THE COMPONENTS OF MEASLES VIRUS 377

Zusatz von Corticoiden in verschiedener Konzentra­ mit Virus infizierten Zellkulturen gemacht wurden, tion. Cortison und Prednisolon zeigten einen Wir­ deuten darauf hin, daß eine direkte Beeinflussung kungsumschlag von Wachstumshemmung bei höhe­ des normalen und des durch Virusvermehrung ver­ rer Dosierung zu deutlicher Wachstumsbeschleuni­ änderten Stoffwechsels von Kulturzellen durch Cor­ gung bei niedriger Konzentration im Nährmedium. tisonzusatz möglich ist. Dabei sind hinsichtlich der Hexadecadron förderte vorübergehend in allen Kon­ Auswirkung jedoch Dosierung und Zeitpunkt der zentrationen das Wachstum. ACTH-Zusatz zeigte Anwendung zu beachten. nur bei niedrigen Konzentrationen ein mehr oder minder gutes Wachstum. Hohe Dosen lösten die Frau M. R e i c h e , Frau H. T r o s c h i e r und Frau E. K u h n danke ich für sorgfältige technische Mitarbeit. Kulturzellen langsam auf. Herr Diplom-Mathemathiker J. K r ü g e r hat midi Alle diese Beobachtungen, die bei Versuchen mit freundlicherweise bei der Auswertung der Versuchs­ cortisonhaltigen Nährmedium auf wachsenden bzw. ergebnisse unterstützt.

The Components of Measles Virus and their Relation to Rinderpest and Distemper

By A. P. W a t e r s o n * and R u d o l f R o t t Max-Planck-Institut für Virusforschung, Tübingen

and G i s e l a R u c k l e -E n d e r s Hygiene-Institut der Universität Marburg (Z. Naturforschg. 18 b, 377—384 [1963] ; eingegangen am 18. Februar 1963)

Measles virus has been disintegrated by treatment with ether and Tween 80. This destroys the infectivity, and physically disintegrates the particle, with the release of an inner component struc­ turally like the nucleoprotein of Newcastle disease virus (NDV), and another structure similar to the haemagglutinin (HA) of NDV. The preparation after ether-tween treatment has an enhanced HA activity. The two components are separable in a CsCl density gradient. The inner component is probably a nucleoprotein. The HA component could be adsorbed by monkey erythrocytes but not eluted from them. The action on it of sodium metaperiodate suggests that a carbohydrate may be in­ volved. The ether-tween preparation could be used as an antigen in the CF-reaction with antisera to rinderpest and distemper. It could also be used in the HA-inhibition test as a sensitive indicator with antisera to rinderpest and distemper, as well as to measles, giving higher titres in the HA- inhibition test than when the untreated virus was used. Injection of the ether-tween preparation stimulated the production of neutralizing, HA-inhibiting and complement-fixing antibodies in the rabbit.

The particle of measles virus has the same fine H o s o k a w a and F u k a i 4. In view of these facts it structure, as seen in the electron microscope, as that seemed reasonable to investigate further the decom­ of the larger myxoviruses, i. e. the Newcastle disease- position of the measles particle by this treatment. mumps-parainfluenza group L When treated with a Such experiments might be expected to provide detergent (Triton X100 ) 1 structures similar to the further evidence of similarity between the measles haemagglutinin (HA) of Newcastle disease virus 2 group and the parainfluenza group. The present (NDV) are released. Measles virus has been shown paper deals with the preparation and properties of to agglutinate erythrocytes of certain primate spe­ the components derived in this way from the measles cies 3. The release of H A from a parainfluenza virus virus, explains the morphological basis of their re­ (Sendai) by means of the combination of ether and lease from the particle, and also describes the practi­ a detergent (Emasol) was reported by H o s a k a , cal usefulness of such a preparation.

* On sabbatical leave from the Department of Pathology, 3 J. R. P e r i e s and C . C h a n y , C . R. hebd. Seances Acad. Sei. Tennis Court Road, Cambridge, England. 251, 820 [I960]. 1 A. P. W a t e r s o n , J. G. C r u ic k s h a n k , G. D. L a u r e n c e and 4 Y. H o sa k a , Y. H o sa k a w a and K . F u k a i , Biken’s J. 2, 367 A. D. K a n a r e k , Virology 15, 379 [1961]. [1959]. 2 R . R o tt and W . S c h ä f e r , Virology 14, 298 [1961]. 378 A. P. WATERSON, R. ROTT AND G. RUCKLE-ENDERS

Materials and Methods c) Distemper Serum taken four weeks after inoculation of a dog 1. Viruses with the Rockborn strain of canine distemper was a) Strains supplied by the Behring-Werke, Marburg. The “nor­ mal” dog serum used had no detectable neutralizing The 1677 strain of measles virus was used through­ activity against distemper virus. out. This strain is identical with the Edmonston strain All sera were inactivated by heating at 56 °C for in its antigenic and biological properties. It was iso­ 30 minutes. Those used for haemagglutination-inhibi- lated in primary cultures of human kidney from a tion titrations were also treated with M/90 sodium lymph-node of a patient in Marburg. During succes­ periodate 6. sive passages at high multiplicity in the human kidney cultures and also in cultures of various continuous cell- 3. Assay methods lines (HeLa, HEp 2 and FL) it caused a predominantly a) Injectivity spindle-cell type of degeneration. A variant of this Infectivity was assayed by detection of CPE and of strain, selected by passage at limit dilution, and the picking of well-separated plaques, induces giant-cell haemadsorption with monkey erythrocytes at 37 °C in formation 6. This variant of the 1677 strain was used HeLa and FL cells, as previously described 5. in a few experiments. b) Neutralization test Titrations of neutralizing antibody were done in the b) Growth HeLa and FL cells by the conventional methods, using The virus was grown in HeLa, HEp 2 or FL cells. the 1677 strain and Edmonston strains. An immune These were cultivated in 0.5% lactalbumin hydrolysate rabbit serum against the latter served as control. Tests in H a n k s’ saline, supplemented with 20% TCM 199, were read after 10 days for cytopathic effect and haem­ and with 10% or 5% calf serum for growth or main­ adsorption. Serum titres were calculated by the method tenance respectively. When the cytopathic effect was of R e e d and M u e n c h . nearly complete, virus was prepared from tissue cul­ c) Haemagglutination test ture fluid, and from cells frozen and thawed three or four times. The material was clarified by centrifugation Serial two-fold dilutions of the material were made at 3000 r.p.m. for 15 minutes, followed by sedimenta­ in 0.9% NaCl, buffered to pn 7.2, in 0.2 ml volumes. tion at pi 7 10a. The sediment was resuspended in phos­ 0.1 ml of a \% suspension of rhesus or cynomolgus mon­ phate-buffered saline with or without the addition of key erythrocytes was added. The results were read 0.5% bovine albumin. This concentrate was centrifuged after two hours at room temperature (21 ~C). The final at 2500 r.p.m. for 10 minutes, and frozen at — 70 °C tube showing clearly detectable haemagglutination was until required. Virus which had been passed three times taken as the end point. Because of the poor end points in FL cells after earlier passes in HeLa cells was used in titrations made in plastic plates, tubes were used in some complement-fixation tests. throughout. d) Haemagglutination-inhibition 2 . Sera Serial two-fold dilutions of the serum in buffered a) Measles saline were made in 0.2 ml volumes. Four haemaggluti- Serum against measles virus was prepared by in­ nating units in 0.2 ml were added. After one hour at jecting rabbits with suspensions of virus in tissue cul­ room temperature 0.1 ml of red cells was added and ture fluid. Human serum taken at various stages from the results read after a further two hours. patients with a typical measles illness was used for e) Complement fixation acute and convalescent sera. Some of these were sup­ plied by Dr. J. F. E nders (Boston). A modification of the micro-method of F u l t o n and D u m b e l l was used 6. b) Rinderpest 4. Caesium chloride density gradient Paired bovine sera taken from a cow before inocula­ centrifugation tion and after recovery (21 days later) from rinderpest (strain RGK/I) and similar sera from a second cow in­ Centrifugation in the caesium chloride density fected from this one were supplied by Mr. W. Plo­ gradient was done in Spinco rotor SW 39. After mixing w r i g h t (East African Veterinary Research Organisa­ the virus suspension with the corresponding amount of tion) . CsCl solution in cellulose tubes, the mixture was co-

5 G. R u c k l e -E n d e r s , Amer. J. Diseases Children 103, 297 7 S. B r e n n e r and R. W. H o r n e , Biochim. biophysica Acta [1962]. [Amsterdam] 34. 103 [1959]. 6 F. M. D a v e n p o r t , R . R o tt and W. S c h ä f e r , J. exp. Medi­ cine 112, 765 [1960]. THE COMPONENTS OF MEASLES VIRUS 379 vered with paraffin and centrifuged for about 16 hours 2. Sedimentation of the ether-tween at 35,000 r.p.m. at + 4'JC. After pricking the bottom preparation in the u 1 1 racentrifuge of the tubes a series of samples of 10 drops each were taken, and the refractive indices determined at room The HA component is less easily sedimented than temperature. the untreated virus in the ultracentrifuge (Fig. 1). Reference to the figure shows that at a centrifugal 5. Electron Microscopy force sufficient to sediment the intact particles much Specimens were sprayed with phosphotungstic acid of the HA component is left in suspension. The sedi­ and examined using the negative staining technique of mented material resuspended itself very quickly on B r e n n e r and H o r n e 7. standing, and therefore centrifugation was carried out at +4 °C, and resuspension was done im­ R esults mediately the centrifuge stopped.

1. Treatment of the virus with ether

Concentrates of virus were mixed with an equal volume of peroxide-free ether and shaken for two hours at room temperature ( 2 1 °C), with or without the addition of Tween 80 in aqueous solution, to give a final concentration of 1.25 mg/ml. Typical results are shown in Table 1.

H A titre C onditions E xp t. before After D ifference N o. treatm en t treatm en t p.i. (60Minutes) —*■ E ther 1 2 - 8 2-4 - 94% Fig. 1. Sedimentation in the ultracentrifuge of untreated measles virus and of ether-tween preparation. Ordinate: E ther 2 2 -io 2 - 8 - 75% % recovery of HA activity in supernatant after centrifugation. E ther + Abscissa: centrifugal force, expressed as performance index 2 -io 2 - 1 2 Tw een 80 3 + 400% (pi)10a for one hour at 21 °C. E ther 2 -io 2-13 T w een 80 4 + 800% 3. U s e o f Caesium chloride density Table 1. Effect of Tween 80 on the yield of haemagglutinin gradients in ether-treated measles concentrates. After centrifugation of the ether-tween prepara­ The major difference in the results of treatment tion in the CsCl density gradient, two different bands with and without tween is that, while the infectivity could be seen (Fig. 2). A sharp band with a density is in either case completely destroyed, the haem- of 1.3530 corresponds to the maximal titre of CF agglutinating activity is not only preserved by the antigen, whereas the broader band with a density of tween, but enhanced several-fold. The biological 1.3425 corresponds to the maximal titre of haem- properties after ether-tween treatment are sum­ agglutinating activity. In the high salt concentration, there was no overall loss of haemagglutinating acti- marized in Table 2 .

Infectivity HA CFT (serum) (TCD 5 0 , H eL a) per ml.

Before 2 - 8 2-8.5 106.3 A fter 2 -io 2-8.5 CIO 1

Table 2. Biological properties of measles virus before and after ether-tween treatment.

The crude material obtained as the aqueous phase will be known as the ether-tween preparation and the carrier of the haemagglutinating activity as the Fig. 2. Separation of bands in CsCl density gradient. Spinco haemagglutinating (HA) component. rotor SW 39, 35,000 r.p.m. 16 hours. 3 8 0 A. P. WATERSON, R. ROTT AND G. RUCKLE-ENDERS vity (density 1.3425), but an appreciable loss of 5. Relation of haemagglutinating complement-fixing activity (density 1.3530). These component to complement fixing results indicate that two different components are antigen obtained after splitting the virus. These components In view of the findings of previous workers 14 it both had a higher density than the virus particle, as was not surprising that virus suspensions, both be­ given by S chluederberg 8, and S chluederberg and fore and after splitting, were somewhat anticomple- R o izm a n 9. mentary, particularly when virus grown in HeLa 4. Electron microscopy cells was used. This was largely abolished by heating for 30 minutes at 56 C for preparations of virus, Examination of a concentrate of the starting but not for the ether-tween preparation. Both pre­ material (i. e. before splitting) revealed typical parations showed high CF activity with measles con­ measles particles, with a membrane bearing short valescent sera. projections, and a coiled elongated inner component After thorough adsorption of HA from the ether- (Fig. 3 a, b*). Some particles were disrupted, and tween preparation with monkey erythrocytes there there were some aggregates of the inner component remained about 50% of the CF activity (Table 3). lying free on the grid. A few short filamentous forms The ultra-violet extinction curve after purification similar to the structures described with rinderpest of this material at pi 100 and pi 0.1 suggests the virus 10 were seen. These bore the surface projec­ presence of nucleoprotein. The curve showed a mini­ tions found on the virus particles. A concentrate of mum at E240 and a maximum at E26o • The E 26o/E 28o the ether-tween preparation was prepared for elec- ratio was about 1,3. It is clearly desirable that tron-microscopy by sedimentation for 6 hours at pi further examination of more highly purified material 75 (cf. Fig. l ) 10a. The sediment was resuspended in should be made. 0.4 ml 1 % ammonium acetate, and the HA titre of this suspension was 2~14. No intact or even partially 6. Interaction between virus pre­ disrupted virus particles were seen. In addition to parations and erythrocytes small unidentifiable background matter, two distinct The HA component, like the intact virus, ag­ structures were found, one corresponding to the glutinated the erythrocytes of rhesus and cynomol- inner component. Pieces of this component were gus monkeys. Both virus and HA component failed seen (Fig. 4 a), and were similar to that previously to agglutinate erythrocytes of guinea-pig, mouse, described n. Some were as long as 3000 Ä. rat. hamster, rabbit, fowl and man (group “0”) at They were about 170 Ä in diameter, and had a cen­ 21 or 37 C. The HA component was quantitatively tral canal of about 40 Ä. and a periodicity of about adsorbed (cf. Table 3) from suspensions by con­ 55 Ä along their axis. The other structures resembled centrated monkey erythrocytes. After washing the closely the haemagglutinin 1 2,13 of NDV, and it erythrocytes, there was no detectable HA released seems reasonable, therefore, to identify them as the from them by incubation for 3 hours at 37 cC, with haemagglutinating component of measles virus. They had a diameter of 500 — 1 0 0 0 Ä, were more HACFT or less circular, and possessed the short surface pro­ Before jections seen on the intact virus (Fig. 4 b. c). Unlike 2-12 2~7 A dsorption the inner component they have not been seen in con­ A fter 2-5 2-6 centrates of untreated virus, and the smaller of them A dsorption resemble the structures previously reported in pre­ Table 3. Complement fixing activity of ether-tween prepara­ parations of detergent-treated measles virus. tion before and after treatment with monkey erythrocytes.

* Figs. 3 — 4 s. Table pag. 380 a. 10;l P. G i e b l e r . Z. Naturforschg. 1 3 b . 238 [1958]. 8 A. E. S chluederberg , Amer. J. Diseases Children 103, 291 11 A. F. H o w a ts o n , Federat. Proc. 2 1 . 947 [1962]. [1962]. 12 R. R ott, H. Frank and W. S c h ä f e r . Z. Naturforschg. 1 6 b , 9 A. E. S chluederberg and B. R o iz m a n , Virology 16, 80 625 [1961]. [1962]. 13 A. P. W a t e r s o n and J. G. Cruickshank, Z. Naturforschg. 10 W. P l o w r ig h t , J. G. C r u ic k s h a n k and A. P . W a t e r s o n , Viro­ 1 8 b . 114 [1963]. logy 17, 118 [1962]. 14 J. F. E n d e r s and T. C. P e e b l e s . Proc. Soc. exp. Biol. Med. 86. 277 [1954], A. P . W A TERSox, R . R o tt a n d G. R u c k l e -E x d e r s , The Components of Measles Virus and their Relation to Rinderpest and Distemper (pag. 377)

Fig. 3 a. Measles virus. Untreated. X 175,000. The line Fig. 4. Measles virus after ether-tween treatment. Structures represents 1000 Ä. seen in resuspended sediment, a) Internal component (NP- Fig. 3 b. Measles virus. Untreated. A partially disrupted parti antigen). b), c) Structures resembling haemagglutinin of cle, and a filamentous structure. X 105,000. The line repre­ NDV. X 175,000. The line represents 1000 Ä. sents 1000 Ä.

Zeitschrift für Naturforschung 18 b. Seite 380 a.

THE COMPONENTS OF MEASLES VIRUS 381 or without added neuraminidase (20U/ml; Behring- 8. Inhibition of haemagglutination Werke, Marburg), nor by incubation with crystal­ by m e a s 1 e s - s p e c i f i c serum line trypsin (0.05%) either for 2 hours at 37 WC or for 18 hours at + 4 °C. The ether-tween preparation could be used in the haemagglutination-inhibition test in the same way Treatment of virus or ether-tween preparation as the intact virus. Both with human measles con­ with M/90 sodium periodate resulted in rapid loss valescent serum and with rabbit serum against intact of most of the haemagglutinating activity. The ma­ virus, there was a two to eight-fold difference in terial was mixed with an equal volume of periodate titre, the HA component always giving a higher solution. Samples were taken at intervals and the titre than the intact virus. periodate neutralized with 0.1 M glucose before titration (Fig. 5). This result is similar to that found 9. Antibody response to injection of with NDV15 and suggests that there is a carbo­ split virus hydrate on the surface of the virus which is respon­ A rabbit was injected with 1.5 ml of fluid con­ sible for the haemagglutinating activity. taining 12,000 HA units of the ether-tween prepara­ tion, mixed with 1.5 ml Freund’s incomplete ad­ juvant1'. Serum taken after 32 and 48 days, com­ pared with serum taken before inoculation, showed the following titres in the HA-inhibition test (Table 4 ).

Tested against : Ether-tween Virus* preparation

Serum before inoculation < 2 - 3 < 2 -3 32 days after inoculation 2-7 2-8 48 days after inoculation 2-9 2 - n Minutes---- Table 4. Production of HA-inhibiting antibodies in rabbit. Fig. 5. Reduction in HA activity on treatment with sodium metaperiodate. Ordinate: % revovery of HA activity. Abscissa: * Identical titres were obtained against the Edmonston strain Time (minutes). of measles virus. This demonstrates not only that the antigenicity Using sialolactose as a substrate, we were unable of the virus survives the ether-tween treatment, but to detect any significant amout of neuraminidase in also that the ether-tween preparation may prove to be a useful antigen for the production of immune the virus preparation before or after ether-tween sera. treatment, by the technique of A m i n o f f 1 6 . 10. Failure to demonstrate haem­ 7. Stability agglutination in a non-haemaggluti- nating variant

The haemagglutinating activity of virus or ether- A variant of the 1677 strain (RZ) which causes tween preparation was not appreciably changed by giant cell formation in cell cultures and which fails heating at 56 °C for 30 minutes. It was abolished to give rise to haemadsorption in infected cells, or completely by heating for 30 minutes at 60 °C. The to detectable haemagglutinating or haemolysing ac­ HA activity of the ether-tween preparation was lost tivity in culture fluids, was treated with ether and only slowly on storage at +4 °C, but the loss dif­ tween. The initial concentrate had an infectivity titre fered appreciably with different specimens. of 10°-5“ 6 0. No haemagglutinating activity was de-

13 R . R ott, Habilitation thesis, University of Giessen [1962]. 17 B . S. B e r l in and R. W. M cK in n e y , J . Lab. clin. Med. 5 2 . 16 D. A m in o f f , Biochem. J. 8 1 , 384 [1961]. 657 [1958], 382 A. P. WATERSON, R. ROTT AND G. RUCKLE-ENDERS tected after treatment, although the titre of infective NDV (strain “Italien’) or fowl plague virus (strain particles, compared with the spindle-cell variant, was “Rostock”), or with bovine serum against foot and enough for this to have been detectable. mouth disease virus, Type “0” (supplied by Prof. 11. Reaction of measles virus and E i s s n e r , Bundesforschungsanstalt für Viruskrank­ ether-tween preparation with rinder­ heiten der Tiere, Tübingen), using either virus or pest and distemper sera ether-tween preparation. The bovine sera (both before and after infection with rinderpest) ag­ a) Haemagglutination-inhibition test glutinated the monkey erythrocytes to a low titre. In Antisera containing neutralizing antibodies to this respect rhesus erythrocytes were agglutinated distemper and rinderpest viruses were tested against less readily than cynomolgus, but this action of the both the virus and the ether-tween preparation. As sera was almost completely removed by treatment can be seen from Table 5, antisera to these two with sodium periodate. The sensitivity of the haem- viruses inhibit haemagglutination by measles virus agglutination-inhibition test depended on the dose and by the ether-tween preparation. Higher titres of virus used, but 4 HA units was chosen as com­ bining sensitivity and reliability. HA-inhibiting dilutions of sera b) Complement fixation test against Neutralizing titre against Each antigen, i. e. the untreated virus and the Serum E ther- homologous ether-tween preparation, was standardized against a Measles tween virus human measles convalescent serum by determining virus prepa­ ration the amount of complement fixed by varying dilutions of antigen in the presence of excess antibody. The Human measles 2“7 2 ~ 9 - dilution of each antigen which fixed a standard convalescent CO V

1 amount of complement (1.6 mm3) was chosen as the Rabbit before < 2 -3 < 2 -3 unoculation standard dose of antigen. Rabbit 32 days 2 -7 2-8 2 -io As can be seen from Table 6 the virus preparation after injection reacts with antisera to distemper and rinderpest to of ether-tw een preparation nearly the same degree after treatment with ether Rabbit 48 days 2 -9 2 - n 2 - n and tween as before. Normal canine and bovine sera after injection did not fix complement in the presence of either of ether-tween antigen. After adsorbing the HA material with mon­ preparation R inderpest key erythrocytes, the CF antigen remaining still re­ before (a) < 2 - 4 < 2 -4 — acted with rinderpest and distemper antisera. injection (b) < 2 - 4 < 2 - 4 — Rinderpest (a) 2-4 2-8 logioSNso = 3.8 , ,. Treatment of measles virus A ntiserum ... , ,, CFT convales­ with tween-ether c e n t1 (b) 2-4 2-8 logioSNso = 2.0 F oot and < 2 - 3 < 2 -3 — Measles before 1:384 mouth disease 2 after 1:384 CO V Normal dog3 <2-3 < 2 -3 1 -p. • , before 1: 22 D istem per3 < 2 - 3 2-4 2-9 Distemper after 1: 25 < 2 - 3 < 2 -3 2 - 1 0 4 NDV -p,. j , before 1: 57 2 ~ U Fow l plague < 2 - 3 < 2 - 3 4 m erpest äftcr 1: 60

Table 5. Inhibition of haemagglutination by various sera. Table 6. Effect of ether-tween treatment on complement fixa­ 1 = Neutralizing titres determined by W. P l o w r ig h t . 2 = tion with various sera. Neutralizing titres determined by Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tübingen. 3 = Neutralizing titres determined by Behring Werke (Marburg). 4 = HA- Discussion inhibition titre. The structure of the particle of measles virus is were always obtained against the ether-tween pre­ indistinguishable in the electron microscope from paration than against the intact virus. No reaction that of the parainfluenza viruses and of most strains was observed with sera prepared in fowls against of NDV. It is not therefore surprising that treatment THE COMPONENTS OF MEASLES VIRUS 383 with ether and Tween 80 has the same effect on In this connection it should be emphasised that measles as on Sendai virus 4> 18 and on NDV 15. The the HA produced from the measles particle by ether inner component is by this means released from the and Tween 80, and described in this paper, is an outer coat. In the case of NDV this inner component artefact, i. e. that the smaller particles found after is known to be a ribonucleoprotein. So far as the treatment are not present in that form in the measles virus is concerned, there is at least sug­ intact particle itself, just as the ether-produced HA’s gestive evidence that this structure is also a nucleo­ of NDV2,12, fowl plague 20 and influenza 21 are also protein. The UV extinction curve of partially puri­ artefacts. However, a somewhat inhomogeneous po­ fied material is consistent with this, and also the pulation of small non-infective haemagglutinating identity of its microscopic appearance and dimen­ particles occurs in infected tissue culture fluids 22, 23, sions with its counterpart in NDV. Moreover it and these particles occur naturally, i. e. they are not clearly forms the central mass of the particle, and, formed from the intact particles by added chemical as was shown by B e n y e s h et al. 19, the inner kernel agents. of the measles particle consists of radiation-sensitive The relationship of these various haemagglutinal- material responsible for the infectivity of the par­ ing particles to each other can best be explained in ticle and assumed to be nucleoprotein. It is proposed terms of morphology. The substance or substances that this structure be known as the nucleoprotein responsible for the agglutination of red cells are on antigen (NP-antigen) by analogy with NDV. the surface of the particle. It is clear from the in­ The other component released by ether-tween complete forms of influenza and fowl plague viruses treatment, i. e. the HA component, is also best con­ and the various non-infective haemagglutinating sidered by analogy with NDV. This treatment breaks forms of NDV, that any structurally variant form the coat into smaller pieces, which retain their haem- of the virus which has part of the viral surface agglutinating activity, as has been demonstrated intact will agglutinate red cells. In addition, viro- with NDV and with influenza and fowl plague microsomes, which are believed to consist of endo­ viruses. In the case of measles, of some strains of plasmic reticulum bearing the viral haemagglutinat­ NDV (e.g. “Italien”), and of Sendai virus, much ing substance, and which can be extracted from cells of the HA activity is lost by treatment with ether infected with myxoviruses, can also haemagglutinate. alone, whereas the addition of Tween 80 protects The various forms which “haemagglutinin” may and increases the HA activity. In the electron micro­ take in the case of NDV are as follows: scope structures similar to those positively identified I. Released by the cell as the ether-produced HA of NDV can be seen after treatment of measles. The finer division of the HA a) Intact material (i. e. the conversion of the relatively large (1) Infective particles, virus particles into a greater number of smaller HA (2) Non-infective particles 24 (smaller). units) probably explains why the ether-tween pre­ paration is a more sensitive reagent for the HA- b) F ragm ented inhibition test, i, e. that less antibody is needed per (1) From spontaneous breakdown of particles, unit of HA activity when the haemagglutinating (2) By ether, or ether-tween, treatment. material is finely divided. It follows that a serum will be active at a higher titre with the small, artifi­ II. Extracted from the cell cially subdivided, HA than with the particle itself. It Viromicrosomes 24 is impossible at present to exclude completely the possibility that another antigen is uncovered during Spontaneously derived fragments [1(b) ( 1 ) ] may the destruction of the particle. be expected to occur, because particles of measles,

18 A. P. W a t e r s o n , R . R o t t , unpublished results. 22 J. R . PERrEs and C . C h a n y , Proc. Soc. exp. Biol. Med. 1 1 0 , 10 M. B e n y e s h , E. C. P o l l a r d , E. M. O p t o n , F. L. B l a c k , W. D. 417 [1962]. B e l la m y and J. L. M e l n ic k , Virology 5, 256 [1958]. 23 E. C. J. N o r r b y , Proc. 8th Internat. Congress Microbiol. 20 A. P. W a t e r s o n , R . R o tt and W . S c h ä f e r , Z. Naturforschg. 1962. 16b, 152 [1961]. 24 R . R o t t , I. M. R e d a and W. S c h ä f e r , Virology 1 6 , 207 21 L. H o y l e , R . W . H o r n e and A. P . W a t e r s o n , Virology 13, [1961], Z. Naturforschg. 1 8 b , 188 [1963], 448 [1961]. 38 4 THE COMPONENTS OF MEASLES VIRUS like those of NDV, the parainfluenzas, mumps, and that the basis of the immunogenic relationship rinderpest and distemper (but unlike influenza and in vivo is a similarity of the surface antigens of these fowl plague viruses) disintegrate spontaneously in three viruses to each other. Furthermore, the result suspension with relative ease cf. Fig. 3 b. The frag­ of the complement-fixation test after adsorption of ments from this disintegration [1(b) (1), above] the ether-tween preparation with red cells suggests may be of many different sizes, and yet may all re­ that the NP-antigen of measles, as well as the HA, tain their capacity to haemagglutinate. is the bearer of an antigen common to rinderpest Non-infective but intact particles as described and distemper also. Preparation of the two com­ with NDV 24 can in the case of measles only be postu­ ponents of measles virus will make it possible to lated. The comparatively small proportion of these discover whether these two components are anti- in the total population (2 — 20%) makes it unlikely genically related to each other. Further experiments that they would be obvious in the electron micro­ to determine the relation between measles-haemag- scope if present in a similar proportion in pre­ glutination-inhibiting activity and neutralizing acti­ parations of measles virus, although there is some vity against these viruses are in progress. inhomogeneity in the size of particles. So far as viro- Besides this, the preparation is potentially valu­ microsomes are concerned, the inability of measles able as a vaccine. One dose can stimulate the pro­ virus to elute from red cells renders the preparation duction of a high titre of haemagglutination-inhibit- of such forms impossible, as they can only be pre­ ing antibodies in the rabbit, and the preparation is pared by adsorption from homogenates of infected of course non-infective. The results of neutralization cells, and elution from the red cells. tests in tissue culture suggest that haemagglutination- The term “haemagglutinin” might be taken to inhibiting antibody titre is also an index of neu­ include any or all of these forms, and has also been tralizing ability, and hence probably of protection applied to the actual substance or substances in the in the inoculated subject. Moreover, it has been viral coat which confer on it the ability to adhere shown that the separated HA of influenza viruses to red cells. However, it should be increasingly pos­ can be used for the production of haemagglutination- sible to explain and describe the various “haemag- inhibiting antibodies in the rabbit 6. and of clinical glutinins” of measles virus in terms of precise struc­ immunity to fowl plague virus in the chicken 28. This ture, and hence of their relationship to the intact in­ viral component was non-toxic for mice when given fective particle. intracerebrally or intranasally, in contrast to the in­ The practical value of the ether-tween preparation tact influenza virus particle. These findings may be is considerable. It is not only more economical of applicable also to measles virus. material in the haemagglutination-inhibition test, but Note Added in Proof: During the preparation of can be used for the detection of antibodies to rinder­ this report our attention has been drawn to the pu­ pest and distemper viruses, a use which is particu­ blication of findings similar to some of those in larly desirable in view of the failure hitherto to de­ this paper, by Dr. E. C. J. N o r r b y (Proc. Soc. exp. monstrate haemagglutination with these two viruses. Biol. Med. Ill, 814 [1962]). Its high sensitivity makes it more valuable for the detection of antibodies than preparations of the in­ tact virus. Hitherto the proof of serological rela­ We are indebted to Prof. W. S chäfer for his interest tionship among these three viruses has depended and criticism. We wish to thank those mentioned in the text for the gift of sera, the Behring-Werke (Marburg) mainly on neutralization tests 25, 26, in vivo or vitro, for a supply of monkey blood, and Frau U. S chäfer and on the CF test 27, but it is now clear that the for technical assistance. The work was supported by relationship can be demonstrated by HAI tests also, the Deutsche Forschungsgemeinschaft.

25 J. W a r r e n , Advances Virus Res. 7, 27 [I960]. 28 W. S c h ä f e r , in: The Nature of Viruses (Ed. A. E. W. 26 G . C a r l st r o m . Amer. J. Diseases Children 103, 287 [1962]. W olstenholme and E. C. P. M il l a r , p. 160, London 1957. 27 V. B e c h , Acta pathol. microbiol. scand. 50, 331 [I960].