence of obvious nuclear damage, and by 14 hours the whole cell showed fluorescence. By contrast, the hemagglutinin was first detected at the fourth hour throughout the cytoplasm, but the nucleus was not stained. The hemagglutinin gradually moved toward the cell margin and at the fourteenth hour the cells showed deeply fluorescing borders. This is the first definite evidence that different viral constituents are synthesized in different cellular sites. Thirdly, Wecker and Schéfer (1957c) have now pro- duced additional evidence that infection by eastern equine encephalitis virus can be initiated by viral ribonucleic acid. They extracted infected mouse brains with phenol and showed that these extracts were infective. Further- more, in contrast to a preparation of virus with the same infective titer, the infectivity of the phenolic extracts was sensitive to very small amounts of ribonuclease (but not deoxyribonuclease); it was not sedimented significantly with high centrifugal forces; and it was not inactivated on precipitation with ethanol. There is, therefore, strong presumptive evidence that the infectivity BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 153 of these extracts is due to viral nucleic acid. When we remember, too, that in a number of animal viruses, as was shown for some plant viruses, the amount of ribonucleic acid in viruses of very different sizes is surprisingly constant, corresponding to a molecular weight of about two million per virus particle (Frisch-Niggemeyer, 1956), it is difficult not to feel that viral nucleic acids will all be found to play a similar role in other animal viruses. The difference between viral ribonucleic and deoxyribonucleic acids and the information contained in the nucleic acids which can induce the cell to synthesize viral constituents and assemble them into mature virus particles are surely the main challenges for future viral research. REFERENCES Ackerman, W. W., and Maassab, H. F. (1954). J. Exptl. Med. 100, 329. Ackermann, W. W., Ishida, N., and Maassab, H. 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Sci. & Biol. 9, 303. Chapter VII Interference between Animal Viruses R. WALTER SCHLESINGER Saint Lows University School of Medicine, Saint Louis, Missouri Pee TOM yo) atetas ake! Chapter IX Variation in Virulence in Relation to Adaptation to New Hosts FRANK FENNER AND JOHN CAIRNS Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia Mowe ETNtTO CLC GIONIN ocr ecc cyais: Sere. o. ovo 61 9 a Sister eleueechaalacctepa vhs w erclajcle wa oasttre aa varra einen 225 ie Mechanisms of Virus) Variation... : 5 t).is< oh cir eiiaists steels citi eels brew: ote Sake 227 Ave ly Bacberial: VarUsesie). steers trols ess. aia Byvsic!s wisi oh sod RES i eishel tee tels « Ale seta sais 228 HES opera Arn tr VTLS ra es Say dest che Sento ate ol pay cp ovared lak lash edel Se RRP al Aeonets es la IIE 228 Me ALIA LION Dy VEDA ELON of: coarse cies Matar crcs ake she are Ee dacop Pere als hae ale eet 229 Septont- Induced! Variation 32.15 8 <0 sacs oa, 2 ao niolere Tualelchn sto sis ie ee ae 229 Bag GENOUICVECOM DIMA IO cists cre f2 soar stet neni ecient Sere eter arses ake ae 229 ie 7Animal Virus- Host Cell Systems.% ss ./.4 ss.aw cae ones hing acs ee eben ents 230 XP SIP IO Cel AS VStEMIS! tartan ats ches igtiecneeaute nuanaeety he Oc etcetera eternal 230 i the-O—D: Change of-Intlienza Wirusiy.4..cnce.= 22s avekete rien Seo) eee 230 2.) Variation: of Poliovirus inuVissue Culture ...%:5/ <4 os. seu as ances 232 3. Various Examples of Coexistence of Virus and Cell.................. 234 Bo Strueburally Complex SVSteMS) 5.0 eccce own /sic oe eat Oats Bees we hes oe ee 234 i Pock vaniantsrOf the POXxviITUSes: .2/s-.es ss Auer eee wee hel bien es eae 236 2. 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InrTRODUCTION Although there is an increasing trend toward the study of animal viruses and the cells they infect as systems of intrinsic biological interest, the science of animal virology has until recently been developed as a branch of medical science, by men concerned primarily with viruses as agents of disease. The essential prerequisite for the experimental investigation of an animal virus or of a virus disease of man or a domestic animal is the production in some laboratory host of recognizable signs of infection associated specifically with the virus in question. Such a result may be observed the first time the virus is inoculated into an experimental host. For example, cowpox virus from natural human or bovine VObs tits) 225 226 F. FENNER AND J. CAIRNS infections produces characteristic lesions on the chorioallantoic membrane or in the rabbit skin, and neither the nature of the lesions nor the “efficiency of plating’ for the passage host changes with serial passage in either of these hosts. In other cases minimal signs of infection are observed on first introduc- tion of the virus into a new host, yet after serial passage, sometimes prolonged, a lethal infection is regularly produced, e.g., the adaptation of poliovirus and dengue virus to mice (Armstrong, 1939; Sabin and Schlesinger, 1945). During serial passage a process of “adaptation”’ of the virus to the new host is said to have occurred. Once a virus has been adapted to an experimental host, the attention of many workers has turned to a frequent by-product of adaptation, namely, the coincident attenuation of the virus for its original host. Thus, after his experiences with the attenuation and exaltation of virulence of bacteria by passage in different animal hosts Pasteur turned his attention to rabies (Pasteur et al., 1884) and found that by serial passage of this virus in rabbit brain it changes from “‘street’’ virus to “fixed” virus, and showed enhanced virulence for the rabbit, and reduced (but still high) virulence for the dog and man. The best-known examples of the production of an attenuated virus vaccine by adaptation to a new host tissue are the yellow fever vaccines. The “French neurotropic” strain was obtained by serial passage in the mouse brain (Peltier e¢ al., 1940) and the 17D vaccine during prolonged passage in tissue culture (Theiler and Smith, 1937). In consequence of the practical requirements just outlined, there has grown up an enormous volume of literature concerned with the growth of animal viruses in different laboratory hosts—involving primary adaptations, secon- dary adaptations from one laboratory host to another or one organ or tissue of a particular host to another, and studies of the effect of such adaptations on the pathogenic capacity of the virus in its original host. Because of the complexity of the experimental material, studies on the adaptation of animal viruses to new hosts have so far yielded little basic information, but have given rise to a great volume of superficial observations. These, although invaluable at the practical level, reveal virtually nothing of the mechanism of adaptation. For most people, maximum virulence of a virus, as a character, indicates that the strain possesses the maximum destructive power which can be associated with the multiplication of a virus of that sort. With bacterial viruses, where the host is a unicellular microorganism, virulence implies cell lysis. The animal host, however, consists of a vast number of individual cells differing widely in many properties. Some may be suited and others unsuited to support virus multiplication, some are of major and others of minor importance in the economy of the host. In animals, therefore, there is no such simple dichotomy and no general agreement as to how the word virulent can VARIATION IN VIRULENCE Paap be defined. A strain of influenza virus which is virulent for man is, in common parlance, either one that spreads rapidly and extensively through the popula- tion, or one that is characteristically associated with a severe morbidity and, for influenza, a relatively high death rate. These effects may obviously be due to widely different and potentially very complex mechanisms. In the same way a strain of poliovirus may be called virulent for man because it paralyzes a high proportion of those it infects, or because it kills a high proportion of those it paralyzes. These vague uses of the term virulence, sanctioned by convention, merely amount to calling any strain virulent if it has high destructive power of the type associated with that virus. There are obviously many grades of virulence, not the simple alternatives we see in bacterial viruses. This usage does emphasize the medically and socially important aspect of the virus diseases of man and his domestic animals, but it deals only with the end product of a multitude of unknown factors, and tells us nothing at all of the possible mechanisms involved. There are more sophisticated definitions, but not one of these can be framed so that it will cover the activity of all animal viruses. With this definition of virulence, it is apparent that nearly all examples of adaptation of viruses to new hosts involve increase in virulence for the new host. The field under review therefore covers nearly all examples of alteration in the biological behavior of viruses. Two ways of treating the subject present themselves. One would be to catalogue all examples of variation in host spectrum among animal viruses. This might be useful if the literature were both interesting and as yet inadequately reviewed, but it would be dull, brut- ish, and long. Catalogues of examples of variation in animal viruses have already been prepared (Findlay, 1936, 1939). The other procedure, which will in fact be followed, is to try to break down the process of “adaptation” into its component parts, starting with the simplest system, i.e., animal cells in suspension, which is analogous to the bacteria which serve as hosts for bacterial viruses. With this start it will be possible to progress steadily through systems of increasing complexity where adaptedness necessarily constitutes a more complex property, and a strain of virus may be “adapted” or “unadapted”’ for any one of several widely different reasons. Ultimately, we shall discuss the only available example where the natural evolution of virulence of virus in virgin territory has been studied. First, we shall discuss briefly what is known of the mechanisms of virus variation. Il. Mecuanisms or Virus VARIATION For all living creatures, natural selection copes with, and occasionally profits by, the instability of the genome. So it is, too, for viruses. Survival of 228 F. FENNER AND J. CAIRNS the line is, as we shall see in the last example of variation in virulence, not assisted by exceptionally great lethality; if the host is rapidly killed, the chance of successful transplantation of the virus to another host may not be high (Burnet, 1945). Throughout this chapter we are dealing with the variation of virulence and the way in which discernibly new varieties of virus arise. Occasionally, evidence is mentioned which indicates that, during the change in character of a virus, one population of virus particles is being replaced by another. But the process of natural selection must be read into everything that follows. At all levels of complexity, the adaptation of a virus to a new situation must be pictured as the product of a single change or succession of changes, augmented by intense selection pressure. What is known of the manner in which these primary changes occur will now be discussed briefly, taking first of all evidence from the field of bacteriophage to provide a basis for what little is known in the case of the animal viruses. A. In Bacterial Viruses First, and simplest, variants may arise by a process of random mutation occurring during virus replication; in this case, variant clones are found in the virus populations yielded by individual cells (Luria, 1945, 1951). The distribution of such clone sizes among the yields of individual cells shows that the change is random rather than directed by the host cell. The second process operates primarily on the host cell; variant clones are distributed randomly among virus populations yielded by large groups of host cells, but these clones arise from particular cells which themselves are variants in that they give rise (uniformly or not) to virus variants (Fredericq, 1950a,b). Although such host-induced changes are usually only phenotypic (Luria and Human, 1952; Bertani and Weigle, 1953), there is apparently one instance of the change being ultimately genotypic (Fredericq, 1950a,b). In many instances, this distinction between primarily virus and primarily host- induced variation disappears, for there is increasing evidence of genetic homogeny between certain bacterial viruses and their hosts (Stent, 1958). Thirdly, genetic recombination may provide a mechanism whereby changes resulting from the operation of the first two mechanisms may be transferred from one virus to another differmg in many aspects of its genetic constitution. It is conceivable that genetic recombination may play an important part in the ecology of bacterial viruses. B. In Animal Viruses Adequately documented examples from the field of animal viruses of the three processes described in the previous section are so rare as to be almost unique. VARIATION IN VIRULENCE 229 1. Variation by Mutation Although random mutation is usually invoked as the basis for variation in animal viruses and, on this basis, mutation rates have been calculated (Burnet and Bull, 1943; Medill-Brown and Briody, 1955; Dulbecco and Vogt, 1958), in only one instance has there been any attempt to demonstrate the essential character of a mutational change—namely, its randomness (Dulbecco and Vogt, 1958). In this instance, the d to d+ mutation of polio- virus, it was possible to show significant fluctuation in the number of mutants present in sister populations of virus grown in aliquots of one cell population. In other words, sister populations of virus show nonrandom variation in proportion of mutants if multiplication has occurred since they were separated, whereas the variation is, of course, random if there has been no multiplication in the interim. 2. Host-Induced Variation There are at least two examples of what is probably host-induced variation of an animal virus. The first, and less conclusive, concerns the behavior of unadapted influenza A virus and Newcastle disease virus (NDV) in mouse brain; after small inocula of non-neurotropic influenza virus into mouse brain, there is a single cycle of virus production, the virus produced being infective for the allantois but apparently not for mouse brain in that multiplication there was confined to one cycle (Cairns, 1951, 1954). The second concerns the behavior of encephalomyocarditis virus in mouse brain; a variant of this virus, adapted to form plaques on sarcoma 180 cells, loses its capacity to form plaques on these cells if passed through a single cycle in mouse brain; this capacity is restored after a single cycle in Krebs 2 carcinoma cells (Sanders and Hoskins, 1958). 3. Genetic Recombination The genetic importance of sex in higher organisms, as a means of producing novel combinations of characters and providing the opportunity for rapid spread of mutant characters through the gene pool, needs no emphasis. The very short generation time and enormous populations of viruses provide a much greater opportunity for the combined interplay of mutation (random or host-induced) and selection to serve as the major basis for evolution. Except possibly with bacteriophages it seems to us unlikely that genetic recombination is of importance in the evolutionary history of viruses, but recent work has shown that it may be a useful method for producing novel combinations of virus characters in the laboratory. In a number of animal viruses only phenotypic mixing has been detected, i.e., the novel combination dissociates to the parental types on passage. Genetic recombination has been unequivocally demonstrated with influenza virus (Burnet, 1955) and with 230 F, FENNER AND J. CAIRNS vaccinia virus (Fenner and Comben, 1958). In both cases characters concerned with pathogenicity or virulence behaved in a complex manner (as would be expected from their nature) and cannot yet be analyzed physiologically or genetically. However, genetic recombination should eventually prove a powerful weapon for the analysis of these most obscure and complex aspects of viral behavior. III. Anrmat Virrus-Host Cet Systems It was noted earlier that all known associations of viruses with animals are much more complex than the relation between a bacterial virus and its host. For analytical purposes, however, it is possible to construct simpler artificial systems or to utilize situations which do not involve the whole complex activity of the animal. We shall consider three types of virus-cell system— simple cell systems, analogous to the bacterial virus-bactertum system; struc- turally complex but relatively self-contained cell systems, such as the chorio- allantoic membrane of the chick embryo; and cell systems involving sequential infection of a variety of organs. As a special case of the latter we shall introduce sequential infection of individual animals, i.e., we shall extend our study to include some epidemiological facets of adaptation and virulence. A. Simple Cell Systems This group comprises those examples of infection of cells by viruses in which spread of infection from cell to cell is not limited by the nature of the system and merely demands that, after infection of a cell, the progeny virus is liberated from that cell in a form which can infect further cells. In this group fall all examples of infection of cells in fluid suspension and also those examples of infection of cells in sheets, where extension of infection from cell to cell can occur freely by way of the overlying fluid. As examples of adaptation and variation in such systems we will discuss the work done on the changes in influenza virus on adaptation to the embryonated egg (the O-D change), and on the variants of poliovirus in tissue culture. In neither of these cases is the problem of spread from cell to cell a deciding issue in the success of virus multiplication. 1. The O-D Change of Influenza Virus This example of variation of an animal virus was the first to receive detailed attention from the point of view both of the nature of the character undergoing change and the manner in which the change occurred. On first isolation, influenza A strains were found to agglutinate human and guinea pig red cells more readily than fowl red cells (Burnet, 1942). On further VARIATION IN VIRULENCE 231 passage, either in the amnion at low dilution or in the allantois, the virus acquired the capacity to agglutinate fowl cells to as high a titer as human cells. This change, from the O (“original”) to the D (“derivative’’) phase, is associated with a change in affinity for various hemagglutinin inhibitors (Stone, 1951) and in position in various receptor gradients (Burnet et al., 1946a, 1949); it is also associated with a marked reduction in virulence for man (Burnet and Foley, 1940) and emergence of capacity to multiply in the allantois (Burnet and Bull, 1948). However, it has been possible to maintain virus in the O phase through repeated passages in the amnion if each passage is initiated at “limit dilution”’ (Burnet and Bull, 1943). This shows that a mere history of prolonged multi- plication im the amnion is not in itself a certain inducer of the change to D phase: for this to happen there has to be full opportunity for minority populations to supplant the original type. It is as if D phase virus arises relatively infrequently during multiplication in the amnion; although endowed with considerable survival advantages over O phase virus, D phase virus must seldom represent the majority type after a single passage in the amnion. These considerations led Burnet to propose that the O-D change represents a relatively rare mutation backed by intense selection pressure, rather than a host-induced change. As we shall see later, an exactly analogous argument has been proposed as evidence for the mutational nature of the attenuation of poliovirus in tissue culture (Sabin et al., 1954). Unfortunately, the O-D change, on more detailed examination, proved to be more complex than originally supposed. The dividing line between agglutinative behavior of O- and D phase is not completely clear-cut. Between the two extremes (no agglutination of fowl cells, even in the cold, and agglutina- tion of fowl and human cells to equal titer) there occur intermediates which are not easily distinguished from mixtures (Burnet and Stone, 1945a). Continued passage of O phase virus at limit dilution in the amnion tends to exaggerate the O character of its agglutination, so that strains which showed slight agglutination of fowl cells when originally isolated, show no agglutina- tion of fowl cells after prolonged passage in the amnion; perhaps it was because of this that the definition of O phase agglutination became progressively more stringent with the passage of time (Burnet and Bull, 1943; Burnet and Stone, 1945a; Burnet, 1950, 1951; Burnet e¢ al., 1949): At least one strain, IAN, has stabilized im the O phase on passage in the amnion so that D mutants are not readily obtained (Burnet e¢ al., 1949); there is at least one instance of conversion of D phase to O phase by prolonged passage in the amnion (Magill and Sugg, 1948). Further, D phase virus (or intermediate phase virus) does not necessarily possess survival advantages over O phase in the amnion, since the only experiment testing the behavior of artificial mixtures in the amnion gave inconclusive results (Burnet and Stone, 1945a). 232 F. FENNER AND J. CAIRNS In fact, the O-D change does not really lend itself to a detailed examination of the processes of mutation and selection, since there is no clear-cut procedure for assaying separately either O or D phase virus. Originally it was supposed that only D phase virus multiplies in the allantois, which therefore could be used as a specific host for assay of D phase virus. However, O phase virus has been shown to multiply in the allantois to a limited extent (Burnet and Stone, 1945b), so that there is no precise procedure for distinguishing mixtures of O and D phase virus from intermediates. In view of these difficulties it is clear that no accurate determination of the relative survival advantages of the various types can be made, and hence no estimate of mutation frequency is possible. Lastly, the idea that agglutinative behavior bears any simple relation to the host range or past history of any particular strain is itself difficult to sustain. Influenza B strains are not in the O phase on first isolation (Burnet et al., 1946b; Hirst, 1947a); indeed, on passage, the agglutinative behavior of B strains tends to proceed in the reverse direction (Ledinko and Perry, 1955). Most recent influenza A strains have on isolation shown qualities closer to D than to O phase. And we shall see, in the section on adaptation of influenza virus to mouse lung, that changes in hemagglutinating behavior, although often associated with the process of adaptation, are not an obligatory step in the change in host range but a secondary phenomenon which may bear little relation to the alteration in host range. 2. Variation of Poliovirus in Tissue Culture The study of the d mutation by Dulbecco and Vogt (1958) has already been discussed in the section of mechanisms of variation. It remains in this section to deal with those cases where there is some relation between neuropathogenicity and behavior in tissue culture. In general, prolonged passage in tissue culture results in a reduction of neuropathogenicity. This, although not invariably true, has been demon- strated repeatedly [Enders et al. (1952) and countless others since] during the search for attenuated strains suitable as vaccines. There is some evidence for the belief that this attenuation occurs by way of selection of spontaneous mutants which have survival advantage in tissue culture and, incidentally, a lowered neuropathogenicity. Thus, passage of Mahoney, Y-SK, and Leon in cynomolgus kidney tissue cultures results in conversion to attenuated variants only if large inocula are used for each passage; if passage is conducted at “limit dilution” the strain keeps its neuropathogenicity, presumably because one passage will seldom provide adequate opportunity for the attenuated mutants to become the majority type (Sabin ef al., 1954). This result is exactly analogous to the findings with the O-D change (Burnet and Bull, 1943). Alteration of neuropathogenicity to mice of a line of type 3 VARIATION IN VIRULENCE 233 poliovirus has been shown to be influenced by the type of cell used for tissue cultures as well as the constitution and pH of the medium (Li, 1957). These studies, however, have so far not been conducted with pure clones of virus; consequently, what is potentially an extremely interesting situation has yet to be exploited. Similarly, it has been shown that cells which are incapable of yielding virus when infected in the host will proceed to develop virus if taken from the animal and grown in vitro (Kaplan, 1955). Thus, environment may have a marked effect on the potentiality for cells to support virus multiplication. Other variants with reduced neuropathogenicity have been demonstrated to arise in tissue cultures. Such are the minute plaque variants of MEF-1 which appear in the presence of bovine serum (Takemori et al., 1957), and the variants of several strains which are capable of multiplying in monkey kidney cells at 30°C. (Dubes and Chapin, 1956). In the latter case, it is tempting to believe that the lowered neuropathogenicity is a simple con- sequence of a development of marked heat sensitivity; certainly it was only in those cases where the mutant proved incapable of multiplying in monkey kidney cells at 37°C. that neuropathogenicity was lowered. Where looked for, there has been ample evidence of inhomogeneity of various lines of poliovirus. Heat-inactivation curves show the concavity which would result from inhomogeneity; part of this is due to stable characters which breed true on further passage (Stanley et al., 1956), and part is due to some phenotypic difference not apparently reflected in genotype (Pohjanpelto, 1958). In this respect, poliovirus, in its heat-stability, is like certain bacterio- phages (Adams, 1953) and viruses of the influenza group (Jones, 1945; Goldman and Hanson, 1955). Just as poliovirus can be shown to vary in its host range, so can certain lines of once-cloned cell lines be shown to contain subpopulations with varying susceptibility to poliovirus which breed true (Vogt and Dulbecco, 1958). The more resistant types have a very low probability of being infected on meeting a virus particle; hence, with such cells, it is possible to establish infected cultures in which cell and virus multiplication can apparently coexist indefinitely unless something disturbs the balance (Vogt and Dulbecco, 1958). Whether it would be possible to select for new virus types, capable of infecting these resistant cells, has not yet been determined; this step has been taken only in a rather more complex situation (Sabin, 1952). However, there is every reason to believe that, as for bacteria and bacteriophages, both host cell and virus enjoy variation in susceptibility and host-range, respectively. The mechanism of cell insusceptibility in this case is probably not dependent upon the absence of adsorption (Vogt and Dulbecco, 1958), and so is not comparable to most examples of bacterial mutation to phage resistance. 234 F. FENNER AND J. CAIRNS 3. Various Examples of Coexistence of Virus and Cell At various times, instances of the coexistence of virus and cell multiplica- tion have been demonstrated. These are of relevance here in that they represent a distinct type of interrelation of virus and cell which could be taken to epitomize one type of avirulence. Production of vaccinia virus in roller tube cultures of fibroblasts was suggested to occur without preventing continued multiplication of the infected cells (Feller et al., 1940). The peaceful existence in Drosophila of the virus of CO,-sensitivity and its transmission through the egg is another example (L’Héritier, 1958), as must be all those cases where there is trans- ovarial transmission in arthropods of viruses pathogenic for some other host (Fukushi, 1933; Syverton and Berry, 1941; Florio and Stewart, 1947; Black, 1950). Indeed, it has yet to be shown that the multiplication of any of the arthropod-borne viruses in their vectors is associated with any cell destruction. None of these cases was amenable to the detailed quantitative investigation which is necessary in order to prove that liberation of virus by the cell is not necessarily associated with cell death. The establishment of a carrier state in tissue culture for poliovirus and NDV has recently been demonstrated (Ackermann, 1957; Cieciura eé al., 1957) and in these cases it should be possible to demonstrate nonlethal virus liberation, if it happens. So far, only for the infection of chicken fibroblasts by Rous sarcoma virus has it been demonstrated that a cell may liberate virus and then give rise to a relatively permanent line of multiplying cells, each of which liberates more virus (Temin, 1958). These examples, some clear-cut, some merely suggestive, demonstrate the difficulties inherent in any definition of virulence. Rous sarcoma virus achieves its virulence for chickens by way of a relation with its host cell that represents an extreme variety of avirulent infection. B. Structurally Complex Systems In none of the examples of virus infection mentioned so far has there been the need to consider the manner of spread of virus infection from cell to cell. This great simplification was justified either because the example concerned only the immediate effects of infection (i.e., the first cycle of multiplication) or because the example concerned a host system in which it was reasonable to assume that there was no limitation in the spread of infection from cell to cell. However, when we consider that animals are characteristically multicel- lular organisms, whose component cells are organized in highly ordered groups often preserved behind a succession of defensive ramparts, it becomes obvious that animal viruses must possess well-ordered powers of infection if they are to spread successfully among the cells of their hosts. VARIATION IN VIRULENCE 235 Even in simple systems, the spread of infection is occasionally seen to be limited. Thus, varicella virus infection spreads among cells in tissue culture only by direct contact between cells and serial passage can be effected only with ground-up cells, not with the fluid supernatant (Weller, 1953). Apart from those numerous instance (dealt with in the next section) where the natural course of infection demands the surmounting of successive barriers, there is ample evidence that the existence of an ordered organization of the host cells into a tissue may impose certain limitations upon the spread of infection. Thus, increasing the area involved by the inoculum by adding hyaluronidase, increases both the infectivity and the size of the resulting lesions of vaccinia virus in the rabbit skin, as well as increasing the proba- bility of subsequent generalized infection (Duran-Reynals, 1929, 1933; Sprunt, 1941); this effect of hyaluronidase is seen also with herpes and vesicular stomatitis infection of rabbit skin (Hoffman, 1931). An enhanced response was found with Borna disease infection of rabbit brain (Hoffman, 1931), and with herpes infection of mouse brain (Levaditi ef al., 1949). Presumably in these infections the spread of virus from cell to cell is limited under normal conditions. Since, in the same tissues, hyaluronidase had no discernible effect upon the progression of infection by rabies, St. Louis encephalitis, or Lansing poliovirus, the infection produced by these viruses does not suffer the same limitations (Levaditi et al., 1949). Perhaps paralleling these observations are those examples in which the severity of a virus infection is increased by the presence of nonspecific irritants (Jones, 1950; Findlay and Howard, 1950a) or by coexistent mnfec- tion by some quite unrelated virus (Lépine and Marcenac, 1948; Findlay and Howard, 1950b). All examples of the production of localized lesions, whether in a two- dimensional structure, such as the monolayer overlaid by agar (Dulbecco, 1952), or a three-dimensional structure, such as the liver (Marchal, 1930), provide evidence for limitation of spread—spread either of the infecting virus particles or, in the case of the neoplastic viruses, of the primarily infected cells (Keogh, 1938). Histological studies have provided evidence for the slowness of spread of certain virus infections in the nervous system where others spread rapidly, and this has been demonstrated also by direct assay of the virus content of various parts of brain (Webster and Fite, 1934; Sabin and Olitsky, 1937; King, 1939). Unfortunately, although this subject of the spread of virus infection through organized systems of cells may loom large in the future when know- ledge of virus infection in simple systems has to be applied to complex hosts, available information at the moment amounts merely to demonstrating that the subject exists. Despite this lack, we shall now discuss certain examples of the variation of viruses infecting complex tissues under their own separate 236 F. FENNER AND J. CAIRNS heading as if the exact limitations imposed by each situation were precisely known. 1. Pock Variants of the Poxviruses Infection of the chorion by the poxviruses results in a mixture of cellular proliferation, infiltration, and necrosis, involving both ectodermal and mesodermal cells (Burnet, 1938). The appearance of the pocks is therefore an index not only of the response of the infected cells but also the extent of spread of the infection and the reaction of the host animal—more complex properties than those that control, say, the morphology of the plaques produced by poliovirus in tissue culture. However, like plaques in tissue culture (Dulbecco and Vogt, 1954), each pock must be the result of infection by a single virus particle, since the number formed is proportional to the inoculum size (Burnet, 1936; Keogh, 1936; and many others since). For this reason the pock-producing viruses make a suitable group in which to study virus variation. Cowpox virus (Downie and Haddock, 1952) and neurovaccinia (Fenner, 1958a) both give rise to white pock variants. These arise with a frequency of 10-? to 10-4, are stable on further passage, and presumably owe their difference from the normal hemorrhagic necrotic pock to a decreased tendency to invade underlying blood vessels and an increased tendency to cause cell infiltration. In both cases, attempts to demonstrate significant fluctuation in the frequency of white variants in sister populations of virus particles have failed, but it is not certain whether the observed absence of significant fluctuation is itself significant evidence that the change is not due to random mutation (Fenner, unpublished). Neither change appears to be host-induced in that the frequency of white variants is independent of the source of virus (pock on chorioallantoic membrane or nodule on rabbit skin) (Fenner, unpublished). In view of the exceptionally high frequency of the change (up to 10-* for some strains) compared to all examples of mutation in other viruses, the possibility must be considered that, as in the case of the r mutation in the T-even bacteriophages, it may be the common phenotypic expression of a large number of different genotypes. Whether the wild type can be obtained from crosses between different white variants has yet to be determined. Associated with this change in pock morphology there is a change in virulence for other hosts (Downie and Haddock, 1952; van Tongeren, 1952). The white variants produce less severe reactions in both rabbits and mice. However, the correlation between the pock morphology of the poxviruses and their virulence is not complete, for some strains of vaccinia and cowpox which produce hemorrhagic pocks have low neuropathogenicity for mice and rabbits, and some which produce white pocks are highly pathogenic. VARIATION IN VIRULENCE 237 Pock variants of herpes (Wildy, 1955) and myxoma viruses (Fenner and Marshall, 1957) have also been described. In both cases strains producing small pocks were usually less virulent for other hosts (mouse and rabbit) but the correlation was not complete, as the Californian strains of myxoma virus, though producing very small pocks, were highly virulent for rabbits. 2. Adaptation of Influenza Virus to Mouse and Hamster Lung Many strains of influenza virus have been adapted by passage to produce fatal pneumonia in mice. The ease with which this process occurs varies greatly from strain to strain; some are apparently in the adapted state on first isolation from man (Francis and Magill, 1937; Clampit and Gordon, 1937), some are adapted readily, some become adapted only if special procedures are employed to lower the resistance of the mice (Jones, 1950), and some have so far defied all attempts. There is some evidence that adaptation occurs more rapidly if preceded by way of a few passages in ferrets (Andrewes et al., 1935), and less readily if preceded by prolonged passage in the allantois (Smith et al., 1951; Ledinko and Perry, 1955). Because most strains of influenza virus were not, in their natural state, adapted to any of the convenient laboratory hosts available in the 1930’s, a large volume of literature has sprung up on the mechanism of adaptation to mouse (or hamster) lung and the nature of unadaptedness. The nature of the tissue being infected is more complex than any so far considered. Perhaps because of this the findings are, in places, singularly difficult to interpret. Once again, the process of virus variation is seen to operate by way of an upsurgence of minority types. Selection of the majority type by passage at limit dilution in the allantois, between each passage in lungs, prevents the process of adaptation (Davenport, 1951). Also, as in the case of the O-D change, adaptation to mouse lung is associated with changes in in vitro behavior; thus, there may be a reduction in capacity to agglutinate fowl cells (Hirst, 1947b; Friedewald and Hook, 1948), change in position in the fowl cell receptor gradient (Ledinko, 1956), alteration in enzyme action on and sensitivity of hemagglutinin to ovomucin (Ledinko, 1956), mouse lung inhibitor (Davenport, 1952), and the f-inhibitor of mouse and ox serum (Chu, 1951; Smith et al., 1951; Brans et al., 1953; Briody et al., 1955) and sheep salivary gland mucoid (Ledinko, 1955). Some of these changes in in vitro behavior have been shown to apply to an increasing proportion of the virus particles as adaptation progresses during passage; indeed, an association can be demonstrated, at any stage in adaptation, between the in vitro behavior of clones isolated from the lungs and their degree of virulence (Ledinko, 1956). Just as an increase in virulence seems to be associated with insensitivity to certain inhibitors, so the converse seems to be true; from a mouse-adapted strain there was isolated, by chance, a variant which was 238 F. FENNER AND J. CAIRNS earlier in the fowl cell gradient, had reduced enzymatic action against ovomucin, and was less virulent to mice (Isaacs and Edney, 1950). This, then, is probably the most liberally documented instance of variation in virulence of a virus being associated with changes in i vitro behavior. There is, however, evidence that the association is not always perfect. Variants with extreme insensitivity to inhibitors may be obtained by passage of virus in eggs in the presence of bovine serum inhibitor; these variants, which arise with a frequency of about 10-* (Medill-Brown and Briody, 1955) are not, however, fully adapted to mice (Chu, 1951). Conversely, during the process of adaptation to mice, clones of virus may be isolated which apparently are fully adapted to mouse lung and yet almost unaltered in their in vitro properties (Ledinko, 1956). Unfortunately, there is more conflict of opinion over the nature of the change in in vivo behavior during adaptation; indeed, the conclusion seems inescapable that not all unadapted strains are unadapted for the same reason. Much of this difficulty is due to the exceedingly complex behavior of un- adapted virus on first introduction to the mouse lung. Inoculation of large amounts of unadapted virus into mice results in extensive lung consolidation; on subinoculation of a suspension of these consolidated lungs little or no lung lesions result, and it is only after several more such passages—usually unaccompanied by any lung lesions and therefore constituting “blind” passages—that lung lesions reappear and become a regular feature of each passage (Anderson and Burnet, 1947; Sugg, 1949). In general, it seems that all unadapted strains are capable of multiplying in mouse lung at the first passage (Burnet and Stone, 1945b; Hirst, 1947b; Wang, 1948; Sugg, 1950; Davenport and Francis, 1951; Ginsberg, 1953a; Ledinko and Perry, 1955; Ledinko, 1956). [There has been only one suggestion that lesions may be produced in the absence of virus multiplication, and this example, the case of the related virus, Newcastle disease virus, is open to very considerable objection (Ginsberg, 1951)]. There is no doubt however that, in terms of hemagglutinin titer or infectivity for the allantois, unadapted strains produce lung consolidation and death much less readily than adapted strains. Part of this difference is probably due to the fact that, in terms of capacity to initiate multiplication, unadapted strains are about one hundred times less infective for mouse lung than adapted strains (Ginsberg, 1953b). But most of the difference in pathogenicity of unadapted and adapted strains almost certainly depends on the difference in their behavior once multiplication has been initiated. Studies on the relative multiplication rates and final yields of adapted and unadapted strains in mouse (or hamster) lung have given conflicting results. In some cases, there is a clear increase in the rate of multiplication with adaptation (Wang, 1948; Ledinko, 1956), in some cases, not (Davenport and VARIATION IN VIRULENCE 239 Francis, 1951). In some cases, there is clearly an increase in the final maximum titer of virus in the lungs (Andrewes and Smith, 1937; Anderson'and Burnet, 1947; Hirst, 1947b; Friedewald and Hook, 1948; Wang, 1948; Sugg, 1949, 1950; Davenport and Francis, 1951; Briody and Cassel, 1955; Ledinko and Perry, 1955; Ledinko, 1956), and in some cases, not (Hirst, 1947b; Davenport, 1952); and in some cases the final yield alters only in that it has a higher infectivity for the allantois in relation to its hemagglutinin content (Ledinko and Perry, 1955) or for mice in relation to its allantoic infectivity (Sugg, 1949; Ginsberg, 1953b). In many cases, too, there was evidence that the final yields of infective virus were high in the first one or two passages, passed through a minimum, and then rose again as the virus became fully adapted (Anderson and Burnet, 1947; Hirst, 1947b; Friedewald and Hook, 1948; Ledinko and Perry, 1955; Ledinko, 1956); this effect was seen even when each passage employed the same dose of virus in terms of hemagglutinin (Ledinko, 1956) and, at least in one instance, was associated with marked formation of incomplete virus in the first few passages but not in later passages (Ledinko and Perry, 1955). All these conflicting reports show that unadapted strains are not always unadapted for the same reason. Some show low multiplication rate, some produce low final yields, some mainly produce incomplete virus, and some (probably most) show a combination of these defects. Part of the complexity of the situation may be a manifestation of the complexity of the mouse lung as a host for virus multiplication. First, although the system can for many purposes be regarded as a simple sheet of susceptible cells capable of adsorbing huge quantities of moculated virus (Fazekas de St. Groth, 1950), there is definite evidence, even in the case of fully adapted strains, that virus multiplication fully exploits the system only after very large inocula. Thus, intranasal instillation of saline, a day or two after inoculation of virus, greatly increases the size of the lesions, so that mice die which would otherwise survive (Straub, 1940; Taylor, 1941); also, the fact that circumscribed lesions are produced by sublethal mocula of adapted strains implies that the system is not normally fully exploited. The second major complication to the interpretation of eventsis, of course, that the effect of antibody production can be demonstrated from about the third day (Donnelly, 1951). The effect of these two factors is that part of the change to the adapted state could, on occasion, represent an increase in capacity to spread through the system or a decrease in speed of evoking antibody formation; either of these changes could be manifest simply as an increase in final yield, without necessarily any alteration in infectivity for mouse lung or multiplication rate. Indeed, there is some evidence that unadaptedness to mouse lung may represent a very complex defect, for the process of adaptation is markedly hastened either by keeping the inoculated mice at 240 F. FENNER AND J. CAIRNS low temperature (Sulkin, 1945; Briody et al., 1953) or by combining the virus inoculum with various nonspecific irritants (Jones, 1950). C. Systems Involving Sequence We have discussed variation and virulence in those systems where either one type of cell or one tissue is being infected. There remain those situations where a definite sequence of tissues is infected as the virus progresses toward that final stage by which it is regarded as virulent or not. skin: Bi small intestine : invasion hie : invasion multiplication KG multiplication t regional lymph node: f=) — mesenteric lymph nodes: Holl ese multiplication ! bloodstream: ; bloodstream: primary viremia 3 | primary viremia spleen & liver: el iplicati central focus of aa a 2 4 | multiplication bloodstream: | 3 bloodstream: secondary viremia 6 (secondary) viremia skin- sis | focal multiplication 7 central nervous system: invasion ?via ga multiplication intra neural spread antibody in _.- g serum ae ° SRS ons early rash 10 antibody in contamination AES serum C0) OO oils : environment arch Paralysis ulceration excretion 12 in faeces MOUSEPOX POLIOMYELITIS Fia. 1. Schematic diagram showing the mode of spread of mousepox virus and polio- virus in the host after infection by natural routes (modified from Fenner, 1956). In a great many infectious diseases there is a stage of initial infection (which may be symptomless), a relatively long incubation period, and then a stage of generalization with toxic symptoms and symptomatic involvement of some particular organs or tissues, such as the skin (the exanthemata), the central nervous system (the encephalitides and poliomyelitis), or certain glands (mumps). In such diseases, the symptoms (and hence the assessment of virulence) do not depend on multiplication of the virus at the site of entry but upon multiplication at a number of distant sites. Mousepox (infectious ectromelia of mice) has provided a model for a study of a sequential infection culminating in a rash (Fenner, 1948a). The scheme proposed, and its later extension to cover poliomyelitis (Fenner, 1956) are shown in Fig. 1. VARIATION IN VIRULENCE 241 Generalizing very broadly from this model, it can be said that in such virus infections several steps must occur in sequence: (1) Initial multiplication at the site of entry—this may eventually produce an apparent lesion, like the primary lesion in mousepox, or it may be inapparent. Sometimes multiplica- tion of this type on a mucous surface (respiratory tract or gut) may produce free virus which can infect others. (2) At an interval after implantation, which may be short or not, virus enters the lymphatics. The local lymph node acts as a barrier to further spread, and a further process of virus multiplica- tion must occur in the lymph node if the infection is to become generalized. (3) Once the lymph node barrier is surmounted, the virus has immediate access to the blood stream, and a wide variety of different sites of multiplica- tion is available. Important among these are the vascular endothelial cells and the scavenger cells of the reticuloendothelial system (in the spleen, bone marrow, lymph nodes, and liver). The low entry rate of virus into the blood stream during this primary viremia, and the abundance of cells susceptible to infection ensure that little free virus can be recovered from the blood at this stage. (4) Progressive multiplication in the cells just entered, perhaps with further distribution of virus via the circulation, leads eventually to frank viremia. By the time this stage is reached changes have occurred in the host reaction due to antibody production (both sensitization, in its broadest sense, and serum antibodies being involved). (5) This combination of factors ushers in the next stage of the disease, i.e., localization and multiplication at the “secondary’’ sites (skin, brain, etc.) leading to the various typical syndromes of the generalized infection. Especially in the case of the central nervous system, there are barriers which may prevent this final step, perhaps in the vast majority of cases of infection. Sucha definite series of obligatory steps, each involving different types of cells, allows ample scope for different final results (i.e., different degrees of virulence) to be due to any combination of a number of variations in virus or host. Some virusstrainsmay be avirulent because they merely have a low ability to multiply in the various tissues involved; others may be defective at some particular point in the sequence. The following examples of virus infections in systems involving sequence provide several instances of both types of defect. 1. Mousepox Some of the less virulent variants of viruses which cause severe generalized diseases go through exactly the same sequence of events as the virulent strains, and are not “blocked” at any stage of the sequence. This is true of two attenuated strains of ectromelia virus, the “Hampstead egg’’ strain (Fenner, 1948b), and a strain passaged in tissue culture by Dr. G. Ruckle (Pittsburgh strain). Comparison of the growth of Moscow and Hampstead egg strain in various organs (Fenner, 1948b) showed that virus was detected VOL. 111—16 242 F. FENNER AND J. CAIRNS in the different organs on the same day with each strain, but that the titer rose more slowly and to a lower level in infections with the Hampstead strain. No detailed investigations have been made with the even less virulent Pittsburgh stram, but the virus certainly multiplies in the liver and spleen after peripheral inoculation and produces a slight rash in some mice. 2. Yellow Fever The pathogenesis of yellow fever has been studied in rhesus monkeys by Theiler (1951). He found that, after intradermal inoculation of a moderately virulent strain, no multiplication of virus could be demonstrated in the skin but early multiplication occurred in the local lymph node. This was followed by invasion of the blood stream and then multiplication throughout the recticuloendothelial system (lymph nodes, spleen, and bone marrow) and, finally, by a stage of multiplication in the parenchymal cells of the liver, in the adrenal gland, kidney, and elsewhere. The attenuated 17D strain, on the other hand, multiplied in the local lymph node, invaded the blood, and was then found in the general lymphoid tissue and bone marrow, but to much lower titers than was the case with more virulent strains. Further, only occasionally was 17D found in the liver and then only in trace amounts. The attenuated strain thus multiplied to a lower titer in all sites and usually failed to infect the vital target site of virulent yellow fever virus, the liver. Strains intermediate in virulence between the 17D and the highly virulent Asibi strain showed intermediate levels of multiplication in the lymphoid and reticuloendothelial tissues, and in the liver. A further effect of attenuation of yellow fever virus and other members of this group of arthropod-borne encephalitides is to lower the efficiency of their transmission by mosquitoes (Whitman, 1939; Hammon and Reeves, 1943). In part this operates merely by the reduction in the titer of circulating virus in infected animals, and in part by some specific inability of mosquitoes, even when infected, to transmit the attenuated virus (Whitman, 1939). For viruses of this group, therefore, the stages comprising the whole sequence of infection includes those taking place in the vector—namely, infection and penetration of the gut wall, circulation in the hemolymph and localization in the salivary glands, and finally multiplication in the salivary glands and excretion in the saliva at the time of feeding. There are at least two instances of transmission being blocked by failure of the virus to surmount one of the barriers in the vector (Merrill and Tenbroeck, 1935; McLean, 1955). 3. Poliomyelitis Poliomyelitis shows perhaps the most complex series of events between initial infection and the final production of symptoms. Figure | is probably an oversimplification, but it illustrates the existence of at least four stages— VARIATION IN VIRULENCE 243 occurring in the intestinal wall, the local lymph nodes, some central extra- neural focus, and the central nervous system. Within the central nervous system there may be further barriers to the spread of virus, for histological examination of orally infected monkeys and chimpanzees has shown that asymptomatic infection of the nervous system may occur in some animals, whereas in others there may be extensive spread through the spinal cord and the brain. Sabin’s extensive studies on variation in the neuropathogenicity of the polioviruses show clearly the great complexity of this property. The impossi- bility of subjecting it to adequate genetic analysis at the present time is further exemplified by the observation that at least three different genetic factors are involved in the determination of neuropathogenicity (Vogt ef al., 1957). Variants of poliovirus have been described which illustrate the failure of virus to multiply in one or other tissue of the sequence illustrated in Fig. 1. First, a high degree of neuropathogenicity of intracerebral inoculation may be dissociated from the ability to establish infection by feeding (Melnick, 1951; Sabin, 1956). Second, variants which are able to multiply in the alimentary mucosa (and provoke antibody formation) may fail to be immunogenic after intramuscular inoculation, 1.e., such variants may fail to multiply in lymphoid tissue (Sabin, 1955a). Third, variants incapable of producing either paralysis or lesions after inoculation of large doses into the lumbar cord of chimpanzees will nevertheless multiply extensively in their alimentary tract (Sabin, 1955b,¢c). During the last few years it has become apparent that the polioviruses are members of a large group of viruses which normally parasitize the enteric tract of man—a group now designated as the “enteroviruses.” From the point of view of survival in nature, all that is required of such viruses is that they should be able to infect the cells of the alimentary tract and be excreted in the feces; this appears to be the limit of the activity of most members of the group. The aim of those who seek attenuated variants of the polioviruses for use as oral vaccines is essentially to obtain virus strains with the limited invasive power of most enteroviruses but the same antigenic constitution as those which occasionally invade the central nervous system of man. The major difficulty appears to be the occasional acquisition by attenuated variants of some degree of neuropathogenicity after multiplication in the intestinal tract of man (Sabin, 1955c; Dick and Dane, 1957). The suggestion made by Dick and Dane that, to counteract this risk, the virus should not be transmissible from vaccinated to nonvaccinated people may well be incom- patible with the requirement that the oral vaccine should be immunogenic. It is perhaps relevant that involvement of the central nervous system is of no evolutionary significance for most viruses, whereas in the exanthemata 244 F. FENNER AND J. CAIRNS transmission is dependent upon the virus produced at the final sites of multiplication (the skin and mucous membranes). Whether a virus is able to invade the nervous system is, possibly with the exception of rabies virus and a few others, immaterial to its survival: the ready multiplication of many viruses, when implanted directly into the brain, contrasts sharply with the rare occurrence of natural infection of the central nervous system. The next section illustrates the effect of this further restriction on the selection of variants of different virulence, i.e., the necessity for efficient transmission between different host animals. 4, Myxomatosis Infectious myxomatosis, in laboratory rabbits, 1s another generalized infection involving a sequential invasion of skin, lymph nodes, and vascular endothelial cells, and culminating in a widespread secondary rash (Fenner and Woodroofe, 1953). Myxoma virus originated in South America, where it is enzootic in the local wild rabbit (Sylvilagus braziliensis). In this host it usually produces only a single localized skin tumor, and is transmitted mechanically by mosquitoes (Aragao, 1943). However, minute doses of myxoma virus, if transferred directly from the skin lesion of a Sylvilagus rabbit to the skin of an Oryctolagus rabbit, cause a very severe generalized disease, which is almost invariably lethal. The deliberate introduction of myxomatosis into populations of wild Oryctolagus rabbits in Australia in 1950 (Ratcliffe et al., 1952) and im Europe in 1952 (Radot and Lépine, 1953) provided an opportunity to see what changes in virulence would occur when a lethal generalized infection was introduced into virgin populations. In the present context it allows us to introduce a further factor into the sequence of events we have been discussing, and one of major importance in natural infections, namely, transmission from one animal in a population to another. Thus we may compare the simplest situation in animal viruses (transfer from one individual cell to another through a fluid menstruum) with one of the most complex (transfer from one mammal to another by an intermediate vector, with a complex sequence of events necessarily occurring in each mammalian host before the virus is available for transfer to another mammal). With myxomatosis it 1s possible to use either the mortality rate or survival time after infection as a direct measure of virulence (Fenner and Marshall, 1957). The reverse procedure, namely, challenge of rabbits from areas with different histories of exposure to myxomatosis with one known strain of virus, made it possible to observe changes in the genetic resistance of wild rabbit populations (Marshall and Fenner, 1958). VARIATION IN VIRULENCE 245 In Australia and in France, myxomatosis is predominantly a summer mosquito-borne disease; in Britain, there is no marked seasonal incidence and the rabbit flea appears to be the important vector. The virus strains originally introduced in Australia and Europe differed in their passage histories and in the symptomatology produced in Oryctolagus rabbits, but both almost invariably produced lethal infections. A single introduction of virus was made in Europe, whereas in Australia there are annual inoculation campaigns with the highly virulent virus, but in both continents the virus has established itself enzootically. Samples of virus from different parts of Australia and from Europe have been collected each year, inoculated into test rabbits, and grouped into one of five categories according to the mortality rates or the mean survival times seen in laboratory rabbits (Fenner, 1958b). In spite of the annual reintroduction into Australia of highly virulent virus (causing a mortality rate of over 99 %) the majority of strains recovered from natural cases have been attenuated to a slight or high degree. Within a year of the first introduction of the virus, strains with a 90 % mortality rate had appeared. These have remained dominant ever since, but still more attenuated strains (causing mortalities sometimes as low as 20 %) have been recovered since 1955. In Europe the fully virulent strain has persisted longer and on a wider scale, but here, also, attenuated strains are becoming common. When viruses are passed repeatedly in a particular host in the laboratory they often become more virulent for that host, although this may be accom- panied by attenuation for some other (perhaps the natural) host. Attenuation for the passage host is virtually unknown. Yet in both Australia and Europe (and in many widely separated areas in each continent) the tendency has been toward a moderate degree of attenuation. The explanation lies in the method of transfer of virus from one host to another. In the laboratory, the usual procedure is to select the animal show- ing the first signs of infection and to use material from this animal for the next passage; this procedure naturally tends to select variants with maximum virulence. In nature, however, the rabbit most likely to act as a source of infection for others is the one which offers large virus-rich skin lesions as feeding grounds for mosquitoes for the greatest length of time—that is, a rabbit infected by a strain of virus which does not terminate the infectivity of its host for others by killing it rapidly. This survival advantage of less virulent strains is presumably greatest when spread of the disease is least efficient—that is, during winter. In short, selection in the laboratory is for rapid multiplication and high final titer; in nature, it is for these properties and also for the property of persistence of the high final titer. The results of titration of the superficial cells of the skin of rabbits infected with several strains of virus of different virulence showed that all naturally 246 F. FENNER AND J. CAIRNS occurring strains (whether highly virulent or not) multiplied to about the same extent and at the same rate. However, the highly virulent strains rapidly killed the host, whereas the common, moderately attenuated strains produced many skin lesions which remained infective for mosquitoes for a prolonged period. A field experiment in Australia, in 1954, demonstrated the high survival advantage of the current strains (Fenner e¢ al., 1957). The highly virulent European strain of virus was introduced on a large scale at the beginning of the transmission season, in a rabbit population harboring the enzootic, attenuated strain. Although the virulent EKuropean strain dominated the peak of the epizootic, the local attenuated strain occurred in appreciable numbers at the end of the epizootic and was the only type of virus to survive through the ensuing winter. There is, of course, a delicate balance between the genetic resistance of the host and the virulence of the virus. Tests on susceptible young wild rabbits, obtained each spring over a period of five years from rabbit popula- tions exposed annually to severe epizootics of myxomatosis, showed that there had been a relatively rapid selection of genetically more resistant rabbits (Marshall and Fenner, 1958). It is interesting to speculate upon the effect of this change in host resistance on the selection of virus virulence: unless a variant appears which multiplies predominantly im the skin, causing very persistent virus-rich lesions, one might expect that more virulent strains will be selected in the future, as they may cause in partially resistant hosts the type of disease caused in susceptible hosts by the moderately attenuated strains of virus. IV. SuMMARY In this article we have traced the factors determining virulence for the host from the simplest virus-host systems to the most complex and elaborate. Wherever possible, we have chosen examples to demonstrate the effect of each increase in order of complexity. But it is obvious that at the present time there can be no over-all synthesis. The type of problem considered in simple systems is quite different from that presented by complex systems, and it is impossible to analyze the virulence of viruses for complex systems in terms of those mechanisms demonstrated to operate in simple systems. This is particularly unfortunate, as most of the work on variation in virulence of animal viruses has, perforce, been conducted with the whole intact animal. All that can be done is to discuss the different levels of complexity offered by the various host systems which have been studied. We feel, however, that only in such terms will it be possible ultimately to create a science of animal virology. VARIATION IN VIRULENCE 247 REFERENCES Ackermann, W. W. (1957). Ann. N.Y. Acad. Sci. 67, 392. Adams, M. H. (1953). Ann. inst. Pasteur 84, 164. 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Serological Variation among Influenza Viruses..............++ee sees eee eee 253 Pee Viner SETUICOUTE serie letras way coke ole legs Sachey ciated ceimuate, Goapele bcietetp ae oye ee cree 253 ES MOnO otic laymen cer tiie te eevee tol ate) Shas ley, sre, Sel cselel ace siahshsttiel> Soa susiene ioe 254 C. Antigenic Analysis of Type A Strains. s/ahapisdintets sateen ste cde eeereat OO 1. Studies with Sera of Experimental aioe Satie on tar AAE Naan ite 254 DEE Stu ciessud hie hiimant SCLa reer mee eitmere ren ee eee 260 Sa Classification.of Ly po-A Strains octets, 46 inplis «oan tees sel wie 263 De Waria tion ana yne i lative zan, VavUs,. foc msche oie cor <.a5s0 9/501 alersraiciacuasheraie ieee 265 Beeserolocical Variation dure Passage sao. oo anciot ete woo navn shes ales ofa ausve Pie oe 265 He ACE beLOM CO.NOW, ELOSUS cress g craleitges ale t eines eimin) (cya) t Ma aihala arias Sorts 265 Deambrra iced AV ATA TIONG oie 2s stenitom ee ies ake eed) oho 'es ais eISe ini Bet oh 8 eee ele loa 267 eee ASS m VPI ALLO MA age Kiccattaeci hier sicleiers abate oS heel a eters eis Gate ay Seales ener 267 Dee Variation im Arthropod-Borne: Viruses... >: )v-iec.s ae 2 * a © Se ° < On aa Oe eb ° ° CG =e Ss MS * ied O,5 4 . ° % cau <> i 9 is ®o % ° ° > oe 4 re, 2 o* . =o San oe - 6 gf 0 Pome fae? e° "© & ° Go a 4 07. pe SHO, Bp. te ee a?“ o BF ag ~ > © & on eo” ° r} o %9 ero, cad wm =e Bhat ey (b) % ° @:o4° OF & oro ge Pes ° e's in 9 Be uo Ses co fas @ s% ee. fe 2 i oe Pe » Ot te Seiad RR Fia. 6. (a) Nuclear polyhedra with 7’. paludosa before treatment from weak alkali ; (b) the same polyhedra after treatment. Note the peculiar elongation of the crystals. (Magnification: < 150.) THE INSECT VIRUSES 31D while the number is much reduced in the polyhedra of Tineola bisselliella and Euvanessa antiopa. There seems little doubt that in the nuclear polyhedra of the Lepidoptera the arrangement of the virus rods within the polyhedron is quite haphazard. This can be demonstrated by thin sections (Morgan et al., 1956) (Fig. 4). In the case of the nuclear polyhedrosis of a fly larva (Tipula paludosa), however, there is some suggestion of a more regular arrangement of the particles. Sections through the polyhedra (Fig. 5) give this impression and the striations visible in the polyhedra on the optical microscope may be an expression of the inner regular array of particles. On several occasions sections through the polyhedra of 7. paludosa have revealed the virus particles lined up along the edge of the crystal; the significance of this 1s not very clear unless it is that the particles failed to become incorporated in the polyhedral body at the time of crystallization. e. The Reaction of Nuclear Polyhedra to Alkalies, etc. Bolle (1898) was the first to show that the polyhedra dissolved in acids and alkalis and Paillot and Gratia (1939) observed the dissolution of polyhedra with weak alkali under dark-ground illumination. Bergold (1953) has worked out a number of concentrations of Na,Co, for the dissolution of nuclear polyhedra from different species under standard conditions. Polyhedra from different species differ greatly in their resistance to alkaline treatment, but the majority are dissolved by treatment for 5 minutes with 4 °/, Na,Co;. However, the polyhedra from larvae of Pterolocera amplicornis Walker withstand this concentration for 30 minutes and a treatment of at least v0 minutes at 56°C. isnecessary to dissolve them completely (Day e¢ al., 1953). The nuclear polyhedra from the dipterous insect Tipula paludosa are in a class apart from all other nuclear polyhedra so far described. They are resistant to trypsin and to dilute and weak acids and alkalis. In 1 NV sodium hydroxide they elongate to six or more times their length, becoming first biconvex spindles and then elongating into crescents or wormlike shapes (Fig. 6). At about three times their normal length this elongation is still completely reversible and in water, at pH 5-8, they return to their normal shape and size. After such treatment, however, the polyhedra are “activa- ted’’; in ether words, they now respond in a similar manner in ammonia, 1-12 °% sodium carbonate, and hydrochloric acid, pH 1-4, but not to 1 N hydrochloric acid or 25 °%% sodium carbonate. The elongation and contraction or return to normal shape, which take place along the same axis, can be repeated indefinitely in these solutions and take place as rapidly as the solutions can be alternated (Smith and Xeros, 1954b). 2. Cytoplasmic Viruses The existence of a separate and distinct type of polyhedral virus, which was spherical instead of rod-shaped, was first demonstrated by Smith and Wyckoff 376 K. M. SMITH (1950), and the differential staining properties of the polyhedra later investi- gated (Smith et al., 1953). Previously, several workers had observed polyhedra in the cytoplasm of the mid-gut (Ishimori, 1934; Lotmar, 1941), but the fact that they were of an entirely different nature from the nuclear polyhedra was not realized. The reaction of the cytoplasmic polyhedra to stains and alkalis differs sharply from that of the nuclear type and is an important aid in diagnosis. In smears made from infected caterpillars, fixed by mild heating and subsequently stained with methylene blue or Giemsa solution, the cytoplasmic polyhedra take up the stain readily. This is in marked contrast to the behavior of the nuclear polyhedra, which under these conditions do not stain at all. Figure 7 shows a smear made from a larva of the privet hawk moth, Sphine ligustri, which had a double infection with both nuclear and cyto- plasmic polyhedroses; note how the latter are clearly differentiated. a. Location of Cytoplasmic Polyhedra in Insect Tissues. The number of cytoplasmic polyhedroses now recorded is extremely large and it seems highly probable that they are more numerous than the nuclear diseases; this, at all events, has been the writer’s experience in his studies at Cambridge. In every case the virus appears to develop in the gut cells, usually the mid- or hind- gut, and in a late stage of the disease the cells become completely filled with polyhedra. These also occur in the lumen of the gut and are excreted in large numbers in the feces (Fig. 8). In a cytoplasmic polyhedrosis of the silkworm, Bombyz mort, the polyhedra are produced in the cylindrical cells of the mid-gut epithelium but not in the goblet or interstitial cells (Tsujita, 1955). In a similar disease of the spruce budworm, Choristoneura fumiferana Clem., the polyhedra occur in the cyto- plasm of the digestive cells of the mid-gut epithelium (Bird and Whalen, 1954). The symptomatology of the cytoplasmic polyhedroses usually differs sharply from that of the nuclear disease. The main difference arises from the fact that the skin is not attacked and, in consequence, the larva, though limp and flaccid, does not disintegrate in the manner so characteristic of nuclear polyhedroses. In some species, the mid-gut is clearly differentiated through the skin by the accumulation of the polyhedra in the cells. This may occur quite early in the disease before the general health of the larva appears to be affected and is most marked in the cytoplasmic polyhedrosis of certain species, notably Phlogophora meticulosa, the large angleshades moth and Diatarazia oleracea, the tomato moth (Fig. 9). b. Development of Cytoplasmic Polyhedra. Xeros (1957) has described the formation of the polyhedra in a cytoplasmic disease of the larva of Thaumato- poea pityocampa. Bodies which are apparently virogenic stromata appear in the epithelial cells before the formation of the polyhedra. Later, small THE DIRECT VIRUSES 377 polyhedra form sparsely over the virogenic masses; these polyhedra vary in size but may be under 0.5 » in diameter. They are best observed in relation to the cytoplasm and virogenic masses in HCl-Giemsa preparations. Further development proceeds by the continuing growth of the virogenic masses and by the increase in the number and size of the polyhedra at their surfaces. Polyhedron formation does not take place equally at all parts of these surfaces; some parts may be packed with well-developed polyhedra 0.5-1 » in diameter, other parts may be completely free of them. During this stage of the disease, large pores develop within the virogenic masses and polyhedra also arise and develop within these pores. In the fully matured colonies of the moribund cell the polyhedra reach a size of 1.5 x in diameter. c. Different Shapes and Sizes of Cytoplasmic Polyhedra. On the whole, the cytoplasmic polyhedra resemble those of the nuclear diseases but there are differences. There seems to be a greater range of size of cytoplasmic polyhedra in an individual smear, from very small to very large indeed, sometimes as much as 10. This was particularly the case in a cytoplasmic disease of Ourapteryx sambucaria. There is also a tendency on the part of the large polyhedra to lose their many-sided character and to appear almost spherical. In the spruce budworm, the polyhedra are chiefly cuboidal and triangular forms have not been observed (Bird and Whalen, 1954). d. Dispersal and Arrangement of Virus Particles inside Polyhedra. It is the writer’s opinion, after long experience of cutting thin sections of both types of polyhedra, that the cytoplasmic type is considerably harder and it is more difficult to get good sections without much compression by the knife. This gives the impression that the viruses are somewhat elongated but in fact this is only an artifact due to compression. Furthermore, some of the cytoplasmic viruses are very small, one affecting the larva of Sphinx lingustri measures 12mp, about half the size of the tomato bushy stunt virus (Fig. 19), and it is difficult to observe them at ali in sections. Indeed, from observation of the sections alone it would appear that little or no virus was present, whereas in actual fact the virus content is very high. Observation is rendered more difficult by the lack of contrast between the virus particle and the surround- ing polyhedral protein (Fig. 10). On the whole, it appears that the particles are arranged haphazardly within the polyhedral crystal. Occasionally, however, sections may reveal what appears to be a regularity of assembly within the crystal (Fig. i118) e. Reaction of Cytoplasmic Polyhedra to Alkalies. It is in their response to treatment with weak sodium carbonate that the two types of polyhedra differ most. We have seen that the nuclear polyhedra dissolve completely, leaving behind a membrane in which the virus rods are contained (Fig. 12). With similar treatment the cytoplasmic polyhedra dissolve only partially and 378 K. M. SMITH a honeycomb or spongelike residue remains; there is no membrane (Fig. 13). (Smith and Wyckoff, 1950). The actual time of application and strength of sodium carbonate used are extremely critical and the margin between liberation and dissolution of the virus particles is very narrow. It may be a matter of a few seconds only in some species. B. Granulosis Viruses The first record of this type of insect virus disease was made by Paillot (1926a), who described what was probably a granulosis in the caterpillars of the large white butterfly, Pieris brassicae, and later (Paillot, 1934) a similar disease in the larvae of the cutworm, Huzxoa segetum. Steinhaus (1947) first characterized the virus by means of the electron microscope from the larvae of the variegated cutworm, Peridroma margaritosa (Haw.). A year later Bergold (1948) observed a similar disease in caterpillars of the fir-shoot roller, Cacoecia murinana Hb. Smith and Rivers (1956) described six granulosis diseases from Euplexia lucipara L., Agrotis segetum Schiff., Melanchra persicareae L., Natada nararia, Pieris brassicae L., and P. rapae, and several more are recorded in the literature. In the granuloses there are no large polyhedral crystals, but their place is taken by the “granule” from which the disease takes its name. The granule is also a crystal (Fig. 14) and usually contains one virus rod, which can be liberated by means of weak alkali in a similar manner to the nuclear polyhedra (Fig. 12). The tissues of affected larvae contain immense numbers of granules which, when stained with Giemsa solution, are just within the limit of resolution of the optical microscope. So far, granuloses have only been recorded from the larvae of Lepidoptera. The following description of the granulosis disease of Pieris brassicae 1s fairly typical of the group. Under laboratory conditions the onset of the disease is very rapid, and young larvae begin to die within 72 hours of infection; older larvae take a little longer. The first indication of infection is loss of appetite; infected larvae immediately stop feeding and remain quies- cent. A pallor next develops, which is most marked in the thoracic region, and death rapidly ensues after this. The larva becomes flaccid and hangs down in a characteristic manner, rather in the shape of an inverted “V.” The skin is extremely fragile and ruptures at a touch, liberating the contents of the body which have become almost entirely liquefied. Studies of sections of cater- pillars at different stages of infection seem to support Paillot’s suggestion that the granules occur in the hypodermis and fat body. Furthermore, it seems as if the virus develops in the nucleus rather than in the cytoplasm. In sections through the fat body of a young larva of P. brassicae, 96 hours after infection, the nuclei appear to be full of granules and there are indications Fic. 9. Larvae of Phlogophora meticulosa killed by a cytoplasmic polyhedrosis. Note the accumu- lations of polvhedra showing through the skin. (Magnification: < 14.) Fia. 8. Transverse section through the mid-gut of a larva of Lithophane lapidea infected with a cytoplasmic polyhedrosis. Note the polyhedra filling the lumen of the gut. (Magnification: 650.) Fic. 11. Part of a section through a cytoplasmic polyhedron from a larva of Arctia caja. Note the apparently composite virus particles. (Magnifi- bh cation: < 57,000.) Fig. 10. Section through a cyto- plasmic polyhedron from a larva of S. ligustri; it is not easy to see the virus particles (compare Fig. 19). (Magnification: x 27,000.) Fie. 12. A nuclear poly- hedron from the silkworm, Bombyx mori, after treat- ment with weak sodium carbonate. Note that the crystal has dissolved and liberated many virus rods, some with and some with- out their capsules. (Magni- fication: < 17,000.) Fie. 13. Cytoplasmic poly- hedra from P. meticulosa after treatment with weak sodium carbonate. Note sponge like residue and absence of virus particles (compare Figs. 11 and 12). (Magnification: x 14,000.) Fie. 14. Three granulosis particles from a larva of Pieris brassicae, frozen-dried. Note the crystalline shape (compare Fig. 22). (Magnification: 15,000.) (Fie. 14 electron micro- graph by R. C. Williams.) THE INSECT VIRUSES 379 that they rupture, liberating a mass of granules into the cytoplasm of the cell (Fig. 15) (Smith and Rivers, 1956). C. Viruses without Intracellular Inclusions As mentioned earlier, the number of viruses so far discovered, occurring freely without crystalline inclusions in the tissues of insects, is not large. There are two in which the virus itself has been well-characterized on the electron microscope, one attacking a lepidopterous larva, Cirphis unipuncta Haworth, and the other infecting the dipterous larva, Tipula paludosa. Larvae of C. unipuncta infected in late third instar appear swollen and somewhat darker than normal insects. The cuticle of the diseased larvae has a waxy appearance, and in some cases the mid-portion is slightly enlarged. The liquefaction and disintegration characteristic of the nuclear polyhedrosis of the same insect are absent. Deaths from the disease may occur in either the larval or pupal stage. As the disease progresses, the Cirphis larvae become sluggish, are soon eating little, and gradually succumb, usually within 6 to 14 days following infection (Wasser, 1952). There seems to be no information as to the site of multiplication of this virus in the insect’s body or whether it is nuclear or cytoplasmic in origin. By the analogy of shape, however, this virus, which is very small and apparently spherical, should multiply in the cell cytoplasm. The second virus of this type, which attacks the larva of the crane fly, T. paludosa, is most unusual in many ways and is of great scientific interest. It was first discovered by the Virus Research Unit at Cambridge in 1954 and was later briefly described (Xeros, 1954; Smith, 1954). The change induced in infected larvae is very striking and enables the disease to be diagnosed with ease. The normal color of these larvae, which are known in England as “leatherjackets,” is a brownish gray, while the diseased specimens exhibit a somewhat opalescent blue-indigo. This color is rendered more striking if the insect is placed in a test tube with moistened sides and viewed through the glass. The site of multiplication of the virus is in the cyto- plasm of the fat body cells and it is here that the blue color originates. When sections of the diseased fat body are viewed in the electron microscope, it is seen that the cells are filled with the darkly staining virus particles. No virus occurs in the cell nuclei and there are no polyhedra such as would be present in a cytoplasmic polyhedrosis. The amount of virus in the fat body is very great and the blue or violet color is due to the optical effect given by the virus, which actually begins to crystallize in the living insect. The quantity of virus produced by each larva is extremely high and measurements suggest that one- quarter of the dry body weight is converted into virus particles. Pellets formed from this virus, which is known as the tipula iridescent virus (TIV), 380 K. M. SMITH are found to have fascinating optical properties. When observed by trans- mitted light they are generally orange in color, but when examined by reflected light the pellets have a beautiful iridescence with the more noticeable colors in the blue and green regions of the spectrum. The origin of the colors is shown by making thin sections of pellets which have been dehydrated and embedded in methacrylate. On the electron microscope the sections show a somewhat bizarre pattern of particle array (Fig. 16). This suggests that the pellet is made up of a mass of small crystals, each crystal being about 10 in diameter. Purified preparations of tipula iridescent virus tend to crystallize out after standing in the cold. A suspension of the virus in a glass tube, if left undis- turbed, will show a layer of small, brilliantly reflecting crystals at the bottom, with a consequent diminution of virus concentration in the superna- tant fluid. It has been found possible to photograph in color an array of crystals on the flat surface of a microscope slide. The crystals, photographed at different angles, give an exquisite kaleidoscope effect. (Smith and Williams, 1958). III. MorpHoLocy oF INSECT VIRUSES AND THEIR ASSOCIATED MEMBRANES A. Nuclear Polyhedral Viruses Insect viruses differ greatly in size and shape; these variations almost equal those occurring in the plant viruses. There are, in addition, to complicate matters, the various membranes and envelopes which in many cases enclose the virus particles. As we have mentioned previously, the shape of the virus particles from the nuclear polyhedroses seems to be exclusively rodlike, but there is very little homogeneity in the sizes of the virus rods and the size may vary even within one nucleus. This is particularly the case with Lymantria monacha, which seems to show the greatest variation in the length of the rods. One individual polyhedral body may contain an almost complete range of virus rods far longer than the average of 290 my to less than one-half this length. Bergold (1953) gives the average dimensions in millimicrons of a number of nuclear polyhedral viruses, of which the following are typical: Bombyx mori L. 279 x 40; Porthetria dispar L. 364 x 41; and Lymantria monacha L. 350 X 57. Half-length rods, single and in bundles, occur commonly in several species, notably Lymantria monacha, L. dispar, Bombyx mori, and others. These are the so-called spherical developmental forms of Bergold (1953). The proportion of half-length to normal-sized rods varies from one species to another, and appears highest in L. monacha and L. dispar, with very few half-sized rods in THE INSECT VIRUSES 381 B. mori and Abraxas grossulariata. In sections of B. mori infected with its nuclear polyhedrosis, extremely long virus rods are sometimes observed of a size never seen among rods obtained from polyhedra. Similar long rods, which appeared to be breaking up into two more normal-sized rods in the nuclear ring zone, have also been observed. Bergold (1950) and Bird (1952) have described V-shaped forms which, in the writer’s opinion, consist of either two half-length rods within one capsule or two bundles of half-length rods, each bundle within its own inner capsule and the two bundles lying alongside each other at an angle (Smith and Xeros, 1954c). As regards the outer membranes and capsules of the nuclear polyhedral viruses, there is, first, the intimate membrane, which actually holds the constituent protein and nucleic acid components of the virus. Then comes the inner capsule, which is acquired by the virus rod after its formation and after it has been liberated into the nuclear ring zone, (Smith and Xeros 1954a). Finally, there is the polyhedral crystal with its own encircling membrane. In the rather atypical nuclear polyhedrosis of Tipula paludosa, the inner capsule behaves in a manner quite different from other nuclear polyhedroses. During the formation of the polyhedral crystal, the virus rod without its inner capsule can be seen in the center of a large vesicle (Fig. 17). This vesicle may be the greatly enlarged capsule, as suggested by Xeros (1957), connected with the peculiar elasticity of the polyhedral crystals. On the other hand, sections of the virus particles in the insect’s tissues (Fig. 18) sometimes reveal a somewhat similar dilation of the capsule in the absence of polyhedral bodies. B. Cytoplasmic Polyhedral Viruses Although there is some variation in the length of virus rods in the nuclear polyhedroses, this does not approach the degree of variation in morphology which occurs in the cytoplasmic polyhedral viruses. The virus particles from three separate cytoplasmic diseases are briefly described to illustrate this diversity of form. The difficulty of isolating some of the cytoplasmic viruses from their enclosing polyhedra has been previously commented upon (Smith and Xeros, 1954c), so it is less easy to make a comparative survey of the virus morphology of this group than with the nuclear polyhedral viruses. The first example is a cytoplasmic polyhedrosis of the larva of Sphinx ligustri, the privet hawk moth. At first, owing to the difficulty of isolating this virus, thin sections were cut of the polyhedra and observed on the electron microscope. As can be seen from Fig. 10, it is difficult to observe virus particles in the sections of the polyhedra. However, after some experimenting with the polyhedra both in bulk and on the electron microscope grid, using very weak alkali for very short periods, the virus was eventually isolated. It 382 K. M. SMITH proved to be present in considerable quantities and of an unexpectedly small size, measuring only 12-15 mp; it is apparently spherical. There is little doubt that this virus can be crystallized; in Fig. 19 a microcrystal can be seen. The second type of virus comes from a cytoplasmic polyhedrosis of one of the tropical silkmoths, Antheraea mylitta Drury. These particles seem to be definitely polyhedral in shape and appear to be 6-sided (see the tipula iridescent virus, p. 383). They measure about 30 my in diameter and in Fig. 20 they can be seen arranged in a regular manner within the matrix of the polyhedral body. Figure 21 shows two virus particles at a very high magnifica- tion in which the polyhedral shape can be made out. The third type of virus was first observed in a cytoplasmic polyhedrosis of the larva of Phlogophora meticulosa, the large angleshades moth. The virus particles were apparently spherical and measured about 60 mp in diameter. The peculiarity about this virus however, is that the particles appear to be composite, consisting of a number, usually four, of very small units, each about 15 my in diameter. The composite bodies were also found loose in the cytoplasm together with some of the small single units (Smith and Xeros, 1954c). A similar state of affairs exists in the cytoplasmic polyhedrosis of Arctia caja, the garden tiger moth. Here again the particles seem to be made up of five or six subunits (Fig. 11) (Smith, 1956). CO. Granulosis Viruses Quite a large number of granuloses have now been recorded, but so far only from lepidopterous larvae. In every case the virus particle is rod-shaped and closely resembles the virus rods of the nuclear polyhedroses. The dimensions of the virus rods occurring in a granulosis of Pieris rapae, which is a typical case, are given as 41-50 x 291-300 my. (Tanada, 1953). The mode of concealment of the virus particle within granule and capsules is rather complicated. As a rule, a single virus rod is thus enclosed, though it is possible there may be two in some cases (‘Tokuyasu, 1953). The number and arrangement of the enclosmg membranes has been worked out in some detail for a granulosis disease of Natada nararia, the “nettle-grub” of tea (Smith and Xeros, 1954c). This process was divided into four steps and viewed on the electron microscope. First come the “granules,” which are actually minute crystals (Fig. 22a) and opaque to the electron beam. Second, after treatment with weak alkali, the granule collapses on the grid, revealing a rod-shaped body within (Fig. 22b); this is the inner capsule. Third, the virus rod can sometimes be caused to emerge from this capsule (Fig. 22c¢); and fourth, further treatment with weak alkali dissolves the actual virus content of the rod, leaving behind the intimate membrane (Fig. 22d). This arrange- ment of occluding membranes can also be demonstrated by thin sectioning THE INSECT VIRUSES 383 for electron microscopy, which also reveals the crystal lattice of the granule, see Fig. 23. D. Viruses without Intracellular Inclusions According to Wasser (1952), the size of the small virus isolated from Cirphis unipuncta (Haworth), the cosmopolitan army worm, is approximately 25 my in diameter. It is described as a more or less regular, spherical to slightly ovoid body. The other virus in this category is the tipula iridescent virus (TIV) and its size and shape have been very carefully studied. In air-dried and frozen- dried preparations the virus appears as a five-or six-sided particle and in sections of the particle itself some degree of nonuniform structure can be seen. There appears to be an outer envelope, inside of which there is a relatively transparent region; the central area of the particle is filled with opaque material, tentatively identified with its nucleic acid portion. An unexpected observation is the fairly frequent appearance of six-sided contours when the virus particles are seen in section. Purified preparations of TIV appear quite monodisperse in the electron microscope, with each virus particle having a diameter of about 130 mp. Even in specimens dried out of a water suspension in the usual way, and hence suffering the distortions brought about by surface tension, the characteristic contour of the particles is six-sided, rather than circular (Fig. 24). This appearance is quite unique among the known viruses of comparable size; vaccinia virus for example, is brick-shaped and nonuniform in size, while influenza virus appears quite circular and heterodisperse. Since the particles of TIV appear to have six sides, it is reasonable to suppose they are polyhedral in external shape. Other cases of polyhedral-shaped virus particles are known (Williams, 1953) among the bacterial and plant viruses. But up to now it has not been possible to arrive at a convincing notion of the exact form of the polyhedron. An indirect approach is to infer the three-dimensional shape of the polyhedra from the shapes of the shadows found by application of the shadow-casting technique (Williams and Wyckoff, 1954). The large size and regular shape of the TIV particles are suitable for the determination of their full polyhedral shape by analysis of shadow shapes (Figs. 25 and 26). This has been done by Williams, who has shown that the virus particles, when frozen-dried and shadowed with azimuth angles 60° apart, cast two shadows, one five-sided and blunt on its end, the other four-sided and pointed. The only polyhedron which will do this is a twenty-sided figure, so that we can conclude that the three-dimensional morphology of one virus is now known (Fig. 25) and that its shape is that of an icosahedron (Smith and Williams, 1958). 384 K. M. SMITH E. The Ultimate Infective Unit The criticism has been made of a too facile assumption that the various types of particles associated with the different insect virus diseases are the actual virus particles. In the writer’s opinion this criticism is not valid, in view of the extreme similarity between these particles and other viruses of proved infectious nature. So far as the nuclear polyhedroses are concerned the criticism does not apply, since Bergold (1953) has demonstrated the high degree of infectivity possessed by the purified rods extracted from the poly- hedra. It is true that the parallel experiment with the purified particles from the cytoplasmic polyhedra does not seem to have yet been done. It is known, however, that the polyhedra are extremely infectious; a glance at Figs. 19 and 20 will show how exactly similar the extracted particles are to other viruses. It is perhaps more legitimate to inquire what the ultimate infective unit is and how far the numerous enclosing capsules and membranes influence the infectivity of some viruses. In certain viruses from cytoplasmic polyhedroses the particle appears to be composite and at very high magnification, a number of apparent subunits are visible (Fig. 11). Experimental evidence as to whether these subunits are infectious has not yet been obtained but they are interesting as affording a possible insight into the build-up of some spherical infectious particles. IV. PATHOLOGICAL CHANGES IN THE INFECTED CELL AND THE DEVELOPMENT OF THE VIRUS PARTICLES In the nuclear polyhedroses the most striking pathological change is the development of the central chromatic mass or net, referred to by Xeros (1956) as a “‘virogenic stroma.” This central mass has been observed also by earlier workers, but the first electron micrograph of a section through this nuclear net was shown in a polyhedrosis of the larva of the privet hawk moth, Sphinx ligustri (Smith et al., 1953). The chromatic mass or net was once thought to be a stage in the further development of a fused aggregation of nucleoli (Mazzocchi, 1908; Glaser, 1927). Another view was that the net was formed primarily by the fusion of chromatin granules of the pathological nucleus (Paillot, 1926c; Heidrenreich, 1940). However, according to Xeros (1955), who made a detailed histological study of a number of nuclear polyhedroses, no evidence was found that the chromatic mass had been formed from the chromatin. He concludes that the chromatic masses or nets do not arise by fusion of chromatin granules but are produced de novo. It seems clear that this chromatic mass is the site of the development of the virus rods, but about the exact method of development of the virus rods there is still a good deal of uncertainty. Bergold (1950, 1953) postulated a life cycle Fic. 15. Section through the fat body of a larva of Melanchra persicariae infected with a granu- losis virus. Note the nucleus on the right ap- parently liberating granules into the cytoplasm. (Magnification: < 660.) Fic. 16. Section through a methacrylate- embedded pellet of the tipula iridescent virus. The pattern results from a transection made through small crystallme regions oriented at random. (Magnification: < 5800.) (Fig. 16 after Wiliams and Smith, 1957). Fie. 17. Section through part of a nucleus of a blood cell from the larva of 7’. paludosa infected with its polyhedrosis. Note the peculiar vesicle surrounding each virus rod. (Magnification: < 20,000.) Fia. 18. Sections through virus particles from the same disease as in Fig. 17. Note a similar dilatation of the membranes surrounding the particles. (Magnific- ation < 40,000.) Fie. 19. Very small virus from a cytoplasmic polyhedrosis of S. ligustri. Note the formation of a micro-crystal. (Magnification: « 40,000.) Fie. 20. Virus from a cytoplasmic polyhedrosis of Antheraea mylitta. Note the regular packing of the virus particles and their polyhedral outlme. (Magnification: 30,000.) Fre.21. The same virus as in Fig. 20 at high magnification to slow the six-sided contour. (Magnification: x 60,000.) Fic. 22. Four stages in the breakdown of a granule from a larva of Natada nararia: (a) the untouched granules; (b) after treatment with weak sodium carbonate, showing the collapse of the outer granule and the inner capsule; (c) a virus rod outside the capsule; (d) dissolution of the virus rod showing the intimate membrane. (Magnification: 30,000.) Fic. 23. Thin section of granules from a larva of P. brassicae, with one virus rod cut longitudinally, showing the inner capsule, and one virus rod cut transversely. Note also the apparent molecular lattice of the outer granule. (Magnification « 80,000.) Fic. 24. Frozen-dried particles of the tipuia iridescent virus showing the six-sided shape. (Magnification: < 40,000.) (Figs. 24, 25, 26 after Smith and Williams 1958.) Fic. 26. Frozen-dried particles of the tipula iridescent virus doubly shadowed. Note simi- larity to shadows in Fig. 25. (Magnification Fia. 25. A model of an icosahedron doubly < 59,000.) shadowed. Note two shadow shapes which can be cast simultaneously only by an icosa- hedron in this orientation. Fic. 27. Section through the nucleus of a blood cell from 7’. paludosa infected with its polyhedrosis. Note the apparent development of the virus rods in the central chromatic mass. (Magnification: < 28,000.) Fira. 28. Later stages of the same Ciscase as in Fig. 27. Note the fully formed virus rods, some with enclosing membranes. (Magnifica- tion: 25,000.) THE INSECT VIRUSES 385 for insect viruses based on a number of apparent ‘““developmental forms” collected at random from dissolved polyhedra observed on the electron microscope. He considered that the virus rod appeared first as a minute spherical body, which gradually increased in size to form an elongated, curved body surrounded by a membrane. Later the rod straightened out, ruptured the membrane, and escaped, leaving an empty spherical membrane behind. Bird (1957) supports this theory of a life cycle; he has investigated a nuclear polyhedrosis of the sawfly, Neodiprion pratt: banksianae Roh., and considers that a study of thin sections of infected nuclei suggests a cycle of virus development, commencing with the attachment of rod-shaped particles to strands of chromatin. According to Bird, the chromatin is then converted to virus in the form of minute spherical bodies surrounded by membranes that increase in size to form rods. The rods may escape from their develop- mental membranes to repeat the cycle, or rods and spheres may be occluded by protein material to form polyhedra, in which case virus development ceases. On the other hand, Xeros (1956) considers that the nuclear nets or virogenic stromata become increasingly proteinaceous and Feulgen-positiveas they grow and develop. Morphologically they are net works, and virus rods differentiate within vesicles in their cords. These begin as fine rodlets about 60 A x 1200 A in size and increase in situ to their final size of 280 A x 2800 A. They are then set free from their vesicles into the pores of the net by disruption of the surrounding cord material and may ultimately reach the ring zone between the centrally placed virogenic mass and the nuclear membrane. The freed virus rods become enveloped by independently formed and still growing capsule membranes, within which capsule protein is deposited. The encapsu- lated rods then become occluded within crystalline protein polyhedra, which arise and grow in the ring zone and later in the enlarged pores of the infected nucleus. In thin sections, cut by the writer, of the nuclei of blood cells of Tipula paludosa, infected with its characteristic polyhedrosis, what appear to be immature virus rods can be seen in large numbers round the periphery of the chromatic mass (Fig. 27). At a later stage of the disease, sections show the fully formed rods, in some cases with their capsule membranes, differentiated out of the central mass (Fig. 28). If we accept Bergold’s thesis that insect viruses are organisms with a ‘life cycle,” we are faced with the anomaly that this life cycle applies only to one group of insect viruses, those of the nuclear polyhedroses. There is still the very large group of cytoplasmic viruses, some of which are of extremely small size and are comparable to the smallest plant viruses. As we have seen, certain of these insect viruses are crystallizable and the particles themselves VOL. I1I—25) 386 K. M. SMITH are minute polyhedral crystals. It would be extremely difficult to postulate any form of developmental cycle for particles of this nature. Xeros (1956) considers that the virus particles of cytoplasmic polyhedroses arise In “‘virogenic stromata’’ essentially similar to those of the nuclear polyhedroses. He has studied a cytoplasmic polyhedrosis in a processionary caterpillar or “army worm,” Thawmatopoea pityocampa, in which he describes virogenic stromata with a micro-net structure. In the cords of the stromata virus bodies arise which are spherical, with an extremely dense center about 35 my in diameter and a less electron-dense cortex about 80 my in diameter. When the virus bodies have been formed, the cord material around them disrupts and liberates them into the larger pores formed as a result of dissolu- tion of the cords. The freed virus bodies become occluded in the polyhedra. V. Latent INFECTION IN INSECTS AND ITS BEARING ON THE Cross-TRANSMISSION OF VIRUSES There is a good deal of confusion caused in the study of latent infections by a lack of uniformity in the use of such words as “latent,’”’ “inapparent,”’ “subclinical,” and “masked.” A symposium on the subject was recently held at Madison, Wisconsin, and the chief findings were reported by Andrewes (1957). It was suggested that the words “latent virus” should not be used but that “‘inapparent infection” at the host-parasite level would cover the whole field of infections which give no overt signs of their presence. “Latent infec- tion” denotes those cases of inapparent infection which are chronic and in which a certain host-virus equilibrium is established. At the cell-virus level, Dulbecco’s term ‘“‘moderate virus” was approved to denote a virus which grew in a cell while still permitting its continued survival and multiplication; “cytocidal”’ described one which killed it; “submoderate” covered inter- mediate cases. There is no doubt that large populations of insects, particularly lepidop- terous larvae, carry latent infections; this fact is lable to cause much confusion in experiments in the cross-transmission of insect viruses. For many years the opinion was held that insect viruses were extremely specific and that true examples of transmission between different species were unknown; examples of apparent cross-infection in the literature were regarded as unproved. In 1953 a paper was published (Smith and Xeros, 1953b) in which a large number of experiments, involving apparently genuine cross-transmission of viruses, was described. The alternative possibility of stimulation of latent infection in some cases was, however, admitted. Up to that date the term “insect virus’ referred mainly to the nuclear polyhedral viruses and possibly to those of the granuloses. With the discovery of the distinct group of cytoplasmic polyhedroses the situation became more complicated. THE INSECT VIRUSES 387 The following experiments on cross-transmission with the larvae of Sphinx ligustri, the privet hawk moth (Smith and Xeros, 1953a), give a fairly characteristic picture of what happens with this type of experiment. Six similar batches of third and early fourth instar larvae of ligustri, which appeared perfectly healthy, and among which no deaths from polyhedrosis had occurred, were fed on the same day with nuclear polyhedra from the following lepidopterous larvae, chosen entirely at random: Telea polyphemus, Lymantria dispar, Philosamia ricini, Panaxia dominula, and Cycnia mendica. The ligustri larvae infected with virus from mendica and dominula died on the seventh and eighth days after infection with a typical nuclear polyhed- rosis. The polyhedra were nonstaining and square in shape. The larvae infected with the polyphemus virus died on the eighth day with identical symptoms, but the polyhedra in this case were either many-sided or triangular in shape and not square. The deaths were extremely regular, occurring together over two or three days, and the symptoms were classic. One larva only, one of those dying on the ninth day, showed some staining cytoplasmic polyhedra among the nuclear type. Of the twelve ligustri larvae infected with virus from ricint, two died on the tenth day, four on the eleventh, one each on the twelfth and thirteenth days, one on the eighteenth, and one each on the twenty-ninth and thirty-second day after infection. The first six larvae to die had fairly typical symptoms of a nuclear polyhedrosis. Their polyhedra were nonstaining, with a tendency to a square shape. The other six larvae com- menced to die on the eleventh day and subsequently; two of these had cyto- plasmic polyhedroses only, while the other four had both types of infection. Those ligustri larvae infected from dispar all died of a cytoplasmic poly- hedrosis, and none developed a nuclear disease. The control larvae for this experiment also died similarly. The conclusion we draw from this type of result is that there was genuine cross-transmission of a foreign nuclear polyhedral virus; but, in addition, there was a latent cytoplasmic polyhedrosis. This latter point has been amply confirmed by subsequent observations on S. ligustri. Provided the incubation period of the nuclear virus was short (mendica, 7 days, dominula and poly- phemus, 8 days) the foreign virus was able to establish itself early and kill the larva before any appreciable quantity of gut polyhedra developed at the molt to the fifth instar. In the case of the ricini infections, the incubation period of 11 days was slightly longer and there appeared to be a suppression of any great development of nuclear polyhedra in those older larvae in which the cytoplasmic disease had got a good start before the nuclear one could do so. With the dispar virus one can conclude either that it is not cross-transmis- sible to ligustri or else that the cytoplasmic disease suppressed its getting a foothold. In fact, we can now go further and say that where a latent cyto- plasmic infection is present this is almost invariably stimulated to development 388 K. M. SMITH by the introduction of a foreign nuclear virus. The reverse phenomena, stimulation of a nuclear virus by introduction of a foreign cytoplasmic virus, does not seem to occur. Some experiments with the larvae of the winter moth, Operophtera brumata, are of interest in this connection. Failure to find a naturally occurring virus disease of this larva led to attempts to infect it with a nuclear virus from the butterfly larva, Vanessa cardw. This induced a high percentage of mortality in the winter moth larvae, all the controls remaining healthy. The disease which resulted, however, was a cytoplasmic one and, once stimulated into activity could be transmitted indefinitely in series (Smith and Rivers, 1956). It may be that the reason for the frequent stimulation of a cytoplasmic virus by a foreign nuclear virus is connected with the location of the cyto- plasmic virus in the cells of the mid-gut. In this position it is likely to come rapidly into contact with the foreign virus swallowed by the larva. There is little doubt that the number of latent infections with cytoplasmic viruses is extremely high and, in the writer’s opinion, higher than the number of latent nuclear polyhedroses. There are other methods by which latent polyhedral viruses can be stimulated to action, notably by feeding with certain chemicals and by keep- ing the larvae under unsuitable conditions. A few other examples of apparently genuine cross-transmission may be quoted. A nuclear polyhedral virus from Vanessa carduz is easily transmissible to Aglais urticae and V. io and is intertransmissible between all three species. With the granulosis virus of the large white butterfly, Pieris brassicae, transmission is easily achieved between this species, P. rapi, and P. napt. It is difficult to say, at this moment, whether it 1s always the same granulosis virus which is involved, since there is no means of differentiating between them until they can be tested serologically. Steinhaus (1952) has demonstrated a similar cross-transmission of a granulosis virus between species of Colzas. It is possible that in the case of the three species of white butterfly, Pzervs, the granulosis virus may change slightly after passage of one or other of the species. It has been found (Smith and Rivers, unpublished) that a considerable resistance on the part of P. brassicae can develop to infection with the granulosis virus, but after passage of the virus through P. napi, this resistance breaks down. Attempts to stimulate development of the granulosis, which is frequently latent in Pieris spp., by feeding with foreign polyhedral viruses have given negative results. VI. Meruops or TRANSMISSION OF INSECT VIRUSES There can be little doubt that the two main methods of transmission of insect viruses are by ingestion of contaminated food and by the inheritance of THE INSECT VIRUSES 389 infection through the parent. This statement should perhaps be modified slightly in so far as it is known to apply to the polyhedroses and granuloses; there is not yet sufficient evidence to say definitely that the viruses without intracellular inclusions are similarly transmitted. We know that TIV is spread by ingestion orally, in the laboratory and presumably also in the field; there is a slight amount of evidence that there may be latent infection with the same virus. Transmission of the polyhedral viruses and those of the granuloses through a given population of insect larvae during the current season is undoubtedly by the ingestion of contaminated foliage. So far as the nuclear polyhedroses are concerned the disease itself greatly facilitates the spread of the polyhedra. The skin, being one of the organs attacked, quickly becomes extremely fragile and easily ruptures, scattering the polyhedra in the liquefied body contents over the foliage. This process is helped by the wind and the rain which carry the polyhedra still further afield; the fact that the polyhedra are quite resistant to the weather is very important in the spread and over- wintering of the virus. The same principles probably hold in the spread of the granuloses, since the skin is also attacked and the liquefied body contents are similarly spread abroad. There is some evidence, however, that the longevity of the granules is much less than that of the polyhedra; the granules from Pieris spp., for example, seem to lose much of their infectivity after storage for one winter. An interesting fact in regard to the granulosis disease of the large white butterfly, Pieris brassicae, is the apparent attraction of the liquefied cadavers for the healthy caterpillars, which can often be seen feeding greedily on the bodies with, of course, disastrous results. The situation regarding the cytoplasmic polyhedroses is slightly different, since the skin of the affected larvae is not attacked and in consequence there is no general liberation of polyhedra. In this case, the polyhedra are excreted in large numbers with the feces and the food plant in consequence is heavily contaminated. This fact is easily demonstrated by examining smears of the droppings of larvae with cytoplasmic polyhedroses, as in Operophtera bruma‘a, the winter moth, for instance, or by examining sections of the gut of infected larvae (see Fig. 8). Since the polyhedra are capable of retaining viable virus within them for many years, fifteen years in the case of the silkworm, Bombyx mort, it is fair to assume that infective material will remain over winter on contaminated food plants. This has been proved on more than one occasion, for example, with the Great Basin tent caterpillar, Malacosoma fragile (Stretch). In 1953, a number of trees in an abandoned orchard in California was sprayed with a polyhedral virus from this species and a second block of trees was left un- treated. It was observed that a number of larvae died of the disease on the 390 K. M. SMITH treated section. In the spring of 1954, from 60 to 80 % of the larval population in the treated block died of polyhedrosis. No evidence of virus was found in the untreated block (Clark, 1956). The second most important method of insect virus dissemination is by passage of the virus through the egg; this applies to both types of polyhedral viruses and is probably more frequent with the cytoplasmic polyhedroses. Sudden outbreaks of polyhedroses in areas where the disease was unknown, (Tanada and Beardsley, 1957), and attacks under conditions where external contamination was impossible are certainly due to the stimulation of a latent infection. Geneticists and others who breed large numbers of lepidopterous larvae in captivity are only too familiar with this phenomenon. There has been some disagreement on the exact mode of transovarial transmission as to whether the virus is inside the egg or is carried mechanically attached to the outside of the shell. There are two facts which suggest that the virus is not merely an external contamination of the outside of the egg: one 1s the sudden appearance of a polyhedrosis in caterpillars which have been reared through a number of generations under conditions where external contamination is ruled out. It is unlikely that virus on the outside of an egg would go through several generations in a latent infection. If the virus can remain latent throughout the larval and adult life, there seems no reason why it should not also be latent in the egg stage. The second fact against the theory of external contamination of the egg surface is the discovery that young larvae may die of a polyhedral disease even before they have left the egg shell (Smith et al., 1953). There are one or two miscellaneous agents which may possibly help to spread virus infections of insects and these are briefly dealt with. Predacious insects may be instrumental in carrying the polyhedra around, either on their mouthparts or possibly by contamination with their feces. Blowflies and similar insects which are frequently attracted by and feed on the cadavers of insect larvae which have died of virus infections are also potential vectors. The same also probably applies to birds, and, as previously mentioned, the wind and the rain no doubt play a part in distributing the polyhedra. Some recent work by Franz et al. (1955) is relevant in this connection. They state that after passing through the intestinal canal of the predatory bug, Rhinocoris annulatus L., and of the robin, Erithacus rubecula L., the polyhedral viruses of the pine sawfly, Neodiprion sertifer (Geoffr.), proved to be still infectious in experiments carried out with the specific host. REFERENCES Acqua, C. (1918-1919). Rend. Ist. bact. Scuola super. agr. Portici 3, 243. Andrewes, C. H. (1957). Nature 180, 788. Bergold, G. H. (1947). Z. 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Landwirtsch. 9, 381. Franz, V. J., Krieg, A., and Langenbuch, R. (1955). Z. Pflanzenkrankh. u. Pflanzenschutz 62 (11), 723. Glaser, R. W. (1927). Ann. Entomol. Soc. Am. 20, 319. Heidenreich, E. (1946). Arch. ges. Virusforsch. 1, 582. Hughes, K. M. (1953). Hilgardia 22, 391. Ishimori, N. (1934). Compt. soc. rend. biol. 114, 1169 Komarek, J., and Breindl, V. (1924). Z. angew Entomol. 10, 99. Krieg, A. (1955). Z. Naturforsch. 10b, 34. Krieg, A. (1957). Z. mikrobiol. Forsch. Methodik 12, 110. Lotmar, R. (1941). Bull. soc. entomol. suisse 18, 372. Maestri, A. (1856). ““Frammenti anatomici fisiologici e patologici sul baco da seta, Fusi, Pavia. Mazzocchi, V. (1908). Arch. Parasitol. 12, 456. Morgan, C., Bergold, G. H., and Rose, H. M. (1956). J. Biophys. Biochem. Cytol. 2, 23. Paillot, A. (1924). Compt. rend. 179, 229. Paillot, A. (1925). Compt. rend. 180, 1139. Paillot, A. (1926a). Compt. rend. acad. agr. France 12, 201. Paillot, A. (1926b). Compt. rend. 182, 180. Paillot, A. (1926c). 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(1949). “‘Principles of Insect Pathology”, p. 426. McGraw-Hill, New York. Steinhaus, E. A. (1952). J. Hcon. Entomol. 45, 897. Tanada, Y. (1953). Proc. Hawaiian Entomol. Soc. 15, 235. Tanada, Y., and Beardsley, J. W. (1957). J. Hcon. Entomol. 50, 118. Tokuyasu, K. (1953). Hnzymologia 16, 62. Tsujita, M. (1955). Proc. Japan Acad. 31, 93. von Prowazek, 8. (1907). Arch. Protistenk. 10, 358. Wasser, H. B. (1952). J. Bacteriol. 64, 787. Williams, R. C. (1953). Huptl. Cell Research 4, 188. Williams, R. C., and Smith, K. M. (1957). Nature 179, 119. Williams, R. C., and Wyckoff, R. W. G. (1945). Proc. Soc. Exptl. Biol. Med. 58, 265. Xeros, N. (1953a). Nature 172, 548. Xeros, N. (1953b). Nature 172, 309. Xeros, N. (1954). Nature 174, 562. Xeros, N. (1955). Nature 175, 588. Xeros, N. (1956). Nature 178, 412. Xeros, N. (1957). Thesis, Cambridge University. N. NE Author Index Numbers in italics refer to the pages on which references are listed in bibliographies at the end of each article. Ackermann, W. W., 16, 19, 94, 105, 123, 124, 140, 153, 155, 166, 182, 189, 201, 203, 205, 206, 209, 211, 212, 213, 214, 217, 218, 219, 220, 222, 223, 234, 247, 254, 272 Acqua, C., 371, 390 Ada, G. L., 30, 32, 33, 45, 47, 49, 56, 57, 58, 59, 60, 128, 143, 145, 153, 180, 189, 208, 222, 276, 283, 305 Adams, J. M., 260, 272 Adams, M. H., 233, 247 Adams, W. BR., 16, 19, 84, 89, 105 Aiston, 8., 27, 47 Alexander, H., 9, 14, 301, 306 Alexander, H. E., 189 Algire, G., 67, 105 Allen, E. G., 75, 107, 119, 120, 154 Allfrey, V. G., 10, 14, 207, 209, 216, 222 Allsopp, C. B., 71, 107 Amies, C. R., 76, 105 Andersen, F., 159, 191 Anderson, K., 164, 189 Anderson, R., 80, 105 Anderson, S. G., 25, 28, 30, 31, 33, 34, 35, DOO Ceo nO Dos Ole Oo st lize 20s 137, 153, 232, 238, 239, 247, 261, 270, 276, 305 Anderson, T. F., 77, 108 Andervont, H. B., 99, 100, 105, 315, 318, 320, 337, 338, 339, 340, 341, 342, 343, 344, 363, 364 Andrewes, C. H., 3, 10, 14, 22, 24, 36, 46, 47, 83, 98, 105, 123, 141, 153, 167, 171, 173, 181, 189, 199, 222, 237, 239, 247, 254, 255, 257, 259, 260, 267, 268, 270, 271, 272, 304, 305, 308, 310, 321, 336, 363, 364, 365, 366, 386, 390 Andrews, B. E., 254, 271 Aoyuma, A., 181, 193 Appleby, J. C., 36, 46, 290, 305 Aragao, H. B., 244, 247 Archetti, I., 85, 105, 267, 270 Armen, D. M., 100, 106 Armstrong, C., 226, 247 393 Arnoult, J., 80, 107 Arquie, E., 226, 249 Backus, R. C., 114, 155 Baker, J. R., 70, 105 Baluda, M., 112, 114, 116, 126, 134, 136, 138, 139, 140, 155 Baluda, M. A., 181, 183, 184, 185, 187, 188, 189 Banfield, W. E., 101, 106 Bang, B. G., 79, 105 Bang, F. B., 3, 14, 22, 28, 46, 67, 69, 70, 13, (1, 18; 19,8), 82, 83,. 84, 85, 86; 87, 88, 89, 95, 97, 98, 99, 100, 103, 104, 105, 106, 107, 108, 109, 143, 155, 162, 163, 167, 168, 172, 173, 189, 192, 214, 223, 323, 364, 366, 367 Bang, O., 308, 365 Bankowski, R. A., 86, 104, 106 Barer, R., 69, 106 Bargmann, W., 68, 106 Baron, S., 178, 189, 281, 283, 284, 285, 288, 305 Barski, G., 92, 93, 106 Bauer, A., 67, 79, 80, 106 Baumeister, L., 55, 59 Beale, A. J., 150, 153 Beard, D., 23, 24, 28, 47, 49, 50, 53, 60 Beard, J. W., 23, 24, 28, 36, 47, 49, 50, 53, G0), Bills Bile, Sls, Vil, SVG) BRE aaBt 334, 364, 367 Beardsley, J. W., 390, 392 Beck, W. S., 349, 351, 366 iBedsony S) Ps 73, 745) 15, 106. V8, 124. 125, 154 Begg, A. M., 128, 154 Behar, A., 80, 110 Bell, J. A., 5, 14, 254, 271 Bellett, A. J. D., 151, 153 Belyavin, G., 27, 49, 237, 249 Benedetti, E. L., 98, 106 Benewolensjkaja, S. W., 71, 110 Benyesh, M., 301, 305 394 Bergold, G. H., 371, 374, 375, 378, 380, 381, 384, 390, 391 Bergs, V. V., 127, 136, 138, 155, 162, 182, 187, 189, 191 Bernhard, W., 67, 79, 80, 97, 98, 99, 100, 106 Bernkopf, H., 144, 147, 153 Berry, G. P., 234, 249, 302, 303, 304, 305, 335, 364 Bertani, G., 228, 247 Bessis, M., 69, 106 Beveridge, W. I. B., 23, 25, 28, 29, 31, 35, 39, 46 Biesele, J., 67, 106 Binkhorst, J. L., 237, 247 Birch-Andersen, A., 145, 154, 179, 180, 191 Bird; He. 32) ol 4s Ol Os oul oOls So; 391 Bird, H. H., 112, 153, 189, 190, 200, 222, 276, 305 Bittner, J. J., 337, 339, 342, 344, 345, 364 Black, F. L., 141, 753, 301, 305 Black, L. M., 234, 247 Blackman, J. R., 17, 19 Blackman, S., 68, 72, 106 BlandsJe OVW nies oss ton lOsiida des 106 Blank, H., 80, 81, 106, 110, 118, 132, 137, 141, 155 Blaskovic, D., 262, 270 Blix, G., 54, 55, 56, 59, 61 Bloch, D. P., 91, 106 Bloch, O., 86, 106 Blyth, J. 8. 8., 318, 566 Boake, W. C., 23, 25, 28, 29, 35, 39, 45, 46 Boand, A. V., Jr., 83, 106 Bodian, D., 91, 93, 106, 165, 189 Bodily, H. L., 27, 46 Bohm, P., 55, 59 Bohnel, E., 27, 47 Bolle, J., 375, 391 Boot, L. M., 340, 364 Borel, A., 96, 106 Borrel, A., 307, 364 Borghese, E., 69, 110 Borghese, N.G., 354, 358, 360, 391, 367, 368 Borsos, T., 323, 364 Borysko, E., 67, 78, 85, 106 Bovarnick, M., 53, 60 AUTHOR INDEX Boyer, G. S., 89, 106, 219, 222 Boyle, P. J., 349, 351, 352, 354, 355, 357, 358, 366 Bozeman, F. M., 72, 110 Bozzo, A., 49 Bradish, C. J., 132, 153 Brandly, C. A., 24, 26, 48, 50 Brans, L. M., 237, 247, 258, 266, 268, 272 Braun, G. A., 55, 61 Breindl, V., 371, 372, 391 Breitenfeld, P. M., 88, 106, 152, 153 Brieger, E. M., 67, 72, 107 Broidy, B. A., 32, 46, 53, 59, 117, 137, faa, 229, 237, 238, 239, 240, 247, 249, 292, 305 Brooksby, J. B., 132, 153 Brossmer, R., 54, 55, 58, 60 Brown, G. C., 215, 217, 218, 222 Brown, L. V., 40, 41, 42, 47, 165, 190, 269, 270 Brown, R. A., 112, 153, 189, 190, 200, 222, 276, 305 Bryan, W. R., 94, 110, 314, 315, 316, 317, 318, 319, 320, 321, 322, 326, 334, 364, 365, 366, 367 Buckingham, M., 24, 48 Buckley, 8S. M., 75, 94, 106 Buescher, E. L., 40, 41, 42, 49 Buescher, L. L., 269, 272 Bull, D. R., 25, 46, 229, 231, 232, 247 Bunting, H., 101, 106, 110 Burgoon, C. F., 80, 110 Burke, D. C., 143, 153, 176, 183, 192 Burmester, B. R., 308, 309, 310, 312, 348, 365 Burnet, H. M., 3, 4, 13) 14, 22235242 be 26, 28, 29, 30, 31, 32, 33, 34.30) a0: 37, 39, 44, 45, 46, 47, 52, 53, 58, 59, 75, 83, 105, 106, 107, 112, 132, 142, 149, 150, 153, 159, 173, 175, 178, 179, 180, 189, 190, 192, 198, 199, 201, 222, 228, 229, 230, 231, 232, 236, 238, 239, 247, 254, 255, 260, 264, 267, 270, 276, 277, 279, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 295, 296, 298, 299, 305, 306 Burnside, C. E., 370, 391 Burnstein, T., 84, 87, 106 Burr, M. M., 44, 48 AUTHOR INDEX Cabasso, V. J., 27, 47 Cairns, H. J. F., 17, 18, 19, 23, 47, 115, 135, 136, 139, 140, 146, 147, 153, 172, 178, 179, 190, 201, 213, 214, 222, 229, 247 Calaby, J. H., 244, 249 Calnan, D., 314, 316, 321, 565 Cameron, G. R., 68, 106 Canti, R. G., 72, 74, 75, 106 Carmichael, N., 164, 194 Caro, L. G:, 72, 110 Carr, J. G., 318, 319, 325, 365, 366 Carré, M. C., 90, 108 Carrel, A., 96, 106, 322, 365 Carter, J. E., 203, 210, 212, 213, 223 Casals, J., 40, 41, 42, 47, 49, 165, 190, 269, 270 Caspar, D., 301, 305 Cassel, W. A., 237, 240, 247, 292, 305 Challice, C. E., 84, 86, 107 Chamberland, C., 226, 249 Chambers, V. C., 168, 181, 182, 190 Chanock, R. M., 40, 41, 47, 50, 163, 181, 190, 254, 269, 271 Chany, C., 90, 108 Chapin, M., 233, 247 Chapronierre, D. M., 104, 106, 336, 364, 365 Chase, M., 112, 154 Chesbro, W. R., 38, 47 Ching, C., 159, 190 Chun Cs M-. 24526. 27, 28; 3, 35, 36; 39, 44. 45, 47, 83, 106, 142, 153, 237, 238, 247, 257, 268, 271 Cieciura, 8. J., 181, 187, 190, 234, 247 Clampit, J. M., 237, 247 Clark, E., 24, 25, 45, 46, 47, 56, 59 Clark, E. C., 390, 391 Clarke, D. H., 40, 41, 47, 49 Claude, A., 97, 106 Coffey, J. H., 167, 109 Coffin, D. L., 104, 106 Cohen, G. N., 189, 190 Cohen, H., 27, 49 Cohen, S. M., 167, 190 Cohen, S. 8., 202, 222 Cohn, M., 189, 190 Colter, J. S., 112, 153, 189, 190, 200, 222, 276, 305 Comb, D. G., 54, 59 Comben, B. M., 230, 248, 299, 306 395 Common, I. F. B., 375, 391 Cook, K., 254, 271 Coons, A. H., 87, 88, 110, 200, 224 Coons, A. L., 103, 106 Cooper, P., 151, 153 Cooper, P. D., 164, 168, 181, 184, 190 Corey, M., 27, 46 Coriell, L. L., 80, 81, 92, 108, 110, 118, 132, 137, 141, 155 Cornalia, E., 371, 391 Cornforth, J. W., 54, 59 Cowdry, E. V., 64, 80, 82, 106 Coxe Hy Rig 2a 2 eall5 9190 Craig, D. E., 215, 222 Craigie, J., 132, 154 Crawford, G. N. C., 117, 154 Crick, F. H. C., 12, 14, 301, 305 Crocker, T. T., 74, 106 Croissant, O., 80, 81, 110 Crouse, H. V., 80, 81, 106 Cryns, W. F., 271 Cunha, R., 28, 47 Curnen, E. C., 44, 47 Curtain, ©; Cy, 36; 37, 27, ba, 06, 99,60 Daines, M. E., 54, 59 Dalldorf, G., 164, 167, 190 Dalmat, H. T., 327, 335, 365, 366 Dalton, A. J., 98, 108, 325, 566 Dane, D. S., 243, 247 das Ferreira, A., 68, 107 Davenport, F. M., 24, 34, 47, 160, 190, 198, 199, 222, 237, 238, 239, 247, 261, 262, 263, 265, 266, 271, 272 Davis, E. V., 201, 223 Dawson, I. M., 28, 47, 77, 83, 106, 107, 142, 153 Day, M. F., 375, 391 de Baan, 43, 50 De Burgh, P. M., 53, 60 Dedrick, H. M., 302, 305, 335, 364 Deinhardt, F., 86, 108, 162, 182, 187, 189, 191 Delbriick, M., 159, 190, 203, 222 Denington, E. M., 308, 365 Depoux, R., 162, 183, 190, 268, 271 Deringer, M. K., 339, 340, 357, 365, 366 DeRobertis, E., 68, 107 DeSanctis, A. N., 203, 210, 212, 213, 223 Dick, G. W. A., 243, 247 396 Dillon, J. F., 132, 153 Dineen, J. K., 80, 82, 109 Dingle, J. H., 5, 14, 23, 24, 49, 50 Dmochowski, L., 99, 100, 107, 308, 319, 337, 339, 344, 365 Dochez, A. R., 44, 49 Doermann, A. H., 113, 154, 159, 190 Doerr, R., 167, 168, 172, 190 Dohi,'S.,; 16, 77, 107, 109 Dolch, M. C., 100, 108 Doljanski, L., 96, 107, 110, 322, 366 Donald, H. B., 23, 47, 131, 142, 144, 146, 154, 155, 277, 295, 305 Donnelley, M., 239, 247 Dontcheff, A., 97, 106 Dorman, D. C., 236, 249 Dorrance, W. R., 163, 165, 195 Dougherty, R. M., 159, 190, 220, 223 Douglass, M., 164, 190 Downie, A. W., 75, 77, 107, 236, 247, 299, 305 Drake, J. W., 164, 181, 190 Dubes, G. R., 233, 247 Dudgeon, J. A., 28, 47 Duffy, C. E., 163, 172, 190, 192 Dulaney, A. D., 349, 351, 355, 356, 357, 360, 361, 365 Dulbecco, R. D., 18, 19, 93, 109, 114, 126, 127,133, 134, 138,139) 142) 754, 105, 159, 190, 199, 200, 208, 214, 222, 223, 299, 232, 233, 235, 236, 243, 247,.249, 302, 305, 306 Dumbell, K. H., 299, 305 Dunkel, V. C., 324, 366 Dunn, T. B., 99, 107, 337, 339, 340, 341, 348, 344, 349, 351, 352, 354, 355, 357, 358, 359, 361, 364, 365, 366 Dunnebacke, T. H., 92, 93, 94, 104, 107 Duplan, J. I., 362, 367 Duran-Reynals, F., 235, 247, 248, 308, 312, 315, 320, 321, 323, 329, 333, 335, 365, 367 Durieux, C., 226, 249 Duryeé, W., 102, 107 Dyce, A. L., 246, 248 Eagle, H., 71, 107 Earle, W. B., 96, 110, 322, 367 Eaton, M. D., 27, 46, 219, 222 Eaves, G., 77, 107 AUTHOR INDEX Ebara, S., 181, 193 Ebeling, A. H., 96, 106, 322, 365 Eckert, E. A., 53; 60, 326, 333, 364 Eddy, B. E., 84, 107, 354, 358, 360, 361, 365, 367, 368 Edney, J. M., 24, 25, 29, 32, 46, 47, 115, 121, 124, 146, 147, 153, 154, 155, 162, 173, 175, 176, 177, 178, 183, 184, 185, 187, 188, 189, 190, 192, 231, 238, 247, 248, 267, 271, 283, 290, 292, 305 Kisenberg-Merling, K. B., 76, 107 Elford, W. J., 24, 28, 47, 83, 106, 142, 153, 171, 189, 199, 222 Elion, G. B., 215, 218, 224 Ellerman, V., 308, 365 Ellis, E. L., 203, 222 Ellison, 8., 77, 80, 81, 82, 109 Enders, J. F., 5, 14, 22, 31, 48, 49, 91, 107, 118, 156, 232, 234, 248 Endo, M., 92, 93, 106 Engelbreth-Holm, J., 308, 362, 365 Epstein, M. A., 97, 98, 107, 325, 365 Erickson, J. O., 100, 108 Ernster, L., 70, 110 Escherich, K., 371, 391 Aspmark, A., 5, 14 Faillard, H., 54, 55, 56, 60 Fairbrother, R. W., 126, 155, 255, 272 Farrant, J. L., 375, 391 Fastier, L. B., 42, 43, 47 Fawcett, D. W., 101, 102, 107 Fazekas de St. Groth, S., 17, 19, 23, 26, 28, 33, 34, 39, 46, 47, 51, 52, 59, 60, 146, 147, 148, 153, 154, 162, 176, 177, 178, 179, 184, 185, 190, 201, 214, 222, 239, 248 Fearing, M., 240, 247 Febvre, H., 80, 107 Fekete, E., 351, 365 Kell BB 67, a. 125107, Feller, A. E., 23, 24, 49, 50, 234, 248 Fenner, F. J., 3, 4, 14, 45, 47, 75, 107, 230, 236, 237, 240, 241, 244, 245, 246, 2458, 249, 299, 303, 305, 306 Fennessy, B. V., 244, 249 Findlay, G. M., 163, 167, 168, 190, 227, 235, 248 Finter, N. B., 17, 19, 132, 135, 136, 139, 148, 150, 153, 154, 176, 178, 180, 190 AUTHOR INDEX Firth, M. E., 54, 59 Fischer, A., 68, 96, 107 Fiset, P., 268, 271 Fite, G. L., 235, 249 Flewett, J. H., 84, 86, 107 Flewett, T. H., 77, 107 Flick, J. A., 28, 47 Florio, L., 234, 248 Florman, A. L., 162, 190 Foard, M., 95, 106 Fogh, J., 208, 213, 222, 277, 306 Foley, M., 231, 247 Folkers, K., 203, 205, 206, 207, 208, 212, 213, 215, 216, 219, 224 Fong, J., 159, 175, 187, 190 Forssmann, O. C., 146, 156 Forster, G. F., 37, 49 Foster, R. A. C., 209, 222 Foulds, L., 99, 107, 308, 365 OxsiJiqpess 4dn 46 Fraenkel-Conrat, H., 112, 154 Francis, T., Jr., 5, 14, 33, 34, 36, 47, 48, 52, 60, 81, 83, 107, 110, 219, 222, 237, 238, 239, 247, 248, 254, 255, 256, 258, 260, 261, 262, 263, 265, 266, 267, 268, 270, 271, 272, 286, 290, 306 Frankel, J. W., 159, 163, 165, 167, 168, 169, 191, 193, 194 Franklin, R., 199, 224 Franklin, R. M., 116, 133, 136, 138, 140, 154, 155 Franz, V. J., 390, 391 Fraser, K. B., 27, 47, 173, 189, 283, 286, 290, 295, 306 Fredericq, P., 228, 248 Frederiksen, O., 362, 365 French, E. L., 4, 14, 32, 33, 36, 45, 47, 53, 60 French, R. C., 188, 197 Friedenreich, V., 30, 48 Friedewald, W. F., 24, 48, 237, 239, 248, 258, 271 Friedrich-Freksa, H., 371, 391 Friend, C., 218, 222, 361, 365 Frisch-Niggemeyer, W., 115, 122, 125, 153, 154,150, 25350141, 202 Fukae, 8., 181, 193 Fukushi, T., 234, 248 Fuller, H., 310, 365 397 Fulton, F., 28, 47, 128, 130, 132, 150, 154, 155, 179, 191 Furesz, J., 259, 273 Furono, 8., 171, 193 Gard, S., 42, 43, 48, 50, 145, 154, 156, 167, 168, 179, 180, 197 Gardner, B. J., 31, 48 Gardner, R. E., 303, 506 Gaylord, W. H., Jr., 73, 77, 78, 97, 107 Gear, J. H.S., 3, 14 Gerber, P., 267, 271 Gerngross, O. G., 254, 271 Gey, G. O., 65, 67, 70, 73, 79, 81, 86, 92, 95, 97, 98, 105, 106, 107, 109, 110, 323, 367 Gey, M. K., 70, 107 Gierer, A., 112, 154, 199, 224 Gifford, G. E., 219, 222 Gildemeister, E., 164, 191 Gillen, A. L., 44, 48 Gilmore, L. K., 89, 110 Ginder, D. R., 327, 328, 329, 365 Ginsberg, H. 8., 17, 19, 35, 37, 48, 88, 89, 106, 108, 141, 154, 159, 162, 163, 175, 179, 191, 193, 200, 203, 204, 205, 212, 219, 220, 222, 238, 239, 245, 268, 271 Girardi, A., 86, 108, 130, 154, 179, 191 Girardi, A. J., 75, 107, 119, 120, 154 Glaser, R. W., 384, 391 Gledhill, A. W., 36, 47, 257, 267, 268, 271, 272 Gochenour, A. M., 354, 358, 360, 361, 367 Gocke, I. M., 219, 222 Godman, G. C., 91, 106 Goebel, W. F., 35, 37, 48, 204, 222 Gogolak, F. M., 44, 48, 75, 107 Golde, A., 98, 110 Goldfield, M., 43, 48 Goldman, E. C., 233, 248 Golub, O. J., 164, 171, 197, 302, 306 Gonzalez-Ramirez, J., 67, 107 Goodpasture, E. W., 79, 80, 86, 107, 108 Gorbunova, A. S., 254, 271 Gordon, F. B., 237, 247, 302, 306 Gordon, I., 163, 165, 193 Goss, M. T., 349, 351, 356, 357, 360, 361, 365 Gostling, J. V. T., 74, 75, 106, 118, 119, 124, 125, 154 398 Gotlieb, T., 159, 162, 173, 174, 175, 178, 180, 191, 192, 276, 277, 281, 283, 286, 287, 288, 290, 306 Gottschalk, A., 29, 36, 48, 53, 54, 55, 56, 57, 09, 60,267, 271 Graffi, A., 362, 365 Graham, A. F., 188, 191 Graham, D. M., 17, 19, 26, 47, 148, 153, 178, 179, 190 Graham, EK. R. B., 57, 60 Granoff, A., 23, 48, 116, 130, 154, 162, 163, 168, 174, 179, 181, 191 Gratia, A., 375, 391 Gray, A., 81, 107, 118, 132, 137, 141, 155 Green, A. S., 92, 108 Green, M., 182, 187, 191, 200, 222 Green, R. G., 167, 191 Green, R. H., 37, 48, 77, 108 Greene, H. 8. N., 330, 365, 366 Greenwood, A. W., 318, 366 Greig, J. R., 345, 366 Griffiths, F., 302, 306 Grist, N. R., 127, 154 Gross, L., 347, 348, 349, 350, 352, 353, 355, 356, 358, 359, 360, 361, 366 Groupé, V., 159, 168, 171, 190, 220, 223, 316, 317, 319, 320, 323, 324, 366, 367 Grubbs, G. E., 354, 358, 360, 361, 367 Guarnieri, G., 75, 108 Guerin, M., 99, 100, 108, 308, 311, 312, 313, 318, 319, 327, 335, 367 Guetier, M., 235, 248 Gye, W. E., 321, 366 Gyorgy, P., 55, 61 Haagenson, C. D., 100, 107 Haas, V. H., 354, 368 Haddock, D. W., 236, 247, 299, 305 Hagenau, F., 67, 98, 106, 108, 110 Haguenau, F., 325, 366 Hahn, E., 189 Hahn, R. G., 43, 48, 204, 223 Haig, D. A., 44, 48 Halberstaedter, L., 322, 366 Haley, R., 98, 105 Hallauer, C., 43, 48, 167, 168, 172, 191 Halvorson, H. O., 218, 223 Hamer, M., 258, 273 Hamlin, A., 84, 86, 87, 89, 90, 108 AUTHOR INDEX Hammon, W. M., 165, 192 Hammon, W. McD., 242, 248, 269, 271 Hampil, B., 203, 210, 212, 213, 223 Hamre, D., 267, 271 Hanes, F. M., 70, 108 Hanig, M., 56, 60 Hanson, R. J., 83, 106 Hanson, R. P., 24, 26, 48, 50, 233, 248 Harding, C. V., 92, 93, 108 Harding, D., 92, 93, 108 Hare, R., 22, 48, 260, 271 Harel, J., 67, 79, 80, 107 Harford, C. G., 84, 86, 87, 89, 90, 108 Harkins, A., 159, 191 Harris, R. J. C., 314, 317, 319, 366 Hartley, J. W., 219, 223 Hays, E. F., 349, 351, 366 Hayward, M. E., 132, 154 Hechter, O., 70, 109 Hedrick, L. R., 38, 47 Heidenreich, E., 384, 391 Heimer, R., 56, 57, 60 Heller, L., 5, 14, 43, 48 Helm, R., 164, 191 Helmold, R. J., 44, 48 Henderson, D. W., 160, 791 Henle, G., 26, 48, 86, 108, 121, 123, 127, 129, 130, 135, 138, 154, 156, 162, 172, 73; 175s VIG Ais U79s 82 Saanlsos 186, 187, 188, 189, 191 Henle, W.., 23, 26, 285 11/5, 121, 122 1238 127, 128, 129) 130; 1315 1325 VeomltaG: 138, 139, 145, 148, 149, 150, 154, 155, 156; W625 V2. WIS a4 lie lad Oo luge 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 214, 223, 278, 306 Hennessen, W. A., 164, 181, 193, 231, 232, 249 Hennessy, A. V., 34, 47, 261, 262, 263, 265, Cilete Henry-Eveno, J., 165, 192 Herrmann, E. C., Jr., 159, 191 Hers, J. F. P., 83, 108 Hershey, A. D., 112, 154, 202, 207, 223 Hertzberger, E., 237, 247 Heston, W. E., 339, 340, 357, 365, 366 Heyl, D., 215, 224 Hilleman, M. R., 5, 14, 44, 48, 89, 91, 108, 110, 257, 259, 261, 272 AUTHOR INDEX 399 Hirst, G. K., 17, 19, 22, 23, 25, 26, 27,29, Hyde, J., 86, 104, 106 34, 35, 36, 38, 39, 48, 52, 58, 60, 159, Hyde, R. R., 303, 306 162, 163, 170, 172, 173, 174, 175, 178, 180, 188, 191, 192, 194, 232, 237, 238 , 188, 191, 192, 194, 232, 237, 238, Tom TZ. E., 165, 192 239, 248, 256, 258, 265, 272, 276, 277, Tease A. 93, 25, 35, 36, 47, 48, 49, 84, 85, eee 88, 89, 105, 110, 121, 122, 124, 125, 130, Hitchings, G. H., 215, 218, 224 131, 142, 143, 144, 146, 150, 153, 154, Hoagland, C. L., 209, 216, 223 155, 156, 160, 162, 173, 175, 176, 177, Hoffman, D. C., 235, 248 183, 184, 185, 187, 188, 190, 191, 192, Hoffstadt, R. E., 304, 306 200, 223, 231, 238, 247, 248, 257, 267, Hogue, M. J., 69, 92, 108 268, 272 Holden, H. F., 131, 155 Tsaaca, I., 277, 295, 305 Hook, E. W., 237, 239, 248 ee popper Eo, 31, Eid Ishida, N., 16, 19, 123, 124, 153, 155 Horii, 8., 76, 109 Ishii, S., 181, 193 Horning, B.6., 71, 108 Ishimori, N., 376, 391 Horsfall, F. L., Jr., 23, 24, 27, 35, 36, 37, 42, 43, 44, 47, 48, 49, 50, 53, 61, 115, 146, 148, 154, 156, 162, 163, 173, 174, Jackson, E. B., 72, 110 175, 176, 177, 183, 191, 194, 196, 198, Jenkins, D. L., 167, 192 199, 200, 202, 203, 204, 205, 206, 207, Jensen, K. E., 34, 48, 178, 189, 254, 258, 208, 212, 213, 214, 215, 216, 219, 222, 259, 262, 264, 266, 268, 272, 281, 283, 223, 224, 261, 267, 268, 270, 271, 272, 284, 285, 288, 305 277, 306 Johnson, C. D., 86, 108 Hoskins, J. M., 115, 155, 229, 249 Johnson, R. B., oi, 222 Hoskins, M., 167, 168, 192 Jonchiere, H., 226, 249 Hotchkin, J. E., 112, 114, 116, 126, 134, Jones, E. E., 346, 366 138, 139, 155 Jones, H. P., 74, 75, 109 Jones, M., 233, 235, 237, 240, 248 Hotchkiss, R. D., 11, 14, 189, 192 Jordan, R. T., 163, 172, 190, 192 Hotta, T., 181, 193 Hotz, G., 83, 84, 85, 87, 88, 108 Joseph, 8., 69, 106 Houser, H. B., 271 Jotz, Ns Cs 257, 272 Howard, E. M., 235, 248 Jungeblut, Cs W., 164, 192 Howe, C., 53, 60, 89, 90, 91, 106, 109, 142, 143, 155 Kalaja, T., 41, 49 Howe, H. E., 91, 108 Kandel, A., 215, 222 Howes, D. W., 92, 93, 110, 134, 139, 154 Kapikian, A. Z., 254, 271 Hoyle, L., 17, 19, 23, 26, 27, 34, 48, 85, Kaplan, A. S., 233, 248 108, 115, 121, 122, 125, 126, 127, 128, Karr, i. Wee 136, 148, 155 132, 154, 155, 253, 255, 271, 272 Karrer, H. ih 66, 75, 108 Hsuing, G. D., 301, 305 Karzon, D. T., 84, 85, 109, 168, 192 Huang, C. H., 163, 164, 181, 192 Kausche, G. A., 72, 108 Huebner, R. J., 5, 14, 89, 110, 254, 271 Kempf, J., 83, 106 Hughes, A. F., 67, 69, 79, 92, 108 Kent, S. 2:7), 109 Hughes, K. M., 373, 391 Keogh, E. V.,-199, 222, 235, 236, 248, 323, Hull, R.N., 7, 14, 196, 201, 214, 217, 220, 223 366 : Human, M. L., 228, 248 Khoobyarian, N., 159, 192 Huppert, J., 115, 155 Kidd, J. G., 333, 366, 367 Hurst, E. W., 196, 201, 214, 217, 223, 303, Kieler, J., 71, 108 306 Kilbourne, E. D., 187, 192 400 Kilham, L., 4, 14, 31, 48, 304, 506, 335, 336, 366 Kam), Se, 10, 109 King, L. 8., 235, 248 Kinosita, R., 100, 108 _Kirber, M. W., 172, 187, 191 Kirschbaum, A., 339, 364 Kitaoka, M., 233, 249 Kjellen, L., 89, 90, 108 Klenk, E., 54, 55, 56, 59, 60 Kloéne, W., 92, 94, 108 Knight, C. A., 24, 48 Knoop, A., 68, 106 Kohler, H., 79, 108 Kojima, Y., 171, 193 Komarek, J., 371, 391 Kon, M., 167, 168, 172, 190 Koprowski, H., 162, 163, 166, 181, 183, 192 Korteweg, R., 337, 366 Krieg, A., 370, 374, 390, 391 Kubota, Y., 74, 110 Kuhn, R., 54, 55, 58, 60 Kuroya, N., 254, 272 Kurtz, H., 81, 94, 105, 107, 166, 182, 189, 203, 209, 212, 218, 220, 222 Kuske, T. T., 187, 193 Kusumoto, K., 181, 193 Kvedar, J. P., 321, 365 Lacour, F., 98, 110 Lagerborg, D. L., 218, 223 Lagermolm, G., 89, 90, 108 Lahelle, O., 42, 43, 48 Laidlaw, P. P., 237, 247, 254, 255, 260, BO GUE Lambert, R. A., 70, 71, 108 Lancaster, M. C., 160, 191 Langenbuch, R., 390, 391 Lanni, F., 36, 49, 53, 60, 199, 223 Lanni, Y. T., 199, 223 Larkin, M., 249 Laser, H., 96, 107 Lasfargues, E. Y., 101, 108 Latarjet, R., 126, 155, 362, 367 Lauffer, M. A., 23, 24, 48 Law, L. W., 349, 351, 352, 354, 355, 357, 358, 359, 366 Lavelle, J. M., 220, 223 AUTHOR INDEX Lavin,.G. 1... 162; 173; 175; 176; 17 ies. 194, 209, 216, 223 Le Bouvier, G. L., 164, 167, 181, 192 Le Brun, J., 80, 82, 108, 133, 155 Le Clerc, J., 187, 192, 276, 306 Ledinko, N., 127, 136, 138, 155, 163, 181, 192, 232, 237, 238, 239, 248, 292, 301, 306 Lehninger, A. L., 70, 108 Leidy, C., 189 Lempfrid, H., 54, 55, 56, 60 Lennette, E. H., 162, 163, 166, 181, 183, 192, 256, 272 Lépine, P., 80, 81, 90, 108, 110, 235, 244, 248, 249 Lesley, S. M., 188, 191 Lettré, R., 67, 79, 108 Leuchtenberger, C., 71, 89, 106, 108, 219, eee Levaditi, C., 165, 192, 235, 248 Levens, J. H., 22, 48 Levenson, C. G., 219, 222 Levillain, W. D., 339, 366 Levine, A. S., 159, 191, 198, 223 Levine, S., 23, 24, 29, 39, 48, 49, 162, 163, 172, 181, 184, 188, 192 Levinthal, C., 296, 306 Levinthal, W., 73, 108 Levy, E., 67, 79, 105 Levy, H. B., 219, 223 Lewis, M. R., 65, 67, 69, 70, 72, 108, 109 Lewis, W. H., 65, 67, 69, 70, 93, 109 L’Héritier, P., 234, 248 Ibis (Onl RRR RE 727 £0} Libert, R., 99, 100, 105 Lieberman, M., 176, 190 Lief, F. S., 128, 129, 131, 145, 154, 155, 179, 180, 197, 192 Likely, G. D., 96, 110, 322, 367 Lim, K., 290, 305 Lind, P. E., 25, 28, 31, 35, 36, 39, 46, 48, 53, 58, 60, 132, 149, 150, 153, 159, 173, 175, 178, 179, 180, 189, 190, 192, 267, 270, 276, 277, 279, 281, 282, 283, 285, 286, 287, 288, 289, 290, 291, 292, 293, 296, 305, 306 Lindberg, E., 54, 55, 59 Lindenmann, J.,83, 109,122, 143, 155, 160, 176, 177, 183, 185, 192 AUTHOR INDEX Liu, O. C., 23, 48, 87, 88, 104, 106, 109, 130; 131, 132; 1335 135, 13651895148; 152, 153, 155, 174; 175, 176; 177, 178, 179, 180, 187, 190, 191, 193, 203, 210, 212, 213, 223, 278, 306 Lo, W. H. Y., 97, 109, 323, 366, 367 Logie, L. C., 71, 109 Loh, P. C., 219, 223 Loosli, C. G., 267, 271 Lotmar, R., 376, 391 Louie, R., 159, 175, 185, 190 Low, B. W., 91, 109 Low, H., 70, 110 Lowell, F. C., 24, 48 Lucké, B., 102, 109 Ludford, R. J., 64, 79, 109, 322, 367 Luft, J., 70, 109 Luria, 8. E., 114, 126, 155, 228, 248 Lush, D., 22, 48, 199, 222, 260, 270 Lwoff, A., 93, 109, 133, 139, 142, 155, 208, 223 Lwoff, M., 93, 109, 133, 139, 142, 155, 208, 223 Lytle, J., 240, 247 Maassab, H. F., 123, 124, 140, 153, 201, 20d. 205420622122. Qld. 214 27 NOM ees: McAllister, R., 92, 108 McCann, G. F., 91, 109 McCarty, M., 203, 204, 223 McClelland, L., 22, 48 MeCreasn Jn bes 29s oO ol Oo soso On ols 39, 46, 47, 48, 249, 52, 53, 57, 59, 60, ADIL, 2342, PR, AV MacCullum, F’. O., 163, 167, 168, 190 Macdonald, F., 40, 49 McEwin, J., 23, 25, 28, 29, 35, 39, 46 McFarlane, A. S., 77, 107 McGaughey, C. A., 4, 14 McGauhey, C. A., 77, 107 McLean, D. M., 242, 248 . McLean, I. W., Jr., 24, 49 McLimans, W. F., 92, 93, 108, 201, 223 Macpherson, I. A., 37, 49 Madsen, T., 132, 155 Maestri, A., 371, 391 Magill, T. P,, 23, 25, 49, 174, 194, 231, 237, 248, 255, 256, 257, 260, 261, 267, 268, Bish, PPS VOL. I1I—26 401 Magrassi, F., 167, 168, 172, 193 Magrath, D. I., 117, 125, 138, 155 Maitland, H. B., 117, 125, 132, 138, 155 Maloney, J. B., 98, 108 Malsberger, R. G., 203, 210, 212, 213, 223 Mamay, H. K., 302, 306 Manabe, K., 73, 109 Manaker, R. A., 323, 324, 366, 367 Mandel, B., 18, 19, 43, 49, 59, 60 Manire, G. P., 159, 180, 193 Maral, R., 104, 109 Marcenac, F., 235, 248 Marchal, J., 235, 249 Marcus, P. I., 71, 109, 181, 187, 190, 234, 247 Markham, F. S., 27, 47 Marmion, B. P., 53, 60 Marshall, I. D., 237, 244, 248, 249, 303, 306 Mason, H. C., 163, 181, 193 Mason, P. J., 140, 153 Mastrota, F. M., 254, 271 Masurel, N., 258, 263, 266, 268, 272 Matthews, R. E. F., 196, 201, 214, 217, 218, 223 Matumoto, 8., 76, 109, 171, 193 Maurin, J., 90, 108 Maver, M. E., 326, 364 Maxey, M., 349, 351, 356, 357, 360, 361, 365 Mazzocchi, V., 384, 391 Medill, M. A., 237, 247, 292, 305 Medill-Brown, M., 229, 238, 249 Meiklejohn, G., 256, 272 Melechen, N. E., 207, 223 Mellors, R. C., 332, 367 Melnick, J., 81, 109 Melnick, J. L., 77, 78, 92, 93, 101, 107, 110, 134, 139, 141, 153, 154, 163, 181, 192; 243, 249, 301, 305 Merrill, M. H., 242, 249 Metcalf, D., 45, 49, 117, 132, 155 Meyer, H. M., 163, 190 Meyer, K. F., 3, 14, 56, 57, 60 Mider, G. B., 339, 361, 365, 367 Milford, J., 323, 367 Miller, G. L., 23, 24, 25, 26, 27, 48, 49 Mills, K. C., 44, 49 Mimms, C. A., 151, 155, 169, 193 Minnegan, D., 95, 106 402 Minner, J. R., 7, 14 Minton, S. A., Jr., 215, 218, 224 Minuse, E., 34, 36, 47, 48, 254, 261, 265, 266, 268, 272 Mirsky, A. E., 12, 14, 207, 216, 222 Mitchell, R. F., 46 Miyagawa, Y., 73, 74, 109 Miyajima, M., 371, 391 Modi, N. L., 118, 132, 155 Mogabgab, W. J., 219, 223 Moloney, J. B., 314, 316, 321, 325, 326, 364, 365, 366 Monod, J., 189, 190 Moore, A. E., 286, 290, 306 Moore, D. H., 77, 80, 81, 82, 84, 85, 89, 90, 91, 100, 107, 109, 142, 143, 155, 214, 223 Morgan, C., 77, 79, 80, 81, 82, 84, 85, 89, 90, 91, 106, 109, 142, 143, 155, 214, 228, 375, 391 Morgan, H. R., 31, 47, 48, 49, 119, 126, 155 Morgan, I. M., 163, 164, 165, 166, 172, 193 Morgan, P. N., 163, 190 Morioka, Y., 233, 249 Morris, M. C., 42, 43, 49 Morton, J. J., 339, 367 Mosely, V. M., 83, 109 Mosley, V. M., 142, 155 Moulder, J. W., 3, 4, 14 Mount, R. A., 86, 109 Mountain, I. M., 9, 74, 301, 306 Miihlbock, O., 340, 364, 367 Mulder, J., 35, 49, 256, 258, 263, 266, 268, Die Biss Munk, K., 116, 123, 130, 155 Murphy, J. B., 323, 367 Murphy, J. 8., 69, 84, 85, 109, 214, 223 Murphy, T. P., Jr., 164, 172, 194 Murray, W. S., 342, 367 Myers, K., 244, 249 Nagano, Y., 171, 193 Nagler, F. P. O., 24, 44, 45, 47, 48, 49, 56, 59, 132, 155 Nakano, M., 233, 249 Naudé, W. du T., 171, 193 Nemes, M. M., 203, 208, 209, 210, 211, PAPAS DAB}, DAI PANT, AS Pe! Nicholson, F. M., 82, 106 AUTHOR INDEX Nikkila, E., 41, 49 Nomura, S., 233, 249 Nomura, Y., 74, 110 Norambuena, M., 132, 153 Noury, H., 165, 192 Noyes, W. F., 78, 95, 109, 332, 367 Nunez-Galvan, A., 67, 107 Oberling, C. A., 67, 79, 80, 97, 98, 99, 100, 106, 308, 311, 312, 313, 31873195320. 335, 367 O’Dea, J. F., 80, 82, 109 Odin, L., 54, 55, 59, 60, 61 Oker-Blom, N., 41, 49 Okuno, Y., 181, 193 Oliphant, J. W., 83, 109 Olitsky, P. K., 43, 48, 49, 163, 164, 166, IP, HDS, PBs Ae Orr, J., 339, 365 Osawa, S., 207, 216, 222 Otis, H. K., 351, 365 Overman, J. R., 78, 109, 118, 1387, 141, 155, 209, 216, 224 Ozaki, Y., 76, 109 Packman, L., 160, 191 Paillot, A., 371, 375, 378, 384, 391 Palay, 8. L., 92, 109 Palade, G., 66, 67, 92, 109 Pallade, G. E., 12, 14 Parker, E., 84, 87, 89, 90, 108 Parrott, R. N., 110, 254, 271 Parry, W. R., 160, 193 Pasteur, L., 168, 193, 226, 249 Patterson, P. R., 163, 165, 193 Paucker, K., 115, 131, 148, 149, 150, 154, 155, 176, 177, 178, 179, 180, 183, 184, 185, 191, 193 Paulydekvss os L4 Payne, A. M. M., 5, 14 Peacock, P. R., 367 Peacock, 8., 160, 191 Pearson, H. E., 215, 218, 223, 224 Pehrson, G. C., 163, 190 Peltier, M., 226, 249 Perlmann, G. E., 36, 49 erring «l. Ee. Soe l09 Perry, B., 128, 143, 145, 153, 180, 189, 208, 222, 232, 237, 238, 239, 248, 276, 283, 292, 305 AUTHOR INDEX Perry, M. E., 219, 222 Peters, D., 13, 14, 77, 109, 209, 216, 223 Petersen, W. D., Jr., 34, 48 Peterson, W. D., Jr., 259, 264, 272 Pickels, E. G., 24, 44, 48, 97, 106 Pikovski, M. A., 346, 367 Pilcher, K. 8., 304, 306 Pinnock, P. R., 91, 109 Pohjanpelto, P., 233, 249 Pollard, E. C., 175, 179, 183, 187, 193 Pollister, A. W., 65, 70, 109 Pollister, P. F., 65, 70, 109 Pomerat, C. M., 71, 109 Polson, A., 132, 155, 156, 171, 193 Ponsford, J., 236, 249 Pontén, G., 325, 367 Poole, W. E., 246, 248 Porter, K. R., 65, 97, 99, 106, 109 Porterfield, B. M., 29, 32, 49 Potter, C., 375, 391 Powell, W. F., 175, 179, 183, 187, 193 Price, M. L., 215, 218, 224 Price, W. H., 270, 272 Prince, A. M., 16, 19, 84, 88, 89, 105, 109, 159, 193, 324, 367 Pucks 28... 23,39): 48, 49,41, 109; 181; 187, 190, 199, 223, 234, 247 Pugh, L. H., 159, 168, 191 Pulido-Villegas, I., 67, 107 Putnam, F. W., 202, 223 Pye, J., 36, 37, 47, 53, 56, 59, 60, 61, 131, 155 Quilligan, J. J., 256, 261, 271, 272 Quilligan, J. J., Jr., 30, 49 Rabson, A., 94, 105, 203, 209, 212, 218, 220, 222 Racker, E., 18, 19, 43, 49, 59, 60 Radot, C., 244, 249 Rafelson, M. E., Jr., 215, 218, 223, 224 Rake, G., 3, 14, 74, 75, 92, 93, 108, 109 Rapp, F., 75, 106 Ratcliffe, F. N., 244, 249 Rathova, V., 262, 270 Rauscher, F. J., 316, 317, 319, 320, 366 Ray, B. L., 70, 108 Reeves, W. C., 4, 14, 242, 248 Reichelderfer, T., 254, 271 Reinié, L., 165, 192 403 Rhodes, A. J., 101, 110 Richardson, K., 71, 108 Rickard, E. R., 260, 261, 272 Riehm, W. C., 260, 271 Riessig, M., 81, 92, 93, 109, 110 Rivers, C. F., 378, 379, 388, 391 Rivers, T. M., 64, 110, 209, 216, 223 Robbins, F. C., 232, 248 Robertson, H. E., 219, 222 Robineaux, R., 92, 93, 106 Robinow, C., 76, 77, 78, 106 Robinow, C. F., 85, 110, 142, 155 Robinson, H..E., 164, 190 Robinson, R. H. M., 329, 367 Roh, H., 301, 305 Rollhauser, H., 68, 110 Rondanelli, E. G., 69, 110 Ropes, M. W., 83, 110 Rose, G. G., 67, 110 Rose, H. M., 77, 80, 81, 82, 84, 85, 89, 90, 91, 106, 109, 142, 143, 155, 214, 223, 375, 391 Roseman, 8., 54, 59 Rosenberg, E. B., 135, 138, 154, 177, 191 Ross, J., 55, 59 Ross, M. R., 44, 48 Ross, R. W., 119, 125, 126, 156 Rouiller, C., 98, 110 Rous, P., 96, 110, 308, 314, 323, 326, 327, Sal, ada, aba, d07 Roux, E., 226, 249 Rowe, W. P., 89, 110, 167, 193, 219, 223 Rowley, D., 160, 193 Rowntree, P. M., 75, 106 Rubin, H., 17, 19, 112, 114, 116, 126, 134, 136, 138, 139, 140, 155, 199, 224, 324, 367 Rudali, G., 362, 367 Ruska, H., 94, 110 Russeff, C. G., 167, 193 Sabin, A. B., 40, 41, 42, 47, 49, 50, 163, 164, 165, 167, 181, 193, 194, 226, 231, 232, 233, 235, 243, 249, 269, 271, 272, 273 Sagik, B. P., 23, 24, 29, 39, 48, 49, 198, 199, 223 Salk, J. E., 165, 193, 256, 261, 271, 272 Sampaio, A. A. de C., 36, 49 Sanders, F. K., 115, 117, 120, 151, 154, 155, 229, 249 404 Sanders, M., 164, 192 Sanford, K. K., 96, 110, 322, 367 Sapras, P., 70, 97, 107, 109 Sawai, Y., 171, 193 Sawin, P. B., 330, 367 Schaecter, M., 72, 110 Schafer, W., 17, 19, 23, 27, 49, 89, 106, 283, 306 Schafer: Wee 12116123. 1285 130, 13: 152, 153, 155, 156, 189, 194 Schaefer, W., 84, 85, 108, see also Shafer, W. Schaeffer, P., 189, 193 Schaffer, F. L., 208, 224 Scheer, J. D., 27, 47 Schillig, M. G., 349, 351, 356, 357, 360, 361, 365 Schindler, R., 167, 193 Schlesinger, R. W., 89, 110, 130, 131, 136, 144, 148, 151, 155, 156, 159, 163, 164, 165, 166, 167, 168, 169, 172, 179, 180, 181, 187, 188, 191, 193, 194, 226, 249 Schmidt, F., 360, 367 Schone, H. H., 55, 60 Schoolman, H. M., 362, 367 Schramm, G., 112, 154, 199, 224 Schreibel, W., 79, 108 Schulz, H., 70, 110 Schwartz, S. O., 362, 367 Schwerdt, C. E., 9, 14, 208, 224, 277, 306 Scott, T. F. McN., 80, 81, 106, 107, 118, 132, 137, 141, 155 Scott, T. M., 80, 110 Seidenberg, 8., 167, 168, 172, 190 Selbie, F. R., 164, 194, 329, 367 Seltsam, J. H., 36, 49 Selzer, G., 132, 155 Setlow, R. B., 175, 187, 193 Shafer, W., 276, 306 Shaffer, M. F., 118, 156 Shapras, P., 323, 367 Sharp, D. G., 23, 24, 28, 47, 49, 50, 326, 333, 364 Shaw, E. W., 101, 110 Shinkawa, E., 171, 193 Shiratori, T., 254, 272 Shope, R. E., 167, 192, 254, 255, 260, 277, 272, 303, 304, 305, 306, 327, 329, 335, 367 Shragg, R. L., 256, 272 AUTHOR INDEX Shunk, C. H., 206, 207, 208, 215, 216, 224 Siem, R. A., 201, 223 Sigel, M. M., 75, 107, 119, 120, 154 Siebs, W., 67, 79, 108 Silver, R. K., 55, 61 Simmons, N, 8., 349, 351, 366 Sjostrand, F. 8., 65, 110 Skinner, H. H., 270, 272 Slater, E. A., 201, 223 Smadel, J. E., 72, 110, 209, 216, 223 Smadel, J. G., 77, 108 Small, M. C., 349, 355, 356, 357, 359, 368 Smiles, J., 28, 47 Smith, C. E. G., 168, 194 Smith, H. H., 226, 249 Smith) J, Dr, 196) 2015 214 ipeelss 223 Smith, J. W., 7, 14 Smith, K. M., 372, 373, 374, 375, 376, 378, 379, 380, 381, 382, 383, 384, 386, 387, 388, 390, 391, 392 Smith, M. H. D., 303, 304, 306, 335, 336, 367 Smith, W., 27, 34, 37, 49, 237, 239, 247, 249, 254, 255, 260, 270, 272 Smith, W. E., 333, 367 Smithburn, K. C., 165, 168, 194, 269, 272 Snellbaker, L. F., 219, 223 Spiegelman, 8., 218, 223 Sprunt, D. H., 235, 249 Sprunt, K., 9, 14, 301, 306 Spurrier, W., 362, 367 Sribongse, 8., 43, 48 Stahmann, M. A., 200, 223 Stanley, N. F., 164, 194, 233, 249 Stanley, W. H., 308, 367 Stanley, W. M., 24, 25, 26, 27, 49 Stannard, C., 117, 137, 153 Steinhaus, E. A., 371, 378, 388, 391 Stent, G. S., 228, 249 Stevens, K. M., 149, 150, 753,178, 179, 180, 190 Stewart, F. S., 30, 49 Stewart, M. O., 234, 248 Stewart, S. E., 349, 354, 358, 360, 361, 365, 367, 368 Stoeckenius, W., 77, 109, 209, 216, 223 Stoker, M. G. P., 119, 125, 126, 156 Stoler, M. K., 92, 110 AUTHOR INDEX Stone, J. D., 17, 19, 25, 26, 29, 30, 32, 33, 34, 35, 36, 37, 39, 44, 45, 46, 47, 49, 52, 53, 56, 58, 59, 61, 201, 222, 224, 231, 232, 238, 247, 249, 298, 306 Straub, M., 239, 249 Straus, M. J., 101, 106, 110 Strickland, A. G. B., 18, 19, 199, 200, 222 Strosselli, E., 69, 170 Stuart, D. C., Jr., 94, 110 Stuart-Harris, C. H., 35, 36, 46, 49, 83, 110, 260, 262, 271, 272, 282, 286, 290, 306 Stulberg, C. 8., 167, 191 Sugai, T., 181, 193 Sugg, J. Y., 25, 49, 174, 194, 231, 238, 239, 248, 249, 261, 266, 272 Sulkin, S. E., 164, 172, 194, 240, 249 Suriano, O. C., 256, 27. Svec, F. A., 37, 49 Svedmyr, A., 89, 90, 108, 145, 154, 156, 178, 180, 791 Swain, R. H. A., 37, 49, 74, 110 Sweet, B. H., 40, 41, 50, 165, 194, 269, 273 Syverton, J. T., 219, 222, 234, 249, 327, 328, 331, 368 Szanto, P. B., 362, 367 Tajima, M., 74, 110 Takatsy, G., 258, 259, 273 Takemori, N., 233, 249 Tamm, I., 35, 36, 38, 43, 29, 50, 53, 61, 78, 109, 118, 121, 123, 137, 141, 146, 155, 156, 187, 194, 196, 197, 199, 200, 202, 203, 205, 206, 207, 208, 209, 210, 211, DiI, DABS OEE OA ay PACs PATI Pale AA 0) DP Beaty pee Tanada, Y., 382, 390, 392 Tang, F. F., 24, 47 Taniguchi, H., 124, 125, 156 Taylor, A., 164, 194 Taylor, A. R., 23, 24, 28, 47, 49, 50 Taylor, C. E., 181, 194 Taylor, R. M., 239, 249 Teague, O., 80, 107 Temin, H., 234, 249 Tenbroeck, C., 242, 249 Tenenbaum, E., 96, 107, 110, 322, 366 Theiler, M., 40, 42, 47, 50, 168, 194, 226, 242, 249 Thigpen, M. P., 260, 27. 405 Thomsen, O., 30, 50 Thomson, H. P., 99, 109 Thompson, R. L., 206, 215, 217, 218, 224 Thorson, K. G., 89, 90, 108 Timofejewsky, A. D., 71, 110 Tobin, B. M., 117, 132, 155 Tobin, J. O’'H., 118, 132, 155 Tokuyasu, K., 382, 392 Tolmach, L. J., 198, 201, 224 Touisimis, A. J., 91, 110 Traub, E., 310, 368 Tsujimoto, N., 181, 193 Tsujita, M., 376, 392 Turner, G. S., 132, 156 Tyrrell, D. A. J., 23, 27, 36, 43, 50, 86, 170, 121, 123, 136, 146, 156, 187, 194, 200, 203, 205, 206, 207, 212, 213, 224, 268, 273 Uhler, M., 145, 156 Ullmann, S., 283, 306 Underwood, G. E., 201, 223 Upton, E., 24, 26, 48, 50 Vaisman, A., 165, 192, 235, 248 Valentine, R. C., 23, 50, 85, 91, 110, 136, 143, 146, 156, 160, 176, 183, 185, 192 van den Ende, M., 132, 156, 292, 306 van der Veen, J., 256, 261, 268, 273 van Ravenswaay, T., 89, 90, 108 van Rooyen, C. E., 101, 110, 188, 191 von Magnus, H., 3, 14 von Magnus, P., 129, 130, 131, 144, 145, 146, 147, 154, 156, 178, 179, 180, 1917, 194 van Tongeren, H. A. E., 236, 249, 299, 306 Vellisto, I., 99, 100, 105 Verlinde, J. D., 43, 50 Vigier, P., 97, 106 Vilches, A., 162, 163, 170, 172, 188, 194 Viriden, P., 5, 14 Visser, D. W., 218, 223 Vogell, W., 68, 110 Vogt, M., 18, 19, 93, 109, 126, 127, 133, 134, 135, 139, 142, 154, 155, 199, 200, RNG, QUES Bee, as), OVAL). 2B. BBB PBL 243, 247, 249, 302, 306 von Prowazek, S8., 371, 392 Wagley, P. F., 31, 49 406 Wagner, J. C., 164, 171, 191 Wagener, R. R., 16, 19, 159, 194 Walker, D. L., 159, 192 Walker, J., 143, 153 Wallis, C., 164, 172, 194 Wang, C., 238, 239, 249 Wang, C. I., 266, 273 Ward, J. P., 100, 108 Ward, T. G., 42, 43, 48, 89, 108 Warner, S. G., 342, 367 Wasser, H. B., 379, 383, 392 Waters, N. F., 309, 318, 368 Watson, B. K., 87, 88, 109, 110, 200, 224 Watson, J. D., 12, 14, 301, 305 Watson, M. L., 78, 110 Webster, L. T., 235, 249 Wecker, E., 17, 19, 112, 152, 156, 189, 194, 276, 306 Weigle, J. J., 228, 247 Weil, M. L., 28, 47 Weiss, D. L., 256, 272 Weiss, E., 4, 14, 75, 107, 110 Weller, T. H., 141, 156, 232, 234, 235, 248, 249 Wenner, A. J., 302, 306 Wenner, H. A., 243, 249 Werner, G. H., 89, 110, 131, 144, 156 Werner, I., 54, 55, 59, 61 Werner, J. A., 89, 108 Westwood, J. C. N., 27, 34, 37, 49, 237, 249 Weygand, F., 55, 60 Whalen, M. M., 372, 374, 376, 377, 391 White, C. L., 326, 364 Whitman, L., 242, 249 Whitney, E., 75, 106, 164, 190 Wiener, F. P., 203, 210, 212, 213, 223 Wiener, M., 129, 156 Wildy, P., 118, 120, 125, 137, 141, 156, 237, 249 AUTHOR INDEX Wilkin, M. L., 217, 224 Wilkinson, J. F., 37, 49 Williams, R. C., 114, 155, 380, 383, 391, 892 Williams, S. E., 25, 46 Wilson, E., 101, 107 Winslow, N. S., 24, 26, 48, 50 Winsser, J., 94, 110, 181, 193, 231, 232, 249 Winter, J. W., 163, 165, 169, 193, 194 Winzler, R. J., 215, 218, 223, 224 Wissig, 8S. L., 72, 110 Wolff, H., 86, 110 Wood, M. T., 326, 364 Woodie, J., 163, 181, 193 Woodroofe, G. M., 244, 248 Woolley, D. W., 37, 48, 50, 349, 355, 356, 357, 359, 368 Wolman, M., 72, 81, 110 Wyckoff, R. W. G., 77, 79, 80, 81, 83, 84, 85, 107, 109, 142, 155, 334, 364, 375, 376, 383, 384, 390, 391, 392 Xeros, N., 372, 373, 374, 375, 376; 379; 381, 384, 385, 386, 387, 390, 391, 392 Yager, R. H., 43, 48, 49 Yamakawa, T., 56, 6/ Yoshino, K., 124, 125, 156 Young, R., 361, 365 Younger, J. 8., 18, 19 Yu Cr be. 60 Zahler, 8. A., 3, 14 Ziegler, J. E., Jr., 162, 173, 174, 175, 176, 177, 183, 194 Zillig, W., 23, 27, 49, 283, 306 Zilliken, F., 55, 61 Subject AMPS, see a-amino-p-methoxyphenyl- methane sulfonic acid Abraxas grossulariara, nuclear polyhedral virus and, 381 Absorption, antibody, influenza A virus and, 258-260 N - Acetyl - O - diacetylmeuraminic acid, bovine submaxillary gland mucoprotein and, 56 N-Acetylglucosamine bovine submaxillary gland mucoprotein and, 57 hemagglutination inhibition and, 53 N-Acetylneuraminic acid chemical synthesis of, 54-55 human erythrocytes, receptor destroy- ing enzyme and, 55-56 urine mucoprotein and, 54 6-a-D-N-Acetylneuraminyl-N-acetylgalac- tosamine bovine submaxillary gland mucoprotein and, 57 receptor-destroying enzyme and, 57 Achromobacter xerosis, 220, see also Xerosin Adaptaticn influenza virus hamster lung and, 237-240 mouse lung and, 237-240 new hosts and, 265-267 Rous sarcoma virus and, 319-320 Adenoviruses, 3 bacteriophage and, 13 crystalline aggregates and, 5, 9 deoxyribonucleic acid and, 12 release of, 141, 142 tissue culture, HeLa cells and, 5 monkey kidney and, 5 Adenocarcinoma, frog and, 101-102, 103, 105 Adenosine, nucleolus effect and, 67, 79 Adenoviruses, 65, 103, 252, 270 cytochemistry, 89-90; deoxyribonucleic acid and, 90, 91 electron microscopy of, 90-91 HeLa cells and, 89, 90, 91 Adsorption Index allantoic cavity and, 15, 16, 17 guinea pig leucocytes, receptor-destroy- ing enzyme and, 17 influenza virus, respiratory cells and, 52 viral eclipse phase and, 16 African horse sickness virus, intracellular soluble antigens and, 132 Agglutination, see Hemagglutination Aglais urticae, nuclear polyhedral virus and, 388 Agratis segetum, granulosis virus and, 378 Allantoic cavity, viral adsorption and, 15, 1617 Amino acids, deficiency, pathologic cells and, 71 a-Amino-p-methoxyphenylmethanesul- fonic acid infection prevention and, 201 influenza virus release inhibition and, 140, 217 Antheraea mylitta, cytoplasmic polyhedral virus and, 382 Antibiotics, psittacosis virus and, 75 Antibody absorption, influenza A virus and, 258- 260 fluorescent, viral antigens and, 64, 75, 78, 82, 83, 87-89, 94, 103, 133, 152 neutralizing, infection prevention and, 199-200 Antigen, see also Soluble antigen complement-fixing, occurrence in viruses and, 5 fowl plague virus, properties of, 128-129 influenza virus, lag period and, 120-122, 123 phenotypic mixtures, enteroviruses and, 3 Antihemagglutination, specific antibodies, influenza viruses and, 34-35 Antimycin A, influenza virus and, 219 Arboviruses, 3, 8, 10-11, 13, 252 coexistence and, 237 hemagglutination and, 22, 40-42 ribonucleic acid and, 12 serological groups and, 6 407 408 Arboviruses serological variation, strain relationships and, 269 successive exposure and, 269-270 structure and, 12-13 Arctia caja, cytoplasmic polyhedral virus and, 382 Arthropod-borne viruses, see Arboviruses Autointerference definition of, 168 dengue encephalitis virus and, 168, 169- 170 egg-adapted influenza virus and, 168 incomplete influenza virus and, 168-169, 178-179 influenza B virus and, 168, 181 rabies virus and, 168 Rift Valley fever virus and, 168, 169 vesicular stomatitis virus and, 168, 181 western equine encephalomyelitis virus and, 168, 181 yellow fever virus and, 168 Avian. pox viruses Gymnorhina and, 4 Avian viruses, see also individual viruses, infectious laryngotracheitis and, 7 B(simiae) virus, see Herpesvirus Bacillus megatherium, vaccinia virus and, 13-14 Bacteriophage deoxribonucleic acid and, 13, 51 Escherichia coli B and, 51 lag period, recoverable infective virus and, 113-114 virulence variation, mechanisms of, 228 Banyamwera encephalitis virus, hemag- glutination and, 40 Bee paralysis virus, 370 Benzimidazole poliovirus and, 215, 219 Theiler’s virus and, 215 vaccinia virus and, 215 Boerlage fibroma virus, transformation and, 304 Bombyx mori cytoplasmic polyhedral virus and, 376, 389 nuclear polyhedral virus and, 370-372, 373, 374, 380, 381, 389 Borna disease virus, infection spread, hyaluronidase and, 235 SUBJECT INDEX Bovine salivary mucin, 54 Bovine submaxillary gland mucoprotein, see also Mucoprotein 6-%-D-N-acetylneuraminyl-N-acetyl- galactosamine and, 57 LEE influenza indicator virus and, 56 N-acetylglucosamine and, 57 N-acetyl-o-diacetylneuraminic acid and, 56 N-glycolylIneuraminic acid and, 56 ON-diacetylneuraminic acid and, 55, 56 PR8 influenza indicator virus and, 56 prosthetic groups of, 56-57 receptor-destroying enzyme and, 56 Bronchitis virus, 200 inactivated infectious bronchitis virus, interference between, 171 Brucella suis, 8, 10, host protection and, 160 Bwamba virus, influenza virus, inter- ference between, 162 Cacoecia murinana, granulosis virus and, 378 Caprochlorone, see levo-y-(o-chlorobenzyl)- §-oxo-y-phenyl caproic acid Canary pox, malignant smallpox and, 4 Canine distemper virus, 7 Carcinoma Lucké, frog and, 65 human epidermoid Egypt 101 encephalitis virus and, 95 vaccinia virus and, 78 Cell damage, modification of, 219-220 interfering virus, association of, 186-187 normal mitochondria and, 67 morphology of, 65-68 nucleus and, 67 osmotic changes and, 68 pinocytosis and, 67 secretion and, 67-68 tissue culture and, 66, 67 nuclei, human papillomas and, 101, 105 pathologic amino acid deficiencies and, 71 cell surfaces and, 69 cytoplasmic degenerative changes and, 71-72 SUBJECT INDEX Cell pathologic giant cells and, 70 hypertrophied cells and, 70 mitochondria and, 70 nuclear degenerative changes and, 71- 72 osmotic imbalance and, 69-70 paranuclear hypertrophy and, 70 respiratory, influenza virus adsorption and, 52 Cell surface virus infection and, 13 Chicken embryo heart cells Rickettsia burneti and, 72 tissue culture and, 71 embryo liver cells, herpesvirus and, 81 fibroblasts Rickettsia burneti and, 7 tissue culture and, 69 lung epithelium, psittacosis virus and, 72-73 Rous sarcoma virus, 65, 96, 103, 105 electron microscopy and, 97-98 tissue culture and, 96-97 virus localization and, 98-99 Chicken erythroblastosis virus, 311-312 Chicken lymphomatosis virus, types of, 310, 312 Chicken myeloblastosis virus, 311-312 Chlamydozoaceae, see Meningopneumon- itis virus and Psittacosis virus 5-Chlorouridine, Theiler’s virus and, 218 Choristoneura fumiferana, cytoplasmic polyhedral virus and, 376 Chu inhibitor antihemagglutination chemical properties of, 35-36 physical properties of, 835-36 relation to virus and, 35 Cirphisunipuncta, insect virus and, 379, 383 Clostridium histolyticum, 42 Clostridium lentoputrescens, 42 Clostridium perfringens, 41 Clostridium welchii, receptor-destroying enzyme of, 32 Coenzyme A, 6-mercaptopurine, mitochon- dria and, 67 Colias philodice eurytheme, nuclear poly- hedral virus and, 373-374 409 Columbia MM virus, hemagglutination and, 43 Columbia SK virus hemagglutination and, 43 CO,-sensitivity virus, Drosophila and, 234 Cowpox virus, 4, 77, 225-226, 251, 299 pock variants and, 236 Coxiella burnetii, 8 Coxsackie virus, 6, 320 hemagglutination and, 43 poliovirus, interference between, 167, 172 Cycnia mendica, nuclear polyhedral virus and, 387 8 forms, human influenza A virus and, 25 DNA, see Deoxyribonucleic acid D phase, human influenza A virus and, 25, 231-232 DRB, see Ribosyl-benzimidazole deriva- tive DSP virus, hemagglutination and, 27 Deficiencies, amino acids, pathologic cells and, 71 “Degenerative” changes cytoplasmic, pathologic cells and, 71-72 nuclear pathological cells and, 71-72 Dengue encephalitis virus arboviruses and, 6 autointerference and, 168, 169-170 avirulent dengue encephalitis virus, interference between, 167 hemagglutination and, 40, 41 heterotypic dengue encephalitis virus, interference between, 163, 165-166 mice, adaptation and, 226 yellow fever virus immunogenicity suppression and, 161 interference between, 163 Deoxyribonucleic acid, 86-87, 141, 153, 209, 216, 283, 304 adenoviruses and, 12, 90, 91 bacteriophage and, 51 pathologic cell, nuclear degeneration and, 71-72 psittacosis viruses and, 3, 4, 12 vaccinia virus and, 4, 8, 12 Development sequence, fowlpox and, 79-80 ON-Diacetylneuraminic acid, bovine sub- maxillary gland mucoprotein and, 55, 56 410 2, 6-Diaminopurine poliovirus and, 218, 219 Russian spring-summer virus and, 218 vaccinia virus and, 218 Diataraxia olexacea, cytoplasmic poly- hedral virus and, 376 5, 6-Dichloro-l-«-D-arabinopyrano- sylbenzimidazole influenza virus and, 211, 216 poliovirus and, 203, 210-211, 213, 216 5, 6-Dichloro-1-B - p - ribofuranosyl- benzimidazole influenza A virus and, 208, 216 influenza B virus and, 203, 206-209, 212, 216 mumps virus and, 208 poliovirus and, 203, 208, 212, 216 5, 6-diethylbenzimidazole, influenza B virus and, 215 2, 5-dimethylbenzimidazole influenza A virus and, 205, 219 influenza B virus and, 203, 205, 212, 213, 219 Diprion hercyniae, virus and, 373 Distemper virus egg-adapted distemper ference between, 167 ferret-adapted distemper virus, inter- ference between, 167 Drosophila, CO.-sensitivity virus and, 237 Ducks, Rous sarcoma virus and, 320 encephalitis nuclear polyhedral virus, inter- EMC, see Encephalomyocarditis viruses Eastern equine encephalomyelitis virus, 112 cytological studies and, 95 hemagglutination and, 42 infective nucleic acid and, 276 ribonucleic acid, infection initiation and, 152-153 ECHO virus, 6, 252, 270 hemagglutination and, 43 Newcastle disease virus, between, 163 Eclipse phase definition of, 112 fowl plague virus and, 16, 17 influenza viruses and, 16 interference SUBJECT INDEX Newcastle disease virus and, 16, 17 polioviruses and, 16, 18 viral reproduction, adsorption and, 16 attached virus liberation and, 16 electron microscopy and, 16 immune serum and, 16 inactivation and, 16 inhibitors and, 16 metaperiodate ion and, 16, 17 receptor-destroying enzyme and, 16 Ectromelia virus inactivated, ectromelia virus, ference between, 171 mice, sequential infection and, 240-242 Egypt 101 encephalomyelitis virus, fluor- escent antibody study and, 95 Ehrlich ascites cells, ectromelia virus, elementary bodies and, 77 Ectromelia virus, 4, 51, 76, 77, 80, 299 Ehrlich ascites cells, elementary bodies and, 77 Ehrlich ascites tumor, Newcastle disease virus and, 16 Electron microscopy, 64 adenoviruses and, 90-91 ballooned cytoplasm and, 69 cellular fluid localization and, 68 fowl plague virus hemagglutinin and, 130, 131 frog adenocarcinoma and, 102 herpesvirus and, 81 “imcomplete” virus and, 89 myxovirus and, 83, 84, 87, 89 normal cell morphology and, 65 nuclear polyhedral virus, Bombyx mori and, 371 pinocytosis and, 67 psittacosis virus elementary bodies and, 74 Rickettsia moosert, yolk sac and, 72 Rous’ chicken sarcoma virus and, 97-98 Shope fibroma and, 80, 102, 105 vaccinia virus and, 77-78 viral eclipse phase and, 16 Elementary bodies, 128, 129 ectromelia virus, Ehrlich ascites cells and, 77 psittacosis virus and, 73-75 vaccinia virus and, 76, 77 inter- SUBJECT INDEX Elution influenza virus and, 29 Newcastle disease virus and, 29 receptor destruction and, 29 stabilized cell production and, 28-29 properties and, 29-31 Embryo heart, chick, tissue culture and, 71 Encephalitide viruses, see also individual viruses hemagglutination cells agglutinated and, 40 hemagglutinin inhibition and, 41-42 hemagglutinin preparation and, 40 history of, 40 hydrogen ion concentration and, 41 serological grouping and, 42 virus particle and, 41 Encephalitis, 65, 95 Encephalomyocarditis virus, 6, 112 equine encephalomyelitis virus, inter- ference between, 164, 181 hemagglutination and, 22, 43 host-induced. variation and, 229 incomplete virus and, 151 lag period recoverable infective virus, and, 115, 120 poliovirus, interference between, 164 Enders’ strain, mumps virus, hemagglu- tination and, 28 Enteric cytopathogenic human orphan, see ECHO viruses Enteroviruses, 6-7, 9, 15, 18 antigens, phenotypic mixtures and, 9 hemagglutination and, 42-43 ribonucleic acid and, 12 Epithelium chick, lung, psittacosis virus and, 72-73 ferret nasal, influenza virus and, 69 mouse bronchial, influenza virus and, 87 rabbit corneal, vaccinia virus and, 76 Equine encephalomyelitis virus, 188 encephalomyocarditis virus, interference between, 164, 181 heterotypic equine encephalomyelitis virus interference between, 163, 166 influenza virus, interference between, 162, 170-171, 181, 184 Japanese B encephalitis virus, inter- ference between, 163 mumps virus, interference between, 163 411 Newcastle disease virus, interference between, 162, 163, 181, 184 St. Louis encephalitis virus, interference between, 163, 172, 181 Theiler’s virus, interference between, 167, 172, 186 vesicular stomatitis virus, interference between, 163 Erithacus rubecula, polyhedral virus and, 390 Erythrocytes attachment prevention influenza virus and, 198, 199, 200 mouse pneumonia virus and, 198, 199 cat, LEE influenza virus and, 38 cow, Newcastle disease virus and, 24 fowl fowl plague virus and, 22 influenza virus and, 22, 25, 52, 230 Japanese B encephalitis virus and, 40 LEE influenza indicator virus and, 34 MEL influenza indicator virus and, 34 mumps virus and, 22, 25 Newcastle disease virus and, 22, 28 West Nile encephalitis virus and, 40 vaccinia virus and, 44-45 guinea pig influenza A virus and, 25, 230 myxovirus and, 24 hamster, mouse pneumonia virus and, 44 horse mumps virus and, 33 Newcastle disease virus and, 33 N-glycolyIneuraminic acid and, 56 human human influenza A virus and, 25 mumps virus and, 28 myxovirus and, 24 N-acetylneuraminic acid and, 55-56 Newcastle disease virus and, 28 mouse meningopneumonitis virus and, 44 pneumonia virus and, 44 ox, Mumps virus and, 33 pigeon influenza virus and, 25 Japanese B encephalitis virus and, 40 Murray Valley encephalitis virus and, 40 vaccinia virus and, 44-45 412 Erythrocytes sheep Newcastle disease virus and, 24 Russian far eastern encephalitis virus and, 40 West Nile encephalitis virus and, 70 Escherichia coli B, 158 T bacteriophage and, 51 pDL-Ethionine influenza virus and, 217 poliovirus and, 217, 219 B-Ethoxy-«-ketobutyra!dehyde infection prevention and, 201 2-Ethyl-5-methylbenzimidazole, influenza B virus and, 215 Euplexia lucipara, granulosis virus and, 378 Euvanessa antiopa, nuclear polyhedral virus and, 375 Euxoa segetum, granulosis virus and, 378 Evolution, animal viruses and, 10-11, 229 hydrate, Far eastern encephalitis virus, hemag- glutination and, 40 Feline pneumonitis virus, hemagglutina- tion and, 44 Ferret mucosa, influenza virus and, 87 nasal epithelium, influenza virus and, 69 Fibroblasts chick Rous’ sarcoma virus and, 234 tissue culture and, 69 human, lymphopathia veneria virus and, 73 rat eastern equine encephalomyelitis virus and, 95 Rickettsia rickettsit infection and, 72 Rickettsia tsutsugamushi infection and, 72 roller tube cultures, and, 237 Fibroma virus rabbits, 4 myxoma virus and, 335-336 squirrel virus and, 335 squirrels and, 4 p-Fluorophenylalanine poliovirus and, 203, 209, 218, 220 vaccinia virus SUBJECT INDEX Theiler’s virus and, 218 Foot-and-mouth disease virus, 65, 251 enteroviruses and, 6-7, 9 intracellular soluble antigens and, 132 vaccinia virus, interference between, 167 Fowl] leunemia, see Visceral lymphomatosis virus Fowl] plague virus, 83 cellular receptors and, 51 eclipse phase and, 16, 17 filament formation and, 85 fluorescent antibody studies and, 88-89, 133 hemagglutination and, 22, 23, 130 lag period radioactive labelling studies and, 152 recoverable infective virus and, 116, 120235125 nuclear masses and, 86 myxovirus and, 5, 8 soluble antigen and, 128-129 Fow!pox virus, 4 cell infection and, 67 chorioallantoic cells, nucleous effect and, 79 development sequence and, 79-80 inclusion and, 79 vaccinia virus, interference between, 164 Friedlander bacillus, see also Klebsiella pneumoniae type B,; mumps virus antihemagglutina- tion and, 35, 37 Frog adenocarcinoma and, 101-102, 103, 105 Lucké’s carcinoma and, 65 Genetic interaction, types of, 276-277 Genetic recombination, see also Recom- bination influenza virus and, 229 vaccinia virus and, 229-230 virulence variation and, 229-230 N-GlycolyIneuraminie acid, bovine sub- maxillary gland mucoprotein and, 56 “Golgi apparatus’, 65, see also Paranuclear hypertrophy Granulosis viruses insects Agratis segetum and, 378 Cacoecia murinana and, 378 SUBJECT INDEX Granulosis viruses insects Euplexia lucipara and, 378 Euxoa segetum and, 378 Melanchra persicareae and, 378 morphology of, 382-383 Natada nararia and, 378, 382 Peridroma margoritosa and, 378 Pieris brassicae and, 378, 379 Pieris rapae and, 378, 382 Guinea pig, leucocytes, viral adsorption, and, 17 Gymnorhina sp., avian pox viruses and, 4 Hamster lung, influenza virus, adaptation and, 237-240 HeLa cells adenoviruses and, 89, 90, 91 fowl plague virus hemagglutinin and, 131 herpes simplex virus and, 119 influenza virus hemagglutinin and, 130- 131 Newcastle disease virus and, 181-182 poliovirus and, 93, 209, 218, 219, 220 Hemagglutination arboviruses and, 22 DSP virus and, 27 encephalitide viruses Bunyamwera and, 40 dengue 1 and 2 and, 40, 41, 42 eastern equine and, 42 far eastern and, 40 Tiheus and, 40, 42 Japanese B and, 38, 40, 41, 42 louping ill and, 40 Mayaro and, 42 Murray Valley and, 40, 42 Ntaya and, 40, 42 Russian far eastern and, 40, 42 Semliki Forest and, 40 42 Sindbis and, 42 St. Louis and, 40, 41, 42 Uganda S and, 42 Venezuelan equine and, 42 western equine and, 40, 41, 42 West Nile and, 40, 41, 42 yellow fever (Asibi) and, 40, 42 Zika and, 42 413 enteroviruses Columbia MM and, 43 Columbia SK and, 43 Coxsackie B3 and, 43 ECHO and, 43 encephalomyocarditis and, 43 6D VII poliovirus and, 42-43 Mengo encephalitis and, 43 fowl] plague virus and, 22, 23 GD VII poliovirus and, 22 Jan D virus and, 25 Jan O virus and, 25 “indicator” virus and, 26, 33-34 influenza A virus and, 23, 25, 26, 230 influenza B virus and, 25 influenza virus and, 22, 24, 25, 28 inhibition genetic marker and, 279 influenza A virus and, 256-258 phase influenza viruses and, 268 inhibitors arboviruses and, 41-42 Chu and, 35-36 mucoid and, 33-34, 36-37, 52, 53 specific antibody and, 34-35 LEE influenza virus and, 24, 26 mechanism enzyme substrate attraction and, 39 physical adsorption and, 38-39 “Melbourne egg” virus and, 25 MEL influenza virus and, 26 mouse pneumonia virus and, 22, 43-44 mumps virus and, 22, 25, 26-27, 28, 31 myxovirus aging and, 26-27 alcohol and, 27 alum and, 27 ether and, 27 formaldehyde and, 26-27, 28 glycerol and, 27 heat and, 26, 27 hemagglutinin titer and, 28 history of, 22 hydrogen-ion concentration and, 26, Pal ionic environment and, 24, 25 periodate ion and, 26, 27-28, 33 red cell species and, 24-25 temperature and, 24 trypsin and, 26 414 Hemagglutination myxovirus ultraviolet irradiation and, 25, 26 urea and, 27 virus particle and, 22-24 Newcastle disease virus, 22, 23, 24, 26, 27 infectious mononucleosis and, 31 Victorian strain of, 28 NWS influenza virus and, 26, 27 poxviruses, 22 neuro-vaccinia and, 45 rabbitpox and, 45 vaccinia and, 44-45 PR8& influenza virus and, 24, 26, 27 psittacosis group, 22 feline pneumonitis and, 44 mouse meningopneumonitis and, 44 psittacosis and, 44 receptor gradient and, 29-30 St. Louis encephalitis virus and, 40, 41 thermal inactivation of virus and, 26 vaccinia virus and, 44-45 western equine encephalitis virus and, 40, 41 West Nile encephalitis virus and, 40, 41 WS influenza A virus and, 25 WSM influenza virus and, 26 yellow fever virus and, 40 Zika encephalitis virus and, 42 Hemagglutinin, myxoviruses and, 129-132 Hepatitis infectious, 7 serum, 7 Herpangina, 9 Herpes B virus cellular changes and, 81-82 release of, 14-1 Herpes simplex virus, 113 fluorescent antibody straining and, 133 growth of, 137 intracellular soluble antigens and, 132 lag period, recoverable infective virus and, 116, 118-119, 120, 123, 124, 126 nonencephalitogenic herpes simplex virus, interference between, 167, 168 Herpesvirus, 5, 65, 72 electron microscopy of, 81 fluorescent antibody straining and, 82 histochemistry of, 81 SUBJECT INDEX infection spread, hyaluronidase and, 235 intranuclear inclusion and, 80 pock variants and, 237 release of, 141 Heterozygotes, 294, 295 influenza A virus MEL influenza A virus NSWE and, 287 influenza A virus WSN and, 287-288 viruses, definition of, 276-277 Hyaluronidase infection spread Borna disease virus and, 235 herpesvirus and, 235 vaccinia virus and, 235 vesicular stomatitis virus and, 235 Hydrocortisone, Rous sarcoma virus and, 317 5-Hydroxyuridine, Theiler’s virus and, 218 Jan D virus, hemagglutination and, 25 Jan O virus, hemagglutination and, 25 Iiheus encephalitis virus, hemagglutina- tion and Inclusion fowlpox and, 79 intranuclear, herpesvirus and, 80 Incomplete virus, 130, 131, 143-144, 267 definition of, 129 electron microscopy of, 89 encephalomyocarditis virus and, 151 fluorescent antibody studies and, 88-89 influenza autointerference and, 168-169, 178- ge) interfering capacity of, 179-181 partial cycle and, 149-150 production of, 146-149 properties of, 144-146 Rift Valley fever virus and, 151 vesicular stomatitis virus and, 151 Indicator virus definition of, 53 hemagglutination and, 26, 33-34 LEE influenza virus and, 33, 34 MEL influenza virus and, 34 mucoid inhibitors and, 33-34, 52 PR8 influenza virus and, 34 receptor-destroying enzyme and, 34 receptor gradient and, 34 WSE influenza virus and, 24 SUBJECT INDEX Infections bacterial, cultured cells and, 72 initiation and, 15-18 rickettsial, cultured cells and, 72 viral, mitosis and, 68 Influenza A virus, 126-127, 200, 201, 254- 265 age distribution and, 261-263 %-amino-p-methoxyphenylmethane- sulfonic acid, release inhibition and, 217 cellular receptors and, 51 5 forms and, 25 D phase and, 25, 231-232 5, 6-dichloro-l-8-p-ribofuranosylbenl- zimidazole and, 208 2, 5-dimethylbenzimidazole and, 205 hemagglutination and, 23, 25, 26, 230 host-induced variation and, 229 influenza B virus, pure clone isolations and, 278 levo-y-(0-chlorobenzyl)-6-oxoy-phenyl- caproic acid and, 203, 210, 213 DL-methoxinine and, 203, 205-206, 212, 213, 217 O phase and, 25, 231-232 ovomucin hemagglutination inhibition and, 53 w forms and, Influenza A virus Alabama, 258 Influenza A virus Asian, 263, 264. Influenza A virus BEL Chu inhibitor and, 35 receptor gradient and, 29 stabilized cells, electrophoretic mobility and, 30 Influenza A virus BEL D, Chu inhibitor and, 35 Influenza A virus CAM, 159, 173, 256, 266 influenza A virus WSE, linkage group interchange and, 285 mouse lung virulence redistribution and, 291, 292 Influenza A virus FMI, 173, 208, 256, 257, 258, 264 Influenza A virus Hume, 257 Influenza A virus KUNZ, neuropatho- genicity transfer and, 290 Influenza A virus MEL, 173, 174, 175, 176, 177, 258, 260 415 hemagglutination and, 26 indicator virus and, 34 influenza A virus NWSE, heterozygosis and, 287 influenza A virus WSE genetic interaction and, 283, 284-285 linkage group interchange and, 285 phenotypic mixture and, 286, 294-295 influenza A virus WSN heterozygosis and, 287-288 phenotypic mixture and, 286-287 mouse lung virulence redistribution and, 291, 292 mucoid inhibitor and, 37 neuropathogenicity transfer and, 290 receptor gradient and, 29 stabilized cells, electrophoretic mobility and, 30 Influenza A virus NWS, 17, 175 influenza virus SW, genetic interaction and, 282-283 Influence A virus NWSE influenza A virus MEL, heterozygosis and, 287 Influenza A virus Oc.I, neuropathogenicity transfer and, 290 Influence A virus PER, mouse lung virulence redistribution and, 291-292 Influenza A virus PR8, 174, 177, 184, 208, 255, 256, 257, 258, 259, 260, 261, 262, 264, 266, 267 bovine submaxillary gland mucoprotein and, 56 fluorescent antibody studies and, 88 hemagglutination and, 24, 26, 27 hemagglutinin of, 130 indicator virus, 34 mucoid inhibitor and, 37 stabilized cells, electrophoretic mobility and, 30 Influenza A virus SW, influenza virus NWS, genetic interaction and, 282-283 Influenza A virus Talmey, 258 Influenza A virus WS, 167, 173, 177, 255, 256, 257, 258, 259, 260, 261, 284 bronchial epithelial cells deoxyribonucleic acid and, 86-87 glycoprotein and, 86 genetic interaction and, 283 hemagglutination and, 25, 26 416 Influenza A virus WS hemagglutination NWS substrain and, 26, 27 WSM substrain and, 26 neuropathogenicity transfer KUNZ influenza virus and, 290 MEL influenza virus and, 290 Oc.I influenza virus and, 290 swine 15 influenza virus and, 290 WSEH influenza virus and, 290 WSM influenza virus and, 290 receptor gradient and, 29 viral eclipse phase and, 16 Influenza A virus WSE indicator virus and, 34 influenza A virus CAM, linkage group interchange and, 285 influenza A virus MEL genetic interaction and, 283, 284-285 linkage group interchange and, 285 phenotypic mixture and, 286, 294-295 mouse lung virulence redistribution and, 291, 292 mucoid inhibition and, 37 neuropathogenicity transfer and, 290 Influenza A virus WSM, neuropatho- genicity transfer and, 290 Influenza A virus WSN influenza A virus MEL heterozygosis and, 287-288 phenotypic mixture and, 286-287 influenza A virus Wr, linkage group interchange and, 285 Influenza A virus Wr influenza A virus WSN, linkage group interchange and, 285 Influenza B virus autointerference and, 168, 181 cellular receptors and, 51 5, 6-dichloro-1-8-p-ribofuranosylbenzi- midazole and, 203, 206-209, 212 5, 6-diethylbenzimidazole and, 215 2, 5-dimethylbenzimidazole and, 203, 205, 212, 213 2-ethyl-5-methylbenzimidazole and, 215 hemagglutination and, 25 influenza A virus, pure clone isolations and, 278 2, 4, 5, 6, 7-pentamethylbenzimidazole and, 215 SUBJECT INDEX receptor gradient and, 29 serological variation in, 265 urine mucoprotein, hemagglutination inhibition and, 53, 57 Influenza B virus GL, 265 Influenza B virus LEE, 174, 175, 176, 185, 208, 265 bovine submaxillary gland mucoprotein and, 56 fluorescent antibody studies and, 88 hemagglutination and, 24, 26 indicator virus and, 33, 34 mouse lung virulence redistribution and, 292, 293 receptor gradient and, 29 stabilized cells, electrophoretic mobility and, 30 Influenza B virus MB, 208 Influenza B virus MIL mouse lung virulence redistribution and, 292, 293 receptor gradient and, 29 stabilized cells, electrophoretic mobility and, 30 Influenza B virus ROB, mouse lung virulence redistribution and, 292 Influenza C virus, 254 cellular receptors and, 51 Influenza D virus, 254. cellular receptors and, 51 Influenza E virus, 254 Influenza virus, 15, 83, 103, 113, 183, 200, 201, 221, adsorption respiratory cells and, 52 antimycin A and, 219 Bwamba virus, interference between, 162 5, 6-Dichloro-l-«-D-arabinopyranosyl- benzimidazole and, 211, 216 eclipse phase and, 16, 18 egg-adapted influenza virus WS, inter- ference between, 167, 173 elution of, 29 equine encephalomyelitis virus, inter- ference between, 162, 170-171, 181, 184 erythrocytes, attachment prevention and, 198, 199, 200 DL-ethionine and, 217 ferret mucosa and, 87 SUBJECT INDEX 5, 6-Dichloro-l-«-D-ara binopyranosyl- benzimidazole and ferret nasal epithelium and, 69 filament formation and, 85 genetic recombination and, 229 growth of, 135-136 hamster lung, adaptation and, 237-240 hemagglutination and, 22, 24, 25, 28 hemagglutinin of, 131 heterotypic influenza virus, interference between, 162, 173, 174 homologous, interference between, 175 lag period, recoverable infective virus and, 115, 116, 120, 123-124, 125 mouse lung, adaptation and, 237-240 mouse pneumonia virus, interference between, 162 mumps virus, interference between, 162, 163, 175 Newcastle disease virus, interference between, 162, 163, 173 O-D change, virulence variation and, 230-232 pentamidine and, 219 P phase, hemagglutination inhibition and, 268 Q phase, hemagglutination inhibition and, 268 ribonuclease, multiplication inhibition and, 276 recombination studies, virulence and, 159 release of, 139-140, 142, 214 R phase, hemagglutination inhibition and, 268 serological variation adaptation to new hosts and, 265-267 during passage and, 265-269 induced and, 267 phase and, 267-269 serological types and, 254 Type A strains and, 254-265 Type B strains and, 265 viral structure and, 253-254 soluble antigen and, 126-129, 206-207 St. Louis encephalitis virus, interference between, 162, 163 structure of, 12 vaccinia virus, interference between, 162 West Nile encephalitis virus, inter- ference between, 162, 163 VOL. TtI—27/ 417 Xerosin, host protection and, 159 yellow fever virus, interference between, 163 Inhibition chemical structure amino acids and, 217-218 benzimidazoles and, 214-217 oxidative metabolism and, 218-219 purine analogs and, 218 pyrimidine analogs and, 218 hemagglutinin, genetic marker and, 279 hemagglutination influenza A virus and, 256-258 phase influenza viruses and, 268 intracellular multiplication 5, 6-dichloro-l-x-D-arabinopyranosyl- benzimidazole and, 203, 210-211, 212, 213, 216, 217 5, 6-dichloro-l-8-p-ribofuranosylbenzi- midazole and, 203, 206-209, 211, 212, 215-216, 217 2, 5-dimethylbenzimidazole and, 203 205, 211, 212, 213 p-fluorophenylalanine and, 203, 209, 211, 212, 218, 220 Klebsiella pneumoniae capsular poly- saccharide and, 203-205, 211, 212 latent period and, 202-211 levo -y-(o- chlorobenzy]) - 6 - oxo -y- phenyl caproic acid and, 203, 210 212, 213 DL-methoxinine and, 203, 205-206, 211, 212, 213, 217 post-latent period and, 211, 213 ribonuclease, influenza virus multiplica- tion and, 276 virus multiplication, current status of, 220-222 virus release and, 213-214 Inhibitors hemagglutination N-acetylglucosamine and, 53 arboviruses and, 41-42 Chu and, 35-36 mucoid and, 33-34, 36-37, 52, 53 specific antibody and, 34-35 Insect viruses, see also Polyhedral viruses, Granulosis viruses and individual insect species cross transmission and, 386-388 418 Insect viruses latent infection and, 386-388 transmission methods and, 388-390 ultimate infective unit and, 384 virus localization and, 372, 376 without intracellular inclusions Cirphis unipuncta and, 379, 383 Tipula paludosa and, 379-380, 383 “Interferon’’, virus interference and, 183 Isolations pure clone, virus genetics and, 277-279 Japanese B encephalitis virus, 6 equine encephalomyelitis virus, inter- ference between, 163 hemagglutination and, 38, 40, 41, 42 poliovirus, interference between, 163, 181 Japanese hemagglutinating Influenza D virus virus, see Kethoxal, see f-ethoxy-«-ketobutyralde- hyde hydrate Klebsiella pneumoniae capsular polysaccharide mouse pneumonia virus and, 203-205, 212 mumps virus and, 204-205 Lag period, definition of, 112 Laryngotracheitis, infectious, avian viruses and, 7 Latent period, intracellular multiplication inhibition and, 202-211 Leucocytes, guinea pig, viral adsorption and, 17 Leukemia, 98 mice, 346-352 adrenal tumors and, 358 parotid tumors and, 352-356 sarcomas and, 356-358 Levo-y-(o-chlorobenzy1)-5-oxo-y-phenyl caproic acid, influenza A virus and, 203, 210, 213 Louping ill virus, hemagglutination and, 40 Lymantria dispar, nuclear polyhedral virus and, 372, 373, 374, 380, 387 Lymantria monacha, nuclear polyhedral virus and, 373, 380 SUBJECT INDEX Lymphocytic choriomeningitis virus nonviscerotropic lymph.-chorio. virus, interference between, 167 poliovirus, interference between, 164 Lymphogranuloma, see Lymphopathia venerea virus Lymphopathia venerea virus, 3 human fibroblasts and, 73 malignant rat cells and, 73 Malacosoma fragile, polyhedrosis and, 389— 390 Malignant smallpox, 251 canary pox and, 4 myxomatosis and, 4 Marner characteristics virus genetics incidental and, 280 morphologic and, 279 reproductive and, 280 somatic and, 279 Mayaro encephalitis virus, hemagglutina- tion and, 42 Measles virus, 7, 251 Meconium mucoprotein, see Mucoprotein Melanchra persicareae, granulosis virus and, 378 ‘““Melbourne egg” virus, hemagglutination and, 25 Melolontha sp. rickettsia-line organism and, 370 Mengo encephalitis virus hemagglutination and, 43 infective nucleic acid and, 276 neutralizing antibody and, 200 Menigopneumonitis virus, lag period, recoverable infective virus and, 119, 120 6-Mercaptopurine coenzyme A, mito- chondria and, 67 Metaperiodate ion, viral eclipse phase and, LOST DL-methoxinine influenza A virus and, 203, 205-206, 212, 213, 217 vaccinia virus and, 206, 217 Mice dengue encephalitis virus adaptation to, 226 poliovirus adaptation to, 226 “Milk factor”, see Mouse mammary carcinoma virus SUBJECT INDEX Mitochondria, 69, 70, 71, 74, 87, 91, 92, 95, 98, 100, 103 normal cells and, 67 pathologic cell and, 70 Mitosis cell surface bubbling and, 69 virus infection and, 68 Miyagawanella, see Psittacosis virus Mobility electrophoretic BEL influenza virus and, 30 LEE influenza virus and, 30 MEL influenza virus and, 30 MIL influenza virus and, 30 mumps virus and, 30 Newcastle disease virus and, 30 PR8 influenza virus and, 30 swine influenza virus and, 30, 58 Moliuscum contagiosum, poxvirus and, 4, 79 Mononucleosis infectious, 7, Newcastle disease virus hemagglutination and, 31 Mouse bronchial epithelium, virus and, 87 Mouse leukemia, 346-352 adrenal tumors and, 358 parotid tumors and, 352-356 sarcomas and, 356-358 Mouse lung influenza virus, adaptation and, 237-240 redistribution, virulence and, 291-292, 296 Mouse lymphocytic choriomeningitis virus, 310 Mouse mammary carcinoma virus, 65, 99- 101, 105 age factor and, 344-346 vaccinia virus, interference between, 164. viral latency and, 341-344 viral role and, 338-341 Mouse meningopneumonitis virus, hemag- glutination and, 44 Mouse pneumonia virus, 221 dextran, multiplication inhibition and, 204 erythrocytes, attachment prevention and, 198, 199 hemagglutination and, 22, 43-44 influenza virus, interference between, 162 influenza VOL. Wi—?27= 419 Klebsiella pneumoniae, capsular poly- saccharide and, 203-205, 212 mumps virus, interference between, 163 streptococcus MG, capsular substance and, 204 Mouse poliovirus GD VII, hemagglutina- tion and infection polysaccharide inhibi- tion and, 18 Mousepox, see Ectromelia virus Mucoid inhibitors hemagglutination biological properties and, 36-37 erythrocyte mucopolysaccharide and, 53 meconium mucoprotein and, 53 ovarian cyst mucin and, 53 ovomucin and, 53 serum mucoprotein and, 53 sheep submaxillary gland protein and, 53 sputum mucoprotein and, 53 sources of, 36, 53 urine mucoprotein and, 53 Mucopolysaccharide, erythrocyte, hemag glutination inhibition and, 53 Mucoprotein hemagglutination inhibition bovine submaxillary gland and, 54, 55 cerival mucus and, 55 meconium and, 53, 55 milk and, 55 ovarian cyst mucin and, 53, 55 ovomucin and, 53, 55 serum and, 53, 55 sheep submaxillary gland and, 53 sputum and, 53 urine and, 53, 54, 55 Munpfs virus, 5, 8, 83, 200, 201 antihemagglutination, Friedlander bacil- lus type B and, 35, 37 Chu inhibitor and, 35 5, 6-dichloro-!-8-p-ribofuranosy]benzi- midazole and, 208 equine encephalomyelitis virus, inter- ference between, 163 false giant cells and, 86 fluorescent antibody studies and, 87, 88 hemagglutination and, 22, 25, 26-27, 28, 31 muco- 420 Mumps virus influenza virus, interference between, 162, 163, 175 Klebsiella pneumoniae, capsular poly- saccharide and, 204-205 mouse pneumonia virus, interference between, 163 receptor-destroying enzyme gradient and, 33 receptor gradient and, 29 stabilized cells, electrophoretic mobility and, 30 Murray Begg endothelioma, 98 Murray Valley encephalitis virus hemagglutination and, 40 infective nucleic acid and, 276 Mutation discontinuous, virulence redistribution and, 292-293 poliovirus and, 229 virulence variation and, 229 Mutual exclusion, virus interference and, 159, i174 Mycobacterium tuberculosis, host protection and, 160 Myxomatosis, see also Myxoma virus malignant smailpox and, 4 Myxoma virus Oryctolagus sp. and, 244-246 pock variants and, 237 rabbit fibroma virus and, 335-336 rabbits and, 4 sequential infection by, 244-246 Shope fibroma virus, transformation and, 302-305 Sylvilagus braziliensis and, 244 Myxoviruses, 5, 8, 15, 16, 65, 83, see also individual viruses active interference and, 174-175 mixed infection and, 174-175 cellular receptors chemistry of, 51-59 fowl plague and, 51 influenza A, B, C and D and, 51, 52 Newcastle disease and, 51 electron microscopy of, 83, 84, 87, 104 filament formation and, 85-86 genetic interactions and, 282—299 SUBJECT INDEX hemagglutination aging and, 26-27 alcohol and, 27 alum and, 27 ether and, 27 formaldehyde and, 26-27, 28 heat and, 26, 27 hemagglutinin titer and, 28 history of, 22 hydrogen ion concentration and, 26, 27 ionic environment and, 24, 25 periodate ion and, 26, 27-28, 33 red cell species and, 24-25 temperature and, 24 trypsin and, 26 ultraviolet irradiation and, 25, 26 urea and, 27 virus particle and, 22-24 hemagglutinin of, 129-132 heterology among, 173-174 homology among, 173-174. inactivated and active genetic interaction and, 175-178 interference between, 175-178 interference among, 173-181 neuraminidase and, 5, 8, 11 release of, 139-141 ribonucleic acid and, 5, 8, 9, 12 structure of, 12-13 Myxovirus influenzae A, see Influenza A virus Myxovirus influenzae B, see Influenza B virus Myxovirus influenzae C, see Influenza C virus Myxovirus multiforme, 173 Myxovirus parotidis, 173 Myxovirus pestis galli, 173 NDV, see Newcastle disease virus Natada nararia, granulosis virus and, 378, 382 Neodiprion americanus banksianae, nuclear polyhedral virus and, 373 Neodiprion pratti banksianae, polyhedral virus and, 385 Neodiprion sertifer, nuclear polyhedral virus and, 372, 390 Neuraminic acid, chemistry of, 54-59 nuclear SUBJECT INDEX Neuraminidase, 22, 29, 32, 57, 58, 121, see also Receptor-destroying enzyme myxoviruses and, 5, 8, 11 Neurolymphomatosis virus, 7 Neuropathogenicity, transfer, and, 290-291, 295-296 Neurovaccinia virus hemagglutination and, 45 pock variants and, 236 Newcastle disease virus, 83, 103, 174, 183, 184, 186, 187, 188, 200, 201, 238 attachment prevention and, 198 avirulent Newcastle disease virus, inter- ference between, 167, 168, 181 cellular receptors and, 51 coexistence and, 234 ECHO virus, interference between, 163 eclipse phase and, 16, 17 Ehrlich ascites tumor and, 16 elution of, 29 equine encephalomyelitis virus, inter- ference between, 162, 163, 181, 184 filament formation and, 85 fluorescent antibody studies and, 87, 89 growth of, 136-137 hemagglutination and, 22, 23, 24, 26, 27, 28, 31 hemagglutinin of, 130 host-induced variation and, 229 inactivated Newcastle disease virus, interference between, 172 influenza virus, interference between, 162, 163, 173 lag period, recoverable infective virus and, 116, 120 myxovirus and, 5 poliovirus, interference between, 163,181 receptor gradient and, 29 release of, 140-141 stabilized cells, electrophoretic mobility and, 30 vesicular stomatitis virus, interference between, 162, 182 Ntaya encephalitis virus, hemagglutina- tion and, 40 Nucleolus, 71 adenosine effect on, 67, 79 chorioallantoic cells, fowlpox and, 79 monkey kidney cells, poliovirus and, 92, 94. virulence 421 normal cells and, 67 Ophase, human influenza A virus and, 25, 231-232 Operophtera brumata, infection of, 388, 389 Oryctolagus sp. myxoma virus and, 244— 246 Osmotic imbalance, pathologic cells and, 69-70 Ourapteryx sambucaria, cytoplasmic poly- hedral virus and, 377 Ovarian cyst mucin, see Mucoprotein Ovomucin, see Mucoprotein P phase, influenza virus and, 268 PVM, see Mouse pneumonia virus Panaxia dominula, nuclear polyhedral virus and, 374, 387 Papillomas, see Warts Papillomatosis virus rabbits papilloma-to-carcinoma sequence and, 331-334 transmission and, 327-331 Paranuclear hypertrophy, pathologic cell and, 70, 93, 94, 103 Pasteurella pestis, host protection and, 160 2, 4, 5, 6, 7-Pentamethylbenzimidazole, influenza B virus and, 215 Penicillium stoloniferum, culture filtrate, polioviruses and, 220 Pentamidine, influenza virus and, 219 Peridroma margaritosa, granulosis virus and, 378 Periodate ion Chu inhibitor and, 36 mucoid inhibitors and, 37 myxovirus hemagglutination and, 26, 27-28, 33 Phagocytosis, 17 pinocytosis and, 67, 83 “viropexis” and, 51 Phase microscopy, 64, 76, 86 poliovirus and, 92-93, 142 Philosamia sicini, nuclear virus and, 387 Phlogophora meticulosa, cytoplasmic poly- hedral virus and, 376, 382 Pieris brassicae, granulosis virus and, 378, 379, 388, 389 polyhedral 422 Pieris napi, granulosis virus and, 388 Pieris sapae, granulosis virus and, 378, 382 Pieris rapi, granulosis virus and, 388 Pinocytosis, 65 chicken macrophages and, 83 electron microscopy and, 67 phagocytosis and, 67, 83 rat macrophages and, 67 tissue culture and, 67, 92 viral entry and, 67, 92 Poliomyelitis, see Poliovirus Poliovirus, 15, 65, 103, 113, 126, 221, 232, 233, 235 benzimidazole and, 215, 219 coexistence and, 234 coxsackie virus, interference between, 164, 172 2, 6-diaminopurine and, 218, 219 5, 6-dichloro-]-«-D-arabinopyranosyl- benzimidazole and, 203, 210-211, 213, 216 5, 6-dichloro-l-8-p-ribofuranosylbenzi- midazole and, 203, 208, 212 eclipse phase and, 16, 18 encephalomyocarditis virus, interference between, 164 epithelial cells living cells and, 92-93 virus release and, 93-94 DL-ethionine and, 217, 219 fluorescent antibody study and, 94 p-fluorophenylalanine and, 203, 209, 218, 220 6D VII, 22, 38, hemagglutination and, 42-43, 59 growth of, 133-134 heterotypic poliovirus, interference be- tween, 163, 164, 165, 181 intracellular soluble antigens and, 132 Japanese B encephalitis virus, inter- ference between, 163, 181 lymphocytic choriomeningitis virus, interference between, 164 mice, adaptation and, 226 mutation of, 229 nerve cells classic findings and, 91-92 tissue culture and, 92 Newcastle disease virus, between, 163, 181 interference SUBJECT INDEX noncytopathogenic poliovirus, inter- ference between, 167 Penicillium stoloniferum, culture filtrate and, 220 recombination and, 301-302 release of, 139, 141 sequential infection by, 240-241, 242- 244 structure of, 12-13 B-2-thienylalanine and, 217, 219 tissue culture, virulence variation and, 232-233 Polyhedral viruses cytoplasmic development of, 376-377 location in tissues and, 376 morphology of, 381-382 reaction to alkalies and, 377-378 shapes and sizes of, 377 virus particle dispersal and, 377 nuclear deveiopment of, 372-374 location in tissues and, 372 morphology of, 380-381 reaction to alkalies and, 375 shapes and sizes of, 374 virus rod dispersal and, 374-375 Polyhedroses, see Polyhedral viruses Polylysine peptides, infection prevention and, 200 Porthetria dispar, nuclear polyhedral virus and, 380 Poxviruses, 2, 4-5, 8, 10, 13, 65, 103, see also individual viruses fowlpox development sequence and, 79-80 inclusion and, 79 nucleolus and, 79 genetic interactions and, 299-305 hemagglutination and, 22, 44-45 molluscum contagiosum and, 4 pock variants and, 236-237 Shope fibroma and, 80 vaccinia, 75 electron microscopy and, 77—78 fluorescent antibody staining and, 78 identification of, 76 Pseudorabies virus, see Herpesvirus Psittacosis-lymphogranuloma venereum viruses SUBJECT INDEX heterotypic psitt.-lgr. ven. viruses, interference among, 164 inactivated psitt.-lgr. ven. viruses, interference among, 171 Psittacosis virus, 2, 3-4, 10, 13, 51, 65 antibiotics and, 75 chemotherapy and, 75 deoxyribonucleic acid and, 3, 4, 12 elementary bodies and, 73-75 fluorescent antibody staining and, 75 hemagglutination and, 22, 44 lag period, recoverable infective virus and, 119-120, 126 recombination and, 302 ribonucleic acid and, 3, 4 tissue culture stages and, 72-73 vaccinia virus and, 4, 8 Pterolocera amplicornis, hedral virus and, 375 Pyknosis, cell surface bubbling and, 69 Pyrrole-2-carboxylic acid bovine submaxillary gland mucoprotein and, 54 chemical synthesis of, 54, 55 nuclear poly- Q phase, influenza virus and, 268 RDE, see Receptor-destroying enzyme RNA, see Ribonucleic acid R phase, influenza virus and, 268 Rabbit corneal cells herpesvirus and, 81 vaccinia virus and, 76 Rabbit fibroma virus, infection and, 67 Rabbit papillomatosis virus papilloma-to-carcinoma sequence and, 331-334 transmission of, 327-331 Rabbitpox virus, 4 hemagglutination and, 45 recombination and, 299-301 Rabies virus, 7, 65, 235 attenuation of, 226 autointerference and, 168 intracellular soluble antigens and, 132 fibroblasts, tissue culture and, 72 Rat ; malignant cells, lymphopathia veneria virus and, 73 335-336, cell 423 Receptor-destroying enzyme, 31 N-acetylneuraminic acid, human eryth- rocytes and, 55-56 6-«-D-N-acetylneuraminyl-N-acetyl- galactosamine and, 57 bovine submaxillary gland mucoprotein and, 56 Chu inhibitor and, 35-36 Clostridium welchii and, 32 host protection and, 159, 188 indicator viruses and, 34 mucoid inhibitors and, 37 siadyl-lactose and, 58 urine mucoprotein and, 56 Vibrio cholerae and, 29, 30, 32-33, 41, 52, 53, 55, 58, 121, 122, 123, 124, 125, 128, 135, 140, 210, 214 viral eclipse phase and, 16, 17 Receptor-destroying enzyme gradient, 58— 59 mumps virus and, 33 Receptor destruction, elution and, 29 Receptor gradient BEL influenza virus and, 29 indicator viruses and, 34 influenza A virus and, 230-231 influenza B virus and, 29 LEE influenza virus and, 29 MEL influenza virus and, 29 MIL influenza virus and, 29 mumps virus and, 29 Newcastle disease virus and, 29 swine influenza virus and, 29 WS influenza A virus and, 29 Receptors myxoviruses chemistry of, 51-59 fowl plague and, 51 influenza A, B, C and D and, 51, 52 Newcastle disease and, 51 Recombination experiments inactive ‘“‘parent’’ and, 281 principles of, 280-282 tissue culture and, 280-281 virulence and, 281-282 influenza viruses and, 282-299 interpretation, 293-294 genetic aspects and, 294 genetic loci and, 297 424 Recombination interpretation heterozygosis and, 294-295 multiplication nature and, 297-299 virulence and, 295-297 nonviable ‘“‘parent’’ and, 288-289 poliovirus and, 301-302 psittacosis virus, 302 rabbitpox virus and, 299-301 vaccinia virus and, 299-301 Red cells, see Erythrocytes Rhinocoris annulatus, polyhedral virus and, 390 Ribonuclease, influenza virus multiplica- tion inhibition and, 276 Ribonucleic acid, 141, 152, 153, 207, 208, 211, 212, 213, 216, 283, 304 arboviruses and, 12 enteroviruses and, 6, 9, 12 myxovirus and, 5, 8, 9 psittacosis viruses and, 2, 4, 12 western equine encephalomyelitis virus and, 6 Ribosyl-benzimidazole derivative, influ- enza virus production and, 12 Rickettsiae, 2, 3-4, 10, 119, 128 Rickettsia burneti chick fibroblast cultures and, 72 chick heart cultures and, 72 Rickettsia mooseri, electron microscopy, yolk sac and, 72 Rickettsia rickettsii, rat fibroblasts and, 72 Rickettsia tsutsugamushi, rat fibroblasts and, 72 Rift Valley fever virus autointerference and, 168, 169 inactivated Rift Valley fever virus, interference between, 171 incomplete virus and, 151 yellow fever virus, interference between, 163 Rinderpest virus, egg-adapted rinderpest virus, interference between, 167 Rous sarcoma virus adaptation of, 319-320 assay of, 322-326 chickens, 65, 96, 103, 105 biological properties and, 319-327 electron microscopy and, 97—98 host-virus relationships and, 315-319 SUBJECT INDEX tissue culture and, 96-97 virus localization and, 98-99 coexistence and, 234 hydrocortisone and, 317 lag period, recoverable infective virus and, 116, 120 masking and, 321-322 multiplication of, 322-326 purification of, 326-327 variation in, 319-320 Xerosin and, 317-318 Rubella, 7 Russian far eastern encephalitis virus, ~ hemagglutination and, 40 Russian spring-summer encephalitis virus, 2, 6-diaminopurine and, 218 Santigen, see Soluble antigen Secretions, normal cell, virus release and, 67-68 Semliki Forest encephalitis virus, hemag- glutination and, 40 Sendai virus, see Influenza D virus Serological reagents, serological variation and, 252-253 Serological types, influenza viruses and, 257 Serological variation arboviruses strain relationships and, 269 successive exposure and, 269-270 influenza viruses adaptation to new hosts and, 265-267 during passage and, 265-269 induced and, 267 phase and, 267-269 serological types and, 254 Type A strains and, 254-265 Type B strains and, 265 viral structure and, 253-254 reagents and, 252-253 Serum, immune, viral eclipse phase and, 16 Serum mucoprotein, see Mucoprotein Sheep dermatitis virus, Shope papilloma virus, interference between, 167 Sheep submaxillary gland mucoprotein, see Mucoprotein Shope fibroma virus, 13 electron microscopy and, 80, 102, 105 SUBJECT INDEX 425 Shope fibroma virus myxoma virus, transformation and, 302— 305 Shope papilloma virus, 13 sheep dermatitis virus, between, 164 Sialic acid, see also N-acetylneuraminic acid, ON-diacetylneuraminic acid and N-glycolyl-neuraminic acid, inhibitory mucoproteins, linkage in, 56 Sialyl-lactose, receptor-destroying enzyme and, 58 Sindbis encephalitis virus, hemagglutina- tion and, 42 Smallpox, see Malignant smallpox Soluble antigen fowl plague virus and, 128-129 influenza virus bacteriophage and, 127 deoxyribonucleic acid and, 128 5, 6-dichloro-l-8-p-ribofuranosyl- benzimidazole and, 206-207 ribonucleic acid and, 128 virus multiplication and, 126-129 intracellular African horse sickness virus and, 132 foot-and-mouth disease virus and, 132 herpes simplex virus and, 132 poliovirus and, 132 rabies virus and, 132 vaccinia virus and, 132 Sparrow pox virus, 4 Sphinx ligustri cytoplasmic polyhedral virus and, 376, 377, 381-382 nuclear polyhedral virus and, 370, 384, 387 Sputum mucoprotein, see Mucoprotein St. Louis encephalitis virus, 235 equine encephalomyelitis virus, inter- ference between, 163, 172, 181 hemagglutination and, 40, 41 influenza virus, interference between, 162, 163 Surfaces cell bubbling and, 69 pathologic cells and, 69 Swine influenza virus, 254-255, 257, 258, 259, 260, 261, 262, 264 electrophoretic mobility and, 30, 58 interference neuropathogenicity transfer and, 290 receptor gradient and, 29 Sylvilagus braziliensis, myxoma virus and, 244 T agglutinin, 30 T agglutinogen, 30, 33 T phage, 158, see also Bacteriophage Telea polyphemus, nuclear polyhedral virus and, 387 Thaumatopoea pityocampa, cytoplasmic polyhedral virus and, 376-377, 386 Theiler’s virus benzimidazole and, 215 5-chlorouridine and, 218 equine encephalomyelitis virus, inter- ference between, 164, 172, 186 p-fluorophenylalanine and, 218 5-hydroxyuridine and, 218 low virulent Theiler’s virus, interference between, 167, 168 Thermal inactivation, virus hemagglutina- tion and, 26 B-2-Thienylalanine poliovirus and, 217, 219 vaccinia virus and, 217 Thyroxine, ballooned mitochondria and, 70 Tineola bisselliella, nuclear polyhedral virus and, 372, 375 Tipula paludosa inclusionless virus and, 379-380, 383 nuclear polyhedral virus and, 372-373, 374, 375, 381, 385 Tissue culture chick fibroblasts and, 69 fibroma-myxoma transformation and, 336 pinocytosis and, 67, 92 poliovirus and, 91, 92, 232-233 rat fibroblasts and, 72 recombination experiments and, 280- 281 Rous chicken sarcoma and, 96-97 varicella virus and, 235 viruses chick embryo chorionic epithelium and, 11 Hela cells and, 11 virus interference growing cell cultures and, 181-182 surviving tissue fragments and, 181 426 Tobacco mosaic virus, 112, 127, 200 Transformation Berry-Dedrick, fibroma-myxoma and, 276, 302-305 Boerlage fibroma virus and, 304 Shope fibroma virus, myxoma virus and, 302-305 Transforming agent, myxoma virus and, 304 Trypsin Chu inhibitor and, 36 influenza virus, soluble antigen and, 128 myxovirus hemagglutination and, 26, 85 receptor-destroying enzyme and, 32, 58 Tumor viruses, 65, see also individual virus Uganda S encephalitis virus, glutination and, 42 Ultraviolet irradiation, 288 myxovirus hemagglutination and, 26, 31 Urea, myxovirus hemagglutination and, 27 Urine mucoprotein, see also Mucoprotein prosthetic groups of, 56 receptor-destroying enzyme and, 56 hemag- Vantigen, see Antigen V, carcinoma, rabbit papilloma virus and, 333 Vaccines, bacterial, host protection and, 159 yellow fever virus, attenuation and, 226 Vaccinia virus, 2, 51, 103, 113, 124, 183, 201, 221, 320 Bacillus megatherium and, 13-14 benzimidazole and, 215 deoxyribonucleic acid and, 4, 8 dermotropic vaccinia virus, interference between, 167 2, 6-diaminopurine and, 218 electron microscopy and, 77—78 elementary bodies and, 7€, 77 fibroblast roller tube cultures and, 234 fluorescent antibody staining and, 78 foot-and-mouth disease virus, inter- ference between, 164 fowlpox virus, interference between, 164 genetic recombination and, 229-230 growth of, 137-138 hemagglutination and, 44-45 human epidermoid carcinoma and, 78 identification of, 76 SUBJECT INDEX inactivated vaccinia virus, interference between, 171 infection spread, hyaluronidase and, 235 influenza virus, interference between, 162 intracellular soluble antigens and, 132 lag period “enhancement effect” and, 117 recoverable infective virus and, 116, 117-118, 125, 126 DL-methoxinine and, 206, 217 mouse mammary carcinoma virus, inter- ference between, 164 psittacosis virus and, 4, 8 rabbit corneal cells and, 76 recombination and, 299-301 release of, 141, 142 B-2-Thienylalanine and, 217 Vanessa cardui, nuclear polyhedral virus and, 388 Vanessa io, nuclear polyhedral virus and, 388 Variation, see Virulence variation Varicella virus release of, 141, 142 tissue culture and, 235 Variola vaccinia virus, 4 Venezuelan equine encephalitis virus hemagglutination and, 42 yellow fever virus, interference between, 163 Vesicular stomatitis virus autointerference and, 168, 181 equine encephalomyelitis virus, inter- ference between, 163 heterotypic vesicular stomatitis virus, interference between, 164, 181 incomplete virus and, 151 infection spread, hyaluronidase and, 235 Newcastle disease virus, interference between, 162, 182 serological types cattle and, 7 swine and, 7 Vibrio cholerae, receptor-destroying en- zyme of, 29, 30, 32-33, 41, 52, 53, 55, 58, 121, 122, 123, 124, 125, 128, 135, 140, 201, 214 Victorian strain, Newcastle disease virus, hemagglutination and, 28 SUBJECT INDEX “Viropexis’’, 17, 18, phagocytosis and, 51 Virulence definition of, 226-227 genetic marker and, 280 mouse lung, redistribution and, 291-292 recombination, influenza virus and, 159 redistribution discontinuous mutation and, 292-293 mouse lung and, 291-292, 296 neuropathogenicity and, 290-291 virus interference and, 158-159 Virulence variation host-induced encephalomyocarditis virus and, 229 influenza A virus and, 229 Newcastle disease virus and, 229 mechanisms bacterial viruses and, 228 genetic recombination and, 229-230 host-induced and, 229 mutation and, 229 virus-host-cell systems coexistence and, 234 hamster lung and, 237-240 influenza virus O-D change and, 230- 232 mouse lung and, 237-240 mousepox and, 241-242 myxomatosis and, 244-246 pock variants and, 236-237 poliomyelitis and, 242-244 poliovirus tissue culture and, 232-233 yellow fever and, 242 Viruses, see also individual viruses Viruses, bacterial, see Bacteriophage classification, 3-7 comparison of, 7-9 definition of, 1-3, 112 evolution of, 10-11, 229 functional unity of, 11-14 heterologous, interference between, 161- 166 homologous, interference between, 166- 170 inactivated, interference between, 170, 171 infection, initiation and, 15-18 marker characteristics incidental and, 280 morphologic and, 279 427 reproductive and, 280 somatic and, 279 technical requirements marker characteristics and, 279-280 pure clone isolations and, 277—279 recombination experiments and, 280- 282 Virus growth eclipse phase, 112-113 bacteriophages and, 113-114 large viruses and, 116-120 medium-sized viruses and, 115-116 small viruses and, 114-115 herpes simplex virus and, 137 incomplete virus partial cycle and, 149 production of, 146 properties of, 144 infected cells antigens and, 126 influenza virus and, 135-136 lag period antigenic properties and, 120-122 recoverable virus and, 113-120, 122- 126 Newcastle disease virus and, 136-137 poliovirus and, 133-134 vaccinia virus and, 137-138 western equine encephalitis virus and, 112, 134-135 Virus infection host cell surface and, 13 prevention extracellular environment and, 198- 199 extracellular virus and, 199-201 host cell alteration and, 201 Virus interference, see also Autointer- ference definition, 160 mutual exclusion and, 159, 174 virulence and, 158-159 dynamics of, 182-185 experimental systems dosage and, 170, 172 growing cell cultures and, 181-182 heterologous viruses and, 161-166 homologous viruses and, 166-170 inactivated viruses and, 170, 171 localized character and, 172-173 428 Virus interference experimental systems myxoviruses and, 173-181 surviving tissue fragments and, 181 timing and, 170, 172 induction specificity and, 182-184 “imterferon” and, 183 mechanisms, 185-188 cell association and, 186-187 superinfecting virus fate and, 187-188 overcoming of, 185 quantitative analysis of, 160-161 quantitative criteria for, 184—185 speed and duration of, 184-185 tissue culture growing cell cultures and, 181-182 surviving tissue fragments and, 181 Virus localization insect viruses and, 372, 376 Rous’ chicken sarcoma and, 98-99 Virus release adenoviruses and, 141, 142 herpes B virus and, 141 herpes simplex virus and, 141 herpesvirus and, 141 influenza virus and, 139-140, 142, 214 inhibition of, 213-214 mechanism of, 142-143 mouse mammary tumor and, 100-101 myxoviruses and, 139-141 poliovirus and, 93-94, 139, 141 vaccinia virus and, 141, 142 varicella virus and, 141, 142 virus filaments and, 142-143 western equine encephalitis virus and, 138-139, 142 Virus structure influenza viruses, serological variation and, 253-254 Visceral lymphomatosis virus chickens age factors and, 308-309 genetic factors and, 309 host-virus relationships and, 369-314 transmission and, 309 w forms, human influenza A virus and, 25 Warts human ceil nuclei and, 101, 105 SUBJECT INDEX Western equine encephalitis virus, 3, 6, 116, 188 autointerference and, 168, 181 growth of, 134-135 hemagglutination and, 40, 41 lag period, recoverable infective virus and, 114, 120 release of, 138-139, 142 structure of, 12 West Nile encephalitis virus hemagglutination and, 40, 41 influenza virus, interference between, 162, 163 Venezuelan equine encephalitis virus, interference between, 163 yellow fever virus, interference between, 163 X irradiation, 318, hypertrophied cells and, 71 Xerosin host protection and, 159 Rous’ sarcoma virus and, 317-318 Yellow fever virus, 3, 6, 9, 242, 251 attenuated vaccine and, 226 autointerference and, 168 hemagglutination and, 40 sequential infection by, 242 17 DD yellow fever virus, 183 interference between, 167 dengue encephalitis virus immunogenicity suppression and, 161 interference between, 163 influenza virus, interference ketween, 163 neurotropic yellow fever virus, inter- ference between, 167, 168 Rift Valley fever virus, interference between, 163 Venezuelan equine encephalomyelitis virus, interference between, 63 West Nile encephalitis virus, interference between, 163 Zika encephalitis virus, hemagglutination and, 42 (5 a Fa eel; ae ee | « ~ oe tes tapaage Bb 2 tae Las ny OBS Npmavage. canoes ats 4 : Rerath sheet aod pe ea Shem SiG at ; ‘se aes : ag cabal eR he Nerd pages eae ataibletel pony japan hatate es