JOURNAL OF VIROLOGY, May 1969, p. 477-483Copyright © 1969 American Society for Microbiology

Vol. 3, No. 5Printed in U.S.A.

Substituted Sialic Acid Prosthetic Groups as

Determinants of Viral HemagglutinationBARBARA LEVINSON,' DUNCAN PEPPER,2 AND GEORGE BELYAVIN

Bacteriology Department, University College Hospital Medical School, London, England

Received for publication 29 January 1969

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolatedstrain of type B influenza virus were separated by sucrose density gradient centrifu-gation and agarose gel filtration from horse serum. Using selected reagents, it wasdemonstrated that the active substituent on the horse serum inhibitor of A2 in-fluenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituenton the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid(NANA). Sodium metaperiodate treatment of a component of horse serum resultedin a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereasthe A2 inhibitor was completely destroyed. Since this enhancement did not occurwith influenza B viruses isolated prior to 1965, it was considered that this sensitivityto an oxidized NANA glycoside may have been a reflection of an antigenic changewhich occurred at that time. The use of different virus strains and selected chemicalreagents to define the important sialic acid prosthetic groups active in inhibition wasdescribed.

Inhibitors of influenza virus hemagglutinationhave been discovered in many naturally occurringsubstances, including various animal sera and se-cretions. These inhibitors, which are capable ofblocking hemagglutination by prior reaction withthe virus, provide a system of competitive inhibi-tion which allows the study of the virus-red cellreaction in considerable chemical detail.

Early work in the field of hemagglutinationinhibitors was instrumental in establishing theimportance of N-acetylneuraminic acid (NANA)as the essential prosthetic group responsible forlinking the influenza virus to the surface of thered cell (7, 11). All of the well-characterized in-hibitors of viral hemagglutination that have beenpurified so far, such as the urinary mucoproteinisolated by Tamm and Horsfall (22) and ovinesubmaxillary gland glycoprotein (8), containNANA as the biologically active grouping. Therecognition of neuraminidase-insensitive inhibi-tors indicates that another receptor grouping,besides NANA, may be implicated in the bindingof some of these viruses (5, 20). The neuramini-dase-resistant inhibitor of influenza A2 virus,present in horse serum, is well known, but theidentity of the inhibitory receptor grouping as a

I Present address: Department of Biochemistry and Biophysics,University of Catifornia School of Medicine, San Francisco, Calif.94122.

2 Present address: Blood Program Research Laboratory, Ameri-can National Red Cross, Washington D.C.

sialic acid other than NANA has only recentlybeen established (16). The isolation of this in-hibitor in a sufficiently purified state to enablethe investigation of the types of neuraminic acidspresent has made this work possible (2, 15).

This report describes the use of chemical rea-gents which make relatively minor alterations ofthe sialic acids but which result in profoundchanges in the inhibition of the virus. Most of thework was done using a component ofhorse serum,equine a-macroglobulin (EcaG), which containsthe inhibitor of influenza A2. Evidence is pre-sented that horse serum also contains an inhibitorto contemporary influenza B viruses which canbe physically separated from the already de-scribed A2 inhibitor.

MATERIALS AND METHODSViruses. The two strains of influenza virus used

in this study were the inhibitor-sensitive variant ofA2/Singapore/57 (5) previously used in this depart-ment and B/England/13/65, originally received fromA. S. Beare. The viruses were grown and purified asalready described (14).

Hemagglutination (HA) and hemagglutination.inhibition (HI). One method used to determine theHA and HI titers was the standard "pattern" methodusing eight agglutination doses of virus in plastictrays (24). A second method, a continuous-flowtechnique utilizing the Technicon Autoanalyzer, wasused to screen column- and density-gradient fractions

477

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

LEVINSON, PEPPER, AND BELYAVIN

for inhibitory activity. The technique is based on thatdescribed by Cohen (4), except that a second channelwas added to allow samples to be screened againsttwo viruses simultaneously. In this technique, a singlesampler module is used, a sample being taken upthrough a pair of probes with each probe feeding intoa separate channel. The results, expressed as opticaldensity of hemoglobin at 420 nm, are continuouslydisplayed on a recorder. One unit of optical density(Technicon unit) is defined as an excursion fromthe virus baseline of 3 mm. This system was usedmainly to give a semiquantitative indication of theinhibitor distribution in column and gradient samples.

Sera and serum fractions. Whole horse serum, froma single horse, was obtained from Burroughs Well-come & Co. (Beckenham, England) and was stored at-15 C.A 5-g sample of EaG, lyophilized and salt-free,

was obtained from Calbiochem (Los Angeles, Calif.;grade B, Cohn fraction IV, batch no. 55391) andstored at 4 C.

Partially purified EaG (PP5) was prepared by pool-ing the inhibitory fraction from six agarose gel-filtration runs, using as starting material EaG. Thepooled fractions were concentrated by ultrafiltrationand rerun on the same column to remove aggregates.The active fractions were pooled and run on a sucrosedensity gradient. The dialyzed and concentratedinhibitor material was examined in the analyticalultracentrifuge and showed one sedimenting peak,which was calculated to be 18S.A number of randomly collected human serum

samples were pooled to form a single 30-ml batch.This was stored untreated at -15 C.

Sucrose density gradients. A sample was layeredonto a 19-ml linear sucrose density gradient (45 to9% sucrose in borate-buffered saline, pH 7.6) andrun in the no. 30 swing-out rotor of the MSE Super-speed 50 ultracentrifuge at 75,000 X g for 16 to 18 hr.Fractions (40 drops/ml) were collected by piercingthe bottom of the tube and collecting drops in suc-cessive tubes by hand.

Molecular exclusion chromatography. A columnof agarose beads (4% gel, 120 mesh, 1.16 by 123 cm)was prepared and calibrated (14). The sample waslayered on the column and eluted at 3.0 ml/hr with0.15 M NaCl containing 0.02% NaN3.

Periodate treatment of inhibitor. The conditions foroxidation of glycoproteins by NaIO4 were thoserecommended for the specific oxidation of straight-chain cis-hydroxyl groups and which reduce non-Malapradian oxidations to a minimum (17, 19).The concentration of sodium metaperiodate was ad-justed to give a ratio of moles of periodate not reducedto moles of periodate consumed of approximately2 on the basis of the available oxidizable groups. Inthe case of whole serum, this consisted of three vol-umes of 0.011 M NaIO4 to one volume of undilutedserum. The reaction was carried out in the dark, atpH 4.5 and at 4 C for 15 min; at that time, the reactionwas stopped by the addition of one volume of 10%glucose. After an additional 15 min at 4 C, the treatedserum or inhibitor was ready for testing. The reagentswere made up in 0.15 M NaCI immediately before

use. Under these conditions, it is unlikely that ex-tensive oxidation of pyranose rings occurs.

Neuraminidase treatment. Purified Vibrio choleraeneuraminidase (Seravac Laboratories, Maidenhead,England; 500 units/ml) was incubated with serum orinhibitor overnight at 37 C. A concentration of 50 to100 units of neuraminidase to 30 mg of protein wasfound to be completely effective in destroying in-hibitor activity. All dilutions were made in calciumacetate buffer at pH 6.2.

Alkali treatment of EaG. A 12-mg sample of EaGwas mixed with 1.0 ml of 0.1 N NaOH and, after 20min at room temperature, the pH was adjusted toneutrality with 1.0 ml of 0.1 N HCl and 0.2 ml of 1 MP04 buffer. The neutralized mixture was then titratedfor inhibitory activity. In a number of experiments,alkali treatment was followed by exposure to bac-terial neuraminidase or periodate. When alkalinehydrolysis was followed by addition of neuramini-dase, the pH was first adjusted to 6.2 with calciumacetate buffer, and the reaction was allowed to pro-ceed for 20 hr at 37 C in the presence of 50 units ofenzyme.

Complement fixation. A microtechnique was usedto determine the presence of antibody to the type-specific V antigen in the human serum pool (1).

RESULTSEffect of neuraminidase and periodate on horse

and human serum inhibitors. It has been shownpreviously that the inhibitory effect of horseserum against A2 influenza viruses is readilyabolished by treatment with sodium metaperio-date, whereas neuraminidase is ineffective indestroying the inhibitor activity (5). In contrastto horse serum, the human serum inhibitor to A2influenza viruses is destroyed by both methods oftreatment.

In view of these results, it was of interest todetermine the effect of these inhibitors and thesetreatments on the hemagglutinating activity of anewly isolated strain of type B influenza virus,B/England/65. As can be seen in Table 1, thetype B virus was relatively insensitive to untreatedhorse and human serum. However, when theserum was treated with NaIO4, the inhibitoryactivity to A2 was lost but the activity againstB/Eng/65 was enhanced 8- to 15-fold. Con-versely, neuraminidase is effective in removal ofthe B inhibitor in both sera. This phenomena hasbeen observed recently in human serum withother contemporary type B strains (13). Comple-ment fixation tests, using the V antigen of boththe A2 and the B virus, confirmed the absence ofantibody in horse serum and demonstrated onlya low level present in the pooled human serumwhich is responsible for the residual inhibitoryactivity after neuraminidase treatment. Alsotested were two preparations of horse seruminhibitor, EaG and a highly purified component

478 J. VIROL.

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

DETERMINANTS OF VIRAL HEMAGGLUTINATION

EaG-PP5. It was confirmed from these experi-ments that the semipurified EaG fraction be-haved identically to whole horse serum and thehighly purified material with respect to thetreatments used; due to its substantially higherinhibitory activity, this material was used in allthe subsequent experiments.

Fractionation of EaG and human serum. Treat-ment of the macroglobulin fraction with neu-raminidase destroyed the periodate enhancementof inhibition of the B virus (Table 2), indicatingthat this effect was dependent on a moleculecarrying a substituted neuraminic acid prostheticgroup. As the inhibitory effect against A2 virusis not destroyed by neuraminidase, it is conceiva-able that two distinct molecular species of in-hibitor are present in horse serum, one active

TABLE 1. Effect ofneuraminidase and NaIO4 on thehemagglutination inhibition potency of horse

and human serum and derived fractions

Serum

Horse

Human

EaG (60 mg/ml)

EaG-PP5(6.1 mg/ml)

Treatment

NoneNaIO4Neuraminidase

NoneNaIO4Neuraminidase

None

NaIO4Neuraminidase

None

NaIO4Neuraminidase

HI titers

A2/Sing/57 B/Eng/65

1,280 20<5 160

1,280 <5

320 48060 3,84080 60

60,000 600

<10 10,00060,000 <10

30,000 8

<10 2430,000 <10

TABLE 2. Effects of multiple treatment with alkali,neuraminidase, and periodate on thehemagglutination inhibition activity

of EaG

Treatment HI titer

1 2 A2/Sing/57 B/Eng/65

Untreated <60, 000 600Neuraminidase 60,000 <10NaTO4 <10 10,0000.1 N NaOH 2,560 1,280Neuraminidase NaIO4 <25 <250.1 N NaOH Neuraminidase 60 <200.1 N-NaOH NaIO4 <36 18,400Neuraminidase 0.1 N NaOH 2,560 960

against A2 and the other active against B/Eng/65.Further evidence to support this hypothesis is thefinding that the highly purified horse serum in-hibitor (EaG-PP5) showed a lower inhibition ofthe type B virus relative to whole serum and EaG(Table 1). Since this fraction has been isolated onthe basis of A2 virus inhibition, this is also sug-gestive of multiple inhibitor populations. In con-trast, human serum would be expected to containonly one inhibitor active against the two viruses,since treatment with neuraminidase results inequal destruction of inhibitory activity againstboth viruses.

In an attempt to separate the inhibitory activitypresent in horse and human serum, sucrosedensity gradient centrifugation and agarose gelfiltration were done. The separation of the A2and B virus inhibitor peaks was achieved inEaeG, substantiating the presence of at least twomolecular species responsible for inhibition(Fig. 1, 2). The inhibitory activity of humanserum was not resolvable into more than onepeak, indicating that most likely only one in-hibitor is responsible for the activity of humanserum against the two viruses (Fig. 3, 4).

Identification of the inhibitory prosthetic groups

.0

0z

zLU1-

zI

Uz

0

I

U

z

J

u

I

2000

N

0z

1500 c;4

zDz0U

1000 zIuU

z

-j500

U

0-0

FRACTION NUMBER

FIG. 1. Comparison of the distribution of inhibitoryactivity against A2/Sing/57 and B/Eng/65 infractionsderived from agarose column separation of untreatedEaG. A 0.4-ml (24 mg) sample of EaG was layeredonto an agarose gel column and eluted at a rate of3.0 ml/hr with 0.15 M NaCI. Fractions of 1.5 ml werecollected, and the HI activity was determined by use ofthe Technicon autoanalyzer.

VOL. 3, 1969 479

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

LEVINSON, PEPPER, AND BELYAVIN

by chemical treatment. The differences in the bio-logical effects of the EaG inhibitors can mostlikely be ascribed to variations in the relevant in-hibitory prosthetic groups present on the respec-

A2/SING/57

10 150-

z

1-

Z 100

z0

zI

l-

>- 50

z

U

C.

0

N

15000

z

l-

1000 z

Dz0

z

u

500>-

z-A

U

0.

0

FIG. 2. Comparison of the distribution of inhibitoryactivity against A2/Sing/57 and B/Eng/65 infractionscollected from sucrose density gradient centrifugationof EaG. A 0.5-ml (30 mg) sample of EaG was layeredonto a 19-ml linear sucrose gradient and run for 18 hrat 75,000 X g. The HI activity was determined by useof the Technicon autoanalyzer.

70-- A2/SING/57o---O B/ENG/65

Z 60

z50

U

zz

u 40 -

30

z

~20

oo

FRACTION NUMBER

FIG. 3. Comparison of the distribution of inhibitoryactivity against A2/Sing/57 and B/Eng/65 infractionsderived from agarose column separation of pooledhuman serum. A 0.5-ml sample of human serum was

layered onto the agarose gel column and eluted with0.15 M NaCl. Fractions of 1.5 ml were collected, andthe HI activity was determined by use of the Techniconautoanalyzer.

60

z

z50--*A2/SING/57

A-BB/ENG/65Z 40 -

I

~ 30

Z 20

<0 10 .>vI-~~~o

0

5 10 15

FRACTION NUMBER

FIG. 4. Comparison of the distribution of inhibitoryactivily against A2/Sing/57 and B/Eng/65 infractionscollectedfrom sucrose density gradient centrifugation ofpooled human serum. The 0.5-ml sample was layeredonto a 19-ml linear sucrose gradient and run for 18 hrat 75,000 X g. Fractions of 1.0 ml were collected,and the HI activity was determinedby use ofthe Techni-con autoanalyzer.

tive inhibitory molecules. Therefore, attemptswere made to further characterize these inhibitorsby chemical means.

It is known from previous studies, performedin this laboratory by Pepper (16), that thesialic acids present in purified horse serum in-hibitor are NANA, N-glycolylneuraminic acid(NGNA), and the O-acetylated derivatives ofthese acids. The latter constitute over 50% of thesialic acids present in horse serum and are resist-ant to the action of bacterial neuraminidase. Thesialic acid in horse serum, active against A2 virus,is most likely the O-acetyl-N-acetylneuraminicacid (O-acetyl NANA) since NGNA is known tohave little inhibitory activity (F. Biddle, personalcommunication) and the glycosidic linkages ofNANA are susceptible to the action of neura-minidase. In addition, dilute alkali, under certainconditions, has been recently shown by Schauerand Faillard (18) and Pepper (16) to remove

O-acetyl groups from substituted neuraminicacids. Thus, when EaG was exposed to diluteNaOH, the inhibitory activity to A2 virus wasreduced to 3% of its former level, and the re-

maining activity was then susceptible to neura-minidase (16). This is presumably due to theremoval of the O-acetyl group, resulting inNANA which has a low degree of inhibitoryactivity against A2 virus. Therefore, it seemeduseful to examine the effects of mild alkalitreatment in combination with enzymatic hy-

480 J. VIROL.

11

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

DETERMINANTS OF VIRAL HEMAGGLUTINATION

drolysis and periodation on the biological prop-erties of the horse serum inhibitors.

In this experiment, a sample of EaG wastreated with dilute NaOH and followed by expo-sure to neuraminidase or periodate. Table 2 showsthe inhibitory titer of EaG to the two virusesafter the various treatments. As can be seen,treatment with alkali, which removes the O-acetylgroup, increases the inhibition of the B virus, pre-sumably due to the increased amount of NANA.As would be expected, subsequent NaIO4 treat-ment resulted in a dramatic rise in the inhibitorytiter. Treatment with neuraminidase, after expo-sure to alkali, removes the nonacetylated neur-aminic acids, thereby removing all inhibitory ac-tivity to the B virus. It is clear that any treatmentwhich results in an increase in available NANAheightens the HI titer to the B virus, and the pro-duction of an oxidized NANA glycoside in-creases the inhibitory titer approximately 10-foldmore. Conversely, the A2 virus is most sensitive toneuraminidase-resistant, acetylated neuraminicacids, and any treatment which results in removalof the acetyl group-i.e., NaOH-or oxidizes themolecule, substantially reduces or destroys theactivity.

DISCUSSIONThe experiments using treated horse serum in-

hibitor lead to the conclusion that horse serumcontains two inhibitor populations which can bephysically separated and which possess at leasttwo different biologically active sialic acids. Theprobable composition of the active, terminalsialic acid groups can be deduced from their re-action with the various reagents used in thisstudy. To more easily comprehend the sites of at-tack of these reagents, Fig. 5 shows the points atwhich the various chemicals are active under thedescribed conditions.

NEURAMINIDASE CHOH

.0 i 104CHOH

_51 104-CH2I° HCH20H

FIG. 5. Sites ofaction ofNaIO4, neuraminidase, anddilute alkali on the 4-0-acetyl-N-acetylneuraminicacid.

The susceptibility of the B-virus horse seruminhibitor to neuraminidase, whereby both the in-hibition and the periodate enhancement effect areabolished, points to the fact that a simple NANAmust be the principle receptor involved in thisreaction. It is also clear that the inhibition is at arelatively low level until the oxidation of a por-tion of the molecule by periodate. Using thedescribed condition for periodate oxidation, thestraight-chain cis-hydroxyl groups are morerapidly oxidized than those occurring within thepyranose ring (19). Assuming that the sialic acidgroup is terminal and is linked at C2 to a hexos-amine, the only straight-chain cis-hydroxylgroups occur at C7, C8, and C9, the oxidationof which would result in an aldehyde group atC7. This new prosthetic group is a much moreeffective inhibitor of hemagglutination than theunoxidized molecule, presumably due to a betterfit with the receptor on the virus surface.The A2 virus inhibition is clearly not due to

NANA because of its insensitivity to neuramini-dase. Most of the evidence indicates that the in-hibition is due to another substituted neuraminicacid, most likely the O-acetyl NANA. Here themost significant fact is the demonstration thatmild-alkali treatment of the horse serum in-hibitor, which removes O-acetyl groups, rendersthe A2 inhibitory component fully susceptible todestruction by bacterial neuraminidase and theresidual activity is almost equally active againstboth viruses. Moreover, it is apparent that alkalitreatment converts the inhibitor into one that isalso susceptible to periodate enhancement, i.e.,NANA. These facts together mean that the anti-A2 component also inhibits by binding througha substituted neuraminic acid group and thatboth viruses are sensitive to NANA, but optimalinhibition is achieved by slight modifications ofthe molecule.

It is possible to localize the position of thesecond acetyl group of the O-acetyl NANA to theC4 position by the use of the B virus and appro-priate chemical reagents. Acetylation at C7 is notpossible, as this substitution would prevent theformation of a chromogen in the Warren thio-barbituric acid assay of sialic acids (23). Since thetotal substituted neuraminic acid content ofEaG and purified equine inhibitor is identical byboth the thiobarbituric acid and Svennerholmresorcinol test (21), an acetyl group at C7 is ex-cluded. Acetylation at C8 is not compatible withthe data, as this form would not be oxidizable byperiodate at all and acetylation at C9 wouldresult, after periodation, in an aldehyde whichwould be indistinguishable from oxidized NANAglycoside. The combined use of neuraminidaseand periodate, which resulted in no effective in-

VOL. 3, 1969 481

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

LEVINSON, PEPPER, AND BELYAVIN

hibition of the B virus, demonstrated that thisvirus is insensitive to an oxidized O-acetylatedNANA. The only remaining possibility is acetyla-tion at C4. On this basis, the essential prostheticgroup on the A2 inhibitor is a 4-0-N-diacetyl-neuraminic acid. This would agree with the workof Blix and Lindberg (3) and Martensson et al.(12), which showed that the predominate sialicacid present in both horse serum and submaxil-lary mucins is the 4-0-acetyl NANA.

In the case of whole human serum, only NANAis responsible for the inhibition of both viruses.Thus neuraminidase is completely effective in re-moval of all activity. As would be expected,sodium metaperiodate oxidizes NANA to a mol-ecule which shows a high affinity for the typeB virus while destroying all the anti-A2 activity.The identification of the biologically active

sialic acids is interesting since if they are to actas effective competitive inhibitors in the virus-redcell interaction, they must be complementary tothe virus surface and reflect its basic configura-tion. Evidence supporting this hypothesis is thatthe appearance of the periodate enhancementeffect, not seen in influenza B viruses isolatedbefore 1965, is associated with a known antigenicchange (13).

In addition to being complementary to thevirus surface, the structure of the inhibitor shouldbe analogous to the structure of the cell surface.An interesting question raised by changes in theinhibitor spectrum of a virus is the continuedaffinity of the virus for the red-cell receptor, whichfor any particular species remains essentially thesame. Kathan and Winzler (9) have characterizedthe receptor substance previously isolated fromhuman red-cell stroma (10). The sialic acidpresent is probably NANA, all of which can beremoved by neuraminidase. It may be possiblethat small amounts of other substituted neur-aminic acid groups are present on human andchick red blood cells. However, more likely, thespecificities of the receptor groups are not abso-lute; for instance, the two inhibitor componentsin horse serum clearly cross-react to some degreebetween the two strains. It may be that slightchanges in the virus surface occur which merelymodify the binding efficiency of the virus with anygiven receptor without necessarily destroying itcompletely. Conceivably, such modification mayhave profound effects on the general biologicaleffectiveness of the virus.

In a recent article, Gottschalk (6) discussed thecriteria that glycoproteins must fulfill to be effec-tive inhibitors of influenza hemagglutination.Work with trypsinized ovine submaxillary glandglycoprotein and the soluble receptor fromhuman red cells has led to the conclusion that the

effectiveness of an inhibitory substance is a func-tion of molecular size, the number of terminalsialic acids, and of the three-dimensional structurecomplementary to an area on the virus surface.It appears, from the work reported here, that afourth criterion must be considered: the structureof the neuraminic acid.

ACKNOWLEDGMENTS

We gratefully acknowledge the financial support of the Rocke-feller Research Fund of University College Hospital MedicalSchool, the Fleming Memorial Trust, and the Wellcome Trust.

LITERATURE CITED

1. Belyavin, G. 1953. Influenza complement-fixation-a simplequantitative micro-method. J. Hyg. 51:492-501.

2. Biddle, F., D. Pepper, and G. Belyavin. 1965. Properties ofhorse serum gamma inhibitor. Nature 207:381-383.

3. Blix, G., and E. Lindberg. 1960. The sialic acids of bovineand equine submaxillary mucins. Acta Chem. S-and. 14:1809-1814.

4. Cohen, A. 1966. Automatic technique for titration of influenzavirus haemagglutination inhibitors. J. Clin. Pathol. 19:589-594.

5. Cohen, A., and G. Belyavin. 1959. Hemagglutination inhibi-tion of asian influenza viruses: a new pattern of response.Virology 7:59-74.

6. Gottschalk, A. 1966. Glycoproteins and viruses. In R. Jeanlozand E. Balazs (ed.), The amino sugars, Vol. IIB. Metabolismand interactions, p. 337-359. Academic Press Inc., NewYork.

7. Gottschalk, A., and P. Lind. 1949. Product of interaction between influenza virus enzyme and ovomucin. Nature 164:232-233.

8. Graham, E., and A. Gottschalk. 1960. Studies on mucopro-teins. I. The structure of the prosthetic group of ovine sub-maxillary gland mucoprotein. Biochim. Biophys. Acta38:513-524.

9. Kathan, R., and R. Winzler. 1963. Structure studies on themyxovirus hemagglutination inhibitor of human erythro-cytes. J. Biol. Chem. 238:21-25.

10. Kathan, R., R. Winzler, and C. Johnson. 1961. Preparation ofan inhibitor of viral hemagglutination from human erythro-cytes. J. Exp. Med. 113:37-45.

11. Klenk, E., H. Faillard, and H. Lempfrid. 1955. Uber dieenzymatische Wirkung von Influenzavirus. Z. Phys. Chem.301:235-246.

12. Martensson, E., A. Raal, and E. Svennerholm. 1958. Sialicacids in blood serum. Biochim. Biophys. Acta 30:124-129.

13. Mascoli, C., M. Leagus, and M. Hilleman. 1966. Influenza Bin the spring of 1965. Proc. Soc. Exp. Biol. Med. 123:952-960.

14. Pepper, D. 1967. A chromatographic procedure for the purifi-cation of influenza virus. J. Gen. Virol. 1:49-55.

15. Pepper, D. 1968. The isolation and properties of gamma in-hibitor: an equine a2-macroglobulin subfraction. Biochim.Biophys. Acta 156:327-339.

16. Pepper, D. 1968. The sialic acids of horse serum with specialreference to their virus inhibitory properties. Biochim.Biophys. Acta 156:317-326.

17. Neuberger, A., and R. Marshall. 1966. Structural analysis ofthe carbohydrate group of glycoproteins. In A. Gottschalk(ed.), Glycoproteins, p. 240-250. Elsevier, Amsterdam.

18. Schauer, R., and H. Faillard. 1968. Das verhalten isomererN-0-Diacetyl-neuraminsaureglykoside im Submaxillaris-mucin von Pferd und Rind bei Einwirung bakteriellerNeuraminidase. Z. Phys. Chem. 349:961-968.

19. Schneir, M., and M. Rafelson. 1966. Isolation and charac-terization of two structural isomers of N-acetylneuraminyl-

482 J. VIROL.

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

VOL. 3, 1969 DETERMINANTS OF VIRAL HEMAGGLUTINATION 483

lactose from bovine colostrum. Biochim. Biophys. Acta 22. Tamm, I., and F. Horsfall. 1952. A mucoprotein derived from130:1-11. human urine which reacts with influenza, mumps, and New-

20. Shimojo, H., A. Sugura, Y. Akao, and C. Enomoto. 1958. castle disease viruses. J. Exp. Med. 95:71-97.Studies of a non-specific haemagglutination inhibitor of 23. Warren, L. 1959. The thiobarbituric acid assay for sialic acids.influenza A/Asian/57 virus. Bull. Inst. Public Health J. Biol. Chem. 234:1971-1975.(Tokyo) 17:219-230.

21. Svennerholm, L. 1957. Quantitative estimation of sialic acids. 24 World Health Organization. 1959. Expert committee onII. A colorimetric resorcinol-hydrochloric acid method. respiratory virus diseases. World Health Organ. Tech. Rep.Biochim. Biophys. Acta 24:604-611. Ser 107:1-59.

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from