Mechanism of hemagglutination by adenovirus types 1, 2, 4, 5 and 6

VIROLOGY

18,

l-8

(1962)

Mechanism

of Hemagglutination Types

H. G. PEREIRA

by Adenovirus

1, 2, 4, 5 and ASD

Accepted

M. V. T.

March

DE

6

FIGUEIREDO’

9. l!I&?

Fractionation of admovirus materials by rhromatography on DEAE-cellulose columns revealed two distinct hemagglutinating factors with elution peaks corresponding to those of antigens B and C previously described. Hemagglutination of rat crythrorytes by factor C requires the presence of a heterotypic virus antiserum in the system whereas factor B is capable of causing hemagglutination in the absence of such an antiserum. Hemagglutination by factor C results from adsorption of this factor to the red cell surface followed by agglutination by the heterotypic serum. Homotypic serum inhibits this renction by removing the sensitizing antigen from the rcll surface.

the relationship between the adenovirus hemagglutinin and either the virus particles The capacity of adenoviruses to agglutior t.lie noninfectious antigenic materials nate erythrocytes from a variety of animal produced by infected cells. Fractionation of species was described by Rosen (1958)) who adenovirus ant.igens by chromatography on also showed that this activity of adcnovirus DEAE-cellulose columns revealed two dispreparations is inhibited type specifically tinct hemagglutinating factors with elution by appropriate antisera. The hemagglutinapatterns corresponding closely t,o those of tion-inhibition technique has been successantigens B and C previously described fully used for the identification of adenoand (Pereira et al., 1959; Klemperer virus serotypes (Rosen, 1960; Bell et aZ., Pereira, 1959). It was further observed that 1960; Rosen et nl., 1961; Bellelli et al., hemagglutination by the factor correspond1961), the results obtained agreeing closely ing to antigen B can take place in the abwith those of the virus neutralization techvirus antiserum sence of heterotypic nique. This finding suggests that the same whereas hemagglutination by antigen C is t,ype-specific antigen is involved both in entirely dependent on the presence of such hemagglutination and in virus infectivity. antisera in the syst,em. Antigen C acts not Zuschek (1961) has recent.ly presented evias a true hemagglutinin, but as a sensitizdence that, t,he hemagglutinin of adenovirus ing factor rendering the red cells agglutintype 3 may be separated from the virus able by appropriate heterotypic ant,isera. particles and suggested the possible existThis sensitization is prevcnt’etl by homoence of 2 types of hemagglut.inins. The virus typic antibody. types included in the present investigation were shown by R.osen (1960) to form a MATERIALS AI\‘D METHODS group characterized by the ability to cause Adenovirus Prepnra tions. Prototype partial agglutination of rat cells, this agstrains of adenovirus types 1, 2, 4, 5, and 6 glutinat’ion becoming more marked when a were propagated in HeLa cells by methods heterotypic serum is added in appropriate previously described (Pereira and Kelly, concentration to the red cell suspension. 1957). The materials used consisted of exThe aim of our studies was to establish tracts of infected cells obtained by t.he tech’ World Health Organization Fellowship. nique of Gessler et al. (1956) using the 1KTRODI:CTIOK

2

PEREIRA AND

fluorocarbon compound Arcton 63 (I.C.I., London) as described by Pereira and Valentine (1958). These materials were fractionated by chromatography on diethylaminoethylcellulose columns at pH 6.5 and stepwise elution with NaCl concentrations varying from 0.01 to 0.5 111in 0.025 iM steps (Klemperer and Pereira, 1959). The volume of each fraction varied between 10 and 20 ml in different experiments. Some of the fractions were concentrated approximately 10 times by dialysis against polyethylene glycol (Carbowax, Union Carbide Ltd., London) as described by Kohn (1959). Some of t~he materials used in the present work had been stored at 4” for over two years either in the liquid state or as freezedried preparations. The latter were reconstituted to original volume with distilled water and stored at 4” without demonstrable loss of activity over a period of 2 months. Antisera. Adult rabbits were inoculated wit.h 2 to 4 intramuscular doses of virus material in Freund’s adjuvant (Freund and McDermott, 1942) at weekly intervals and bled 10 days after the last inoculation. Sera were inactivated at 56” for 30 minutes and stored at -10”. Before use, sera were absorbed with kaolin and with rat erythrocytes as recommended by Rosen (1960). Rat erythrocytes. White rats (SpragueDawley strain) were bled from the heart into a syringe containing 1 ml of a 0.001% solution of heparin. The erythrocytes were washed three times with normal saline (0.85% NaCl in deionized water) and stored at 4” as packed cells for up to 1 week. Before use, a volume of the packed cells was transferred to a graduated centrifuge tube, washed with saline by repeated cycles of centrifugation at 400 g for 5 minutes until no color was seen in the washing fluid. The volume of red cells was read from the tube graduation and an equal volume of saline was added to give a 50% cell suspension. For use in hemagglutination tests this was further diluted in saline to a final concentration of 1%. Some cell suspensions failed to sediment into a compact button under conditions of the hemagglutination test, but this could be overcome by adding a small amount of normal rabbit serum to

DE

FIGUEIREDO

such suspensions. To determine the concentration of serum necessary to cause this effect, 0.2-ml volumes of the cell suspension were added to 0.4 ml volumes of serial twofold dilutions of normal rabbit serum and allowed to settle at 37”. The highest dilution of serum necessary for the formation of a compact button of sedimented cells was added to the suspension. This dilution varied bet,ween 1:200 and 1: 1600, above which the addition of serum was unneccssary. Hemagglutination tests. Twofold dilutions of the virus material in normal saline were added in 0.4-ml volumes in Perspex hemagglutination plates. To each dilution, 0.2 ml of the red cell suspension was added and the plates were transferred to a hot room at 37” and left undisturbed until the formation of a negative pattern of sedimentation in the saline controls. In agreement with the findings reported by Rosen (1960), we observed that the agglutination of rat erythrocytes by the viruses included in the present study was incomplete throughout the range of dilutions tested giving rise to the formation of a ring of sedimented cells rather than to the typical shield seen in the case of complete agglut,ination. The latter could, however, be obtained by adding an adequate concentrat,ion of a heterotypic virus antiserum (within the types included in this study) to the red cell suspension used in the tests. By this procedure we observed not only a change of the sedimentation pattern from partial to complet,e hemagglutination, but also an increase in hemagglutination end point. It was also observed, as will be mentioned in detail below, that some of the virus materials fractionated by chromatography were able to cause hemagglutination only when tested in the presence of heterotypic antiserum. Hemagglutination titrations were therefore performed in duplicate, one series of dilutions receiving red cell suspension containing a heterotypic ant,iserum at a dilution (usually 1:200) known to enhance hemagglutination and a second series receiving a comparable suspension without heterotypic antiserum. Except when stated otherwise, a type 6 antiserum was added to red cell suspensions used for titrations of

materials derived from adenovirus types 1, 2, 4, and 5, and a type 5 antiserum for t.itrations of adenovirus type 6 materials. The titration end point was taken as the highest dilution causing approximately 50% hemaggiutination. Hemagglu tina tion inhibition ( HI) test. To 0.2-ml volumes of serial twofold dilutions of serum in saline, starting from 1: 20, was added 0.2 ml of a virus dilution containing 4-8 hemagglutination doses. After 1 hour’s contact at room temperature, 0.2 ml of red cell suspension containing 1: 200 hcterotypic antiserum was added to each cup and the plate was transferred to 37”. Each series of tests included titrations of t,he virus dilutions used and a test for possible residual hcmagglutinat,ing activity of the lowest dilutions of each serum. When the latter was positive the serum was reabsorbed with rat erythrocytes. The hemagglutinat,ion-inhibition titer was taken as the highest serum dilution causing a 50% reduction of hemagglut’ination. Heterotypic hemagglutination-enhancement (HE) test. The technique used was the same as for hemagglut’ination-inhibition except that, the serum-virus mixtures were not kept for 1 hour before addition of the red cell suspension and the latter did not contain heterotypic antiserum. Under these conditions the virus doses used in these tests cause at the most a trace of hemagglutination. The HE titer was t.aken as the highest serum dilut.ion showing complete or nearly complete hemagglutination. Complement fixation. Tests were performed by the technique described by Pereira i 1956). Digestion by trypsin. The virus materials were treated wit,h 0.1% crystalline trypsin (Armour and Company, Ltd. j in 0.07 M phosphate buffer pH 7.0 for 1 hour at 37” followed by addition of an equivalent amount of cryst,alline trypsin inhibitor (Worthington Biochemical Corporation). RESCLTS

Hemagglutinating Actizlity of Virus Materials Fractionated by Chromatography Fractions of adenovirus type 5 preparaon tion obtained by chromat,ography

DEAE-cellulose were tested for hemagglutinating activity in the presence and absence of heterot’ypic antiserum. From the results shown in the top part of Fig. 1, it is seen that hemagglutinat,ion activity was elutcd from t,he columns in two distinct ranges of NaCl concentrations, with peaks at 0.075 M and 0.175 M. It is further seen that whereas the hemagglutinating activity of the fractions making up the first peak was entirely dependent, on the presence of hcterotypic serum in the system, that of the second peak occurred both in the presence and, to a lesser extent, in the absence of such serum. The two elution peaks correspond closely to those of antigens B and C revealed by complement fixation (see Fig. 4, Klemperer and Pereira, 1959). and this was confirmed by testing the fractions obtained in the present experiment against antisera for type 5 antigens A and C used in opt,imal dilutions. The results shown in the lower part of Fig. 1 re\-eal a close parallelism between hemagglutination and reactivity with anti-C, but not with anti-A sera. These findings suggest that the factor responsible for hemagglutination dependent on heterotypic antiserum is identical wit,h the type-specific antigen C. This suggestion is strengthened by the fact t,hat. hemagglutination by this factor is inhibited by typespecific adenovirus antisera as shown by Rosen (1958, 1960) and confirmed in the present invest,igation. On the other hand, the fact,or causing hemagglutination in the absence of heterotypic antiserum corrcsponds in cliromatographic behavior to antigen B, which, as shown by Pereira (1960) is composed of a complex containing the type-specific component y and the early cytopathic factor p. To compare this hemagglutinin with antigen B, preparations of adenovirus types 1, 2, and 5 containing antigen B were digested by trppsin. This t,reatmcnt caused complete inactivation of both hemagglutinating activity in the nbsence of heterotypic antiserum and early cytopathic action, leaving unaltered the capacity to cause hemagglutination in the presence of heterotypic antiserum.

4

PEREIRA

-with

heterotypic

AND

antiserum

FIG. 1. Fractionat.ion of adenovirus type 5 by chromatography on DEAE-cellulose column. Stepwise elution with increasing concentrations of NaCI. Unconcentrated fractions (15 ml each) were tested for hemagglutinating and complement-fixing activities.

Adsorption of Hemagglutinating Rat Erythrocytes

Factors to

Attempts to demonstrate adsorption of hemagglutinating factors derived from adenovirus types 1, 2,5, and 6 to rat erythrocytes were carried out at different temperatures. Results obtained with adenovirus type 2 are representative of the whole group and are presented in Table 1. The factor causing hemagglutination in the absence of heterotypic serum is shown to be removed after contact with red cells at 37” but not at 4”. On the other hand, adsorption of the hemagglutinating activity dependent on heterotypic antiserum could not be demonstrated by this method. This point was further investigated by treating rat erythrocytes with adenovirus materials and subsequently washing the cells thoroughly with saline and testing their agglut,inability by

DE FIGCEIR,EDO

heterotypic and homotypic sera. From the results shown in Table 2 it is seen that treatment by adenovirus type 5 antigen C renders the erythrocytes agglutinable by heterotypic, but not by homotypic, antisera. Similar results were obtained using antigen C preparations derived from adenovirus types 1, 2, and 6. This alteration in agglutinability of rat eryt.hrocytes by adenovirus antisera could conceivably be due to a change in the cell surface brought about by the action of the material or to adsorption of an antigen which would sensitize the cells rendering them agglutinable by heterotypic antiserum. Lack of agglutination by homotypic antiserum might be due to a higher affinity of this serum for the antigen in question resulting in its removal from the cell surface. Results of the experiment presented in Table 3 reveal that incubation of antigen C-treated erythrocytes with homotypic antiserum results in loss of agglutinability of the red cells by heterotypic antiserum suggesting removal or blockage of the sensitizing antigen responsible for this reaction. It was subsequently shown that the erythrocytes treated with antigen C followed by homotypic antiserum were identical to normal red cells in their agglutinTABLE

1

ADSORPTION OF ADENOVIRUS TYPE 2 HEMAGGLUTININS TO RAT ERYTHROCYTESQ Temperature of adsorption (“Cl 4 22 37 Unadsorbed controls (40, 220, 37”)

HA titer* With type 6 antiserum 480 480 480 480

Without type 6 antiserum 60 40
a Adenovirus type 2 was diluted 1: 10 in normal saline; l-ml volumes were transferred to 6 tubes, 3 of which received 0.1 ml of packed rat erythrocytes; pairs of tubes (1 with and 1 without, erythrocytes) were incubated for 1 hour, each at 4”, 22”, and 37”, respectively; cells were removed by centrifugation at 400 9 for 5 minutes, and fluids were tested for hemagglutinating activity. * Reciprocal of highest dilution causing 50YC hemagglutination.

HEMAGGLUTINATION

BY SDENOVIRUSES

TABLE ,~GGI,~TISATI~~

2

OF 11.4~ ERYTHROCYTES TREATED WITH TYPE 5 ANTIGEN .~NT) HETEROTYPIC ANTISERA"

C BY HOMOTYPK

Serum dilutions Antiserum

Ad. type 1

5C-treated Untreated

-lb 0

4 0

4 0

4 0

4 0

2 0

0 0

0 0

Ad. type 2

X-treated Untreated

4 0

4 0

4 0

4 0

4 0

4 0

3 0

0 0

Ad. type 5

5Ctreated Untreated

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

e Antigen C obtained from adenovirus type 5 by chromatographic fractionation was diluted 1:80 in saline; packed erythrocytes a-ere added to give a cell concentration of lGjO;incubated at 37” for 1 hour. Cells were sedimented by centrifugation, washed 4 times, each with twice the original volume of saline, and resuspended in saline to a concentration of 1%; after addition of 1:200 normal rabbit serum, 0.2.ml volumes of the cell suspension were added to 0.4.ml volumes of twofold dilutions of antisera. An untreated erythrocyte suspension was tested alongside. * Numbers: 4 = complet,e agglutination; 1 to 3 = increasing degrees of partial agglut,ination; 0 = absence of agglutination. TABLE EFFECT OF HOMOTYPIC ANTISERUM

3

ON AGGLVTINABILITY

OF ANTIGEN

C-TREATED

ERYTHROCYTES~

Type 2 antiserum dilutions Erythrocptes 5C-treated 5Ctreated + type 5 antiserum Untreated Untreated + type 5 antiserum

1:80

1:160

1:320

1:&&O

1:1280

1:2560

1:5120

1:10240

4h 0

4 0

4 0

4 0

4 0

3 0

1 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

a Rat erythrocytes were treated with type 5 antigen C as described in Table 2. Final 1% suspension was divided into 2 aliquots, to one of which was added 1% type 5 antiserum. Both aliquots were incubated for 1 hour at 37”, washed 3 times each with 10 ml of saline, and resuspended in saline with 1:X@ normal rabbit serum to give a 17o suspension. Two aliquots of normal erythrocytes (untreated u-ith 5C antigen) were carried through the same procedures and tested alongside. Agglutination of each cell suspension by type 2 antiserum was tested by adding 0.2 ml of suspension to 0.4 ml of serum dilutions. * See Table 2.

by both homotypic and heterotypic adenovirus preparations. Thus, red cells treated with type 5 antigen C followed by type 5 antiserum as described above were agglutinated to the same extent as normal cells by adenovirus types 1, 2, 5, and 6. Therefore, sensitization of erythrocytes by antigen C seems to be completely reversed by treatment with homotypic antiserum. ability

Hemagglutination

Inhibition

Sera from rabbits immunized with unfractionated virus materials or with antigens A or C obtained by chromatographic fractionation were tested against homologous and hcterologous virus types. The results shown in Table 4 are in complete agreement with the findings reported by Rosen (1958, 1960) demonstrating the type

6

PEREIRA

AND

FIGUEIREDO

DE

TABLE

Serotype

1

2

4 4 4

5 5 5 6 6

HI Titers.* virus type

Antigen

1

160 <40 <40 <40

<40 <40

<40 <40

<40 <40

(40 (1-O <40

2

4

5

6

<40 <40 640 <40 320 <40 <40 <40 <40 <40

<40 <40 <40 <40

<40 <40 <40 <40 <40 <40 <40

<40 <40 <40 <40 <40 <40 <40 (40 <40 <40 C40 320 160

<40 <40 <40

NT 640 1280 LO240 <40 <40 <40 <40 <40

<-lo 1280 <40 240 <40 <40

TITERS

OF

HE Titers,’ virus type

-

Unfractionated Antigen A Unfractionated Antigen A Antigen C Unfractionated Unfractionated Unfractionated Unfractionated Antigen A Antigen C Unfractionated Antigen C

1 2 2

(HE)

AND HETEROTYPIC-ENHANCEMENT RABBIT ANTISERA

7-

Antiserum’

4

(HI)

HEMAGGLUTINATION-INHIBITION

1 2560 <40 640 40 40 2560 640

<40 320 640 640

6

2

4

5

480 <40

160 <40 640

480

480

<40 1280

<40 1280 <40 640 <40 <40 1280 640 (40 640

NTd NT <40 40 2560 320 (40 320 640 640

<40 640

<40 <40 1280

160 <43

-

160

320

XT

640 1280

(1Sera from rabbits immunized with unfractionated virus materials or with antigen A or C obtained by chromatography. 6 Reciprocal of highest serum dilution showing 507& reduction of hemagglutination. c Reciprocal of highest serum dilution showing complete or nearly complete hemagglutination. d NT = Not tested.

specificity of the HI tests. Our results show in addition that the antibodies responsible for this reaction are, as expected from the type specificity of the reaction, those directed against antigen C. Several antisera from rabbits immunized with purified antigen A had no demonstrable HI activity. These anti-A sera tested by complement fixation or by gel-diffusion precipitation against homologous or heterologous adenovirus antigens were shown to contain high levels of antibody reactive with the groupspecific antigen A. Heterotypic ment

Hemagglutination

Enhance-

From the results presented in Table 4 it is seen that the capacity of adenovirus antisera to enhance hemagglutination by heterologous virus types is on the whole correlated wit.h homologous HI activity. This is particularly clear within the subgroup formed by adenovirus types 1, 2, 5, and 6, where HE titers are as a rule slightly higher than HI titers. Adenovirus type 4, on the other hand, shows certain differences in behavior.

Thus, HE titers

determined

against

this type tend to be lower than those against types 1, 2, 5, and 6, and conversely HE titers of type 4 antisera are significantly lower than HI titers. It should be noted that, of the three type 4 antisera tested, only the one with an exceptionally high HI titer was capable of enhancing hemagglutination by all the heterologous types included in the test. COMMEKTS

It has been previously demonstrated that antigens A, B, and C extracted from cells infected with certain adenovirus types contain different antigenic components forming complexes with each other or with deoxyribonucleic acid (Allison et al., 1960; Pereira, 1960). The antigenic components present in antigens A and C were named antigens O(and 7, respectively. Component 7 is also present in antigen B, where it forms a complex with component p. The latter corresponds to the protein factor responsible for cell detachment (Rowe et al., 1958) and early cytopathic effects caused by adenoviruses (Pereira, 1958; Everett and Ginsberg, 1958).

HEMAGGLUTINATION

In the present investigation it has been shown that the hemagglutinating activity of the adenovirus types studied is associated w&h antigens B and C, both of which contain the type-specific component 7. This finding was to be expected in view of the type specificity of the adenovirus hemagglut.ination-inhibition test (Rosen, 1958, 1960). Our results offer in addition a possible explanation for the enhancing effect of hcterotypic sera on the hemagglutinating activity of t’he adenoviruses under study. This phenomenon was described by Rosen ( 1960), who pointed out’ that reciprocal enhancement of hemagglutination is limited to a subgroup of adenoviruses including types 1,2,5, and 6. According to our findings adenorirus type 4 should bc added to this subgroup although its behavior in this, as in other respects, differs from that of the other types. We have been able to demonstrate that heterotypic antisera act’ in this reaction by agglutinating red cells sensitized by antigen C. Homotypic antisera, on the other hand, are capable of removing the sensitizing ant,igen from the red cell surface, thus preventing agglutination by lieterotypic serum. According to these findings it must be presumed that the antigen C involved in this reaction contains antigenic determinants capable of reacting both with type-specific and with subgroup specific antibodies. The type-specific determinant corresponds to component y previously described and the subgroup-specific determinant will be referred to as component 6. The mechanism of hemagglutination by antigen B remains to bc determined, but one might speculate that’ the combination between components p and 7 present in this antigen may result in aggregates with a similar to those spatial configuration formed by the combination of C antigen with heterotypic antibody. Evidence for the presence of a subgroupspecific component 6 in antigen C has only been obt,ained so far by means of the hetcrotypic hemagglutination-enhancement test here described. Attempts to demonstrate hcterotypic reactions between purified antigen C and respective antisera by complement fixation or gel-diffusion precipitation have been ent,irely negative. It is impossible

BY ADENOVIRUSES

i

for us to say at present whether this failure is due to lack of sensitivity of the techniques used or to peculiarities of the antigen-antibody reaction taking place in this system. The mechanism of hemagglutination mediated by heterotypic sera corresponds essentially to that of indirect liemagglutination reactions described in many antigenantibody systems. Application of this type of reaction to the study of adenoviruses has been rcportcd by several workers (Friedman and Bennett, 1957; Prier and Le Beau, 1958; Ross and Ginsberg, 1958; Carmichael and Barnes, 1961)) who showed that sheep erythrocytes treated with tannic acid and sensitized with adenovirus antigens arc agglutinated by both type-specific and groupapccific adenorirus antisera. These results suggest that t’lie antigen mainly involved in this reaction is the group-specific antigen A rather than the antigen C responsible for the reactions studied in the present, inveetigation. Further work is required before the extent to which the present findings are applicable to other adenovirus types may be determined. It is likely that, hemagglutination by different adenoviruses may depend on different mechanisms and even within a given type various factors may be involved in this reaction, depending on experimental conditions and the kind of red cells used. Studies carried out with many types of adenoviruses of human origin (Rosen, 1958, 1960; Bell et nl., 1960; Rosen et nl., 1961; Zuschek, 1961) and with infectious canine hepatitis virus (Fastier, 1957) demonst.rate a wide variation in hemagglutinating properties of different adcnoviruses. More extensive studies may allow the identification of further subgroups of adenoviruses on the basis of hemagglutinnt,ing and other properties. ACKNOWLEDGMENTS WC are inckbtrd to V. G. Law and .I. D. Haswell for valuable twhnical xssistanw. REFERESCES A. C., PEHEIRA, H. G., and FARTEIIR.G, C. P. (1960). Investigation of adenovirus antigens by agar gel diffusion techniqws. Virolo~?~ 10, 316328. BELL, S. D. JR., ROTA. T. R., and MCCOIIB. D. E. ALLISON,

8

PERE1R.A

AND

(1960). Adenoviruses isolated from Saudi Brabia. III. Six new serotypes. Am. J. Trap. Med. Hyg. 9,523-526. BELLELLI, E., VITALI, M. 4., and Moiuc~, V. (1961). Sulla tipizzazione degli adenovirus con la prova di I.E.A. Ann. Sclavo 3, 161-170. CARMICH.AEL, L. E., and BARSES, F. D. (1961). Serological comparisons between canine hepatitis virus and human adenovirus types. Proc. Sot. Exptl. Biol. Med. 107,214-218. EVERETT, S. F., and GINSBERG, H. S. (1958). A toxinlike material separable from type 5 adenovirus particles. Virology 6, 770-771. FASTIER, L. B. (1957). Studies on the haemagglutinin of infectious canine hepatitis. J. Immunol. 78, 413-418. FREUXD, J., and MCDERMOTT, K. (1942). Sensitizabon to horse serum by means of adjuvants. Proc. Sot. Exptl. Biol. Med. 49, 54%553. FRIEDMAN, M., and BENNETT, C. R. (1957). A hemagglutination test for detection of adenovirus antibodies. Proc. Sot. Exptl. Biol. .Wed. 94,712-717. GESSLER, A. E., BENDER, C. E., and PARKINSON, M. C. (1956). B new rapid method for isolating viruses by selective fluorocarbon deproteinization. Trans. N. Y. Acad Sci. 18, 701-703. KLEMPERER, H. G., and PEREIRA, H. G. (1959). Study of adenovirus antigens fractionated by chromatography on DEAE-cellulose. Virology 9,536-545. KOHS, J. (1959) A simple method for the concentration of fluids containing protein. Nature 183, 1055. PEREIRA, H. G. (1956). Typing of adenoidalpharyngeal-conjunctival (BPC) viruses by complement fixation. J. Pathol. Bactekol. 72, 10% 109. PEREIRA, H. G. (1958). A protein factor responsible

DE FIGUEIREDO

for the early cytopathic effect of adenoviruses. Virology 6,601-611. PEREIRA, H. G. (1960). Antigenic structure of noninfectious adenovirus materials. Nature 186, 571-572. PEREIRA, H. G., and KELLY, B. (1957). Doseresponse curves of toxic and infective actions of adenovirus in HeLa cell cultures. J. Gcn. Microbiol. 17,517-524. PEREIRA, H. G., and VALENTINE, R. C. (1958). Infectivity titrations and particle counts of adenovirus type 5. J. Gen. Microbial. 19, 178-181. PEREIHA, H. G., ALLISON, A. C., and FARTHIN<;, C. P. (1959). Study of adcnovirus antigens by immunoelectrophoresis. Nature 183, 895-896. PRIER, J. E., and LE BEAU, R. W. (1958). Studies on t,he serologic identification of adenovirus. I. Comparison of the hemagglutination, conglutinating complement adsorption and complement fixation tests. J. Lab. Clin. Med. 51, 495-500. ROSES, L. (1958). Hemagglutination hy adenoviruses. Virology 5, 574- 577. ROSEN, L. (1960). A hemagglutination-inhibition technique for typing adenoviruses. Am. J. Hyg. 71, 120-128. ROSEN, I,., BAROS, S., and BELL, J. A. (1961). Foul newly recognized adenoviruses. Proc. Sot. Exptl. Biol. -Wed. 107, 434-437. Ross, E., and GISSBERG, H. S. (1958). Hemagghitinat,ion with adenoviruses. Nature of viral antigen. Proc. Sot. Exptl. Biol. Med. 98, 501-505. ROWE, W. P., HARTLEY, J. W., ROIZMAN, B., and LEVY, H. B. (1958). Characterization of a factor formed in the course of adenovirus infection of tissue cultures causing detachment of cells from glass. J. Exptl. Med. 108, 713-729. ZUSCHEK, F. (1961). Studies of the hemagglutinins of type 3, 4 and 7 adenovirus. Proc. Sot. Exptl. Biol. Med. 107,27-30.